{"id":599,"date":"2022-08-27T11:20:51","date_gmt":"2022-08-27T11:20:51","guid":{"rendered":"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/?page_id=599"},"modified":"2023-12-27T18:27:17","modified_gmt":"2023-12-27T18:27:17","slug":"despre-medici","status":"publish","type":"page","link":"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/index.php\/despre-medici\/","title":{"rendered":"FOR DOCTORS"},"content":{"rendered":"\t\t<div data-elementor-type=\"wp-page\" data-elementor-id=\"599\" class=\"elementor elementor-599\">\n\t\t\t\t\t\t<section class=\"elementor-section elementor-top-section elementor-element elementor-element-ed98015 elementor-section-full_width elementor-section-height-default elementor-section-height-default\" data-id=\"ed98015\" data-element_type=\"section\">\n\t\t\t\t\t\t<div class=\"elementor-container elementor-column-gap-default\">\n\t\t\t\t\t<div class=\"elementor-column elementor-col-100 elementor-top-column elementor-element elementor-element-fc9a055\" data-id=\"fc9a055\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap elementor-element-populated\">\n\t\t\t\t\t\t<div class=\"elementor-element elementor-element-0eaddc3 elementor-widget elementor-widget-image\" data-id=\"0eaddc3\" data-element_type=\"widget\" data-widget_type=\"image.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t\t\t\t\t\t\t\t\t<img fetchpriority=\"high\" decoding=\"async\" width=\"1920\" height=\"900\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2021\/10\/Untitled-design-4.jpg\" class=\"attachment-2048x2048 size-2048x2048 wp-image-144\" alt=\"\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2021\/10\/Untitled-design-4.jpg 1920w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2021\/10\/Untitled-design-4-300x141.jpg 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2021\/10\/Untitled-design-4-1024x480.jpg 1024w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2021\/10\/Untitled-design-4-768x360.jpg 768w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2021\/10\/Untitled-design-4-1536x720.jpg 1536w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2021\/10\/Untitled-design-4-228x107.jpg 228w\" sizes=\"(max-width: 1920px) 100vw, 1920px\" \/>\t\t\t\t\t\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<div class=\"elementor-element elementor-element-b386068 elementor-widget elementor-widget-text-editor\" data-id=\"b386068\" data-element_type=\"widget\" data-widget_type=\"text-editor.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t\t\t<p><a href=\"http:\/\/www.interreg-rohu.eu\/\">www.interreg-rohu.eu<\/a><\/p>\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<section class=\"elementor-section elementor-top-section elementor-element elementor-element-749f98d elementor-section-boxed elementor-section-height-default elementor-section-height-default\" data-id=\"749f98d\" data-element_type=\"section\">\n\t\t\t\t\t\t<div class=\"elementor-container elementor-column-gap-default\">\n\t\t\t\t\t<div class=\"elementor-column elementor-col-100 elementor-top-column elementor-element elementor-element-89a98a2\" data-id=\"89a98a2\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap elementor-element-populated\">\n\t\t\t\t\t\t<div class=\"elementor-element elementor-element-073fb3f elementor-widget elementor-widget-text-editor\" data-id=\"073fb3f\" data-element_type=\"widget\" data-widget_type=\"text-editor.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t\t\t<p><span style=\"text-decoration: underline;\"><a href=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/index.php\/international-medical-conference\/\"><strong>International Medical Conference<\/strong><\/a><\/span><\/p>\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<section class=\"elementor-section elementor-top-section elementor-element elementor-element-99bfde5 elementor-section-boxed elementor-section-height-default elementor-section-height-default\" data-id=\"99bfde5\" data-element_type=\"section\">\n\t\t\t\t\t\t<div class=\"elementor-container elementor-column-gap-default\">\n\t\t\t\t\t<div class=\"elementor-column elementor-col-100 elementor-top-column elementor-element elementor-element-15cd842\" data-id=\"15cd842\" data-element_type=\"column\">\n\t\t\t<div class=\"elementor-widget-wrap elementor-element-populated\">\n\t\t\t\t\t\t<div class=\"elementor-element elementor-element-50fdbee elementor-widget elementor-widget-toggle\" data-id=\"50fdbee\" data-element_type=\"widget\" data-widget_type=\"toggle.default\">\n\t\t\t\t<div class=\"elementor-widget-container\">\n\t\t\t\t\t<div class=\"elementor-toggle\">\n\t\t\t\t\t\t\t<div class=\"elementor-toggle-item\">\n\t\t\t\t\t<div id=\"elementor-tab-title-8491\" class=\"elementor-tab-title\" data-tab=\"1\" role=\"button\" aria-controls=\"elementor-tab-content-8491\" aria-expanded=\"false\">\n\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon elementor-toggle-icon-left\" aria-hidden=\"true\">\n\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-closed\"><i class=\"fas fa-caret-right\"><\/i><\/span>\n\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-opened\"><i class=\"elementor-toggle-icon-opened fas fa-caret-up\"><\/i><\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t\t<\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t<a class=\"elementor-toggle-title\" tabindex=\"0\">Protocol for identification and characterization of isolated strains from pregnant, infertile women and newborn babies using automatic identification system for microorganisms after molecular fingerprint<\/a>\n\t\t\t\t\t<\/div>\n\n\t\t\t\t\t<div id=\"elementor-tab-content-8491\" class=\"elementor-tab-content elementor-clearfix\" data-tab=\"1\" role=\"region\" aria-labelledby=\"elementor-tab-title-8491\"><h6>MALDI-TOF Working Protocol<\/h6><p><strong>Process<\/strong> <strong>of<\/strong> <strong>rapid<\/strong> <strong>identification<\/strong> <strong>of<\/strong> <strong>pathogenic<\/strong> <strong>bacterial<\/strong> <strong>species<\/strong> <strong>using<\/strong> <strong>MALDI-TOF<\/strong> <strong>MS<\/strong> <strong>(Matrix-assisted<\/strong> <strong>laser<\/strong> <strong>desorption\/ionization<\/strong> <strong>&#8211;<\/strong> <strong>time<\/strong> <strong>of<\/strong> <strong>flight<\/strong> <strong>mass<\/strong> <strong>spectrometry)<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><p>The MALDI-TOF system can be used to identify clinically significant pathogens (bacteria, fungi) isolated from vaginal, cervical secretions, blood cultures (of pregnant or infertile patients, hospitalized in the Department of Obstetrics), as well as from hypopharyngeal aspirates, cerebrospinal fluid, blood cultures, etc. (of newborns hospitalized in the Department of Neonatology).<\/p><p>MALDI-TOF MS is a technique used in mass spectrometry that allows the analysis of biomolecules (such as DNA, ribosomal proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), and can be used to analyze the protein composition of a bacterial cell. The identification of bacterial proteins is based on their molecular weight.<\/p><p>MALDI-TOF MS involves a laser that acts on a matrix of small molecules to transform the analyte molecules into the gaseous phase without fragmenting or breaking them down. A mass spectrum is generated and automatically compared to a database of mass spectra by the software, resulting in the identification of the organism.<\/p><p>This technique has been shown to be effective due to its reproducibility, speed and sensitivity, with the advantage of MALDI-TOF MS over other methods of identification being that results are available in minutes to a few hours rather than days.<\/p><h6>Equipment and consumables:<\/h6><ul><li>Matrix HCCA (alpha-Cyano-4-hydroxycinnamic acid), ported<\/li><li>Standard solvent (acetonitrile 50%, water 5% and trifluoroacetic acid 2.5%)<\/li><li>Wooden chopsticks for transferring biological material or plastic inoculation loops<\/li><li>Pipette tips 5-10 \u03bcL, 2-200 \u03bcL, 50\u20131000 \u03bcL and corresponding pipettes<\/li><li>MALDI target plate<\/li><li>Tubes Shaker (vortexor)<\/li><li>Formic acid 70%<\/li><\/ul><p><\/p><h6>Preparation of HCCA matrix solution:<\/h6><ul><li>add 250 \u03bcL of standard solvent prepared in advance in a HCCA tube<\/li><li>dissolve HCCA by shaking\/vortexing at room temperature until the solution is clear\/transparent<\/li><\/ul><p><\/p><h6>Principle:<\/h6><ul><li>ideally, cultures not older than 24 hours should be used for identification, except for slow-growing germs, in which case older cultures (a few days) can also be<\/li><li>transfer biological material (e.g. an isolated bacterial colony) from a bacterial culture and apply as a thin film directly to a free position on the MALDI target plate<\/li><li>using the pipette and the 5-10 \u03bcL tips, add 1.0 (\u00b1 0.1) \u03bcL of the Matrix solution<\/li><li>leave to dry at room temperature (a homogeneous preparation must be obtained)<\/li><li>the MALDI-TOF-MS measurement is<\/li><\/ul><p><\/p><p><\/p><p><strong>Note:<\/strong> Although most bacteria will be easily identified by direct application to the Maldi target plate, some organisms possess the capsule that prevents effective lysis of the bacterial cells and obtaining identification. In these situations (e.g. in the case of fungi) a protein extraction procedure is required to identify them correctly. So when initial attempts at direct identification fail, the material can be superimposed with formic acid before the matrix is added.<\/p><p><\/p><h6>References<\/h6><ol><li>UK Standards for Microbiology Investigations Matrix-assisted laser desorption\/ionisation &#8211; time of flight mass spectrometry (MALDI-TOF MS) test procedure; Issued by the Standards Unit, Microbiology Services, PHE; Bacteriology \u2013 Test Procedures | TP 40 | Issue no: 1 | Issue date: 27.11.19<\/li><\/ol><p><br \/>MALDI Biotyper Bruker Standard Operating Procedure<\/p><\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t\t\t<div class=\"elementor-toggle-item\">\n\t\t\t\t\t<div id=\"elementor-tab-title-8492\" class=\"elementor-tab-title\" data-tab=\"2\" role=\"button\" aria-controls=\"elementor-tab-content-8492\" aria-expanded=\"false\">\n\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon elementor-toggle-icon-left\" aria-hidden=\"true\">\n\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-closed\"><i class=\"fas fa-caret-right\"><\/i><\/span>\n\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-opened\"><i class=\"elementor-toggle-icon-opened fas fa-caret-up\"><\/i><\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t\t<\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t<a class=\"elementor-toggle-title\" tabindex=\"0\"> Protocol for serological screening of pathogens from pregnant, infertile women and newborn babies using Advia Centaur XP \/ XPT<\/a>\n\t\t\t\t\t<\/div>\n\n\t\t\t\t\t<div id=\"elementor-tab-content-8492\" class=\"elementor-tab-content elementor-clearfix\" data-tab=\"2\" role=\"region\" aria-labelledby=\"elementor-tab-title-8492\"><p>WORKING PROTOCOL CHEMILUMINESCENTA<\/p><p>Chemiluminescence determinations (Anti-HAV IgM , Anti-HCV Ac, Anti-HIV Ac, HBsAg,<br \/>Toxo-IGG, Toxo-IGM, Rub-IGG, Rub-IGM, CMV-IGG, CMV-IGM, EPB-IGG,EPB-IGM)<\/p><p>Viral and parasitic infectious pathology occupy an important place in the management<br \/>of pregnancy at risk, as well as neonatal infections. In this regard, viral hepatitis, HIV infection,<br \/>toxoplasmosis, rubella, CMV and EBV infection are some of the most common infections<br \/>diagnosed.<\/p><p>Anti-HAV IgM<\/p><p>a) Purpose of the examination: qualitative determination of IgM antibodies against<br \/>hepatitis A virus (anti-HAV IgM) using the chemiluminescent method; the<br \/>determination is used as a support for the diagnosis of an acute or recent (usually<br \/>less than 6 months) infection with hepatitis A virus<br \/>b) Principle and method of procedure used for examinations: the method is based<br \/>on two-site immunometry; the light signals emitted by the system are directly<br \/>proportional to the amount of anti-HAV IgM in the sample<br \/>c) Performance characteristics: linearity: 0.02 \u2013 7.0 S\/CO; specificity = 100%,<br \/>sensitivity = 100%, CV = 7.5%<br \/>d) Sample type:<br \/>\u2022 serum, heparinized plasma or EDTA plasma<br \/>\u2022 test as soon as possible after harvesting.<br \/>\u2022 primary samples can be stored closed in the refrigerator until 7 days after<br \/>collection<br \/>\u2022 serum\/plasma samples can be frozen up to 180 days after collection<br \/>e) Type of container and additives: red stopper container without additive or green<br \/>stopper container with heparin or purple stopper container with EDTA<br \/>f) Calibration procedures<br \/>\u2022 Calibration is performed when changing the lot of reagent and calibrator,<br \/>once every 28 days,after major service interventions, in case of failure of<br \/>internal quality control<\/p><p>g) Interference<br \/>\u2022 Haemoglobin &gt; 500 mg\/dL<br \/>\u2022 Triglycerides &gt;3000 mg\/dL<br \/>\u2022 Bilirubin &gt; 60 mg\/dL<br \/>\u2022 Proteinaemia: &gt; 12 g\/dL<br \/>h) Reference biological ranges: Non-reactive<\/p><p>Translation from Romanian<\/p><p>i) Clinical Laboratory Interpretation:<br \/>\u2022 Samples with value &lt; 0.8 S\/CO (signal to cutoff) are considered non-reactive<br \/>\u2022 Samples with a value of \u2265 0.8 S\/CO but less than 1.20 S\/CO are<br \/>considered inconclusive and must be repeated; it is recommended that the<br \/>repeat be done in duplicate and the result released be based on the results of<br \/>the repeat; if the result of the repeat is still inconclusive, it is recommended to<br \/>obtain a new specimen and test it<br \/>\u2022 Samples \u2265 1.20 S\/CO are considered reactive<br \/>j) Potential sources of variation:<br \/>a) Heterophilic antibodies present in serum or plasma may interfere with<br \/>immunological tests<br \/>b) The determination is not validated for other biological products<br \/>c) igM anti-HAV are usually detectable between 3 and 6 months after the onset of the<br \/>disease (anti-HAV IgG persists throughout life)<\/p><p>Anti-HCV needle<\/p><p>a) Purpose of examination: qualitative detection of IgG antibodies against hepatitis C<br \/>virus (anti-HCV) using the chemiluminescent method;determination may be used in<br \/>conjunction with other serological tests and clinical information for diagnostic<br \/>purposes in patients with symptoms of hepatitis and in individuals at risk of HCV<br \/>infection; the product is not intended for screening or testing of serum pools, other<br \/>blood types or plasma from more than one individual<br \/>b) Principle and method of the procedure used for examinations: the method is based<br \/>on two-site immunometry (sandwich technique); the light signals emitted by the<br \/>system are directly proportional to the anti-HCV in the sample<br \/>c) Performance characteristics: linearity: 0.0 \u2013 11.0 index value; specificity = 99.90%,<br \/>sensitivity = 100%, CV = 6.6%<br \/>d) Sample type:<br \/>\u2022 serum, heparinized plasma or EDTA plasma<br \/>\u2022 samples are handled as potentially infectious<br \/>\u2022 the samples are tested as soon as possible after collection (they can be kept<br \/>uncentrifuged for up to 24 hours at room temperature)<br \/>\u2022 primary samples can be kept for up to 7 days in the refrigerator<br \/>\u2022 serum\/plasma samples can be frozen<br \/>e) Type of container and additives: red stopper container without additive or green<br \/>stopper container with heparin or purple stopper container with EDTA<br \/>f) Calibration procedures<br \/>\u2022 Calibration is performed when changing the lot of reagent and calibrator, once<br \/>every 28 days,<br \/>after major service interventions, in case of failure of internal quality control<\/p><p>g) Interference<br \/>\u2022 Haemoglobin &gt; 500 mg\/dL<br \/>\u2022 Triglycerides &gt; 1000 mg\/dL<br \/>\u2022 Bilirubin &gt; 60 mg\/dL<br \/>\u2022 Protein level 3-12 g\/dL<br \/>h) Reference biological ranges: Index value &lt; 0.8 (Non-reactive)<br \/>i) Clinical Laboratory Interpretation:<br \/>\u2022 Samples with index value less than 0.8 are considered non-reactive<br \/>\u2022 Samples with index value \u2265 0.8 but less than 1.0 are considered inconclusive;<br \/>testing is repeated in duplicate; if 2 of the 3 are with index value less than 0.8,<br \/>the sample is considered non-reactive; if 2 of the 3 have index value \u22651.0, the<br \/>sample is considered reactive and confirmatory tests are recommended; if 2 of<br \/>the 3 are with index value \u2265 0.8 and &lt;1 it is recommended to perform additional<br \/>tests<br \/>\u2022 Samples with index value \u2265 1.0 are considered reactive; testing is repeated in<\/p><p>duplicate; if 2 of the 3 are with index value &lt; 0.8, the sample is considered non-<br \/>reactive; if 2 of the 3 have index value \u22651.0, the sample is considered reactive<\/p><p>and confirmatory tests are recommended; if 2 of the 3 are with index value \u2265 0.8<br \/>and &lt;1.0, additional tests are recommended<br \/>\u2022 The results are considered invalid and must be repeated if the controls are<br \/>outside the allowed limits<br \/>j) Potential sources of variation:<br \/>\u2022 Heterophilic antibodies present in serum or plasma may interfere with<br \/>immunological tests<br \/>\u2022 The determination is not validated for other biological products<br \/>\u2022 If the result is used for diagnostic purposes, its interpretation should be made in<br \/>conjunction with the clinical examination, the patient&#8217;s history or other types of<br \/>tests<br \/>\u2022 A negative result does not exclude the possibility of exposure or infection with<br \/>HCV; HCV antibodies may be undetectable at some stages of infection and in<br \/>some clinical situations<br \/>\u2022 Test performance has not been studied in immunocompromised populations and<br \/>patients<\/p><p>Anti-HIV needle<\/p><p>a) Purpose of the examination: Qualitative detection of antibodies against human<br \/>immunodeficiency virus type 1, including subtype O and\/or type 2 (anti-HIV) using the<br \/>chemiluminescent method<\/p><p>b) Principle and method of the procedure used for examinations: the method is<br \/>based on two-site immunometry (sandwich technique); the light signals emitted by the<br \/>system are directly proportional to the anti-HIV in the sample<br \/>c) Performance characteristics: linearity: 0.05 &#8211; 50 index value; specificity = 99.87%,<br \/>sensitivity = 100%, CV = 8.8 &#8211; 11.6%<br \/>d) Sample type:<br \/>\u2022 serum, heparinized plasma or EDTA plasma<br \/>\u2022 samples are handled as potentially infectious<br \/>\u2022 the samples are tested as soon as possible after collection (primary samples can be<br \/>kept uncentrifuged for up to 24 hours at room temperature)<br \/>\u2022 primary samples can be kept for up to 7 days in the refrigerator<br \/>\u2022 serum\/plasma samples can be frozen<br \/>e) Type of container and additives: red stopper container without additive or green<br \/>stopper container with heparin or purple stopper container with EDTA<br \/>f) Calibration procedures<br \/>\u2022 Calibration is performed when changing the lot of reagent and calibrator, once every<br \/>28 days, after major service interventions, in case of failure of internal quality<br \/>control.<br \/>g) Interference<br \/>\u2022 Haemoglobin &gt; 500 mg\/dL<br \/>\u2022 Triglycerides &gt;3000 mg\/dL<br \/>\u2022 Bilirubin &gt; 30 mg\/dL<br \/>\u2022 Protein level 3-12 g\/dL<br \/>h) Reference biological ranges: Index value &lt; 1.0 (Non-reactive)<br \/>i) Clinical Laboratory Interpretation:<br \/>\u2022 Samples with index value &lt; 1.0 are considered non-reactive<br \/>\u2022 Samples with index value \u2265 1.0 are initially considered reactive and are rewritten<br \/>in duplicates after centrifugation; if one or both duplicates are reactive, the<br \/>sample is considered repeatedly reactive and confirmatory tests are<br \/>recommended; if confirmatory tests are positive, then the sample is considered<br \/>to be positive<br \/>\u2022 Initially reactive samples where both duplicates are non-reactive (index value<br \/>&lt;1.0) are considered negative<br \/>\u2022 The results are considered invalid and must be repeated if the controls are<br \/>outside the allowed limits<br \/>j) Potential sources of variation:<br \/>\u2022 Heterophilic antibodies present in serum or plasma may interfere with<br \/>immunological tests<\/p><p>\u2022 The determination is not validated for other biological products<br \/>\u2022 It is not recommended to test blood pools or products derived from such<br \/>pools<br \/>\u2022 Test performance has not been studied in immunocompromised populations<br \/>and patients<br \/>\u2022 The test may not detect anti-HIV antibodies in all infected individuals; a<br \/>negative result does not exclude the possibility of exposure or infection with<br \/>HIV; anti-HIV antibodies may be undetectable at some stages of infection or<br \/>in some clinical situations<\/p><p>AgHBs<\/p><p>a) Purpose of examination: qualitative detection of hepatitis B virus surface antigen<br \/>(HBsAg) using the chemiluminescent method; determination may be used in<br \/>conjunction with other serological tests and clinical information for the diagnosis of<br \/>acute and chronic hepatitis B virus infection; determination may also be used for<br \/>screening for hepatitis B virus infection in pregnant women to identify newborns at risk<br \/>of infection in the neonatal period<br \/>b) Principle and method of the procedure used for examinations: the method is based on<br \/>two-site immunometry (sandwich technique); the light signals emitted by the system<br \/>are directly proportional to the AgHBs in the sample<br \/>c) Performance characteristics: linearity: 0.1 \u2013 1000 index value; initial specificity =<br \/>99.51%, specificity after retesting = 99.91%, sensitivity = 100%, CV = 3.6%<br \/>d) Sample type:<br \/>\u2022 serum, heparinized plasma or EDTA plasma<br \/>\u2022 samples are handled as potentially infectious<br \/>\u2022 the samples are tested as soon as possible after collection (they can be kept<br \/>uncentrifuged for up to 24 hours at room temperature)<br \/>\u2022 primary samples can be stored closed in the refrigerator until 7 days after<br \/>collection<br \/>\u2022 serum\/plasma samples can be stored in the refrigerator for up to 14 days or can<br \/>be frozen<\/p><p>e) Type of container and additives: red stopper container without additive or green stopper<br \/>container with heparin or purple stopper container with EDTA<br \/>f) Calibration procedures<br \/>\u2022 Calibration is performed when changing the lot of reagent and calibrator, once every<br \/>21 days, after major service interventions, in case of failure of internal quality control<br \/>g) Interference<br \/>\u2022 Haemoglobin &gt; 500 mg\/dL<br \/>\u2022 Triglycerides &gt; 1000 mg\/dL<br \/>\u2022 Bilirubin &gt; 40 mg\/dL<br \/>\u2022 Cholesterol &gt; 400 mg\/dL<\/p><p>\u2022 Protein level 3-12 g\/dL<br \/>h) Reference biological ranges: Index value &lt; 1.0 (Negative)<br \/>i) Clinical Laboratory Interpretation:<br \/>\u2022 Samples with an index value of less than 1.0 are considered non-reactive (negative)<br \/>\u2022 Samples with index value \u2265 1.0 but less than or equal to 50 are considered reactive<br \/>(positive); the test is repeated in duplicate; if 2 of the 3 results are non-reactive<br \/>(negative) the result is negative for HBsAg; if 2 of the 3 results are reactive (positive)<br \/>the result is considered to be repeated reactive (positive) for HBsAg and confirmatory<br \/>tests are recommended, other markers of HBB infection<br \/>\u2022 Samples with index value &gt; 50 or signaled by the device as \u201c&gt;Index Range\u201d are<br \/>considered reactive (positive) for HBsAg and confirmatory tests are recommended<br \/>\u2022 The results are considered invalid and must be repeated if the controls are outside<br \/>the allowed limits<br \/>j) Potential sources of variation:<br \/>\u2022 Heterophilic antibodies present in serum or plasma may interfere with<br \/>immunological tests<br \/>\u2022 The determination is not validated for other biological products<br \/>\u2022 If the result is used for diagnostic purposes, its interpretation should be made in<br \/>conjunction with the clinical examination, the patient&#8217;s history or other types of tests<br \/>\u2022 A negative result does not exclude the possibility of exposure or infection with HBV; a<br \/>negative result in a patient exposed to HBV may be generated by HBsAg levels below<br \/>the limit of detection of the HBsAg method or lack of reactivity with antibodies from<br \/>the reaction<\/p><p>TOXO IgG<\/p><p>a) Purpose of the examination: ToxoplasmaG (Toxo G) is an IgG antibody that captures<br \/>microparticles directly chemiluminometric for the in vitro diagnosis of the quantity and<br \/>quality of Toxoplasma gondii IgG antigens in serum or plasma (EDTA heparin). The test<br \/>can also be used to screen for Toxoplasma G infection in pregnant women to identify<br \/>newborns at risk of infection during the neonatal period.<br \/>b) Principle and method of the procedure used for examinations: the method is based on<br \/>direct immunometry with two sites (sandwich technique); human monoclonal antibodies<br \/>are covalently coupled with paramagnetic particles in the solid phase.<br \/>c) Performance characteristics: linearity: 0.5 \u2013 700 index value; initial specificity = 96.5%,<br \/>specificity after retesting = 99.91%, sensitivity = 96.5%,<br \/>d) Sample type:<\/p><p>\u2022 Serum, heparinized plasma or EDTA plasma<br \/>\u2022 Samples are handled as potentially infectious<br \/>\u2022 Store samples at 2\u20138\u00b0C for up to 7 days<br \/>\u2022 Freeze samples, free of red blood cells, at or below -20\u00b0C for longer storage<\/p><p>\u2022 Samples stored at room temperature for up to 7 days or refrigerated for up to<br \/>14 days<\/p><p>e) Type of container and additives: red stopper container without additive or green<br \/>stopper container with heparin or purple stopper container with EDTA<br \/>f) CALIBRATION PROCEDURES<br \/>\u2022 Calibration is performed when changing the lot of reagent and calibrator, once every<br \/>14 days, after major service interventions, in case of failure of internal quality control<br \/>g) Interference:<br \/>\u2022 Haemoglobin &gt; 500 mg\/dL<br \/>\u2022 Triglycerides &gt; 1000 mg\/dL<br \/>\u2022 Bilirubin &gt; 40 mg\/dL<br \/>\u2022 Protein level 3-12 g\/dL<br \/>h) Reference biological ranges: Index value &lt; 10.0 (Negative)<br \/>i) Clinical Laboratory Interpretation:<\/p><p>\u2022 Samples with a calculated value of less than 6.4 IU\/mL are considered non-<br \/>reactive<\/p><p>\u2022 Samples with a calculated value between 6.4 and 9.9 IU\/mL are equivocal<br \/>\u2022 Samples with a calculated value greater than or equal to 10.0 IU\/mL are<br \/>reactive<br \/>\u2022 The results are considered invalid and must be repeated if the controls are<br \/>outside the allowed limits<br \/>\u2022<\/p><p>j) Potential sources of variation:<br \/>\u2022 Heterophilic, antinuclear (AAN), and antimytochondrial (AMA) antibodies present<br \/>in serum or plasma may interfere with immunological tests.<br \/>\u2022 The determination is not validated for other biological products<br \/>\u2022 If the result is used for diagnostic purposes, its interpretation should be made in<br \/>conjunction with the clinical examination, the patient&#8217;s history or other types of<br \/>tests<br \/>\u2022 Pay attention to the interpretation of the results for patients who have been<br \/>transfused recently or for several months.<\/p><p>TOXO-IgM<\/p><p>a) Purpose of the examination: ToxoplasmaM (Toxo M) is an IgM antibody that captures<br \/>microparticles directly chemiluminometric for the in vitro diagnosis of the quantity and<br \/>quality of IgM antigens of Toxoplasma gondii in serum or plasma (EDTA heparin). The<br \/>determination can also be used to screen for Toxoplasma M infection in pregnant<br \/>women to identify newborns at risk of infection during the neonatal period.<\/p><p>b) Principle and method of the procedure used for examinations: the method is based on<br \/>direct immunometry with two sites (sandwich technique); human monoclonal antibodies<br \/>are covalently coupled with paramagnetic particles in the solid phase.<br \/>c) Performance characteristics: linearity: 0.5 \u2013 700 index value; initial specificity = 99%,<br \/>specificity after retesting = 99.91%, sensitivity = 99.2%,<br \/>d) Sample type:<br \/>\u2022 Serum, heparinized plasma or EDTA plasma<br \/>\u2022 Samples are handled as potentially infectious<br \/>\u2022 Store samples at 2\u20138\u00b0C for up to 7 days<br \/>\u2022 Freeze samples, free of red blood cells, at or below -20\u00b0C for longer storage<br \/>\u2022 Samples stored at room temperature for up to 7 days or refrigerated for up to 14<br \/>days<\/p><p>e) Type of container and additives: red stopper container without additive or green<br \/>stopper container with heparin or purple stopper container with EDTA<br \/>f) Calibration procedures:<br \/>\u2022 Calibration is performed when changing the lot of reagent and calibrator, once<br \/>every 14 days, after major service interventions, in case of failure of internal<br \/>quality control<br \/>g) Interference:<br \/>\u2022 Haemoglobin &gt; 500 mg\/dL<br \/>\u2022 Triglycerides &gt; 1000 mg\/dL<br \/>\u2022 Bilirubin &gt; 40 mg\/dL<br \/>\u2022 Protein level 3-12 g\/dL<br \/>h) Reference biological ranges: Index value &lt; 1.0 (Negative)<br \/>i) Clinical Laboratory Interpretation:<\/p><p>\u2022 Samples with a calculated value less than &lt;0.9 IU\/mL are considered non-<br \/>reactive<\/p><p>\u2022 Samples with a calculated value between 0.9 and 0.99 IU\/mL are equivocal<br \/>\u2022 Samples with a calculated value greater than or equal to 1.0 IU\/mL are reactive<br \/>\u2022 The results are considered invalid and must be repeated if the controls are<br \/>outside the allowed limits<br \/>j) Potential sources of variation:<br \/>\u2022 Heterophilic antibodies present in serum or plasma may interfere with<br \/>immunological tests<br \/>\u2022 The determination is not validated for other biological products<br \/>\u2022 If the result is used for diagnostic purposes, its interpretation should be made in<br \/>conjunction with the clinical examination, the patient&#8217;s history or other types of<br \/>tests<\/p><p>\u2022 Pay attention to the interpretation of the results for patients who have been<br \/>transfused recently or for several months.<\/p><p>RUB-IgG<\/p><p>a) Purpose of examination: In vitro diagnosis for the quantitative and qualitative detection<br \/>of Rubella virus IgG antibodies in serum or plasma (EDTA heparin)<br \/>b) Principle and method of the procedure used for examinations: the method is based on<br \/>direct immunometry with two sites (sandwich technique); human monoclonal<br \/>antibodies are covalently coupled with paramagnetic particles in the solid phase.<br \/>c) Performance characteristics: linearity: 0.2 \u2013 500 index value; initial specificity = 98.8%,<br \/>specificity after retesting = 99.91%, sensitivity = 99.4%,<br \/>d) Sample type:<\/p><p>\u2022 Serum, heparinized plasma or EDTA plasma<br \/>\u2022 Samples are handled as potentially infectious<br \/>\u2022 Freeze samples, free of red blood cells, at or below -20\u00b0C for longer storage<br \/>\u2022 Samples stored at room temperature for up to 7 days or refrigerated for up to<br \/>14 days<\/p><p>e) Type of container and additives: red stopper container without additive or green<br \/>stopper container with heparin or purple stopper container with EDTA<br \/>f) CALIBRATION PROCEDURES<br \/>\u2022 Calibration is performed when changing the lot of reagent and calibrator, once<br \/>every 14 days, after major service interventions, in case of failure of internal<br \/>quality control<br \/>g) Interference:<br \/>\u2022 Haemoglobin &gt; 500 mg\/dL<br \/>\u2022 Triglycerides &gt; 1000 mg\/dL<br \/>\u2022 Bilirubin &gt; 40 mg\/dL<br \/>\u2022 Protein level 3-12 g\/dL<br \/>h) Reference biological ranges: Index value &lt; 5.0 IU\/mL (Negative)<br \/>i) Clinical Laboratory Interpretation:<\/p><p>\u2022 Samples with a calculated value less than &lt; 5.0 IU\/mL are considered non-<br \/>reactive<\/p><p>\u2022 Samples with a calculated value between 5 and 9.9 IU\/mL are equivocal<br \/>\u2022 Samples with a calculated value greater than or equal to 10.0 IU\/mL are<br \/>reactive<br \/>\u2022 The results are considered invalid and must be repeated if the controls are<br \/>outside the allowed limits<br \/>j) Potential sources of variation:<\/p><p>\u2022 The determination is not validated for other biological products<br \/>\u2022 If the result is used for diagnostic purposes, its interpretation should be made<br \/>in conjunction with the clinical examination, the patient&#8217;s history or other<br \/>types of tests<\/p><p>RUB-IgM<\/p><p>a) Purpose of the examination: In vitro diagnosis for the quantitative and qualitative<br \/>detection of IgM antibodies of Rubella virus, in serum or plasma (EDTA heparin)<br \/>b) Principle and method of the procedure used for examinations: the method is based on<br \/>direct immunometry with two sites (sandwich technique); human monoclonal<br \/>antibodies are covalently coupled with paramagnetic particles in the solid phase.<br \/>c) Performance characteristics: linearity: 0.2 \u2013 500 index value; initial specificity = 99.1%,<br \/>specificity after retesting = 99.91%, sensitivity = 91.0%,<br \/>d) Sample type:<br \/>\u2022 Serum, heparinized plasma or EDTA plasma<br \/>\u2022 Samples are handled as potentially infectious<br \/>\u2022 Freeze samples at or below -20\u00b0C for longer storage<br \/>\u2022 Samples stored at room temperature for up to 7 days or refrigerated for up to 14<br \/>days<\/p><p>e) Type of container and additives: red stopper container without additive or green<br \/>stopper container with heparin or purple stopper container with EDTA<br \/>f) CALIBRATION PROCEDURES<\/p><p>\u2022 Calibration is performed when changing the lot of reagent and calibrator,<br \/>once every 14 days, after major service interventions, in case of failure of<br \/>internal quality control<\/p><p>g) Interference:<\/p><p>\u2022 Haemoglobin &gt; 500 mg\/dL<br \/>\u2022 Triglycerides &gt; 1000 mg\/dL<br \/>\u2022 Bilirubin &gt; 40 mg\/dL<br \/>\u2022 Protein level 3-12 g\/dL<br \/>\u2022 Hypergamaglobulinaemia &gt;3 mg\/mL<br \/>h) Reference biological ranges: Index value &lt; 0.80 IU\/mL(Negative)<br \/>i) Clinical Laboratory Interpretation:<\/p><p>\u2022 Samples with a calculated value less than &lt;0.80 IU\/mL are considered<br \/>non-reactive<br \/>\u2022 Samples with a calculated value between 0.80 and 0.99 IU\/mL are<br \/>equivocal<\/p><p>\u2022 Samples with a calculated value greater than or equal to 1.0 IU\/mL are<br \/>reactive<br \/>\u2022 The results are considered invalid and must be repeated if the controls<br \/>are outside the allowed limits<br \/>j) Potential sources of variation:<br \/>\u2022 The determination is not validated for other biological products<br \/>\u2022 If the result is used for diagnostic purposes, its interpretation should be made in<br \/>conjunction with the clinical examination, the patient&#8217;s history or other types of<br \/>tests<br \/>\u2022 Specimens taken early during the acute phase of infection may not contain<br \/>detectable levels of Rubella IgM antibodies, this does not rule out a primary<br \/>infection.<\/p><p>CMV IgG<\/p><p>a) Purpose of the examination: qualitative determination of IgG antibodies for<br \/>Cytomegalovirus (CMV) in human serum in order to determine the immune status to<br \/>Cytomegalovirus.<br \/>b) Principle and method of procedure used for examinations: Chemiluminescence.<br \/>c) Performance characteristics:.<br \/>\u2022 Sensitivity : 100%<br \/>\u2022 Specificity: 100%<br \/>d) Sample type:<br \/>\u2022 human serum or plasma (heparinised or EDTA).<br \/>\u2022 in case of lipemic serums, ultracentrifugation is recommended<br \/>\u2022 The primary samples can be stored closed in the refrigerator until 3 days after<br \/>collection, at 2\u20138\u00b0C,<br \/>\u2022 Serum samples can be stored in the freezer for up to 6 months at \u201320\u00b0C.<br \/>g) Interference<br \/>\u2022 Bilirubin: The presence of direct or indirect bilirubin in concentrations up to 200 mg\/L<br \/>does not affect the result.<br \/>\u2022 Hemolysis: the presence of hemoglobin in concentrations up to 539 mg\/dL does not<br \/>affect the result.<br \/>\u2022 Lipemia: The presence of triglycerides in concentrations up to 3000 mg\/dL does not<br \/>affect the result .<br \/>h) Clinical Laboratory Interpretation:<\/p><p>\u2022 Reagent: A ratio of \u2265 1.1 S\/CO indicates that IgG antibodies have been detected in<br \/>the patient&#8217;s serum.<br \/>\u2022 Non-reactive: a ratio &lt; 0.9 S\/CO indicates that IgG antibodies have not been<br \/>detected in the patient&#8217;s serum<br \/>\u2022 Inconclusive: a ratio of 0.9 to 1.1 S\/CO, requires retesting.<br \/>i) Potential sources of variation:<br \/>\u2022 Heterophilic antibodies in human serum may interfere with immunological tests.<\/p><p>\u2022 An increased level of IgG antibodies can be found in the case of measles, varicella-<br \/>zoster virus, Epstein-Barr.<\/p><p>CMV IgM<\/p><p>a) Purpose of examination: qualitative determination of IgM antibodies against<br \/>Cytomegalovirus(CMV) for the purpose of diagnosis of acute cytomegalovirus infection.<br \/>b) Principle and method of procedure used for examinations: Chemiluminescence .<br \/>c) Type of sample:<br \/>\u2022 serum or plasma (EDTA or heparinized)<br \/>\u2022 The primary samples can be stored closed in the refrigerator until 3 days after<br \/>collection, at 2\u20138\u00b0C,<br \/>\u2022 Serum samples can be stored in the freezer for up to 6 months at \u201320\u00b0C.<br \/>\u2022<br \/>d) Interference:<br \/>Bilirubin: The presence of bilirubin in concentrations up to 200 mg\/L does not affect the<br \/>result.<br \/>Hemolysis: The presence of hemoglobin in concentrations up to 522 mg\/dL does not affect<br \/>the result<br \/>Lipemia: The presence of triglycerides in concentrations up to 3000 mg\/dL does not affect<br \/>the result.<br \/>e) Reference biological ranges: Non-reactive<br \/>f) Clinical Laboratory Interpretation:<br \/>\u2022 Reagent: A ratio of \u2265 1.1 S\/CO indicates that IgM antibodies have been detected in<br \/>the patient&#8217;s serum. The presence of IgM antibodies indicates that the patient had a<br \/>recent exposure to Cytomegalovirus.<br \/>\u2022 Non-reactive: a ratio &lt; 0.9 S\/CO indicates that IgM antibodies have not been<br \/>detected in the patient&#8217;s serum<br \/>\u2022 Inconclusive: a ratio of 0.9 to 1.1 S\/CO, requires retesting.<br \/>g) Potential sources of variation:<\/p><p>\u2022 An increased level of antibodies can be found in the case of measles, varicella-zoster<br \/>virus, Epstein-Barr.<br \/>\u2022 Samples containing antinuclear antibodies may give false reactive results<\/p><p>EBV-EBNA IgG<\/p><p>a) Purpose of the examination: Qualitative detection of IgG antibodies to the Epstein-Barr<br \/>virus (EBNA) nuclear antigen in human serum or plasma. This test can also be used to help<br \/>diagnose EBV-related diseases.<br \/>(b) Principle and method of the procedure used for examinations: two-stage solid-state<br \/>chemiluminescent enzyme immunoassay.<br \/>d) Sample type:<br \/>\u2022 human serum or plasma (heparinised or EDTA)<br \/>\u2022 In the case of lipemic serums it is recommended to use an ultracentrifuge.<br \/>\u2022 Hemolysed samples may indicate maltreatment of a sample prior to receipt by the<br \/>laboratory; therefore results should be interpreted with caution.<br \/>\u2022 The primary samples can be stored closed in the refrigerator until 3 days after<br \/>collection, at 2\u20138\u00b0C,<br \/>\u2022 Serum samples can be stored in the freezer for up to 6 months at \u201320\u00b0C.<br \/>g) Interference<br \/>\u2022 Bilirubin: The presence of conjugated and unconjugated bilirubin in concentrations<br \/>up to 200 mg\/L has no effect on the results.<br \/>\u2022 Haemolysis: The presence of haemoglobin in concentrations up to 537 mg\/dL has no<br \/>effect on the results.<br \/>\u2022 Lipemia: The presence of triglycerides in concentrations up to 3000 mg\/dL has no<br \/>effect on the results<br \/>h) Reference biological ranges<br \/>\u2022 Reagent : a ratio of \u2265 1.1 S\/CO . The report indicates that EBV-EBNA IgG antibodies<br \/>were detected in the patient sample.<br \/>The presence of IgG antibodies to EBV-EBNA is an indication of prior exposure to the<br \/>virus.<br \/>\u2022 Non-reactive: a ratio of &lt; 0.9. Indicates that EBV-EBNA IgG antibodies were not<br \/>detected in the patient sample.<br \/>\u2022 Inconclusive: a ratio of 0.9 to 1.1 S\/CO, requires retesting. Alternative testing is<br \/>recommended, or a second sample taken, within a reasonable period of time , e.g.<br \/>one week.<br \/>j) Potential sources of variation:<br \/>\u2022 Samples containing antinuclear antibodies or other anti-cellular antibodies may give<br \/>false positive results.<\/p><p>EBV-EBNA IgM<\/p><p>a) Purpose of the examination: for the qualitative detection of IgM antibodies to<br \/>EpsteinBarr virus viral capsid antigen in human serum or plasma.<br \/>b) Principle and method of the procedure used for examinations: chemiluminescent IgM<br \/>capture immunoassay, solid phase.<br \/>c) Performance characteristics: total concordance: 91.1%, sensitivity: 89.4%, specificity:<br \/>96.4%.<br \/>d) Sample type:<br \/>\u2022 serum or plasma (with EDTA and heparinized) In the case of lipemic serums it is<br \/>recommended to use an ultracentrifuge.<br \/>\u2022 Hemolysed samples may indicate maltreatment of a sample prior to receipt by the<br \/>laboratory; therefore results should be interpreted with caution.<br \/>\u2022 The primary samples can be stored closed in the refrigerator until 3 days after<br \/>collection, at 2\u20138\u00b0C,<br \/>\u2022 Serum samples can be stored in the freezer for up to 6 months at \u201320\u00b0C.<br \/>f) Interference:<br \/>\u2022 Biotin: Specimens containing biotin at a concentration of 1500 ng\/ml demonstrate a<br \/>change of less than or equal to 10% in the results. Biotin concentrations higher than<br \/>this may result in incorrect results for patient samples.<br \/>\u2022 Bilirubin: The presence of conjugated and unconjugated bilirubin in concentrations<br \/>up to 200 mg\/L has no effect on the results.<br \/>\u2022 Haemolysis: The presence of haemoglobin in concentrations up to 269 mg\/dl has no<br \/>effect on the results.<br \/>\u2022 Lipemia: The presence of triglycerides in concentrations up to 3000 mg\/dL has no<br \/>effect on the results.<br \/>g) Biological reference ranges:<br \/>\u2022 Reagent : a ratio of \u2265 1.1 S\/CO . The report indicates that IgM EBV-EBNA antibodies<br \/>were detected in the patient sample.<br \/>\u2022 Non-reactive: a ratio of &lt; 0.9. Indicates that IgM EBV-EBNA antibodies were not<br \/>detected in the patient sample.<br \/>\u2022 Inconclusive: a ratio of 0.9 to 1.1 S\/CO, requires retesting. Alternative testing is<br \/>recommended, or a second sample taken, within a reasonable period of time , e.g.<br \/>one week.<br \/>h) Potential sources of variation:<br \/>\u2022 Samples containing antinuclear antibodies or other anti-cellular antibodies may give<br \/>false positive results.<\/p><\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t\t\t<div class=\"elementor-toggle-item\">\n\t\t\t\t\t<div id=\"elementor-tab-title-8493\" class=\"elementor-tab-title\" data-tab=\"3\" role=\"button\" aria-controls=\"elementor-tab-content-8493\" aria-expanded=\"false\">\n\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon elementor-toggle-icon-left\" aria-hidden=\"true\">\n\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-closed\"><i class=\"fas fa-caret-right\"><\/i><\/span>\n\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-opened\"><i class=\"elementor-toggle-icon-opened fas fa-caret-up\"><\/i><\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t\t<\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t<a class=\"elementor-toggle-title\" tabindex=\"0\">Protocol for MIC identification and interpretative reading of antimicrobial sensitivity tests for the most important bacteria and fungi species isolated from pregnant, infertile women and newborn babies using Vitek 2C and Sensititre plates. <\/a>\n\t\t\t\t\t<\/div>\n\n\t\t\t\t\t<div id=\"elementor-tab-content-8493\" class=\"elementor-tab-content elementor-clearfix\" data-tab=\"3\" role=\"region\" aria-labelledby=\"elementor-tab-title-8493\"><h6>Vitek 2C System Work Protocol<\/h6><p><strong>\u00a0<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><p>The Vitek 2 Compact System can be used both to identify and to test the sensitivity to antibiotics of pathogens of clinical significance (bacteria, fungi) isolated from vaginal, cervical secretions, blood cultures (of pregnant or infertile patients, hospitalized in the Department of Obstetrics), as well as from hypopharyngeal aspirates, cerebrospinal fluid, blood cultures, etc. (of newborns hospitalized in the Department of Neonatology).<\/p><p>The Vitek 2 Compact System uses a fluorogenic methodology for the identification of microorganisms and a turbidimetric method for antibiotic sensitivity testing (AST) using appropriate 64-well cards.<\/p><h6>1.\u00a0\u00a0\u00a0\u00a0\u00a0 Identification process on the Vitek 2 system<\/h6><p>Aseptically transfer 3 ml of sterile saline (0.45% &#8211; 0.50% NaCl, pH 4.5 &#8211; 7.0) into a transparent plastic tube. With a sterile loop or swab, the isolated colonies are suspended in the saline solution tube, from the 24-hour culture on blood-geloza. Homogenise the bacterial suspension until a density equivalent to 0,5 \u2013 0,63 McFarland is obtained for unpretentious bacteria, 1,8-2,2 McFarland for fungi and 2,7-3,3 McFarland for the identification of neisserias, species of the genera <em>Haemophilus<\/em> and <em>Corynebacterium<\/em> or anaerobes. The tube with the suspension is placed in the cassette, then the Vitek card is inserted. Transferring the inoculum into the card is done automatically in the filling chamber of the device, after which it is transferred to the reader \/ incubator room.<\/p><p>Identification cards are read based on changes in bacterial growth and metabolism during incubation; they are read every 15 minutes with an optical reader. The results of the biochemical tests are automatically compared with the information in the database, selecting the bacterial species corresponding to the obtained biochemical profile<strong>.<\/strong><\/p><h6>2.\u00a0\u00a0\u00a0\u00a0\u00a0 The process of establishing antibiotic sensitivity on the Vitek 2C system<\/h6><p>Vitek AST cards are used to automatically test the sensitivity to antibiotics with the determination of the minimum inhibitory concentration (mic) for pathogens with clinical significance isolated from vaginal secretions, cervical secretions, hemocultures, from patients hospitalized in the department of Obstetrics or hypopharyngeal aspirates, CSF cultures, hemocultures from<\/p><p><\/p><p>newborns hospitalized in the department of Neonatology of SCJUPBT. The tested germs are: Gram negative bacilli, <em>Staphylococcus<\/em> species, <em>Enterococcus<\/em>, <em>Streptococcus<\/em> <em>agalactiae,<\/em> <em>Streptococcus<\/em> <em>pneumoniae<\/em>, fungi, etc.<\/p><p><strong>Principle<\/strong>: Determination of CMI by the microdilution method. Each AST card contains 64 microcells with antibiotics in different concentrations, as well as the control cell.<\/p><p><strong>Technique:<\/strong> a quantity of 145 \u00b5l (for gram-positive bacteria) or 280 \u00b5l (for gram-negative bacteria) of the suspension of 0.5-0.63 Mc Farland from the bacterial strain, to be tested, is resuspended in 0.45% saline. Insert the antibiogram card into the tube and insert it into the apparatus (incubator\/reader).<\/p><p>The growing conditions are optimized to a microaerophilic atmosphere.<\/p><p>The instrument monitors the growth in each card cell over a defined period of time (up to 18 hours). Throughout the incubation period, CMI values are determined every 15 minutes for each antibiotic in the card.<\/p><p>Once the CMI result is obtained, the valid interpretation standards (breakpoint) specified in the Vitek2 software (CLSI-Clinicaland Laboratory Standards Institute) are applied to provide the clinical categories of sensitive, intermediate and resistant<strong>.<\/strong><\/p><p>For testing the sensitivity of gram-negative bacteria, AST-N204, <strong>ASTN-222<\/strong> and <strong>AST<\/strong> <strong>N-397<\/strong><\/p><p>cards are <strong>used.<\/strong><\/p><p>The <strong>AST-N204<\/strong> card contains: ampicillin, amoxicillin\/clavulanic acid, ceftazidime, cefotaxime, cefepim, piperacillin\/tazobactam, ESBL screening (extended spectrum beta- lactamases) imipenem, meropenem, ertapenem, colistin, phosphomycin, ciprofloxacin, norfloxacin, gentamicin, amikacin.<\/p><p>The <strong>AST-N222<\/strong> card contains: ticarcillin, ticarcillin \/clavulanic acid, piperacillin, piperacillin<\/p><p>\/clavulanic acid, ceftazidim, cefepim, aztreonam, imipenem, meropenem, amikacin, gentamicin, tobramycin, minocycline, colistin, ciprofloxacin, trimethoprim\/sulfamethoxazole.<\/p><p><\/p><p><strong>AST<\/strong> <strong>card<\/strong> <strong>N-397<\/strong> contains: amoxicillin, cefotaxime, ceftazidime, ceftazidime\/avibactam, piperacillin\/tazobactam, colistin, ciprofloxacin, gentamicin, tobramycin, amikacin, imipenem, meropenem, trimethoprim\/sulfamethoxazole.<\/p><p><\/p><p>For testing the antibiotic sensitivity of gram-positive bacteria (<em>Staphylococcus<\/em> <em>aureus<\/em>, coagulase-negative staphylococci, <em>Enterococcus<\/em> spp and <em>Streptococcus<\/em> <em>agalactiae<\/em>, the AST-GP67 card is used <strong>(<\/strong>card content in the table):<\/p><table><tbody><tr><td width=\"151\"><p>Antimicrobial<\/p><\/td><td width=\"38\"><p>Code<\/p><\/td><td width=\"103\"><p>Concentration<\/p><p>\u00a7<\/p><\/td><td colspan=\"2\" width=\"135\"><p>Calling Range<\/p><p>\u0e00<\/p><\/td><td width=\"177\"><p>FDA Indications for Use<\/p><\/td><\/tr><tr><td width=\"151\"><p>Ampicillin<\/p><p><em>Enterococcusspp<\/em>.<\/p><p><em>S.<\/em> <em>agalactiae<\/em><\/p><\/td><td width=\"38\"><p>AM<\/p><\/td><td width=\"103\"><p>0.5, 4, 8, 32<\/p><\/td><td width=\"68\"><p>2<\/p><p>0.2<\/p><p>5<\/p><\/td><td width=\"68\"><p>&#8211; 3<\/p><p>2<\/p><p>16<\/p><\/td><td width=\"177\"><p><em>Enterococcusspp<\/em>., <em>S.<\/em> <em>agalactiae<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Benzylpenicillin <em>Enterococcusspp<\/em>. <em>Staphylococcusspp<\/em>.<\/p><p><em>S.<\/em> <em>agalactiae<\/em><\/p><\/td><td width=\"38\"><p>P<\/p><\/td><td width=\"103\"><p>0.125, 0.25, 1,<\/p><p>2, 8, 64<\/p><\/td><td width=\"68\"><p>0-<\/p><p>12<\/p><p>% 0.0<\/p><p>3<\/p><p>0.1<\/p><p>2<\/p><\/td><td width=\"68\"><p>\u2265<\/p><p>6<\/p><p>4<\/p><p>0.5<\/p><p>64<\/p><\/td><td width=\"177\"><p><em>Staphylococcusspp<\/em>., <em>Enterococcus<\/em> pp.,<\/p><p><em>S.<\/em> <em>agalactiae<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Cefoxitin Screen<\/p><\/td><td width=\"38\"><p>OXSF<\/p><\/td><td width=\"103\"><p>6<\/p><\/td><td width=\"68\"><p>NEG<\/p><\/td><td width=\"68\"><p>POS<\/p><\/td><td width=\"177\"><p><em>Staphylococcus<\/em>spp.<\/p><\/td><\/tr><tr><td width=\"151\"><p>Ciprofloxacin<\/p><\/td><td width=\"38\"><p>CIP<\/p><\/td><td width=\"103\"><p>1, 2, 4<\/p><\/td><td width=\"68\"><p>0.5<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p><em>Staphylococcusspp<\/em>., <em>Enterococcusspp<\/em>.<\/p><\/td><\/tr><tr><td width=\"151\"><p>Clindamycin<\/p><\/td><td width=\"38\"><p>CM<\/p><\/td><td width=\"103\"><p>0.5, 1, 2<\/p><\/td><td width=\"68\"><p>0.25<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p><em>Staphylococcusspp<\/em>., <em>S.<\/em> <em>agalactiae<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Erythromycin<\/p><\/td><td width=\"38\"><p>E<\/p><\/td><td width=\"103\"><p>0.25, 0.5, 2<\/p><\/td><td width=\"68\"><p>0.2<\/p><p>5<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p><em>Staphylococcusspp<\/em>., <em>Enterococcus<\/em> pp.,<\/p><p><em>S.<\/em> <em>agalactiae<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Gentamycinum<\/p><\/td><td width=\"38\"><p>GM<\/p><\/td><td width=\"103\"><p>8, 16, 64<\/p><\/td><td width=\"68\"><p>0.5<\/p><\/td><td width=\"68\"><p>16<\/p><\/td><td width=\"177\"><p><em>Staphylococcus<\/em>spp.<\/p><\/td><\/tr><tr><td width=\"151\"><p>Gentamicin High Level (synergy)<\/p><\/td><td width=\"38\"><p>HLG<\/p><\/td><td width=\"103\"><p>500<\/p><\/td><td width=\"68\"><p>S<\/p><\/td><td width=\"68\"><p>R<\/p><\/td><td width=\"177\"><p><em>Enterococcusspp<\/em>.<\/p><\/td><\/tr><tr><td width=\"151\"><p>Inducible Clindamycin Resistance<\/p><\/td><td width=\"38\"><p>ICR<\/p><\/td><td width=\"103\"><p>CM 0.5, CM\/E 0.25\/0.5<\/p><\/td><td width=\"68\"><p>NEG<\/p><\/td><td width=\"68\"><p>POS<\/p><\/td><td width=\"177\"><p><em>Staphylococcus<\/em>spp.<\/p><\/td><\/tr><tr><td width=\"151\"><p>Levofloxacin<\/p><\/td><td width=\"38\"><p>LEV<\/p><\/td><td width=\"103\"><p>0.25, 2, 8<\/p><\/td><td width=\"68\"><p>0.12<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p><em>Staphylococcusspp<\/em>., <em>Enterococcus<\/em> pp.,<\/p><p><em>S.<\/em> <em>agalactiae<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Linezolid<\/p><\/td><td width=\"38\"><p>LNZ<\/p><\/td><td width=\"103\"><p>0.5, 1, 2<\/p><\/td><td width=\"68\"><p>0.5<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p><em>S.<\/em> <em>agalactiae<\/em>, <em>E.<\/em> <em>faecalis<\/em>,<\/p><p><em>E.<\/em> <em>faecium<\/em>,<\/p><p><em>S.<\/em> <em>aureus<\/em>, <em>S.<\/em> <em>epidermidis<\/em>,<\/p><p><em>S.<\/em> <em>haemolyticus<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Moxifloxacin<\/p><\/td><td width=\"38\"><p>MXF<\/p><\/td><td width=\"103\"><p>0.25, 2, 8<\/p><\/td><td width=\"68\"><p>0.25<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p>MSSA**<\/p><\/td><\/tr><tr><td width=\"151\"><p>Nitrofurantoin<\/p><\/td><td width=\"38\"><p>FT<\/p><\/td><td width=\"103\"><p>16, 32, 64<\/p><\/td><td width=\"68\"><p>16<\/p><\/td><td width=\"68\"><p>512<\/p><\/td><td width=\"177\"><p><em>Staphylococcusspp<\/em>., <em>Enterococcusspp<\/em>.<\/p><\/td><\/tr><\/tbody><\/table><p><\/p><table><tbody><tr><td width=\"151\"><p>Oxacillin<\/p><\/td><td width=\"38\"><p>OX1<\/p><\/td><td width=\"103\"><p>0.5, 1, 2<\/p><\/td><td width=\"68\"><p>0.25<\/p><\/td><td width=\"68\"><p>4<\/p><\/td><td width=\"177\"><p><em>Staphylococcus<\/em>spp.<\/p><\/td><\/tr><tr><td width=\"151\"><p>Quinupristin\/ Dalfopristin<\/p><\/td><td width=\"38\"><p>QD A<\/p><\/td><td width=\"103\"><p>0.25, 0.5, 2<\/p><\/td><td width=\"68\"><p>0.25<\/p><\/td><td width=\"68\"><p>16<\/p><\/td><td width=\"177\"><p>MSSA**, <em>S.<\/em> <em>epidermidis<\/em>, VREF**,<\/p><p><em>S.<\/em> <em>agalactiae<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Rifampicin<\/p><\/td><td width=\"38\"><p>RA<\/p><\/td><td width=\"103\"><p>0.25, 0.5, 2<\/p><\/td><td width=\"68\"><p>0.5<\/p><\/td><td width=\"68\"><p>32<\/p><\/td><td width=\"177\"><p><em>Staphylococcus<\/em>spp.<\/p><\/td><\/tr><tr><td width=\"151\"><p>Streptomycin High Level (synergy)<\/p><\/td><td width=\"38\"><p>HLS<\/p><\/td><td width=\"103\"><p>1000<\/p><\/td><td width=\"68\"><p>S<\/p><\/td><td width=\"68\"><p>R<\/p><\/td><td width=\"177\"><p><em>Enterococcusspp<\/em>.<\/p><\/td><\/tr><tr><td width=\"151\"><p>:<\/p><\/td><td width=\"38\"><p>TE<\/p><\/td><td width=\"103\"><p>0.5, 1, 2<\/p><\/td><td width=\"68\"><p>1<\/p><\/td><td width=\"68\"><p>16<\/p><\/td><td width=\"177\"><p><em>Staphylococcusspp<\/em>., <em>Enterococcus<\/em> pp.,<\/p><p><em>S.<\/em> <em>agalactiae<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Tigecycline<sup>NS<\/sup><\/p><\/td><td width=\"38\"><p>TGC<\/p><\/td><td width=\"103\"><p>0.25, 0.5, 1<\/p><\/td><td width=\"68\"><p>0.12<\/p><\/td><td width=\"68\"><p>2<\/p><\/td><td width=\"177\"><p><em>E.<\/em> <em>faecalis<\/em>, <em>E.<\/em> <em>faecium<\/em>,<\/p><p><em>S.<\/em> <em>aureus<\/em>,<\/p><p><em>S.<\/em> <em>epidermidis<\/em>, <em>S.<\/em> <em>haemolyticus<\/em>,<\/p><p><em>S.<\/em> <em>agalactiae<\/em>, <em>E.<\/em> <em>casseliflavus<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Trimethoprim<\/p><p>\/Sulfamethoxazolce<\/p><\/td><td width=\"38\"><p>SXT<\/p><\/td><td width=\"103\"><p>2\/38, 8\/152,<\/p><p>16\/304<\/p><\/td><td width=\"68\"><p>10(0.5\/<\/p><p>9.5)<\/p><\/td><td width=\"68\"><p>320(16\/<\/p><p>304)<\/p><\/td><td width=\"177\"><p><em>S.<\/em> <em>aureus<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Vancomycin<\/p><\/td><td width=\"38\"><p>VA<\/p><\/td><td width=\"103\"><p>1, 2, 4, 8, 16<\/p><\/td><td width=\"68\"><p>0.5<\/p><\/td><td width=\"68\"><p>32<\/p><\/td><td width=\"177\"><p><em>Enterococcusspp<\/em>., <em>Staphylococcusspp<\/em>.,<\/p><p><em>S.<\/em> <em>agalactiae<\/em><\/p><\/td><\/tr><\/tbody><\/table><p><\/p><p>For the anthribiogram of various streptococcal species, the AST-ST03 <strong>card<\/strong> is used (table below):<\/p><p><\/p><p><\/p><table><tbody><tr><td width=\"151\"><p>Antimicrobial<\/p><\/td><td width=\"38\"><p>Code<\/p><\/td><td width=\"103\"><p>Concentration<\/p><p>\u00a7<\/p><\/td><td colspan=\"2\" width=\"135\"><p>Calling Range<\/p><p>\u0e00\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u0e00<\/p><\/td><td width=\"177\"><p>FDA Indications for Use<\/p><\/td><\/tr><tr><td width=\"151\"><p>AmpicillinNS2<\/p><\/td><td width=\"38\"><p>AM<\/p><\/td><td width=\"103\"><p>0.5, 1, 4, 8<\/p><\/td><td width=\"68\"><p>0.25<\/p><\/td><td width=\"68\"><p>16<\/p><\/td><td width=\"177\"><p>grpBetaHS**, grpVIR**<\/p><\/td><\/tr><tr><td width=\"151\"><p>BenzylpenicillinNS2<\/p><\/td><td width=\"38\"><p>P<\/p><\/td><td width=\"103\"><p>0.06, 0.12, 0.5,<\/p><p>2<\/p><\/td><td width=\"68\"><p>0.06<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p>grpC\/G**, <em>S.<\/em> <em>pyogenes<\/em>,<\/p><p><em>S.<\/em> <em>agalactiae<\/em>,<\/p><p><em>S.<\/em> <em>pneumoniae<\/em>, grpVIR**<\/p><\/td><\/tr><tr><td width=\"151\"><p>Cefotaxime<\/p><\/td><td width=\"38\"><p>CTX<\/p><\/td><td width=\"103\"><p>0.25, 0.5, 1, 2<\/p><\/td><td width=\"68\"><p>0.12<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p><em>S.<\/em> <em>pneumoniae<\/em>, SpyA**,<\/p><p><em>Streptococcussp<\/em> <em>p<\/em>.<\/p><\/td><\/tr><tr><td width=\"151\"><p>CeftriaxoneNS2<\/p><\/td><td width=\"38\"><p>CRO<\/p><\/td><td width=\"103\"><p>0.12, 0.25, 1.4<\/p><\/td><td width=\"68\"><p>0.12<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p>grpVIR**, <em>S.<\/em> <em>agalactiae<\/em>, <em>S.<\/em> <em>pneumoniae<\/em>,<\/p><p><em>S.<\/em> <em>pyogenes<\/em><\/p><\/td><\/tr><\/tbody><\/table><p><\/p><table><tbody><tr><td width=\"151\"><p>Chloramphenicol<\/p><\/td><td width=\"38\"><p>C<\/p><\/td><td width=\"103\"><p>1, 4, 8<\/p><\/td><td width=\"68\"><p>1<\/p><\/td><td width=\"68\"><p>16<\/p><\/td><td width=\"177\"><p>N\/A**<\/p><\/td><\/tr><tr><td width=\"151\"><p>Clindamycinc<\/p><\/td><td width=\"38\"><p>CM<\/p><\/td><td width=\"103\"><p>CM 0.12, CM<\/p><p>0.25, CM<\/p><p>0.5, CM\/E<\/p><p>0.5\/0.1<\/p><\/td><td width=\"68\"><p>0.25<\/p><\/td><td width=\"68\"><p>1<\/p><\/td><td width=\"177\"><p>SP(Pen(S))**, <em>S.<\/em><\/p><p><em>pyogenes<\/em>,<\/p><p><em>S.<\/em> <em>agalactiae<\/em>, <em>S.<\/em> <em>mitis<\/em>,<\/p><p><em>S.<\/em> <em>oralis<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Erythromycin<\/p><\/td><td width=\"38\"><p>E<\/p><\/td><td width=\"103\"><p>1, 2, 4, 16<\/p><\/td><td width=\"68\"><p>0.12<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p><em>S.<\/em> <em>pneumoniae<\/em>, <em>S.<\/em> <em>pyogenes<\/em>, grpVIR**<\/p><\/td><\/tr><tr><td width=\"151\"><p>Gentamycinum<\/p><\/td><td width=\"38\"><p>GM<\/p><\/td><td width=\"103\"><p>512, 1024<\/p><\/td><td width=\"68\"><p>64<\/p><\/td><td width=\"68\"><p>512<\/p><\/td><td width=\"177\"><p>N\/A**<\/p><\/td><\/tr><tr><td width=\"151\"><p>Inducible Clindamycin Resistance<\/p><p><em>S.<\/em> <em>agalactiae,<\/em> <em>S.<\/em><\/p><p><em>pyogenes<\/em><\/p><\/td><td width=\"38\"><p>ICR<\/p><\/td><td width=\"103\"><p>CM 0.5, CM\/E 0.25\/0.5<\/p><\/td><td width=\"68\"><p>&#8211; NE G<\/p><\/td><td width=\"68\"><p>&#8211; PO S<\/p><\/td><td width=\"177\"><p><em>S.<\/em> <em>agalactiae<\/em>, <em>S.<\/em> <em>pyogenes<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Levofloxacin<\/p><\/td><td width=\"38\"><p>LEV<\/p><\/td><td width=\"103\"><p>1, 2, 4, 16<\/p><\/td><td width=\"68\"><p>0.25<\/p><\/td><td width=\"68\"><p>16<\/p><\/td><td width=\"177\"><p>SP+MDRSP* <em>S<\/em> *,<\/p><p><em>.pyogenes,<\/em> grpC\/F**, grpG**, <em>S.<\/em> <em>agalactiae<\/em>,<\/p><p>grpVIR**, <em>S.<\/em> <em>milleri<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>LinezolidNS<\/p><\/td><td width=\"38\"><p>LNZ<\/p><\/td><td width=\"103\"><p>2, 4<\/p><\/td><td width=\"68\"><p>2<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p>SP+MDRSP** <em>S<\/em>,<em>.<\/em><\/p><p><em>agalactiae<\/em>,<\/p><p><em>S.<\/em> <em>pyogenes<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Moxifloxacin<\/p><\/td><td width=\"38\"><p>MXF<\/p><\/td><td width=\"103\"><p>0.5, 1, 4, 8<\/p><\/td><td width=\"68\"><p>0.06<\/p><\/td><td width=\"68\"><p>4<\/p><\/td><td width=\"177\"><p><em>S.<\/em> <em>anginosus<\/em>, <em>S.<\/em> <em>constellatus<\/em>, SP+MDRSP** <em>S<\/em>,<em>.<\/em> <em>pyogenes<\/em>,<\/p><p><em>S.<\/em> <em>agalactiae<\/em>, grpVIR**<\/p><\/td><\/tr><tr><td width=\"151\"><p>Rifampicin<\/p><\/td><td width=\"38\"><p>RA<\/p><\/td><td width=\"103\"><p>0.015, 0.03,<\/p><p>0.12, 0.5<\/p><\/td><td width=\"68\"><p>0.06<\/p><\/td><td width=\"68\"><p>4<\/p><\/td><td width=\"177\"><p>N\/A**<\/p><\/td><\/tr><tr><td width=\"151\"><p>Teicoplanin<\/p><\/td><td width=\"38\"><p>TEC<\/p><\/td><td width=\"103\"><p>0.5, 1, 4<\/p><\/td><td width=\"68\"><p>0.12<\/p><\/td><td width=\"68\"><p>4<\/p><\/td><td width=\"177\"><p>N\/A**<\/p><\/td><\/tr><tr><td width=\"151\"><p>Tetracycline<\/p><\/td><td width=\"38\"><p>TE<\/p><\/td><td width=\"103\"><p>0.12, 0.5, 1.4<\/p><\/td><td width=\"68\"><p>0.25<\/p><\/td><td width=\"68\"><p>16<\/p><\/td><td width=\"177\"><p><em>S.<\/em> <em>pneumoniae<\/em>, <em>S.<\/em> <em>pyogenes<\/em>, grpVIR**<\/p><\/td><\/tr><tr><td width=\"151\"><p>Tigecycline<sup>NS<\/sup><\/p><\/td><td width=\"38\"><p>TGC<\/p><\/td><td width=\"103\"><p>0.12, 0.25, 0.5<\/p><\/td><td width=\"68\"><p>0.06<\/p><\/td><td width=\"68\"><p>1<\/p><\/td><td width=\"177\"><p>SP(Pen(S))**, <em>S.<\/em><\/p><p><em>anginosus<\/em><\/p><p>grp. (<em>includesS.<\/em> <em>anginosus<\/em> <em>,<\/em> S.intermedius, <em>andS.constellatus<\/em>),<\/p><p><em>S.<\/em> <em>agalactiae<\/em>, <em>S.<\/em> <em>pyogenes<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Trimethoprim\/Sulfam ethoxazolce<\/p><\/td><td width=\"38\"><p>SXT<\/p><\/td><td width=\"103\"><p>8\/152, 16\/304,<\/p><p>64\/1216<\/p><\/td><td width=\"68\"><p>10(0.5\/9<\/p><p>.5)<\/p><\/td><td width=\"68\"><p>320(16\/3<\/p><p>04)<\/p><\/td><td width=\"177\"><p><em>S.<\/em> <em>pneumoniae<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>VancomycinNS<\/p><\/td><td width=\"38\"><p>VA<\/p><\/td><td width=\"103\"><p>0.5, 1, 2, 4<\/p><\/td><td width=\"68\"><p>0.12<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p>grpVIR**, <em>S.<\/em> <em>pyogenes<\/em>, <em>S.<\/em> <em>agalactiae<\/em><\/p><\/td><\/tr><\/tbody><\/table><p><\/p><p>For antifungigram, the AST-YS08 card <strong>is<\/strong> used, table below:<\/p><p><\/p><table><tbody><tr><td width=\"151\"><p>Antimicrobial<\/p><\/td><td width=\"38\"><p>Code<\/p><\/td><td width=\"103\"><p>Concentration<\/p><p>\u00a7<\/p><\/td><td colspan=\"2\" width=\"135\"><p>Calling Range<\/p><p>\u0e00\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u0e00<\/p><\/td><td width=\"177\"><p>FDA Indications for Use<\/p><\/td><\/tr><tr><td width=\"151\"><p>Amphotericin B<\/p><\/td><td width=\"38\"><p>AB<\/p><\/td><td width=\"103\"><p>1, 4, 16, 32<\/p><\/td><td width=\"68\"><p>0.25<\/p><\/td><td width=\"68\"><p>16<\/p><\/td><td width=\"177\"><p><\/p><\/td><\/tr><tr><td width=\"151\"><p>Caspofungin<\/p><\/td><td width=\"38\"><p>CAS<\/p><\/td><td width=\"103\"><p>0.12, 0.5, 2, 8<\/p><\/td><td width=\"68\"><p>0.125<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p>N\/A**<\/p><\/td><\/tr><tr><td width=\"151\"><p>Fluconazole,<\/p><\/td><td width=\"38\"><p>FLU<\/p><\/td><td width=\"103\"><p>2, 4, 8, 16, 32,<\/p><p>64<\/p><\/td><td width=\"68\"><p>0.5<\/p><\/td><td width=\"68\"><p>64<\/p><\/td><td width=\"177\"><p><em>C.<\/em> <em>dubliniensis<\/em>, <em>C.<\/em> <em>albicans<\/em>,<\/p><p><em>C.<\/em> <em>parapsilosis<\/em>, <em>C.<\/em> <em>tropicalis<\/em>,<\/p><p><em>C.<\/em> <em>guilliermondi<\/em>, <em>i.c.<\/em> <em>lusitaniae<\/em><\/p><\/td><\/tr><tr><td width=\"151\"><p>Flucytosine<sup>,<\/sup><\/p><\/td><td width=\"38\"><p>FCT<\/p><\/td><td width=\"103\"><p>1, 4, 16, 32<\/p><\/td><td width=\"68\"><p>1<\/p><\/td><td width=\"68\"><p>64<\/p><\/td><td width=\"177\"><p><em>C.<\/em> <em>albicans<\/em>, <em>C.<\/em> <em>dubliniensis<\/em>, <em>C.<\/em> <em>glabrata<\/em>,<\/p><p><em>C.<\/em> <em>guilliermondi<\/em>, <em>i.c.<\/em> <em>lusitaniae<\/em>,<\/p><p><em>C.<\/em> <em>parapsilosis<\/em>, <em>C.<\/em> <em>tropicalis<\/em>,<\/p><\/td><\/tr><tr><td width=\"151\"><p>Micafungin<\/p><\/td><td width=\"38\"><p>MCF<\/p><\/td><td width=\"103\"><p>0.06, 0.25, 1.4<\/p><\/td><td width=\"68\"><p>0.06<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p>N\/A**<\/p><\/td><\/tr><tr><td width=\"151\"><p>VoriconazoleSDD<\/p><\/td><td width=\"38\"><p>VRC<\/p><\/td><td width=\"103\"><p>0.5, 1, 4, 8<\/p><\/td><td width=\"68\"><p>0.1<\/p><p>2<\/p><\/td><td width=\"68\"><p>8<\/p><\/td><td width=\"177\"><p><em>C.<\/em> <em>albicans<\/em>, <em>C.<\/em> <em>krusei<\/em>, <em>C.<\/em> <em>parapsilosis<\/em>,<\/p><p><em>C.<\/em> <em>tropicalis<\/em>, <em>C.<\/em> <em>lusitaniae<\/em>,<\/p><p><em>C.<\/em> <em>guilliermondii<\/em><\/p><\/td><\/tr><\/tbody><\/table><p><\/p><p>The reference strains (ATCC American Type Culture Collection) recommended by the card maker will be used as test control witnesses: <em>Escherichia<\/em> <em>coli<\/em> ATCC 25922, <em>Escherichia<\/em> <em>coli<\/em> ATCC 35218, <em>Pseudomonas<\/em> <em>aeruginosa<\/em> ATCC 27853, <em>Klebsiella<\/em> <em>pneumoniae<\/em> ssp <em>pneumoniae<\/em> ATCC 700603, <em>Enterococcus<\/em> <em>faecalis<\/em> ATCC 29212, <em>Enterococcus<\/em> <em>faecalis<\/em> ATCC 51299, <em>Staphylococcus<\/em> <em>aureus<\/em> ATCC 29213, <em>Staphylococcus<\/em> <em>aureus<\/em> ATCC BAA-1026, <em>Staphylococcus<\/em> <em>aureus<\/em> ATCC BAA-976, <em>Staphylococcus<\/em> <em>aureus<\/em> ATCC BAA-977.<\/p><p>To identify fungi<\/p><p><\/p><p>Bibliography: Vitek 2. User Manuals.Biomerieux tool. France, 2009<\/p><\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t\t\t<div class=\"elementor-toggle-item\">\n\t\t\t\t\t<div id=\"elementor-tab-title-8494\" class=\"elementor-tab-title\" data-tab=\"4\" role=\"button\" aria-controls=\"elementor-tab-content-8494\" aria-expanded=\"false\">\n\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon elementor-toggle-icon-left\" aria-hidden=\"true\">\n\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-closed\"><i class=\"fas fa-caret-right\"><\/i><\/span>\n\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-opened\"><i class=\"elementor-toggle-icon-opened fas fa-caret-up\"><\/i><\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t\t<\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t<a class=\"elementor-toggle-title\" tabindex=\"0\">Customized protocol for detection of aneuploid embryos derived from in vitro fertilisation (PGS preimplantation genetic screening)<\/a>\n\t\t\t\t\t<\/div>\n\n\t\t\t\t\t<div id=\"elementor-tab-content-8494\" class=\"elementor-tab-content elementor-clearfix\" data-tab=\"4\" role=\"region\" aria-labelledby=\"elementor-tab-title-8494\"><p>PGS, preimplantaon genec screening, refers to removing one or more cells from an in vitro ferlized embryo to test for chromosomal normalcy.<\/p><p>An abnormal number of chromosomes is a major cause of in vitro ferlizaon (IVF) failure as most embryos with aneuploidy will not implant or will miscarry during the first trimester of pregnancy. PGS is performed for selecng those embryos with a normal number of chromosomes, increases the chance that a viable embryo will be selected for transfer and the likelihood of successful implantaon and pregnancy.<\/p><p>Our lab has the possibility to use next-generaon sequencing (NGS) technology to provide comprehensive, accurate screening of all 24 chromosomes for selecng euploid embryos. Illumina plaorm that we use enables massively parallel sequencing of millions of DNA fragments, detecng single bases as they are incorporated into growing DNA strands. \u00a0<\/p><p>General workflow<\/p><p><img decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/02\/Picture1-300x31.png\" alt=\"\" width=\"300\" height=\"31\" class=\"alignnone size-medium wp-image-708\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/02\/Picture1-300x31.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/02\/Picture1-228x23.png 228w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/02\/Picture1.png 625w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><\/p><h4>DNA extracon and amplificaon<\/h4><p><strong>\u00a0<\/strong><\/p><p><strong>DNA sample<\/strong> -provided by IVF clinics must be suspended in molecular grade 1x PBS (Phosphate Buffered Saline) and, if used, with a maximum concentraon of 0.5% PVP (Polyvinylpyrrolidone) in a volume of 2.5 \u03bcl. Samples must be stored at -65 &#8211; 85\u00b0C following biopsy to preserve integrity and quality. Cell lysis and amplificaon must be completed within 14 days from the harvesng. \u00a0<\/p><p><em>Materials required for sample preparation<\/em>: SurePlex DNA amplificaon system, 20x PBS (dilute to 1x with nuclease-free water), genomic control DNA, PCR tubes (0.2 ml) or 96-well PCR plate and adhesive plate seals, microcentrifuge tube (1.5 ml), genomic DNA 100ng\/ \u00b5l. Sample and control required \u00a0<\/p><table width=\"623\"><tbody><tr><td width=\"623\"><p>Sample provided by IVF clinics \u2013 single cell in 2.5 \u03bcl PBS \u00a0<\/p><\/td><\/tr><tr><td width=\"623\"><p>Posive control 1: 15 pg genomic DNA in 2.5 \u00b5l 1x PBS<\/p><\/td><\/tr><tr><td width=\"623\"><p>Posive control 2: 60 pg genomic DNA in 2.5 \u00b5l 1x PBS<\/p><\/td><\/tr><tr><td width=\"623\"><p>Negave control: 2.5 \u00b5l of 1x PBS \u00a0<\/p><\/td><\/tr><tr><td width=\"623\"><p>Negave control: 2.5 \u00b5l of collecon buffer control<\/p><\/td><\/tr><\/tbody><\/table><p><\/p><p>Control Tubes for Sample Preparaon<\/p><table width=\"623\"><tbody><tr><td width=\"54\"><p>No.<\/p><\/td><td width=\"198\"><p>Descripon<\/p><\/td><td width=\"126\"><p>Volume PBS 1X<\/p><\/td><td width=\"246\"><p>Volume of Female Genomic DNA<\/p><p>\/Stock Soluon<\/p><\/td><\/tr><tr><td width=\"54\"><p>1<\/p><\/td><td width=\"198\"><p>1x PBS negave<\/p><\/td><td width=\"126\"><p>100 \u00b5l<\/p><\/td><td width=\"246\"><p>None<\/p><\/td><\/tr><tr><td width=\"54\"><p>2<\/p><\/td><td width=\"198\"><p>2.5 ng\/\u00b5l posive<\/p><\/td><td width=\"126\"><p>195 \u00b5l<\/p><\/td><td width=\"246\"><p>5 \u00b5l of 100 ng\/\u00b5l stock<\/p><\/td><\/tr><tr><td width=\"54\"><p>3<\/p><\/td><td width=\"198\"><p>25 pg\/\u00b5l posive<\/p><\/td><td width=\"126\"><p>495 \u00b5l<\/p><\/td><td width=\"246\"><p>5 \u00b5l of 2.5 ng\/\u00b5l stock (from tube 2)<\/p><\/td><\/tr><tr><td width=\"54\"><p>4<\/p><\/td><td width=\"198\"><p>6.25 pg\/\u00b5l posive<\/p><\/td><td width=\"126\"><p>15 \u00b5l<\/p><\/td><td width=\"246\"><p>5 \u00b5l of 25 pg\/\u00b5l stock (from tube 3)<\/p><\/td><\/tr><\/tbody><\/table><p>PCR Tubes for Sample Preparaon<\/p><table width=\"623\"><tbody><tr><td width=\"102\"><p>PCR tube no. \u00a0<\/p><\/td><td width=\"174\"><p>Label on Tube Cap<\/p><\/td><td width=\"348\"><p>Contents<\/p><\/td><\/tr><tr><td width=\"102\"><p>1<\/p><\/td><td width=\"174\"><p>15.6 pos<\/p><\/td><td width=\"348\"><p>2.5 \u00b5l of control tube 4 (6.25 pg\/\u00b5l posive)<\/p><\/td><\/tr><tr><td width=\"102\"><p>2<\/p><\/td><td width=\"174\"><p>62.5 pos<\/p><\/td><td width=\"348\"><p>2.5 \u00b5l of control tube 3 (25 pg\/\u00b5l posive)<\/p><\/td><\/tr><tr><td width=\"102\"><p>3<\/p><\/td><td width=\"174\"><p>Neg<\/p><\/td><td width=\"348\"><p>2.5 \u00b5l of control tube 1 (1x PBS)<\/p><\/td><\/tr><\/tbody><\/table><p>Store the control PCR tubes in a 96-well rack on ice unl required. <strong>Cell Lysis\/Extracon steps\u00a0 <\/strong><\/p><p>1.Collect samples in 2.5 \u00b5l of 1x PBS and 2.5 \u00b5l of collecon buffer control. Centrifuge the tubes\/plate at 200 \u00d7 g for 3 minutes at 4\u00b0C<\/p><p>2.Add 2.5 \u00b5l of Cell Extracon Buffer to each sample (including controls) and store at 2\u00b0C to 8\u00b0C.<\/p><ol start=\"3\"><li>For every sample or control, add 5 \u00b5l of the freshly prepared Extracon Cocktail (4.8 \u00b5l Extracon<\/li><\/ol><p>Enzyme Diluon Buffer + 0.2 \u00b5l Cell Extracon Enzyme\/ sample) \u00a0<\/p><ol start=\"4\"><li>Briefly centrifuge samples to get all contents to the botom of the tube and incubate samples in a PCR thermal cycler for the following program:<\/li><\/ol><table width=\"623\"><tbody><tr><td width=\"208\"><p>Number of Cycles<\/p><\/td><td width=\"208\"><p>Temperature of Cycle<\/p><\/td><td width=\"208\"><p>Incubaon Time<\/p><\/td><\/tr><tr><td width=\"208\"><p>1<\/p><\/td><td width=\"208\"><p>75\u00b0C<\/p><\/td><td width=\"208\"><p>10 min<\/p><\/td><\/tr><tr><td width=\"208\"><p>1<\/p><\/td><td width=\"208\"><p>95\u00b0C<\/p><\/td><td width=\"208\"><p>4 min<\/p><\/td><\/tr><tr><td width=\"208\"><p>1<\/p><\/td><td width=\"208\"><p>22\u00b0C<\/p><\/td><td width=\"208\"><p>Hold<\/p><\/td><\/tr><\/tbody><\/table><h4>Preamplificaon \u00a0<\/h4><p>For each 10 \u00b5l sample prepared in Cell Lysis\/Extracon, add 5 \u00b5l of SurePlex Pre-amp Cocktail (4.8 \u00b5l SurePlex Pre-amp Buffer + 0.2 \u00b5l SurePlex Pre-amp Enzyme). Briefly centrifuge and incubate samples according to the following thermal cycler program:<\/p><table width=\"623\"><tbody><tr><td width=\"208\"><p>Number of Cycles<\/p><\/td><td width=\"208\"><p>Temperature of Cycle<\/p><\/td><td width=\"208\"><p>Incubaon Time<\/p><\/td><\/tr><tr><td width=\"208\"><p>1<\/p><\/td><td width=\"208\"><p>95\u00b0C<\/p><\/td><td width=\"208\"><p>2 min<\/p><\/td><\/tr><tr><td width=\"208\"><p>12<\/p><\/td><td width=\"208\"><p>95\u00b0C 15\u00b0C<\/p><p>25\u00b0C<\/p><p>35\u00b0C<\/p><p>65\u00b0C 75\u00b0C<\/p><\/td><td width=\"208\"><p>15 sec 50 sec<\/p><p>40 sec<\/p><p>30 sec<\/p><p>40 sec 40 sec<\/p><\/td><\/tr><tr><td width=\"208\"><p>1<\/p><\/td><td width=\"208\"><p>4\u00b0C<\/p><\/td><td width=\"208\"><p>Hold<\/p><\/td><\/tr><\/tbody><\/table><p>Following preamplificaon the products are maintained on ice \u00a0<strong>Amplificaon steps <\/strong><\/p><ol><li>Combine the Amplificaon Cocktail components<\/li><\/ol><table width=\"618\"><tbody><tr><td width=\"306\"><p>Amplificaon Cocktail<\/p><\/td><td width=\"312\"><p>Volume per Single Sample<\/p><\/td><\/tr><tr><td width=\"306\"><p>SurePlex Amplificaon Buffer<\/p><\/td><td width=\"312\"><p>25 \u00b5l<\/p><\/td><\/tr><tr><td width=\"306\"><p>SurePlex Amplificaon Enzyme<\/p><\/td><td width=\"312\"><p>0.8 \u00b5l<\/p><\/td><\/tr><tr><td width=\"306\"><p>Nuclease-free water<\/p><\/td><td width=\"312\"><p>34.2 \u00b5l<\/p><\/td><\/tr><tr><td width=\"306\"><p>Total Volume<\/p><\/td><td width=\"312\"><p>60 \u00b5l<\/p><\/td><\/tr><\/tbody><\/table><p>2.Add 60 \u03bcl of the freshly prepared Amplificaon Cocktail to the 15 \u03bcl pre-amplificaon reacon product, cap the tube, and invert to mix. Centrifuge briefly.<\/p><ol start=\"3\"><li>Amplify samples according to the thermal cycler program.<\/li><\/ol><table width=\"623\"><tbody><tr><td width=\"208\"><p>Number of Cycles<\/p><\/td><td width=\"208\"><p>Temperature of Cycle<\/p><\/td><td width=\"208\"><p>Incubaon Time<\/p><\/td><\/tr><tr><td width=\"208\"><p>1<\/p><\/td><td width=\"208\"><p>95\u00b0C<\/p><\/td><td width=\"208\"><p>2 min<\/p><\/td><\/tr><tr><td width=\"208\"><p>14<\/p><\/td><td width=\"208\"><p>95\u00b0C 65\u00b0C 75\u00b0C<\/p><\/td><td width=\"208\"><p>15 sec<\/p><p>1 min 1 min<\/p><\/td><\/tr><tr><td width=\"208\"><p>1<\/p><\/td><td width=\"208\"><p>4\u00b0C<\/p><\/td><td width=\"208\"><p>Hold<\/p><\/td><\/tr><\/tbody><\/table><p>4 Keep amplified SurePlex products on ice before proceeding with the VeriSeq PGS assay protocol.<\/p><p><\/p><h4>VeriSeq PGS Library Prep<\/h4><p><strong>\u00a0<\/strong><\/p><p>The VeriSeq PGS Library Prep Kit protocol is opmized for 1 ng of input SurePlex amplified DNA. Illumina\u00ae strongly recommends quanfying the starng SurePlex amplified dsDNA. Steps for quanficaon are included in this protocol. \u00a0<em>DNA Input Quantification <\/em><\/p><p>To obtain an accurate quanficaon of the DNA library, quanfy the starng DNA library using a fluorometric based method specific for duplex DNA. \u00a0<\/p><h4>Consumables and Equipment\u00a0<\/h4><p>96-well PCR plate, molecular grade water, Qubit dsDNA HS Assay Kit, Qubit Assay Tubes (1 tube per sample) Qubit 2.0 or greater Fluorometer, Adhesive PCR seal,\u00a0<\/p><p>Procedure \u00a0<\/p><p>Prepare 1\/10 Diluons of SurePlex Sample and Controls \u00a01 Vortex each sample and control. \u00a0<\/p><ul><li>Centrifuge at 280 \u00d7 g for 1 minute.<\/li><li>In a new PCR plate, add 45 \u03bcl molecular-grade water to the required wells.<\/li><li>Add 5 \u03bcl sample or control to the wells containing molecular-grade water.<\/li><li>Seal the plate and briefly vortex to mix.<\/li><li>Centrifuge at 280 \u00d7 g for 1 minute.<\/li><li>Set aside on wet ice.<\/li><\/ul><p><\/p><p>Qubit Method \u00a0<\/p><ul><li>Prepare the working soluon according to the manufacturer instrucons.<\/li><li>To calibrate the Qubit fluorometer, add 10 \u03bcl of each standard to 190 \u03bcl of working soluon.<\/li><li>Add 10 \u03bcl of the 1\/10 diluted SurePlex sample and 190 \u03bcl working soluon to each assay tube. Briefly vortex to mix.<\/li><li>For opmal fluorescence, incubate the assay tubes for 2 minutes.<\/li><li>Calculate the concentraon of each 1\/10 diluted SurePlex sample as described by the Qubit dsDNA HS Assay Kit user guide. Convert the units to ng\/\u03bcl.<\/li><\/ul><p><\/p><h4>Tagment Input DNA \u00a0<\/h4><p><\/p><p>In this step, the SurePlex amplificaon product is tagmented (tagged and fragmented) by the VeriSeq PGS transposome. \u00a0<\/p><p>Consumables ATM (Amplicon Tagment Mix), TD (Tagment DNA Buffer), NT (Neutralize Tagment Buffer), SurePlex amplificaon product (diluted at 0.2 ng\/\u00b5l), 96-well PCR plate, Adhesive PCR seal, PCR 8-tube strips<\/p><p><\/p><h4>Tagmentation of SurePlex WGA Product\u00a0<\/h4><ul><li>Label a new PCR plate VTA (VeriSeq Tagment Amplicon Plate).<\/li><li>Calculate the total volume of TD for all reacons. Using a mulchannel pipete, divide the volume equally among the wells of a PCR 8-tube strip, or use a reservoir.<\/li><li>Add 10 \u00b5l TD Buffer to each well. 4 Add 5 \u00b5l ATM to the wells containing TD Buffer.<\/li><li>Add 5 \u00b5l SurePlex amplificaon product (diluted at 0.2 ng\/\u00b5l) to each sample well.<\/li><li>Mix at 1,800 rpm for 1 minute centrifuge at 280 \u00d7 g for 1 minute.<\/li><\/ul><ol start=\"7\"><li>Make sure that each well contains a volume of 20 \u00b5l. Record any nonuniform volumes.<\/li><\/ol><p>8 Immediately place on a thermal cycler with a heated lid and run the following program: 55\u00b0C for 5 minutes\/ Hold at 10\u00b0C<\/p><h4>Neutralization of the Tagmented SurePlex DNA\u00a0<\/h4><ul><li>Calculate the total volume of NT buffer required for all reacons. Using a mulchannel pipete, divide the volume equally among the wells of a PCR 8-tube strip.<\/li><li>Add 5 \u00b5l NT Buffer to each well. 3 Mix at 1800 rpm for 1 minute.<\/li><li>Centrifuge at 280 \u00d7 g for 1 minute.<\/li><li>Make sure that each well contains a volume of 25 \u00b5l. Record any nonuniform volumes.<\/li><li>Incubate at room temperature for 5 minutes.<\/li><\/ul><p><\/p><h4>Amplify Tagmented DNA<\/h4><p>Consumables and Equipment NPM (Nextera\u00ae PCR Master Mix), index 1 primers (N701 to N712), index 2 primers (S503 and S504), TruSeq\u00ae Index Plate Fixture, Adhesive PCR seal, Plate sealer<\/p><h4>Procedure<\/h4><ul><li>Print the sample assay plate layout using the BlueFuse Workflow Manager.<\/li><li>Arrange the index primers in the TruSeq Index Plate Fixture, as follows:<\/li><\/ul><p>-Index 1 (i7) adapters: N701\u2013N712 in columns 1\u201312 \u00a0-Index 2 (i5) adapters: S503 in row A, S504 in row C<\/p><ul><li>Place the plate on the TruSeq Index Plate Fixture.<\/li><li>Add index adapters according to the sample assay plate layout.<\/li><\/ul><p>-Add 5 \u03bcl of each Index 1 (i7) adapter to each column. \u00a0<\/p><p>-Add 5 \u03bcl of each Index 2 (i5) adapter to each row.<\/p><ul><li>Add 15 \u03bcl NPM to each well.<\/li><li>Mix at 1800 rpm for 1 minute.<\/li><li>Centrifuge at 280 \u00d7 g for 1 minute.<\/li><li>Make sure that each well contains a volume of 50 \u00b5l. Record any nonuniform volumes.<\/li><li>Place on the thermal cycler and run the following program on a thermal cycler with a heated lid:<\/li><\/ul><p><\/p><table width=\"623\"><tbody><tr><td width=\"174\"><p>Number of Cycles<\/p><\/td><td width=\"246\"><p>Temperature of Cycle<\/p><\/td><td width=\"204\"><p>Incubaon Time<\/p><\/td><\/tr><tr><td width=\"174\"><p>1<\/p><\/td><td width=\"246\"><p>72\u00b0C<\/p><\/td><td width=\"204\"><p>3 min<\/p><\/td><\/tr><tr><td width=\"174\"><p>12<\/p><\/td><td width=\"246\"><p>95\u00b0C \u00a0<\/p><p>55\u00b0C 72\u00b0C<\/p><\/td><td width=\"204\"><p>10 sec 30 sec 30 sec<\/p><\/td><\/tr><tr><td width=\"174\"><p>1<\/p><\/td><td width=\"246\"><p>72\u00b0C<\/p><\/td><td width=\"204\"><p>5 min<\/p><\/td><\/tr><tr><td width=\"174\"><p>1<\/p><\/td><td width=\"246\"><p>4\u00b0C<\/p><\/td><td width=\"204\"><p>Hold<\/p><\/td><\/tr><\/tbody><\/table><p><\/p><h4>Clean Up PCR -steps<\/h4><p>Step that removes both the short library fragments and Primers from the populaon. \u00a0<\/p><ul><li>Centrifuge the VTA plate at 280 \u00d7 g for 1 minute to collect condensaon.<\/li><li>Add an appropriate volume of beads to a trough.<\/li><li>Add 45 \u00b5l AMPure XP beads to each required well of a clean deep well plate.<\/li><li>Transfer 45 \u00b5l PCR product from the VTA plate to the plate containing beads.<\/li><li>Mix at 1800 rpm for 1 minute.<\/li><li>Incubate at room temperature for 5 minutes. Do not shake the plate.<\/li><li>Pulse centrifuge. To prevent magnec bead aggregaon, do not centrifuge longer than a pulse. 8 Place on a magnec stand and wait unl the liquid is clear (~2 minutes). Keep the plate on the stand during the following steps.<\/li><li>Discard the supernatant from each well.<\/li><li>Wash 2 mes, as follows. Using a mulchannel pipete, add 200 \u00b5l freshly prepared 80% EtOH to each row on the opposite side of the aggregated beads, being careful not to disturb the bead pellet at the botom of the well. Do not resuspend the beads. Incubate on the magnec stand for \u2264 30 seconds. Start the mer aer dispensing 80% EtOH into the first well being careful to not exceed 30 seconds for each row. Immediately remove and discard all supernatant from each well and discard to appropriate waste<\/li><li>Using a mulchannel pipete and fine pipete ps, remove residual EtOH from each well.<\/li><li>Air-dry on the magnec stand for 15 minutes, or unl beads are completely dry.<\/li><li>Add 50 \u00b5l RSB to each well.<\/li><li>Remove the plate from the magnec stand.<\/li><li>Mix at 1800 rpm for 1 minute.<\/li><li>Centrifuge at 280 \u00d7 g for 1 minute.<\/li><li>Place on a magnec stand and wait unl the liquid is clear (~2 minutes).<\/li><li>Transfer 45 \u00b5l of each supernatant from each well to a new PCR plate.<\/li><li>Run a Quality Control check to determine the success of the library preparaon.<\/li><\/ul><p><\/p><h4>Normalize Libraries &#8211; steps<\/h4><p><\/p><p>1 In a new 15 ml conical tube, prepare the LNA1\/LNB1 mix according to the number of reacons. 2 Vortex thoroughly unl LNA1\/LNB1 mix is homogenized.<\/p><ul><li>Label a new deep-well plate LNP (Library Normalizaon Plate).<\/li><li>Pour the LNA1\/LNB1 mix into a reservoir.<\/li><li>Transfer 45 \u00b5l LNA1\/LNB1 mix to each well.<\/li><li>Add 20 \u00b5l dsDNA from the Clean-Up PCR procedure to each well.<\/li><li>Mix at 1800 rpm for 30 minutes.<\/li><li>Pulse centrifuge to collect any droplets. To prevent magnec bead aggregaon, do not centrifuge longer than a pulse.<\/li><li>Place on a magnec stand and wait unl the liquid is clear (~2 minutes). Keep the plate on the stand during the following steps.<\/li><li>Remove and discard all supernatant from each well.<\/li><li>Discard the ps in an appropriate hazardous waste container.<\/li><li>Wash beads 2 mes as follows. Keep on the magnec stand and add 45 \u00b5l LNW1 to each well. Seal and shake at 1800 rpm for 5 minutes. To prevent magnec bead aggregaon, pulse centrifuge to collect any droplets. Place on a magnec stand and wait unl the liquid is clear (~2 minutes). Remove and discard all supernatant from each well.<\/li><li>Add 30 \u00b5l 0.1 N NaOH to each well.<\/li><li>Remove from the magnec stand.<\/li><li>Mix at 1800 rpm for 5 minutes.<\/li><li>Centrifuge at 280 \u00d7 g for 1 minute.<\/li><li>Place on a magnec stand and wait unl the liquid is clear (~2 minutes).<\/li><li>Add 25 \u00b5l of LNS1 to each well of a new PCR plate.<\/li><li>Transfer 25 \u00b5l of supernatant from the LNP plate to the new PCR plate containing LNS1.<\/li><li>Vortex, and then centrifuge the PCR plate containing LNS1 and supernatant at 280 \u00d7 g for 1 minute.<\/li><\/ul><p><\/p><h4>Pool Libraries for the MiSeq System<\/h4><p><\/p><p>Equal volumes of normalized libraries are combined, diluted in HT1, and heat-denatured before being transferred to the flow cell for cluster generaon and sequencing.<\/p><ul><li>Centrifuge the plate at 280 \u00d7 g for 1 minute.<\/li><li>Transfer 5 \u00b5l of each normalized library to pool into a LoBind tube.<\/li><li>Vortex and centrifuge the pooled library.<\/li><li>Transfer 15 \u03bcl library pool to a new PCR tube or PCR 8-tube strip. The recommended cluster density of the MiSeq VeriSeq PGS workflow ranges from 1,100 K\/mm2 to 1,600 K\/mm2. Adjust the volume of the library pool to keep the cluster density in the recommended cluster density range.<\/li><li>Add 85 \u00b5l HT1 record the volumes of library pool and HT1.<\/li><li>Gently vortex and centrifuge the pool\/HT1 mixture.<\/li><li>Immediately place on the preprogrammed thermal cycler and run the DENATURE program.<\/li><li>Transfer 600 \u03bcl of HT1 into a second clean LoBind tube. Set aside in an ice-water bath.<\/li><li>When the denaturaon is complete, immediately transfer 100 \u00b5l of denatured pool\/HT1 mixture to the LoBind tube with HT1. Set aside on wet ice.<\/li><li>Sequence your library<\/li><\/ul><p><\/p><h4>MiSeq Sequencing VeriSeq PGS workflow<\/h4><p><\/p><p><em>Thaw Reagent Cartridge Inspect the Reagent Cartridge <\/em>1 Remove the reagent cartridge from -25\u00b0C to -15\u00b0C storage.<\/p><ul><li>Place the reagent cartridge in a water bath containing enough room temperature deionized water to submerge the base of the reagent cartridge. Do not allow the water to exceed the maximum water line printed on the reagent cartridge.<\/li><li>Allow the reagent cartridge to thaw in the room temperature water bath unl it is thawed completely. 4 Remove the cartridge from the water bath and gently tap it on the bench to dislodge water from the base of the cartridge. Dry the base of the cartridge.<\/li><\/ul><p>5.Inspect the reagents in posions 1, 2, and 4 to make sure that they are fully mixed and free of precipitates.<\/p><p>6 Place the reagent cartridge on ice for up to six hours, or set aside at 2\u00b0C to 8\u00b0C unl ready to set up the run. For best results, proceed directly to loading the sample and seng up the run.<\/p><p><\/p><h4>Load Sample Libraries<\/h4><ul><li>Clean the foil seal covering the reservoir labeled Load Samples with a low-lint lab ssue.<\/li><li>Pierce the foil seal with a clean 1 ml pipete.<\/li><li>Pipete 600 \u00b5l prepared libraries into the reservoir Load Samples. Avoid touching the foil seal<\/li><li>Proceed directly to the run setup steps using the MiSeq Control Soware (MCS) interface.<\/li><\/ul><p><\/p><h4>Clean\/ Load the Flow Cell<\/h4><p><\/p><ul><li>Using plasc forceps, grip the flow cell by the base of the plasc cartridge and remove it from the flow cell container<\/li><li>Lightly rinse the flow cell with laboratory-grade water unl both the glass and plasc cartridge are thoroughly rinsed of excess salts.<\/li><li>Using care around the black flow cell port gasket, thoroughly dry the flow cell and cartridge with a linree lens cleaning ssue. Gently pat dry in the area of the gasket and adjacent glass.<\/li><li>Clean the flow cell glass with an alcohol wipe. Make sure that the glass is free of streaks, fingerprints, and lint or ssue fibers.<\/li><li>Dry excess alcohol with a lint-free lens cleaning ssue.<\/li><li>Make sure that the flow cell ports are free of obstrucons and that the gasket is well-seated around the flow cell ports<\/li><\/ul><p>7.Raise the flow cell compartment door, and then press the release buton to the right of the flow cell clamp.<\/p><p>8.Make sure that the flow cell stage is free of lint. If lint or other debris is present, clean the flow cell stage using an alcohol wipe or a lint-free ssue moistened with ethanol or isopropanol. Carefully wipe the surface of the flow cell stage unl it is clean and dry.<\/p><p>9.Holding the flow cell by the edges, place it on the flow cell stage, gently press down on the flow cell clamp to close it over the flow cell. Close the flow cell compartment door.<\/p><p><\/p><h4>Load Reagents and Start the Run<\/h4><p><\/p><ul><li>Remove the botle of PR2 from 2\u00b0 to 8\u00b0C storage. Invert to mix, and then remove the lid.<\/li><li>Open the reagent compartment door.<\/li><li>Raise the sipper handle unl it locks into place.<\/li><li>Remove the wash botle and load the PR2 botle.<\/li><li>Empty the contents of the waste botle into the appropriate waste container.<\/li><li>Slowly lower the sipper handle. Make sure that the sippers lower into the PR2 and waste botles.<\/li><\/ul><ol start=\"7\"><li>Open the reagent chiller door.<\/li><li>Hold the reagent cartridge on the end with the Illumina label, and slide the reagent cartridge into the reagent chiller unl the cartridge stops.<\/li><li>Close the reagent chiller door.<\/li><li>Aer loading the flow cell and reagents, review the run parameters and perform a pre-run check before starng the run. When all items successfully pass the pre-run check, select Start Run. During a run, monitor run detail using the Sequencing screen on the instrument.<\/li><\/ol><p><\/p><p><\/p><h4>Data Analysis<\/h4><p><\/p><p>Aneuploidy screening embryos prior to transfer has been shown to deliver significant improvements IVF success. Preimplantaon genec diagnosis (PGD) enables screening of embryos for specific genec condions prior to transfer. Following sequencing sample informaon is uploaded directly from the MiSeq System, saving me and allowing sample tracking. BlueFuse Mul is a sophiscated soware for analyzing and interpreng results from molecular cytogenomic studies.<\/p><p><img decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/02\/Picture2-300x126.png\" alt=\"\" width=\"300\" height=\"126\" class=\"alignnone size-medium wp-image-712\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/02\/Picture2-300x126.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/02\/Picture2-1024x431.png 1024w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/02\/Picture2-768x323.png 768w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/02\/Picture2-1536x647.png 1536w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/02\/Picture2-2048x862.png 2048w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/02\/Picture2-228x96.png 228w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><\/p><p>Sample informaon includes sample type (e.g., blood, CVS, amnioc fluid), date of birth, pedigree number, and phenotype. Related samples can be overlaid for data comparisons and regions of change compared to phenotype. Data are permanently available and can be revisited and interrogated at any me ensuring that any follow-up quesons can be readily answered. NGS provides reliable, comprehensive screening of 24-chromosome aneuploidy, achieving results with a high degree of concordance to those obtained using established array-based PGS techniques. NGS offers a readily available, high-throughput method for PGS in the clinic with the potenal benefits of reduced costs and enhanced precision.<\/p><\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t\t\t<div class=\"elementor-toggle-item\">\n\t\t\t\t\t<div id=\"elementor-tab-title-8495\" class=\"elementor-tab-title\" data-tab=\"5\" role=\"button\" aria-controls=\"elementor-tab-content-8495\" aria-expanded=\"false\">\n\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon elementor-toggle-icon-left\" aria-hidden=\"true\">\n\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-closed\"><i class=\"fas fa-caret-right\"><\/i><\/span>\n\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-opened\"><i class=\"elementor-toggle-icon-opened fas fa-caret-up\"><\/i><\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t\t<\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t<a class=\"elementor-toggle-title\" tabindex=\"0\">Customized protocol for detection of monogenic inherited disorders in embryos derived from in vitro fertilisation (PGD preimplantation genetic diagnosis)<\/a>\n\t\t\t\t\t<\/div>\n\n\t\t\t\t\t<div id=\"elementor-tab-content-8495\" class=\"elementor-tab-content elementor-clearfix\" data-tab=\"5\" role=\"region\" aria-labelledby=\"elementor-tab-title-8495\"><p>Karyomapping is a molecular karyotyping method that can be used on a single cell or few cells from embryo biopsy samples. The method provides a comprehensive test for preimplantation genetic diagnosis (PGD) of single gene defects. It can be used where there is a risk of severe genetic disorders being inherited from parents. A couple could have a family history of a genetic disorder or have had a child with a genetic disease. PGD screening with karyomapping can be used to identify embryos that do not carry defective genes and can be safely transferred.<\/p><p>The assay requires single or multi-cell embryo biopsy samples amplified by Multiple Displacement Amplification (MDA) as starting material. The use of an MDA-based whole genome amplification (WGA) method is essential to amplify DNA from embryo biopsy samples (blastomere and trophectoderm biopsied cells) to quantities suitable for use in the Infinium Karyomapping Assay.<\/p><p>The first step of the Infinium Karyomapping Assay protocol is the WGA of the DNA generated from MDA amplified embryo biopsies and the genomic DNA from parents and reference. The preparation for this amplification is included in Prepare and Incubate the MSA3 Plate, this step should be performed in a pre-amp area specific for the WGA of the Infinium Karyomapping Assay.<\/p><p>The remaining Infinium Karyomapping Assay protocol steps can be performed in a postamplification (post-amp) area that is a separate laboratory space from the amplification areas.<\/p><p>Sample Input Requirements<\/p><p>Single and multi-cell biopsy samples do not have the required quantities of genomic DNA to be used as starting material in the Infinium Karyomapping Assay. To overcome this limitation, an initial WGA of the embryo biopsy samples using MDA is essential. In this process, genomic DNA starting from as low as 6.5 pg is amplified &gt;1000-fold resulting in several micrograms of amplified DNA. A suitable MDA kit should be used for amplifying DNA from embryo biopsy samples. End products of MDA should meet the following criteria to be suitable for their use in the Infinium Karyomapping Assay:<\/p><p>-Product length after MDA should be in the range of 2 to 100 kb<\/p><p>&#8211; Amplified product mass of 1600 to 6400 ng in a total of 8 \u03bcl (200 to 800 ng\/\u03bcl of the completed MDA reaction).<\/p><p>Prepare and Incubate the MSA3 Plate<\/p><p>Good quality parental and reference genomic DNA is required for karyomapping. The DNA is added to the MSA3 DNA plate at this stage together with the MDA amplified biopsy samples. This step denatures and neutralizes the samples, preparing them for WGA. During incubation, the genomic and amplified DNA is further amplified and prepared for downstream procedures<\/p><p><\/p><p>Consumables<\/p><table><tbody><tr><td width=\"312\"><p>MA1<\/p><\/td><td width=\"312\"><p>1 tube<\/p><\/td><\/tr><tr><td width=\"312\"><p>MSM<\/p><\/td><td width=\"312\"><p>1 tube<\/p><\/td><\/tr><tr><td width=\"312\"><p>MA2<\/p><\/td><td width=\"312\"><p>1 tube<\/p><\/td><\/tr><tr><td width=\"312\"><p>0.1N NaOH<\/p><\/td><td width=\"312\"><p>1 tube<\/p><\/td><\/tr><tr><td width=\"312\"><p>96-well 0.8ml storage plate (MIDI)<\/p><\/td><td width=\"312\"><p>1 plate<\/p><\/td><\/tr><tr><td width=\"312\"><p>DNA plate with genomic DNA samples (50ng\/ \u03bcl)<\/p><\/td><td width=\"312\"><p>1 plate or tubes<\/p><\/td><\/tr><tr><td width=\"312\"><p>DNA plate with MDA amplified products<\/p><\/td><td width=\"312\"><p>1 plate or tubes<\/p><\/td><\/tr><\/tbody><\/table><p><\/p><p>Steps<\/p><p>1 Dispense 40\u00a0\u03bcl MA1 into the MSA3 plate wells of the midi plate. Fill wells according to the plate layout diagram where columns 1 and 2 contain samples for a single BeadChip.<\/p><p>2 Transfer 8\u00a0\u03bcl (50 ng\/\u03bcl) of gDNA from parents and reference, followed by 8 \u03bcl of amplified gDNA from the MDA plate, to the corresponding wells of the MSA3 plate.<\/p><p>3 In the Lab Tracking Form, record the original DNA sample ID for each well in the MSA3 plate.<\/p><p>4 Dispense 8\u00a0\u03bcl 0.1N NaOH into each well of the MSA3 plate that contains MA1 and sample.<\/p><p>5 Seal the MSA3 plate with a 96-well cap mat, vortex the plate at 1600\u00a0rpm for 1\u00a0minute, pulse centrifuge at 280\u00a0\u00d7 g, incubate for 10\u00a0minutes at room temperature<\/p><p>6 Dispense 68\u00a0\u03bcl\u00a0MA2 into each well of the MSA3 plate containing sample.<\/p><p>7 Dispense 76\u00a0\u03bcl MSM into each well of the MSA3 plate containing sample.<\/p><p>8 Reseal the MSA3 plate with the cap mat, vortex the sealed MSA3 plate at 1600\u00a0rpm for 1\u00a0minute, pulse centrifuge at 280\u00a0\u00d7 g, incubate in the heat block with midi insert for 2 hours at 37\u00b0C.<\/p><p><strong>Fragment the DNA<\/strong><\/p><p>Consumables<\/p><table><tbody><tr><td width=\"312\"><p>FMS<\/p><\/td><td width=\"312\"><p>1 tube<\/p><\/td><\/tr><\/tbody><\/table><p>Steps<\/p><p>1 Pulse centrifuge the MSA3 plate to 280\u00a0\u00d7 g.<\/p><p>2 Add 50\u00a0\u03bcl FMS to each well containing sample.<\/p><p>3 Seal the MSA3 plate with the 96-well cap mat, vortex the plate at 1600 rpm for 1 minute, pulse centrifuge the plate to 280 \u00d7 g.<\/p><p>4 Place the sealed plate on the 37\u00b0C heat block for 30 minutes.<\/p><p><\/p><p><strong>Precipitate the DNA<\/strong><\/p><p>Consumables<\/p><table><tbody><tr><td width=\"312\"><p>PM1<\/p><\/td><td width=\"312\"><p>1 tube<\/p><\/td><\/tr><tr><td width=\"312\"><p>100% 2 propanol<\/p><\/td><td width=\"312\"><p>15 ml<\/p><\/td><\/tr><\/tbody><\/table><p><\/p><p>Steps<\/p><p>1 Remove the 96-well cap mat and add 100\u00a0\u03bcl PM1 to each MSA3 plate well containing sample.<\/p><p>2 Seal the plate with the cap mat, vortex the sealed plate at 1600\u00a0rpm for 1\u00a0minute.<\/p><p>3 Place the sealed plate on the 37\u00b0C heat block for 5\u00a0minutes, pulse centrifuge at 280 \u00d7 g<\/p><p>4 Add 310\u00a0\u03bcl 100% 2-propanol to each well containing sample.<\/p><p>5 Carefully seal the MSA3 plate with a new, dry cap mat, taking care not to shake the plate in any way until the cap mat is fully seated, invert the plate at least 10 times to mix contents thoroughly.<\/p><p>6 Place the sealed MSA3 plate in the centrifuge opposite another plate of equal weight, centrifuge at 1000\u00a0\u00d7 g at 4\u00b0C for 20\u00a0minutes, remove the MSA3 plate from centrifuge.<\/p><p>7 Over an absorbent pad, decant the supernatant by quickly inverting the MSA3 plate. Drain liquid onto the absorbent pad and then smack the plate down, avoiding the liquid that was drained onto the pad.<\/p><p>8 Tap firmly several times for 1 minute or until all wells are devoid of liquid.<\/p><p>9 Leave the uncovered, inverted plate on the tube rack for 15 minutes at room temperature to air dry the pellet. After drying, make sure that blue pellets are present at the bottoms of the wells.<\/p><p><\/p><p><strong>Resuspend the DNA<\/strong><\/p><p>Consumables<\/p><table><tbody><tr><td width=\"312\"><p>RA1<\/p><\/td><td width=\"312\"><p>17 \u03bcl \/sample<\/p><\/td><\/tr><\/tbody><\/table><p>Steps<\/p><p>1 Add 17\u00a0\u03bcl\u00a0RA1 to each well of the MSA3 plate containing a DNA pellet.<\/p><p>2 Apply a foil heat seal (with the dull side facing down) to the MSA3 plate by firmly and evenly holding the heat sealer sealing block down for 5\u00a0seconds.<\/p><p>3 Immediately remove the MSA3 plate from the heat sealer and forcefully roll the rubber plate sealer over the plate until you can see all 96 well indentations through the foil. Repeat application of the heat sealer if all 96 wells are not defined.<\/p><p>4 Place the sealed plate in the Illumina Hybridization Oven and incubate for 15 minutes at 48\u00b0C.<\/p><p>5 Vortex the plate at 1800\u00a0rpm for 1\u00a0minute, pulse centrifuge to 280\u00a0\u00d7 g.<\/p><p><\/p><p><strong>Hybridize DNA to the BeadChip<\/strong><\/p><p>Consumables<\/p><table><tbody><tr><td width=\"312\"><p>PB2<\/p><\/td><td width=\"312\"><p>1 tube<\/p><\/td><\/tr><tr><td width=\"312\"><p>Bead Chips<\/p><\/td><td width=\"312\"><p>1<\/p><\/td><\/tr><tr><td width=\"312\"><p>Hyb Chambers<\/p><\/td><td width=\"312\"><p>1<\/p><\/td><\/tr><tr><td width=\"312\"><p>Hyb Chambers gaskets<\/p><\/td><td width=\"312\"><p>1<\/p><\/td><\/tr><tr><td width=\"312\"><p>Hyb Chambers inserts<\/p><\/td><td width=\"312\"><p>1<\/p><\/td><\/tr><tr><td width=\"312\"><p>EtOH<\/p><\/td><td width=\"312\"><p>330ml<\/p><\/td><\/tr><\/tbody><\/table><p><\/p><p>Prepare the Hybridization Chambers<\/p><p>1 Place the resuspended MSA3 plate on the heat block to denature the samples at 95\u00b0C for 20\u00a0minutes. On the Lab Tracking Form, enter the start and stop times.<\/p><p>2 After the 20 minute incubation, remove the MSA3 plate from the heat block and place it on the benchtop at room temperature for 5 minutes to cool.<\/p><p>Load BeadChips<\/p><p>1 After the 5 minute cool down, pulse centrifuge the MSA3 plate to 280 \u00d7 g.<\/p><p>2 Just before loading DNA samples, remove all BeadChips from their ziplock bags and mylar packages.<\/p><p>3 Place each BeadChip in a Hyb Chamber insert, orienting the barcode end so that it matches the barcode symbol on the Hyb Chamber insert.<\/p><p>4 Remove the foil seal from the MSA3 plate, and then, using a multichannel precision pipette, dispense 15\u00a0\u03bcl of each DNA sample onto the appropriate BeadChip section.<\/p><p>5 After loading all DNA onto the BeadChip, wait for the sample to disperse over the entire surface.<\/p><p>6 Inspect the loading port to see if a large bolus of liquid remains. Excess sample volume in the BeadChip loading port helps prevent low-intensity areas resulting from evaporation.<\/p><p><\/p><p><strong>Set up the BeadChips for Hybridization<\/strong><\/p><p>1 Load the Hyb Chamber inserts containing BeadChips into the Illumina Hyb Chamber. Position the barcode end over the ridges indicated on the Hyb Chamber.<\/p><p>2 Place the back side of the lid onto the Hyb Chamber and then slowly bring down the front end to avoid dislodging the Hyb Chamber inserts.<\/p><p>3 Close the clamps on both sides of the Hyb Chamber so that the lid is secure and even on the base (no gaps). It is best to close the clamps in a kitty-corner fashion, closing first the top left clamp, then the bottom right, then the top right followed by the bottom left.<\/p><p>4 Place the Hyb Chamber in the 48\u00b0C Illumina Hybridization Oven so that the Illumina logo on top of the Hyb Chamber is facing you.<\/p><p>5 Incubate at 48\u00b0C for 12 hours or overnight but no more than 24\u00a0hours.<\/p><p><\/p><p><\/p><p><\/p><p><strong>Wash the BeadChip<\/strong><\/p><p>Consumables<\/p><table><tbody><tr><td width=\"312\"><p>PB1<\/p><\/td><td width=\"312\"><p>550 ml (up to 4 BeadChips)<\/p><\/td><\/tr><tr><td width=\"312\"><p>MultiCample BeadChip Alignment Fixture<\/p><\/td><td width=\"312\"><p>1 (per 4 BeadChips)<\/p><\/td><\/tr><tr><td width=\"312\"><p>Te- Flow flow -through chambers<\/p><\/td><td width=\"312\"><p>1 \/ BeadChip<\/p><\/td><\/tr><tr><td width=\"312\"><p>Wash dish<\/p><\/td><td width=\"312\"><p>2<\/p><\/td><\/tr><tr><td width=\"312\"><p>Wash rack<\/p><\/td><td width=\"312\"><p>1<\/p><\/td><\/tr><\/tbody><\/table><p><\/p><p>Steps<\/p><p>1 Attach the wire handle to the rack and submerge the wash rack in the first wash dish containing 200\u00a0ml PB1.<\/p><p>2 Remove a BeadChip from the Hyb Chamber and then remove its cover seal.<\/p><p>3 Immediately and carefully slide the BeadChip into the wash rack, making sure that the BeadChip is submerged in the PB1.<\/p><p>4 When all the BeadChips are in the wash rack, move the wash rack up and down for 1\u00a0minute, breaking the surface of the PB1 with gentle, slow agitation.<\/p><p>5 Move the wash rack to the other wash dish containing clean PB1. Make sure the BeadChips are submerged.<\/p><p>6 Move the wash rack up and down for 1 minute, breaking the surface of the PB1 with gentle, slow agitation.<\/p><p>8 When you remove the BeadChips from the wash rack, inspect them for remaining residue.<\/p><p><\/p><p>Assemble Flow-Through Chambers<\/p><p>1 Fill the BeadChip Alignment Fixture with 150 ml PB1.<\/p><p>2 For each BeadChip to be processed, place a black frame into the BeadChip Alignment Fixture.<\/p><p>3 Place each BeadChip into a black frame, aligning its barcode with the ridges stamped onto the Alignment Fixture. Make sure that each BeadChip is fully immersed in PB1.<\/p><p>4 Place a clear spacer onto the top of each BeadChip. Use the Alignment Fixture grooves to guide the spacers into proper position.<\/p><p>5 Place the Alignment Bar onto the Alignment Fixture. Make sure that the groove in the Alignment Bar fits over the tab on the Alignment Fixture.<\/p><p>6 Place a clean glass back plate on top of the clear spacer covering each BeadChip. Make sure that the plate reservoir is at the barcode end of the BeadChip, facing inward to create a reservoir against the BeadChip surface.<\/p><p>7 Attach the metal clamps onto each flow-through chamber as follows:<\/p><p>a Gently push up the glass back plate against the Alignment Bar with one finger.<\/p><p>b Place the first metal clamp around the flow-through chamber so that the clamp is 5 mm from the top edge.<\/p><p>c Place the second metal clamp around the flow-through chamber at the barcode end, 5 mm from the bottom of the reagent reservoir<\/p><p>8 Remove the assembled flow-through chamber from the alignment fixture and, using scissors, trim the spacer at the nonbarcode end of the assembly. Slip the scissors up over the barcode to trim the other end.<\/p><p><\/p><p><\/p><p><\/p><p><\/p><p><\/p><p><strong>Extend and Stain (XStain) BeadChips<\/strong><\/p><p>Consumables<\/p><table><tbody><tr><td width=\"312\"><p>RA1<\/p><\/td><td width=\"312\"><p>5\u00a0ml (per 4 BeadChips)<\/p><\/td><\/tr><tr><td width=\"312\"><p>XC1<\/p><\/td><td width=\"312\"><p>1 tube (per 4 BeadChips)<\/p><\/td><\/tr><tr><td width=\"312\"><p>XC2<\/p><\/td><td width=\"312\"><p>1 tube (per 4 BeadChips)<\/p><\/td><\/tr><tr><td width=\"312\"><p>TEM<\/p><\/td><td width=\"312\"><p>1 tube (per 4 BeadChips)<\/p><\/td><\/tr><tr><td width=\"312\"><p>XC3<\/p><\/td><td width=\"312\"><p>25\u00a0ml (per 4 BeadChips)<\/p><\/td><\/tr><tr><td width=\"312\"><p>STM<\/p><\/td><td width=\"312\"><p>1 tube (per 4 BeadChips)<\/p><\/td><\/tr><tr><td width=\"312\"><p>ATM<\/p><\/td><td width=\"312\"><p>1 tube (per 4 BeadChips)<\/p><\/td><\/tr><tr><td width=\"312\"><p>PB1<\/p><\/td><td width=\"312\"><p>310\u00a0ml (up to 4 BeadChips)<\/p><\/td><\/tr><tr><td width=\"312\"><p>XC4<\/p><\/td><td width=\"312\"><p>310\u00a0ml (up to 4 BeadChips)<\/p><\/td><\/tr><tr><td width=\"312\"><p>Alconox Power Detergent<\/p><\/td><td width=\"312\"><p>as needed<\/p><\/td><\/tr><tr><td width=\"312\"><p>EtOH<\/p><\/td><td width=\"312\"><p>as needed<\/p><\/td><\/tr><tr><td width=\"312\"><p>95% formamide \/ 1mM EDTA<\/p><\/td><td width=\"312\"><p>10\u00a0ml (up to 4 BeadChips)<\/p><\/td><\/tr><\/tbody><\/table><p><\/p><p>Single-Base Extension<\/p><p>1 When the Chamber Rack reaches 44\u00b0C \u00b1 0.5\u00b0C, quickly place each flow-through chamber assembly into the Chamber Rack.<\/p><p>2 Into the reservoir of each flow-through chamber, dispense:<\/p><p>a150\u00a0\u03bcl RA1. Incubate for 30 seconds. Repeat 5 times.<\/p><p>b 450\u00a0\u03bcl XC1. Incubate for 10 minutes.<\/p><p>c 450\u00a0\u03bcl XC2. Incubate for 10 minutes.<\/p><p>d 200\u00a0\u03bcl TEM. Incubate for 15 minutes.<\/p><p>e 450\u00a0\u03bcl 95% formamide\/1 mM EDTA. Incubate for 1 minute. Repeat one time.<\/p><p>f Incubate 5 minutes.<\/p><p>g Begin ramping the Chamber Rack temperature to the temperature indicated on the STM tube \u00b1 0.5\u00b0C.<\/p><p>h 450\u00a0\u03bcl XC3. Incubate for 1 minute. Repeat one time.<\/p><p>Stain BeadChip<\/p><p>1 Into the reservoir of each flow-through chamber, dispense:<\/p><p>a 250 \u03bcl STM. Incubate for 10 minutes.<\/p><p>b 450 \u03bcl XC3. Incubate for 1 minute. Repeat one time.<\/p><p>c Wait 5 minutes. d 250 \u03bcl ATM. Incubate for 10 minutes.<\/p><p>e 450 \u03bcl XC3. Incubate for 1 minute. Repeat one time.<\/p><p>f Wait 5 minutes. g 250 \u03bcl STM. Incubate for 10 minutes.<\/p><p>h 450 \u03bcl XC3. Incubate for 1 minute. Repeat one time.<\/p><p>i Wait 5 minutes.<\/p><p>j 250 \u03bcl ATM. Incubate for 10 minutes.<\/p><p>k 450 \u03bcl XC3. Incubate for 1 minute. Repeat one time.<\/p><p>l Wait 5 minutes.<\/p><p>m 250 \u03bcl STM. Incubate for 10 minutes.<\/p><p>n 450 \u03bcl XC3. Incubate for 1 minute. Repeat one time.<\/p><p>o Wait 5 minute<\/p><p><strong>Wash and Coat BeadChips<\/strong><\/p><p>Steps<\/p><p>1 Set up 2 top-loading wash dishes, labeled &#8222;PB1&#8221; and &#8222;XC4&#8221;, pour 310\u00a0ml PB1 into the wash dish labeled \u201cPB1.\u201d, submerge the unloaded staining rack into the wash dish with the locking arms and tab facing towards you.<\/p><p>2 Disassemble each flow-through chamber, use the dismantling tool to remove the 2 metal clamps, remove the glass back plate by lifting the glass straight up, set the glass back plate aside, remove the spacer, remove the BeadChip from the black frame.<\/p><p>3 Place the BeadChips in the staining rack while it is submerged in PB1, slowly move the staining rack up and down 10 times, breaking the surface of the reagent, soak for 5\u00a0minutes.<\/p><ol start=\"4\"><li>Shake the XC4 bottle vigorously to ensure complete resuspension. If necessary, vortex until dissolved, pour 310\u00a0ml XC4 into the dish labeled \u201cXC4,\u201d and cover the dish to prevent any lint or dust from falling into the solution.<\/li><\/ol><p>5 Remove the staining rack from the PB1 dish and place it directly into the wash dish containing XC4, slowly move the staining rack up and down 10\u00a0times, breaking the surface of the reagent, soak for 5\u00a0minutes.<\/p><p>6 Remove the staining rack in one smooth, rapid motion and place it directly on the prepared tube rack, place the tube rack in the vacuum desiccator. Start the vacuum, using at least 675\u00a0mm Hg (0.9 bar). Dry under vacuum for 50\u201355 minutes<\/p><ol start=\"7\"><li>Scan the BeadChips within 72 hours.<\/li><\/ol><p><\/p><p>Perform analysis using BlueFuse Multi software, which requires that scanning data are available in a genotype call (GTC) file format. By default, the NextSeq 550 system generates normalized data and associated genotype calls in the format of a GTC file. The iScan can be configured to produce GTC files, in addition to the standard file types (such as IDAT files).<\/p><\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t\t\t<div class=\"elementor-toggle-item\">\n\t\t\t\t\t<div id=\"elementor-tab-title-8496\" class=\"elementor-tab-title\" data-tab=\"6\" role=\"button\" aria-controls=\"elementor-tab-content-8496\" aria-expanded=\"false\">\n\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon elementor-toggle-icon-left\" aria-hidden=\"true\">\n\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-closed\"><i class=\"fas fa-caret-right\"><\/i><\/span>\n\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-opened\"><i class=\"elementor-toggle-icon-opened fas fa-caret-up\"><\/i><\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t\t<\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t<a class=\"elementor-toggle-title\" tabindex=\"0\">Customized protocols for genetic screening and testing for couples who have had multiple failed IVF transfers and women with reoccurring miscarriage<\/a>\n\t\t\t\t\t<\/div>\n\n\t\t\t\t\t<div id=\"elementor-tab-content-8496\" class=\"elementor-tab-content elementor-clearfix\" data-tab=\"6\" role=\"region\" aria-labelledby=\"elementor-tab-title-8496\"><p>When couples have experienced multiple failed IVF transfers or women have had recurrent miscarriages, it&#8217;s crucial to consider customized genetic screening and testing to identify potential underlying causes. Customized protocols can help pinpoint specific issues and guide treatment decisions. Here&#8217;s a suggested approach:<\/p><p><\/p><ol><li><strong> <\/strong><strong>Initial Consultation:<\/strong><\/li><\/ol><p>Start with a comprehensive consultation with a reproductive specialist or fertility expert who can assess the medical history, previous fertility treatments, and genetic background of both partners.<\/p><ol start=\"2\"><li><strong> <\/strong><strong>Chromosomal Testing:<\/strong><ol><li>Karyotype Analysis: Assess the chromosomal makeup of both partners to identify any structural or numerical chromosomal abnormalities that might contribute to recurrent miscarriages.<\/li><li>Preimplantation Genetic Testing (PGT-A or PGT-M): If you&#8217;re pursuing IVF, consider PGT-A (aneuploidy screening) or PGT-M (monogenic disorder screening) to select embryos with the highest chance of success.<\/li><\/ol><\/li><li><strong> <\/strong><strong>Hormonal and Autoimmune Testing:<\/strong><\/li><\/ol><p>Evaluate hormone levels, thyroid function, and autoimmune markers. Imbalances or autoimmune issues can affect fertility and may lead to recurrent miscarriages.<\/p><ol start=\"4\"><li><strong> <\/strong><strong>Genetic Carrier Screening:<\/strong><\/li><\/ol><p>Both partners should undergo genetic carrier screening to identify potential recessive genetic conditions. Carrier status can impact the likelihood of genetic disorders in offspring.<\/p><ol start=\"5\"><li><strong> <\/strong><strong>Thrombophilia and Blood Clotting Disorders:<\/strong><\/li><\/ol><p>Investigate for thrombophilia and blood clotting disorders, as these conditions may lead to recurrent miscarriages.<\/p><ol start=\"6\"><li><strong> <\/strong><strong>Immunological Factors:<\/strong><\/li><\/ol><p>Explore potential immunological factors affecting fertility and pregnancy, including antiphospholipid syndrome and natural killer cell activity.<\/p><ol start=\"7\"><li><strong> <\/strong><strong>Endometrial Receptivity Testing:<\/strong><\/li><\/ol><p>Assess the uterine lining to ensure it is receptive for embryo implantation. Endometrial receptivity testing may include a receptive window assessment.<\/p><ol start=\"8\"><li><strong> <\/strong><strong>Hysteroscopy:<\/strong><\/li><\/ol><p>A hysteroscopy can detect uterine abnormalities or polyps that might interfere with implantation or cause recurrent miscarriages.<\/p><ol start=\"9\"><li><strong> <\/strong><strong>Lifestyle and Environmental Factors:<\/strong><\/li><\/ol><p>Evaluate lifestyle factors like diet, exercise, and stress, as well as environmental exposures, which may impact fertility and pregnancy outcomes.<\/p><ol start=\"10\"><li><strong> <\/strong><strong>Multidisciplinary Team:<\/strong><\/li><\/ol><p>Consider involving a multidisciplinary team of fertility experts, genetic counselors, and specialists to develop a tailored plan for each couple.<\/p><ol start=\"11\"><li><strong> <\/strong><strong>Treatment Options:<\/strong><\/li><\/ol><p>Based on the findings, develop a personalized treatment plan, which may include adjusting IVF protocols, lifestyle modifications, medications, or surgical interventions.<\/p><ol start=\"12\"><li><strong> <\/strong><strong>Counseling and Emotional Support:<\/strong><\/li><\/ol><p>Recurrent IVF failures and miscarriages can be emotionally challenging. Provide psychological support and counseling to help couples cope with the stress.<\/p><ol start=\"13\"><li><strong> <\/strong><strong>Ongoing Monitoring:<\/strong><\/li><\/ol><p>Regularly monitor the progress and adjust as needed, based on the response to treatment.<\/p><p><\/p><p>Customized genetic screening and testing protocols should be adapted to each couple&#8217;s unique situation. It&#8217;s essential to work closely with a fertility specialist to address specific concerns and tailor the testing and treatment plan accordingly. Genetic counseling can help couples understand their genetic risks and make informed decisions regarding family planning.<\/p><p>We created an advanced next-generation sequencing (NGS) panel designed to examine genes linked to infertility. This panel specifically focused on exons and their surrounding regions, incorporating selected introns within the CFTR gene. Additionally, the analysis of the FMR1 gene and Y chromosome microdeletions was carried out using other recommended techniques.<\/p><p><strong>DNA<\/strong><\/p><ul><li>DNA extracted from blood and saliva using the QIAamp Mini Kits (Qiagen) following the manufacturer&#8217;s instructions;<\/li><li>DNA quantification using Qubit dsDNA BR Assay Kit and the Qubit\u00ae 3.0 fluorometer;<\/li><li>genes with proven disease-associated variants related to infertility disorders included in the targeted NGS panel (&#8222;diagnostic genes&#8221;). The panel also included &#8222;candidate genes&#8221; which their association with infertility needs further study;<\/li><li>selection of the genes based mainly on relevant literature referenced in PubMed and Online Mendelian Inheritance in Man (OMIM) and guided by ClinGen Clinical Validity Classifications (Strande et al., 2017);<\/li><li>designated panel should encompass the coding exons and adjacent regions, extending at least 10 nucleotides upstream and downstream of each exon (in accordance with the RefSeq database);<\/li><li>clinically significant noncoding (intronic) areas harboring known pathogenic variants in the CFTR gene should also be part of the panel;<\/li><li>specific regions for determining the sample&#8217;s gender should be incorporated as an internal control.<\/li><\/ul><p><strong>Next-generation DNA sequencing<\/strong><\/p><ul><li>DNA samples prepared for sequencing following the manufacturer&#8217;s instructions described in the SureSelectQXT Target Enrichment for Illumina Multiplexed Sequencing manual (Agilent Technologies, Inc.), a transposase-based library preparation technology;<\/li><li>DNA libraries quantification performed using Qubit dsDNA Assay with the Qubit\u00ae 3.0 fluorometer. DNA Libraries were sequenced on a MiSeq platform (Illumina, San Diego, CA) using a 300 cycles-MiSeq Reagent Micro Kit v2 following the manufacturer&#8217;s instructions.<\/li><\/ul><p><strong>Sequence analysis and variant interpretation<\/strong><\/p><p>Sequence data analysis was carried out using an in-house developed bioinformatic pipeline, built on the best practices in the field.<\/p><ul><li>data demultiplexed and converted to Fastq format simultaneously using bcl2fastq software (Illumina, 2017);<\/li><li>quality control of the data performed using FastQC software (Babraham Bioinformatics, 2017). Reads aligned against the human reference genome hg19 using Burrows-Wheeler Aligner v0.7.17 (Li &amp; Durbin, 2009) and in order to detect single-nucleotide variants (SNVs) and small insertions and deletions, Genome Analysis Toolkit v4.0 was used (Van der Auwera et al., 2013);<\/li><li>for obtaining biological and functional information, variants annotated against several databases, such as ClinVar, dbSNP, 1000Genomes, dbNSFP and ExAC, among others, using SnpEff software v4.3i (Cingolani et al., 2012).<\/li><\/ul><p>We employed allele frequency as a criterion for categorizing identified variants into common or rare groups. Variants with an allele frequency of \u2265 5% in ExAC were designated as common and subsequently excluded, with a few exceptions. Among the included genes, some were associated with pharmacogenetic effects, particularly those encoding receptors that could influence the response to ovarian hyperstimulation treatment. For these genes, variants with an allele frequency higher than 5% were also scrutinized. Additionally, the filtering process took into account the outcomes from various bioinformatic predictors regarding pathogenicity and the variant&#8217;s type of effect.<\/p><p>Finally, filtered variants were classified as pathogenic, likely pathogenic, variant of unknown significance (VUS), likely benign or benign, according to the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines (Richards et al., 2015). The report of the variants was made according to the Human Genome Variation Society (HGVS) nomenclature (den Dunnen et al., 2016).<\/p><p>FMR1 testing FMR1 testing was performed using 20-80 ng of genomic DNA per sample. The FMR1 CGG repeat region was amplified using the AmplideX\u00ae PCR\/CE FMR1 kit (TP-PCR), following the manufacturer&#8217;s instructions. PCR products were then separated by capillary electrophoresis on an ABI 3500xL. Manual annotation was performed using GeneMapper\u00ae 5.0 software (Applied BiosystemsTM).<\/p><p><strong>CFTR, T\/TG tract analysis.<\/strong> The amplification of intron 9 &#8211; exon 10 region was performed using the following conditions: genomic DNA was amplified in a reaction that contained 2\u00b5L 50\u00b5M of each primer (Invitrogen), 0.4\u00b5L 10 mM dNTP Set (Invitrogen), 2,5\u00b5L 50 mM MgCl, 0,2\u00b5L 5U\/\u00b5L Taq DNA polymerase (Invitrogen), 5\u00b5L 10x buffer and 2\u00b5L 20-60 ng\/\u00b5L genomic DNA. It was completed with sterile distilled water up to a volume of 50\u00b5L. The primers used were: 5\u00b4CCATGTGCTTTTCAAACTAATTGT3\u00b4 (forward), 5\u00b4TAAAGTTATTGAA TGCTC GCCATG 3\u00b4(reverse).<\/p><p>The diagnosis of Y chromosome microdeletion was made by two multiplexes PCRs. Both reactions amplify three AZF loci and the SRY control fragment. DNA samples from a fertile male and female were used as an internal controls in each multiplex assay according to the recommendations of the European Academy of Andrology (EAA) and the European Molecular Genetics Quality Network (EMQN) guidelines (Krausz et al, 2014).<\/p><p>The 50 \u00b5L PCR reaction mix contained: 0.4\u00b5L 10 mM dNTP Set (Invitrogen), 1.85\u00b5L 50 mM MgCl, 0.4\u00b5L 5U\/\u00b5L Taq DNA polymerase (Invitrogen), 2.5\u00b5L 10x buffer, 4\u00b5L 100 ng\/\u00b5L template DNA and sterile distilled water.<\/p><p><strong>Amplification conditions: <\/strong><\/p><ul><li>start with an initial step of 94\u00b0C 2 min;<\/li><li>35 cycles at 94\u00b0C for 30 sec, 60\u00b0C for 30 sec and 72\u00b0C for 30 sec;<\/li><li>ended by a last elongation step of 7 min at 72\u00baC using a Veriti 96-well thermal cycler (Applied Biosystems, Foster City, CA);<\/li><li>reaction products separated by 3% agarose gel electrophoresis.<\/li><\/ul><p><strong>Fertility gene panel design<\/strong><\/p><p><\/p><p>After the selection and classification of genes associated with infertility, a custom sequencing panel was designed including 75 genes. Genes were grouped into two sub-panels: a female fertility panel and a male fertility panel (Lorenzi et al., 2020).<\/p><p><\/p><table><tbody><tr><td width=\"246\"><p><strong>Female conditions<\/strong><\/p><\/td><td width=\"378\"><p><strong>\u00a0<\/strong><\/p><\/td><\/tr><tr><td width=\"246\"><p><strong>Primary ovarian insufficiency\u00a0<\/strong><\/p><\/td><td width=\"378\"><p><strong><em>BMP15, ESR1, FGFR1, FIGLA, FMR1, FOXL2, FSHB, FSHR, GALT, GDF9, HFM1, KISS1R, LHB, MCM8, MCM9, NOBOX, NR5A1, POF1B, PSMC3IP, SOHLH1, STAG3, SYCE1,<\/em><\/strong><em>\u00a0DIAPH2, DMC1, FOXO3, LHCGR, LHX8, MSH4, NANOS3, PGRMC1, REC8, SMC1B<\/em><\/p><\/td><\/tr><tr><td width=\"246\"><p><strong>Oocyte maturation defects<\/strong><\/p><\/td><td width=\"378\"><p><strong><em>TUBB8, ZP1<\/em><\/strong><\/p><\/td><\/tr><tr><td width=\"246\"><p><strong>Embryo development arrest<\/strong><\/p><\/td><td width=\"378\"><p><strong><em>PADI6, TLE6<\/em><\/strong><\/p><\/td><\/tr><tr><td width=\"246\"><p><strong>Ovarian hyperstimulation syndrome<\/strong><\/p><\/td><td width=\"378\"><p><strong><em>FSHR,\u00a0<\/em><\/strong><em>AMH, AMHR2, CAPN10, DENND1A, GDF9 LHCGR, SULT2A1, THADA\u00a0<\/em><\/p><\/td><\/tr><tr><td width=\"246\"><p><strong>Recurrent pregnancy loss<\/strong><\/p><\/td><td width=\"378\"><p><em>F2, F5, MTHFR, PROC, PROS1 SERPINC1, SYCP3<\/em><\/p><\/td><\/tr><tr><td width=\"246\"><p><strong>Hormone receptors<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7169920\/table\/t1\/?report=objectonly#TFN1\"><sup>*<\/sup><\/a><\/strong><\/p><\/td><td width=\"378\"><p><strong><em>AR, ESR1, FSHR, LHCGR<\/em><\/strong><\/p><\/td><\/tr><\/tbody><\/table><p><\/p><p><\/p><table><tbody><tr><td width=\"246\"><p><strong>Male conditions<\/strong><\/p><\/td><td width=\"378\"><p><strong>\u00a0<\/strong><\/p><\/td><\/tr><tr><td width=\"246\"><p><strong>Non-obstructive azoospermia\/ Severe oligospermia<\/strong><\/p><\/td><td width=\"378\"><p><strong><em>AR, AZF, FGFR1, FSHB, KAL1\/ANOS1, KISS1R, KLHL10, LHB, NANOS1, NR5A1, SOHLH1, SYCE1, SYCP3, TAF4B, TEX11, TEX15, USP9Y, ZMYND15,\u00a0<\/em><\/strong><em>HSF2\u00a0<\/em><\/p><\/td><\/tr><tr><td width=\"246\"><p><strong>Obstructive azoospermia<\/strong><\/p><\/td><td width=\"378\"><p><strong><em>ADGRG2, CFTR<\/em><\/strong><\/p><\/td><\/tr><tr><td width=\"246\"><p><strong>Oocyte activation failure<\/strong><\/p><\/td><td width=\"378\"><p><strong><em>PLCZ1<\/em><\/strong><\/p><\/td><\/tr><tr><td width=\"246\"><p><strong>Asthenozoospermia<\/strong><\/p><\/td><td width=\"378\"><p><strong><em>CATSPER1, DNAH1, DNAH5, DNAI1 SEPT12, SLC26A8, SUN5,\u00a0<\/em><\/strong><em>CATSPER2 GALNTL5\u00a0<\/em><\/p><\/td><\/tr><tr><td width=\"246\"><p><strong>Sperm morphology alterations\u00a0<\/strong><\/p><\/td><td width=\"378\"><p><strong><em>AURKC, DPY19L2, SEPT12 SPATA16, SUN5,<\/em><\/strong>\u00a0<em>PICK1, ZPBP\u00a0<\/em><\/p><\/td><\/tr><tr><td width=\"246\"><p><strong>Hormone receptors<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7169920\/table\/t1\/?report=objectonly#TFN1\"><sup>*<\/sup><\/a><\/strong><\/p><\/td><td width=\"378\"><p><strong><em>AR, ESR1, FSHR, LHCGR<\/em><\/strong><\/p><\/td><\/tr><\/tbody><\/table><p><\/p><p><sup>*<\/sup>Hormone receptors are evaluated in both panels.<\/p><p><\/p><p>Genes associated with female and male infertility included in the NGS panel. Genes in bold font have proven associations with infertility (diagnostic genes). Candidate genes are shown in normal font.<\/p><p><\/p><p><strong>\u00a0<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><p><strong>References:<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><ol><li>Strande NT, Riggs ER, Buchanan AH, Ceyhan-Birsoy O, DiStefano M, Dwight SS, Goldstein J, Ghosh R, Seifert BA, Sneddon TP, Wright MW, Milko LV, Cherry JM, Giovanni MA, Murray MF, O&#8217;Daniel JM, Ramos EM, Santani AB, Scott AF, Plon SE, Rehm HL, Martin CL, Berg JS. Evaluating the Clinical Validity of Gene-Disease Associations: An Evidence-Based Framework Developed by the Clinical Genome Resource.\u00a0<em>Am J Hum Genet.\u00a0<\/em>2017; 100: 895-906. doi:\u00a010.1016\/j.ajhg.2017.04.015.<\/li><li>Illumina, Inc. Bcl2fastq Conversion Software. San Diego: Illumina; 2017. Version 2.17.1.14.<\/li><li>Babraham Bioinformatics.\u00a0FastQC: a quality control tool for high throughput sequence data.<em>Cambridge: Babraham Bioinformatics<\/em>; 2017. Version 0.11.7.<\/li><li>Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler Transform.\u00a0<em><\/em>2009; 25: 1754\u20131760. doi:\u00a010.1093\/bioinformatics\/btp324.<\/li><li>Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, Levy-Moonshine A, Jordan T, Shakir K, Roazen D, Thibault J, Banks E, Garimella KV, Altshuler D, Gabriel S, DePristo MA. From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline.\u00a0<em>Curr Protoc Bioinformatics.\u00a0<\/em>2013; 43: 11.10.1\u201311.1033. doi:\u00a010.1002\/0471250953.bi1110s43.<\/li><li>Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3.\u00a0<em>Fly (Austin)\u00a0<\/em>2012; 6: 80-92. doi:\u00a010.4161\/fly.19695.<\/li><li>Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL, ACMG Laboratory Quality Assurance Committee Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.\u00a0<em>Genet Med.\u00a0<\/em>2015; 17: 405-423. doi:\u00a010.1038\/gim.2015.30.<\/li><li>den Dunnen JT, Dalgleish R, Maglott DR, Hart RK, Greenblatt MS, McGowan-Jordan J, Roux AF, Smith T, Antonarakis SE, Taschner PE. HGVS Recommendations for the Description of Sequence Variants: 2016 Update.\u00a0<em>Hum Mutat.\u00a0<\/em>2016; 37: 564-569. doi:\u00a010.1002\/humu.22981.<\/li><li>Krausz C, Hoefsloot L, Simoni M, T\u00fcttelmann F. European Academy of Andrology; European Molecular Genetics Quality Network. EAA\/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions: state-of-the-art 2013.\u00a0<em><\/em>2014; 2: 5-19. doi:\u00a010.1111\/j.2047-2927.2013.00173.x.<\/li><li>Lorenzi D, Fernandez C, Bilinski M, Fabbro M, Galain M, Menazzi S, Miguens M, Perassi PN, Fulco MF, Kipelman S, Fiszbajn G, Nodar F, Papier S. First custom next-generation sequencing infertility panel in Latin America: design and first results. <em>JBRA Assist Reprod.<\/em> 2020; 24(2): 104-114. doi: 10.5935\/1518-0557.20190065<\/li><\/ol><\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t\t\t<div class=\"elementor-toggle-item\">\n\t\t\t\t\t<div id=\"elementor-tab-title-8497\" class=\"elementor-tab-title\" data-tab=\"7\" role=\"button\" aria-controls=\"elementor-tab-content-8497\" aria-expanded=\"false\">\n\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon elementor-toggle-icon-left\" aria-hidden=\"true\">\n\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-closed\"><i class=\"fas fa-caret-right\"><\/i><\/span>\n\t\t\t\t\t\t\t\t<span class=\"elementor-toggle-icon-opened\"><i class=\"elementor-toggle-icon-opened fas fa-caret-up\"><\/i><\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t\t<\/span>\n\t\t\t\t\t\t\t\t\t\t\t\t<a class=\"elementor-toggle-title\" tabindex=\"0\">Study on the Regional Microbiologic and Genetic Profile of Infertility<\/a>\n\t\t\t\t\t<\/div>\n\n\t\t\t\t\t<div id=\"elementor-tab-content-8497\" class=\"elementor-tab-content elementor-clearfix\" data-tab=\"7\" role=\"region\" aria-labelledby=\"elementor-tab-title-8497\"><p>A joint study on the regional genetic and epidemiological profile and the most common microbial species in symptomatic or asymptomatic infertile persons was developed using the data collected during the project from both sides of the cross-border region.<\/p><p>Infertility is a multifactorial health issue affecting couples worldwide, and its etiology varies across populations. This study aims to explore the regional microbiologic and genetic profiles of infertility in Romania and Hungary, shedding light on the unique factors contributing to reproductive challenges in these two Central European countries in the cross-border area.<\/p><p>According to WHO, addressing infertility is an important component of sexual and reproductive health and rights, and is central to achieving SDG 3 (Ensure healthy lives and promote well-being for all at all ages) and SDG 5 (Achieve gender equality and empower all women and girls) (SDG \u2013 Sustainable Development Goal). Addressing infertility is also central to achieving the human rights to the enjoyment of the highest attainable standard of physical and mental health and to decide the number, timing and spacing of one\u2019s children. Understanding the magnitude of infertility is critical for developing appropriate interventions, for monitoring access to quality fertility care, and for mitigating risk factors for and consequences of infertility. Yet there is considerable variation in estimates of infertility. Differences in how infertility is defined and measured partly contribute to this variation.<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture1-300x147.png\" alt=\"\" width=\"300\" height=\"147\" class=\"alignnone size-medium wp-image-767\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture1-300x147.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture1-768x377.png 768w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture1-228x112.png 228w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture1.png 936w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p>In the WHO report, 75 health for all, Human Reproduction Programme, Infertility prevalence estimates between 1990-2021, regional infertility prevalence estimates are defined as lifetime infertility prevalence and period of infertility prevalence. These statistics indicate that the European region has the highest prevalence estimates ranges, between 5.0-34.0 %.<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture2-300x249.png\" alt=\"\" width=\"300\" height=\"249\" class=\"alignnone size-medium wp-image-768\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture2-300x249.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture2-768x638.png 768w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture2-140x116.png 140w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture2.png 988w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture3-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-769\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture3-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture3-768x461.png 768w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture3-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture3.png 986w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><ol><li><strong> <\/strong><strong>Study Design:<\/strong><\/li><\/ol><ul><li>Cross-sectional study design<\/li><li>Inclusion criteria: Couples seeking fertility evaluation and treatment in selected regions of Romania and Hungary<\/li><li>Exclusion criteria: Couples with known medical conditions affecting fertility unrelated to microbiologic or genetic factors.<\/li><\/ul><ol start=\"2\"><li><strong> <\/strong><strong>Data Collection:<\/strong><\/li><\/ol><ul><li>Microbiologic Profile:<\/li><li>Collection of genital samples from both partners for microbiological analysis, including bacterial and viral assessments.<\/li><li>Investigation of sexually transmitted infections (STIs) and other relevant microbial factors.<\/li><li>Genetic Profile:<\/li><li>Blood samples obtained from both partners for genetic testing.<\/li><li>Analysis of common genetic mutations associated with infertility.<\/li><li>Assessment of chromosomal abnormalities.<\/li><li>Demographic and Clinical Data:<\/li><li>Collection of demographic information, medical history, lifestyle factors, and previous fertility treatments.<\/li><\/ul><ol start=\"3\"><li><strong> <\/strong><strong>Laboratory Analysis:<\/strong><\/li><\/ol><ul><li>Microbiologic Analysis:<\/li><li>Culture and sensitivity testing for bacterial and viral isolates.<\/li><li>Polymerase chain reaction (PCR) assays for specific pathogens.<\/li><li>Quantitative analysis of microbial load.<\/li><li>Genetic Analysis:<\/li><li>DNA extraction and purification.<\/li><li>Genotyping for known genetic markers associated with infertility.<\/li><li>Karyotyping for chromosomal abnormalities.<\/li><\/ul><ol start=\"4\"><li><strong> <\/strong><strong>Statistical Analysis:<\/strong><\/li><\/ol><ul><li>Descriptive statistics for demographic and clinical variables.<\/li><li>Comparison of microbiologic and genetic profiles between regions using appropriate statistical tests.<\/li><li>Correlation analysis between microbiologic and genetic factors with infertility outcomes.<\/li><\/ul><p>The study&#8217;s results provided insights into the prevalence of microbial infections and genetic factors contributing to infertility in different regions of Romania and Hungary. Comparative analyses identified potential regional variations and patterns.<\/p><p>Understanding the regional microbiologic and genetic profiles of infertility in Romania and Hungary is crucial for developing targeted interventions and personalized treatment strategies. This study contributes valuable data to the global understanding of infertility, helping healthcare professionals tailor interventions to the specific needs of the population in these Central European countries.<\/p><p><strong>\u00a0<\/strong><\/p><p><strong>Microorganisms capable of interfering with the reproductive function of women<\/strong><\/p><p>The top microorganisms involved in UGT infections, and considered as causes of female cervicitis, PID, infertility, and preterm delivery are:<\/p><ul><li><em> trachomatis<\/em><\/li><li><em> gonorrhoeae<\/em>,<\/li><li>bacterial vaginosis (<em>Gardnerella vaginalis<\/em> infections)<\/li><li><em>Ureaplasma urealyticum<\/em> and <em>Mycoplasma hominis<\/em><\/li><\/ul><p>An important role in female infertility: <em>Treponema pallidum, Candida <\/em>spp., <em>Cytomegalovirus, Trichomonas vaginalis, Toxoplasma gondii, <\/em>etc.<\/p><p><em>GNB, GPC: <\/em><em>s<\/em>ome other causes of the acute inflammatory diseases of the UGT, in infertile, pregnant women or neonate infections.\u00a0<\/p><p><strong>Infections occurring around the time of birth<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><p><strong>Effects on the foetus and neonates<\/strong><\/p><ul><li><strong>Viral infections:<\/strong><\/li><li>rubella, CMV &#8211; less damaging to the foetus when maternal infection takes place late in pregnancy;<\/li><li>varicella-zoster virus at this time can lead to limb deformities and other severe lesions in the newborn.<\/li><li><strong>Bacterial infections<\/strong><strong>:<\/strong><\/li><li>group B streptococci, <em> coli, Klebsiella, Proteus,Bacteroides, <\/em>staphylococci, <em>Mycoplasma hominis) <\/em><\/li><\/ul><p>These infections are originating from the vagina and perineum are more important, occurring especially when fetal membranes have been ruptured for more than 1-2 days, and resulting in chorioamnionitis, maternal fever, premature deliver. They can be acquired after delivery, giving later onset disease. Infants of low birth weight (less than 1500g) tend to be more severely affected. Neonatal septicemia often progresses to meningitis \u2013 frequently fatal unless treated.<\/p><p>Clinical diagnosis is difficult because the infant shows generalized signs: respiratory distress, poor feeding, diarrhea and vomiting, but early diagnosis is essential and requires emergency treatment.<\/p><p><strong>Miscellaneous neonatal infections<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><p>Infection may reach the newborn infant during the first week or two after birth, rather than during delivery.<\/p><ul><li>Group B beta-haemolytic streptococci and GNB can still cause serious infection at this time, often with meningitis;<\/li><li>Herpes simplex may come from cold sores of attending adults;<\/li><li>During the first week or two of life the nose of the neonate becomes colonized with <em>aureus <\/em>which can enter the nipple during feeding to cause a breast abscess.<\/li><\/ul><p>These infections are preventable when hospital staff pay vigorous attention to hand washing and aseptic techniques.<\/p><p><strong>Effects on the mother<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><p>Puerperal sepsis was a major cause of maternal death in Europe in the 19th Century. In 1843 Oliver Wendell Holmes made the unpopular suggestion that it was carried on the hands of doctors, and four years later Ignaz Semmelweiss in Vienna showed how it could be prevented if doctors washed their hands before attending a woman in labor and practiced aseptic techniques.<\/p><p>Major culprits for puerperal sepsis were:<\/p><ul><li>Group A beta-hemolytic streptococci,<\/li><li>Other possible organisms include anaerobes such as <em>Clostridium perfringens <\/em>or <em>Bacteroides, and E. coli<\/em>.<\/li><\/ul><p>The streptococci came from the nose, throat or skin of hospital attendants whereas the others were derived from the mother&#8217;s own fecal flora. Puerperal sepsis, which carried up to 10% mortality until the 1930s, is now, like septic abortion, less common in developed countries. Predisposing factors include: premature rupture of the membranes, instrumentation and retained fragments of membrane or placenta. Where there is postnatal pyrexia or massive discharge, high vaginal swabs and blood cultures should be taken.<\/p><p><strong>Infections in newborns<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><p>Infections remain one of the most significant problems in modern medicine. Once the fetus is infected it is susceptible, its immune defenses are poor, IgM and lgA antibodies are not produced in significant amounts until the second half of pregnancy and cell-mediated immune responses are poorly developed or absent, with inadequate production of the necessary cytokines. The newborn&#8217;s microbiome contributes to the development of its immune system. Antibiotics administered during pregnancy alter the microbiome and may influence disease risks in newborns.<\/p><p><strong>\u00a0<\/strong><\/p><p><strong>Vaginal colonization with bacteria that can cause infections in newborns<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><p>The predominant flora in the vagina is represented by <em>Lactobacillus<\/em> and <em>Streptococcus <\/em>spp. However, the presence of other bacteria such as <em>E. coli, Klebsiella <\/em>spp. can be very important, although not necessarily synonymous with infection. <em>E. coli<\/em> in the vagina can cause symptomatic or asymptomatic infections and is associated with neonatal sepsis. These strains possess several virulence factors that allow vaginal and\/or endocervical colonization.<em> E. coli <\/em>&#8211; important cause of neonatal respiratory tract infections and is associated with high mortality. High prevalence of <em>Klebsiella<\/em> ESBL colonization that can be transmitted to newborns at birth have also been reported.<\/p><p>Maternal-to-newborn transmission of <em>E. coli <\/em>can cause early infections in newborns and can occur after infection of the amniotic fluid, after rupture of membranes, or during the passage of the newborn through the vaginal canal during birth. <em>E. coli <\/em>and group B streptococcus are cited as the two most frequent bacteria isolated from neonates with sepsis or meningitis. Neonates born at preterm gestational ages are more likely to develop neonatal sepsis and meningitis. The rates of vaginal colonization with <em>Staphylococcus<\/em> spp. range from 5 to 26% and there is increasing incidence of infections in pregnant women as well as neonates caused by these organisms. <em>S. aureus <\/em>is more prevalent in vaginal tract of pregnant women when compared to the other species.<\/p><p><strong>\u00a0<\/strong><\/p><p><strong>The study of microbial infections\/colonization during pregnancy<\/strong><\/p><p>This is an observational, retrospective study which included all strains isolated from pregnant women hospitalized in our hospital, due to any infectious disease between 01.09.2021-01.09.2022 during one-year period. \u201cPius Brinzeu\u201d Clinical County Emergency Hospital Timi\u0219oara (SCJUPBT), is a tertiary medical care unit with 1,174 beds, of which 82 beds are in Obstetrics-Gynecology Departments. The laboratory analyses were performed as follows:<\/p><ul><li>Microbial identification &#8211; MALDI-TOF<\/li><li>Antimicrobial sensitivity tests &#8211; VITEK system with MIC determination (2021 CLSI standards)<\/li><li>Genes identification with Unyvero system in one case of new-born BSI<\/li><\/ul><p>A total number of 260 pregnant women under medical follow-up were monitored for: maternal diseases (hypertension, kidney diseases, thyroiditis, anemia, etc.) and maternal diseases associated with pregnancy (preeclampsia, pathological pregnancy, placenta previa).<\/p><p><strong>Table 1.<\/strong> Patients from OG<\/p><table><tbody><tr><td width=\"208\"><p><strong>Total no. of patients from OG<\/strong><\/p><\/td><td width=\"208\"><p><strong>Pregnant women<\/strong><\/p><\/td><td width=\"208\"><p><strong>Gynecological pathology<\/strong><\/p><\/td><\/tr><tr><td width=\"208\"><p><strong>506<\/strong><\/p><\/td><td width=\"208\"><p>260<\/p><\/td><td width=\"208\"><p>246<\/p><\/td><\/tr><tr><td width=\"208\"><p><strong>%<\/strong><\/p><\/td><td width=\"208\"><p>51.38<\/p><\/td><td width=\"208\"><p>48.62<\/p><\/td><\/tr><\/tbody><\/table><p><strong>Table 2.<\/strong> Sample types<\/p><table><tbody><tr><td width=\"89\"><p><strong>Samples<\/strong><\/p><\/td><td width=\"89\"><p><strong>Cervical samples<\/strong><\/p><\/td><td width=\"89\"><p><strong>Urine cultures<\/strong><\/p><\/td><td width=\"89\"><p><strong>Amniotic fluids<\/strong><\/p><\/td><td width=\"89\"><p><strong>Peritoneal fluids<\/strong><\/p><\/td><td width=\"89\"><p><strong>Wound secretions<\/strong><\/p><\/td><td width=\"89\"><p><strong>Sputum<\/strong><\/p><\/td><\/tr><tr><td width=\"89\"><p><strong>275<\/strong><\/p><\/td><td width=\"89\"><p>250<\/p><\/td><td width=\"89\"><p>15<\/p><\/td><td width=\"89\"><p>3<\/p><\/td><td width=\"89\"><p>2<\/p><\/td><td width=\"89\"><p>4<\/p><\/td><td width=\"89\"><p>1<\/p><\/td><\/tr><\/tbody><\/table><p><strong>\u00a0<\/strong><\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture4-207x300.jpg\" alt=\"\" width=\"207\" height=\"300\" class=\"alignnone size-medium wp-image-772\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture4-207x300.jpg 207w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture4-80x116.jpg 80w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture4.jpg 452w\" sizes=\"(max-width: 207px) 100vw, 207px\" \/><\/p><p><strong>Fig. 1.<\/strong> Microscopical examination from our own collection, Zeiss Primo Star microscope with digital video camera.<\/p><p><strong>\u00a0<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><p><strong>Table 3.<\/strong> Microorganisms and their AMR patterns from clinical samples (other than cervical)<\/p><table><tbody><tr><td width=\"125\"><p><strong>Sample type<\/strong><\/p><\/td><td width=\"55\"><p><strong>Sample no.<\/strong><\/p><\/td><td width=\"194\"><p><strong>Microorganism<\/strong><\/p><\/td><td width=\"125\"><p><strong>Resistance phenotypes<\/strong><\/p><\/td><td width=\"125\"><p><strong>Diagnosis\/treatment<\/strong><\/p><\/td><\/tr><tr><td width=\"125\"><p><strong>Urine<\/strong><\/p><\/td><td width=\"55\"><p>15<\/p><\/td><td width=\"194\"><p>E. coli<\/p><p>Streptococcus group B<\/p><p>Enterococcus spp.<\/p><\/td><td width=\"125\"><p>ESBL E. coli<\/p><\/td><td width=\"125\"><p>UTI<\/p><\/td><\/tr><tr><td width=\"125\"><p><strong>Amniotic fluid<\/strong><\/p><\/td><td width=\"55\"><p>3<\/p><\/td><td width=\"194\"><p>S. epidermidis<\/p><p>S. lugdunensis<\/p><p>E. faecalis<\/p><\/td><td width=\"125\"><p>Without acquired resistance phenotypes<\/p><\/td><td width=\"125\"><p>&#8211;<\/p><\/td><\/tr><tr><td width=\"125\"><p><strong>Peritoneal fluid<\/strong><\/p><\/td><td width=\"55\"><p>2<\/p><\/td><td width=\"194\"><p>P. aeruginosa and A. baumannii<\/p><p>S. aureus<\/p><\/td><td width=\"125\"><p>A. baumannii (CR)<\/p><p>S. aureus \u2013 BL producing phenotype<\/p><\/td><td width=\"125\"><p>Post cesarean complications solved by total hysterectomy<\/p><\/td><\/tr><tr><td width=\"125\"><p><strong>Sputum<\/strong><\/p><\/td><td width=\"55\"><p>1<\/p><\/td><td width=\"194\"><p>C. albicans<\/p><\/td><td width=\"125\"><p>&#8211;<\/p><\/td><td width=\"125\"><p>SARS-CoV-2 infection<\/p><\/td><\/tr><tr><td width=\"125\"><p><strong>Wound secretions<\/strong><\/p><\/td><td width=\"55\"><p>4<\/p><\/td><td width=\"194\"><p>S. aureus<\/p><p>Klebsiella spp.<\/p><\/td><td width=\"125\"><p>S. aureus (BL\/MLSB)<\/p><p>Klebsiella spp. (CASE)<\/p><\/td><td width=\"125\"><p>Post cesarean complications<\/p><\/td><\/tr><\/tbody><\/table><p><strong> <img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture5-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-773\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture5-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture5-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture5.png 384w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/strong><\/p><p><strong>Fig. 2.<\/strong> Microorganisms from cervical samples &#8211; 250<\/p><p><strong>\u00a0<\/strong><\/p><p><strong>\u00a0<\/strong><\/p><p><strong>Table 4.<\/strong> E. coli identification in various samples<\/p><table><tbody><tr><td width=\"125\"><p><strong>E. coli<\/strong><\/p><\/td><td width=\"79\"><p><strong>Abortion<\/strong><\/p><\/td><td width=\"120\"><p><strong>Premature birth<\/strong><\/p><\/td><td width=\"132\"><p><strong>Ectopic pregnancies<\/strong><\/p><\/td><td width=\"168\"><p><strong>Pregnancy in evolution\/full-term<\/strong><\/p><\/td><\/tr><tr><td width=\"125\"><p>96<\/p><\/td><td width=\"79\"><p>33<\/p><\/td><td width=\"120\"><p>8<\/p><\/td><td width=\"132\"><p>7<\/p><\/td><td width=\"168\"><p>48<\/p><\/td><\/tr><tr><td width=\"125\"><p>%<\/p><\/td><td width=\"79\"><p>34.37%<\/p><\/td><td width=\"120\"><p>8.33%<\/p><\/td><td width=\"132\"><p>7.29%<\/p><\/td><td width=\"168\"><p>50%<\/p><\/td><\/tr><\/tbody><\/table><p><strong>\u00a0<\/strong><\/p><p><strong> <img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture6-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-774\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture6-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture6-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture6.png 384w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/strong><\/p><p><strong>Fig. 3.<\/strong> E. coli identification in different samples<\/p><p><strong>\u00a0<\/strong><\/p><p><strong>Table 5.<\/strong> Group B Streptococcus identification in various samples<\/p><table><tbody><tr><td width=\"125\"><p><strong>Group B Streptococcus<\/strong><\/p><\/td><td width=\"125\"><p><strong>Abortion<\/strong><\/p><\/td><td width=\"125\"><p><strong>Premature birth<\/strong><\/p><\/td><td width=\"125\"><p><strong>Ectopic pregnancies<\/strong><\/p><\/td><td width=\"125\"><p><strong>Pregnancy in evolution\/full term<\/strong><\/p><\/td><\/tr><tr><td width=\"125\"><p>80<\/p><\/td><td width=\"125\"><p>12<\/p><\/td><td width=\"125\"><p>4<\/p><\/td><td width=\"125\"><p>4<\/p><\/td><td width=\"125\"><p>60<\/p><\/td><\/tr><tr><td width=\"125\"><p>%<\/p><\/td><td width=\"125\"><p>15%<\/p><\/td><td width=\"125\"><p>5%<\/p><\/td><td width=\"125\"><p>5%<\/p><\/td><td width=\"125\"><p>75%<\/p><\/td><\/tr><\/tbody><\/table><p><strong> <img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture7-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-775\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture7-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture7-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture7.png 384w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/strong><\/p><p><strong>Fig. 4.<\/strong> Group B Streptococcus identification in different samples<\/p><p><strong>\u00a0<\/strong><\/p><p><strong>Table 6.<\/strong> Group B Streptococcus antibiotic resistance pattern<\/p><table><tbody><tr><td width=\"104\"><p><strong>Group B Streptococcus<\/strong><\/p><\/td><td width=\"104\"><p><strong>R-Te<\/strong><\/p><\/td><td width=\"104\"><p><strong>MLSB<\/strong><\/p><\/td><td width=\"104\"><p><strong>R-FQ<\/strong><\/p><\/td><td width=\"104\"><p><strong>R-P<\/strong><\/p><\/td><td width=\"104\"><p><strong>MDR<\/strong><\/p><\/td><\/tr><tr><td width=\"104\"><p>80<\/p><\/td><td width=\"104\"><p>49<\/p><\/td><td width=\"104\"><p>20<\/p><\/td><td width=\"104\"><p>9<\/p><\/td><td width=\"104\"><p>6<\/p><\/td><td width=\"104\"><p>22<\/p><\/td><\/tr><tr><td width=\"104\"><\/td><td width=\"104\"><p>61.25%<\/p><\/td><td width=\"104\"><p>25%<\/p><\/td><td width=\"104\"><p>11.25%<\/p><\/td><td width=\"104\"><p>7.5%<\/p><\/td><td width=\"104\"><p>27.16%<\/p><\/td><\/tr><\/tbody><\/table><p><strong> <img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture8-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-776\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture8-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture8-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture8.png 384w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/strong><\/p><p><strong>Fig. 5.<\/strong> Group B Streptococcus antibiotic resistance in different samples<\/p><p><strong>\u00a0<\/strong><\/p><p>A percentage of 27% (22 samples) of the strains were MDR phenotype, associating resistance to BL \/ FQ \/ MLSB. These MDR strains were identified from pregnant women in 7-19 weeks of pregnancy, in the stage of imminent abortion or metrorrhagia.<\/p><p><strong>\u00a0<\/strong><\/p><p><strong>Table 7.<\/strong> S. aureus identification in various samples<\/p><table><tbody><tr><td width=\"125\"><p><strong>S. aureus<\/strong><\/p><\/td><td width=\"125\"><p><strong>Abortion<\/strong><\/p><\/td><td width=\"125\"><p><strong>Premature birth<\/strong><\/p><\/td><td width=\"125\"><p><strong>Ectopic pregnancies<\/strong><\/p><\/td><td width=\"125\"><p><strong>Pregnancy in evolution\/full term<\/strong><\/p><\/td><\/tr><tr><td width=\"125\"><p>49<\/p><\/td><td width=\"125\"><p>6<\/p><\/td><td width=\"125\"><p>5<\/p><\/td><td width=\"125\"><p>1<\/p><\/td><td width=\"125\"><p>38<\/p><\/td><\/tr><tr><td width=\"125\"><p>%<\/p><\/td><td width=\"125\"><p>12.24%<\/p><\/td><td width=\"125\"><p>10.20%<\/p><\/td><td width=\"125\"><p>2.04%<\/p><\/td><td width=\"125\"><p>75.51%<\/p><\/td><\/tr><\/tbody><\/table><p><strong> <img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture9-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-777\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture9-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture9-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture9.png 384w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/strong><\/p><p><strong>Fig. 6.<\/strong> S. aureus identification in different samples<\/p><p><strong>\u00a0<\/strong><\/p><p><strong>Table 8.<\/strong> S. aureus antibiotic resistance pattern<\/p><table><tbody><tr><td width=\"78\"><p><strong>S. aureus<\/strong><\/p><\/td><td width=\"78\"><p><strong>Beta-lactamase<\/strong><\/p><\/td><td width=\"78\"><p><strong>MRSA<\/strong><\/p><\/td><td width=\"78\"><p><strong>MLSB<\/strong><\/p><\/td><td width=\"78\"><p><strong>R-M<\/strong><\/p><\/td><td width=\"78\"><p><strong>R-FQ<\/strong><\/p><\/td><td width=\"78\"><p><strong>R-SXT<\/strong><\/p><\/td><td width=\"78\"><p><strong>MDR<\/strong><\/p><\/td><\/tr><tr><td width=\"78\"><p>92<\/p><\/td><td width=\"78\"><p>22<\/p><\/td><td width=\"78\"><p>15<\/p><\/td><td width=\"78\"><p>13<\/p><\/td><td width=\"78\"><p>13<\/p><\/td><td width=\"78\"><p>5<\/p><\/td><td width=\"78\"><p>5<\/p><\/td><td width=\"78\"><p>19<\/p><\/td><\/tr><tr><td width=\"78\"><p>%<\/p><\/td><td width=\"78\"><p>44.89%<\/p><\/td><td width=\"78\"><p>30.61%<\/p><\/td><td width=\"78\"><p>26.53%<\/p><\/td><td width=\"78\"><p>26.53%<\/p><\/td><td width=\"78\"><p>10.2%<\/p><\/td><td width=\"78\"><p>10.2%<\/p><\/td><td width=\"78\"><p>38.77%<\/p><\/td><\/tr><\/tbody><\/table><p><strong>\u00a0<\/strong><\/p><p><strong> <img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture10-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-778\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture10-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture10-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture10.png 384w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/strong><\/p><p><strong>Fig. 7.<\/strong> S. aureus antibiotic resistance in different samples<\/p><p><strong>\u00a0<\/strong><\/p><p>A percentage of 30.61% of the strains were MRSA and 38.77% were MDR phenotype, associating resistance to BL \/ FQ \/ MLSB. Of the pregnant women with MRSA, approximately 1\/4 were hospitalized for abortion in progress or imminent abortion and the rest of 3\/4, for monitoring \/ delivery.<\/p><p><strong>Table 9.<\/strong> K. pneumoniae identification in various samples<\/p><table><tbody><tr><td width=\"125\"><p><strong>K. pneumoniae<\/strong><\/p><\/td><td width=\"125\"><p><strong>Abortion<\/strong><\/p><\/td><td width=\"125\"><p><strong>Premature birth<\/strong><\/p><\/td><td width=\"125\"><p><strong>Ectopic pregnancies<\/strong><\/p><\/td><td width=\"125\"><p><strong>Pregnancy in evolution\/full term<\/strong><\/p><\/td><\/tr><tr><td width=\"125\"><p>32<\/p><\/td><td width=\"125\"><p>6<\/p><\/td><td width=\"125\"><p>4<\/p><\/td><td width=\"125\"><p>1<\/p><\/td><td width=\"125\"><p>21<\/p><\/td><\/tr><tr><td width=\"125\"><p>%<\/p><\/td><td width=\"125\"><p>18.75%<\/p><\/td><td width=\"125\"><p>12.5%<\/p><\/td><td width=\"125\"><p>3.12%<\/p><\/td><td width=\"125\"><p>65.62%<\/p><\/td><\/tr><\/tbody><\/table><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture11-300x181.png\" alt=\"\" width=\"300\" height=\"181\" class=\"alignnone size-medium wp-image-779\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture11-300x181.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture11-192x116.png 192w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture11.png 374w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 8.<\/strong> K. pneumoniae identification in different samples<\/p><p><strong>Table 10.<\/strong> K. pneumoniae antibiotic resistance pattern<\/p><table><tbody><tr><td width=\"114\"><p><strong>K. pneumoniae<\/strong><\/p><\/td><td width=\"94\"><p><strong>ESBL<\/strong><\/p><\/td><td width=\"104\"><p><strong>FQ-R<\/strong><\/p><\/td><td width=\"104\"><p><strong>SXT-R<\/strong><\/p><\/td><td width=\"104\"><p><strong>AG-R<\/strong><\/p><\/td><td width=\"104\"><p><strong>MDR<\/strong><\/p><\/td><\/tr><tr><td width=\"114\"><p>32<\/p><\/td><td width=\"94\"><p>2<\/p><\/td><td width=\"104\"><p>2<\/p><\/td><td width=\"104\"><p>5<\/p><\/td><td width=\"104\"><p>1<\/p><\/td><td width=\"104\"><p>1<\/p><\/td><\/tr><tr><td width=\"114\"><p>%<\/p><\/td><td width=\"94\"><p>6.25%<\/p><\/td><td width=\"104\"><p>6.25%<\/p><\/td><td width=\"104\"><p>15.62%<\/p><\/td><td width=\"104\"><p>3.12%<\/p><\/td><td width=\"104\"><p>3.12%<\/p><\/td><\/tr><\/tbody><\/table><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture12-300x181.png\" alt=\"\" width=\"300\" height=\"181\" class=\"alignnone size-medium wp-image-780\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture12-300x181.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture12-192x116.png 192w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture12.png 374w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 9.<\/strong> K. pneumoniae antibiotic resistance in different samples<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture13-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-781\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture13-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture13-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture13.png 384w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 10.<\/strong> Comparative analysis of clinical condition and identified pathogens<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture14-188x300.jpg\" alt=\"\" width=\"188\" height=\"300\" class=\"alignnone size-medium wp-image-783\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture14-188x300.jpg 188w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture14-642x1024.jpg 642w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture14-73x116.jpg 73w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture14.jpg 672w\" sizes=\"(max-width: 188px) 100vw, 188px\" \/><\/p><p><strong>Fig. 11.<\/strong> Blood culture result in a newborn with XDR A. baumannii<\/p><p>It is necessary to develop a universal method for screening vaginal-rectal GBS colonization\u00a0in\u00a0pregnant\u00a0women\u00a0worldwide. Streptococcus agalactiae could cause severe\u00a0infections in\u00a0neonates. Group B Streptococcus (GBS), are often associated with GBS transmission from their mothers during labor or birth. The proposed MALDI detection method with analysis of peaks of TOF (MDAPT) detects GBS directly from cultured broth with high sensitivity. Therefore, it can be an alternative method for GBS screening\u00a0in\u00a0pregnant\u00a0women, thereby contributing to the prevention of severe GBS infectious diseases\u00a0in\u00a0neonates. As previously reported, 10%-30% of\u00a0pregnant\u00a0women\u00a0carry Streptococcus agalactiae, Group B Streptococcus (GBS),\u00a0in\u00a0their vagina or rectum, and approximately 50% of them vertically transmit GBS to their neonates during labor or birth. Moreover, 1%-2% of the GBS-transmitted neonates develop severe GBS infectious diseases, which have a mortality rate of 19.2%\u00a0in\u00a0a preterm infant and 2.1%\u00a0in\u00a0a full-term infant.<\/p><p>Administration of antibiotics during pregnancy leads to changes in the vaginal microbial ecology before birth, with potential for morbidity, and long-term effects on the early microbial colonization of the newborn. Women who received oral antibiotics during any trimester of pregnancy had an increased rate of colonization with staphylococcal species in vaginal samples, compared with samples obtained from women without antibiotic treatment during pregnancy. Oral antibiotic administration for urinary tract infections in the third trimester was also associated with increased colonization by Staphylococcus species. Women treated in the third trimester for respiratory tract infection &#8211; were more often colonized with Escherichia coli than women without antibiotic treatment. Significant changes in vaginal colonization with Streptococcus agalactiae (group B streptococcus) following antibiotic treatment during pregnancy were not observed.<\/p><p>Regarding GNB, we can conclude that low incidence of resistant strains with clinical significance are: UTI with 1 ESBL-E. coli; SSI with 2 CASE-Klebsiella sp.; Peritonitis with 1 CR-Acinetobacter baumannii. The high incidence of E. coli strains in cervical secretions was noted, but taking into account the clinical significance of the presence of this strain in the cervix (considered colonization) as well as the low antimicrobial resistance (they are community strains), it can be appreciated that treatment in these cases &#8211; is necessary only if the clinical condition of the patients requires such an approach but their resistance pattern must be known in the case of detecting early infections in newborns.<\/p><p>What is noteworthy is the fact that GPC registered high frequencies, and the isolated strains show resistance to antimicrobials that describe resistance phenotypes that pose therapeutic problems. Group B streptococcus registers high frequencies in pregnant women and those hospitalized for full-term birth (35.08%) but also in those hospitalized for premature birth (19.04%). Colonization with S. aureus should also be mentioned, as long as most of the strains were of the MRSA type or associated multiple resistance mechanisms, so adequate supervision and treatment due to the potential for illness in newborns is required.<\/p><p>Empiric and definitive antimicrobial treatment in early and late onset infections in newborns should be applied. Infections with MDR pathogens, especially those caused by ESBL\/CRE Enterobacterales or MRSA are associated with increased risk of morbidity and mortality. Treatment options for these infections are limited and efficacy and safety of novel antibiotics are currently extrapolated from adult data, requiring dose optimization in clinical trials for Ceftazidime\/avibactam, Meropenem\/vaborbactam, Imipenem\/cilastatin\/relebactam, cefiderocol.<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture15-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-784\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture15-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture15-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture15.png 384w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 12.<\/strong> Microbiological tests performed for identification of bacterial-induced infertility<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture16-300x216.png\" alt=\"\" width=\"300\" height=\"216\" class=\"alignnone size-medium wp-image-785\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture16-300x216.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture16-161x116.png 161w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture16.png 385w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 13.<\/strong> Granulocytes presence in the specimen indicating a bacterial infection<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture17-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-786\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture17-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture17-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture17.png 385w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 14.<\/strong> Identification of lactobacilli in different specimens<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture18-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-787\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture18-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture18-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture18.png 384w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 15.<\/strong> Bacterial vaginosis (BV) identified in samples received during a period of 2 years<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture19-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-788\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture19-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture19-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture19.png 384w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 16.<\/strong> Samples distributed by several medical departments<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture20-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-789\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture20-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture20-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture20.png 384w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 17. <\/strong>Diagnostic of female infertiliry<\/p><p>The most frequent isolated microorganisms in 2021 and 2022 were: <em>E. coli<\/em>, <em>C. albicans <\/em>and <em>S. agalactiae.<\/em><\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture21-300x180.jpg\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-791\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture21-300x180.jpg 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture21-193x116.jpg 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture21.jpg 576w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 18.<\/strong> Prevalence of isolated pathogens<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture22-300x180.png\" alt=\"\" width=\"300\" height=\"180\" class=\"alignnone size-medium wp-image-797\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture22-300x180.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture22-193x116.png 193w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture22.png 384w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 19.<\/strong> Most frequent pathogens isolated from different samples<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture23-300x137.png\" alt=\"\" width=\"300\" height=\"137\" class=\"alignnone size-medium wp-image-801\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture23-300x137.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture23-768x351.png 768w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture23-228x104.png 228w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture23.png 975w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 20.<\/strong> Antibiotic resistance rates by pathogen and drug<\/p><p>Ceftazidime and cefriaxone susceptibility testing were performed only for the ESBL producing or AmpC positive <em>E. coli <\/em>isolates. ESBL producing or AmpC positive <em>E. coli <\/em>isolates were resistant to cefixime also. Half part of the ampicillin and amoxicillin-clavulanic acid resistant <em>E. coli <\/em>isolates\u00a0 are susceptible and half part of them\u00a0 were resistant to cefixime.<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture24-300x165.png\" alt=\"\" width=\"300\" height=\"165\" class=\"alignnone size-medium wp-image-803\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture24-300x165.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture24-768x421.png 768w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture24-211x116.png 211w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture24.png 975w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 21.<\/strong> Antibiotic resistance rates by pathogen and drug<\/p><p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture25-300x174.png\" alt=\"\" width=\"300\" height=\"174\" class=\"alignnone size-medium wp-image-804\" srcset=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture25-300x174.png 300w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture25-768x446.png 768w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture25-200x116.png 200w, https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/uploads\/2023\/12\/Picture25.png 975w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p><p><strong>Fig. 22.<\/strong> Differences in antibiotic resistance rates between 2022 and 2021<\/p><p>In the case of patients with abnormal vaginal microscopical result, aerobic vaginitis and bacterial vaginosis were more likely to occur.<\/p><p><strong>Table 11.<\/strong> Correlation between the presence of granulocytes and lactobacilli or pathogens<\/p><table><tbody><tr><td width=\"59\"><p><strong>\u00a0<\/strong><\/p><\/td><td width=\"85\"><p><strong>Granulocytes<\/strong><\/p><\/td><td width=\"89\"><p><strong>Lactobacilli<\/strong><\/p><\/td><td width=\"78\"><p><strong>Path<\/strong><\/p><\/td><td width=\"156\"><p><strong>Correlation Granulocytes\/Lactobacilli<\/strong><\/p><\/td><td width=\"156\"><p><strong>Correlation Granulocytes\/Path<\/strong><\/p><\/td><\/tr><tr><td width=\"59\"><p><strong>\u00a0<\/strong><\/p><\/td><td width=\"85\"><p>141<\/p><\/td><td width=\"89\"><p>1019<\/p><\/td><td width=\"78\"><p>309<\/p><\/td><td width=\"156\"><p>-1<\/p><\/td><td width=\"156\"><p>1<\/p><\/td><\/tr><tr><td width=\"59\"><p><strong>\u00a0<\/strong><\/p><\/td><td width=\"85\"><p>1248<\/p><\/td><td width=\"89\"><p>365<\/p><\/td><td width=\"78\"><p>1080<\/p><\/td><td width=\"156\"><\/td><td width=\"156\"><\/td><\/tr><\/tbody><\/table><p>There is a positive correlation between the presence of granulocytes and a positive vaginal culture results. And there is a negative correlation between the presence of lactobacilli and the symtoms and the positive vaginal culture results.<\/p><p><strong>Table 12.<\/strong> Correlation between the diagnosis of bacterial vaginosis (BV) and the presence of lactobacilli or pathogens<\/p><table><tbody><tr><td width=\"104\"><\/td><td width=\"104\"><p><strong>BV<\/strong><\/p><\/td><td width=\"104\"><p><strong>Lactobacilli<\/strong><\/p><\/td><td width=\"104\"><p><strong>Path<\/strong><\/p><\/td><td width=\"104\"><p><strong>Corellation BV vs. Lacto<\/strong><\/p><\/td><td width=\"104\"><p><strong>Correlation BV vs. Path<\/strong><\/p><\/td><\/tr><tr><td width=\"104\"><\/td><td width=\"104\"><p>99<\/p><\/td><td width=\"104\"><p>1019<\/p><\/td><td width=\"104\"><p>308<\/p><\/td><td width=\"104\"><p>-1<\/p><\/td><td width=\"104\"><p>1<\/p><\/td><\/tr><tr><td width=\"104\"><\/td><td width=\"104\"><p>1282<\/p><\/td><td width=\"104\"><p>357<\/p><\/td><td width=\"104\"><p>1073<\/p><\/td><td width=\"104\"><\/td><td width=\"104\"><\/td><\/tr><\/tbody><\/table><p>Infertility, defined as the inability to conceive after a year of regular unprotected intercourse, affects millions of couples worldwide. While numerous factors contribute to reproductive challenges, recent advancements in medical science have underscored the importance of genetic testing in understanding and addressing infertility. This comprehensive exploration delves into the significance, types, methodologies, and ethical considerations associated with genetic testing in the context of infertility.<\/p><p><strong>\u00a0<\/strong><\/p><ol><li><strong> Genetic Basis of Infertility<\/strong><\/li><\/ol><p>Hereditary Factors. Infertility often has a genetic basis, with hereditary factors playing a crucial role. Genetic mutations, chromosomal abnormalities, and variations in specific genes can significantly impact fertility in both men and women.<\/p><p>Genetic Disorders and Infertility. Certain genetic disorders, such as Turner syndrome, Klinefelter syndrome, and cystic fibrosis, are known to be associated with infertility. Genetic testing allows for the identification of these conditions, enabling targeted interventions and family planning.<\/p><p><strong>\u00a0<\/strong><\/p><ol><li><strong> Types of Genetic Testing in Infertility<\/strong><\/li><\/ol><p>Preconception Genetic Testing. Preconception genetic testing involves assessing the genetic compatibility of prospective parents. This form of testing can identify carriers of specific genetic conditions, allowing couples to make informed decisions about family planning.<\/p><p>Carrier Screening.Carrier screening is particularly relevant for couples with a family history of genetic disorders. By identifying carriers of recessive genetic conditions, couples can understand their risk of passing on such conditions to their offspring.<\/p><p>Preimplantation Genetic Testing (PGT). PGT is a groundbreaking technique used in assisted reproductive technologies (ART). It involves the genetic analysis of embryos before implantation, allowing the selection of embryos free from chromosomal abnormalities or specific genetic mutations.<\/p><p>Genetic Testing in Male Infertility. Genetic testing is instrumental in diagnosing male infertility. Y chromosome microdeletions, karyotype analysis, and testing for specific gene mutations associated with sperm production are commonly employed to identify genetic factors contributing to male infertility.<\/p><p>Genetic Testing in Female Infertility. For women, genetic testing can help identify conditions such as polycystic ovary syndrome (PCOS) and premature ovarian insufficiency (POI). Additionally, chromosomal analysis can uncover abnormalities affecting egg quality.<\/p><p><strong>\u00a0<\/strong><\/p><p><strong>III. Methodologies in Genetic Testing for Infertility<\/strong><\/p><p>Karyotyping. Karyotyping involves the visualization and analysis of an individual&#8217;s chromosomes. This technique is crucial for identifying numerical and structural chromosomal abnormalities that may contribute to infertility.<\/p><p>Polymerase Chain Reaction (PCR). PCR is a widely used technique for amplifying specific DNA sequences. It is employed in genetic testing to identify mutations associated with infertility, such as those linked to sperm production or hormonal regulation.<\/p><p>Next-Generation Sequencing (NGS). NGS allows for the rapid and cost-effective sequencing of DNA. In the context of infertility, NGS is employed for comprehensive genetic analysis, including the screening of multiple genes simultaneously.<\/p><p>Comparative Genomic Hybridization (CGH). CGH is utilized in PGT to assess chromosomal abnormalities in embryos. By comparing the genetic material of embryos, CGH helps identify those with a higher likelihood of successful implantation.<\/p><ol><li><strong> Ethical Considerations in Genetic Testing for Infertility<\/strong><\/li><\/ol><p>Informed Consent. Given the sensitive nature of genetic information, obtaining informed consent from individuals undergoing genetic testing is imperative. This ensures that individuals are aware of the potential implications of the results.<\/p><p>Privacy and Confidentiality. Protecting the privacy and confidentiality of genetic information is paramount. Healthcare providers and researchers must adhere to strict ethical guidelines to safeguard individuals&#8217; genetic data.<\/p><p>Reproductive Autonomy. Genetic testing raises questions about reproductive autonomy, as individuals may be faced with decisions about family planning, embryo selection, and the potential use of assisted reproductive technologies based on genetic information.<\/p><p>Genetic testing has emerged as a powerful tool in unraveling the complexities of infertility. From identifying hereditary factors to enabling targeted interventions through assisted reproductive technologies, genetic testing offers hope to couples grappling with reproductive challenges. However, ethical considerations must accompany the advancements in this field to ensure the responsible and informed use of genetic information. As technology continues to advance, genetic testing will likely play an increasingly pivotal role in the holistic management of infertility, fostering a new era of personalized and precise reproductive medicine.<\/p><p>Infertility stands as a worldwide health concern impacting millions of individuals in their reproductive years across the globe. Existing data indicates that one in six people globally grapple with infertility at some point in their lives. It is characterized as a condition affecting the male or female reproductive system, marked by the inability to achieve pregnancy after 12 months or more of consistent unprotected sexual intercourse. Primary infertility denotes the incapacity to experience any pregnancy, while secondary infertility pertains to the inability to conceive after having previously achieved successful conception. The International Classification of Diseases by the World Health Organization (WHO) offers comprehensive insights into the various primary and secondary factors contributing to infertility in both women and men.<\/p><p>Infertility can stem from male factors, female factors, a combination of both, or may remain unexplained. Regardless of the cause, lifestyle choices such as smoking, excessive alcohol consumption, and obesity have been linked to an increased risk of infertility for both men and women.<\/p><ul><li>Infertility is defined as a malfunction in the male or female reproductive system, characterized by the inability to achieve pregnancy after 12 months or more of consistent unprotected sexual intercourse.<\/li><li>This condition has a profound impact on millions of individuals, affecting not only their lives but also their families and communities. Approximately one in every six people of reproductive age worldwide is estimated to experience infertility at some point in their lifetime.<\/li><li>In the male reproductive system, infertility is commonly attributed to issues with semen ejaculation, absence or low levels of sperm, or abnormalities in sperm shape (morphology) and movement (motility).<\/li><li>In the female reproductive system, infertility may result from various abnormalities in the ovaries, uterus, fallopian tubes, and the endocrine system, among other factors.<\/li><li>Infertility is categorized as primary when an individual has never achieved pregnancy and as secondary when at least one prior pregnancy has been attained.<\/li><li>Fertility care involves the prevention, diagnosis, and treatment of infertility. However, equal and equitable access to fertility care remains a challenge in most countries, especially in low and middle-income nations. Fertility care is seldom prioritized in national universal health coverage benefit packages.<\/li><\/ul><p>Why is it crucial to address infertility? Every individual is entitled to the highest achievable standard of physical and mental health, encompassing the right to make decisions about the number, timing, and spacing of their children. Infertility can undermine the realization of these fundamental human rights. Consequently, addressing infertility becomes a vital component of ensuring individuals and couples exercise their right to establish a family.<\/p><p>A diverse range of individuals, including heterosexual couples, same-sex partners, older individuals, those not in sexual relationships, and those with specific medical conditions such as some HIV sero-discordant couples and cancer survivors, may find themselves in need of infertility management and fertility care services. Disparities and inequities in access to these services disproportionately impact vulnerable populations, including the poor, unmarried, uneducated, unemployed, and other marginalized groups.<\/p><p>Tackling infertility also has the potential to alleviate gender inequality. While both men and women can encounter infertility, societal perceptions often attribute infertility issues to women in relationships, irrespective of their actual fertility status. The social ramifications of infertility significantly impact the lives of infertile couples, particularly women, who may face violence, divorce, social stigma, emotional stress, depression, anxiety, and diminished self-esteem. In certain contexts, the fear of infertility can dissuade individuals from using contraception, especially if they feel compelled by societal expectations to demonstrate fertility early in life due to the high social value placed on childbearing. In such scenarios, it becomes imperative to implement educational and awareness initiatives to enhance understanding of the prevalence and determinants of fertility and infertility.<\/p><p><strong>Addressing Challenges <\/strong><\/p><p>Challenges persist in the availability, accessibility, and quality of interventions to tackle infertility in the majority of countries. The diagnosis and treatment of infertility often lack prioritization in national population and development policies, as well as reproductive health strategies, and are seldom covered by public health financing. Additionally, a shortage of trained personnel, inadequate equipment and infrastructure, and the current high costs of treatment medicines pose significant barriers, even for countries actively addressing the needs of individuals with infertility.<\/p><p>Despite assisted reproduction technologies (ART) being accessible for over three decades, with more than 5 million children worldwide born from ART interventions such as in vitro fertilization (IVF), these technologies remain largely inaccessible and unaffordable in numerous parts of the world, especially in low and middle-income countries (LMIC).<\/p><p>In addition, enabling laws and policies that regulate third party reproduction and ART are essential to ensure universal access without discrimination and to protect and promote the human rights of all parties involved. Once fertility policies are in place, it is essential to ensure that their implementation is monitored, and the quality of services is continually improved.<\/p><p><strong>WHO response<\/strong><\/p><p>WHO acknowledges that delivering high-quality family planning services, which encompass fertility care, is a fundamental component of reproductive health. In recognition of the significance and impact of infertility on people&#8217;s overall quality of life and well-being, WHO is dedicated to addressing infertility and fertility care through the following:<\/p><ol><li>Collaborating with partners to conduct global epidemiological and etiological research on infertility.<\/li><li>Engaging in and facilitating policy dialogues with countries worldwide to situate infertility within a supportive legal and policy framework.<\/li><li>Supporting the generation of data on the burden of infertility to guide resource allocation and service provision.<\/li><li>Developing guidelines covering the prevention, diagnosis, and treatment of male and female infertility, integrating them into global norms and standards for quality care in fertility services.<\/li><li>Regularly revising and updating normative products, including the WHO laboratory manual for the examination and processing of human semen.<\/li><li>Collaborating with relevant stakeholders, such as academic centers, ministries of health, other UN organizations, non-state actors (NSAs), and additional partners, to enhance political commitment, availability, and health system capacity for delivering fertility care on a global scale.<\/li><li>Offering country-level technical support to member states for the development or enhancement of national fertility policies and services.<\/li><\/ol><\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t<\/div>\n\t\t","protected":false},"excerpt":{"rendered":"<p>www.interreg-rohu.eu International Medical Conference Protocol for identification and characterization of isolated strains from pregnant, infertile women and newborn babies using automatic identification system for microorganisms after molecular fingerprint MALDI-TOF Working Protocol Process of rapid identification of pathogenic bacterial species using MALDI-TOF MS (Matrix-assisted laser desorption\/ionization &#8211; time of flight mass [&hellip;]<\/p>\n<div class=\"pvc_clear\"><\/div>\n<p id=\"pvc_stats_599\" class=\"pvc_stats all  \" data-element-id=\"599\" style=\"\"><i class=\"pvc-stats-icon medium\" aria-hidden=\"true\"><svg aria-hidden=\"true\" focusable=\"false\" data-prefix=\"far\" data-icon=\"chart-bar\" role=\"img\" xmlns=\"http:\/\/www.w3.org\/2000\/svg\" viewBox=\"0 0 512 512\" class=\"svg-inline--fa fa-chart-bar fa-w-16 fa-2x\"><path fill=\"currentColor\" d=\"M396.8 352h22.4c6.4 0 12.8-6.4 12.8-12.8V108.8c0-6.4-6.4-12.8-12.8-12.8h-22.4c-6.4 0-12.8 6.4-12.8 12.8v230.4c0 6.4 6.4 12.8 12.8 12.8zm-192 0h22.4c6.4 0 12.8-6.4 12.8-12.8V140.8c0-6.4-6.4-12.8-12.8-12.8h-22.4c-6.4 0-12.8 6.4-12.8 12.8v198.4c0 6.4 6.4 12.8 12.8 12.8zm96 0h22.4c6.4 0 12.8-6.4 12.8-12.8V204.8c0-6.4-6.4-12.8-12.8-12.8h-22.4c-6.4 0-12.8 6.4-12.8 12.8v134.4c0 6.4 6.4 12.8 12.8 12.8zM496 400H48V80c0-8.84-7.16-16-16-16H16C7.16 64 0 71.16 0 80v336c0 17.67 14.33 32 32 32h464c8.84 0 16-7.16 16-16v-16c0-8.84-7.16-16-16-16zm-387.2-48h22.4c6.4 0 12.8-6.4 12.8-12.8v-70.4c0-6.4-6.4-12.8-12.8-12.8h-22.4c-6.4 0-12.8 6.4-12.8 12.8v70.4c0 6.4 6.4 12.8 12.8 12.8z\" class=\"\"><\/path><\/svg><\/i> <img loading=\"lazy\" decoding=\"async\" width=\"16\" height=\"16\" alt=\"Loading\" src=\"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/wp-content\/plugins\/page-views-count\/ajax-loader-2x.gif\" border=0 \/><\/p>\n<div class=\"pvc_clear\"><\/div>\n","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"page-templates\/template-pagebuilder-full-width.php","meta":{"footnotes":""},"class_list":["post-599","page","type-page","status-publish","hentry"],"aioseo_notices":[],"_links":{"self":[{"href":"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/index.php\/wp-json\/wp\/v2\/pages\/599","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/index.php\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/index.php\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/index.php\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/index.php\/wp-json\/wp\/v2\/comments?post=599"}],"version-history":[{"count":45,"href":"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/index.php\/wp-json\/wp\/v2\/pages\/599\/revisions"}],"predecessor-version":[{"id":807,"href":"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/index.php\/wp-json\/wp\/v2\/pages\/599\/revisions\/807"}],"wp:attachment":[{"href":"https:\/\/healthypregnancy-ro-hu.oncogen.ro\/index.php\/wp-json\/wp\/v2\/media?parent=599"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}