Multiplex Real-time Polymerase Chain Research Articles

Comparison of prevalence estimates of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum determined by conventional PCR and multiplex qPCR and implications for surveillance and monitoring

ObjectivesThe accuracy of malaria rapid diagnostic tests is threatened by Plasmodium falciparum with pfhrp2/3 deletions. This study compares gene deletion prevalence determined by multiplex real time polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR) using existing samples with clonality previously determined by microsatellite genotyping. MethodsMultiplex qPCR was used to estimate prevalence of pfhrp2/3 deletions in three sets of previously collected patient samples from Eritrea and Peru. The qPCR was validated by multiplex digital polymerase chain reaction. Sample classification was compared with cPCR, and receiver operating characteristic curve analysis was used to determine the optimal ΔCq threshold that aligned the results of the two assays. ResultsqPCR classified 75% (637 of 849) of samples as single, and 212 as mixed-pfhrp2/3 genotypes, with a positive association between clonality and proportion of mixed-pfhrp2/3 genotype samples. The sample classification agreement between cPCR and qPCR was 75.1% (95% confidence interval [CI] 68.6-80.7%) and 47.8% (95% CI 38.9-56.9%) for monoclonal and polyclonal infections. The qPCR prevalence estimates of pfhrp2/3 deletions showed almost perfect (κ = 0.804, 95% CI 0.714-0.895) and substantial agreement (κ = 0.717, 95% CI 0.562-0.872) with cPCR for Peru and 2016 Eritrean samples, respectively. For 2019 Eritrean samples, the prevalence of double pfhrp2/3 deletions was approximately two-fold higher using qPCR. The optimal threshold for matching the assay results was ΔCq = 3. ConclusionsMultiplex qPCR and cPCR produce comparable estimates of gene deletion prevalence when monoclonal infections dominate; however, qPCR provides higher estimates where multi-clonal infections are common.

Comparing single versusmultiple virus detection in pediatric acute gastroenteritis postimplementation of routine multiplex RT-PCR diagnostic testing.

Utilizing multiplex real time polymerase chain reaction (RT-PCR) for rapid diagnosis of gastroenteritis, enables simultaneous detection of multiple pathogens. A comparative analysis of disease characteristics was conducted between cases with single and multiple viruses. Rotavirus vaccine was introduced in 2010, reaching a 70% coverage in 2 years. All rectal swabs collected from diarrheic children (<5 years) between December 2017 and March 2022 were included. Detection of the same viruses within 2 months was considered a single episode. Episodes with positive stool bacterial PCR were excluded. A total of 5879 samples were collected, revealing 86.9% (1509) with single virus detection and 13.1% (227) with multiple viruses. The most frequent combination was rotavirus and norovirus (27.8%), these infections followed a winter-spring seasonality akin to rotavirus. Children with multivirus infections exhibited higher immunodeficiency (OR 2.06) rates, but lower food allergy (OR 0.45) and prematurity rates (OR 0.55) compared to single infections. Greater disease severity, evaluated by the Vesikari score, was observed in multivirus episodes (p < 0.001, OR 1.12). Multivirus infections accounted for 13.1% of symptomatic cases in hospitalized young children. Despite vaccination efforts, rotavirus remained prominent, frequently in co-infections with norovirus. Overall, multivirus infections were linked to more severe diseases than single virus cases.

Surveillance of respiratory viruses at health facilities from across Kenya, 2014

Background: Acute respiratory illnesses (ARI) are a major cause of morbidity and mortality globally. With (re)emergence of novel viruses and increased access to childhood bacterial vaccines, viruses have assumed greater importance in the aetiology of ARI. There are now promising candidate vaccines against some of the most common endemic respiratory viruses. Optimal delivery strategies for these vaccines, and the need for interventions against other respiratory viruses, requires geographically diverse data capturing temporal variations in virus circulation. Methods: We leveraged three health facility-based respiratory illness surveillance platforms operating in 11 sites across Kenya. Nasopharyngeal (NP) and/or oropharyngeal (OP) specimens, patient demographic, and clinical characteristics were collected in 2014 from individuals of various ages presenting with respiratory symptoms at the surveillance facilities. Real time multiplex polymerase chain reaction was used to detect rhinoviruses, respiratory syncytial virus (RSV), influenza virus, human coronaviruses (hCoV), and adenoviruses. Results: From 11 sites, 5451 NP/OP specimens were collected and tested from patients. Of these, 40.2% were positive for at least one of the targeted respiratory viruses. The most frequently detected were rhinoviruses (17.0%) and RSV A/B (10.5%), followed by influenza A (6.2%), adenovirus (6.0%) and hCoV (4.2%). RSV was most prevalent among infants aged &lt;12 months old (18.9%), adenovirus among children aged 12–23 months old (11.0%), influenza A among children aged 24–59 months (9.3%), and rhinovirus across all age groups (range, 12.7–19.0%). The overall percent virus positivity varied by surveillance site, health facility type and case definition used in surveillance. Conclusions: We identify rhinoviruses, RSV, and influenza A as the most prevalent respiratory viruses. Higher RSV positivity in inpatient settings compared to outpatient clinics strengthen the case for RSV vaccination. To inform the design and delivery of public health interventions, long-term surveillance is required to establish regional heterogeneities in respiratory virus circulation and seasonality.

Platelet and coagulation hemostasis status in patients with atrial fibrillation depending on warfarin dosing method

The aim. To evaluate indicators of induced platelet aggregation and D-dimer level in patients with atrial fibrillation (AF) depending on the method of warfarin (WF) dosing. Materials and methods. The study involved 110 patients with AF (mean age 68.72 ± 0.79; men – 57, women – 53). Patients with AF were divided into two groups: the main group – 50 patients with AF and genotype-guided dosing method, the control group – 60 patients with AF and traditional dosing method. CYP2C9, CYP4F2, VKORC1 genetic polymorphisms were determined using multiplex real time polymerase chain reaction, D-dimer concentration – by the method of solid-phase enzyme immunoassay; ADP- and epinephrine-induced aggregation indicators – by the method of G. Born. Results. The percentage (by 24 %), time (by 3 min 6 s) and rate in 30 s (by 19.5 %/min) of ADP-induced platelet aggregation were significantly lower in patients with AF and genotype-guided WF dosing method. D-dimer concentration in patients of the main and control groups did not differ significantly. However, significantly fewer patients with elevated D-dimer values were registered in the group with genotype-guided WF dosing method compared to the group with traditional dosing method: 0 (0.00 %) vs. 7 (11.67 %) at 500 ng FEU/ml cut-off (χ2 = 4.43, P &lt; 0.05), 1 (2.00 %) vs. 9 (15.00 %) at 390 ng FEU/ml cut-off (χ2 = 4.16, P &lt; 0.05), 0 (0.00 %) vs. 7 (11.67 %) at age-adjusted cut-off (χ2 = 4.43, P &lt; 0.05). Conclusions. The obtained results may indicate a potentially lower risk of thrombotic events in patients with AF and genotype-guided WF dosing method compared to the group with traditional dosing method, which confirms the benefit of the widespread use of the pharmacogenetic testing in clinical practice.

2196. Frequencies of Adenovirus Types in U.S. Children with Acute Respiratory Illness, 2016–2019

Abstract Background Adenovirus (AdV) is a common cause of acute respiratory illness (ARI). Multiple respiratory AdV types have been identified in humans, but it remains unclear which are the most common in U.S. children with ARI. Methods We conducted a multicenter, prospective viral surveillance study at seven U.S. children’s hospitals, the New Vaccine Surveillance Network, during 12/1/16–11/30/19, prior to the COVID-19 pandemic. Children &amp;lt; 18 years of age seen in the emergency department or hospitalized with fever and/or respiratory symptoms were enrolled, and mid-turbinate nasal +/- throat swabs were tested using multiplex respiratory pathogen assays or real time polymerase chain reaction (PCR) test for AdV, respiratory syncytial virus (RSV), human metapneumovirus, rhinovirus/enterovirus (RV), influenza, parainfluenza viruses, and endemic coronaviruses. AdV-positive specimens were subsequently typed using single-plex qPCR assays targeting sequences in the hexon gene specific for types 1-7, 11, 14, 16 and 21. Demographics, clinical characteristics, and outcomes were compared between AdV types. Results Of 29,381 enrolled children, 2,106 (7.2%) tested positive for AdV. The distribution of types among the 1,330 (63.2%) successfully typed specimens were as follows: 31.7% AdV-2, 28.9% AdV-1, 15.3% AdV-3, 7.9% AdV-5, 5.9% AdV-7, 1.4% AdV-4, 1.2% AdV-6, 0.5% AdV-14, 0.2% AdV-21, 0.1% AdV-11, and 7.0% ≥1 AdV type. Most children with AdV-1 or AdV-2 detection were &amp;lt; 5 years of age (Figure 1a). Demographic and clinical characteristics varied by AdV types, including age, race/ethnicity, smoke exposure, daycare/school attendance, and hospitalization (Table 1). Co-detection with other viruses was common among all AdV types, with RV and RSV being the most frequently co-detected (Figure 1b). Fever and cough were the most common symptoms for all AdV types (Figure 2). Children with AdV-7 detected as single pathogen had higher odds of hospitalization (adjusted odds ratio 6.34 [95% CI: 3.10, 12.95], p= 0.027). Conclusion AdV-2 and AdV-1 were the most frequently detected AdV types among children over the 3-year study period. Notable clinical heterogeneity of the AdV types warrants further surveillance studies to identify AdV types that could be targeted for pediatric vaccine development. Disclosures Rangaraj Selvarangan, BVSc, PhD, D(ABMM), FIDSA, F(AAM), BioFire: Grant/Research Support|Luminex: Grant/Research Support John Williams, MD, GlaxoSmithKline: Advisor/Consultant|Quidel: Advisor/Consultant Mary A. Staat, MD, MPH, Centers for Disease Control and Prevention: Grant/Research Support|Cepheid: Grant/Research Support|National Institute of Health: Grant/Research Support|Uptodate: Royalties Christopher J Harrison, MD, Astellas: Grant/Research Support|GSK: Grant/Research Support|Merck: Grant/Research Support|Pediatric news: Honoraria|Pfizer: Grant/Research Support Mary E. Moffatt, M.D., Becton and Dickinson and Company: Stocks/Bonds|Biogen: Stocks/Bonds|Coloplast B: Stocks/Bonds|Express Scripts: Stocks/Bonds|Novo Nordisk A/S Spons ADR: Stocks/Bonds|Novo Nordisk A/S-B: Stocks/Bonds|Steris PLC: Stocks/Bonds|Stryker Corp: Stocks/Bonds|Thermo Fisher Scientific: Stocks/Bonds Geoffrey A. Weinberg, MD, Merck &amp; Co.: Honoraria|Merck &amp; Co.: Honoraria for composing and reviewing textbook chapters, Merck Manual of Therapeutics Janet A. Englund, MD, AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant Natasha B. Halasa, MD, Quidel: Grant/Research Support|Quidel: equipment donation|Sanofi: Grant/Research Support|Sanofi: HAI testing and vaccine donation.

California's experience with SMA newborn screening: A successful path to early intervention.

Universal spinal muscular atrophy (SMA) newborn screening was implemented in California on June 24, 2020. We describe California's experience with the first 18 months of SMA newborn screening, including our assay methodology, timeliness of screening and follow-up milestones, and clinical and epidemiological outcomes observed. Dried blood spots are screened for SMA using multiplex real time polymerase chain reaction (RT-PCR) to detect deletions of exon 7 in the survival of motor neuron 1 (SMN1) gene. Short-term follow-up data is collected from clinical staff via an online data collection tool. In the first 18 months, 628,791 newborns from California's diverse population were tested for SMA. Thirty-four screened positive and were confirmed to have the disorder. Infants were referred, diagnosed, and treated at a median of 8, 12, and 33 days of life, respectively. Nearly all infants received the desired treatment modality, and 62% received treatment while still asymptomatic. SMA newborn screening is a highly sensitive and specific test which identifies infants with SMA early when treatment is most effective. Even with newborn screening's success in facilitating early intervention, there is still work to be done to expedite treatment, especially for infants with the most severe form of the disease.

Surveillance of respiratory viruses at health facilities from across Kenya, 2014

Background: Acute respiratory illnesses (ARI) are a major cause of morbidity and mortality globally. With (re)emergence of novel viruses and increased access to childhood bacterial vaccines, viruses have assumed greater importance in the aetiology of ARI. There are now promising candidate vaccines against some of the most common endemic respiratory viruses. Optimal delivery strategies for these vaccines, and the need for interventions against other respiratory viruses, requires geographically diverse data capturing temporal variations in virus circulation. Methods: We leveraged three health facility-based respiratory illness surveillance platforms operating in 11 sites across Kenya. Nasopharyngeal (NP) and/or oropharyngeal (OP) specimens, patient demographic, and clinical characteristics were collected in 2014 from individuals of various ages presenting with respiratory symptoms at the surveillance facilities. Real time multiplex polymerase chain reaction was used to detect rhinoviruses, respiratory syncytial virus (RSV), influenza virus, human coronaviruses (hCoV), and adenoviruses. Results: From 11 sites, 5451 NP/OP specimens were collected and tested from patients. Of these, 40.2% were positive for at least one of the targeted respiratory viruses. The most frequently detected were rhinoviruses (17.0%) and RSV A/B (10.5%), followed by influenza A (6.2%), adenovirus (6.0%) and hCoV (4.2%). RSV was most prevalent among infants aged &lt;12 months old (18.9%), adenovirus among children aged 12–23 months old (11.0%), influenza A among children aged 24–59 months (9.3%), and rhinovirus across all age groups (range, 12.7–19.0%). The overall percent virus positivity varied by surveillance site, health facility type and case definition used in surveillance. Conclusions: We identify rhinoviruses, RSV, and influenza A as the most prevalent respiratory viruses. Higher RSV positivity in inpatient settings compared to outpatient clinics strengthen the case for RSV vaccination. To inform the design and delivery of public health interventions, long-term surveillance is required to establish regional heterogeneities in respiratory virus circulation and seasonality.

Surveillance of respiratory viruses at health facilities from across Kenya, 2014

Background: Acute respiratory illnesses (ARI) are a major cause of morbidity and mortality globally. With (re)emergence of novel viruses and increased access to childhood bacterial vaccines, viruses have assumed greater importance in the aetiology of ARI. There are now promising candidate vaccines against some of the most common endemic respiratory viruses. Optimal delivery strategies for these vaccines, and the need for interventions against other respiratory viruses, requires geographically diverse data capturing temporal variations in virus circulation. Methods: We leveraged three health facility-based respiratory illness surveillance platforms operating in 11 sites across Kenya. Nasopharyngeal (NP) and/or oropharyngeal (OP) specimens, patient demographic, and clinical characteristics were collected in 2014 from individuals of various ages presenting with respiratory symptoms at the surveillance facilities. Real time multiplex polymerase chain reaction was used to detect rhinoviruses, respiratory syncytial virus (RSV), influenza virus, human coronaviruses (hCoV), and adenoviruses. Results: From 11 sites, 5451 NP/OP specimens were collected and tested from patients. Of these, 40.2% were positive for at least one of the targeted respiratory viruses. The most frequently detected were rhinoviruses (17.0%) and RSV A/B (10.5%), followed by influenza A (6.2%), adenovirus (6.0%) and hCoV (4.2%). RSV was most prevalent among infants aged &lt;12 months old (18.9%), adenovirus among children aged 12–23 months old (11.0%), influenza A among children aged 24–59 months (9.3%), and rhinovirus across all age groups (range, 12.7–19.0%). The overall percent virus positivity varied by surveillance site, health facility type and case definition used in surveillance. Conclusions: We identify rhinoviruses, RSV, and influenza A as the most prevalent respiratory viruses. Higher RSV positivity in inpatient settings compared to outpatient clinics strengthen the case for RSV vaccination. To inform the design and delivery of public health interventions, long-term surveillance is required to establish regional heterogeneities in respiratory virus circulation and seasonality.

Efficacy and safety of warfarin therapy in patients with atrial fibrillation using genotype-guided dosing method

The aim. To evaluate the efficacy and safety of warfarin (WF) therapy in patients with atrial fibrillation (AF) using pharmacogenetic dosing method at an anticoagulant monitoring office according to the results of a one-year prospective follow-up. Materials and methods. The study involved 110 patients with AF (mean age 68.72 ± 0.79; men – 57, women – 53). By the method of stratified randomization, the patients with AF were divided into two groups: the main group – 50 patients with AF and genotype-guided dosing method, the control group – 60 patients with AF and clinical dosing method. The outcomes were examined after the one-year follow-up: excessive hypocoagulation episodes (INR &gt; 4) and bleeding. CYP2C9, CYP4F2, VKORC1 gene polymorphisms were determined using multiplex real time polymerase chain reaction; INR was controlled monthly; CHA2DS2-VASC, HAS-BLED, SAMe-TT2R2 scale scores were evaluated; TTR was calculated using the Rosendaal method. Results. The distribution of CYP2C9, CYP4F2, VKORC1 genotypes in the main and control groups were in accordance with the Hardy–Weinberg equilibrium. In patients with AF and genotype-guided dosing, the frequency and risk of excessive hypocoagulation episodes (χ2 = 5.11; P &lt; 0.05; RR = 0.50 (CI 0.27; 0.94)) and bleeding (χ2 = 9.57; P &lt; 0.05; RR = 0.41 (CI 0.22; 0.77)) were significantly lower. However, the groups did not differ in TTR. The validity of genetic-guided dosing was confirmed by the comparability of the medians and the direct correlation between the calculated and therapeutic WF doses (r = +0.57; P &lt; 0.05). There were no relationships between TTR, excessive hypocoagulation episodes, hemorrhagic complications and clinical and genetic factors in the main group. Conclusions. In patients with AF, the use of genotype-guided dosing method in the anticoagulant monitoring office helped to reduce the frequency and risk of excessive hypocoagulation episodes and bleeding as well as eliminate the influence of endogenous and exogenous factors in the personalized management of patients.

Comparison of the Multiplex Real Time Polymerase Chain Reaction Method Directly from Whole Blood and from Blood Culture Bottle with the Conventional Method for the Etiological Diagnosis in Patients with Pre-Diagnosis of Sepsis

In this study, it was aimed to evaluate the use of multiplex real time polymerase chain reaction (RtPCR) method, directly from whole blood and blood culture bottles of intensive care unit patients with a pre-diagnosis of sepsis for the determination of the causative agent by comparing it with the conventional method. The study was carried out prospectively between November 2019 and August 2020. Ninety patients hospitalized with a pre-diagnosis of sepsis were included in the study. Samples were collected simultaneously with aseptic technique as whole blood and at least two sets of blood cultures. Blood culture bottles were monitored by the BACT/ALERT 3D (bioMerieux, France) instrument. Blood culture bottles were studied both by Sepsis Pathogens Panel Kit v1 (Anatolia, Turkey) using the multiplex Rt-PCR and with the conventional culture methods. The duration of detection and reporting of the multiplex Rt-PCR methods were compared with the conventional method. In this study, a total of 90 whole blood samples and 280 blood culture bottles were collected from the patients. It was found that 73 (81%) patients had already been administered antibiotic treatment at the time of blood sampling. Conventional blood culture was accepted as the gold standard in the diagnosis of sepsis. Rt-PCR performed from blood culture bottles was found to have a sensitivity of 89.58%; specificity of 57.14%; positive predictive value of 70.49%; negative predictive value of 82.76% and accuracy of 74.44% (p= 0.019). On the basis of the bacterial species, the sensitivity of the Rt-PCR method was determined to be between 66.7-100% and the specificity was between 86.6-100%. The Rt-PCR performed from whole blood, was found to have a sensitivity of 11.67% and a specificity of 96.67%; positive predictive value of 87.50%; negative predictive value of 35.37% and accuracy of 40% (p= 1.684). In this study, the duration of reporting of blood culture results was in average 116:50 hours, that of PCR from blood culture bottles was 80:57 hours, and that of PCR from whole blood samples was 15:38 hours (p<0.001). It was determined that the PCR from whole blood shortened the reporting time by an average of 101:12 hours (approximately four days), and that the PCR from blood culture bottles shortened the reporting time by an average of 35:53 hours (1.5 days) compared to the conventional method. In this study, the results determined by the Sepsis Pathogens Panel Kit v1 (Anatolia, Turkey) performed from blood culture bottles were superior compared to the conventional method. It has been determined that improvement is required for the usage of the sepsis kit directly from whole blood. On the other hand, considering the species detected by the blood culture method and not by the assay, the species range detected by the multiplex assay panel needs to be improved.

The influence of clinical and genetic factors on the stability of warfarin’s anticoagulant effect in patients with atrial fibrillation

The aim. To investigate the influence of clinical and genetic factors on the stability of warfarin’s anticoagulant effect in patients with atrial fibrillation (AF) during the year. Materials and methods. The study involved 60 patients with AF, age 70.50 (64.25; 76.25) years (32 men and 28 women). Coagulogram indexes with International Normalized Ratio (INR) were determined using Coag Chrome 3003 monthly; the CHA2DS2-VASC, HAS-BLED, SAMe-TT2R2 scales scores were evaluated; the calculation of TTR was performed using the Rosendaal method. CYP2C9, CYP4F2, VKORC1 genes polymorphisms were determined using multiplex real time polymerase chain reaction in CFX-96 thermocycler (BioRad). Results. Median TTR in groups of patients with SAMe-TT2R2 score &lt;2 (n = 33) and ≥2 (n = 27) did not differ significantly (74 % versus 68 % respectively, P &gt; 0.05). There were significantly more patients with TTR &lt;70 % in the group with predicted labile INR (59.36 % versus 30.30 %; χ2 = 5.07, P &lt; 0.05). SAMe-TT2R2 score ≥2 increased the risk of poor INR control by 1.96 times (CI 1.05–3.63). No association of TTR with CYP2C9, CYP4F2 and VKORC1 gene polymorphisms was found. Episodes of excessive hypocoagulation (INR &gt;4) were detected in 21 (40 %) patients during the year. Excessive hypocoagulation was significantly more common in patients carrying the allele A of the VKORC1 gene in comparison with non-carriers (51.43 % versus 24.00 %; χ2 = 4.57, P &lt; 0.05). The presence of mutant allele A was associated with 2.14-fold higher risk of excessive hypocoagulation (RR = 2.14; CI 1.06–4.69). Taking amiodarone (χ2 = 3.13; P &lt; 0.05) had a significant effect on the development of excessive hypocoagulation with a relative risk RR = 1.83 (CI 1.01–3.35). Conclusions. SAMe-TT2R2 score can be useful to predict poor INR control, while VKORC1 genotype estimating – to predict excessive hypocoagulation episodes. An integrated approach using clinical and genetic methods is needed to determine the potential efficacy and safety of warfarin therapy.

A Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay With Enhanced Capacity to Detect Vesicular Stomatitis Viral Lineages of Central American Origin.

Vesicular stomatitis virus (VSV) causes a disease in susceptible livestock that is clinically indistinguishable from foot-and-mouth disease. Rapid testing is therefore critical to identify VSV and rule out FMD. We previously developed and validated a multiplex real-time reverse transcription polymerase chain reaction assay (mRRT-PCR) for detection of both VS New Jersey virus (VSNJV) and VS Indiana virus (VSIV). However, it was subsequently apparent that this assay failed to detect some VSNJV isolates in Mexico, especially in genetic group II, lineage 2.1. In order to enhance the sensitivity of the mRRT-PCR for VSNJV, parts of the assay were redesigned and revalidated using new and improved PCR chemistries. The redesign markedly improved the assay by increasing the VSNJV detection sensitivity of lineage 2.1 and thereby allowing detection of all VSNJV clades. The new assay showed an increased capability to detect VSNJV. Specifically, the new mRRT-PCR detected VSNJV in 100% (87/87) of samples from Mexico in 2006-2007 compared to 74% for the previous mRRT-PCR. Furthermore, the analytical sensitivity of the new mRRT-PCR was enhanced for VSNJV. Importantly, the modified assay had the same sensitivity and specificity for VSIV as the previously published assay. Our results highlight the challenges the large genetic variability of VSV pose for virus detection by mRRT-PCR and show the importance of frequent re-evaluation and validation of diagnostic assays for VSV to ensure high sensitivity and specificity.

Cross sectional study to evaluate microbiological spectrum of RTI/STI and co-infections among women with cervicitis or cervico-vaginitis from a community clinic in Mumbai

Cervicitis is an inflammatory condition of cervix, when presented along with vaginal discharge; it is termed as cervico-vaginitis. These can be infective, hence important to diagnose due to risk of spreading to upper genital tract. This cross-sectional study was undertaken to evaluate the microbiological spectrum in cervicitis or cervico-vaginitis among 100 sexually active women by Gram stain and Multiplex Real time polymerase chain reaction. Bacterial vaginosis 21(21%) was the most common RTI. Among STIs, genital mycoplasmas were the predominant infections hence further research is required to understand their pathogenesis.

Changes in Methylation Patterns of Tumor Suppressor Genes during Extended Human Embryonic Stem Cell Cultures.

While studies on embryonic stem cells have been actively conducted, little is known about the epigenetic mechanisms in human embryonic stem cells (hESCs) in extended culture systems. Here, we investigated whether CpG island (CGI) methylation patterns of 24 tumor suppressor genes could be maintained during extended hESC cultures. In total, 10 hESC lines were analyzed. For each cell line, genomic DNA was extracted from early and late passages of cell cultures. CGI methylation levels of 24 tumor suppressor genes were analyzed using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), pyrosequencing, and real-time polymerase chain reaction (PCR). Different CGI methylation patterns of CASP8, FHIT, and CHFR genes were identified in between early and late passages in some hESC lines. CGI methylation levels of CASP8 significantly increased at late passage in CHA-36, CHA-40, and CHA-42 cell lines compared to those at early passage. The CGI methylation of the FHIT gene was higher at late passage than at early passage in CHA-15, CHA-31, CHA-32, and iPS (FS)-1 cell lines but decreased at the late passage in CHA-20 and H1 cell lines. Different CGI methylation patterns were detected for the CHFR gene only in iPS (FS)-1, and the level significantly increased at late passage. Thus, our findings show that CGI methylation patterns could be altered during prolonged ESC cultures and examining these epigenetic changes is important to assess the maintenance, differentiation, and clinical usage of stem cells.

Frequencies of polymorphisms in genes affecting the pharmacokinetics of warfarin in the Zaporizhzhia region

The aim of the study: to establish the frequencies of polymorphisms in VKORC1, CYP2C9 and CYP4F2 genes among residents of the Zaporizhzhia region. Materials and methods. A total of 150 persons (62 male, 88 female) with a median age of 46 years (26; 58) undergoing preventive examination at the Medical Educational and Scientific Center “University Clinic” of Zaporizhzhia State Medical University (ZSMU). The CYP2C9, CYP4F2, VKORC1 genes polymorphisms in atrial fibrillation patients were determined in the Department of Molecular Genetic Studies of the ZSMU Medical Laboratory Center. Amplification of DNA fragments containing polymorphic regions was performed using multiplex real time polymerase chain reaction with Warfarin Pharmacogenetics kits (LLC NPO DNA Technology) in a CFX-96 thermocycler (BioRad) with a fluorescence detection scheme. Results. It was determined that among Zaporizhzhia region residents, the frequencies of CYP2C9*2 genotypes were: C/C – 77.3 %, C/T – 22.7 %, T/T – 0; CYP2C9*3 genotypes: A/A – 88.7 %, A/C – 10.7 %, C/C – 0.6 %; CYP4F2 genotypes: C/C – 56.0 %, C/T – 35.3 %, T/T – 8.7 %; VKORC1 genotypes: G/G – 38.0 %, G/A – 50.0 %, A/A – 12.0 %. There were no statistically significant differences in the distribution of genotype frequencies between males and females and between different age groups. The frequencies of CYP2C9, CYP4F2, VKORC1 genotypes in different populations were compared. Their variability in different geographic regions was established. Conclusions. CYP4F2 and VKORC1 genes polymorphisms are more common in the Zaporizhzhia region, while the prevalence of CYP2C9*2 and CYP2C9*3 genes polymorphisms is much lower. It is necessary to take into account the prevalence of genes polymorphisms that affect warfarin metabolism for each individual population to select its dose by pharmacogenetic testing.

Molecular Typing of Pathogenic Leptospira Species Isolated from Wild Mammal Reservoirs in Sardinia.

Simple SummaryLeptospirosis is caused by pathogenic spirochaetes of the genus Leptospira. Humans can become infected with these bacteria through direct contact with urine from infected animals or indirectly through interaction with a urine contaminated environment. Among wildlife species, rodents are considered the primary reservoir hosts for leptospirosis in rural and urban environments. Epidemiological data, regarding leptospirosis in various wild species in Europe, suggest that these animals play a different role in leptospiral persistence. Unfortunately, studies on the presence and typing of Leptospira species in wild mammals are lacking in Sardinia. The aim of the present study was to investigate the prevalence of Leptospira species in wild mammals. Kidneys collected from carcasses were analyzed by culture and molecular testing. Greater positivity was found in hedgehogs, followed by weasels and rodents. The results obtained suggest that Sardinian fauna may play a possible sentinel role in the transmission cycle of leptospirosis to humans. Gathering this information in different wildlife species is crucial for better understanding of the epidemiology of leptospirosis and for the development of appropriate prevention measures.Leptospirosis is a global zoonosis caused by pathogenic species of Leptospira that infect a large spectrum of domestic and wild animals. This study is the first molecular identification, characterization, and phylogeny of Leptospira strains with veterinary and zoonotic impact in Sardinian wild hosts. All samples collected were cultured and analyzed by multiplex real time polymerase chain reaction (qPCR). Sequencing, phylogenetic analyses (based on rrs and secY sequences), and Multilocus Sequence Typing (MLST) based on the analysis of seven concatenated loci were also performed. Results revealed the detection of Leptospira DNA and cultured isolates in 21% and 4% of the samples examined, respectively. Sequence analysis of Leptospira positive samples highlighted the presence of the interrogans and borgpetersenii genospecies that grouped in strongly supported monophyletic clades. MLST analyses identified six different Sequence Types (ST) that clustered in two monophyletic groups specific for Leptospira interrogans, and L. borgpetersenii. This study provided about the prevalence of leptospires in wild mammals in Sardinia, and increased our knowledge of this pathogen on the island. Monitoring Leptospira strains circulating in Sardinia will help clinicians and veterinarians develop strategic plans for the prevention and control of leptospiral infections.

Increased frequency of blaNDM in a tertiary care hospital in southern Brazil.

Resistance to carbapenems due to metallo-beta-lactamase NDM-1 was first described in Brazil in 2013. To date, only a few scattered reports of the prevalence of NDM-1 in the country have been reported, and most of them indicated a very low prevalence of this metalloenzyme. In the present study, we report a steady increase in the frequency of NDM among Enterobacterales resistant to carbapenems in a tertiary care hospital in southern Brazil. Carbapenemase genes were evaluated by multiplex real-time polymerase chain using high-resolution melting analysis among 3501 isolates of 8 different species of Enterobacterales recovered from January 2015 to May 2020. The blaKPC-like was identified in 3003 isolates (85.8%) and the blaNDM-like was the second most common gene (351 isolates-10%). There was a steady increase in frequency of blaNDM-like, from 4.2% in 2015 to 24% in 2020. The increase of blaNDM frequency raises an important matter as novel therapeutic options are currently very limited for the treatment of patients infected by bacteria carrying the blaNDM.

Differentiating Viral and Bacterial Pneumonia by Clinical Manifestations among Children in Chiang Mai, Thailand

Background: Viral pneumonia is a common complication of influenza like illness including COVID-19. Virus is the most common cause of pneumonia in children. While there are important cues in history, the laboratory examination that can help differentiate viral and bacterial causes. There are limited clinical manifestations to differentiate viral and co-infection bacterial and viral pneumonia. Objective: To determine basic clinical manifestations and laboratory performed at bedside as predictors to differentiate viral and co-infection bacterial and viral pneumonia. Materials and Methods: A retrospective study was conducted in pediatric patients with radiographic evidence of severe pneumonia and admitted in Nakornping Hospital, Chiang Mai, Thailand between October 2017 and April 2020. Multiplex real time polymerase chain reactions (PCRs) (RP-24-26) were used to identify the cause of the pneumonia. Demographic data and basic clinical predictors such as age, comorbidity, symptoms, and physical findings, and basic laboratory such as complete blood count were collected. Patients were divided into three groups, no infection detected, viral infection, and bacterial and viral co-infection. Polytomous logistic regression was performed to investigate predictors for type of infection. Area under the receiver operating characteristic curve (AuROC) and 95% confidence interval (CI) were further determined for a final model to differentiate type of infection. Results: Two hundred eight cases participated in this study and included 122 males (58.7%). The etiology of pneumonia identified by RP-24-26, pathogen was detected among 166 cases (79.8%). A virus was detected in 141 cases (67.8%), co-infection was detected in 25 cases (12%), and no infection was detected in 42 cases (20.2%). The statistically significant predictor for viral pneumonia was cough, with an adjusted relative risk ratio (RRR) of 4.42 (1.41 to 13.82). The statistically significant predictors for co-infection were age 13 to 24 months and lymphocytes at 40.0% or greater with adj. RRR 14.28 (1.47 to 138.52), and 4.60 (1.24 to 17.04), respectively. Pneumonia patients with cough were 4.42 times more to have viral cause. Those with lymphocytes of 40.0% or greater were 4.60 times more to be co-infected with both virus and bacteria, especially in the age group 13 to 24 months compared with 1 to 12 months. The age group13 to 24 months, cough, and lymphocytes at 40.0% or greater were 74% (95% CI 0.66 to 0.82) correctly predicted to viral pneumonia. Conclusion: Coughing is a predominant symptom for childhood pneumonia caused by virus. In addition, pneumonia patients age 13 to 24 months who have lymphocyte of more than 40% should start and continue antibiotics until complete course. Keywords: Childhood pneumonia, Respiratory panel 24, Respiratory panel 26, Co-infection bacterial pneumonia, Predictor

Довжина теломер у хворих на цукровий діабет 2-го типу та її зв’язок із метаболічним профілем

To study leukocyte telomere length and its relationship with metabolic profile 35 patients with metabolic syndrome and type 2 diabetes mellitus (T2DM) and 21 healthy people of middle age (35-59 years) were examined. The anthropometric characteristics of obesity, indicators of lipid and glucose metabolism, alanin aminotransferase (ALT) and high sensitive C-reactive protein levels were studied. The relative average telomere length was determined by the method of monochrome multiplex quantitative real time polymerase chain reaction. Patients with T2DM had higher BMI, waist circumference, higher high sensitive C-reactive protein, ALT and glucose levels and a worse lipid profile (p &lt;0.05). At the same time, the median telomere length did not differ between groups. Nevertheless, in the T2DM group the telomere length inversely correlated with body weight (r = –0.35; p &lt; 0.05), BMI (r = –0.36; p &lt; 0.05), waist circumference (r = –0.34; p &lt; 0.05) and ALT level (r = –0,44; p&lt;0,05) in contrast to healthy subjects. No relationship was found between the telomere length and the level of fasting glycemia, as well as the age of the participants of both groups. Thus, in T2DM patients increase in BMI, waist circumference and ALT level were associated with a shorter leukocyte telomere length. Despite the worse metabolic profile, the telomere length in middle-aged T2DM patients did not differ from that in the control group. This indicates that the leukocyte telomere length is influenced not only by the presence of T2DM and the metabolic profile indicators, but, obviously, by other factors as well.

Quantitative Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3

(1) Background: KIR2DL4/KIR3DL3 are the framework genes present in all KIR haplotypes, with unique expression patterns being present only in women and CD56bright NK cells. KIR genes have a high degree of DNA sequence identity. Consequently, they are one of the most challenging genes for molecular detection—especially regarding expressions; (2) Methods: We developed an effective method to determine KIR3DL3/KIR2DL4 expressions based on a multiplex quantitative real-time Reverse transcription polymerase chain reaction (qRT-PCR )with fluorescent probes using NK92; (3) Results: Standardizations of the singleplex KIR2DL4 and KIR3DL3 were performed to evaluate the sensitivity and specificity for further development of the multiplex assay. The limit of detection was at 500 copies each. There was cross-amplification with the presence of related KIR genes at a level of 5 × 107 copies. This is not biologically significant because this high level of KIR expression has not been found in clinical samples. The multiplex assay was reproducible equivalent to its singleplex (KIR2DL4; R2 = 0.995, KIR3DL3; R2 = 0.996, but lower sensitivity of 103 copies). Furthermore, the validation of the developed method on samples of blood donors showed high sensitivity (100%) and specificity (99.9%); (4) Conclusions: The developed method is reliable and highly specific suitable for evaluation of the KIR2DL4/3DL3 mRNA expressions in further applications.