Quantitative Polymerase Chain Reaction Test Research Articles

Genomic surveillance during the first two years of the COVID-19 pandemic – country experience and lessons learned from Türkiye

BackgroundTürkiye confirmed its first case of SARS-CoV-2 on March 11, 2020, coinciding with the declaration of the global COVID-19 pandemic. Subsequently, Türkiye swiftly increased testing capacity and implemented genomic sequencing in 2020. This paper describes Türkiye’s journey of establishing genomic surveillance as a middle-income country with limited prior sequencing capacity and analyses sequencing data from the first two years of the pandemic. We highlight the achievements and challenges experienced and distill globally relevant lessons.MethodsWe tracked the evolution of the COVID-19 pandemic in Türkiye from December 2020 to February 2022 through a timeline and analysed epidemiological, vaccination, and testing data. To investigate the phylodynamic and phylogeographic aspects of SARS-CoV-2, we used Nextstrain to analyze 31,629 high-quality genomes sampled from seven regions nationwide.ResultsTürkiye’s epidemiological curve, mirroring global trends, featured four distinct waves, each coinciding with the emergence and spread of variants of concern (VOCs). Utilizing locally manufactured kits to expand testing capacity and introducing variant-specific quantitative reverse transcription polymerase chain reaction (RT-qPCR) tests developed in partnership with a private company was a strategic advantage in Türkiye, given the scarcity and fragmented global supply chain early in the pandemic. Türkiye contributed more than 86,000 genomic sequences to global databases by February 2022, ensuring that Turkish data was reflected globally. The synergy of variant-specific RT-qPCR kits and genomic sequencing enabled cost-effective monitoring of VOCs. However, data analysis was constrained by a weak sequencing sampling strategy and fragmented data management systems, limiting the application of sequencing data to guide the public health response. Phylodynamic analysis indicated that Türkiye’s geographical position as an international travel hub influenced both national and global transmission of each VOC despite travel restrictions.ConclusionThis paper provides valuable insights into the testing and genomic surveillance systems adopted by Türkiye during the COVID-19 pandemic, proposing important lessons for countries developing national systems. The findings underscore the need for robust testing and sampling strategies, streamlined sample referral, and integrated data management with metadata linkage and data quality crucial for impactful epidemiological analysis. We recommend developing national genomic surveillance strategies to guide sustainable and integrated expansion of capacities built for COVID-19 and to optimize the effective utilization of sequencing data for public health action.

Concordance between ER, PR, Ki67, and HER2-low expression in breast cancer by MammaTyper RT-qPCR and immunohistochemistry: implications for the practising pathologist.

There are limited data on the role of multigene tests and their correlation with immunohistochemistry (IHC), especially on core biopsy. MammaTyper is a quantitative conformite Europeeanne (CE) marked, National Institute for Health and Care excellence (NICE) approved, in in vitro diagnostic quantitative real-time polymerase chain reaction (RT-qPCR) test for assessment of mRNA expression of four biomarkers (ESR1, PGR, ERBB2, MKI67). We evaluated the concordance of MammaTyper with oestrogen receptor (ER), progesterone receptor(PR), HER2, and Ki67 by IHC on 133 core needle biopsies of breast cancer. HER2 was positive if IHC 3+ or 2+ and fluorescence in situ hybridization (FISH)-amplified. Global and hotspot Ki67 expression was analysed using a cutoff of ≥20% assessed manually and by digital image analysis. Agreements were expressed as overall percent agreement (OPA), positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa. RT-qPCR results of ESR1 were highly concordant with IHC with OPA of 94.7% using 1% cutoff and 91.7% when the low ER-positive category was included. The PPA and NPA between RT-qPCR and IHC for PR was 91.5% and 88.0%, respectively, when using the 1% cutoff. For ERBB2/HER2, the OPA was 95% and the PPA was 84.6%. 40 of 72 HER2 IHC score 0 tumours were classified as ERBB2 low. Best concordance between MKI67 by MammaTyper and Ki67 IHC was achieved using hotspot digital image analysis (OPA: 87.2%, PPA: 90.6%, NPA: 80%). RT-qPCR-based assessment of the mRNA expression of ESR1, PGR, ERBB2, and MKI67 showed high concordance with IHC, suggesting that the MammaTyper test on core needle biopsies represents a reliable, efficient, and reproducible alternative for breast cancer classification and refining HER2 low categorisation.

Faster detection of asymptomatic COVID-19 cases among care home staff in England through the combination of SARS-CoV-2 testing technologies

To detect SARS-CoV-2 amongst asymptomatic care home staff in England, a dual-technology weekly testing regime was introduced on 23 December 2020. A lateral flow device (LFD) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) test were taken on the same day (day 0) and a midweek LFD test was taken three to four days later. We evaluated the effectiveness of using dual-technology to detect SARS-CoV-2 between December 2020 to April 2021. Viral concentrations derived from qRT-PCR were used to determine the probable stage of infection and likely level of infectiousness. Day 0 PCR detected 1,493 cases of COVID-19, of which 53% were in the early stages of infection with little to no risk of transmission. Day 0 LFD detected 83% of cases that were highly likely to be infectious. On average, LFD results were received 46.3 h earlier than PCR, enabling removal of likely infectious staff from the workplace quicker than by weekly PCR alone. Demonstrating the rapidity of LFDs to detect highly infectious cases could be combined with the ability of PCR to detect cases in the very early stages of infection. In practice, asymptomatic care home staff were removed from the workplace earlier, breaking potential chains of transmission.

Exploring the potential of VGLL3 methylation as a prognostic indicator for intracranial aneurysm with gender-specific considerations.

DNA methylation is widely recognized to play a role in intracranial aneurysm (IA) pathogenesis. We investigated the levels of methylation of vestigial-like 3 (VGLL3) in IA and explored its potential as a prognostic indicator. A total of 48 patients with IA and 48 healthy controls were included in the present study. Methylation levels of CpG sites were assessed using bisulfite pyrosequencing, and levels of VGLL3, TEAD, and YAP in the blood were measured by real-time quantitative polymerase chain reaction testing. VGLL3 methylation was significantly higher in controls than in IA patients (P=0.001), and this phenomenon was more pronounced in females (P<0.001). Compared with the control group, the expression levels of VGLL3 and TEAD in the blood of IA patients were significantly increased, while YAP was significantly decreased. VGLL3 methylation was positively correlated with HDL (P=0.003) and female Lpa concentration (r = 0.426, P=0.03), and was also negatively correlated with age (P=0.003), APOE (P=0.005), and VGLL3 mRNA expression (P<0.001). Methylation and mRNA expression of VGLL3 may serve as indicators of IA risk in females (AUC = 0.810 and 0.809). VGLL3 methylation may participate in the pathogenesis of IA by regulating the expression of the VGLL3/TEAD/YAP pathway, and its gene methylation and expression levels have IA risk prediction value.

Identification and expression analysis of TPS family gene in Cannabis sativa L

Terpene synthases (TPSs) regulate plant growth, development, and stress response. TPS genes have been identified in Arabidopsis thaliana and Zea mays. Cannabis sativa TPS genes were identified and analyzed using bioinformatics. Genomic data were downloaded from Plant Transcription Factor Database and National Center for Biotechnology Information database, and TPS genes were predicted, analyzed, and visualized using ExPASy, PlantCare, and other online websites along with TBtools, MEGA software, and other software. To verify its role, quantitative real-time polymerase chain reaction (qRT-PCR) tests were conducted. The Cannabis sativa TPS family comprises 41 elements distributed over 8 chromosomes and a single scaffold segment. The isoelectric point varied between 4.96 and 7.03, while the molecular weight spanned from 20705.90 to 102324.64 Da. The majority of genes were found in the cytoplasm and chloroplasts, with the remainder situated in the peroxisome, nucleus, plasma membrane, and mitochondria. Several cis-acting components associated with stress response were present in the gene's upstream promoter region. Data from RNA sequencing and qRT-PCR revealed specific expression of TPS genes in all five organs of female Cannabis sativa plants. Collinearity analysis showed 4 homologous gene pairs between the Cannabis sativa and Arabidopsis thaliana, with many pairs of homologous genes in other species, which was consistent with the dicotyledons evolutionary relationship. Furthermore, some genes may participate in Cannabis sativa growth and development and play a role in secondary metabolite synthesis. Therefore, bioinformatics analysis of the Cannabis sativa TPS gene family provides a theoretical basis for future research on the volatile terpene compounds of Cannabis sativa.

New Manual Quantitative Polymerase Chain Reaction Assay Validated on Tongue Swabs Collected and Processed in Uganda Shows Sensitivity That Rivals Sputum-based Molecular Tuberculosis Diagnostics.

Sputum-based testing is a barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce sputum, and sputum processing increases assay complexity and cost. Tongue swabs are emerging as an alternative to sputum, but performance limits are uncertain. From June 2022 to July 2023, we enrolled 397 consecutive adults with cough >2 weeks at 2 health centers in Kampala, Uganda. We collected demographic and clinical information, sputum for TB testing (Xpert MTB/RIF Ultra and 2 liquid cultures), and tongue swabs for same-day quantitative polymerase chain reaction (qPCR) testing. We evaluated tongue swab qPCR diagnostic accuracy versus sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared 2 swab types (Copan FLOQSWAB and Steripack spun polyester). Among 397 participants, 43.1% were female, median age was 33 years, 23.5% were diagnosed with human immunodeficiency virus, and 32.0% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% (95% confidence interval [CI]: 96.2-99.1) of participants. Tongue swab qPCR sensitivity was 92.6% (95% CI: 86.5 to 96.0) and specificity was 99.1% (95% CI: 96.9 to 99.8) versus microbiological reference standard. A single tongue swab recovered a 7-log range of TB copies, with a decreasing recovery trend among 4 serial swabs. Swab types performed equivalently. Tongue swabs are a promising alternative to sputum for molecular diagnosis of TB, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics.

The role of human rhinovirus in COPD exacerbations in Abu Dhabi: molecular epidemiology and clinical significance

ABSTRACT This study aimed to describe the molecular epidemiology and seasonality of human rhinovirus (HRV) in chronic obstructive pulmonary disease (COPD) and its association with COPD exacerbations in Abu Dhabi, the United Arab Emirates (UAE). Sputum specimens were collected for analysis from all COPD patients who visited a medical center from November 2021 to October 2022. The real-time quantitative polymerase chain reaction (qPCR) test was used to detect HRV. Of the 78 COPD patients included in the study, 58 (74%) patients presented with one or more exacerbation episodes. The incidence of COPD exacerbation peaked over the winter and substantially decreased during the summer. HRV positivity in patients during exacerbation (E1) was 11/58 (19%) and 15/58 (26%) two weeks after the exacerbation episode (E2). There was no significant difference in the HRV load in these patients. No statistically significant difference was observed in the detection of HRV during exacerbation compared to patients with stable COPD. This is the first study to assess the association between HRV detection by qPCR and COPD exacerbations in the UAE. The high sensitivity of the detection technology helped collect reliable epidemiologic data. Few studies have provided similar Middle East data. This study’s pattern of COPD exacerbations and HRV detection parallels that of temperate countries. This information can help with future, more extensive surveillance of respiratory viruses in the UAE and the Middle East and their association with COPD exacerbations.

Natural history of adenoviral conjunctivitis in a US-based population: Viral load, signs, and symptoms

PurposeTo report the clinical signs, symptoms, and viral clearance in individuals in the United States with adenoviral conjunctivitis (Ad-Cs). MethodsIndividuals ≥ 18 years presenting within 4 days of symptoms of Ad-Cs who met eligibility criteria and tested positive with both point-of-care immunoassay antigen and quantitative polymerase chain reaction (qPCR) testing were enrolled. Patient-reported symptoms, clinician-graded signs, and qPCR viral titers were collected at baseline, days 1–2, 4 (days 3–5), 7 (days 6–10), 14 (days 11–17) and 21 (days 18–21). ResultsThere was no detectable viral titers by the day 14 visit in 6/8 patients. By day 21, there was no detectable viral titers in the 7 participants who completed the visit; however, signs and symptoms persisted including: blurry vision (5/7), discomfort (2/7) or redness (1/7). Masked clinicians also noted conjunctival redness (4/7), follicular conjunctivitis (4/7) and bulbar edema (3/7). ConclusionMany patient-reported symptoms and clinical signs persist after viral titers are no longer detectable by qPCR. Using clinical signs and symptoms to determine quarantine duration may result in patients being furloughed longer than the time that the patient is infectious.

A Portable and Disposable Electrochemical Sensor Utilizing Laser-Scribed Graphene for Rapid SARS-CoV-2 Detection.

The COVID-19 pandemic caused by the virus SARS-CoV-2 was the greatest global threat to human health in the last three years. The most widely used methodologies for the diagnosis of COVID-19 are quantitative reverse transcription polymerase chain reaction (RT-qPCR) and rapid antigen tests (RATs). PCR is time-consuming and requires specialized instrumentation operated by skilled personnel. In contrast, RATs can be used in-home or at point-of-care but are less sensitive, leading to a higher rate of false negative results. In this work, we describe the development of a disposable, electrochemical, and laser-scribed graphene-based biosensor strips for COVID-19 detection that exploits a split-ester bond ligase system (termed 'EsterLigase') for immobilization of a virus-specific nanobody to maintain the out-of-plane orientation of the probe to ensure the efficacy of the probe-target recognition process. An anti-spike VHH E nanobody, genetically fused with the EsterLigase domain, was used as the specific probe for the spike receptor-binding domain (SP-RBD) protein as the target. The recognition between the two was measured by the change in the charge transfer resistance determined by fitting the electrochemical impedance spectroscopy (EIS) spectra. The developed LSG-based biosensor achieved a linear detection range for the SP-RBD from 150 pM to 15 nM with a sensitivity of 0.0866 [log(M)]-1 and a limit of detection (LOD) of 7.68 pM.

Association of Demographic, Clinical, and Vaccination Characteristics with COVID-19 Viral Load Assessed by qRT-PCR.

The effect of vaccination on the SARS-CoV-2 baseline viral load and clearance during COVID-19 infection is debatable. This study aimed to assess the effects of demographic and vaccination characteristics on the viral load of SARS-CoV-2. We included the patients referred for outpatient SARS-CoV-2 qRT-PCR (reverse transcriptase quantitative polymerase chain reaction) test between July and September 2022. Cycle threshold (Ct) data were compared based on the demographic and vaccination characteristics. A generalized linear model was used to determine the factors associated with the SARS-CoV-2 PCR Ct value. Of 657 participants, 390 (59.4%) were symptomatic and 308 (47.1%) were COVID-19 positive. Among 590 individuals with known vaccination status, 358 (60.6%) were booster vaccinated, 193 (32.6%) were fully vaccinated, 13 (2.2%) were partially vaccinated, and 26 (4.4%) were unvaccinated. Most vaccinated patients received inactivated vaccines (70.5%). The median Ct value was 20 [IQR: 18-23.75] with no significant difference between individuals with different vaccination statuses (P value = 0.182). There were significant differences in Ct value in terms of both symptom presence and onset (both P values < 0.001). Our regression model showed that inactivated vaccines (P value = 0.027), mRNA vaccines (P value = 0.037), and the presence and onset of symptoms (both P values < 0.001) were independent factors significantly associated with the viral load. The SARS-CoV-2 baseline viral load is unaffected by vaccination status, yet vaccination might accelerate viral clearance. Furthermore, we demonstrated that the presence and onset of symptoms are independent variables substantially associated with the patient's viral load.

Preliminary Results of a Phase 2a Clinical Trial to Evaluate Safety, Tolerability and Antiviral Activity of Intravenous Brincidofovir (BCV IV) in Immunocompromised Patients with Adenovirus Infection

Background: Adenovirus (AdV) may cause life threatening or fatal infections in immunosuppressed patients including recipients of allogeneic hematopoietic cell transplant (allo-HCT). Brincidofovir (BCV) is a lipid conjugate of cidofovir, that crosses target cell membranes by means of facilitated and passive diffusion and has a long intracellular half-life. The safety, tolerability and antiviral activity of intravenous BCV (BCV IV) were evaluated in a dose ascending Phase 2a study (BCV-PA01: ATHENA study, NCT04706923) in sequential cohorts. Methods: Eligible patients aged 2 months or older who were immunocompromised due to allo-HCT, organ transplant, immunosuppressive medications, or congenital immunodeficiency, and had AdV viremia or disseminated AdV disease were included. AdV viremia was defined as 1) AdV viral load in the blood &amp;gt; 10,000 copies/mL OR 2) two samples greater than 48 hours apart with the second result higher than the first and both greater than 1000 copies/mL, within 7 days prior to initiation of BCV IV treatment. BCV IV was administered intravenously twice weekly for 4 weeks or until resolution of AdV viremia up to 14 weeks, whichever occurred later. The dose escalation was as follows: 0.2 mg/kg (Cohort 1), 0.3 mg/kg (Cohort 2), or 0.4 mg/kg (Cohort 3) for patients weighing &amp;lt; 50 kg and 10 mg/dose, 15 mg/dose, or 20 mg/dose, respectively for patients weighing ≥ 50 kg. The patients were subsequently followed up for 30 days after the last BCV IV dosing. AdV viral loads in blood were monitored weekly by quantitative real-time polymerase chain reaction tests (lower limit of detection (LOD) 25 copies/mL, lower limit of quantification 190 copies/mL, upper limit of quantification 1 x 10 9 copies/mL). Viral clearance was defined as two consecutive plasma viral load test results below the LOD. Results: Of 27 patients treated with BCV IV, 8 were in Cohort 1, 9 in Cohort 2 and 10 in Cohort 3. Baseline characteristics of patients are shown in Table 1. Treatment-related adverse events (TRAE) were observed in 7 patients, as shown in Table 2. No serious TRAEs including gastrointestinal and hepatic toxicities were observed. All TRAEs were reversible and resolved after the completion of the treatment. AE-related discontinuations of treatment were observed in 3 of 8 patients in Cohort 1; 2 of 9 patients in Cohort 2; and 1 of 10 patients in Cohort 3 (Table 2). The reason for discontinuation of treatment beyond 4 weeks of treatment in Cohort 3 was successful clearance of AdV viremia. Antiviral activity was dose-dependent, and viral clearance was achieved in 25% (2/8) in Cohort 1; 56 % (5/9) in Cohort 2; and 100% (10/10) in Cohort 3. The duration of treatment with BCV IV and viral responses are summarized in Table 2. In Cohort 3, it is notable that 90 % of patients achieved AdV viremia clearance in ≤ 4 weeks of treatment with BCV IV. In Cohorts 2 and 3, all patients who achieved AdV viremia clearance maintained undetectable AdV in the blood through the follow-up period. Conclusions: BCV IV was found to be safe and well tolerated in immunocompromised patients. Notably, the potentially serious gastrointestinal and hepatic toxicities described with the oral BCV formulation were not observed. BCV IV was highly effective in a dose-dependent manner, led to rapid clearance of AdV viremia within a short treatment period and provided sustained virologic response. In view of promising results and in the absence of any other approved treatments for AdV infection, our results support the need for further exploration and additional clinical trials of BCV IV as a treatment for AdV infection.

Predictive Value of BCR::ABL1 Transcripts Kinetics in the Molecular Relapse of Chronic Myeloid Leukemia Patients after Tyrosine Kinase Inhibitors Discontinuation: Results from the DES-CML Study

Duration of previous treatment, duration and depth of molecular response, previous resistance, BCR::ABL1 transcript, and lymphocyte subtypes are known predictive factors of molecular recurrence after tyrosine kinase inhibitors (TKI) discontinuation in chronic myeloid leukemia (CML) patients (pts). Few studies explored the kinetics of BCR::ABL1 during a dose de-escalation phase before discontinuation in predicting molecular relapse. This study aimed to evaluate the kinetics of BCR::ABL1 transcripts during the de-escalation phase of the DES-CML trial and to correlate with molecular relapse after TKI discontinuation. Methods: This prospective, multicenter, single-arm, phase 2 study included CML pts treated DES-CML Study. Inclusion criteria: Adult CML pts treated with TKI for a minimum of 3 years that achieved sustained deep molecular response (DMR), defined by a minimum duration of MR4.5 for two years, confirmed by four real-time quantitative reverse-transcription polymerase chain reaction (qPCR) tests with six months interval. Exclusion criteria: pts with atypical BCR::ABL1 transcripts, previous or current advanced stage CML, previous resistance to TKI or ABL mutations. The TKI dose was reduced by 50% for six months before discontinuation (de-escalation phase). Molecular monitoring was performed by qRT-PCR, using ABL as the control gene. The results were reported using the International Scale. BCR::ABL1 transcripts were evaluated monthly during the de-escalation phase. After discontinuation, assessments were performed monthly for six months, every two months at months 7-24, and then every three months. Therapy was restarted at the same dose before dose reduction if loss of MMR (qPCR&amp;lt;0.1%). Statistical analysis was performed by using SPSS software. Rates of MRFS were calculated by Kaplan-Meier analysis, and groups were compared using a log-rank test. P values of &amp;lt;.05 were considered statistically significant. This trial is registered at ReBEC (RBR-6f5xbq; UTN code: U1111-1252-7312) and was approved by the ethics committees of the participating centers. All pts signed informed consent. Results: This study is ongoing. Forty-one pts were enrolled from September 2020 to July 2023, with a median age of 61 years (23-85), 60% men, 33% Sokal low risk, 50% intermediate, 17% high; 78% with b3a2 BCR::ABL1 transcript type; 85% using imatinib, 7.5% dasatinib, 5% nilotinib, and 2.5% bosutinib. For 3 pts, it was the second attempt at stopping TKI. The median duration of TKI treatment until discontinuation was 11 years (3-20). The cutoff date was July 2023, with a median follow-up of six months (1-26) after TKI discontinuation. One patient had a screen failure; another was enrolled but has not started the de-escalation phase yet. Five patients are in the de-escalation phase, and 25 are in the discontinuation phase. Two patients did not complete the de-escalation phase due to a lack of medication and went to the discontinuation phase. One patient died from causes unrelated to CML in the discontinuation phase and was censored in the MRFS analysis. 15/33 (45%) patients lost MR4.5 at any time in the de-escalation phase, and 4/15 (31%) lost MMR after discontinuation. Three of the 18 patients who did not lose MR4.5 during de-escalation relapsed (16%, P=0.05). The two patients who did not undergo de-escalation were censored in this analysis. Nine pts lost MMR: one during de-escalation and 8/39 (20%) after discontinuation. The median time between discontinuation and loss of MMR was two months (0-10). The median time to MMR recovery was 1.5 months (1-5). The overall MRFS was 72% (95% CI: 56-88%)(Figure A) and 81% in the first-attempt discontinuation group. All pts in the second attempt relapsed (3 patients) (P&amp;lt;0.0001). There was no difference in MRFS by gender, BCR::ABL1 transcript type, Sokal, and ELTS. Molecular response status at discontinuation: 23 (74%) had MR4.5, 6 (19.5%) MR4.0, and 2 (6.5%) MMR. Patients with MR4.5 at discontinuation had a higher MRFS than those in MR4.0 and MMR (78% vs. 62% vs. 0%, respectively, P=0.012)(Figure B). No patient lost hematologic response or had disease progression. Conclusions: Loss of MR4.5 during the de-escalation phase or molecular status not in MR4.5 before discontinuation was predictive of subsequent loss of MMR. Treatment-free remission was not successful in the second attempt of discontinuation.

Pre-Emptive Use of Rituximab in Epstein-Barr Virus Reactivation: Incidence, Predictive Factors, Monitoring, and Outcomes.

Post-transplant lymphoproliferative disease (PTLD) is a fatal complication of hematopoietic cell transplantation (HCT) associated with the Epstein-Barr virus (EBV). Multiple factors such as transplant type, graft-versus-host disease (GVHD), human leukocyte antigens (HLA) mismatch, patient age, and T-lymphocyte-depleting treatments increase the risk of PTLD. EBV reactivation in hematopoietic cell transplant recipients is monitored through periodic quantitative polymerase chain reaction (Q-PCR) tests. However, substantial uncertainty persists regarding the clinically significant EBV levels for these patients. Guidelines recommend initiating EBV monitoring no later than four weeks post-HCT and conducting it weekly. Pre-emptive therapies, such as the reduction of immunosuppressive therapy and the administration of rituximab to treat EBV viral loads are also suggested. In this study, we investigated the occurrence of EBV-PTLD in 546 HCT recipients, focusing on the clinical manifestations and risk factors associated with the disease. We managed to identify 67,150 viral genomic copies/mL as the cutoff point for predicting PTLD, with 80% sensitivity and specificity. Among our cohort, only 1% of the patients presented PTLD. Anti-thymocyte globulin (ATG) and GVHD were independently associated with lower survival rates and higher treatment-related mortality. According to our findings, prophylactic measures including regular monitoring, pre-emptive therapy, and supportive treatment against infections can be effective in preventing EBV-related complications. This study also recommends conducting EBV monitoring at regular intervals, initiating pre-emptive therapy when viral load increases, and identifying factors that increase the risk of PTLD. Our study stresses the importance of frequent and careful follow-ups of post-transplant complications and early intervention in order to improve survival rates and reduce mortality.

Children's scabies survey indicates high prevalence and misdiagnosis in Auckland educational institutions.

Here, we present results of a survey of scabies prevalence in childcare centres and primary schools in Auckland. Children whose parents agreed to take part in participating centres in the Auckland region were examined for scabies by general practitioners and given questionnaires of relevant symptoms. Diagnoses of clinical or suspected scabies were made according to the International Alliance for the Control of Scabies (IACS) criteria. The survey was a stratified random sample of schools and early childcare centres. A quantitative polymerase chain reaction (PCR) test was also used to complement the IACS criteria. A total of 181 children were examined, with 145 children with history information, 16 of whom (11.0%) met the criteria for 'clinical' or 'suspected' scabies. Weighted analysis, accounting for the survey design, indicated that the prevalence of scabies in early childcare centres was 13.2% (95% CI: 4.3 to 22.1), with no school-aged children fulfilling these criteria. A higher proportion had clinical signs of scabies with 23 (12.7%) having typical scabies lesions and a further 43 (23.8%) had atypical lesions. A total of 64 PCR tests were taken and 15 (23%) were positive. None of these cases were receiving treatment for scabies. Five were undergoing topical skin treatment: three with topical steroid and two with calamine lotion. The prevalence of children with scabies is high in early childcare centres in Auckland. Misdiagnosis is suggested by several PCR positive cases being treated by topical agents used to treat other skin conditions.

Canine respiratory disease outbreak involving canine pneumovirus at an American animal shelter

AbstractAn outbreak of canine infectious respiratory disease complex was attributed to canine pneumovirus in a municipal animal shelter in the northeastern United States. The outbreak comprised eight fatalities and over 100 cases in dogs of all signalments. Cases were characterised by mild to severe signs indicating upper airway, lower airway, and systemic involvement. Nasopharyngeal samples submitted to one commercial and one university laboratory for quantitative polymerase chain reaction testing revealed several canine infectious respiratory disease complex pathogens. However, only the university lab detected canine pneumovirus: multiple nasopharyngeal and lung samples tested positive. Vaccination status, lack of response to antimicrobials, and improvement after implementation of canine infectious respiratory disease outbreak management strategies tailored to canine pneumovirus characteristics indicate substantial involvement of this virus. The outbreak resolved within 1 month of adherence to clean break procedures, limitation of incoming dogs, and deep clean of the premises. This case highlights the difficulty in reliably detecting canine pneumovirus and how population management deficits permit canine infectious respiratory disease complex outbreaks.

Validating Tools to Detect and Inactivate Monkeypox Virus in Human Milk.

Objectives: Breastfeeding and human milk (HM) improve maternal and infant morbidities and mortality. Therefore, monitoring the safety of breastfeeding and access to HM is of critical importance. In this study, we assessed tools to monitor the presence of monkeypox virus (MPXV) in HM and whether standard Holder pasteurization inactivates MPXV. Materials and Methods: Heat-inactivated MPXV was added to HM or viral transport media (VTM) and analyzed using both research and clinical MPXV quantitative polymerase chain reaction (qPCR) tests. Infectious MPXV was added to HM and was exposed to 1 cycle of freeze-thaw, incubation for 1 hour at room temperature, or conditions of Holder pasteurization (62.5°C for 30 minutes) followed by infectious unit quantification by plaque assay. Results: Research and clinical nucleic acid tests detect MPXV that was added to HM but with reduced sensitivity compared with equivalent samples in VTM at low virus inoculum. MPXV added to HM to achieve a starting concentration of 225,000 plaque forming units (pfu)/mL remains infectious after freeze-thaw or 1 hour storage at room temperature. However, Holder pasteurization reduced infectious virus below the limit of detection, >2,000-fold reduction in viral titer. Conclusion: MPXV can be detected when added to HM using a clinical laboratory-developed qPCR test without modification, but the detection limit is reduced compared with equivalent samples in VTM. MPXV remains viable in HM should the virus ever gain access to HM, but Holder pasteurization reduces infectious MPXV to below detection limits and can be used to reduce the risk of MPXV transmission to infants who receive pasteurized (donor) HM.

Molecular epidemiological analyses of Clostridioides difficile isolates in a university hospital in Japan

BackgroundWe performed molecular epidemiological analyses of Clostridioides difficile isolates in a university hospital in Japan to reveal the risk of C. difficile infection. MethodsCultured isolates from 919 stool samples from 869 patients obtained from July 2015 to August 2016 were subjected to toxin gene detection, ribotyping, multilocus sequence typing, antimicrobial susceptibility testing, and quantitative real-time polymerase chain reaction testing for C. difficile toxin gene expression. ResultsOf the 919 stool samples from 869 patients, C. difficile was isolated from 153 samples (16.6%), of which 49 (32%) and 104 (68%) were from patients with and without C. difficile infection, respectively. Analyses showed genetic diversity, with ST8 and ST17 strains of healthcare-associated infections, some of which caused C. difficile infections. There was no significant difference in the transcription levels of C. difficile toxin genes between isolates from patients with and without C. difficile infection. ConclusionsMajor Japanese clonal strains, ST8 and ST17, have been in the hospital environment for a long time and cause healthcare-associated C. difficile infections. The C. difficile toxin genes were transcribed in the isolates from both patients with and without C. difficile infection but were no significant relationship with the development of C. difficile infection.

Identifying Predominant Causes of Death Among Hospitalized COVID-19 Patients During Poland's Second and Third Waves.

BACKGROUND Number of confirmed COVID-19 deaths per million population in Poland between November 2020 and May 2021 was one of the largest in Europe. This retrospective study was conducted at a single center in Poland between November 2020 and May 2021to evaluate the morbidity and mortality rates in 581 patients hospitalized with COVID-19. MATERIAL AND METHODS A retrospective single-center study was conducted in a dedicated COVID-19 hospital from November, 2020 to May, 2021. The data of 581 hospitalized patients were analyzed. Multimorbidity was assessed using the Charlson Comorbidity Index, including chronic kidney, respiratory, cardiovascular diseases, diabetes mellitus, cancer, and dementia. The observation period covered admission to the hospital for severe COVID-19 until discharge or death. Diagnosis of COVID-19 was confirmed by quantitative reverse transcription polymerase chain reaction test. Statistical analysis was carried out in the IBM SPSS Statistics program. RESULTS The mortality rate was 35% of all admitted patients. Lung damage was the cause of death in 60%, bacterial superinfection in 26%, arterial thrombosis or thromboembolism in 9%, and heart failure in 5% of patients. The chi-square test showed a significant relationship between sex and the cause of death related to COVID-19 pneumonia and bacterial ventilator-associated pneumonia (VAP). CONCLUSIONS The findings from this study supports findings from other countries that between November 2020 and May 2021, before SARS-CoV-2 vaccination programs were fully implemented and before effective medications and antiviral agents were developed, patients with severe COVID-19 had high rates of morbidity and mortality.

SaeR as a novel target for antivirulence therapy against Staphylococcus aureus

ABSTRACT Staphylococcus aureus is a major human pathogen responsible for a wide range of clinical infections. SaeRS is one of the two-component systems in S. aureus that modulate multiple virulence factors. Although SaeR is required for S. aureus to develop an infection, inhibitors have not been reported. Using an in vivo knockdown method, we demonstrated that SaeR is targetable for the discovery of antivirulence agent. HR3744 was discovered through a high-throughput screening utilizing a GFP-Lux dual reporter system driven by saeP1 promoter. The antivirulence efficacy of HR3744 was tested using Western blot, Quantitative Polymerase Chain Reaction, leucotoxicity, and haemolysis tests. In electrophoresis mobility shift assay, HR3744 inhibited SaeR-DNA probe binding. WaterLOGSY-NMR test showed HR3744 directly interacted with SaeR’s DNA-binding domain. When SaeR was deleted, HR3744 lost its antivirulence property, validating the target specificity. Virtual docking and mutagenesis were used to confirm the target’s specificity. When Glu159 was changed to Asn, the bacteria developed resistance to HR3744. A structure–activity relationship study revealed that a molecule with a slight modification did not inhibit SaeR, indicating the selectivity of HR3744. Interestingly, we found that SAV13, an analogue of HR3744, was four times more potent than HR3744 and demonstrated identical antivirulence properties and target specificity. In a mouse bacteraemia model, both HR3744 and SAV13 exhibited in vivo effectiveness. Collectively, we identified the first SaeR inhibitor, which exhibited in vitro and in vivo antivirulence properties, and proved that SaeR could be a novel target for developing antivirulence drugs against S. aureus infections.

Metagenomic next-generation sequencing assistance in identifying non-tuberculous mycobacterial infections.

The advent of metagenomics next-generation sequencing (mNGS) has garnered attention as a novel method for detecting pathogenic infections, including Non-Tuberculous Mycobacterial (NTM) and tuberculosis (TB).However, the robustness and specificity of mNGS in NTM diagnostics have not been fully explored. In this retrospective study, we enrolled 27 patients with NTM genomic sequences via mNGS and conducted a comprehensive clinical evaluation. Pulmonary NTM disease was the most commonly observed presentation, with a subset of patients also presenting with extrapulmonary NTM infections.mNGS analysis identified six distinct NTM species, primarily Mycobacteriumavium complex (MAC), followed by Mycobacterium intracellulare andMycobacterium abscessus. Conventional routine culture methods encountered challenges, resulting in negative results for all available 22 samples. Among the 10 patients who underwent quantitative polymerase chain reaction (qPCR) testing, five tested positive for NTM. It is important to note that further species typing is necessary to determine the specific NTM type, as traditional pathogen detection methods serve as an initial step. In contrast, when supplemented with pathogen data, enables the identification of specific species, facilitating precise treatment decisions. In conclusion, mNGS demonstrates significant potential in aidingthe diagnosis of NTMdisease by rapidly detecting NTM pathogens and guiding treatment strategies. Its enhanced performance, faster turnaround time (TAT), and species identification capabilities make mNGS a promising tool for managing NTM infections.