Real-time PCR Test Research Articles

Comparison of RT.PCR and ELISA Methods in the Diagnosis of Human Cytomegalovirus in Kidney Transplant Patients

Background: In kidney transplant recipients prone to infections like cytomegalovirus (CMV), a vital need for an accurate diagnostic method is evident. This study at Khorshid Laboratory in Tehran rigorously compares real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) for CMV detection in transplant patients. Methods: In January to March 1400, 70 kidney transplant recipients were assessed for CMV DNA using RT-PCR and concurrent ELISA tests, with statistical analysis aided by SPSS. Results: In kidney transplant patients (average age: 49.40 ± 13.64 years), 4.3% tested positive for CMV via PCR. Strong correlation between serological and molecular methods. IgG and IgM antibody detection both showed high sensitivity and specificity, advocating for efficient CMV diagnosis at Khorshid Laboratory, Tehran. Conclusion: This study emphasizes the need for a quick and efficient CMV diagnostic approach in kidney transplant patients. The strong correlation between molecular and serological methods favors using the faster RT-PCR method, crucial for timely management, especially with the increasing age- related CMV incidence. The findings strongly recommend RT-PCR integration for enhanced sensitivity in kidney transplant patients at Khorshid Laboratory, Tehran.

Inter-Laboratory Comparison of qPCR Assays for Piscirickettsia salmonis in Atlantic Salmon (Salmo salar L.) in 11 Chilean Laboratories.

Real-time PCR (qPCR) testing is an essential component of early detection surveillance systems for Piscirickettsia salmonis infection in Atlantic salmon farms in Chile. Currently, all 11 laboratories in the authorised diagnostic laboratory network use assays based on published protocols. Compared with other P. salmonis qPCR assays, these assays have the advantage of targeting two different genes, that is, the 16S ribosomal gene and the internal transcribed spacer (ITS), potentially allowing for higher diagnostic accuracy. However, variation and lack of harmonisation of qPCR testing systems (e.g., primers/probe, RNA/DNA as target, extraction methods, etc.) may contribute to among-laboratory variation in qPCR results and an increased frequency of false-negative and false-positive results. The purpose of the ring trial reported herein was to compare qPCR results from 11 laboratories in Chile routinely testing Atlantic salmon for P. salmonis as part of a national control program. The panel of 14 samples included duplicates of three concentrations of P. salmonis in a homogenised head kidney, LF89 and EM90 bacteria and two negative controls (blank and a suspension of Flavobacterium psychrophilum). The sample order was randomised across labs, samples were tested blinded and analysed without knowledge of the source lab. Of the laboratories, 8 (72.7%) had at least one incorrect result out of 14 tested samples. Low-concentration samples (Ct of about 30) were more often incorrectly classified by reverse transcription-qPCR (RT-qPCR) (3/6 labs) than by qPCR (0/5). Six (54.5%) labs had at least one false-positive result indicating that cross-contamination was likely during sample processing. Affected laboratories are advised to conduct internal investigations to confirm the causes of false-positive results and recommendations for design and implementation of future ring trials are discussed.

Molecular detection and sequencing of beet necrotic yellow vein virus and beet cryptic virus 2 in sugar beet from Kazakhstan

IntroductionBeet necrotic yellow vein virus (BNYVV) is a common viral pathogen that causes considerable economic loss globally. In the present study, a commercial realtime PCR test system and custom loop mediated amplification primers were used to detect the virus in asymptomatic sugar beet samples.MethodsA total of 107 of 124 samples tested positive for the presence of the A type BNYVV coat protein gene. Near complete sequences of RNA-3 and RNA-4 were obtained using reverse transcription, followed by nanopore sequencing of 14 samples.Results and discussionA comparison with available sequences, including previously published isolates Kas2 and Kas3 from Kazakhstan, identified RNA-3 as similar to such of the P-type isolates Puthiviers and Kas3. RNA-5 was not detected using real-time PCR or cDNA amplification. Unique variable sites were identified in the p25 protein sequence translated from RNA-3. Another virus, beet cryptic virus 2 (BCV2), was identified and sequenced in samples infected with BNYVV. With 85.28% genome coverage, the identified BCV2 samples were very similar to the previously reported isolates from Hungary and Germany

Detection of equine parvovirus-hepatitis and efficacy of governmental regulation for equine biologics purity.

In 2018, a new virus, named equine parvovirus-hepatitis (EqPV-H), was discovered in a biologic product that had been administered to horses that subsequently developed clinical signs of equine serum hepatitis (Theiler disease). Further correlation of the virus with the disease sparked federal requirements that all equine biologics be free of EqPV-H. The initial quantitative real-time PCR (qPCR) test for EqPV-H has proved to be sensitive to co-extracted PCR inhibitors in template nucleic acids, causing false-negative results. We investigated the use of digital PCR (dPCR) as a more robust test. Examination of 227 equine biologic product lots available for purchase both before and after regulatory implementation using both detection methods indicated that dPCR is a more reliable platform. Nevertheless, use of the qPCR method for product screening had reduced the fraction of serials with EqPV-H detected from 39.6% prior to regulation to 6.8% after regulatory implementation. Adoption of dPCR testing is an opportunity to further decrease the prevalence of EqPV-H in equine biologics.

A New Real-Time PCR Test (Flora Select™) and Nugent Score for the Diagnosis of Bacterial Vaginosis During Pregnancy.

This prospective cohort study aimed to evaluate the performance of Flora select™ (FS), a newly developed real-time PCR test, for the assessment of the vaginal microbiome during early pregnancy. Five hundred and fifty-six pregnant women underwent examinations of FS, Nugent score-a Gram-staining scoring system for the diagnosis of bacterial vaginosis (BV)-and conventional bacterial culture between 8 weeks and 12 gestational weeks. Nugent scores of 0-3, 4-6, and ≥7 were found in 469 (84.2%), 41 (7.4%), and 47 (8.5%) of the women, respectively. Relative dominance rates of Lactobacillus species of high (≥80% medium (50%≤, <80%), and low (0.1≤, <50%), and no detection (<0.1%) were 63.0%, 8.8%, 17.1%, and 11.2%, respectively. Gardnerella, Prevotella, Atopobium, Streptococcus, Ureaplasma, and Mycoplasma species were detected in 23.9%, 17.6%, 17.1%, 7.0%, 23.0%, and 4.9% of the women, respectively. Gardnerella species were detected in all women with Nugent scores ≥7 and Ureaplasma were detected in 40.4% of them. BV-associated bacterial species were also detected in 70.7% of women with Nugent scores of 4-6. Gardnerella, Prevotella, Atopobium, Streptococcus, Ureaplasma, and Mycoplasma species were highly prevalent in women with Nugent scores ≥4 or Lactobacillus species <50%. FS detected Gardnerella, Prevotella, and Atopobium species more effectively than conventional bacterial culture. FS could determine relative dominance rates of Lactobacillus species in the vaginal microbiome, and simultaneously detect four kinds of BV-associated bacteria, Ureaplasma and Mycoplasma species. Therefore, FS may be clinically useful for the screening of the vaginal microbiome during pregnancy to prevent preterm birth and for the assessment of the vaginal microbiome after BV treatments.

Serological and molecular analysis of Leishmania infection in a recent outbreak of visceral leishmaniasis in South Omo Zone, Ethiopia.

Ethiopia has a high burden of visceral leishmaniasis. Recently, there was a significant increase in cases in the South Omo Zone. This study aims to assess the prevalence of Leishmania donovani infection and its associated factors. A household-based cross-sectional study was carried out in January 2023 in the South Omo Zone in Ethiopia. Dried blood spot samples were collected from 382 randomly selected study participants. Direct agglutination test (DAT) and kinetoplast DNA real-time PCR tests were performed to detect L. donovani infection. Participants' sociodemographic, clinical and risk factors for L. donovani infection data were collected using questionnaires. Bivariate and multivariate logistic regressions were used to analyze the data. Febrile cases were checked for malaria with a multiplex PCR assay. Overall prevalence of L. donovani infection among the sampled population was 32.5% (n=124), of which 41.1% (n=51) was detected by PCR, 33.9% (n=42) by DAT and 25.0% (n=31) by both tests. The majority of the positives were from the Logira (28.2%; n=35) and Dilbayne (29.0%; n=36) villages. Participants residing in Logira (adjusted OR [AOR]: 5.80; 95% CI 1.85 to 18.15) and Dilbayne (AOR: 3.38; 95% CI 1.15 to 9.96) villages and owning cows (AOR: 2.31; 95% CI 1.03 to 5.15) showed an association with Leishmania infection. Plasmodium falciparum was detected in 3.4% (n=2) of 59 febrile participants. The prevalence of L. donovani infection in the South Omo Zone is high. Further research on the role of cows in the transmission cycle is needed to design the best strategy to control Leishmania infection in the South Omo Zone. Such interventions should focus on the Logira and Dilbayne villages, where most of the infections were identified.

Rapid detection of SARS-CoV-2 variants by molecular-clamping technology-based RT-qPCR.

We have developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) testing platform for the rapid detection of SARS-CoV-2 variants using the xenonucleic acid (XNA)-based molecular-clamping technology. The XNA-based RT-qPCR assay can achieve high sensitivity with a limit of detection of about 100 copies/mL for variant detection which is much better than the next-generation sequencing (NGS) assay. Its turnaround time is about 4 hours with lower cost and a lot of Clinical Laboratory Improvement Amendments (CLIA) labs own the instrument and meet skillset requirements. This assay provides a rapid, reliable, and cost-effective testing platform for rapid detection and monitoring of known and emerging SARS-CoV-2 variants. This testing platform can be adopted by laboratories that perform routine SARS-CoV-2 PCR testing, providing a rapid and cost-effective method in lieu of NGS-based assays, for detecting, differentiating, and monitoring SARS-CoV-2 variants. This assay is easily scalable to any new variant(s) should it emerge.

B-305 High-risk HPV Scenario Among Young People: Data From a Clinical Laboratory (2019-2023)

Abstract Background Human papillomavirus (HPV) infection is the most common sexually transmitted infections and occurs early after the start of sexual life. There are more than 150 types of HPV, classified considering the affected area, symptoms, and oncogenic potential. The types of HPV that cause cancerous lesions are classified as high-risk HPV types. HPV is the main cause of cervical cancer in women and is strongly associated with penile cancer in men. Despite the existence of vaccination, it is still not easily accessible to the entire population of Brazil. In this context, the authors are periodically analyzing the positivity of high-risk HPV infection in secretion and cell scraping samples. Methods 14,806 results were analyzed from a laboratory in São Paulo/Brazil. 4,935 real-time PCR tests were carried out from January/2019 to December/2023 for the detection of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 high risk types. In this study, there was a special focus on HPV types 16 and 18, as they are the main high-risk types for cervical cancer. The other high-risk genotypes were detected, but not differentiated. Results The most ages affected by the infection in both sexes were 25 to 40 years old. Infections with other high-risk types HPV were more frequent in relation to types 16 and 18, Table 1. Conclusions Although the male population represented only 1.19% of processes tests, a higher frequency of HPV positive results was detected (9.04%) compared to female patients (7.55%).

A-264 Validation of a Novel Real-Time PCR Assay for Identification of Mycobacterium species Isolates Recovered from Clinical Culture

Abstract Background Historically, clinical mycobacteriology laboratories used the nucleic acid hybridization-based AccuProbe assay manufactured by Hologic, Inc., to rapidly rule in or rule out Mycobacterium tuberculosis (MTB) and members of the M. avium-intracellulare complex (MAC). The clinical value of this test was in its ability to rapidly rule in/out MTB from positive broth cultures and allow the initiation of appropriate antimicrobial therapy and infection prevention practices. Recent discontinuation of this commercial assay left a gap in testing available for the rapid identification of these organisms, which allowed us to develop an in-house real-time PCR test. Methods A replacement assay was developed by Labcorp R &amp; D based on real-time PCR amplification of extracted nucleic acids from positive culture growth. Aliquots from positive AFB VersaTREK broth cultures were heat inactivated, and nucleic acids were extracted using the MagNA Pure 24 (MP24) instrument (Roche). Real-time PCR using published primer probe sets (Rocchetti, et al 2016) was performed on the QuantStudio™ 7 (QS7) instrument (Applied Biosystems®). The assay was designed with three independent primer/probe sequence targets: one for MTB Complex (IS6110), one for MAC (ITS), and one pan-target for the Mycobacterium genus (16s rRNA). Results Procedural verification was performed using 100 residual clinical specimens previously identified using the Hologic AccuProbe® assay. Categorical concordance was 97% (97/100). The three discrepant results were identified as MAC positive by PCR; however, these were not identified as MAC on the predicate assay. Analytical sensitivity for the PCR assay was established to be 1,000 CFU/mL for each target. Analytical specificity was assessed in vitro for nine Mycobacterium species, with no cross-reactivity observed. In vitro analysis of target detection in the presence of yeast (Candida glabrata) was also performed, and we saw no inhibition. Analysis of the primer/probe sets for all targets was performed in silico using XploreDB database of microbial genomes, with confirmed 100% matches for both MAC and MTB genomes compared to hundreds of species. By comparison, the PAN-Myco primer set showed decreased performance, having three mismatches (two on forward primer and one on the probe). As this target sequence is used as an Internal Control (IC) this was not problematic for overall assay performance. The assay demonstrated excellent precision (%CV) for each target: MTB = 0.74%, MAC = 1.10%, PAN-Myco = 3.19%. Conclusions Utilizing the depth of molecular methodologies available in our laboratories, we developed a rapid method for identification of MAC and MTB from positive broth cultures, which had excellent performance compared to the predicate assay. The PCR ID LDT test is now available and allows testing to continue with no interruption in patient care.

Evaluation of Antioxidant Effects of Coenzyme Q10 against Hyperglycemia-Mediated Oxidative Stress by Focusing on Nrf2/Keap1/HO-1 Signaling Pathway in the Liver of Diabetic Rats.

Hyperglycemia-induced oxidative stress can damage the liver and lead to diabetes complications. Coenzyme Q10 (CoQ-10) reduces diabetes-related oxidative stress. However, its molecular mechanisms are still unclear. This study aimed to examine CoQ-10's antioxidant capabilities against hyperglycemia-induced oxidative stress in the livers of diabetic rats, specifically targeting the Nrf2/Keap1/ARE signaling pathway. This study was conducted between 2020-2021 at Arak University of Medical Sciences. A total of 30 male adult Wistar rats (8 weeks old) weighing 220-250 g were randomly assigned to five groups (n=6 in each group): control healthy, sesame oil (CoQ-10 solvent), CoQ-10 (10 mg/Kg), diabetic, and diabetic+CoQ-10. Liver oxidative stress indicators, including malondialdehyde, catalase, glutathione peroxidase, and glutathione, were estimated using the spectrophotometry method. Nrf2, Keap1, HO-1, and NQO1 gene expressions were measured using real-time PCR tests in the liver tissue. All treatments were conducted for 6 weeks. Statistical analysis was performed using SPSS software. One-way ANOVA followed by LSD's or Tukey's post hoc tests were used to compare the results of different groups. P<0.05 was considered statistically significant. The findings showed that induction of diabetes significantly increased Keap1 expression (2.1±0.9 folds, P=0.01), and significantly inhibited the mRNA expression of Nrf2 (0.38±0.2 folds, P=0.009), HO-1 (0.27±0.1 folds, P=0.02), and NQO1 (0.26±0.1 folds P=0.01), compared with the healthy group. In the diabetic group, the activity of glutathione peroxidase, catalase enzymes, and glutathione levels was decreased with an increase in malondialdehyde level. CoQ-10 supplementation significantly up-regulated the expressions of Nrf2 (0.85±0.3, P=0.04), HO-1 (0.94±0.2, P=0.04), NQO1 (0.88±0.5, P=0.03) genes, and inhibited Keap1 expression (1.1±0.6, P=0.02). Furthermore, as compared to control diabetic rats, CoQ-10 ameliorated oxidative stress by decreasing malondialdehyde levels and increasing catalase, glutathione peroxidase activities, and glutathione levels in the liver tissues of the treated rats in the treatment group. The findings of this study revealed that CoQ-10 could increase the antioxidant capacity of the liver tissue in diabetic rats by modulating the Nrf2/Keap1/HO-1/NQO1 signaling pathway.

Assessment of real-time PCR test in oral fluid samples for screening the serotypes of Actinobacillus pleuropneumoniae circulating in swine herds

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, causing remarkable economic losses in the global swine industry. The diversity of A. pleuropneumoniae is generally determined through serotype identification, which is commonly employed for control strategies and surveillance. However, serological methods currently in use still have significant limitations. This study explores the use of real-time polymerase chain reaction (qPCR) to detect circulating serotypes of A. pleuropneumoniae in non-diseased swine herds through testing of oral fluids.The study included three A. pleuropneumoniae-positive and three A. pleuropneumoniae-negative farms located in Quebec, Canada. Tonsil brushings, microbiological growths, and oral fluids were analyzed using qPCR to detect A. pleuropneumoniae and its distinct serotypes. Serological tests were performed using the LPS ELISA available at that time. In negative farms the absence of A. pleuropneumoniae and any serotype confirmed the specificity of the method. Positive farms, on the other hand, confirmed also the sensitivity of the analysis, with oral fluid samples consistently yielding positive results for the serotypes identified by ELISA.The qPCR test conducted on oral fluids offers a noninvasive and cost-effective method for monitoring, complementing traditional serological techniques. It provides qualitative information about serotype distribution, facilitating proactive surveillance and control strategies.

The Impact of Wharton's Jelly-derived Exosomes on the Production of Inflammatory Mediators from HIG-82 Synoviocytes.

Osteoarthritis (OA) is the most common joint disease worldwide. Routine treatment options are limited, and total knee replacement surgeries often come with complications. In recent years, the use of biologics, such as Wharton's jelly (Wj) derived from the umbilical cord (UC), has gained popularity. While mesenchymal stem cells (MSCs) derived from Wj show promise in restoring articular cartilage, they also have some limitations. Recent studies have indicated that exosomes isolated from acellular Wj may offer advantages under certain conditions. To investigate the anti-inflammatory properties of exosomes isolated from Wj in synoviocytes. Decellularization of Wj was performed using sterile umbilical cords obtained from patients. Next, the exosomes were isolated from Wj using ultracentrifugation. After characterizing the exosomes, they were co-cultured with inflammatory synovial fibroblast cells (HIG-82) for 24 hours. Then, the gene expression levels and protein contents of some important inflammatory mediators including metalloproteinase-13 (MMP-13), cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) were measured in the cells using real-time PCR and ELISA tests, respectively. The expression levels of MMP-13, COX-2, and iNOS genes were significantly reduced in the cultured cells treated with exosomes compared to untreated cells. Moreover, the content of MMP-13, COX-2, and iNOS proteins were significantly lower in the supernatant of the cultured cells compared to the control. Wj-derived exosomes exhibit notable anti-inflammatory properties, which can help mitigate inflammation in the synovial environment of joints. However, further research is required to fully understand their benefits and potential applications in treating osteoarthritis.

Evaluation of molecular and bacteriological detection methods performed on the formalin-fixed paraffin-embedded biopsy samples collected from endometrial and lymph node tuberculosis suspected patients

BackgroundEndometrial Tuberculosis is one of the most common gynecological problems known to have serious implications for the quality of life like infertility. The commonly practiced histopathology solely relies on the suggestive feature of Tuberculosis (TB) with low specificity. Regarding the alternative bacteriological and molecular detection tools, little evidence was generated on their utility in the diagnosis of endometrial tuberculosis in Ethiopia. Therefore, we aim to investigate the detection rate of molecular and bacteriological detection methods on formalin-fixed paraffin-embedded biopsy samples for the diagnosis of endometrial and lymph node TB.MethodsA retrospective cross-sectional study was conducted on 90 formalin fixed paraffin embedded biopsy samples from patients with gynecologic and lymph problems collected between 2018 and 2022 at St. Paul’s Hospital Millennium Medical College, Addis Ababa, Ethiopia. SPSS version 26 was used for statistical analysis. The diagnostic performance was calculated using the histopathology method as the reference standard. Cohen’s Kappa value was used to measure the level of agreement. A test with a P-value of < 0.05 was considered statistically significant.ResultsA total of 90 samples were analyzed in the current study. Auramine O, GeneXpert MTB/RIF assay, and Real-Time PCR tests have shown a detection rate of 32/90 (36%), 43/90 (47.8%), and 54/90 (60%) respectively (P ≤ 0.01). The sensitivity and specificity of AO were 38.1% and 95% respectively. RT PCR showed superior sensitivity followed by GeneXpert MTB/RIF assay, 70% and 58.6%. AO and molecular methods have shown a similarly low level of agreement with histopathology (Kappa value = 0.2).ConclusionsIn a resource-limited setting, the selection of diagnostic tools needs careful attention. Putting the patients on anti-TB treatments based solely on histopathological findings may lead to undesired and adverse complications. Therefore, applying molecular and bacteriological detection methods along with histopathology, could help minimize inappropriate antimicrobial use.

A droplet digital PCR assay to detect Chinese rice-field eels rhabdovirus.

Chinese rice-field eels rhabdovirus (CrERV) causes haemorrhagic disease in Chinese rice-field eels (Monopterus albus), leading to significant mortality and economic losses. Sensitive detection of CrERV nucleic acids is essential to control the spread of this pathogen and to treat infected individuals. Herein, we developed an efficient and sensitive droplet digital PCR (ddPCR) method to rapidly detect and quantify CrERV. The ddPCR assay optimal conditions were an annealing temperature of 53°C, and primer and probe concentrations of 0.5 and 0.25 μM, respectively. The assay had a diagnostic sensitivity of 0.23 copies/μL, and was highly specific, showing no cross reactivity with other viruses (infectious haematopoietic necrosis virus, grass carp reovirus, spring viremia of carp virus, largemouth bass ranavirus, carp edema virus, Chinese giant salamander iridovirus, and white spot syndrome virus). Real-time quantitative PCR testing of 30 Chinese rice-field eels samples detected CrERV in 7 samples (23.3%), whereas ddPCR detected CrERV in 12 samples (40%), demonstrating its higher sensitivity. Thus, ddPCR represents an advanced method to absolutely quantify CrERV in infected fish with low virus concentrations, providing a valuable tool to manage the spread and impact of CrERV.

Promoting molecular diagnostic equity: assessing in-house real-time PCR for Neisseria gonorrhoeae in anal samples from MSM recruited in an outpatient setting in Morocco.

Gonorrhea is a prevalent sexually transmitted infection among men who have sex with men (MSM). In Morocco, the basic laboratory diagnosis of Neisseria gonorrhoeae (NG) is based on microscopy and, in some settings, on culture. However, no nucleic acid amplification test (NAAT) has been implemented for routine diagnosis of gonorrhoeae.The aim of this study is to assess the effectiveness of an in-house real-time PCR test for detecting N. gonorrhoeae DNA in anal swabs samples collected during an Integrated Behavioral and Biological survey. Samples from 245 MSM, recruited using a Respondent Driven Sampling, were collected and tested for NG infection using GeneXpert CT/NG assay (Cepheid, USA). An In-House real-time PCR technique targeting the pseudo gene porA was developed and used for a parallel investigation of the same infection. The reliability of the in-house RT-PCR was validated through tests of reproducibility, repeatability, limit of detection, and cross-reactivity with other bacteria. The intrinsic performance characteristics of the qRT-PCR were assessed, namely, the sensitivity, the specificity, the positive predictive value (PPV), and the negative predictive value (NPV). The GeneXpert CT/NG assay was adopted as a reference method. For N. gonorrhoeae detection, the in-house real-time PCR assay showed a sensitivity and specificity of 80% and 100%, respectively. The PPV of the assay was 100% and the NPV was 97.3%. The in-house real-time PCR assay has high specificity and sensitivity, and it emerges as a promising approach for detecting N. gonorrhoeae in clinical specimens, particularly in decentralized settings such as regional laboratories.

Early biological markers of post-acute sequelae of SARS-CoV-2 infection

To understand the roles of acute-phase viral dynamics and host immune responses in post-acute sequelae of SARS-CoV-2 infection (PASC), we enrolled 136 participants within 5 days of their first positive SARS-CoV-2 real-time PCR test. Participants self-collected up to 21 nasal specimens within the first 28 days post-symptom onset; interviewer-administered questionnaires and blood samples were collected at enrollment, days 9, 14, 21, 28, and month 4 and 8 post-symptom onset. Defining PASC as the presence of any COVID-associated symptom at their 4-month visit, we compared viral markers (quantity and duration of nasal viral RNA load, infectious viral load, and plasma N-antigen level) and host immune markers (IL-6, IL-10, TNF-α, IFN-α, IFN-γ, MCP, IP-10, and Spike IgG) over the acute period. Compared to those who fully recovered, those reporting PASC demonstrated significantly higher maximum levels of SARS-CoV-2 RNA and N-antigen, burden of RNA and infectious viral shedding, and lower Spike-specific IgG levels within 9 days post-illness onset. No significant differences were identified among a panel of host immune markers. Our results suggest early viral dynamics and the associated host immune responses play a role in the pathogenesis of PASC, highlighting the importance of understanding early biological markers in the natural history of PASC.

First report of Erwinia pyrifoliae causing flower blight and fruit rot on greenhouse-grown Fragaria × ananassa in the United States.

Erwinia pyrifoliae causes disease of pear (Pyrus spp.), apple (Malus spp.), and strawberry (Fragaria × ananassa) (Wenneker and Bergsma-Vlami 2015), which are economically important commodities in the US. Disease symptoms on pear and apple are indistinguishable from those caused by the non-quarantine fire blight pathogen, E. amylovora (Kim et al. 1999), which also causes disease on strawberries (Atanasova et al. 2005). Samples of greenhouse-grown strawberry 'Albion' from Ohio were submitted to the Purdue Plant and Pest Diagnostic Lab in December 2023. Fruits were stunted with brown lesions, while sepals and pedicels had brown-black water-soaked lesions. Cut fruit exuded bacterial ooze from the main vascular bundle. Bacterial streaming was observed microscopically from symptomatic tissue which tested positive with the E. amylovora ImmunoStrip® (Agdia Inc., Elkhart, IN); reported by the manufacturer to cross-react with E. pyrifoliae. Isolation from symptomatic tissue produced pure cultures on sucrose peptone agar and Kings medium B after incubation at 27°C for 48 hr, and colonies appeared circular and white/opaque. Crude DNA extractions were prepared by boiling colony suspensions in Tris-EDTA buffer. Two independent real-time PCR tests specific for E. pyrifoliae (Lehman et al. 2008; Yasuhara-Bell et al. 2024) produced positive results. Conventional PCR using an E. pyrifoliae-specific primer set targeting a divergent region between pstS and glmS genes (Wensing et al. 2011) also produced positive results. The amplicon was Sanger-sequenced and deposited into NCBI GenBank (Accession PP757383). BLASTn analysis using the Nucleotide collection and Whole-genome shotgun contigs revealed top matches (100% query coverage; 97.5% identity to type strain DSM 12163) with E. pyrifoliae only; next closest match was E. amylovora (53% query coverage). To confirm Koch's postulates, immature fruit of six healthy strawberry 'Albion' plants were wounded with a sterile pipette tip and then submersed in a bacterial suspension in sterile deionized water (DI H2O) (3.1×107 cells/ml). Fruit of six additional plants were mock inoculated using sterile DI H2O. Plants were placed in plastic bags for 48 hr at room temperature with a 12-hr photoperiod. Symptoms were first observed on inoculated plants 1.5 days post-inoculation (DPI). Brown discoloration was observed within fruit and as spreading lesions on fruit pedicels by 4 DPI; mock-inoculated plants remained asymptomatic. Bacterial streaming from symptomatic tissue allowed successful re-isolation of the bacterium. Molecular testing confirmed isolates to be E. pyrifoliae, thus completing Koch's postulates. Following initial confirmation, additional samples of infected strawberry ('Albion' and 'GB96') from the same greenhouse were confirmed positive for E. pyrifoliae by molecular testing and sequencing. To our knowledge this is the first time Erwinia pyrifoliae was detected in the US. There are many known pathways of introduction from Asia and Europe; however, pstS-glmS sequence comparison with strawberry isolates from the Netherlands (sequences provided by M.J.C. Pel) suggests this US strawberry strain is unique, but most closely related to Japanese strains (98.5% identity). Potential origin of this strain is unknown, but comparative genomics studies to investigate relatedness among strains are planned.

Diagnostic evaluation of Tru-Nat MTB/Rif test in comparison with microscopy for diagnosis of pulmonary tuberculosis at tertiary care hospital of eastern Uttar Pradesh.

This study evaluated the efficacy of the TrueLab™ Real Time mini-PCR system in providing rapid and accurate diagnostic results for tuberculosis (TB) detection in India. The goal is to improve case detection and accelerate treatment in settings with limited resources. This prospective study was conducted by the Department of Microbiology on 120 patients, age ranging from >=15 years with at least two clinical symptoms of pulmonary TB. Molbio and Universal Cartridge Based Sample Prep were the 2 methods used for processing sputum samples. The diagnosis was based on the MTB Real Time PCR test, which has a detection limit of 100 CFU/mL. Patients under 15 years, samples lacking clinical background, saliva specimens or extra-pulmonary TB cases were excluded from the study. A total of 44.17% samples were positive for TB with maximum positivity in the age group 31-45 years. Positivity rate was found to be higher in females. In 4.17% of cases there was rifampicin resistance, which was significantly high in previously treated cases. Comparison of Truenat with Ziehl-Neelsen and fluorescent method revealed that it was more sensitive and less time consuming. Truenat MTB/RIF is a sensitive detection system for TB with rapid results, which serves as an important tool in the early management of tuberculosis patients and drug-resistant-TB cases.

Multiplex real-time PCR in non-invasive respiratory samples to reduce antibiotic use in community-acquired pneumonia: a randomised trial

We assessed whether multiplex real-time PCR plus conventional microbiological testing is safe and more effective than conventional microbiological testing alone for reducing antibiotic use in community-acquired pneumonia (CAP). In this randomised trial, we recruited adults hospitalised with CAP at four Spanish hospitals. Patients were randomly assigned (1:1) to undergo either multiplex real-time PCR in non-invasive respiratory samples plus conventional microbiological testing or conventional microbiological testing alone. The primary endpoint was antibiotic use measured by days of antibiotic therapy (DOT). Between February 20, 2020, and April 24, 2023, 242 patients were enrolled; 119 were randomly assigned to multiplex real-time PCR plus conventional microbiological testing and 123 to conventional microbiological testing alone. All but one of the patients allocated to multiplex real-time PCR plus conventional microbiological testing underwent PCR, which was performed in sputum samples in 77 patients (65.2%) and in nasopharyngeal swabs in 41 (34.7%). The median DOT was 10.04 (IQR 7.98, 12.94) in the multiplex PCR plus conventional microbiological testing group and 11.33 (IQR 8.15, 16.16) in the conventional microbiological testing alone group (difference −1.04; 95% CI, −2.42 to 0.17; p = 0.093). No differences were observed in adverse events and 30-day mortality. Our findings do not support the routine implementation of multiplex real-time PCR in the initial microbiological testing in hospitalised patients with CAP. Clinicaltrials.gov registration: NCT04158492.

Diagnosis with Metagenomic Next-Generation Sequencing (mNGS) technology and real-time PCR for SARS-CoV-2 Omicron detection using various nasopharyngeal swabs in SARS-CoV-2 Omicron.

The SARS-CoV-2 Omicron variant, with the main subtypes BA.5.2 and BF.7 in China, led to off-target effects on the S and N genes from December 1, 2022, to January 31, 2023. The kits used for studying and developing these agents were not adequately and independently evaluated. It is important to verify the performance of commercial Real-Time quantitative PCR (RT-qPCR) tests. We conducted a clinical evaluation of two Real Time SARS-CoV-2 Omicron assays to verify their performance using various detection reagents and clinical specimens. We performed clinical evaluations of two existing Chinese SARS-CoV-2 Omicron RT-qPCR kits 2019-nCoV nucleic acid diagnostic kits (Fosun Biotechnology, National instrument registration 20203400299, Shanghai, China) and COVID-19 nucleic acid detection kits (eDiagnosis Biomedicine, National instrument registration 20203400212, Wuhan, China) and using BSD (Bondson) (Guangzhou Bondson Biotechnology Co. Ltd, batch number 2022101), quality controls provided by the inspection center and a large number of clinically confirmed specimens. The concordance rates for the Fosun and eDiagnosis kits were 95% and 100%, respectively. The detection limit for the Fosun and eDiagnosis kits was verified to be 300 copies/mL and 500 copies/mL. The Fosun assay exhibited the largest coefficient of variation (CV) for ORF1ab and N gene at the detection limit concentration (4.80%, 3.49%), whereas eDiagnosis showed a smaller CV (0.93%, 1.10%). In the reference product from the Hangzhou Clinical Laboratory Center test, it was found that Fosun had the lowest sensitivity of 93.47% and a specificity of 100%, while eDiagnosis exhibited 100% for both sensitivity and specificity. The lowest single target gene detection rate of Fosun reagents was 68.7% for the ORF1ab gene and 87.5% for the N gene, while eDiagnosis detection rate was 100%. Among the clinical group S specimens, the missed detection rate of the Fosun reagent was 10.9%, which was higher than the 3.9% of eDiagnosis. However, there was no significant difference in the clinical diagnostic efficiency of the two reagents. The ORF1ab and N assays of SARS-CoV-2 Omicron on the eDiagnosis platform yielded higher values compared to those on the Fosun platform. Consequently, the eDiagnosis kit has also been used as standard detection reagents. Considering that the Fosun reagent has a relatively low detection limit and targets three single genes, it is more advantageous as a confirmatory reagent for the new museum.