A rapid and highly sensitive multiple detection of human adenovirus type 3, type 7 and respiratory syncytial virus by recombinase-aided reverse transcription PCR.

BACKGROUND: Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase-aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology. METHODS: In this study, we developed and validated the multiplex reverse transcription recombinase-aided PCR (multiplex RT-RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process. RESULTS: Using recombinant plasmids, the sensitivity of multiplex RT-RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT-RAP showed no cross-reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT-RAP and the results were found to be consistent with those of corresponding RT-qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT-RAP was two to eightfold higher than that of corresponding RT-qPCR. CONCLUSION: We conclude the multiplex RT-RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load.

Clinical influence of multiplex polymerase chain reaction routine uses in urgent pediatric admissions.

BACKGROUND: The coronavirus disease 2019 outbreak has prompted some hospitals to implement screening tests upon admission since 2020. FilmArray® Respiratory 2.1 Panel (FilmArray) is a multiplex polymerase chain reaction (PCR) test with high sensitivity and specificity for detecting respiratory pathogens. We aimed to assess the clinical influence of the routine use of FilmArray for pediatric patients, including those without symptoms suggestive of an infection. METHODS: We conducted a single-center retrospective observational study, which investigated patients aged ≤15 years who underwent FilmArray on admission in 2021. We collected the patients' epidemiological information, symptoms, and FilmArray results from their electronic health records. RESULTS: A positive result was observed in 58.6% of patients admitted to the general ward or intensive care unit (ICU) but only in 1.5% of patients in the neonatal ward. Among the patients admitted to the general ward or ICU who tested positive, 93.3% had symptoms suggestive of infections, 44.6% had a sick contact before admission, and 70.5% had siblings. However, 62 (28.2%) out of 220 patients without the four (fever, respiratory, gastrointestinal, and dermal) symptoms also had positive results. Among them, 18 patients with adenovirus and three with respiratory syncytial virus were isolated to private rooms. However, 12 (57.1%) patients were discharged without symptoms suggestive of viral infection. CONCLUSION: Multiplex PCR routine use for all inpatients may lead to excessive management of positive cases because FilmArray cannot quantify microorganisms. Thus, targets for testing should be considered carefully based on patients' symptoms and histories of sick contacts.

Viral loads of parainfluenza virus type 3 and severity of respiratory diseases in children.

BACKGROUND: Parainfluenza virus type 3 (PIV-3) is one of the common pathogens for respiratory infections in children. Whether viral load of PIV-3 is associated with severity of respiratory diseases in children is not yet known. Our aim was to determine significance of PIV-3 viral load among infected children. METHODS: We conducted a single-center, retrospective study at Tokyo Metropolitan. Children's Medical Center, Japan, from June to August 2021. Hospitalized children were screened with a posterior nasal swab for multiplex PCR, and viral load was subsequently measured from remained samples by real-time PCR. Demographic data were collected from digital charts. PIV-3 positive patients were categorized into mild group with no oxygen demand, moderate group with low-flow oxygen demand and severe group with high-flow nasal cannula oxygen or non-invasive positive pressure ventilation or mechanical ventilation. Viral loads were compared among mild, moderate and severe groups. RESULTS: 151 patients were positive for PIV-3. We found no statistically significant association among PIV-3 viral load and severity of respiratory diseases (p = 0.35), and no statistically significant association between severity of illness and co-detection of other viruses. In each severity group, relatively high viral load per posterior nasal swab was observed at the time of testing. CONCLUSION: Among PIV-3 patients, we could not find statistically significant between viral load and their severity, therefore we could not conclude that viral load is a good surrogate marker for clinical severity of PIV-3.

A novel anterior nasal swab to detect respiratory viruses: a prospective study of diagnostic accuracy.

Detection of respiratory viruses requires testing of the upper respiratory tract to obtain specimens for analysis. However, nasal and throat swabs can cause discomfort and procedural anxiety in children. Respiratory sampling methods which are accurate and less invasive are needed. We aim to determine the positive and negative percentage agreement of a novel anterior nasal swab (ANS) compared with the combined throat and anterior nasal swab (CTN), the reference standard, for detection of respiratory viruses. Children 5 - 18 years of age presenting to a tertiary paediatric hospital with respiratory symptoms were tested with both swabs in randomised order. Respiratory samples were tested on a multiplex RT-PCR panel. Viral detections, RT-PCR cycle-threshold values and child/parent/clinician experience of the swab were recorded. There were 157 viral detections from 249 participant CTN swabs. In comparison with the CTN, the overall positive and negative percentage agreement of ANS for detection of respiratory viruses was 96.2% (95% CI, 91.8-98.3%) and 99.8% (95% CI, 99.6-99.9%), respectively. The ANS was "extremely comfortable", or only a "little uncomfortable" for 90% of children compared with 48% for CTN. 202 children (84%) rated the ANS as the preferred swab, and 208 (87%) indicated they would prefer ANS for future testing. The ANS required additional laboratory handling processes compared to the CTN. The ANS has high positive percentage agreement and is comparable to the current standard of care. The high acceptability from the less invasive ANS provides a more comfortable method for respiratory virus testing in children.Trial registrationClinicalTrials.gov ID NCT05043623.

An end-point multiplex PCR/reverse transcription-PCR for detection of five agents of bovine neonatal diarrhea.

Neonatal calf diarrhea (NCD) is frequently associated with single or mixed viral, bacterial and/or protozoal infections. Consequently, laboratory diagnostic of NCD usually requires specific tests for each potential agent; a time-consuming, laborious and expensive process. Herein, we describe an end-point multiplex PCR/reverse transcription-PCR (RT-PCR) for detection of five major NCD agents: bovine rotavirus (BRV), bovine coronavirus (BCoV), Escherichia coli K99 (E. coli K99), Salmonella enterica (S. enterica) and Cryptosporidium parvum (C. parvum). Initially, we selected and/or designed high-coverage primers. Subsequently, we optimized multiplex PCR/RT-PCR conditions. Next, we evaluated the analytical sensitivity of the assay and assessed the performance of the reaction by testing 95 samples of diarrheic calf feces. The analytical specificity was evaluated against bovine viral diarrhea virus (BVDV), E. coli heat-stable enterotoxin (STa) and Eimeria spp. The detection limit of our assay was about 10 infectious units of BRV, 10-2 dilution of a BCoV positive sample pool, about 5 × 10-4 CFU for S. enterica, 5 × 10-6 CFU for E. coli K99 and 50 oocysts for C. parvum. No non-specific amplification of other bovine diarrhea agents was detected. Out of 95 samples analyzed, 50 were positive for at least one target, being 35 single and 15 mixed infections. BRV was the most frequent agent detected in single infections (16/35), followed by Cryptosporidium spp. (11/35), which was the most frequent in mixed infections (11/15). Positive and negative multiplex results were confirmed in individual reactions. In conclusion, we described an end-point multiplex PCR/RT-PCR for faster and easier NCD diagnosis, which may be useful for routine diagnosis and surveillance studies.

Simultaneous detection of SARS-CoV-2, influenza A, respiratory syncytial virus, and measles in wastewater by multiplex RT-qPCR.

A multiplex quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based method was designed for the simultaneous detection of influenza A, SARS-CoV-2, respiratory syncytial virus, and measles virus. The performance of the multiplex assay was compared to four monoplex assays for relative quantification using standard quantification curves. Results showed that the multiplex assay had comparable linearity and analytical sensitivity to the monoplex assays, and the quantification parameters of both assays demonstrated minimal differences. Viral reporting recommendations for the multiplex method were estimated based on the corresponding limit of quantification (LOQ) and the limit of detection at 95 % confidence interval (LOD) values for each viral target. The LOQ was determined by the lowest nominal RNA concentrations where %CV values were ≤35 %. Corresponding LOD values for each viral target were between 15 and 25 gene copies per reaction (GC/rxn), and LOQ values were within 10 to 15 GC/rxn. The detection performance of a new multiplex assay was validated in the field by collecting composite wastewater samples from a local treatment facility and passive samples from three sewer shed locations. Results indicated that the assay could accurately estimate viral loads from various sample types, with samples collected from passive samplers showing a greater range of detectable viral concentrations than composite wastewater samples. This suggests that the sensitivity of the multiplex method may be improved when paired with more sensitive sampling methods. Laboratory and field results demonstrate the robustness and sensitivity of the multiplex assay and its applicability to detect the relative abundance of four viral targets among wastewater samples. Conventional monoplex RT-qPCR assays are suitable for diagnosing viral infections. However, multiplex analysis using wastewater provides a fast and cost-effective way to monitor viral diseases in a population or environment.

Rapid multiplex PCR for respiratory viruses reduces time to result and improves clinical care: Results of a systematic review and meta-analysis.

OBJECTIVES: The clinical impact of rapid sample-to-answer "syndromic" multiplex polymerase chain reaction (PCR) testing for respiratory viruses is not clearly established. We performed a systematic literature review and meta-analysis to evaluate this impact for patients with possible acute respiratory tract infection in the hospital setting. METHODS: We searched EMBASE, MEDLINE, and Cochrane databases from 2012 to present and conference proceedings from 2021 for studies comparing clinical impact outcomes between multiplex PCR testing and standard testing. RESULTS: Twenty-seven studies with 17,321 patient encounters were included in this review. Rapid multiplex PCR testing was associated with a reduction of - 24.22 h (95% CI -28.70 to -19.74 h) in the time to results. Hospital length of stay was decreased by -0.82 days (95% CI -1.52 to -0.11 days). Among influenza positive patients, antivirals were more likely to be given (RR 1.25, 95% CI 1.06-1.48) and appropriate infection control facility use was more common with rapid multiplex PCR testing (RR 1.55, 95% CI 1.16-2.07). CONCLUSIONS: Our systematic review and meta-analysis demonstrates a reduction in time to results and length of stay for patients overall along with improvements in appropriate antiviral and infection control management among influenza-positive patients. This evidence supports the routine use of rapid sample-to-answer multiplex PCR testing for respiratory viruses in the hospital setting.

Relationship between Diagnostic Method and Pathogen Detection, Healthcare Resource Use, and Cost in U.S. Adult Outpatients Treated for Acute Infectious Gastroenteritis.

A retrospective observational study was performed to assess the relationship between diagnostic method (traditional work-up [TW], multiplex PCR panel with < 12 target pathogens [PCR < 12], or multiplex PCR panel with ≥ 12 target pathogens [PCR12]), and diagnostic yield, health care resource use (HRU), and cost in adult outpatients visiting U.S. hospitals for acute infectious gastroenteritis (AGE). Using data from PINC AI Healthcare Database during January 1, 2016-June 30, 2021, we analyzed adult patients with an AGE diagnosis and stool testing performed during an outpatient visit. Detection rates for different pathogens were analyzed for those with microbiology data available. Among 36,787 patients, TW was most often performed (57.0%). PCR12 testing was more frequent in patients from large, urban, and teaching hospitals, compared to TW (all P < 0.01). PCR12 was associated with a higher mean index visit cost (by $97) but lower mean 30-day AGE-related follow-up cost (by $117) than TW. Patients with PCR12 had a lower 30-day AGE-related hospitalization risk than TW (1.7% versus 2.7% P < 0.01). Among the 8,451 patients with microbiology data, PCR12 was associated with fewer stool tests per patient (mean 1.61 versus 1.26), faster turnaround time (mean 6.3 versus 25.7 h) and lower likelihood of receiving in-hospital antibiotics (39.4% versus 47.1%, all P < 0.01) than TW. A higher percentage of patients with PCR12 had a target pathogen detected (73.1%) compared to PCR < 12 (63.6%) or TW (45.4%, P < 0.01). Thus, we found that large multiplex PCR panels were associated with lower 30-day AGE-related follow-up cost and risk of AGE-related hospitalization, and increased diagnostic yield compared to TW.

Evaluation of BioFire Respiratory Panel 2 plus for Detection of Bordetella pertussis in Nasopharyngeal Swab Specimens from Children with Clinically Suspected Pertussis.

The objective of this study was to compare the performances of BioFire Respiratory Panel 2 (RP2) plus, quantitative real-time PCR (qPCR), and culture for the detection of Bordetella pertussis in nasopharyngeal swab (NPS) specimens. Consecutive NPS specimens were collected from patients with clinically suspected pertussis from 1 March 1 to 31 July 2018 in Shenzhen Children's Hospital. All the specimens were tested in parallel by RP2 plus, qPCR, and culture methods. A total of 464 children were enrolled in this study. The positive pertussis rates of culture, RP2 plus, and qPCR were 23.1%, 39.0%, and 38.4%, respectively. Compared to the combined reference standard, the sensitivity, specificity, positive predictive value, and negative predictive values were, respectively, 56.6% (95% confidence interval [CI], 49.2 to 63.7%), 100% (98.3 to 100%), 100% (95.7 to 100%), and 77.0% (72.2 to 81.2%) for culture, 89.9% (84.5 to 93.7%), 96.0% (92.8 to 97.9%), 93.9% (89.1 to 96.8%), and 93.3% (89.5 to 95.8%) for RP2 plus, and 86.8% (80.9 to 91.1%), 94.9% (91.4 to 97.1%), 92.1% (86.9 to 95.5%), and 91.3% (87.2 to 94.2%) for qPCR. The most prevalent codetected pathogen was human rhinovirus/enterovirus (n = 99, 52.4%), followed by parainfluenza virus (n =32, 16.9%) and respiratory syncytial virus (n = 29, 15.3%), in children with B. pertussis present, which was consistent with the top three pathogens previously found in children with B. pertussis absent. Turnaround times for RP2 plus, qPCR, and culture were 2 h, 8 h, and 120 h, respectively. RP2 plus quickly and accurately detected B. pertussis, providing valuable information for an early clinical diagnosis and optimal choice of therapy. IMPORTANCE In recent years, there have been some epidemic or local outbreaks of pertussis in countries with high vaccination rates. One of the crucial factors in controlling pertussis is early diagnosis, which is based on specific laboratory measurements, including culture, serological tests, and PCR assays. Compared to culture and serological tests, PCR is more suitable for clinical application, with a fast detection speed of several hours independent of the disease stage and individual vaccination status. BioFire Respiratory Panel 2 plus, a multiplex PCR assay for simultaneously detecting 22 respiratory pathogens, facilitates the quick detection of Bordetella pertussis and coinfecting respiratory pathogens. It also provides valuable information for an early clinical diagnosis and optimal choice of therapy for children with clinically suspected pertussis.

Characterization of viral pathogens associated with symptomatic upper respiratory tract infection in adults during a low COVID-19 transmission period.

Background: The epidemiology of respiratory tract infections (RTI) has dramatically changed over the course of the COVID-19 pandemic. A major effort in the clinical management of RTI has been directed toward diagnosing COVID-19, while the causes of other, common community RTI often remain enigmatic. To shed light on the etiological causes of RTI during a low COVID-19 transmission period in 2021, we did a pilot study using molecular testing for virologic causes of upper RTI among adults with respiratory symptoms from Almaty, Kazakhstan. Methods: Adults presenting at two public hospitals with respiratory symptoms were screened using SARS-CoV-2 PCR on nasopharyngeal swabs. A subset of RTI+, COVID-19-negative adults (n = 50) was then tested for the presence of common RTI viruses and influenza A virus (IAV). Next generation virome sequencing was used to further characterize the PCR-detected RTI pathogens. Results: Of 1,812 symptomatic adults, 21 (1.2%) tested SARS-CoV-2-positive. Within the COVID-19 negative outpatient subset, 33/50 subjects (66%) had a positive PCR result for a common community RTI virus, consisting of human parainfluenza virus 3-4 (hPIV 3-4) in 25/50 (50%), rhinovirus (hRV) in 2 (4%), hPIV4-hRV co-infection in four (8%) and adenovirus or the OCR43/HKU-1 coronavirus in two (4%) cases; no IAV was detected. Virome sequencing allowed to reconstruct sequences of most PCR-identified rhinoviruses and hPIV-3/human respirovirus-3. Conclusions: COVID-19 was cause to a low proportion of symptomatic RTI among adults. Among COVID-negative participants, symptomatic RTI was predominantly associated with hPIV and hRV. Therefore, respiratory viruses other than SARS-CoV-2 should be considered in the clinical management and prevention of adult RTI in the post-pandemic era.

A Multiplexed, Tiled PCR Method for Rapid Whole-Genome Sequencing of Infectious Spleen and Kidney Necrosis Virus (ISKNV) in Tilapia.

Tilapia farming is one of the most important sectors in aquaculture worldwide and of major importance to global food security. Infectious spleen and kidney necrosis virus (ISKNV) has been identified as an agent of high morbidity and mortality, threatening tilapia aquaculture. ISKNV was detected in Lake Volta, Ghana, in September 2018 and spread rapidly, with mortality rates between 60 and 90% and losses of more than 10 tonnes of fish per day. Understanding the spread and evolution of viral pathogens is important for control strategies. Here, we developed a tiled-PCR sequencing approach for the whole-genome sequencing of ISKNV, using long read sequencing to enable field-based, real-time genomic surveillance. This work represents the first use of tiled-PCR for whole genome recovery of viruses in aquaculture, with the longest genome target (>110 kb dsDNA) to date. Our protocol was applied to field samples collected from the ISKNV outbreaks from four intensive tilapia cage culture systems across Lake Volta, between October 2018 and May 2022. Despite the low mutation rate of dsDNA viruses, 20 single nucleotide polymorphisms accumulated during the sampling period. Droplet digital PCR identified a minimum requirement of template in a sample to recover 50% of an ISKNV genome at 275 femtograms (2410 viral templates per 5 µL sequencing reaction). Overall, tiled-PCR sequencing of ISKNV provides an informative tool to assist in disease control in aquaculture.

The epidemiological features of respiratory tract infection using the multiplex panels detection during COVID-19 pandemic in Shandong province, China.

Respiratory tract infection is one of the most common reasons for both morbidity and mortality worldwide. High attention has been paid to the etiological tracing of respiratory tract infection since the advent of COVID-19. In this study, we aimed to evaluate the epidemiological features of pathogens in respiratory tract infection, especially during COVID-19 pandemic. A total of 7668 patients with respiratory tract infection who admitted to Qilu Hospital of Shandong University from March 2019 to Dec 2021 were retrospectively included. The respiratory tract specimens were detected using a commercial multiplex PCR-based panel assay for common respiratory pathogens including influenza A virus (Flu-A), influenza A virus H1N1 (H1N1), influenza A virus H3N2 (H3N2), influenza B virus (Flu-B), parainfluenza virus (PIV), respiratory syncytial virus (RSV), adenovirus (ADV), Boca virus (Boca), human Rhinovirus (HRV), Metapneumovirus (MPV), Coronavirus (COV), Mycoplasma pneumoniae (MP), and Chlamydia (Ch). The positive rates were compared using a chi-square test. Compared with 2019, the positive rate of pathogen detection during from January 2020 to December 2021 was significantly lower, especially the detection of Flu-A. The positive rate of respiratory pathogen strains was 40.18% during COVID-19 pandemic, and a total of 297 cases (4.69%) of mixed infection with two or more pathogens were detected. There was no statistical difference in the positive rate between male and female patients. However, the positive rates of infection were different among different age groups, with higher incidence of RSV in infancy and toddler group, and MP infection in children and teenager group. While, HRV was the most common pathogen in the adult patients. Moreover, Flu-A and Flu-B were higher in winter, and MP and RSV were higher in spring, autumn and winter. The pathogens such as ADV, BOCA, PIV, and COV were detected without significant seasonal distribution. In conclusion, respiratory pathogen infection rates may vary by age and season, regardless of gender. During the COVID-19 epidemic, blocking transmission routes could help reduce the incidence of respiratory tract infection. The current prevalence of respiratory tract infection pathogens is of great significance for clinical prevention, diagnosis and treatment.

TaME-seq2: tagmentation-assisted multiplex PCR enrichment sequencing for viral genomic profiling.

BACKGROUND: Previously developed TaME-seq method for deep sequencing of HPV, allowed simultaneous identification of the human papillomavirus (HPV) DNA consensus sequence, low-frequency variable sites, and chromosomal integration events. The method has been successfully validated and applied to the study of five carcinogenic high-risk (HR) HPV types (HPV16, 18, 31, 33, and 45). Here, we present TaME-seq2 with an updated laboratory workflow and bioinformatics pipeline. The HR-HPV type repertoire was expanded with HPV51, 52, and 59. As a proof-of-concept, TaME-seq2 was applied on SARS-CoV-2 positive samples showing the method's flexibility to a broader range of viruses, both DNA and RNA. RESULTS: Compared to TaME-seq version 1, the bioinformatics pipeline of TaME-seq2 is approximately 40× faster. In total, 23 HPV-positive samples and seven SARS-CoV-2 clinical samples passed the threshold of 300× mean depth and were submitted to further analysis. The mean number of variable sites per 1 kb was ~ 1.5× higher in SARS-CoV-2 than in HPV-positive samples. Reproducibility and repeatability of the method were tested on a subset of samples. A viral integration breakpoint followed by a partial genomic deletion was found in within-run replicates of HPV59-positive sample. Identified viral consensus sequence in two separate runs was > 99.9% identical between replicates, differing by a couple of nucleotides identified in only one of the replicates. Conversely, the number of identical minor nucleotide variants (MNVs) differed greatly between replicates, probably caused by PCR-introduced bias. The total number of detected MNVs, calculated gene variability and mutational signature analysis, were unaffected by the sequencing run. CONCLUSION: TaME-seq2 proved well suited for consensus sequence identification, and the detection of low-frequency viral genome variation and viral-chromosomal integrations. The repertoire of TaME-seq2 now encompasses seven HR-HPV types. Our goal is to further include all HR-HPV types in the TaME-seq2 repertoire. Moreover, with a minor modification of previously developed primers, the same method was successfully applied for the analysis of SARS-CoV-2 positive samples, implying the ease of adapting TaME-seq2 to other viruses.

Extraction-free clinical detection of SARS-CoV-2 virus from saline gargle samples using Hamilton STARlet liquid handler.

As part of the COVID-19 pandemic, clinical laboratories have been faced with massive increases in testing, resulting in sample collection systems, reagent, and staff shortages. We utilized self-collected saline gargle samples to optimize high throughput SARS-CoV-2 multiplex polymerase chain reaction (PCR) testing in order to minimize cost and technologist time. This was achieved through elimination of nucleic acid extraction and automation of sample handling on a widely available robotic liquid handler, Hamilton STARlet. A customized barcode scanning script for reading the sample ID by the Hamilton STARlet's software system was developed to allow primary tube sampling. Use of pre-frozen SARS-CoV-2 assay reaction mixtures reduced assay setup time. In both validation and live testing, the assay produced no false positive or false negative results. Of the 1060 samples tested during validation, 3.6% (39/1060) of samples required retesting as they were either single gene positive, had internal control failure or liquid aspiration error. Although the overall turnaround time was only slightly faster in the automated workflow (185 min vs 200 min), there was a 76% reduction in hands-on time, potentially reducing staff fatigue and burnout. This described process from sample self-collection to automated direct PCR testing significantly reduces the total burden on healthcare systems in terms of human resources and reagent requirements.

Respiratory viruses in medicolegal autopsies during the winter season 2021/2022: observations after reduction of coronavirus disease-19 (COVID-19) pandemic restrictions.

In the context of the coronavirus disease (COVID-19) pandemic, measures were taken to protect the population from infection. These were almost completely lifted in several countries in the spring of 2022. To obtain an overview of the spectrum of respiratory viruses encountered in autoptical routine case work, and their infectivity, all autopsy cases at the Institute of Legal Medicine in Frankfurt/M. with flu-like symptoms (among others) were examined for at least 16 different viruses via multiplex PCR and cell culture. Out of 24 cases, 10 were virus-positive in PCR: specifically, 8 cases with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 1 with respiratory syncytial virus (RSV), and 1 with SARS-CoV-2 and the human coronavirus OC43 (HCoV-OC43), as a double infection. The RSV infection and one of the SARS-CoV-2 infections were only detected due to the autopsy. Two SARS-CoV-2 cases (postmortem interval of 8 and 10 days, respectively) showed infectious virus in cell culture; the 6 other cases did not show infectious virus. In the RSV case, virus isolation by cell culture was unsuccessful (Ct value of 23.15 for PCR on cryoconserved lung tissue). HCoV-OC43 was measured as non-infectious in cell culture, with a Ct value of 29.57. The detection of RSV and HCoV-OC43 infections may shed light on the relevance of respiratory viruses other than SARS-CoV-2 in postmortem settings; however, further, more extensive studies are needed for a robust assessment of the hazard potential due to infectious postmortem fluids and tissues in medicolegal autopsy settings.

The usefulness of a nasopharyngeal multiplex polymerase chain reaction system to screen pediatric patients with preoperative fever.

BACKGROUND: When children have a preoperative fever, anesthesiologists must help determine whether to postpone or proceed with surgery, as fever may be a sign of upper respiratory tract infection (URTI). Such infections are a known risk factor for perioperative respiratory adverse events (PRAEs), which are still one of the prime causes of anesthetic mortality and morbidity in pediatric patients. Since the COVID-19 pandemic, preoperative assessments have become drastically more complex as hospitals strive to balance practicality and safety. In our facility, if pediatric patients presented with preoperative fever, we used the FilmArray® Respiratory Panel 2.1 to determine whether to postpone or proceed with surgery. METHODS: This is a single-center retrospective observational study evaluating the efficacy of the FilmArray® Respiratory Panel 2.1 as a preoperative screening test. This study included pediatric patients scheduled for elective surgeries between March 2021 and February 2022. FilmArray was used if a patient had a preoperative fever (determined by axillary temperature, ≥38°C for <1-year-old, ≥37.5°C for ≥1-year-old) between hospital admission and before surgery. We excluded patients if they had apparent symptoms of URTI. RESULTS: In the FilmArray positive group, 11 of 25 (44%) cases developed subsequent symptoms after surgery was canceled. No patients in the negative group developed symptoms. The proportion of the development of subsequent symptoms between the FilmArray positive and negative groups was statistically significant (p < .001, odds ratio: 29.6, 95% confidence interval: [3.80-1356.01]). CONCLUSIONS: Our retrospective observational study revealed that 44% of the FilmArray positive group subsequently developed symptoms, and no PRAEs were observed in the FilmArray negative group. We suggest that FilmArray could be useful as a screening test for pediatric patients with preoperative fever.

Alsó légúti minták molekuláris mikrobiológiai vizsgálata a koronavírus-járvány idoszakában.

INTRODUCTION: BioFire FilmArray Pneumonia plus Panel (bioMérieux) is a PCR method for microbiological diagnostics of lower respiratory infections. It can detect 18 bacteria, 9 viruses and 7 antibiotic resistance genes in real time. It can help the differential diagnosis and the choice of therapy of pneumonia, by giving results in two hours. OBJECTIVE: Reviewing the results of pneumonia PCR tests performed in our laboratory, and comparing them with the results of conventional culturing. METHOD: From October 2020 to September 2021, 820 lower respiratory tract samples were analyzed from inpatients with suspected pneumonia. Beside the PCR test, culturing was also performed. Oropharyngeal swabs were used for supplementary SARS-CoV-2 PCR. RESULTS: 40% of samples were collected from SARS-CoV-2-positive patients. In 60% of the samples, the PCR test detected pathogens or resistance genes. The most commonly detected pathogens were Pseudomonas aeruginosa, Staphylococcus aureus and Acinetobacter baumannii. 44% of the bacteria detected by PCR were not verified by culturing, whereas by culturing, several other bacteria, fungi and antibiotic resistance mechanisms were detected, which were not shown in the results of the multiplex PCR tests. In SARS-CoV-2-positive inpatients, 25.8% of the detected bacteria was S. aureus. The most common resistance gene was mecA/C (MRSA). In this group, other respiratory virus genes were detected in 2% of SARS-CoV-2-positive patients, whereas in 13% in samples of SARS-CoV-2-negative patients. CONCLUSIONS: Because of the importance of pathogens excluded from the PCR targets and multifactorial mechanisms of antibiotic resistance, culturing is recommended to perform beside pneumonia-specific multiplex PCR tests. Orv Hetil. 2022; 163(33): 1295-1302.

Cost-Benefit of Real-Time Multiplex PCR Testing of SARS-CoV-2 in German Hospitals.

BACKGROUND: The current Omicron COVID-19 pandemic has significant morbidity worldwide. OBJECTIVE: Assess the cost-benefit relation of implementing PCR point-of-care (POCT) COVID-19 testing in the emergency rooms (ERs) of German hospitals and in the case of inpatient admission due to other acute illnesses. METHODS: A deterministic decision-analytic model simulated the incremental costs of using the Savanna® Multiplex RT-PCR test compared to using clinical judgement alone to confirm or exclude COVID-19 in adult patients in German ERs prior to hospitalization or just prior to discharge. Direct and indirect costs were evaluated from the hospital perspective. Nasal or nasopharyngeal swabs of patients suspected to have COVID-19 by clinical judgement, but without POCT, were sent to external labs for RT-PCR testing. RESULTS: In probabilistic sensitivity analysis, assuming a COVID-19 prevalence ranging between 15.6-41.2% and a hospitalization rate between 4.3-64.3%, implementing the Savanna® test saved, on average, €107 as compared to applying the clinical-judgement-only strategy. A revenue loss of €735 can be avoided when SARS-CoV-2 infection in patients coming unplanned to the hospital due to other acute illnesses are excluded immediately by POCT. CONCLUSIONS: Using highly sensitive and specific PCR-POCT in patients suspected of COVID-19 infection at German ERs may significantly reduce hospital expenditures.

Using Multiplex Amplicon PCR Technology to Efficiently and Timely Generate Rift Valley Fever Virus Sequence Data for Genomic Surveillance.

Rift Valley fever (RVF) is a febrile vector-borne disease endemic in Africa and continues to spread in new territories. It is a climate-sensitive disease mostly triggered by abnormal rainfall patterns. The disease is associated with high mortality and morbidity in both humans and livestock. RVF is caused by the Rift Valley fever virus (RVFV) of the genus Phlebovirus in the family Phenuiviridae. It is a tripartite RNA virus with three genomic segments: small (S), medium (M) and large (L). Pathogen genomic sequencing is becoming a routine procedure and a powerful tool for understanding the evolutionary dynamics of infectious organisms, including viruses. Inspired by the utility of amplicon-based sequencing demonstrated in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Ebola, Zika and West Nile viruses, we report an RVFV sample preparation based on amplicon multiplex polymerase chain reaction (amPCR) for template enrichment and reduction of background host contamination. The technology can be implemented rapidly to characterize and genotype RVFV during outbreaks in a near-real-time manner. To achieve this, we designed 74 multiplex primer sets covering the entire RVFV genome to specifically amplify the nucleic acid of RVFV in clinical samples from an animal tissue. Using this approach, we demonstrate achieving complete RVFV genome coverage even from samples containing a relatively low viral load. We report the first primer scheme approach of generating multiplex primer sets for a tripartite virus which can be replicated for other segmented viruses.

Universal two-dimensional labelled probe-mediated melting curve analysis based on multiplex PCR for rapid typing of Plasmodium in a single closed tube.

Currently, malaria is still one of the major public health problems commonly caused by the four Plasmodium species. The similar symptoms of malaria and the COVID-19 epidemic of fever or fatigue lead to frequent misdiagnosis. The disadvantages of existing detection methods, such as time-consuming, costly, complicated operation, need for experienced technicians, and indistinguishable typing, lead to difficulties in meeting the clinical requirements of rapid, easy, and accurate typing of common Plasmodium species. In this study, we developed and optimized a universal two-dimensional labelled probe-mediated melting curve analysis (UP-MCA) assay based on multiplex and asymmetric PCR for rapid and accurate typing of five Plasmodium species, including novel human Plasmodium, Plasmodium knowlesi (Pk), in a single closed tube following genome extraction. The assay showed a limit of detection (LOD) of 10 copies per reaction and could accurately distinguish Plasmodium species from intra-plasmodium and other pathogens. Additionally, we proposed and validated different methods of fluorescence quenching and tag design for probes that are suitable for UP-MCA assays. Moreover, the clinical performance of the Plasmodium UP-MCA assay using a base-quenched universal probe was evaluated using 226 samples and showed a sensitivity of 100% (164/164) and specificity of 100% (62/62) at a 99% confidence interval, with the microscopy method as the gold standard. In summary, the UP-MCA assay showed excellent sensitivity, specificity, and accuracy for genotyping Plasmodium species spp. Additionally, it facilitates convenient and rapid Plasmodium detection in routine clinical practice and has great potential for clinical translation.