Commercial Real-time PCR Assay Research Articles

A commercial whole blood polymerase chain reaction assay failed to diagnose histoplasmosis in cats confirmed by cytology or urine antigen detection.

To determine the sensitivity and specificity of a commercial whole blood real-time PCR assay (RT-PCR) for the diagnosis of histoplasmosis when compared to direct organism identification and/or urine antigen quantification by enzyme immunoassay (UA-EIA). A secondary objective was to compare the sensitivity and specificity of RT-PCR to anti-Histoplasma immunoglobulin G antibody detection by enzyme immunoassay (IgG-EIA) and IgG-EIA to UA-EIA. Cats presented to the Kansas State University Veterinary Health Center from February through September of 2023 in which histoplasmosis was diagnosed or suspected. From February through September of 2023, cats were tested by RT-PCR, IgG-EIA, and UA-EIA if histoplasmosis was diagnosed cytologically or was a differential diagnosis for the presenting clinical signs. Cats were excluded if all 3 tests were not submitted or if the diagnosis of histoplasmosis could not be excluded despite a negative UA-EIA result. Cats with cytologically or histologically confirmed histoplasmosis were designated as proven histoplasmosis cases, and cats with a positive UA-EIA result without cytological or histological confirmation were designated as probable histoplasmosis cases. 10 cats were diagnosed with either proven (n = 6) or probable (4) histoplasmosis, and 10 cats were considered true negatives. Whole blood RT-PCR results were negative in all 20 cats (sensitivity, 0%; 95% CI, 0% to 30.85%). The IgG-EIA was 90% sensitive (95% CI, 55.50% to 99.75%) and 70% specific (95% CI, 34.75% to 93.33%). The UA-EIA results were positive in all cats with proven histoplasmosis. This commercial RT-PCR is insensitive when used on whole blood collected in EDTA and should not be used to diagnose feline histoplasmosis. Further studies are required to determine whether alternate RT-PCR protocols for EDTA-collected whole blood could be useful for diagnosing histoplasmosis in cats.

Evaluation of different molecular systems for detection and quantification of SARS-CoV-2 RNA from wastewater samples

Wastewater-based epidemiology has proved to be a suitable approach for tracking the spread of epidemic agents including SARS-CoV-2 RNA. Different protocols have been developed for quantitative detection of SARS-CoV-2 RNA from wastewater samples, but little is known on their performance. In this study we compared three protocols based on Reverse Transcription Real Time-PCR (RT-PCR) and one based on Droplet Digital PCR (ddPCR) for SARS-CoV-2 RNA detection from 35 wastewater samples. Overall, SARS-CoV-2 RNA was detected by at least one method in 85.7 % of samples, while 51.4 %, 22.8 % and 8.6 % resulted positive with two, three or all four methods, respectively. Protocols based on commercial RT-PCR assays and on Droplet Digital PCR showed an overall higher sensitivity vs. an in-house assay. The use of more than one system, targeting different genes, could be helpful to increase detection sensitivity.

A-293 Assessment of the Detection of Gastrointestinal Viruses Using Two Commercial Real-Time PCR Assays

Abstract Background Acute Gastroenteritis (AGE) are the second most common infectious diseases, only overtaken by Respiratory Tract Infections. Sign and symptoms of AGE illness include nausea, diarrhoea, vomits, fever and abdominal pain. In infants and children, gastroenteritis is one of the most important causes of morbidity and mortality all around the Globe. Since their identification in the 1970s, enteric viruses are considered a leading cause of gastroenteritis worldwide. Among these, rotaviruses (RV) are considered to be the main cause of AGE in young children, whereas noroviruses (NoV) affect people of all ages. In addition, sapoviruses (SaV), human astroviruses (hAstV), human adenoviruses (AdV) are also relevant. In the recent years, molecular methods, such as real-time PCR, have been widely used for detection of viral genome of enteric viruses due to their high sensitivity and specificity, becoming the gold standard. The aim of this study is the comparison of different commercial real-time PCR assays for the detection of human enteric viruses. Material and methods Three hundred and eighteen clinical stool samples were selected from different groups collected between 2016 and 2019, including specimens from symptomatic adults and symptomatic and asymptomatic children. Samples were stored at −20°C. The collection included a minimum of 25 samples positive for RV, 55 positives for AdV, 16 positives for AstV, 22 positives for SaV and 65 positives for NoV (31 for GI and 34 for GII). All specimens were extracted using the Maxwell®RSC Blood DNA Kit on the Maxwell RSC equipment (Promega). Samples were analysed using the following assays: RIDA®GENE Viral Stool Panel II, RIDA®GENE Sapovirus and RIDA®GENE Norovirus I & II (R-Biopharm), Vitassay qPCR kits for the detection of Rotavirus, Astrovirus, Norovirus GI, Norovirus GII, and Sapovirus. Results obtained with Vitassay were compared to R-Biopharm assays. Sensitivity and specificity were calculated using the software MetaDisc 1.4. Discrepant samples were tested in triplicate. Results After assessing the discrepant results, 29/114 samples were positive for RV in both RIDA®GENE and Vitassay kit. This latter reported 2 false negative (FN) and 4 false positive (FP) results when compared to RIDA®GENE. Sensitivity was 93.5% (78.6–99.2) and specificity was 95.2% (88.1–98.7). For AstV, 27/138 were positive in both kits. Vitassay qPCR reported 1 FN and 2 FP, obtaining sensitivity and specificity values of 96.4% (81.7–99.9) and 98.6% (94.9–99.8), respectively. Regarding SaV, Vitassay reported 100% (88.8%-100) of sensitivity and 97.8% (88.5–99.9) of specificity, as 1 FP was obtained. 30/89 samples tested for Norovirus GI were detected by Vitassay qPCR Norovirus GI and 25/89 RIDA®GENE Norovirus I & II (R-Biopharm). On the other hand, Vitassay qPCR Norovirus GII reported 31/81 positive samples for NoV I, whereas RIDA®GENE obtained 30 positive results. In both cases, sensitivity was of 100%, whereas specificity was over 92%. Conclusions Vitassay real-time PCR assays for the detection of different enteric viruses present high specificity and sensitivity, which is essential for a proper diagnosis and epidemiology assessment. In addition, Vitassay qPCR kit presents the advantage of being a ready-to-use lyophilised kit.

Quick versus Quantitative: Evaluation of Two Commercial Real-Time PCR Assays for the Detection of Pneumocystis jirovecii from Bronchoalveolar Lavage Fluids.

Two commercial real-time PCR assays for the detection of Pneumocystis jirovecii were compared, the quantitative RealStar P. jirovecii assay and the qualitative DiaSorin P. jirovecii assay, the latter of which can be used without nucleic acid extraction. Archived bronchoalveolar lavage (BAL) specimens (n = 66), previously tested by molecular methods, were tested by both assays, and the results were compared to the respective original result. The RealStar P. jirovecii assay demonstrated good positive percent agreement (PPA) (90% [95% confidence interval (CI), 72 to 97%]; 27/30) and negative percent agreement (NPA) (100% [95% CI, 88 to 100%]; 36/36) with the reference method. The DiaSorin P. jirovecii assay concordantly detected P. jirovecii in 19 of 24 positive BAL samples (PPA = 73% [95% CI, 52 to 88%]). All negative BAL samples gave concordant results (NPA = 100% [95% CI, 87 to 100%]; 34/34). Discordant results occurred mostly in samples with low fungal loads. In conclusion, the RealStar assay demonstrated good concordance with reference results, and the DiaSorin P. jirovecii assay performed well for negative BAL and positive BAL samples with P. jirovecii concentrations of greater than 260 copies/mL. IMPORTANCE Pneumonia, caused by the opportunistic fungus Pneumocystis jirovecii, poses a significant risk for immunocompromised individuals. Laboratory testing for P. jirovecii is progressively shifting toward the use of molecular tests such as real-time PCR; however, this is often performed at reference laboratories. Many frontline laboratories are looking into improving their service and reducing turnaround times for obtaining P. jirovecii results by bringing molecular P. jirovecii testing in-house. We evaluated and compared two commercial real-time PCR assays with different workflows for the detection of P. jirovecii from bronchoalveolar lavage specimens. The RealStar P. jirovecii assay requires nucleic acid extraction and provides a quantification of fungal load for positive samples. The DiaSorin P. jirovecii assay offers a simple workflow without nucleic extraction from patient samples and qualitative results. Results from this study provide valuable information on performance and workflow considerations for laboratories that wish to implement P. jirovecii molecular testing.

Comparative Assessment of Two Commercial Real-Time PCR Assays for the Diagnosis of Trypanosoma cruzi DNA in Serum.

This study was performed to comparably assess two commercial real-time PCR assays for the identification of Trypanosoma cruzi DNA in serum. A total of 518 Colombian serum samples with high pre-test probability for infections with either T. cruzi or apathogenic Trypanosoma rangeli were assessed. The assessment comprised the NDO real-time PCR (TIB MOLBIOL, ref. no. 53-0755-96, referred to as the TibMolBiol assay in the following) with specificity for T. cruzi and the RealStar Chagas PCR Kit 1.0 (altona DIAGNOSTICS, order no. 611013, referred to as the RealStar assay in the following) targeting a kinetoplast sequence of both T. cruzi and T. rangeli without further discrimination. To discriminate between T. cruzi- and T. rangeli-specific real-time PCR amplicons, Sanger sequencing results were available for a minority of cases with discordant real-time PCR results, while the amplicons of the remaining discordant samples were subjected to nanopore sequencing. The study assessment indicated a proportion of 18.1% (n = 94) T. cruzi-positive samples next to 24 samples (4.6%) containing DNA of the phylogenetically related but apathogenic parasite T. rangeli. The observed diagnostic accuracy as expressed by sensitivity and specificity was 97.9% (92/94) and 99.3% (421/424) with the TibMolBiol assay and 96.8% (91/94) and 95.0% (403/424) with the RealStar assay, respectively. Reduced specificity resulted from cross-reaction with T. rangeli in all instances (3 cross-reactions with the TibMolBiol assay and 21 cross-reactions with the RealStar assay). DNA from the six discrete typing units (DTUs) of T. cruzi was successfully amplified by both real-time PCR assays. In summary, both assays showed a comparable diagnostic accuracy for the diagnosis of T. cruzi from human serum, with a slightly higher specificity seen for the TibMolBiol assay. The pronounced co-amplification of DNA from apathogenic T. rangeli according to the RealStar assay may be a disadvantage in areas of co-circulation with T. cruzi, while the test performance of the two compared assays will be quite similar in geographic settings where T. rangeli infections are unlikely.

Transmission of monkeypox virus through the saliva and air that need careful management.

Transmission of monkeypox virus through the saliva and air that need careful management.

Proficiency testing of PIK3CA mutations in HR+/HER2-breast cancer on liquid biopsy and tissue

Precision oncology based on specific molecular alterations requires precise and reliable detection of therapeutic targets in order to initiate the optimal treatment. In many European countries—including Germany—assays employed for this purpose are highly diverse and not prescribed by authorities, making inter-laboratory comparison difficult. To ensure reproducible molecular diagnostic results across many laboratories and different assays, ring trials are essential and a well-established tool. Here, we describe the design and results of the ring trial for the detection of therapeutically relevant PIK3CA hotspot mutations in HR+/HER2-breast cancer tissue and liquid biopsy (LB). For PIK3CA mutation detection in tissue samples, 43 of the 54 participants (80%) provided results compliant with the reference values. Participants using NGS-based assays showed higher success rate (82%) than those employing Sanger sequencing (57%). LB testing was performed with two reference materials differing in the length of the mutated DNA fragments. Most participants used NGS-based or commercial real-time PCR assays (70%). The 167 bp fragments led to a successful PIK3CA mutation detection by only 31% of participants whereas longer fragments of 490 bp were detectable even by non-optimal assays (83%). In conclusion, the first ring trial for PIK3CA mutation detection in Germany showed that PIK3CA mutation analysis is broadly established for tissue samples and that NGS-based tests seem to be more suitable than Sanger sequencing. PIK3CA mutation detection in LB should be carried out with assays specifically designed for this purpose in order to avoid false-negative results.

Assessment of a Commercial Real-Time PCR Assay (Vitassay qPCR Malaria 5 Test) to Detect Human Malaria Infection in Travelers Returning to France.

Malaria is the most common human parasitic disease in the world with the highest morbidity and mortality. Due to the severity of malaria caused by Plasmodium falciparum and the urgency of therapeutic management, quick and reliable diagnosis is required for early detection. Blood smear microscopy remains the gold standard for malaria diagnosis. Molecular diagnosis techniques are the most sensitive and specific in cases of low parasitaemia and in the detection of mixed infections. The purpose of this study was to evaluate a new commercial test involving the molecular diagnostic technique to detect the five human Plasmodium species. The Vitassay qPCR Malaria 5 test is based on the multiplex real-time PCR of a conserved target region of the 18S rRNA gene for the five human Plasmodium species. A total of 190 samples collected from imported cases of malaria were diagnosed using this test and compared against a homemade reference real-time PCR. The sensitivities of the Vitassay qPCR Malaria 5 test for all Plasmodium species ranged from 93.8% to 100% and specificity ranged from 97.7% to 100%. Based on these criteria, this test is recommended for the diagnosis of the human Plasmodium species.

EVALUATION OF THE STABILITY OF RNA SARS-CoV-2 IN MULTIPLE FREZEE-THAW CYCLES USING REAL-TIME PCR METHOD

Introduction/ Objectives: According to WHO, clinical samples collected in retrospective clinical studies are required to provide stability claims for each storage condition, such as duration at different temperatures and freeze/thaw cycles[1]. This study was conducted to evaluate the effects of multiple freeze/thaw (FT) cycles on the concentration of RNA SARS-CoV-2 (extracted from retrospective clinical sample) by using Real-time PCR method.
 Methods: Descriptive laboratory study. We used a commercial Real-time PCR assay and reference standard for quantification of RNA concentration after each FT cycle.
 Results: We observed that the extracted RNA SARS-CoV-2 remained stable and did not show significantly different Ct values (equivalent to viral loads) over 10 FT cycles (p >0.05), compared to those Ct values at time point 0. Variations of Ct values over 10 FT cycles ranged from 0.64% to 1.64%. The mean difference in viral load was 0,12log10IU/ml.
 Conclusions: Our study results proved that RNA SARS-CoV-2 extracted from clinical samples can remain stable over 10 freeze-thaw cycles and can be used as a evaluation panel for quality control of in vitro diagnostic test kits detecting SARS-CoV-2.

Universal Newborn Screening for Congenital Cytomegalovirus Infection - From Infant to Maternal Infection: A Prospective Multicenter Study.

Introduction:Most infants at risk for cytomegalovirus (CMV)-associated sensorineural hearing loss (SNHL) are unrecognized because of the absence of a universal neonatal CMV screening. The search of CMV-DNA by molecular methods in salivary swabs was demonstrated to be a reliable approach. This study describes the results obtained by carrying out a universal screening for congenital CMV (cCMV) infection including all live-born newborns in three Italian sites, as well as the therapeutic interventions and clinical outcome of the CMV-infected neonates. Moreover, CMV maternal infection's characteristics were evaluated.MethodsTo confirm or exclude cCMV infection, a CMV-DNA-positive result on a first salivary swab was followed by repeated saliva and urine samples collected within 21 days of age. Breast milk samples were also collected. The search of CMV-DNA was performed with a single automated quantitative commercial real-time PCR assay, regardless of the type of samples used.ResultsA total of 3,151 newborns were enrolled; 21 (0.66%) of them were congenitally infected (median saliva viral load at screening, 6.65 [range, 5.03–7.17] log10 IU/ml). Very low/low viral load in screening saliva samples (median value, 1.87 [range, 1.14–2.59] log10 IU/ml) was associated with false-positive results (n = 54; 1.7%). CMV-DNA was detected in almost half of the breast milk samples of mother–infant pairs with a false-positive result, suggesting that contamination from breast milk may not be the only explanation in the study population. cCMV infection confirmation with the search of CMV-DNA in a urine sample proved to be the gold standard strategy, since false-positive results were observed in 4/54 (7.5%) of the repeated saliva samples. Symptomatic cCMV infection was observed in 3/21 (14.3%) infants; notably, one (4.7%) developed moderate unilateral SNHL at 5 months after birth. Finally, two symptomatic cCMV infections were associated with primary maternal infection acquired in the first trimester of gestation; one newborn with severe cCMV symptoms was born to a mother with no CMV checkups in pregnancy.ConclusionWithout universal neonatal CMV screening, some infected infants who develop late neurological sequelae may not be recognized and, consequently, they are not able to benefit early from instrumental and therapeutic interventions to limit and/or treat CMV disease.

Evaluation of the Multiplex Real-Time PCR DermaGenius® Assay for the Detection of Dermatophytes in Hair Samples from Senegal.

For the successful treatment of dermatophytoses, especially tinea capitis, there is a need for accurate and rapid diagnostic methods. A lot of recent literature has focused on the detection of dermatophytes directly on sample material such as nails, hair and skin scrapings. Molecular tools offer the ability to rapidly diagnose dermatophytosis within 48 h. This study aimed to compare the results of a commercial real-time PCR (real-time PCR) assay DermaGenius®(DG) 2.0 complete multiplex kit with those of conventional diagnostic methods (direct microscopy and culture). A total of 129 hair samples were collected in Dakar (Senegal) from patients suspected of dermatophytosis. DG was applied for the molecular detection of Candida albicans, Trichophyton rubrum/soudanense, T. interdigitale, T. tonsurans, T. mentagrophytes, T. violaceum, Microsporum canis, M. audouinii, Epidermophyton floccosum, T. benhamiae and T. verrucosum. Dermatophytes species and C. albicans were differentiated by melting curve analysis. The sensitivity and specificity of the PCR assay were 89.3% and 75.3%, respectively. DG PCR was significantly more sensitive than culture (p < 0.001). DG PCR is fast and robust to contamination. In this paper, the main questions discussed were the replacement of culture by a broad-spectrum fungal real-time PCR and the implementation of DG PCR into a routine laboratory in Senegal.

Performance of four commercial real-time PCR assays for the detection of bacterial enteric pathogens in clinical samples

ObjectivesMany laboratories use culture-independent diagnostic tests for bacterial gastroenteritis (i.e. real-time polymerase chain reaction, RT-PCR) instead of culture because of better sensitivity, automation, and faster turnaround times. To address some gaps in initial evaluations and lack of intraassay comparisons for many commercial RT-PCRs, this study compared the ability of four commercially available RT-PCR tests (Ridagene, Fast Track Diagnostics, BD Max, and Prodesse Progastro) to detect five major bacterial enteric pathogens: Campylobacter, Salmonella, Shiga-toxin producing Escherichia coli (STEC), Shigella, and Yersinia. MethodsClinical stool specimens and contrived samples comprising commonly circulating species, serotypes, biovars, and/or toxin subtypes were used for the comparison. ResultsConcordance rates for RT-PCR and culture using culture-positive and culture-negative clinical stools were >90% for Campylobacter (97.5-100%), Salmonella (97.5-100%), Shigella (100%), and STEC (90-100%). However, the agreement between RT-PCR and culture for Y. enteroccolitica ranged from 70-90%. For the contrived sample set, stx2f was detected by one of four assays. Of note, no assay could detect Yersinia non-enterocolitica and Campylobacter upsaliensis. ConclusionsDepending on the prevalence of certain stx sub-types, Yersinia species, and Campylobacter species in a laboratory's jurisdiction, without further improvement in culture-independent tests, culture methods remain critical for the detection of these pathogens.

Tenosynovitis caused by Mycobacterium marseillense, initially identified as Mycobacterium avium complex using AccuProbe and COBAS TaqMan

BackgroundMycobacterium marseillense is a new species of the Mycobacterium avium complex. There has been only a few human infections caused by M. marseillense worldwide.Case presentationWe report a case of tenosynovitis caused by M. marseillense in an immunocompetent adult in Japan. The isolate was initially identified as M. intracellulare using commercial real time polymerase chain reaction assays and later identified as M. marseillense with sequencing of the the rpoB and hsp65 regions, and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS).ConclusionsThis is the first case reporting on M. marseillense generating a positive result with commercial real time PCR assays targeting MAC. Human infections associated by M. marseillense might be underreported due to similarities with Mycobacterium intracellulare. To accurately identify M. marseillese, MALDI-TOF MS might provide a rapid and reliable method.

A New PCR-Based Assay for Testing Bronchoalveolar Lavage Fluid Samples from Patients with Suspected Pneumocystis jirovecii Pneumonia.

To support the clinical laboratory diagnosis of Pneumocystis jirovecii (PJ) pneumonia (PCP), an invasive fungal infection mainly occurring in HIV-negative patients, in-house or commercial PJ-specific real-time quantitative PCR (qPCR) assays are todays’ reliable options. The performance of these assays depends on the type of PJ gene (multi-copy mitochondrial versus single-copy nuclear) targeted by the assay. We described the development of a PJ-PCR assay targeting the dihydrofolate reductase (DHFR)-encoding gene. After delineating its analytical performance, the PJ-PCR assay was used to test bronchoalveolar lavage (BAL) fluid samples from 200 patients (only seven were HIV positive) with suspected PCP. Of 211 BAL fluid samples, 18 (8.5%) were positive and 193 (91.5%) were negative by PJ-PCR. Of 18 PJ-PCR-positive samples, 11 (61.1%) tested positive and seven (38.9%) tested negative with the immunofluorescence assay (IFA). All (100%) of the 193 PJ-PCR-negative samples were IFA negative. Based on IFA/PCR results, patients were, respectively, classified as having (n = 18) and not having (n = 182) proven (PJ-PCR+/IFA+) or probable (PJ-PCR+/IFA−) PCP. For 182 patients without PCP, alternative infectious or non-infectious etiologies were identified. Our PJ-PCR assay was at least equivalent to IFA, fostering studies aimed at defining a qPCR-based standard for PCP diagnosis in the future.

Mycoplasma detection in cell therapy products: GMP-compliant implementation &amp; validation of a commercial real-time PCR assay for routine quality control &amp; lot release

<b>Watch the video or read the poster to learn:</b> <ul><li>Regulatory guidance around mycoplasma testing in advanced cell therapy medicinal products</li><li>Factors to consider when choosing a rapid mycoplasma detection solution for use in lot-release testing for cell therapy products</li><li>GMP-compliant validation study design and results in a hMSC cell therapy product</li></ul><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Valentina photo 200x200.jpg" data-image="1"></div><div class="author-det"> Valentina has a master's degree in medical and pharmaceutical biotechnologies, and recently gained a PhD in biochemistry and clinical pathology at the University of Florence, Italy. She has worked since 2010 as a senior&nbsp; scientist in the field of advanced therapy medicinal products (ATMPs) with special focus on drafting and execution of analytical validation protocols, in compliance with international requirements. During her 10 years as medical and pharmaceutical biotechnologist, Valentina has gained experience in process development, technology transfer, process validation, manufacturing, and biological characterization of cell-based products.&nbsp; </div></div></div>

Performance of the currently available DIVA real-time PCR assays in classical and recombinant lumpy skin disease viruses.

The use of live homologous vaccines to protect against lumpy skin disease virus (LSDV) infection requires the use of molecular tools to differentiate between infected and vaccinated animals (DIVA). In this study, the commercial real-time PCR assays; ID Gene™ LSD DIVA Triplex kit and Bio-T kit® LSD - DIVA, as well as published assays targeting the GPCR gene (Journal of Virological Methods, 249, 48-57) and ORF008 and ORF126 (Sel'skokhozyaistvennaya Biologiya, 54, 347-358) were evaluated. These assays correctly identified classical field isolates (European lineage) and vaccine (Neethling vaccine). In contrast, when tested using vaccine-like recombinant viruses, the commercial and published assays were not able to correctly identify recombinant isolates. At the same time, the recombinant viruses were detected as either field and/or vaccine, or not detected at all depending on the assay. The different gene sequences present in recombinant viruses cause these DIVA assays to incorrectly assign recombinant viruses as either a field or vaccine virus. This observation has implications for using these assays and for identification of LSDV vaccine.

Respiratory microbes detected in hospitalized adults with acute respiratory infections: associations between influenza A(H1N1)pdm09 virus and intensive care unit admission or fatal outcome in Vietnam (2015\u20132017)

BackgroundAcute respiratory tract infection (ARI) is a leading cause of hospitalization, morbidity, and mortality worldwide. Respiratory microbes that were simultaneously detected in the respiratory tracts of hospitalized adult ARI patients were investigated. Associations between influenza A(H1N1)pdm09 virus (H1N1pdm) detection and intensive care unit (ICU) admission or fatal outcome were determined.MethodsThis prospective observational study was conducted between September 2015 and June 2017 at Bach Mai Hospital, Hanoi, Vietnam. Inclusion criteria were hospitalized patients aged ≥15 years; one or more of symptoms including shortness of breath, sore throat, runny nose, headache, and muscle pain/arthralgia in addition to cough and fever > 37.5 °C; and ≤ 10 days from the onset of symptoms. Twenty-two viruses, 11 bacteria, and one fungus in airway specimens were examined using a commercial multiplex real-time PCR assay. Associations between H1N1pdm detection and ICU admission or fatal outcome were investigated by univariate and multivariate logistic regression analyses.ResultsThe total of 269 patients (57.6% male; median age, 51 years) included 69 ICU patients. One or more microbes were detected in the airways of 214 patients (79.6%). Single and multiple microbes were detected in 41.3 and 38.3% of patients, respectively. Influenza A(H3N2) virus was the most frequently detected (35 cases; 13.0%), followed by H1N1pdm (29 cases; 10.8%). Hematological disease was associated with ICU admission (p < 0.001) and fatal outcomes (p < 0.001) using the corrected significance level (p = 0.0033). Sex, age, duration from onset to sampling, or number of detected microbes were not significantly associated with ICU admission or fatal outcomes. H1N1pdm detection was associated with ICU admission (odds ratio [OR] 3.911; 95% confidence interval [CI] 1.671–9.154) and fatal outcome (OR 5.496; 95% CI 1.814–16.653) after adjusting for the confounding factors of comorbidities, bacteria/Pneumocystis jirovecii co-detection, and age.ConclusionsH1N1pdm was associated with severe morbidity and death in adult patients hospitalized with respiratory symptoms. The diagnosis of subtype of influenza virus may be epidemiologically important.

Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples.

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

Validation and implementation of a commercial real-time PCR assay for direct detection of Candida auris from surveillance samples.

Rapid and reliable laboratory methods are required for detecting the nosocomial yeast Candida auris. AurisID® (Olm Diagnostics) is a real-time PCR assay approved for detecting C. auris in fungal cultures and directly from blood samples, involving a nucleic acid extraction as a prior step. The purpose of this study is to validate the AurisID® kit for direct detection of C. auris from surveillance samples without prior DNA extraction and to analyse the results of implementing this methodology to our daily laboratory routine protocol for C. auris surveillance studies. Our PCR method using the AurisID® kit was compared with our routine protocol, consisting of culture in CHROMagar® Candida and identification by mass spectrometry. A total of 113 swabs were used for validation and 136 pair of surveillance samples were tested. Limit of detection (LOD) was determined by using swabs in Amies transport medium, which were spiked in a series of dilutions of a C. auris standardised suspension (0.5 McFarland). The PCR method showed high sensitivity, specificity, predictive positive value and predictive negative value (96.6%, 100%, 100% and 98.2%, respectively) when compared with the routine protocol. LOD was 500CFU/ml, which corresponds to approximately 1CFU/PCR. Our PCR method using the AurisID® kit allows a reduction in the turnaround time for surveillance of C. auris compared with other methods. These results are expected to contribute to control C. auris outbreaks, allowing isolation of patients and cleaning of environmental surfaces in advance.

SARS-CoV-2 lineage B.6 was the major contributor to early pandemic transmission in Malaysia.

Malaysia had 10,219 confirmed cases of COVID-19 as of September 20, 2020. About 33% were associated with a Tablighi Jamaat religious mass gathering held in Kuala Lumpur between February 27 and March 3, 2020, which drove community transmission during Malaysia's second wave. We analysed genome sequences of SARS-CoV-2 from Malaysia to better understand the molecular epidemiology and spread. We obtained 58 SARS-CoV-2 whole genome sequences from patients in Kuala Lumpur and performed phylogenetic analyses on these and a further 57 Malaysian sequences available in the GISAID database. Nine different SARS-CoV-2 lineages (A, B, B.1, B.1.1, B.1.1.1, B.1.36, B.2, B.3 and B.6) were detected in Malaysia. The B.6 lineage was first reported a week after the Tablighi mass gathering and became predominant (65.2%) despite being relatively rare (1.4%) globally. Direct epidemiological links between lineage B.6 viruses and the mass gathering were identified. Increases in reported total cases, Tablighi-associated cases, and community-acquired B.6 lineage strains were temporally linked. Non-B.6 lineages were mainly travel-associated and showed limited onward transmission. There were also temporally correlated increases in B.6 sequences in other Southeast Asian countries, India and Australia, linked to participants returning from this event. Over 95% of global B.6 sequences originated from Asia Pacific. We also report a nsp3-C6310A substitution found in 47.3% of global B.6 sequences which was associated with reduced sensitivity using a commercial diagnostic real-time PCR assay. Lineage B.6 became the predominant cause of community transmission in Malaysia after likely introduction during a religious mass gathering. This event also contributed to spikes of lineage B.6 in other countries in the Asia-Pacific. Mass gatherings can be significant causes of local and global spread of COVID-19. Shared genomic surveillance can be used to identify SARS-CoV-2 transmission chains to aid prevention and control, and to monitor diagnostic molecular assays. Clinical Trial Registration: COVID-19 paper.