Repeat testing of mpox specimens with late CTs improves detection of potential false positive cases.

The mpox pandemic necessitated the rapid development of clinical assays for monkeypox virus detection. While the majority of mpox specimens have high viral loads with corresponding early cycle threshold (CT) values, reports have indicated some specimens with late CT values can represent false positive results. To mitigate this risk, the Centers for Disease Control and Prevention (CDC) published an advisory recommending repeat testing of all specimens with CT values ≥34. However, limited experimental data were available to support this specific cutoff. In this study, we examine whether a more conservative approach in which all specimens with CT values ≥29 are repeated would improve the detection of potential false positive results. Compared to the CDC algorithm, our approach identified an additional 20% (5/25) of potential false positive results. To assess the impact of this cutoff on laboratory workload, we modeled the expected increase in test volume and turnaround time (TAT) relative to the CDC method. Using a lower repeat threshold, test volume increased by 0.7% while mean TAT increased by less than 15 minutes. Overall, a lower threshold than recommended by the CDC for repeating late CT mpox specimens may reduce the number of false positives reported while minimally impacting testing volume and TAT.

Development and validation of a next-generation sequencing assay with open-access analysis software for detecting resistance-associated mutations in CMV.

Cytomegalovirus (CMV) resistance testing by targeted next-generation sequencing (NGS) allows for the simultaneous analysis of multiple genes. We developed and validated an amplicon-based Ion Torrent NGS assay to detect CMV resistance mutations in UL27, UL54, UL56, and UL97 and compared the results to standard Sanger sequencing. NGS primers were designed to generate 83 overlapping amplicons of four CMV genes (~10 kb encompassing 138 mutation sites). An open-access software plugin was developed to perform read alignment, call variants, and interpret drug resistance. Plasmids were tested to determine NGS error rate and minor variant limit of detection. NGS limit of detection was determined using the CMV WHO International Standard and quantified clinical specimens. Reproducibility was also assessed. After establishing quality control metrics, 185 patient specimens previously tested using Sanger were reanalyzed by NGS. The NGS assay had a low error rate (<0.05%) and high accuracy (95%) for detecting CMV-associated resistance mutations present at ≥5% in contrived mixed populations. Mutation sites were reproducibly sequenced with 40× coverage when plasma viral loads were ≥2.6 log IU/mL. NGS detected the same resistance-associated mutations identified by Sanger in 68/69 (98.6%) specimens. In 16 specimens, NGS detected 18 resistance mutations that Sanger failed to detect; 14 were low-frequency variants (<20%), and six would have changed the drug resistance interpretation. The NGS assay showed excellent agreement with Sanger and generated high-quality sequence from low viral load specimens. Additionally, the higher resolution and analytic sensitivity of NGS potentially enables earlier detection of antiviral resistance.

Multi-center clinical evaluation of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab specimens from symptomatic individuals.

The symptomology is overlapping for respiratory infections due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A/B viruses, and respiratory syncytial virus (RSV). Accurate detection is essential for proper medical management decisions. This study evaluated the clinical performance of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab (NPS) specimens from individuals of all ages with signs and symptoms of respiratory infection consistent with COVID-19, influenza, or RSV. Retrospective known-positive and prospectively obtained residual NPS specimens were collected during two respiratory seasons in the USA. Clinical performance was established by comparing Panther Fusion SARS-CoV-2/Flu assay results to a three-molecular assay composite comparator interpretation for SARS-CoV-2 and to the FDA-cleared Panther Fusion Flu A/B/RSV assay results for all non-SARS-CoV-2 targets. A total of 1,900 prospective and 95 retrospective NPS specimens were included in the analyses. The overall prevalence in prospectively obtained specimens was 20.7% for SARS-CoV-2, 6.7% for influenza A, and 0.7% for RSV; all influenza B-positive specimens were retrospective specimens. The positive percent agreement of the Panther Fusion assay was 96.9% (378/390) for SARS-CoV-2, 98.0% (121/123) for influenza A virus, 95.2% (20/21) for influenza B virus, and 96.6% (57/59) for RSV. The negative percent agreement was ≥98.5% for all target viruses. Specimens with discordant Panther Fusion SARS/Flu/RSV assay results all had cycle threshold values of ≥32.4 (by comparator or by Panther Fusion SARS/Flu/RSV assay). Only five co-infections were detected in the study specimens. The Panther Fusion SARS-CoV-2/Flu/RSV assay provides highly sensitive and specific detection of SARS-CoV-2, influenza A virus, influenza B virus, and RSV in NPS specimens.

HIV-1 Drug Resistance Assay Using Ion Torrent Next Generation Sequencing and On-Instrument End-to-End Analysis Software.

HIV-1 antiretroviral therapy management requires sequencing the protease, reverse transcriptase, and integrase portions of the HIV-1 pol gene. Most resistance testing is performed with Sanger sequencing, which has limited ability to detect minor variants. Next generation sequencing (NGS) platforms enable variant detection at frequencies as low as 1% allowing for earlier detection of resistance and modification of therapy. Implementation of NGS assays in the clinical laboratory is hindered by complicated assay design, cumbersome wet bench procedures, and the complexity of data analysis and bioinformatics. We developed a complete NGS protocol and companion analysis and reporting pipeline using AmpliSeq multiplex PCR, Ion Torrent S5 XL sequencing, and Stanford's HIVdb resistance algorithm. Implemented as a Torrent Suite software plugin, the pipeline runs automatically after sequencing. An optimum variant frequency threshold of 10% was determined by comparing Sanger sequences of archived samples from ViroSeq testing, resulting in a sensitivity of 98.2% and specificity of 99.0%. The majority (91%) of drug resistance mutations were detected by both Sanger and NGS, with 1.7% only by Sanger and 7.3% only by NGS. Variant calls were highly reproducible and there was no cross-reactivity to VZV, HBV, CMV, EBV, and HCV. The limit of detection was 500 copies/mL. The NGS assay performance was comparable to ViroSeq Sanger sequencing and has several advantages, including a publicly available end-to-end analysis and reporting plugin. The assay provides a straightforward path for implementation of NGS for HIV drug resistance testing in the laboratory setting without additional investment in bioinformatics infrastructure and resources.

Evaluation of a Novel High-Definition PCR Multiplex Assay for Simultaneous Detection of Tick-Borne Pathogens in Human Clinical Specimens.

The incidence of tick-borne infections in the United States has risen significantly in the past decade. Ticks can transmit a variety of pathogens, including bacteria, protozoa, and viruses, that can cause serious illnesses. Therefore, the use of rapid, sensitive, and specific multiplex tests is important to identify the pathogen(s) in the acute phase and determine appropriate treatment to minimize the severity of the disease. The purpose of this study was to evaluate ChromaCode's research use only (RUO) nine-target high-definition PCR (HDPCR) tick-borne pathogen (TBP) panel using 379 retrospective, remnant whole-blood and synovial fluid specimens previously submitted to Associated Regional and University Pathologists (ARUP) Laboratories and tested by clinically validated real-time PCR assays for Ehrlichia spp., Anaplasma phagocytophilum, Babesia spp., or Lyme Borrelia spp. The performance characteristics evaluated included positive percent agreement (PPA) and negative percent agreement (NPA) with the ARUP laboratory-developed tests (LDTs). All tested targets had an initial PPA greater than 97.0%, except Ehrlichia ewingii, with a PPA of 88.9%. The NPAs for all targets were between 98.8% and 100%. The TBP panel detected three coinfections, with two of Babesia microti and A. phagocytophilum and one of B. microti and E. chaffeensis, which were confirmed by the LDTs. There were 16 samples with discordant results compared to the LDT results, five of which were resolved by repeat testing on the TBP panel and bidirectional sequencing. Following discrepant resolution, the final PPA and NPA for the TBP panel were 97.7% (95% confidence interval [CI], 95.2% to 99.0%) and 99.6% (95% CI, 99.3% to 99.8%), respectively, with an overall agreement of 99.5% (95% CI, 99.2% to 99.7%) with the LDTs.

Community-acquired pneumonia requiring hospitalization among U.S. children.

BACKGROUND: Incidence estimates of hospitalizations for community-acquired pneumonia among children in the United States that are based on prospective data collection are limited. Updated estimates of pneumonia that has been confirmed radiographically and with the use of current laboratory diagnostic tests are needed. METHODS: We conducted active population-based surveillance for community-acquired pneumonia requiring hospitalization among children younger than 18 years of age in three hospitals in Memphis, Nashville, and Salt Lake City. We excluded children with recent hospitalization or severe immunosuppression. Blood and respiratory specimens were systematically collected for pathogen detection with the use of multiple methods. Chest radiographs were reviewed independently by study radiologists. RESULTS: From January 2010 through June 2012, we enrolled 2638 of 3803 eligible children (69%), 2358 of whom (89%) had radiographic evidence of pneumonia. The median age of the children was 2 years (interquartile range, 1 to 6); 497 of 2358 children (21%) required intensive care, and 3 (<1%) died. Among 2222 children with radiographic evidence of pneumonia and with specimens available for bacterial and viral testing, a viral or bacterial pathogen was detected in 1802 (81%), one or more viruses in 1472 (66%), bacteria in 175 (8%), and both bacterial and viral pathogens in 155 (7%). The annual incidence of pneumonia was 15.7 cases per 10,000 children (95% confidence interval [CI], 14.9 to 16.5), with the highest rate among children younger than 2 years of age (62.2 cases per 10,000 children; 95% CI, 57.6 to 67.1). Respiratory syncytial virus was more common among children younger than 5 years of age than among older children (37% vs. 8%), as were adenovirus (15% vs. 3%) and human metapneumovirus (15% vs. 8%). Mycoplasma pneumoniae was more common among children 5 years of age or older than among younger children (19% vs. 3%). CONCLUSIONS: The burden of hospitalization for children with community-acquired pneumonia was highest among the very young, with respiratory viruses the most commonly detected causes of pneumonia. (Funded by the Influenza Division of the National Center for Immunization and Respiratory Diseases.).

Serology Enhances Molecular Diagnosis of Respiratory Virus Infections Other than Influenza in Children and Adults Hospitalized with Community-Acquired Pneumonia.

Both molecular and serological assays have been used previously to determine the etiology of community-acquired pneumonia (CAP). However, the extent to which these methods are correlated and the added diagnostic value of serology for respiratory viruses other than influenza virus have not been fully evaluated. Using data from patients enrolled in the Centers for Disease Control and Prevention (CDC) Etiology of Pneumonia in the Community (EPIC) study, we compared real-time reverse transcription-PCR (RT-PCR) and serology for the diagnosis of respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza virus 1 to 3 (PIV1, PIV2, and PIV3), and adenovirus (AdV) infections. Of 5,126 patients enrolled, RT-PCR and serology test results were available for 2,023, including 1,087 children below the age of 18 years and 936 adults. For RSV, 287 (14.2%) patients were positive by RT-PCR and 234 (11.6%) were positive by serology; for HMPV, 172 (8.5%) tested positive by RT-PCR and 147 (7.3%) by serology; for the PIVs, 94 (4.6%) tested positive by RT-PCR and 92 (4.6%) by serology; and for AdV, 111 (5.5%) tested positive by RT-PCR and 62 (3.1%) by serology. RT-PCR provided the highest number of positive detections overall, but serology increased diagnostic yield for RSV (by 11.8%), HMPV (by 25.0%), AdV (by 32.4%), and PIV (by 48.9%). The method concordance estimated by Cohen's kappa coefficient (κ) ranged from good (for RSV; κ = 0.73) to fair (for AdV; κ = 0.27). Heterotypic seroresponses observed between PIVs and persistent low-level AdV shedding may account for the higher method discordance observed with each of these viruses. Serology can be a helpful adjunct to RT-PCR for research-based assessment of the etiologic contribution of respiratory viruses other than influenza virus to CAP.

Testing and genotyping of high-risk human papillomavirus by the cobas HPV Test and the Hybrid Capture 2 high-risk HPV DNA test using cervical and vaginal samples.

The accurate detection and typing of high-risk human papillomavirus (HPV) are critical for cervical cancer screening. The Hybrid Capture 2 (hc2) and cobas HPV tests showed high agreement for cervical samples (94.4%, κ=0.72, n=693) and moderate agreement for vaginal samples (κ=0.62, n=108). The HPV16 and HPV18 results were highly consistent between the cobas and Linear Array tests (κ≥0.96, n=197). Three hc2-negative vaginal samples were repeatedly invalid by the cobas test due to ß-globin control failures, highlighting amplification control benefits. No cross-contamination was detected in a challenge experiment.

Large-scale analysis of the prevalence and geographic distribution of HIV-1 non-B variants in the United States.

The genetic diversity of human immunodeficiency virus type 1 (HIV-1) has significant implications for diagnosis, vaccine development, and clinical management of patients. Although HIV-1 subtype B is predominant in the United States, factors such as global travel, immigration, and military deployment have the potential to increase the proportion of non-subtype B infections. Limited data are available on the prevalence and distribution of non-B HIV-1 strains in the United States. We sought to retrospectively examine the prevalence, geographic distribution, diversity, and temporal trends of HIV-1 non-B infections in samples obtained by ARUP Laboratories, a national reference laboratory, from all regions of the United States. HIV-1 pol sequences from 24,386 specimens collected from 46 states between 2004 and September 2011 for drug resistance genotyping were analyzed using the REGA HIV-1 Subtyping Tool, version 2.0. Sequences refractory to subtype determination or reported as non-subtype B by this tool were analyzed by PHYLIP version 3.5 and Simplot version 3.5.1. Non-subtype B strains accounted for 3.27% (798/24,386) of specimens. The 798 non-B specimens were received from 37 states and included 5 subtypes, 23 different circulating recombinant forms (CRFs), and 39 unique recombinant forms (URFs). The non-subtype B prevalence varied from 0% in 2004 (0/54) to 4.12% in 2011 (201/4,884). This large-scale analysis reveals that the diversity of HIV-1 in the United States is high, with multiple subtypes, CRFs, and URFs circulating. Moreover, the geographic distribution of non-B variants is widespread. Data from HIV-1 drug resistance testing have the potential to significantly enhance the surveillance of HIV-1 variants in the United States.

Evaluation of the FilmArray® Respiratory Panel for clinical use in a large children's hospital.

BACKGROUND: Respiratory pathogens are a leading cause of hospital admission and traditional detection methods are time consuming and insensitive. Multiplex molecular detection methods have recently been investigated in hope of replacing these traditional techniques with rapid panel-based testing. OBJECTIVES: This study evaluated the FilmArray(®) Respiratory Panel ([FARP], Idaho Technology Inc., Salt Lake City, UT) as a replacement for direct fluorescent antibody (DFA) testing in a pediatric hospital. METHODS: Eleven of the 21 FARP analytes (Adenovirus, Bordetella pertussis, human Metapneumovirus, Influenza A, Influenza A H1N1 2009, Influenza B, Parainfluenza [1, 2, & 3], Respiratory Syncytial Virus, and rhinovirus) were evaluated using nasopharyngeal specimens. Positive samples were pooled in groups of 5. Samples identified by reference methods as positive for respiratory pathogens were used for the majority of positive samples. DFA was the reference method for ten analytes; Luminex™ xTAG Respiratory Virus Panel (RVP) was the reference method for rhinovirus. Discrepant results were resolved by positive culture and fluorescent antibody stain and/or laboratory-developed real-time polymerase chain reaction (PCR) assays (LDT). RESULTS: The agreement for most analytes was in concordance with the established reference methods with the exception of Adenovirus. Additionally, the FARP detected several pathogens not previously detected by DFA, and most were confirmed by LDT. Several DFA-positive analytes were confirmed as true-negatives by the FARP and LDT. CONCLUSION: FARP overall performed better than DFA with the exception of Adenovirus, making the FARP an attractive alternative to laboratories looking to replace DFA with a rapid, user-friendly, multiplex molecular assay.

Standardization of SARS-CoV-2 Cycle Threshold Values: Multisite Investigation Evaluating Viral Quantitation across Multiple Commercial COVID-19 Detection Platforms.

The demand for testing during the coronavirus disease 2019 (COVID-19) pandemic has resulted in the production of several different commercial platforms and laboratory-developed assays for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This has created several challenges, including, but not limited to, the standardization of diagnostic testing, utilization of cycle threshold (CT) values for quantitation and clinical interpretation, and data harmonization. Using reference standards consisting of a linear range of SARS-CoV-2 concentrations quantitated by viral culture-based methods and droplet digital PCR, we investigated the commutability and standardization of SARS-CoV-2 quantitation across different laboratories in the United States. We assessed SARS-CoV-2 CT values generated on multiple reverse transcription-PCR (RT-PCR) platforms and analyzed PCR efficiencies, linearity, gene targets, and CT value agreement. Our results demonstrate the inappropriateness of using SARS-CoV-2 CT values without established standards for viral quantitation. Further, we emphasize the importance of using reference standards and controls validated to independent assays, to compare results across different testing platforms and move toward better harmonization of COVID-19 quantitative test results. IMPORTANCE From the onset of the COVID-19 pandemic, the demand for SARS-CoV-2 testing has resulted in an explosion of analytical tests with very different approaches and designs. The variability in testing modalities, compounded by the lack of available commercial reference materials for standardization early in the pandemic, has led to several challenges regarding data harmonization for viral quantitation. In this study, we assessed multiple commercially available RT-PCR platforms across different laboratories within the United States using standardized reference materials characterized by viral culture methods and droplet digital PCR. We observed variability in the results generated by different instruments and laboratories, further emphasizing the importance of utilizing validated reference standards for quantitation, to better harmonize SARS-CoV-2 test results.

Comparison of three Roche hepatitis B virus viral load assay formats.

Two FDA-approved (in vitro diagnostic [IVD]) hepatitis B virus (HBV) viral load assays, the manual Cobas TaqMan HBV Test for use with the High Pure System (HP) and the automated Cobas AmpliPrep/Cobas TaqMan HBV Test v2.0 (CAP/CTM), were compared to a modified (not FDA-approved) version of the HP assay by automating the DNA extraction using the Total Nucleic Acid Isolation (TNAI) kit on the Cobas AmpliPrep. On average, CAP/CTM measurements were 0.08 log IU/ml higher than HP results (n = 206), and TNAI results were 0.17 log IU/ml higher than HP results (n = 166). The limit of detection (LOD), as determined by probit analysis using dilutions of the 2nd HBV international standard, was 10.2 IU/ml for CAP/CTM. The data sets for HP and TNAI were insufficient for probit analysis; however, there was 100% detection at ≥ 5 or ≥ 10 IU/ml for TNAI and HP, respectively. Linearity was demonstrated between 60 and 2,000,000 IU/ml, with slopes between 0.95 and 0.99 and R(2) values of >0.99 for all assays. Total precision (log percent coefficient of variance [CV]) was between 0.8% and 2.1% at 4.3 log IU/ml and between 1.4% and 4.9% at 2.3 log IU/ml. Correlation of samples, reproducibility, linearity, and LOD were acceptable and similar in all assays. The CAP/CTM assay and, to a lesser extent, the TNAI assay reduced hands-on time due to automation. There were no instances of contamination detected in negative samples during the course of the study, despite testing several samples up to 9.6 log IU/ml. The incidence of false-positive negative controls in HP and CAP/CTM clinical testing was <0.5% over 6 to 7 months of testing.

Evaluation of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test and identification of rare polymorphisms potentially affecting assay performance.

We evaluated the FDA-approved Roche Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) HIV-1 viral load assay for sensitivity, reproducibility, linearity, HIV-1 subtype detection, and correlation to the Roche Amplicor HIV-1 monitor test, version 1.5 (Amplicor). The limit of detection calculated by probit analysis was 23.8 copies/ml using the 2nd International WHO Standard and 30.8 copies/ml using Viral Quality Assurance (VQA) standard material. Serial dilutions of six patient samples were used to determine inter- and intra-assay reproducibility and linearity, which were very good (<8% coefficient of variation [CV]; between approximately 1.7 and 7.0 log(10) copies/ml). Subtype detection was evaluated in the CAP/CTM, Amplicor, and Bayer Versant HIV-1 bDNA 3.0 (Versant) assays using a commercially available panel. Versant averaged 0.829 log(10) copies/ml lower than CAP/CTM and Amplicor averaged 0.427 log(10) copies/ml lower than CAP/CTM for the subtype panel. Correlation with samples previously tested by Amplicor was excellent (R(2) = 0.884; average difference [Amplicor value minus CAP/CTM value], 0.008 log(10) copies/ml). Of the 305 HIV samples tested, 7 samples generated CAP/CTM titers between 1.0 and 2.75 log(10) copies/ml lower than those for Amplicor. Three of these samples revealed primer and probe mismatches that could account for the discrepancies. Otherwise, the CAP/CTM assay exhibits excellent sensitivity, dynamic range, reproducibility, and correlation with Amplicor in an automated format.

Decreasing median age of COVID-19 cases in the United States-Changing epidemiology or changing surveillance?

BACKGROUND: Understanding and monitoring the demographics of SARS-CoV-2 infection can inform strategies for prevention. Surveillance monitoring has suggested that the age distribution of people infected with SARS-CoV-2 has changed since the pandemic began, but no formal analysis has been performed. METHODS: Retrospective review of SARS-CoV-2 molecular testing results from a national reference laboratory was performed. Result distributions by age and positivity were compared between early period (March-April 2020) and late periods (June-July 2020) of the COVID-19 pandemic. Additionally, a sub-analysis compared changing age distributions between inpatients and outpatients. RESULTS: There were 277,601 test results of which 19320 (7.0%) were positive. The median age of infected people declined over time (p < 0.0005). In March-April, the median age of positive people was 40.8 years (Interquartile range (IQR): 29.0-54.1). In June-July, the median age of positive people was 35.8 years (IQR: 24.0-50.2). The positivity rate of patients under 50 increased from 6.0 to 10.6 percent and the positivity rate for those over 50 decreased from 6.3 to 5.0 percent between the early and late periods. The trend was only observed for outpatient populations. CONCLUSIONS: We confirm that there is a trend toward decreasing age among persons with laboratory-confirmed SARS-CoV-2 infection, but that these trends seem to be specific to the outpatient population. Overall, this suggests that observed age-related trends are driven by changes in testing patterns rather than true changes in the epidemiology of SARS-CoV-2 infection. This calls for caution in interpretation of routine surveillance data until testing patterns stabilize.

In Search of Covariates of HIV-1 Subtype B Spread in the United States-A Cautionary Tale of Large-Scale Bayesian Phylogeography.

Infections with HIV-1 group M subtype B viruses account for the majority of the HIV epidemic in the Western world. Phylogeographic studies have placed the introduction of subtype B in the United States in New York around 1970, where it grew into a major source of spread. Currently, it is estimated that over one million people are living with HIV in the US and that most are infected with subtype B variants. Here, we aim to identify the drivers of HIV-1 subtype B dispersal in the United States by analyzing a collection of 23,588 pol sequences, collected for drug resistance testing from 45 states during 2004-2011. To this end, we introduce a workflow to reduce this large collection of data to more computationally-manageable sample sizes and apply the BEAST framework to test which covariates associate with the spread of HIV-1 across state borders. Our results show that we are able to consistently identify certain predictors of spread under reasonable run times across datasets of up to 10,000 sequences. However, the general lack of phylogenetic structure and the high uncertainty associated with HIV trees make it difficult to interpret the epidemiological relevance of the drivers of spread we are able to identify. While the workflow we present here could be applied to other virus datasets of a similar scale, the characteristic star-like shape of HIV-1 phylogenies poses a serious obstacle to reconstructing a detailed evolutionary and spatial history for HIV-1 subtype B in the US.

Evaluation of the Abbott investigational use only RealTime hepatitis C virus (HCV) assay and comparison to the Roche TaqMan HCV analyte-specific reagent assay.

The accurate and sensitive measurement of hepatitis C virus (HCV) RNA is essential for the clinical management and treatment of infected patients and as a research tool for studying the biology of HCV infection. We evaluated the linearity, reproducibility, precision, limit of detection, and concordance of viral genotype quantitation of the Abbott investigational use only RealTime HCV (RealTime) assay using the Abbott m2000 platform and compared the results to those of the Roche TaqMan Analyte-Specific Reagent (TaqMan) and Bayer Versant HCV bDNA 3.0 assay. Comparison of 216 samples analyzed by RealTime and TaqMan assays produced the following Deming regression equation: RealTime = 0.940 (TaqMan) + 0.175 log(10) HCV RNA IU/ml. The average difference between the assays was 0.143 log(10) RNA IU/ml and was consistent across RealTime's dynamic range of nearly 7 log(10) HCV RNA IU/ml. There was no significant difference between genotypes among these samples. The limit of detection using eight replicates of the World Health Organization HCV standard was determined to be 7.74 HCV RNA IU/ml by probit analysis. Replicate measurements of commercial genotype panels were significantly higher than TaqMan measurements for most samples and showed that the RealTime assay is able to detect all genotypes with no bias. Additionally, we showed that the amplicon generated by the widely used Roche COBAS Amplicor Hepatitis C Virus Test, version 2.0, can act as a template in the RealTime assay, but potential cross-contamination could be mitigated by treatment with uracil-N-glycosylase. In conclusion, the RealTime assay accurately measured HCV viral loads over a broad dynamic range, with no significant genotype bias.

Evaluation of Nanogen MGB Alert Detection Reagents in a multiplex real-time PCR for influenza virus types A and B and respiratory syncytial virus.

A multiplex real-time RT-PCR assay that detects influenza A, influenza B and respiratory syncytial virus (RSV) using the MGB Alert Influenza A&B/RSV Detection Reagent RUO (Nanogen, San Diego, CA) was developed. The Nanogen detection reagents consist of PCR primers and minor groove binder-conjugated hybridization probes for each virus and an internal control. Virus typing was determined by post-PCR melt curve analysis. A non-competitive armored RNA internal control was co-extracted with each sample to monitor nucleic acid extraction and RT-PCR. The assay was evaluated using a collection of culture, DFA and RT-PCR (Hexaplex, Prodesse, Waukesha, WI) positive and negative samples. The real-time multiplex assay detected 236 of 237 positive specimens for a 99% correlation. Of 30 Hexaplex negative samples tested, the multiplex real-time assay detected an additional 7 positives confirmed using additional PCR assays. Melt curve analysis for each virus produced average melting peaks of 60.4 degrees C, 66.7 degrees C and 69.4 degrees C for influenza A, influenza B and RSV respectively. Sequence analysis of 7 influenza A samples producing aberrant melt curves, confirmed the presence of a single nucleotide polymorphism beneath the influenza A probe. The limit of detection of each virus in 4 different sample types was measured to be between 7 and 806 copies. Overall, the multiplexed real-time RT-PCR assay was sensitive, robust and easy to use.

The use of Armored RNA as a multi-purpose internal control for RT-PCR.

Real time reverse transcriptase-PCR (RT-PCR) is now used commonly for the detection of viral pathogens in respiratory samples. However, due to potential inhibition of the RT-PCR or inefficient extraction, this sample type can present significant challenges to accurate patient testing. The goal of this study was to create an internal control to be multiplexed in a real time RT-PCR assay for detecting a viral target in respiratory samples. This report describes an Armored RNA (aRNA) internal control developed originally to be multiplexed in a real time RT-PCR assay for detecting SARS-associated Coronavirus, but can be incorporated into any RT-PCR assay. The internal control primers and probe target a region in the coat protein gene of the E. coli F-specific bacteriophage ms2, which is contained within the aRNA.

Clinical course and spectrum of intensive care unit patients reactivating herpes simplex-1 virus: a retrospective analysis.

BACKGROUND: Herpes simplex-1 virus (HSV-1) reactivation in the respiratory tract is common in intensive care unit (ICU) patients. However, susceptible ICU populations are poorly defined. Clinical recognition of HSV infection of the respiratory tract is difficult and the impact of such reactivation is not understood. MATERIALS AND METHODS: A retrospective analysis of HSV-1 positive patients encountered over a 5-year period at a multispecialty ICU was carried out. HSV-1 was identified in respiratory secretions using a qualitative polymerase chain reaction (PCR) technique. Patient charts were reviewed for clinical features that would typify HSV-1 respiratory involvement, and the morbidity and mortality risks found with HSV-1 respiratory involvement. RESULTS: A review of 48 HSV-1 positive ICU patients showed that patients reactivating HSV in the respiratory tract fell into one of the three categories: (1) septic elderly patients with and without ARDS, (2) immunosuppressed patients, especially those receiving high-dose steroids, and (3) post-thoracotomy patients. Abnormalities suggestive of HSV-1 reactivation in the respiratory tract included, haemorrhagic or excessive respiratory secretions, concomitant orofacial herpes (42%), and bronchoscopic abnormalities (hemorrhagic ulcers and mucosal friability) (83%). Twenty eight percent of the HSV-1 infected patients experienced postextubation stridor. HSV-1 reactivation was associated with extended ventilator stays, significant mortality (42%), and ventilator-associated pneumonias (52%). CONCLUSIONS: Identification of susceptible populations and definition of clinical features of HSV-1 related respiratory disease can enable diagnosis of HSV-1 infection in ICU patients. Although detection by a PCR technique can rapidly diagnose HSV-1 reactivation, prospective studies are required to clarify HSV disease versus mere shedding, and understand the impact of HSV-1 reactivation in hospitalized patients.