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.

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.

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 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.

Evaluation of the Abbott RealTime HCV genotype II plus RUO (PLUS) assay with reference to core and NS5B sequencing.

BACKGROUND: HCV genotyping remains a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. Current commercial genotyping assays may have difficulty identifying 1a, 1b and genotype 6. OBJECTIVE: To evaluate the concordance for identifying 1a, 1b, and genotype 6 between two methods: the PLUS assay and core/NS5B sequencing. STUDY DESIGN: This study included 236 plasma and serum samples previously genotyped by core/NS5B sequencing. Of these, 25 samples were also previously tested by the Abbott RealTime HCV GT II Research Use Only (RUO) assay and yielded ambiguous results. The remaining 211 samples were routine genotype 1 (n=169) and genotype 6 (n=42). Genotypes obtained from sequence data were determined using a laboratory-developed HCV sequence analysis tool and the NCBI non-redundant database. RESULTS: Agreement between the PLUS assay and core/NS5B sequencing for genotype 1 samples was 95.8% (162/169), with 96% (127/132) and 95% (35/37) agreement for 1a and 1b samples respectively. PLUS results agreed with core/NS5B sequencing for 83% (35/42) of unselected genotype 6 samples, with the remaining seven "not detected" by the PLUS assay. Among the 25 samples with ambiguous GT II results, 15 were concordant by PLUS and core/NS5B sequencing, nine were not detected by PLUS, and one sample had an internal control failure. CONCLUSIONS: The PLUS assay is an automated method that identifies 1a, 1b and genotype 6 with good agreement with gold-standard core/NS5B sequencing and can aid in the resolution of certain genotype samples with ambiguous GT II results.

HCV RNA measurement in samples with diverse genotypes using versions 1 and 2 of the Roche COBAS® AmpliPrep/COBAS® TaqMan® HCV test.

BACKGROUND: Accurate HCV RNA measurement is required for monitoring treatment. Underquantification has been reported with some genotypes, particularly genotype 4, using version 1 of the Roche COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HCV Test. OBJECTIVES: Compare the viral loads of clinical specimens representing diverse genotypes from across the United States using versions 1 (V1) and 2 (V2) of the Roche COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HCV Test. Assess the frequency of nt145 and nt165 variants in the 5' UTR associated with detection failures and underquantification in a large clinical sample database. STUDY DESIGN: Three hundred archived clinical samples were measured using V1 and V2. Bland-Altman analysis was performed on log-transformed results and compared by genotype. The frequencies of nt145 and nt165 variants from 15,817 sequences were calculated. RESULTS: On average, V2 results were 0.16 logIU/mL lower than V1 results. The average genotype 4 sample was 0.08 logIU/mL higher in V1 than V2. The largest individual sample differences were -2.10 (genotype 2b) and 1.57 (genotype 2a) logIU/mL. For genotype 4 samples, the greatest underquantification by V1 was 1.46 logIU/mL. There were 13 (0.082%) variants at nt145 and 24 variants at nt165 (0.152%), including one sequence with variants at both positions (0.0063%). CONCLUSIONS: Genotype 4 samples from the U.S. are rarely underquantified and not disproportionately so compared to other genotypes using the COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HCV Tests. Variants at the nt145 and nt165 positions are uncommon in the U.S. and double variants are exceedingly rare. Underquantification of HCV samples with the V1 assay is likely a very rare occurrence in U.S.

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.).

Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay with reference to 5'UTR, core and NS5B sequencing.

BACKGROUND: HCV genotyping is a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. OBJECTIVE: To evaluate the concordance between the Abbott GT II assay and genotyping by sequencing subregions of the HCV 5'UTR, core and NS5B. STUDY DESIGN: The Abbott assay was used to genotype 127 routine patient specimens and 35 patient specimens with unusual subtypes and mixed infection. Abbott results were compared to genotyping by 5'UTR, core and NS5B sequencing. Sequences were genotyped using the NCBI non-redundant database and the online genotyping tool COMET. RESULTS: Among routine specimens, core/NS5B sequencing identified 93 genotype 1s, 13 genotype 2s, 15 genotype 3s, three genotype 4s, two genotype 6s and one recombinant specimen. Genotype calls by 5'UTR, core, NS5B sequencing and the Abbott assay were 97.6% concordant. Core/NS5B sequencing identified two discrepant samples as genotype 6 (subtypes 6l and 6u) while Abbott and 5'UTR sequencing identified these samples as genotype 1 with no subtype. The Abbott assay subtyped 91.4% of genotype 1 specimens. Among the 35 rare specimens, the Abbott assay inaccurately genotyped 3k, 6e, 6o, 6q and one genotype 4 variant; gave indeterminate results for 3g, 3h, 4r, 6m, 6n, and 6q specimens; and agreed with core/NS5B sequencing for mixed specimens. CONCLUSIONS: The Abbott assay is an automated HCV genotyping method with improved accuracy over 5'UTR sequencing. Samples identified by the Abbott assay as genotype 1 with no subtype may be rare subtypes of other genotypes and thus require confirmation by another method.

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 Roche COBAS AmpliPrep/COBAS TaqMan HCV Test.

The performance of the Roche COBAS AmpliPrep/COBAS TaqMan HCV Test (CAP/CTM) was evaluated. The limit of detection was 9.5 IU/mL using the 3rd International Standard. Serial dilutions of each genotype demonstrated good reproducibility and linearity. Correlation with samples previously tested with the Roche Analyte Specific Reagent (ASR) was very good, with the CAP/CTM assay measuring 0.24 log IU/mL higher on average than ASR. Genotype inclusivity evaluated in the CAP/CTM, ASR, and Siemens Versant HCV RNA 3.0 Assay (bDNA) assay using a commercially available panel showed higher measurements than ASR or bDNA. The differences in observed CAP/CTM and ASR results for genotype 3 patient samples were significantly different (P < 0.05) from those for both genotype 1 and 2 samples. Common inhibitory substances had no more than a 0.25 log IU/mL affect. Overall, the automated CAP/CTM assay exhibits excellent sensitivity, reproducibility, and dynamic range. Its performance is compatible with its use in guiding therapy using direct acting antivirals such as boceprevir and telaprevir.

Application of a new informatics tool for contamination screening in the HIV sequencing laboratory.

BACKGROUND: Current HIV-1 sequencing-based methods for detecting drug resistance-associated mutations are open and susceptible to contamination. Informatic identification of clinical sequences that are nearly identical to one another may indicate specimen-to-specimen contamination or another laboratory-associated issue. OBJECTIVES: To design an informatic tool to rapidly identify potential contamination in the clinical laboratory using sequence analysis and to establish reference ranges for sequence variation in the HIV-1 protease and reverse transcriptase regions among a U.S. patient population. STUDY DESIGN: We developed an open-source tool named HIV Contamination Detection (HIVCD). HIVCD was utilized to make pairwise comparisons of nearly 8000 partial HIV-1 pol gene sequences from patients across the United States and to calculate percent identities (PIDs) for each pair. ROC analysis and standard deviations of PID data were used to determine reference ranges for between-patient and within-patient comparisons and to guide selection of a threshold for identifying abnormally high PID between two unrelated sequences. RESULTS: The PID reference range for between-patient comparisons ranged from 83.8 to 95.7% while within-patient comparisons ranged from 96 to 100%. Interestingly, 48% of between-patient sequence pairs with a PID>96.5 were geographically related. The selected threshold for abnormally high PIDs was 96 (AUC=0.993, sensitivity=0.980, specificity=0.999). During routine use, HIVCD identified a specimen mix-up and the source of contamination of a negative control. CONCLUSIONS: In our experience, HIVCD is easily incorporated into laboratory workflow, useful for identifying potential laboratory errors, and contributes to quality testing. This type of analysis should be incorporated into routine laboratory practice.

Large-scale comparison of Roche Cobas AmpliPrep/Cobas TaqMan and Abbott RealTime HIV assays.

Significant underquantitation of HIV RNA has been reported with the Roche Cobas AmpliPrep/Cobas TaqMan HIV Test (Version 1) compared to other assays. However, these studies have generally involved limited numbers of samples from select patient populations or analysis of samples that were undetectable in the TaqMan assay. Random plasma samples submitted from throughout the United States for HIV RNA quantitation (n=1263) were compared in the Roche TaqMan and Abbott RealTime assays. Twenty-four samples (1.9%) were discrepant, with a maximum difference between the two assays of 1.9logcopies/mL. These data indicate that both tests may be susceptible to underquantitation, but the incidence is low in this large cohort of samples from across the United States.

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.

Development and validation of a hepatitis B virus DNA sequencing assay for assessment of antiviral resistance, viral genotype and surface antigen mutation status.

The objective of this study was to develop a DNA sequencing assay that examines sensitively and reliably all conserved domains of the reverse transcriptase-encoding region of the HBV genome for antiviral resistance-associated mutations while simultaneously producing ample information for precise genotyping and determination of HBsAg mutation. This assay was used to examine 1000 de-identified HBV DNA positive samples with known viral loads from a broad-based, unselected patient population from across the United States. Of these, 946 were assayed successfully. Antiviral resistance-associated mutations were identified in 104 samples. The escape mutation sG145R in the surface antigen was identified in 0.8% of patient samples. Infections with genotypes A, B, C, D, E, F, G and H were observed in 36.6%, 19.6%, 21.7%, 13.5%, 3.6%, 0.7%, 2.2%, and 0.5% of patient samples respectively. Fifteen samples (1.6%) appeared to harbor infections with multiple genotypes as shown by the presence of double peaks throughout sequence electropherograms. The limit of detection of this assay was approximately 150IU/mL.

A novel capillary electrophoresis-based multiplex PCR assay for detection of respiratory pathogens.

The field of infectious disease testing has recently experienced rapid expansion in the number of multiplexed PCR-based assays available for detecting respiratory pathogens. This study provides a preliminary evaluation of a multiplex assay from Seegene that uses capillary electrophoresis as the detection platform for viral and bacterial respiratory pathogens. We compared this technology to a real-time PCR assay for 3 viral targets. Thirty respiratory samples were collected that had previously tested positive for either Flu A, Flu B, or RSV (ten of each). The Seegene assay detected 9/10 Flu A samples, 9/10 Flu B, and 10/10 RSV, for a total detection rate of 93%. The two samples that were undetected by the Seegene assay both generated late-crossing thresholds on the real-time platform, consistent with low viral loads. The Seeplex assay provides a promising alternative for multiplex respiratory 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.

Development of a multiplex real-time RT-PCR assay for detection of influenza A, influenza B, RSV and typing of the 2009-H1N1 influenza virus.

A high-throughput real-time RT-PCR assay was developed to amplify and detect a conserved region of the hemagglutinin gene of the 2009-H1N1 influenza A virus using a minor groove binder-conjugated hybridization probe. The assay was paired with a separate triplex real-time assay that detects influenza A via the matrix gene, influenza B and RSV in a multiplex format and compared with the Centers for Disease Control and Prevention (CDC) rRT-PCR assay using 143 samples. The 2009-H1N1 portion of the multiplex assay had 100% correlation with the CDC assay, while the triplex assay had a 99% agreement. An additional 105 samples collected from October to November 2009 were also tested using both the individual 2009-H1N1 and triplex assays. Of these 105 samples, eight were positive for the hemagglutinin target in the H1N1 assay and negative for the matrix target in the triplex assay. Discrepant analysis revealed single nucleotide polymorphisms within the matrix gene of 2009-H1N1 virus-positive samples. The limit of detection for the 2009-H1N1 assay was between 750 and 1,500 copies/reaction and no cross-reactivity with other respiratory pathogens was observed. Overall, this multiplexed format proved to be sensitive, robust and easy to use and serves as a useful tool for pandemic testing.