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.

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.

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.

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.

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.

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.

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.