Nucleic Acid Amplification Test Assay Research Articles

False-Positive Human Immunodeficiency Virus Nucleic Acid Amplification Test After Chimeric Antigen Receptor T-Cell Therapy With Ciltacabtagene Autoleucel.

Chimeric antigen receptor (CAR) T-cell therapy has emerged as a novel therapeutic option for hematologic malignancies. Human immunodeficiency virus (HIV) nucleic acid amplification tests (NAATs) amplifying 5' long terminal repeat and gag genes cross-react with lentiviral vector-based CAR T-cell products. Cross-reactivity between CAR T-cell products and HIV NAATs may lead to false-positive test results.

Comparison of fifteen SARS-CoV-2 nucleic acid amplification test assays used during the Canadian Laboratory Response Network's National SARS-CoV-2 Proficiency Program, May 2020 to June 2021.

On March 11, 2020, the World Health Organization declared a pandemic caused by the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This led to increased clinical testing and decentralizing of this testing from provincial health laboratories to regional and private facilities. Leveraging the results from the Canadian Laboratory Response Network's National SARS-CoV-2 Proficiency Test (PT) Program, this study compares multiple commercial and laboratory-developed nucleic acid amplification tests, assessing both sensitivity and specificity across multiple users. Each panel consisted of six blinded, contrived-clinical samples. Panels were distributed to international, provincial and territorial laboratories and subsequently to partner facilities. Participating laboratories were asked to run these sample through their respective extraction/PCR workflows and submit results to the National Microbiology Laboratory, outlining the nucleic acid extraction platform and nucleic acid amplification test employed, as well as the viral gene target and Ct values or equivalent obtained. Data were compiled for each molecular platform and gene target used. The PT schemes were deployed in May 2020, November 2020 and June 2021, resulting in 683 data sets using 37 different nucleic acid amplification tests. Over the course of three PT schemes, the average score obtained was 99.3% by participants demonstrating consistent testing between laboratories and testing platforms. This study confirmed the rapid and successful implementation of a Canadian PT Program and provided comparative analysis of the various emergency use authorized and laboratory developed tests employed for the detection of SARS-CoV-2 and demonstrated an overall 99.3% test concordance nationwide.

A Novel, Highly Sensitive Nucleic Acid Amplification Test Assay for the Diagnosis of Loiasis and its Use for Detection of Circulating Cell-Free DNA.

Mass drug administration programs targeting filarial infections depend on diagnostic tools that are sensitive and specific. The coendemicity of Loa loa with other filarial species often hampers the control programs. LL2634 was identified as the most promising target among several highly repeated targets, with sensitivity between 500 ag and 1 fg of genomic DNA. Using DNA from infected individuals, LL2643 quantitative polymerase chain reaction (qPCR) was positive in all individuals. LL2643 was detected in plasma-derived circulating cell-free DNA (ccfDNA) from 48 of 53 microfilariae-positive patients. Detection of ccfDNA in urine was possible, but it occurred rarely among those tested. Importantly, LL2643 ccfDNA became undetectable within 1 month following diethylcarbamazine (DEC) treatment and remained negative for at least a year. LL2643 offers a more sensitive and specific target for detection of L. loa infection and would be easily configurable to a point-of-contact assay. Clinical Trials Registration. NCT00001230 and NCT00090662.

Performance of saliva compared with nasopharyngeal swab for diagnosis of COVID-19 by NAAT in cross-sectional studies: Systematic review and meta-analysis

Nucleic acid amplification testing (NAAT) is the preferred method to diagnose coronavirus disease 2019 (COVID-19). Saliva has been suggested as an alternative to nasopharyngeal swabs (NPS), but previous systematic reviews were limited by the number and types of studies available. The objective of this systematic review and meta-analysis was to assess the diagnostic performance of saliva compared with NPS for COVID-19. We searched Ovid MEDLINE, Embase, Cochrane, and Scopus databases up to 24 April 2021 for studies that directly compared paired NPS and saliva specimens taken at the time of diagnosis. Meta-analysis was performed using an exact binomial rendition of the bivariate mixed-effects regression model. Risk of bias was assessed using the QUADAS-2 tool. Of 2683 records, we included 23 studies with 25 cohorts, comprising 11,582 paired specimens. A wide variety of NAAT assays and collection methods were used. Meta-analysis gave a pooled sensitivity of 87 % (95 % CI = 83–90 %) and specificity of 99 % (95 % CI = 98–99 %). Subgroup analyses showed the highest sensitivity when the suspected individual is tested in an outpatient setting and is symptomatic. Our results support the use of saliva NAAT as an alternative to NPS NAAT for the diagnosis of COVID-19.

Avoiding False-Positive SARS-CoV-2 Rapid Antigen Test Results with Point-of-Care Molecular Testing on Residual Test Buffer.

ABSTRACTAntigen-based rapid diagnostic tests (Ag-RDTs) have been widely used for the detection of SARS-CoV-2 during the coronavirus disease 2019 (COVID-19) pandemic. In settings of low disease prevalence, such as asymptomatic community testing, national guidelines recommend confirmation of positive Ag-RDT results with a nucleic acid amplification test (NAAT). This often requires patients to be recalled for repeat specimen recollection and subsequent testing in reference laboratories. This project assessed the use of a point-of-care molecular NAAT for SARS-CoV-2 detection (i.e., ID NOW), which was performed on-site at a volunteer-led asymptomatic community testing site on the residual test buffer (RTB) from positive Ag-RDTs. The ID NOW NAAT assay was performed on RTB from two Ag-RDTs: the Abbott Panbio and BTNX Rapid Response assays. Results of ID NOW were compared to real-time RT-PCR at a reference laboratory. Along with investigations into the clinical performance of ID NOW on RTB, analytical specificity was assessed with a panel of various respiratory organisms. Of the Ag-RDTs results evaluated, all 354 Ag-RDTs results characterized as true positives by RT-PCR were accurately identified with ID NOW testing of RTB. No SARS-CoV-2 detections by ID NOW were observed from 10 specimens characterized as false-positive Ag-RDTs, or from contrived specimens with various respiratory organisms. The use of on-site molecular testing on RTB provides a suitable option for rapid confirmatory testing of positive Ag-RDTs, thereby obviating the need for specimen recollection for molecular testing at local reference laboratories.IMPORTANCE During the COVID-19 pandemic, rapid antigen tests have been widely used for the detection of SARS-CoV-2. These simple devices allow rapid test results. However, false-positive results may occur. As such, individuals with positive rapid tests often must return to testing centers to have a second swab collected, which is then transported to a specialized laboratory for confirmation using molecular tests. As an alternative to requiring a repeat visit and a prolonged turn-around time for result confirmation, this project evaluated whether the leftover material from rapid antigen tests could be confirmed directly on a portable point-of-care molecular instrument. Using this approach, molecular confirmation of positive antigen tests could be performed in less than 15 min, and the results were equivalent to laboratory-based confirmation. This procedure eliminates the need for individuals to return to testing centers following a positive rapid antigen test and ensures accurate antigen test results through on-site confirmation.

Network-wide analysis of the Serosep EntericBio Gastro Panel 2 for the detection of enteric pathogens in Public Health Wales microbiology laboratories.

Introduction. A retrospective data analysis of 34 months (spanning 2016-2020) of 961573 diagnostic results obtained before and after nucleic acid amplification testing (NAAT) implementation, across the Public Health Wales microbiology network.Hypothesis / Gap Statement. This is the first network-wide analysis of the implementation of enteric NAAT in diagnostic microbiology.Aim. To assess the outcome of replacing microscopy and bacterial culture with NAAT as the primary test in the diagnosis of: Campylobacter spp., Salmonella sp., Shigella spp., Shiga toxin-producing Escherichia coli (STEC), Cryptosporidium spp. and Giardia duodenalis infections.Methodology. Following NAAT introduction, bacterial culture was performed as a secondary test, to provide further information from NAAT positive samples for epidemiological purposes. Primary detection rates and overall bacterial culture rates were calculated for each target pathogen using both testing regimes (Stage I) including a comparison of in-patient and out-patient diagnoses (Stage II).Results. Stage I analysis showed that the primary detection rate significantly increased for Campylobacter spp. (P<0.0001), Salmonella sp. (P=0.0151), Shigella spp. (P<0.0001), STEC (P<0.0001), Cryptosporidium spp. (P<0.0001) and Giardia duodenalis (P<0.0001) when using NAAT compared to microscopy or bacterial culture. A significant decrease was seen in the overall rate of Campylobacter spp. isolation by bacterial culture (P<0.0001), whilst other targets remained unaffected. Stage II analysis showed that NAAT positive out-patient samples were more likely to be supplemented by a positive bacterial culture than NAAT positive in-patient samples for Campylobacter spp. (P<0.0001), Salmonella sp. (P=0.0004) and STEC (P=0.0039). However, Shigella spp. was more frequently isolated from NAAT positive in-patient samples (P=0.0005). A notable increase was seen for G. duodenalis detection from in-patient samples (P=0.0002). Reference laboratory data showed the NAAT assay can detect at least 53 serotypes of STEC but may not be able to detect some of the rarer species of Cryptosporidium seen in human infections.Conclusion. The implementation of NAAT has significantly increased the primary detection rate of all target enteric pathogens in Wales and information gleaned previously from direct culture is largely unaffected.

Suitability of Different Diagnostic Platforms for Virological Testing of Blood Samples from Cornea Donors

Background: To minimize the risk of disease transmission in cornea transplantation, donor screening for blood-derived viral infections is mandatory. Ideally, pre-mortem blood samples are used, but based on availability, cadaveric blood samples of cornea donors may also be used. However, serological and nucleic acid amplification tests (NATs) need to be validated for the use of cadaveric specimens. Methods: Hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV) 1/2, and Treponema pallidum (syphilis)-specific serological and/or NAT assays were validated on different platforms (Abbott Alinity i, Alinity m, Roche Cobas 6800, and Roche Cobas AmpliPrep/Cobas TaqMan (CAP/CTM)) using (un)spiked paired pre- and post-mortem cornea donor blood samples from the same individual (up to 23.83 h after death) of 28 individuals in accordance with the specifications of the German Federal Institute for Vaccines and Biomedicines (Paul-Ehrlich-Institut [PEI]). In addition, routinely HBV-, HCV- and HIV-PCR-negative tested post-mortem blood samples of 24 individuals were used to assess NAT specificity. Results: For the majority of serological parameters on the Abbott Alinity i (HBsAg, anti-HBc, anti-HBs, anti-HCV, anti-HIV, anti-HTLV 1/2, and anti-Treponema pallidum), ratios of generated test results of (un)spiked paired pre- and post-mortem blood samples differed ≤25%, with an agreement of qualitative pre- and post-mortem test results ranging from 91.2 to 100%. For NAT parameters (HBV, HCV, and HIV) on the Cobas 6800, Alinity m, and CAP/CTM, no significant deviation in virus concentrations (factor >5) of spiked pre- and post-mortem blood samples could be observed. Ct-values of corresponding internal controls did also not differ significantly (>1.5 Ct-values). In addition, no false-positive test results were generated when specificity was assessed. Conclusion: Overall, fluctuations of test results for serological and NAT parameters in pre- and post-mortem blood samples examined in this study, were only limited and within the range of what is also observed when routinely testing fresh patient specimens. We conclude that all examined assays are eligible for the screening of blood samples taken up to about 24 h after the occurrence of death.

Comparative analysis of point-of-care, high-throughput and laboratory-developed SARS-CoV-2 nucleic acid amplification tests (NATs)

Multiple nucleic acid amplification tests (NATs) are available for the detection of SARS-CoV-2 in clinical specimens, including Laboratory Developed Tests (LDT), commercial high-throughput assays and point-of-care tests. Some assays were just recently released and there is limited data on their clinical performance. We compared the Xpert® Xpress SARS-CoV-2 (Cepheid) and Vivalytic VRI Panel (Schnelltest COVID-19) (Bosch) point-of-care tests with four high-throughput assays and one LDT, the cobas® SARS-CoV-2 test (Roche), the Allplex™ 2019-nCoV Assay (Seegene), the SARS-CoV-2 AMP (Abbott) Kit, the RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (altona) as well as an assay using a SARS-CoV-2 RdRP gene specific primer and probe set. Samples from patients with confirmed SARS-CoV-2 infection, samples from the first and second SARS-CoV-2-PCR External Quality Assessment (EQA) (INSTAND e.V.) and a 10-fold serial dilution of a SARS-CoV-2 cell culture (SARS-CoV-2 Frankfurt 1) supernatant were examined. We determined that the NAT assays examined had a high specificity. Assays using the N gene as target demonstrated the highest sensitivity in the serial dilution panel, while all examined NAT assays showed a comparable sensitivity when testing clinical and EQA samples.

Comparative Performance of Four Nucleic Acid Amplification Tests for SARS-CoV-2 Virus.

COVID-19 is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was discovered in 2019 and spread around the world in a short time. SARS-CoV-2 nucleic acid amplification tests (NAATs) have been rapidly developed and quickly applied to clinical testing of COVID-19. Aim of this study was to evaluate the performance of four NAAT assays. Limit of detection (LOD), precision, accuracy, analytical specificity and analytical interference studies on four NAATs (Daan, Sansure, Hybribio, and Bioperfectus) were performed according to Clinical Laboratory Standards Institute protocols and guidelines. The four NAATs were compared using 46 clinical samples. The LOD of the N gene for Daan, Sansure, and Hybribio was 500 copies/mL, and that for Bioperfectus was 1,000 copies/mL. The LOD of the ORF1ab gene for Daan, Bioperfectus, and Hybribio was 3,000 copies/mL, and that for Sansure was 2,000 copies/mL. A good precision was shown at the concentration above 20% of the LOD for all four NAATs, with all individual coefficients of variation below 3.6%. Satisfactory results were also observed in the accuracy, analytical specificity, and analytical interference tests. The results of the comparison test showed that Daan, Sansure, and Hybribio NAATs could detect the samples with a specificity of 100% (30/30) and a sensitivity of 100% (16/16), whereas Bioperfectus NAAT detected the samples with a specificity of 100% (30/30) and a sensitivity of 81.25% (13/16). However, no significant difference in sensitivity was found between Bioperfectus NAAT and the three other NAATs (p > 0.05). The four SARS-CoV-2 NAATs showed comparable performance, with the LOD of the N gene lower than the LOD of the ORF1ab gene.

Use of a NAT-based assay to improve the surveillance system and prevent transfusion-transmitted malaria in blood banks

BackgroundMalaria can be transmitted by blood transfusion through donations collected from asymptomatic donors. Transfusion-transmitted malaria (TTM) poses a great risk to blood services worldwide. A good screening tool for Plasmodium spp. detection in blood banks must have a high sensitivity for prevention of TTM. However, in Brazilian blood banks, screening for malaria still relies on microscopy.MethodsIn Brazil, screening for human immunodeficiency virus type 1 (HIV), RNA/DNA for hepatitis C (HCV) and hepatitis B (HBV) viruses is mandatory for every blood donation and uses nucleic acid amplification testing (NAT). The aim of this study was to evaluate the inclusion of an assay for malaria to identify Plasmodium sp. from total nucleic acid (TNA; DNA/RNA) by targeting the 18S rRNA gene of the parasite.ResultsConsidering the limitations of microscopy and the wide availability of the Brazilian NAT platform in the screening of blood units for HIV, HCV, and HBV, a molecular diagnostic tool was validated for detection of Plasmodium sp. in blood banks; a pilot study showed that using this novel NAT assay could reduce the risk of TTM.ConclusionThe prototype HIV/HCV/HBV/malaria NAT assay was effective in detecting infected candidate donors and has good prospects to be applied in routine screening for preventing TTM.

Performance and Utilization of a Laboratory-Developed Nucleic Acid Amplification Test (NAAT) for the Diagnosis of Pulmonary and Extrapulmonary Tuberculosis in a Low-Prevalence Area.

Tuberculosis (TB) is a significant global health problem. In low-prevalence areas and low clinical suspicion, nucleic acid amplification tests (NAAT) for direct detection of Mycobacterium tuberculosis complex (MTBC) can speed therapy initiation and infection control. An NAAT assay (TBPCR) targeting MTBC IS6110 is used for detecting MTBC in our low-prevalence population. Fifteen-year review of patient records identified 146 patients with culture-positive pulmonary tuberculosis (PTB) or extrapulmonary tuberculosis (EPTB). Laboratory-developed TBPCR was retrospectively compared with standard stain and cultures for PTB and EPTB diagnoses. TBPCR assay was used in 57% of patients with PTB and 33% of patients with EPTB. TBPCR detected 88.4% of all TB (smear-positive, 97%; smear-negative, 79%) with 100% specificity. Low bacterial load was indicated in TBPCR-negative PTB (P = .002) and EPTB (P < .008). TBPCR performance was optimum but significantly underused. Guidelines are proposed for mandated use of TBPCR that capture patients with clinically suspected PTB. Focused TBPCR use in low prevalence populations will benefit patient care, infection prevention, and public health.

A methodology for deriving the sensitivity of pooled testing, based on viral load progression and pooling dilution

BackgroundPooled testing, in which biological specimens from multiple subjects are combined into a testing pool and tested via a single test, is a common testing method for both surveillance and screening activities. The sensitivity of pooled testing for various pool sizes is an essential input for surveillance and screening optimization, including testing pool design. However, clinical data on test sensitivity values for different pool sizes are limited, and do not provide a functional relationship between test sensitivity and pool size. We develop a novel methodology to accurately compute the sensitivity of pooled testing, while accounting for viral load progression and pooling dilution. We demonstrate our methodology on the nucleic acid amplification testing (NAT) technology for the human immunodeficiency virus (HIV).MethodsOur methodology integrates mathematical models of viral load progression and pooling dilution to derive test sensitivity values for various pool sizes. This methodology derives the conditional test sensitivity, conditioned on the number of infected specimens in a pool, and uses the law of total probability, along with higher dimensional integrals, to derive pooled test sensitivity values. We also develop a highly accurate and easy-to-compute approximation function for pooled test sensitivity of the HIV ULTRIO Plus NAT Assay. We calibrate model parameters using published efficacy data for the HIV ULTRIO Plus NAT Assay, and clinical data on viral RNA load progression in HIV-infected patients, and use this methodology to derive and validate the sensitivity of the HIV ULTRIO Plus Assay for various pool sizes.ResultsWe demonstrate the value of this methodology through optimal testing pool design for HIV prevalence estimation in Sub-Saharan Africa. This case study indicates that the optimal testing pool design is highly efficient, and outperforms a benchmark pool design.ConclusionsThe proposed methodology accounts for both viral load progression and pooling dilution, and is computationally tractable. We calibrate this model for the HIV ULTRIO Plus NAT Assay, show that it provides highly accurate sensitivity estimates for various pool sizes, and, thus, yields efficient testing pool design for HIV prevalence estimation. Our model is generic, and can be calibrated for other infections.

Utility of reference materials for Zika Virus nucleic acid testing

The emergence of Zika virus (ZIKV) in the Americas has resulted in increased nucleic acid amplification testing (NAT) of clinical samples and blood donations. New molecular diagnostic assays have been developed resulting in a corollary requirement for ZIKV reference material. To address this we have produced and calibrated two African lineage ZIKV reference materials: a highly concentrated secondary standard (NIBSC: 16/110) and a lower concentration external quality control (QC) reagent (NIBSC: 16/124) and compared their performance in three ZIKV NAT assays in relation with the First International Standard (IS) for Zika Virus NAT assays (PEI: 11468/16). In summary the African lineage ZIKV reference materials were detected by all three assays. The ZIKV lineage did not affect the performance of the secondary standard. The external QC reagent (16/124) was detected by all three assays highlighting its suitability for use as a low positive control to monitor assay performance on a regular basis. The relative potency of 16/110 to the IS was 5.49E+06IU/mL (95% CI: 1.46E+06–2.06E+07) and 16/124 to 16/110 was 8.36E+03 (95% CI: 7.83E+03–8.92E+03). The global availability of African lineage ZIKV reference materials will facilitate standardization of ZIKV molecular diagnostic assays between and within laboratories whilst preserving the IS.

Reliable Rapid Assay for Gonorrhea and Chlamydia in the Emergency Department

BackgroundChlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are common sexually transmitted infections seen in the emergency department (ED). Due to an inability to reliably make accurate diagnosis by physical examination, concern for unreliable follow-up, and current delays in diagnostic nucleic acid amplification testing (NAAT), presumptive treatment active against CT and NG, as described by Centers for Disease Control clinical practice guidelines, is often performed. ObjectivesThe purpose of this study was to determine whether a rapid, urine NAAT performed in the ED is noninferior in its diagnostic sensitivity compared with a traditional, swab NAAT assay. MethodsWe performed a prospective, noninferiority study comparing two U.S. Food and Drug Administration-approved NAAT assays for CT and NG: a 90-min rapid assay, the Xpert CT/NG Assay (Cepheid, Sunnyvale, CA) using a urine sample vs. a traditional assay, the Aptima Combo 2 Assay (Gen-Probe Incorporated, San Diego, CA) using a swab sample. This study was registered on Clinicaltrials.gov (NCT02386514). ResultsA total of 1162 patient samples were included in the primary analysis. We observed excellent kappa agreement between assays: NG for men, 1.00 (95% confidence interval [CI] 1.00–1.00); NG for women, 0.87 (95% CI 0.79–0.94); CT for men, 0.81 (95% CI 0.59–1.00); and CT for women: 0.85 (95% CI 0.80–0.90), as well as excellent negative and positive predictive values for the rapid assay. ConclusionAlthough the rapid Xpert CT/NG assay's diagnostic sensitivity did not meet our prespecified threshold for noninferiority, the diagnostic characteristics are robust enough to fit into a management pathway that may reduce unnecessary antibiotic use. There may be an opportunity to utilize the rapid Xpert CT/NG assay to improve accuracy of treatment in the ED.

Rapid detection and strain typing of Chlamydia trachomatis using a highly multiplexed microfluidic PCR assay.

Nucleic acid amplification tests (NAATs) are recommended by the CDC for detection of Chlamydia trachomatis (Ct) urogenital infections. Current commercial NAATs require technical expertise and sophisticated laboratory infrastructure, are time-consuming and expensive, and do not differentiate the lymphogranuloma venereum (LGV) strains that require a longer duration of treatment than non-LGV strains. The multiplexed microfluidic PCR-based assay presented in this work simultaneously interrogates 13 loci to detect Ct and identify LGV and non-LGV strain-types. Based on amplified fragment length polymorphisms, the assay differentiates LGV, ocular, urogenital, and proctocolitis clades, and also serovars L1, L2, and L3 within the LGV group. The assay was evaluated in a blinded fashion using 95 clinical swabs, with 76 previously reported as urogenital Ct-positive samples and typed by ompA genotyping and/or Multi-Locus Sequence Typing. Results of the 13-plex assay showed that 51 samples fell within urogenital clade 2 or 4, 24 samples showed both clade 2 and 4 signatures, indicating possible mixed infection, gene rearrangement, or inter-clade recombination, and one sample was a noninvasive trachoma biovar (either a clade 3 or 4). The remaining 19 blinded samples were correctly identified as LGV clade 1 (3), ocular clade 3 (4), or as negatives (12). To date, no NAAT assay can provide a point-of-care applicable turnaround time for Ct detection while identifying clinically significant Ct strain types to inform appropriate treatment. Coupled with rapid DNA processing of clinical swabs (approximately 60 minutes from swab-in to result-out), the assay has significant potential as a rapid POC diagnostic for Ct infections.

Calibration of BK Virus Nucleic Acid Amplification Testing to the 1st WHO International Standard for BK Virus.

Significant interassay variability in the quantification of BK virus (BKV) DNA precludes establishing broadly applicable thresholds for the management of BKV infection in transplantation. The 1st WHO International Standard for BKV (primary standard) was introduced in 2016 as a common calibrator for improving the harmonization of BKV nucleic acid amplification testing (NAAT) and enabling comparisons of biological measurements worldwide. Here, we evaluated the Altona RealStar BKV assay (Altona) and calibrated the results to the international unit (IU) using the Exact Diagnostics BKV verification panel, a secondary standard traceable to the primary standard. The primary and secondary standards on Altona had nearly identical linear regression equations (primary standard, Y = 1.05X - 0.28, R2 = 0.99; secondary standard, Y = 1.04X - 0.26, R2 = 0.99) and conversion factors (primary standard, 1.11 IU/copy; secondary standard, 1.09 IU/copy). A comparison of Altona with a laboratory-developed BKV NAAT assay in IU/ml versus copies/ml using Passing-Bablok regression revealed similar regression lines, no proportional bias, and improvement in the systematic bias (95% confidence interval of intercepts: copies/ml, -0.52 to -1.01; IU/ml, 0.07 to -0.36). Additionally, Bland-Altman analyses revealed a clinically significant reduction of bias when results were reported in IU/ml (IU/ml, -0.10 log10; copies/ml, -0.70 log10). These results indicate that the use of a common calibrator improved the agreement between the two assays. As clinical laboratories worldwide use calibrators traceable to the primary standard to harmonize BKV NAAT results, we anticipate improved interassay comparisons with a potential for establishing broadly applicable quantitative BKV DNA load cutoffs for clinical practice.

O10.4 Performance of self-collected penile swabs for the detection ofchlamydia trachomatis,neisseria gonorrhoeae,trichomonas vaginalis, andmycoplasma genitalium

Introduction Urethral swabs are used for culture of gonorrhoea (NG) in males and for detection of chlamydia (CT) and NG by nucleic acid amplification tests (NAATs). We hypothesised self-collected penile swabs would perform as well as urethral swabs for detection of CT, NG, trichomonas (TV) and mycoplasma (MG). Methods Men having urethral swabs obtained for NG culture in the STD clinic volunteered to collect penile swabs. Urethral swabs were placed into NAAT transport media; then self-collected penile swabs were placed in transport media. NAATs were performed for CT, NG, TV, and MG for urethral and penile swabs. Acceptability questionnaires were given. Results For 203 urethral/penile pairs, there were 32 penile positive for CT (15.8%), 31 urethral positive for CT (15.3%); [sensitivity 96.8% and specificity 98.8% compared to urethral swabs]. There were 29 penile positives for NG (14.3%) and 27 urethral positives for NG (13.3%); [sensitivity 100%, specificity 98.9%]. 25 were Gram stain positive; 21 by culture. For TV, there were 23 penile positives (11.3%), 20 urethral positives (9.9%); [sensitivity 85.0%, specificity 96.7%]. For MG, 24 penile swabs were positive (11.8%); and 29 urethral were positive (14.3%); [sensitivity 79.3%, specificity 99.4%]. CT: 2 samples were penile+/urethral-, 1 was penile-/urethral+. NG: 2 samples were penile+/urethral-. TV: 6 samples were penile+/urethral-, 3 were penile-/urethral+. MG: 1 pair was penile+/urethral-, 6 were penile-/urethral+. There were no significant differences between self-collected penile swabs and clinician-collected urethral swabs for NAATs (p = 0.625 for CT; p = 0.248 for NG; p = 0.344 for TV; and P = 0.070 for MG). 100% of men preferred penile swabs for diagnosis. Conclusions Self-collected penile swabs were as accurate as urethral swabs for the detection of sexually transmitted infections for NAAT assays and could expedite express visits in a busy STD clinic. Penile swabs show promise as a method of utilising one sample for multiple STIs. Disclosure of interest statement The research group has previously received research funding from GenProbe/Hologic. No pharmaceutical grants were received in the development of this study.

Diagnostic value of nucleic acid amplification tests on bronchoalveolar lavage fluid for smear-negative pulmonary tuberculosis: a meta-analysis.

The diagnosis of smear-negative pulmonary tuberculosis (SNPT) remains a clinical challenge. Many studies suggest that nucleic acid amplification tests (NAATs) on bronchoalveolar lavage fluid (BALF) plays a role in diagnosing SNPT, but with considerable varying results. The current study aimed to summarize the overall diagnostic accuracy of NAATs assay on BALF for SNPT. A systematic literature search was performed and data were retrieved. Pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio (DOR) were calculated. A summary receiver operating characteristic curve and area under the curve (AUC) were used to evaluate the overall diagnostic performance. All the statistical analysis was performed by using STATA 12.0 and Meta-DiSc 1.4 software. A total of nine studies with 1214 subjects were included this meta-analysis. The pooled sensitivity, specificity, PLR, NLR and DOR were 0.54 [95% CI (confidence interval): 0.48-0.59], 0.97 (95% CI: 0.95-0.98), 12.13 (95% CI: 8.23-17.88), 0.36 (95% CI: 0.23-0.56) and 44.71 (95% CI: 22.30-89.63) respectively. The AUC was 0.96. Estimated positive and negative post-probability values for a SNPT prevalence of 20% were 82% and 7% respectively. No publication bias was identified. Current available evidence indicated that NAATs on BALF may play a role in diagnosing SNPT, whereas the results should be interpreted in parallel with clinical information of patients and the results of traditional tests. Further studies should be performed to confirm our findings.

Prevalence and trends of markers of hepatitis B virus, hepatitis C virus and human Immunodeficiency virus in Argentine blood donors.

BackgroundTransfusion-transmitted infections are a major problem associated with blood transfusion. The aim of this study was to determine prevalence and trends of HBV, HCV and HIV in blood donors in Argentina.MethodsA retrospective study was carried out in blood donors of 27 transfusion centers covering the whole country over a period of eight years (2004-2011). Serologic screening assays for HBsAg, anti-HBc, anti-HCV, and anti-HIV were performed in all centers and nucleic acid amplification testing (NAT) was performed in 2 out of the 27 centers.ResultsThe 2,595,852 samples tested nationwide from 2004 to 2011 showed that the prevalence of HBsAg decreased from 0.336% to 0.198% (p < 0.0001), that of anti-HBc from 2.391% to 2.007% (p < 0.0001), that of anti-HCV from 0.721% to 0.460%, (p < 0.0001) and that of anti-HIV from 0.208% to 0.200 (p = 0.075). The prevalence of HBV, HCV and HIV was unevenly distributed among the different regions of the country. Two out of 74,838 screening- negative samples were positive in NAT assays (1 HIV-RNA and 1 HCV-RNA); moreover, HBV-DNA, HCV-RNA and HIV-RNA were detected in 60.29, 24.54 and 66.67% of screening-positive samples of the corresponding assays. As regards donors age, positive HBV-DNA and HCV-RNA donors were significantly older than healthy donors (46.6, 50.5 and 39.5 y respectively, p < 0.001).ConclusionsArgentina has a low prevalence of HBsAg, anti-HCV and anti-HIV in blood donors, with a decreasing trend for HBsAg, anti-HBc and anti-HCV but not for anti-HIV over the last 8 years. The uneven distribution of transfusion-transmitted infections prevalence among the different regions of the country highlights the need to implement regional awareness campaigns and prevention. The discrepancy between samples testing positive for screening assays and negative for NAT assays highlights the problem of blood donors who test repeatedly reactive in screening assays but are not confirmed as positive upon further testing. The uneven distribution of age between healthy donors and NAT-positive donors could be related to changes in risks of these pathogens in the general population and might be attributed to a longer exposure to transmission risk factors in elderly people.

A pilot study on screening blood donors with individual-donation nucleic acid testing in China.

Nucleic acid amplification testing (NAT) is not yet obligatory in China for blood donor screening and the risk of enzyme immunoassay (EIA)-negative, NAT-reactive donations in Chinese blood donors has rarely been reported. The aim of this study was to screen a population of Chinese blood donors using a triplex individual-donation (ID)-NAT assay and assess the safety benefits of implementing NAT. Between 1st August, 2010 and 31st December, 2011 all donations at a Chinese blood centre were screened individually using the Procleix® Ultrio® assay, a multiplex NAT assay for the detection of hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA and human immunodeficiency virus-1 (HIV-1) RNA. All donations were also screened for HBsAg, anti-HIV and anti-HCV using two different EIA for each marker. Samples with discordant results between NAT and EIA were further tested with an alternative NAT assay (Cobas® TaqMan®). Potential yield cases (serologically negative/NAT-reactive donors) were further evaluated when possible. During the study period a total of 178,447 donations were screened by NAT and EIA, among which 169 HBV NAT yield cases (0.095%) were detected. No N AT yield cases were found for HIV-1 or HCV. For the HBV NAT yield cases, follow-up results showed that 11 (6.51%) were probable or confirmed HBV window period infections, 5 (2.96%) were chronic HBV carriers and 153 (90.53%) were probable or confirmed occult HBV infections. There was a statistically significant difference between the NAT-positive rates for first-time vs repeat donations (0.472% vs 0.146%, respectively; P<0.001). Our data demonstrate that the potential HBV yield rate was 1:1,056 for blood donations in the Zhejiang province of China. Implementation of NAT will provide a significant increment in safety relative to serological screening alone.