Commercial Nucleic Acid Amplification Tests Research Articles

Guidelines for the laboratory diagnosis of trichomoniasis in East European countries

The laboratory diagnosis of sexually transmitted infections in many Eastern European countries remains suboptimal. The main objective of the present evidence-based guidelines is to provide comprehensive information regarding the laboratory diagnosis of infections caused by Trichomonas vaginalis in East European countries. In particular, the present guidelines recommend: (i) to encourage examination of the wet mounts of vaginal exudates, instead of stained smears, at all clinical settings; (ii) nucleic acid amplification tests (NAATs) or culture could be employed if no trichomonads are detected on microscopic examination of the wet preparation and there is a strong indication of infection and (iii) the use of NAATs is encouraged in screening, using non-invasive specimens, or high volume testing situations. In the absence of internationally recognized commercial NAAT systems, tests developed in-house should be validated using obtainable international standards and quality assured strictly. Individual East European countries may be required to make minor national adjustments to these guidelines as a result of lack of accessibility to some reagents or equipment, or laws in a specific country.

Nucleic Acid Amplification Tests for Diagnosis of Neisseria gonorrhoeae and Chlamydia trachomatis Rectal Infections

It is uncertain which methods for the diagnosis of rectal gonococcal and chlamydial infection are optimal. This study evaluated the performance of culture and nucleic acid amplification tests (NAATs) for rectal chlamydial and gonococcal diagnosis. From July 2003 until February 2007, 441 rectal test sets were collected from individuals attending a sexually transmitted disease clinic and three HIV clinics who gave a history of anal intercourse or were women at high risk for Neisseria gonorrhoeae or Chlamydia trachomatis infections. Rectal swab specimens were tested using culture and commercial NAATs employing transcription-mediated amplification (TMA), strand displacement amplification (SDA), and PCR amplification. Test performance was evaluated using a rotating standard by which patients were classified as infected if either two or three comparator tests were positive. Test sensitivities for the detection of N. gonorrhoeae ranged from 66.7% to 71.9% for culture to 100% for TMA. Specificities were 99.7% to 100% for culture and greater than 95.5% for all three NAATs. Test sensitivities for C. trachomatis ranged from 36.1% to 45.7% for culture and among NAATS from 91.4% to 95.8% for PCR to 100% for TMA. Specificities of the NAATs ranged from 95.6% to 98.5% (two-of-three standard) and from 88.8% to 91.8% (three-of-three standard). Over 60% and 80% of gonococcal and chlamydial infections, respectively, among men who have sex with men and over 20% of chlamydial infections in women would have been missed if the rectal site had not been tested. Currently available NAATs are more sensitive for the detection of chlamydial and gonococcal infection at the rectal site than is culture.

Guidelines for the Laboratory Diagnosis of Mycoplasma genitalium Infections in East European Countries

The present guidelines aim to provide comprehensive information regarding laboratory diagnosis of Mycoplasma genitalium infections in East European countries. These guidelines are intended primarily for laboratory professionals testing specimens from patients at sexual health care clinics, but may also be useful for community-based screening programmes. Diagnosis of M. genitalium infection is performed exclusively using nucleic acid amplification tests (NAATs), owing to the poor and slow growth of the bacterium in culture. Because no internationally validated and approved commercial NAAT for M. genitalium detection is presently available, it is necessary that laboratories performing M. genitalium diagnostics not only carefully evaluate and validate their in-house PCRs before using them routinely, but also use comprehensive internal controls and take part in external quality assessment programmes. The guidelines were elaborated as a consensus document of the Eastern European Sexual and Reproductive Health (EE SRH) Network, and comprise one element of a series of guidelines aimed at optimizing, standardizing, and providing guidance on quality laboratory testing for reproductive tract infections.

Can E-Technology Through the Internet be Used as a New Tool to Address the Chlamydia trachomatis Epidemic by Home Sampling and Vaginal Swabs?

The Internet provides patients, as well as clinicians with immediate, confidential access to information about sexually transmitted diseases (STDs).1,2 Young individuals are avid users of the Internet and usage has been considered to be a risk factor for STDs.3–7 Data indicate that there are 246,402,574 North American Internet users.8

The Swedish new variant of Chlamydia trachomatis (nvCT) remains undetected by many European laboratories as revealed in the recent PCR/NAT ring trial organised by INSTAND e.V., Germany

The May 2009 round of INSTAND's ring trial "Chlamydia trachomatis detection PCR/NAT" included a sample with high amount of the Swedish new variant of C. trachomatis (nvCT). A spectrum of at least 12 different commercial diagnostic nucleic acid amplification tests (NAATs) and many different in house NAATs were applied by the 128 participating laboratories which reported 152 results. Approximately 80% of the results correctly reported the presence of C. trachomatis in the nvCT specimen. The nvCT sample was mainly missed, as expected, by participants using the Roche COBAS Amplicor CT/NG (15.5% of reported results) but also by several participants using in house NAATs. The trend towards using nvCT-detecting NAATs is obvious and in addition to the new dual-target NAATs from Roche and Abbott, and BD ProbeTec ET, also a number of new CE mark-certified commercial tests from smaller diagnostic companies as well as many different in house NAATs were used. Laboratories using commercial or in house NAATs that do not detect the nvCT are encouraged to carefully monitor their C. trachomatis incidence, participate in appropriate external quality assurance and controls schemes, and consider altering their testing system. The reliable detection of low amounts of the wildtype C. trachomatis strain in other samples of the ring trial set indicates a good diagnostic performance of all applied commercial NAATs while also detecting the nvCT strain.

Reproducibility and specificity concerns associated with nucleic acid amplification tests for detecting Chlamydia trachomatis

Commercial nucleic acid amplification tests (NAATs) have become one of the most frequently used tests for detecting Chalmydia trachomatis. However, published studies have raised important concerns regarding the NAAT evaluation process in general and their reproducibility and clinical specificity in particular. This is because for many infectious diseases including chlamydia, a true gold standard simply does not exist and, as a result, estimation of test performance parameters in the absence of a gold standard is a difficult and challenging task. In this manuscript, we will attempt to address some issues pertaining to the evaluation of NAATs including NAAT reproducibility, test validity, and the manner in which positive NAAT results are confirmed. Finally, we will discuss some of the potential clinical and public health implications of testing by NAATs.

Commercial Nucleic-Acid Amplification Tests for Diagnosis of Pulmonary Tuberculosis in Respiratory Specimens: Meta-Analysis and Meta-Regression

BackgroundHundreds of studies have evaluated the diagnostic accuracy of nucleic-acid amplification tests (NAATs) for tuberculosis (TB). Commercial tests have been shown to give more consistent results than in-house assays. Previous meta-analyses have found high specificity but low and highly variable estimates of sensitivity. However, reasons for variability in study results have not been adequately explored. We performed a meta-analysis on the accuracy of commercial NAATs to diagnose pulmonary TB and meta-regression to identify factors that are associated with higher accuracy.Methodology/Principal FindingsWe identified 2948 citations from searching the literature. We found 402 articles that met our eligibility criteria. In the final analysis, 125 separate studies from 105 articles that reported NAAT results from respiratory specimens were included. The pooled sensitivity was 0.85 (range 0.36–1.00) and the pooled specificity was 0.97 (range 0.54–1.00). However, both measures were significantly heterogeneous (p<.001). We performed subgroup and meta-regression analyses to identify sources of heterogeneity. Even after stratifying by type of commercial test, we could not account for the variability. In the meta-regression, the threshold effect was significant (p = .01) and the use of other respiratory specimens besides sputum was associated with higher accuracy.Conclusions/SignificanceThe sensitivity and specificity estimates for commercial NAATs in respiratory specimens were highly variable, with sensitivity lower and more inconsistent than specificity. Thus, summary measures of diagnostic accuracy are not clinically meaningful. The use of different cut-off values and the use of specimens other than sputum could explain some of the observed heterogeneity. Based on these observations, commercial NAATs alone cannot be recommended to replace conventional tests for diagnosing pulmonary TB. Improvements in diagnostic accuracy, particularly sensitivity, need to be made in order for this expensive technology to be worthwhile and beneficial in low-resource countries.

APTIMA® transcription-mediated amplification assays for Chlamydia trachomatis and Neisseria gonorrhoeae

The APTIMA® transcription-mediated amplification assays for Chlamydia trachomatis and Neisseria gonorrhoeae have the greatest sensitivity of all the commercial nucleic acid amplification tests for the diagnosis of infections from noninvasive samples that may contain small amounts of nucleic acid. They have received extensive attention in male and female populations of varying prevalences of infection. Vulvovaginal swabs appear to be the specimen of choice (either self-collected or physician collected) in women and first-catch urine in men. Gen-Probe Inc. has created alternate amplification primers for confirmatory or initial single organism testing. With automation of the TIGRIS® instrument, the assays should prove to be useful in high-volume laboratories.

Current evidence on diagnostic accuracy of commercially based nucleic acid amplification tests for the diagnosis of pulmonary tuberculosis

Even though commercial nucleic acid amplification tests (NAATs) have become the most frequently used molecular tests for laboratory diagnosis of pulmonary tuberculosis (TB), published studies report variable estimates of their diagnostic accuracy. We analysed the accuracy of commercial NAATs for the diagnosis of pulmonary TB in smear positive and smear negative respiratory samples using culture as a reference standard. English language studies reporting data sufficient for calculating sensitivity and specificity of commercial NAATs on smear positive and/or smear negative respiratory samples were included. Meta-regression was used to analyse associations with reference test quality, the prevalence of TB, sample and test type. Predictive values for different levels of pre-test probability were quantified using Bayes' approach. Sixty three journal articles published between 1995 and 2004 met the inclusion criteria. Pooled sensitivity and specificity were 0.96 and 0.85 among smear positive samples and 0.66 and 0.98 among smear negative samples. The number of culture media used as reference test, the inclusion of bronchial samples, and the TB prevalence were found to influence the reported accuracy. The test type had no effect on the diagnostic odds ratio but seemed to be correlated with sensitivity or specificity, probably via a threshold effect. Commercial NAATs can be confidently used to exclude TB in patients with smear positive samples in which environmental mycobacteria infection is suspected and to confirm TB in a proportion of smear negative cases. The methodological characteristics of primary studies have a considerable effect on the reported diagnostic accuracy.

Use of Flocked Swabs and a Universal Transport Medium To Enhance Molecular Detection of Chlamydia trachomatis and Neisseria gonorrhoeae

The use of new flocked swabs, compared to kit swabs, enhanced the ability of three commercial nucleic acid amplification tests to detect low levels of Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids when the organisms were diluted in a universal transport medium as mocked specimens.

Relative sensitivities of licensed nucleic acid amplification tests for detection of viremia in early human immunodeficiency virus and hepatitis C virus infection

Screening donors for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNA is primarily performed on minipools (MPs) with one of two commercial nucleic acid amplification tests (NAT; Roche Molecular Systems; or Gen-Probe/Chiron). We compared these assays with respect to detection of RNA in early HIV and HCV infection. Twelve HIV plasma donor panels (116 serial samples) and 12 HCV panels (180 serial samples) were selected to optimally represent early viremia. Initial testing was performed in singlicate or triplicate on separately coded aliquots, both neat and at dilutions corresponding to MP screening (1:16 for Gen-Probe; 1:24 for Roche); 20 additional replicates were performed when discordant results were observed. Odds ratios (ORs) comparing detection of RNA by different assays were derived with logistic regression models. Differences in window-period closure and yields of assays in MP or individual-donation (ID) format were estimated. Differences in detection rates between Roche and Gen-Probe NAT assays were small and only observed with samples with very-low-level viremia. ORs for detecting RNA by the Gen-Probe versus the Roche assay were significant for HIV if conducted on MPs (1.8; 95% confidence interval [CI], 1.3-2.5) but not neat (1.0; 95% CI, 0.72-1.4). Odds of detecting HCV RNA were higher if the Gen-Probe assay was conducted either neat (2.3; 95% CI, 1.6-3.2) or on MPs (4.0; 95% CI, 2.8-5.8). These differences translated to <1 day window-period closure and <or=1 infection per 20 million donations. In contrast, comparisons of ID versus MP assays were highly significant for both viruses (ORs for ID vs. MP ranged from 45.3 to 93.4), with projected yields of one to two additional infections per 10 million donations. Differences in sensitivities of licensed NAT assays for HIV and HCV are very small and clinically insignificant, particularly when compared to differences of MP versus ID NAT screening.

Nucleic acid amplification tests in the diagnosis of tuberculous pleuritis: a systematic review and meta-analysis.

BackgroundConventional tests for tuberculous pleuritis have several limitations. A variety of new, rapid tests such as nucleic acid amplification tests – including polymerase chain reaction – have been evaluated in recent times. We conducted a systematic review to determine the accuracy of nucleic acid amplification (NAA) tests in the diagnosis of tuberculous pleuritis.MethodsA systematic review and meta-analysis of 38 English and Spanish articles (with 40 studies), identified via searches of six electronic databases, hand searching of selected journals, and contact with authors, experts, and test manufacturers. Sensitivity, specificity, and other measures of accuracy were pooled using random effects models. Summary receiver operating characteristic curves were used to summarize overall test performance. Heterogeneity in study results was formally explored using subgroup analyses.ResultsOf the 40 studies included, 26 used in-house ("home-brew") tests, and 14 used commercial tests. Commercial tests had a low overall sensitivity (0.62; 95% confidence interval [CI] 0.43, 0.77), and high specificity (0.98; 95% CI 0.96, 0.98). The positive and negative likelihood ratios for commercial tests were 25.4 (95% CI 16.2, 40.0) and 0.40 (95% CI 0.24, 0.67), respectively. All commercial tests had consistently high specificity estimates; the sensitivity estimates, however, were heterogeneous across studies. With the in-house tests, both sensitivity and specificity estimates were significantly heterogeneous. Clinically meaningful summary estimates could not be determined for in-house tests.ConclusionsOur results suggest that commercial NAA tests may have a potential role in confirming (ruling in) tuberculous pleuritis. However, these tests have low and variable sensitivity and, therefore, may not be useful in excluding (ruling out) the disease. NAA test results, therefore, cannot replace conventional tests; they need to be interpreted in parallel with clinical findings and results of conventional tests. The accuracy of in-house nucleic acid amplification tests is poorly defined because of heterogeneity in study results. The clinical applicability of in-house NAA tests remains unclear.

Genotyping of Chlamydia trachomatis strains from cultured isolates and nucleic acid amplification test-positive specimens

AbstractUrogenital strains of Chlamydia trachomatis are divided into several serogroups (D – K). Since these serovars are represented with differing prevalence in the population a serotyping of strains is necessary, when characterising the epidemiological situation. The aim of this study was the genotyping of C. trachomatis strains, the comparison of the results with those of serotyping, and the genotyping of positive specimens using commercial nucleic acid amplification tests (NAAT). The Chlamydia trachomatis major outer membrane protein gene (omp1) from 55 isolated strains and 36 NAAT-positive specimens was amplified by polymerase chain reaction (PCR). The restriction fragment length polymorphism (RFLP) patterns of these amplicons were compared with those of reference strains. The genotypes E and F were found to be most prevalent. The results are discussed considering other studies, genovariants and epidemiology.

Evaluation of sequential testing strategies using non-amplified and amplified methods for detection of Chlamydia trachomatis in endocervical and urine specimens from women

Nucleic acid amplification tests (NAAT) are more sensitive than other methods for the diagnosis of Chlamydia trachomatis (CT) genital infections. Two unique sequential testing strategies that employed two different commercial NAAT methods to detect CT in a population of women with widely varying infection risk were evaluated. Specimens from 504 women aged 15 to 75 years were studied. Two endocervical swabs and a urine sample were collected from each woman. One swab was initially tested using the Access enzyme immunoassay (EIA) (Beckman). An aliquot from the EIA extraction was subsequently amplified using the COBAS AMPLICOR CT assay (PCR) (Roche). The second swab was initially tested using the PACE 2 CT hybridization assay (Gen-Probe). An aliquot was pipetted off prior to performing the PACE 2 assay and also amplified using the AMP-CT assay (TMA) (Gen-Probe). Urine samples were tested for CT using both NAAT methods. True CT infections were defined as any woman that was confirmed to be positive on both NAAT results from endocervical swabs. The results of all other CT assays were compared against this expanded gold standard. 28 women were confirmed to have CT infection giving an overall prevalence of 5.6%; low-risk women had a rate of 1.3% while high-risk women had a rate of 9.8%. NAAT methods have a higher sensitivity for detecting CT cervicitis when swabs are tested compared to urine. The positive predictive value of NAAT is decreased when testing low risk women. Limited automation makes it difficult to test a high volume of samples (i.e., > 100 swabs and/or urines) using either of these NAAT methods and continue to provide same day results. Laboratories performing CT testing must define the female population served so that appropriate diagnostic strategies can be employed.

LCx Mycobacterium tuberculosis assay is valuable with respiratory specimens, but provides little help in the diagnosis of extrapulmonary tuberculosis

BACKGROUND. Commercial nucleic acid amplification tests, designed for the detection of Mycobacterium tuberculosis DNA/RNA in respiratory samples, are often applied also in nonrespiratory specimens in order to verify the diagnosis of extrapulmonary tuberculosis.AIM. To evaluate the value of the Abbott LCx Mycobacterium tuberculosis assay for the diagnosis of pulmonary and extrapulmonary tuberculosis based on routine clinical laboratory results.METHODS. The assay was used to analyse 350 respiratory and 826 nonrespiratory specimens from 961 patients, of whom 3.6% had culture-proven tuberculosis. The results obtained by the LCx assay were compared with the records on mycobacterial isolates of the national reference laboratory and, in the case of positive findings, with clinical data.RESULTS. In comparison with culture, the sensitivity, specificity and positive/negative predictive value of the assay on respiratory specimens were 87.5%, 99.7%, 93.3% and 99.4%, respectively. With nonrespiratory specimens, the overall sensitivity, specificity and positive/negative predictive value of the LCx assay were 73.3%, 98.0%, 40.7% and 99.5%, respectively. When clinical and histological data were also included, the positive predictive value of LCx with nonrespiratory specimens was 45.8%.CONCLUSION. Critical interpretation of the nucleic acid amplification results obtained from nonrespiratory specimens is necessary in both laboratory and clinical settings.

Molecular techniques in mycobacterial detection.

Abstract Objective.—To assess the clinical utility of the commercial nucleic acid amplification (NAA) tests (ie, Amplified Mycobacterium Tuberculosis Direct Test, Gen-Probe, Inc and AMPLICOR Mycobacterium tuberculosis Test, Roche Molecular Systems, Inc) for direct detection of Mycobacterium tuberculosis complex. Data Sources.—Review of the English-language literature. Conclusions.—The performance of both NAA tests is excellent (sensitivity, ≥95%; specificity, 100%) when testing respiratory specimens that are smear-positive for acid-fast bacilli (AFB). Only the Gen-Probe assay is approved for testing respiratory specimens regardless of the AFB smear result. Data from 3 studies showed that the sensitivity of the Mycobacterium Tuberculosis Direct Test in smear-negative patients ranged from 83% to 85%, and that the specificity was 99%. Both NAA tests have been used to test nonrespiratory specimens; in some studies, the performance was comparable to the performance obtained for respiratory specimens, whereas i...