Background 
Hundreds 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 Findings 
We 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/Significance 
The 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.

/pdf/commercial-nucleic-acid-amplification-tests-for-diagnosis-of-gvhpq0ce63.pdf

Commercial nucleic-acid amplification tests for diagnosis of pulmonary tuberculosis in respiratory specimens: meta-analysis and meta-regression.

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

https://thorax.bmj.com/content/thoraxjnl/61/9/783.full.pdf

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

Mycobacteria are a group of acid-fast, aerobic, slow-growing organisms whose genus includes more than 90 different species. The causative agents of tuberculosis (TB), which is currently considered a global emergency, with more than 2 million people dying every year and 8 million new cases, belong to the Mycobacterium tuberculosis complex (MTB). Moreover, although of lesser public health importance, many other species referred to as nontuberculous mycobacteria (NTM) have also been associated with human disease with increasing frequency worldwide (65). As MTB is highly infectious for humans, it is of paramount importance that TB be diagnosed as early as possible to stop the spread of the disease. Active TB is currently diagnosed by conventional laboratory procedures including specimen digestion and decontamination, microscopic examination for the presence of acid-fast bacilli (AFB), isolation by culture on solid and/or liquid media, and identification and drug susceptibility testing of the recovered isolate. Because of the slow growth of mycobacteria, the above-reported laboratory procedures may require turnaround times of 3 to 4 weeks or longer.

During the last decade, several molecular methods have been developed for direct detection and identification of MTB in clinical specimens. These methods, being able to potentially reduce the diagnostic time from weeks to days, have been acquiring greater and greater relevance in the field of laboratory TB diagnosis. The basic principle of any molecular diagnostic test is the detection of a specific nucleic acid sequence by hybridization to a complementary sequence, a probe, followed by detection of the hybrid. However, the sensitivity of nucleic acid probe tests that do not involve amplification is much lower than that of amplified ones. Any portion of nucleic acid can be copied by using the specific polymerase, provided that some sequence data are known for the setup of appropriate primers. In general, amplification of target nucleic acid sequences is composed of three parts: denaturation, primer annealing, and primer extension. Discovery of PCRs in 1986 made this process reiterative, leading to an exponential increase in the production of the amplified target. Soon after, alternative amplification techniques were developed and patented by companies, which used different enzymes and strategies, but they are all based on reiterative reactions. Many different amplification targets including both DNA or RNA fragments have been proposed. The target most frequently amplified in MTB is the IS6110 (31) repetitive element, of which 10 to 16 copies are present in most clinical isolates. Numerous techniques for nucleic acid extraction have been proposed, as have different types of controls for monitoring the efficacy of nucleic acid extraction and amplification procedures. Currently, the U.S. Food and Drug Administration (FDA) requires that culture (still considered the “gold standard” for TB diagnosis) must be done in conjunction with the performance of each amplification-based test. In this paper, we review and discuss the currently available commercial methods which are capable of detecting MTB directly from clinical samples.

Relevance of Commercial Amplification Methods for Direct Detection of Mycobacterium tuberculosis Complex in Clinical Samples

Background: Current HIV-1 viral-load assays are too expensive for resource-limited settings. In some countries, monitoring of antiretroviral therapy is now more expensive than treatment itself. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes.

Methods: We evaluated real-time reverse transcription-PCR with internal control targeting the conserved long terminal repeat (LTR) domain of HIV-1 on reference panels and patient samples from Brazil (n = 1186), South Africa (n = 130), India (n = 44), and Germany (n = 127).

Results: The detection limit was 31.9 IU of HIV-1 RNA/mL of plasma (>95% probability of detection, Probit analysis). The internal control showed inhibition in 3.7% of samples (95% confidence interval, 2.32%–5.9%; n = 454; 40 different runs). Comparative qualitative testing yielded the following: Roche Amplicor vs LTR assay (n = 431 samples), 51.7% vs 65% positives; Amplicor Ultrasensitive vs LTR (n = 133), 81.2% vs 82.7%; BioMerieux NucliSens HIV-1 QT (n = 453), 60.5% vs 65.1%; Bayer Versant 3.0 (n = 433), 57.7% vs 55.4%; total (n = 1450), 59.0% vs 63.8% positives. Intra-/interassay variability at medium and near-negative concentrations was 18%–51%. The quantification range was 50–10 000 000 IU/mL. Viral loads for subtypes A–D, F–J, AE, and AG yielded mean differences of 0.31 log10 compared with Amplicor in the 103–104 IU/mL range. HIV-1 N and O were not detected by Amplicor, but yielded up to 180 180.00 IU/mL in the LTR assay. Viral loads in stored samples from all countries, compared with Amplicor, NucliSens, or Versant, yielded regression line slopes (SD) of 0.9 (0.13) ( P <0.001 for all).

Conclusions: This method offers all features of commercial assays and covers all relevant genotypes. It could allow general monitoring of antiretroviral therapy in resource-limited settings.

/pdf/ultrasensitive-monitoring-of-hiv-1-viral-load-by-a-low-cost-3vno45ibon.pdf

Ultrasensitive Monitoring of HIV-1 Viral Load by a Low-Cost Real-Time Reverse Transcription-PCR Assay with Internal Control for the 5′ Long Terminal Repeat Domain

A novel, robust, reproducible, and multipurpose universal sample processing (USP) methodology for highly sensitive smear microscopy, culturing on solid and liquid media, and inhibition-free PCR which is suitable for the laboratory diagnosis of both pulmonary and extrapulmonary tuberculosis (TB) has been developed. This method exploits the chaotropic properties of guanidinium hydrochloride for sample processing and involves incubating the specimen with USP solution, concentrating bacilli by centrifugation, and using the processed specimen for smear microscopy, culture, and PCR. The detection limit for acid-fast bacilli in spiked sputum by smear microscopy is approximately 300 bacilli per ml of specimen. USP solution-treated specimens are fully compatible with culturing on solid and liquid media. High-quality, PCR-amplifiable mycobacterial DNA can be isolated from all types of clinical specimens processed with USP solution. The method has been extensively validated with both pulmonary and extrapulmonary specimens. Furthermore, the USP method is also compatible with smear microscopy, culture, and PCR of mycobacteria other than tubercle bacilli. In summary, the USP method provides smear microscopy, culture, and nucleic acid amplification technologies with a single sample-processing platform and, to the best of our knowledge, is the only method of its kind described to date. It is expected to be useful for the laboratory diagnosis of TB and other mycobacterial diseases by conventional and modern methods.

Novel Multipurpose Methodology for Detection of Mycobacteria in Pulmonary and Extrapulmonary Specimens by Smear Microscopy, Culture, and PCR

A cooperative study was conducted among six laboratories to compare the performance of the Cobas Amplicor (CA) polymerase chain reaction (PCR) system (Roche Molecular Systems, USA) for the detection of Mycobacterium tuberculosis with that of microscopy and culture in routine clinical laboratory diagnosis. A total of 5,221 decontaminated respiratory specimens were tested. The use of an internal control allowed detection of PCR inhibition in 144 (2.8%) specimens. Only two culture-positive samples were CA PCR inhibitory and therefore could not be detected by PCR testing. Of the 333 culture-positive specimens, 278 (83.5%) were positive by the CA PCR. Of the 4,744 culture-negative specimens, 52 (1.1%) were positive by the CA PCR. After analysis of discrepancies, 40 of the 52 culture-negative, CA PCR-positive specimens were classified as true positive. Thus, the overall sensitivities of culture, CA PCR and microscopy were 89.3%, 85.2% and 55.5%, respectively. The overall specificity of the CA PCR was 99.7%. Five of the six centers found similar performances for the CA PCR, with sensitivities ranging from 85.7 to 90.9%. The CA PCR was more sensitive for smear-positive samples, exhibiting overall sensitivities of 96.1% and 71.7% for smear-positive and smear-negative specimens, respectively. These results indicate that the Cobas Amplicor system enables microbiology laboratories with reasonable previous experience in molecular biology testing to perform PCR and to detect Mycobacterium tuberculosis in more than 70% of specimens obtained from infected patients.

Multicenter study of a commercial, automated polymerase chain reaction system for the rapid detection of Mycobacterium tuberculosis in respiratory specimens in routine clinical practice.

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Multicenter study of a commercial, automated polymerase chain reaction system for the rapid detection of Mycobacterium tuberculosis in respiratory specimens in routine clinical practice.