cTreating extensively drug-resistant (XDR) tuberculosis (TB) is a serious challenge. Culture-based drug susceptibility testing (DST) may take 4 weeks or longer from specimen collection to the availability of results. We developed a pyrosequencing (PSQ) assay including eight subassays for the rapid identification of Mycobacterium tuberculosis complex (MTBC) and concurrent detection of mutations associated with resistance to drugs defining XDR TB. The entire procedure, from DNA extraction to the availability of results, was accomplished within 6 h. The assay was validated for testing clinical isolates and clinical specimens, which improves the turnaround time for molecular DST and maximizes the benefit of using molecular testing. A total of 130 clinical isolates and 129 clinical specimens were studied. The correlations between the PSQ results and the phenotypic DST results were 94.3% for isoniazid, 98.7% for rifampin, 97.6% for quinolones (ofloxacin, levofloxacin, or moxifloxacin), 99.2% for amikacin, 99.2% for capreomycin, and 96.4% for kanamycin. For testing clinical specimens, the PSQ assay yielded a 98.4% sensitivity for detecting MTBC and a 95.8% sensitivity for generating complete sequencing results from all subassays. The PSQ assay was able to rapidly and accurately detect drug resistance mutations with the sequence information provided, which allows further study of the association of drug resistance or susceptibility with each mutation and the accumulation of such knowledge for future interpretation of results. Thus, reporting of false resistance for mutations known not to confer resistance can be prevented, which is a significant benefit of the assay over existing molecular diagnostic methods endorsed by the World Health Organization. Regional increases in the prevalence of tuberculosis (TB) with drug resistance and a broad distribution of multidrug-resistant (MDR) TB and extensively drug-resistant (XDR) TB (1, 2) may reverse recent gains in global TB control (1). Molecular detection of mutations associated with drug resistance has facilitated rapid detection of drug resistance in the Mycobacterium tuberculosis complex (MTBC) (3-7), and the use of such molecular tools has become increasingly important in TB control and TB patient management (5, 7-10). Recognizing the advantages and disadvantages of various molecular methods will help in the selection of optimal methods for improved prediction of drug resistance in MTBC, which is critical in advancing molecular diagnostic approaches for this defined purpose.In 2003, the Microbial Diseases Laboratory (MDL) at the California Department of Public Health developed a real-time, probe-based assay using molecular beacons for screening for MDR TB (11). Remarkable improvements in turnaround times for predicting resistance to isoniazid (INH) and rifampin (RIF) and significant impacts on the management of MDR TB (9) were realized. However, several incidents of detection of rpoB mutations not conferring RIF resistance resulted in erroneous reporting of false RIF resi...

fIn an effort to update and clarify policies on tuberculosis drug susceptibility testing (DST), the World Health Organization (WHO) commissioned a systematic review evaluating WHO-endorsed diagnostic tests. We report the results of this systematic review and meta-analysis of the diagnostic accuracy and reproducibility of phenotypic DST for first-line and second-line antituberculosis drugs. This review provides support for recommended critical concentrations for isoniazid and rifampin in commercial broth-based systems. Further studies are needed to evaluate critical concentrations for ethambutol and streptomycin that accurately detect susceptibility to these drugs. Evidence is limited on the performance of DST for pyrazinamide and second-line drugs.T he global epidemic of drug-resistant tuberculosis (DR-TB), particularly multidrug-resistant TB (MDR-TB, defined as resistance to at least isoniazid and rifampin), is one of the most serious problems facing TB care and control efforts. In their most recent worldwide survey, the World Health Organization (WHO) documented the highest rates of MDR-TB ever reported (1). Effective management of drug-resistant TB relies on multiple components, including detection, treatment, prevention, surveillance, and continuous program evaluation (2). Expanding the capacity to diagnose cases of drug-resistant TB is a priority for global TB control, requiring clear policies on the use of diagnostic tests and strengthened laboratories in which testing can be safely and effectively carried out (3).Conventional phenotypic drug susceptibility testing (DST) using the proportion method (PM) on solid media has been well studied for isoniazid and rifampin, with a general consensus achieved regarding methodology, critical concentrations, and expected performance (4). However, the diagnostic accuracy and reproducibility of DST for other first-line and second-line drugs are inadequate (5). DST for second-line anti-TB drugs has not been standardized internationally, which is reflected in the wide variability of practices among supranational reference laboratories, underscoring the need for standardization of the methods and interpretive criteria for second-line DST (4, 6). Further complicating the lack of consensus is the increasing number of different DST methods that are available. In 2008, the WHO Stop TB Department published interim laboratory policy guidance for DST of second-line anti-TB drugs (4). Guideline recommendations for a specific DST method should ideally be based on the diagnostic test accuracy, reproducibility, ease of use, cost, and rapidity of result availability.In an attempt to address gaps in the evidence base, WHO initiated an update to the 2008 interim guidelines with an expanded scope to include DST for all first-line and second-line drugs. As part of the update, we conducted a systematic review to determine the diagnostic accuracy and reproducibility of WHO-endorsed phenotypic DST methods and commercial genotypic DST methods for first-line and second-line anti-TB drugs. Whi...

The Bactec MGIT 960 system for testing susceptibility to second-line drugs was evaluated with 117 clinical strains in a multicenter study. The four drugs studied were levofloxacin, amikacin, capreomycin, and ethionamide. The critical concentration established for levofloxacin and amikacin was 1.5 g/ml, that established for capreomycin was 3.0 g/ml, and that established for ethionamide was 5.0 g/ml. The overall level of agreement between the agar proportion method and the MGIT 960 system was 96.4%, and the levels of agreement for the individuals drugs were 99.1% for levofloxacin, 100% for amikacin, 97.4% for capreomycin, and 88.9% for ethionamide. The rate of reproducibility of the drug susceptibility testing results between the participating laboratories was 99.5%.The increase in the incidence of multidrug-resistant tuberculosis (MDR TB) and the emergence of extensively drugresistant tuberculosis present tremendous challenges to the global efforts to combat tuberculosis (1,5,16,21). Rapid methods enabling accurate susceptibility testing of first-line and second-line drugs are critical for the early diagnosis of MDR TB and extensively drug-resistant tuberculosis and the initiation of effective regimens. Various drug susceptibility testing (DST) methods that use solid media, including the agar proportion method (AP) and other methods, have the drawback of prolonged turnaround times (TATs). The World Health Organization and the U.S. Centers for Disease Control and Prevention have recommended the use of liquid culture systems for the diagnosis of tuberculosis and DST to improve TATs (22,25). The Bactec 460 (Becton Dickinson Diagnostic Systems, Sparks, MD), a radiometric liquid system, provided an excellent alternative for testing of the susceptibilities of Mycobacterium tuberculosis complex (MTBC) isolates to streptomycin, isoniazid, rifampin (rifampicin), and ethambutol (SIRE) and to pyrazinamide (PZA) with improved TATs. The MGIT 960 liquid, nonradiometric SIRE DST (Becton Dickinson Diagnostic Systems), whose performance is comparable to that of the Bactec 460 system, has been commercially available since April 2002 (4, 20, 23). The Microbial Diseases Laboratory (MDL) of the California Department of Public Health implemented SIRE DST with the MGIT 960 system in 2004. With confidence in the SIRE DST with the MGIT 960 system, a study that used the same platform to test the susceptibilities of MTBC isolates to four classes of second-line drugs, levofloxacin (LVX), amikacin (AMK), capreomycin (CAP), and ethionamide (ETH), was initiated in November 2004. The study was conducted at two laboratories: MDL and the TB Reference Laboratory of the Veteran Affairs Medical Center (VA) in West Haven, CT. Here we report the results of the multicenter study, in which the critical concentrations of the test drugs were established, the performance of the MGIT 960 system was compared to that of AP, and the interlaboratory reproducibility of the method was evaluated.(Part of this work was presented at the 46th Interscience Conference...

Malachite green, a synthetic antimicrobial dye, has been used for over 50 years in mycobacterial culture medium to inhibit the growth of contaminants. The molecular basis of mycobacterial resistance to malachite green is unknown, although the presence of malachite green-reducing enzymes in the cell envelope has been suggested. The objective of this study was to investigate the role of lipoproteins in resistance of Mycobacterium tuberculosis to malachite green. The replication of an M. tuberculosis lipoprotein signal peptidase II (lspA) mutant (⌬lspA::lspA mut ) on Middlebrook agar with and without 1 mg/liter malachite green was investigated. The lspA mutant was also compared with wild-type M. tuberculosis in the decolorization rate of malachite green and sensitivity to sodium dodecyl sulfate (SDS) detergent and first-line antituberculosis drugs. The lspA mutant has a 10 4 -fold reduction in CFU-forming efficiency on Middlebrook agar with malachite green. Malachite green is decolorized faster in the presence of the lspA mutant than wild-type bacteria. The lspA mutant is hypersensitive to SDS detergent and shows increased sensitivity to first-line antituberculosis drugs. In summary, lipoprotein processing by LspA is essential for resistance of M. tuberculosis to malachite green. A cell wall permeability defect is likely responsible for the hypersensitivity of lspA mutant to malachite green.

Five Mycobacterium tuberculosis complex isolates in California were identified as M. africanum by spoligotyping, single nucleotide polymorphisms, a deletion mutation, and phenotypic traits, confirming it as a cause of tuberculosis in the United States. Three of the five patients from whom M. africanum was isolated had lived in Africa.M ycobacterium africanum is a member of the M. tuberculosis complex, which has been isolated from humans in equatorial Africa. The disease produced by M. africanum is similar to that caused by M. tuberculosis or M. bovis, and like M. tuberculosis, this organism is likely spread by aerosol transmission (1). Human tuberculosis caused by M. africanum has been reported in Europe (2,3). However, we are unaware of previous reports of disease caused by M. africanum in the United States. The StudyM. africanum may be identified by spoligotyping (4), by specific deletion mutations (5), DNA fingerprinting by IS6110 restriction fragment length polymorphisms (RFLP) (4), or a combination of these methods. Isolates were initially identified as M. tuberculosis complex by using the AccuProbe system (Gen-Probe; San Diego, CA). The isolates then underwent IS6110-based RFLP fingerprinting. The RFLP analyses were performed according to the method of van Embden et al. (6). In addition to providing genotyping results, RFLP fingerprinting confirmed the identification obtained with Accuprobe.All strain typing was performed in-house at the Microbial Diseases Laboratory, California Department of Health Services. This laboratory has compiled a database (Genomic Solutions BioImage) of approximately 7,000 DNA fingerprints, typed by IS6110 RFLP (6,7) from throughout California; most are from the San Francisco Bay area. Three isolates (from patients A, B, and C) were initially suspected of being M. africanum because of an epidemiologic association with Africa. These isolates were fingerprinted by IS6110 RFLP and by spoligotyping (8).All three were found to have the "signature" spoligotype described by Viana-Niero et al. as being characteristic of M. africanum (4), namely, they were missing spacers 8, 9, and 39 but had spacers 40-43. Using BioImage software, we searched the laboratory's database for IS6110 fingerprints that matched those of the three cases with an African connection. This search yielded an additional two matches, cases D and E. Isolates from cases D and E were then genotyped by using spoligotyping and found to have the M. africanum signature spoligotype.The five M. africanum isolates were further characterized by performing standard biochemical identification tests and testing for susceptibility to pyrazinamide (PZA). Niacin production and nitrate reduction were detected as described by Kent and Kubica (9). Susceptibility to PZA was determined by using the BACTEC radiometric assay performed according to the method of Salfinger et al. (10). The M. africanum isolates were then examined to determine whether they had the RD9 deletion and specific oxyR and katG sequence mutations.Brosch et al. had repor...

To assess the clinical impact of a molecular beacon (MB) assay that detects multidrug-resistant tuberculosis (MDR TB), we retrospectively reviewed records of 127 MDR TB patients with and without MB testing between 2004 and 2007. Use of the MB assay reduced the time to detection and treatment of MDR TB.The emergence and spread of multidrug-resistant tuberculosis (MDR TB) threatens TB control worldwide. MDR TB is resistant to the most effective first-line agents, isoniazid (INH) and rifampin (RIF). The cornerstone of MDR TB diagnosis is culture and drug susceptibility testing (DST) of Mycobacterium tuberculosis isolates. Conventional methods for culture and first-line DST require weeks, causing delays in MDR TB diagnosis and treatment, which in turn are associated with advanced disease (6, 8), treatment failure (4), and transmission. Rapid molecular methods have been developed for the detection of mutations that confer drug resistance (3, 5, 7), and several public health organizations have endorsed molecular DST for TB (2, 9).The California Department of Public Health has been performing a molecular beacon (MB) assay in which real-time PCR is performed on acid-fast bacilli (AFB) smear-positive sputa or M. tuberculosis cultures to detect mutations conferring resistance to INH and RIF. TB providers in California are encouraged to submit specimens for MB testing from patients in whom drug-resistant TB is suspected (i.e., patients with a history of prior TB treatment, patients born in nations with high rates of resistant TB, patients failing TB treatment, and those who are known contacts to MDR TB cases). The assay is not FDA approved but is highly sensitive and specific (5), detects silent mutations infrequently, and shows Ͼ95% agreement with phenotypic DST results after testing on nearly 200 unique samples (unpublished). We performed a retrospective cohort study among MDR TB patients to assess the clinical impact of the MB assay in a public health setting in California.This study used deidentified demographic and clinical data that were collected by the California Department of Public Health for TB surveillance. Standard TB treatment was defined as any regimen containing at least 3 drugs, including INH and RIF. MDR treatment was defined as any regimen containing at least 4 drugs and including at least 2 second-line anti-TB medications (e.g., a fluoroquinolone, injectable agent [amikacin, kanamycin, capreomycin], para-aminosalicylic acid, cycloserine, ethionamide, or linezolid) (1, 10). The time to sputum culture conversion was defined as the interval between the collection dates of the first positive sputum culture and the first consistently negative sputum culture. Median times were compared using Wilcoxon's two-sample test. Proportions were tested using chi-square testing or Fisher's exact test. The MB assay was performed as previously described (5).Of 139 culture-positive MDR TB cases reported in California from 2004 through 2007, 12 cases were excluded because the MDR TB treatment start date could not be determi...

BackgroundRapid molecular diagnostics, with their ability to quickly identify genetic mutations associated with drug resistance in Mycobacterium tuberculosis clinical specimens, have great potential as tools to control multi- and extensively drug-resistant tuberculosis (M/XDR-TB). The Qiagen PyroMark Q96 ID system is a commercially available pyrosequencing (PSQ) platform that has been validated for rapid M/XDR-TB diagnosis. However, the details of the assay’s diagnostic and technical performance have yet to be thoroughly investigated in diverse clinical environments.MethodsThis study evaluates the diagnostic performance of the PSQ assay for 1128 clinical specimens from patients from three areas of high TB burden. We report on the diagnostic performance of the PSQ assay between the three sites and identify variables associated with poor PSQ technical performance.ResultsIn India, the sensitivity of the PSQ assay ranged from 89 to 98 % for the detection of phenotypic resistance to isoniazid, rifampicin, fluoroquinolones, and the injectables. In Moldova, assay sensitivity ranged from 7 to 94 %, and in South Africa, assay sensitivity ranged from 71 to 92 %. Specificity was high (94–100 %) across all sites. The addition of eis promoter sequencing information greatly improved the sensitivity of kanamycin resistance detection in Moldova (7 % to 79 %). Nearly all (89.4 %) sequencing reactions conducted on smear-positive, culture-positive specimens and most (70.8 %) reactions conducted on smear-negative, culture-positive specimens yielded valid PSQ reads. An investigation into the variables influencing sequencing failures indicated smear negativity, culture negativity, site (Moldova), and sequencing of the rpoB, gyrA, and rrs genes were highly associated with poor PSQ technical performance (adj. OR > 2.0).ConclusionsThis study has important implications for the global implementation of PSQ as a molecular TB diagnostic, as it demonstrates how regional factors may impact PSQ diagnostic performance, while underscoring potential gene targets for optimization to improve overall PSQ assay technical performance.Trial registrationClinicalTrials.gov (#NCT02170441). Registered 12 June 2014.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1781-y) contains supplementary material, which is available to authorized users.

Among the bacterial factors studied, East-Asian lineage and rpoB S531L mutation were independently associated with genotypic clustering, suggesting that bacterial factors have an impact on the ability of M. tuberculosis to cause secondary cases.