Resistance to the first-line anti-tuberculosis (TB) drug, isoniazid (INH), is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole genome sequencing (WGS) to analyze 52 phenotypically INH-R Mycobacterium tuberculosis complex (MTBC) clinical isolates that lacked the common katG S315T or inhA promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. Use of WGS minimized the chances of missing infrequent INH resistance mutations outside commonly targeted hotspots. We used recombineering to generate 12 observed clinical katG mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. Our functional genetic experiments have confirmed the role of seven suspected INH resistance mutations and discovered five novel INH resistance mutations. All recombineered katG mutations conferred resistance to INH at a minimum inhibitory concentration of ≥0.25 μg/mL and should be added to the list of INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a superior method for detection of INH-R MTBC compared to current targeted molecular testing methods and could provide earlier diagnosis of drug-resistant TB.

Bedaquiline Drug Resistance Emergence Assessment in Multidrug-resistant-tuberculosis (MDR-TB) (DREAM) was a 5-year (2015-2019) phenotypic drug-resistance surveillance study across 11 countries. DREAM assessed the susceptibility of 5036 MDR-TB isolates of bedaquiline-treatment-naïve patients to bedaquiline and other anti-tuberculosis drugs by the 7H9 broth microdilution (BMD) and 7H10/7H11 agar dilution (AD) minimal inhibitory concentration (MIC) methods. Bedaquiline AD MIC quality control (QC) range for the H37Rv reference strain was unchanged, but the BMD MIC QC range (0.015-0.12 μg/ml) was adjusted compared with ranges from a multilaboratory, multicountry reproducibility study conforming to Clinical and Laboratory Standards Institute Tier-2 criteria. Epidemiological cut-off values of 0.12 μg/ml by BMD and 0.25 μg/ml by AD were consistent with previous bedaquiline breakpoints. An area of technical uncertainty or Intermediate category was set at 0.25 μg/ml and 0.5 μg/ml for BMD and AD, respectively. When applied to the 5036 MDR-TB isolates, bedaquiline-susceptible, intermediate and bedaquiline-resistant rates were 97.9%, 1.5% and 0.6%, respectively, for BMD, and 98.8%, 0.8% and 0.4% for AD. Resistance rates were: ofloxacin 35.1%, levofloxacin 34.2%, moxifloxacin 33.3%, 1.5% linezolid and 2% clofazimine. Phenotypic cross resistance between bedaquiline and clofazimine was 0.4% in MDR-TB and 1% in pre-extensively drug-resistant (pre-XDR-TB)/XDR-TB populations. Co-resistance to bedaquiline and linezolid, and clofazimine and linezolid, were 0.1% and 0.3%, respectively, in MDR-TB, and 0.2% and 0.4% in pre-XDR-TB/XDR-TB populations. Resistance rates to bedaquiline appear to be low in the bedaquiline-treatment-naïve population. No treatment-limiting patterns for cross-resistance and co-resistance have been identified with key TB drugs to date.

BACKGROUND: With the rapid development of new advanced molecular detection methods, identification of new genetic mutations conferring pathogen resistance to an ever-growing variety of antimicrobial substances will generate massive genomic datasets for public health and clinical laboratories. Keeping up with specialized standard coding for these immense datasets will be extremely challenging. This challenge prompted our effort to create a common molecular resistance Logical Observation Identifiers Names and Codes (LOINC) panel that can be used to report any identified antimicrobial resistance pattern. OBJECTIVE: To develop and utilize a common molecular resistance LOINC panel for molecular drug susceptibility testing (DST) data exchange in the U.S. National Tuberculosis Surveillance System using California Department of Public Health (CDPH) and New York State Department of Health as pilot sites. METHODS: We developed an interface and mapped incoming molecular DST data to the common molecular resistance LOINC panel using Health Level Seven (HL7) v2.5.1 Electronic Laboratory Reporting (ELR) message specifications through the Orion Health™ Rhapsody Integration Engine v6.3.1. RESULTS: Both pilot sites were able to process and upload/import the standardized HL7 v2.5.1 ELR messages into their respective systems; albeit CDPH identified areas for system improvements and has focused efforts to streamline the message importation process. Specifically, CDPH is enhancing their system to better capture parent-child elements and ensure that the data collected can be accessed seamlessly by the U.S. Centers for Disease Control and Prevention. DISCUSSION: The common molecular resistance LOINC panel is designed to be generalizable across other resistance genes and ideally also applicable to other disease domains. CONCLUSION: The study demonstrates that it is possible to exchange molecular DST data across the continuum of disparate healthcare information systems in integrated public health environments using the common molecular resistance LOINC panel.

INTRODUCTION: Phase 2 clinical trials of tuberculosis treatment have shown that once-daily regimens in which rifampin is replaced by high dose rifapentine have potent antimicrobial activity that may be sufficient to shorten overall treatment duration. Herein we describe the design of an ongoing phase 3 clinical trial testing the hypothesis that once-daily regimens containing high dose rifapentine in combination with other anti-tuberculosis drugs administered for four months can achieve cure rates not worse than the conventional six-month treatment regimen. METHODS/DESIGN: S31/A5349 is a multicenter randomized controlled phase 3 non-inferiority trial that compares two four-month regimens with the standard six-month regimen for treating drug-susceptible pulmonary tuberculosis in HIV-negative and HIV-positive patients. Both of the four-month regimens contain high-dose rifapentine instead of rifampin, with ethambutol replaced by moxifloxacin in one regimen. All drugs are administered seven days per week, and under direct observation at least five days per week. The primary outcome is tuberculosis disease-free survival at twelve months after study treatment assignment. A total of 2500 participants will be randomized; this gives 90% power to show non-inferiority with a 6.6% margin of non-inferiority. DISCUSSION: This phase 3 trial formally tests the hypothesis that augmentation of rifamycin exposures can shorten tuberculosis treatment to four months. Trial design and standardized implementation optimize the likelihood of obtaining valid results. Results of this trial may have important implications for clinical management of tuberculosis at both individual and programmatic levels. TRIAL REGISTRATION: NCT02410772. Registered 8 April 2015,https://www.clinicaltrials.gov/ct2/show/NCT02410772?term=02410772&rank=1.

Resistance to the first-line anti-tuberculosis (TB) drug, isoniazid (INH), is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole genome sequencing (WGS) to analyze 52 phenotypically INH-R Mycobacterium tuberculosis complex (MTBC) clinical isolates that lacked the common katG S315T or inhA promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. Use of WGS minimized the chances of missing infrequent INH resistance mutations outside commonly targeted hotspots. We used recombineering to generate 12 observed clinical katG mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. Our functional genetic experiments have confirmed the role of seven suspected INH resistance mutations and discovered five novel INH resistance mutations. All recombineered katG mutations conferred resistance to INH at a minimum inhibitory concentration of >/=0.25 mug/mL and should be added to the list of INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a useful tool for detecting uncommon INH resistance mutations that would otherwise be missed by current targeted molecular testing methods, and suggest that its use (or use of expanded conventional or NGS-based targeted sequencing) may provide earlier diagnosis of INH-R TB.

Hurricane Maria made landfall in Puerto Rico on September 20, 2017, causing major damage to infrastructure and severely limiting access to potable water, electric power, transportation, and communications. Public services that were affected included operations of the Puerto Rico Department of Health (PRDOH), which provides critical laboratory testing and surveillance for diseases and other health hazards. PRDOH requested assistance from CDC for the restoration of laboratory infrastructure, surveillance capacity, and diagnostic testing for selected priority diseases, including influenza, rabies, leptospirosis, salmonellosis, and tuberculosis. PRDOH, CDC, and the Association of Public Health Laboratories (APHL) collaborated to conduct rapid needs assessments and, with assistance from the CDC Foundation, implement a temporary transport system for shipping samples from Puerto Rico to the continental United States for surveillance and diagnostic and confirmatory testing. This report describes the initial laboratory emergency response and engagement efforts among federal, state, and nongovernmental partners to reestablish public health laboratory services severely affected by Hurricane Maria. The implementation of a sample transport system allowed Puerto Rico to reinitiate priority infectious disease surveillance and laboratory testing for patient and public health interventions, while awaiting the rebuilding and reinstatement of PRDOH laboratory services.

Because tuberculosis is caused by an infectious organism that is spread from person to person through the air, public health measures are essential to control the disease. There are three priority strategies for tuberculosis prevention and control in the United States: (i) identifying and treating persons who have tuberculosis disease; (ii) finding persons exposed to infectious tuberculosis patients, evaluating them for Mycobacterium tuberculosis infection and disease, and providing subsequent treatment, if appropriate; and (iii) testing populations at high risk for latent tuberculosis infection (LTBI) and treating those persons who are infected to prevent progression to disease. These strategies for prevention and control of tuberculosis are discussed in a framework containing the following important topics: historical and epidemiological context of tuberculosis control, organization of public health tuberculosis control programs, legal basis for public health authority, conducting overall planning and development of policy, identifying persons who have clinically active tuberculosis, evaluation of immigrants, managing persons who have or who are suspected of having disease, medical consultation, interjurisdictional referrals, identifying and managing persons infected with Mycobacterium tuberculosis, providing laboratory and diagnostic services, collecting and analyzing data, and providing training and education. This chapter describes the role of the health department in the context of these components. This discussion is primarily applicable to tuberculosis prevention and control programs in the United States.

OBJECTIVES: To establish standardized drug susceptibility testing (DST) methodologies and reference minimal inhibitory concentration (MIC) quality control (QC) ranges for bedaquiline, a diarylquinoline antimycobacterial, used in the treatment of adults with multidrug-resistant tuberculosis. METHODS: Two tier-2 QC reproducibility studies of bedaquiline DST were conducted in eight laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Agar dilution and broth microdilution methods were evaluated. Mycobacterium tuberculosis H37Rv was used as the QC reference strain. Bedaquiline MIC frequency, mode, and geometric mean were calculated. When resulting data occurred outside predefined CLSI criteria, the entire laboratory dataset was excluded. RESULTS: For the agar dilution MIC, a 4-dilution QC range (0.015-0.12 mug/ml) centered around the geometric mean included 95.8% (7H10 agar dilution; 204/213 observations with one dataset excluded) or 95.9% (7H11 agar dilution; 232/242) of bedaquiline MICs. For the 7H9 broth microdilution MIC, a 3-dilution QC range (0.015-0.06 mug/ml) centered around the mode included 98.1% (207/211 with one dataset excluded) of bedaquiline MICs. Microbiological equivalence was demonstrated for bedaquiline MICs determined using 7H10 agar and 7H11 agar, but not for bedaquiline MICs determined using 7H9 broth and 7H10 agar or 7H9 broth and 7H11 agar. CONCLUSIONS: Bedaquiline DST methodologies and MIC QC ranges against H37Rv M. tuberculosis reference strain have been established: 0.015-0.12 mug/ml for 7H10 and 7H11 agar dilution MICs, and 0.015-0.06 mug/ml for the 7H9 broth microdilution MIC. These methodologies and QC ranges will be submitted to CLSI and EUCAST to inform future research and provide guidance for routine clinical bedaquiline DST in laboratories worldwide.

OBJECTIVES: To establish reference minimal inhibitory concentration (MIC) quality control (QC) ranges for drug susceptibility testing of antimycobacterials, including first-line agents, second-line injectables, fluoroquinolones and World Health Organization Category 5 drugs for multidrug-resistant tuberculosis, using a 7H9 broth microdilution MIC method. METHODS: A Tier-2 reproducibility study was conducted in eight participating laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Three lots of custom-made frozen 96-well polystyrene micro titer plates were used and pre-prepared with 2X pre-diluted drugs in 7H9 broth/oleic acid albumin dextrose catalase. The QC reference strain was Mycobacterium tuberculosis (MTB) H37Rv. MIC frequency, mode and geometric mean were calculated for each drug. QC ranges were derived, based on predefined, strict CLSI criteria. Any data lying outside CLSI criteria resulted in exclusion of the entire laboratory dataset. RESULTS: Data from one laboratory were excluded due to higher MIC values than for other laboratories. QC ranges were established for eleven drugs: isoniazid (0.03-0.12 mug/ml), rifampin (0.03-0.25 mug/ml), ethambutol (0.25-2 mug/ml), levofloxacin (0.12-1 mug/ml), moxifloxacin (0.06-0.5 mug/ml), ofloxacin (0.25-2 mug/ml), amikacin (0.25-2 mug/ml), kanamycin (0.25-2 mug/ml), capreomycin (0.5-4 mug/ml), linezolid (0.25-2 mug/ml) and clofazimine (0.03-0.25 mug/ml). QC ranges could not be established for nicotinamide (pyrazinamide surrogate), prothionamide or ethionamide, which were assay non-performers. CONCLUSIONS: Using strict CLSI criteria, QC ranges against the MTB H37Rv reference strain were established for the majority of commonly used antituberculosis drugs, with a convenient 7H9 broth microdilution MIC method suitable for use in resource-limited settings.

Ethambutol (EMB) is used as a part of drug regimens for treatment of tuberculosis (TB). Susceptibility of Mycobacterium tuberculosis complex (MTBC) isolates to EMB can be discerned by DNA sequencing to detect mutations in the embB gene associated with resistance. US Public Health Laboratories (PHL) primarily use growth-based drug susceptibility test (DST) methods to determine EMB resistance. The Centers for Disease Control and Prevention (CDC) provides a service for molecular detection of drug resistance (MDDR) by DNA sequencing and concurrent growth-based DST using agar proportion. PHL and CDC test results were compared for 211 MTBC samples submitted to CDC from September 2009 through February 2011. Concordance between growth-based DST results from PHL and CDC was 88.2%. A growth-based comparison of 39 samples, where an embB mutation associated with EMB resistance was detected, revealed a higher percentage of EMB resistance by CDC (84.6%) than by PHL (59.0%) which was significant (P value = 0.002). Discordance between all growth-based test results from PHL and CDC was also significant (P value = 0.003). Most discordance was linked to false susceptibility using the BACTEC MGIT 960 (MGIT) growth-based system. Our analysis supports coalescing growth-based and molecular results for an informed interpretation of potential EMB resistance.

BACKGROUND: Xpert MTB/RIF(Xpert) is a rapid nucleic acid amplification test widely used in high tuberculosis(TB) prevalence settings to detect tuberculosis as well as rpoB mutations associated with rifampin resistance. Data are needed on the diagnostic performance of Xpert in lower prevalence settings to inform appropriate use for both tuberculosis detection and the need for respiratory isolation. METHODS: Xpert was compared to two sputa, each evaluated with AFB smear and mycobacterial culture using liquid and solid culture media, from participants with suspected pulmonary TB from the US, Brazil, and South Africa. RESULTS: Of 992 participants enrolled with evaluable results, 22% had culture-confirmed TB. In 638(64%) US participants, one Xpert demonstrated sensitivity of 85.2%(96.7% in participants with AFB smear-positive(AFB+) sputum, 59.3% with AFB- sputum),specificity of 99.2%, NPV 97.6%, and PPV 94.9%. Results did not differ between higher and low prevalence settings. A second Xpert increased overall sensitivity to 91.1%(100% if AFB+, 71.4% if AFB-), with specificity of 98.9%. In US participants, a single negative Xpert predicted the absence of AFB+/culture+ tuberculosis with an NPV of 99.7%; NPV of two Xperts was 100%, suggesting a role in removing patients from airborne infection isolation. Xpert detected TB DNA and mutations associated with rifampin resistance in five of seven participants with rifampin-resistant, culture+ tuberculosis. Specificity for rifampin resistance was 99.5%,NPV was 98.9%. CONCLUSIONS: In the US, Xpert testing performed comparably to two higher TB prevalence settings. These data support the use of Xpert in the initial evaluation of TB suspects and in algorithms assessing need for respiratory isolation.

Newer generation fluoroquinolones moxifloxacin (MXF) and levofloxacin (LVX) are becoming more common components of TB treatment regimens. However, the critical concentrations for testing susceptibility of Mycobacterium tuberculosis to MXF and LVX are not yet well established. Additionally, the degree of cross-resistance between ofloxacin (OFX) and these newer fluoroquinolones has not been thoroughly investigated. In this study, minimal inhibitory concentrations (MICs) for MXF and LVX and susceptibility to the critical concentration of OFX were determined using the agar proportion method for 133 isolates of M. tuberculosis. Most isolates resistant to OFX had LVX MICs >1 mug/mL and MXF MICs >0.5mug/mL. The presence of mutations within the GyrA QRDR correlated well with increased MICs, and the level of LVX and MXF resistance was dependent on the specific GyrA mutation present. Substitutions Ala90Val, Asp94Ala and Asp94Tyr resulted in low-level MXF resistance (MICs >0.5 but ≤2 mug/mL) while other mutations led to MXF MIC >2mug/mL. Based on these results, a critical concentration of 1 mug/mL is suggested for LVX and 0.5 mug/ml for MXF drug susceptibility testing by agar proportion with reflex testing for MXF at 2 mug/mL.

Crucial to interrupting the spread of tuberculosis (TB) is prompt implementation of effective treatment regimens. We evaluated satisfaction, comfort with interpretation, and use of molecular results from a public health service provided by the Centers for Disease Control and Prevention (CDC) for the molecular detection of drug resistant Mycobacterium tuberculosis complex (MTBC). An electronic survey instrument was used to collect information anonymously from U.S. Public Health Laboratories (PHL) that submitted at least one isolate of MTBC to CDC from September 2009 through February 2011. Over 97% of those responding expressed satisfaction with the turnaround time for receiving results. Twenty-six PHL (74%) reported molecular results to healthcare providers in less than two business days. When comparing the molecular results from CDC with their own phenotypic drug susceptibility testing, 50% of PHL observed discordance. No respondents found the molecular results difficult to interpret and 82% were comfortably discussing them with TB program officials and healthcare providers. Survey results indicate PHL were satisfied with CDC's ability to rapidly provide interpretable molecular results for isolates of MTBC submitted for determination of drug resistance. To develop educational materials and strategies for service improvement, reasons for discordant results and areas of confusion need to be identified.

DNA sequencing of rpoB and culture-based drug susceptibility results were evaluated for samples referred for confirmation of rifampin resistance detected by Cepheid Xpert(R) MTB/RIF. Silent mutations and mutations associated with low-level resistance were found in the study population. These data support CDC recommendations to confirm Xpert rifampin resistance results.

Multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis complex (MTBC) are defined by resistance to at least rifampin (RMP) and isoniazid (INH). Rapid and accurate detection of multidrug resistance is essential for effective treatment and interruption of disease transmission of tuberculosis (TB). Over-diagnosis of MDR TB may result in treatment with second-line drugs that are more costly, less effective, and more poorly tolerated than first-line drugs. CDC offers rapid confirmation of MDR TB by molecular detection of mutations associated with resistance (MDDR) to RMP and INH along with analysis for resistance to other first-line and second-line drugs. Simultaneously, CDC does growth-based phenotypic drug susceptibility testing (DST) by the indirect agar proportion method for a panel of first-line and second-line antituberculous drugs. We reviewed discordance between molecular and phenotypic DST for INH and RMP for 285 isolates submitted as MTBC to CDC September 2009-February 2011. We compared CDC's results with those from the submitting public health laboratories (PHL). Concordance between molecular and phenotypic testing at CDC was 97.4% for RMP and 92.5% for INH resistance. Concordance between CDC's molecular testing and PHL DST results was 93.9% for RMP and 90.0% for INH. Overall concordance between CDC molecular and PHL DST results was 91.7% for RMP and INH collectively. Discordance was primarily attributable to absence of known INH-resistance mutations in isolates INH resistant by DST and detection of mutations associated with low-level RMP resistance in isolates that were RMP susceptible by phenotypic DST. Both molecular and phenotypic test results should be considered for diagnosis of MDR TB.

Multidrug-resistant tuberculosis (MDR TB), defined by resistance to the 2 most effective first-line drugs, isoniazid and rifampin, is on the rise globally and is associated with significant morbidity and mortality. Despite the increasing availability of novel rapid diagnostic tools for Mycobacterium tuberculosis (Mtb) drug susceptibility testing, the clinical applicability of these methods is unsettled. In this study, the mechanisms of action and resistance of Mtb to isoniazid and rifampin, and the utility, advantages and limitations of the available Mtb drug susceptibility testing tools are reviewed, with particular emphasis on molecular methods with rapid turnaround including line probe assays, molecular beacon-based real-time polymerase chain reaction and pyrosequencing. The authors conclude that neither rapid molecular drug testing nor phenotypic methods are perfect in predicting Mtb drug susceptibility and therefore must be interpreted within the clinical context of each patient.

Based on data from 14 Supranational Tuberculosis (TB) Reference Laboratories worldwide, the proportion of rifampicin (RMP) resistant isolates that were isoniazid (INH) susceptible by phenotypic drug susceptibility testing varied widely (0.5-11.6%). RMP-resistant isolates that were INH-susceptible had significantly lower rates of resistance to other first- and second-line anti-tuberculosis drugs (except rifabutin) compared to multidrug-resistant isolates. RMP resistance is not a lways a good proxy for a presumptive diagnosis of multidrug-resistant TB, which has implications for use of molecular assays that identify only RMP resistanceassociated DNA mutations. (2012 The Union.)

TO THE EDITOR: Multidrug-resistant tuberculosis (MDR TB), i.e., TB resistant to at least the 2 most effective first-line antituberculous drugs (isoniazid [INH] and rifampin [RIF]), is increasing globally. World Health Organization estimations of 390,000-510,000 new MDR TB cases and 150,000 related deaths in 2008 highlight the need for timely drug susceptibility testing and improved therapies (1). Although novel rapid drug susceptibility testing tools are increasingly available, their clinical applicability is unsettled. We report a patient with pulmonary TB relapse with discordant genotypic and in vitro phenotypic drug susceptibility testing results associated with a mutation outside the RIF resistance determining region (RRDR) of the rpoB gene.

We present a statistical summary of results from the Model Performance Evaluation Program (MPEP) for Mycobacterium tuberculosis Drug Susceptibility Testing, 1994-2008 implemented by the U.S. Centers for Disease Control and Prevention (CDC). During that period, a total of 57,733 test results for culture isolates were reported by 216 participating laboratories for the first-line anti-tuberculosis drugs used in the United States- isoniazid (INH), rifampin (RMP), ethambutol (EMB), and pyrazinamide (PZA). Using Clinical Laboratory and Standards Institute (CLSI) recommended concentrations for one or more of three methods, agar proportion (AP), BACTEC460 (BACTEC), and MGIT-960 (MGIT), yielded overall agreement of 97.0% for first-line drugs. For susceptible strains, agreement was 98.4%; for resistant strains, agreement was 91.0%, with significantly lower accuracy (Chi-sq p<0.0001). For resistant strains, overall agreement by methods was: AP 91.3%; BACTEC 93.0%; and MGIT 82.6% and by drugs was: INH 92.2%; RMP 91.5%; EMB 79.0%; and PZA 97.5%. For some strains, performance by method varied significantly. Use of duplicate strains in the same shipment and repeat strains over time, revealed consistent performance even for strains with higher levels of inter-laboratory discordance. No overall differences in performance between laboratories were observed based on volume of testing or type of facility (e.g., health department, hospital, independent). By all methods, decreased performance was observed for strains with low-level INH resistance, RMP resistance, and EMB-resistant strains. These results demonstrate a high level of performance in detection of drug resistant M. tuberculosis in U.S. laboratories.

SETTING: The mycobacterial laboratory is assuming an increasingly important role in tuberculosis (TB) control in the United States today. OBJECTIVE: To assess mycobacterial laboratory capacity and practices in the New England states, USA. DESIGN: We surveyed 143 hospital and commercial laboratories and five of the six state public health laboratories in New England that offer testing services for Mycobacterium tuberculosis. The survey captured information on types of services offered and volume of testing, use of state laboratories for testing, and promptness of reporting results to TB control programs. RESULTS: State laboratories perform the majority of testing services, particularly for more specialized tests. All state laboratories surveyed perform species identification of acid-fast isolates, culture and first-line drug susceptibility testing. Less than 20% of hospital and commercial laboratories offer these services, and 78.6% of hospitals and commercial laboratories refer specimens to state laboratories for culture. CONCLUSION: Surveys of M. tuberculosis testing capacities in a region can help decision makers ensure maintenance of essential services. Hospital and commercial laboratories with lower testing volume might increase efficiency by referring more specimens to state laboratories. State health departments might consider organizing regional laboratory service networks to monitor the provision of services, improve efficiency and oversee quality improvement initiatives. 2011 The Union.

BACKGROUND: Tuberculosis Trials Consortium Study 28, was a double blind, randomized, placebo-controlled, phase 2 clinical trial examining smear positive pulmonary Mycobacterium tuberculosis. Over the course of intensive phase therapy, patients from African sites had substantially delayed and lower rates of culture conversion to negative in liquid media compared to non-African patients. We explored potential explanations of this finding. METHODS: In TBTC Study 28, protocol-correct patients (n = 328) provided spot sputum specimens for M. tuberculosis culture in liquid media, at baseline and weeks 2, 4, 6 and 8 of study therapy. We compared sputum culture conversion for African and non-African patients stratified by four baseline measures of disease severity: AFB smear quantification, extent of disease on chest radiograph, cavity size and the number of days to detection of M. tuberculosis in liquid media using the Kaplan-Meier product-limit method. We evaluated specimen processing and culture procedures used at 29 study laboratories serving 27 sites. RESULTS: African TB patients had more extensive disease at enrollment than non-African patients. However, African patients with the least disease by the 4 measures of disease severity had conversion rates on liquid media that were substantially lower than conversion rates in non-African patients with the greatest extent of disease. HIV infection, smoking and diabetes did not explain delayed conversion in Africa. Some inter-site variation in laboratory processing and culture procedures within accepted practice for clinical diagnostic laboratories was found. CONCLUSIONS: Compared with patients from non-African sites, African patients being treated for TB had delayed sputum culture conversion and lower sputum conversion rates in liquid media that were not explained by baseline severity of disease, HIV status, age, smoking, diabetes or race. Further investigation is warranted into whether modest variation in laboratory processes substantially influences the efficacy outcomes of phase 2 TB treatment trials or if other factors (e.g., nutrition, host response) are involved. TRIAL REGISTRATION: ClinicalTrials.gov NCT00144417.

The emergence of multi and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure patients receive effective treatment and become non-infectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs, isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB), and the second-line drugs, amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin, (CIP) and ofloxacin (OFX). Nine loci were sequenced: rpoB for resistance to RIF, katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical M. tuberculosis complex isolates, representing a variety of antibiotic resistance patterns, genotypes and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values (as percentages) for the first-line drug loci were rpoB (97.1, 93.6), katG (85.4, 100), inhA (16.5, 100), katG and inhA together (90.6, 100) pncA (84.6, 85.8), and embB (78.6, 93.1). The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates, support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.