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Tuberculosis caused byMycobacterium tuberculosisis one of the leading causes of death from a single infectious agent. Identifying dominant epitopes and comparing their reactivity in different tuberculosis (TB) infection states can help design diagnostics and vaccines. We performed a proteome-wide screen of 20,610Mtbderived peptides in 21 Active TB (ATB) patients 3-4 months post-diagnosis of pulmonary TB (mid-treatment) using an IFNγ and IL-17 Fluorospot assay. Responses were mediated exclusively by IFNγ and identified a total of 137 unique epitopes, with each patient recognizing, on average, 8 individual epitopes and 22 epitopes (16%) recognized by 2 or more participants. Responses were predominantly directed against antigens part of the cell wall and cell processes category. Testing 517 peptides spanning TB vaccine candidates and ESAT- 6 and CFP10 antigens also revealed differential recognition between ATB participants mid-treatment and healthy IGRA+ participants of several vaccine antigens. An ATB-specific peptide pool consisting of epitopes exclusively recognized by participants mid-treatment, allowed distinguishing participants with active pulmonary TB from healthy interferon-gamma release assay (IGRA)+/- participants from diverse geographical locations. Analysis of longitudinal samples indicated decreased reactivity during treatment for pulmonary TB. Together, these results show that a proteome-wide screen of T cell reactivity identifies epitopes and antigens that are differentially recognized depending on theMtbinfection stage. These have potential use in developing diagnostics and vaccine candidates and measuring correlates of protection.

While the goal of universal drug susceptibility testing has been a key component of the WHO End TB Strategy, in practice, this remains inaccessible to many. Rapid molecular tests for tuberculosis (TB) and antituberculosis drug resistance could significantly improve access to testing.

Background As part of its End TB strategy, the WHO has identified the need for non-sputum-based diagnostics that meet target product profiles (TPP) of 90% sensitivity and 70% specificity for diagnosis of Active TB (ATB) and 75% sensitivity and specificity for predicting progression from Latent TB (LTB) to ATB. The successful translation of a 3-gene blood-based signature, identified using diverse datasets, into a prototype point-of-care diagnostic, that meets the WHO TPPs, has demonstrated the power of integrating large amounts of heterogeneous data to identify generalizable disease signatures. We hypothesized that integration of more diverse datasets, comprising patients with ATB or other inflammatory lung diseases, would identify novel, robust signatures, for diagnosing ATB and predicting progression from LTB to ATB, that meet the WHO TPPs. Methods Using multi-cohort analyses, we integrated and analyzed data from 3,615 peripheral blood samples, in 49 publicly available transcriptomic datasets (discovery cohorts), from healthy controls and patients with LTB, ATB, and other diseases (COPD, viral infections, sarcoidosis, etc.). We used data from (1) 3,836 blood samples in 28 retrospective datasets and (2) 360 prospectively collected blood samples, from a household contact study in Moldova, as validation cohorts. Results Using the discovery cohorts we identified a 9-gene signature for diagnosing ATB patients from healthy controls, or individuals with LTB or other diseases. The signature achieved 90% sensitivity and 82% specificity in retrospective validation (Figure 1A) and 90% sensitivity and 69% specificity in the prospective cohort from Moldova (Figure 1B). In a longitudinal cohort of adolescents, the 9-gene signature predicted progression from LTB to ATB up to 1 year prior to sputum conversion with 76% sensitivity and 83% specificity (Figure 1C). Finally, the signature predicted prolonged lung inflammation post-treatment in the Catalysis Treatment Response Cohort (Figure 1D). 9-gene TB signature validates in independent retrospective and prospective cohorts. (A) ROC curves for comparing ATB vs. all other samples (All), or individually comparing ATB vs. Healthy, LTBI or Other Disease (OD) samples in independent retrospective validation and (B) a prospective Moldova cohort. (C) ROC curves comparing progressor and non-progressor samples collected at different time points in the Adolescent Cohort Study (ACS), for the 9-gene signature in solid lines and a previous 3-gene signature in dashed lines, (D) Distribution of the 9-gene score at different time points post-treatment in the Catalysis Treatment Response Cohort Study, for individuals with persistent lung inflammation at 24 weeks compared with those who had clear lungs at 24 weeks. Conclusion Overall, the 9-gene signature meets the WHO TPPs required for the End TB strategy. Disclosures Purvesh Khatri, PhD, Cepheid, Inc.: Advisor/Consultant|Inflammatix, Inc.: Advisor/Consultant|Inflammatix, Inc.: Inflammatix is in negotiations to license the 9-gene signature discussed here.|Inflammatix, Inc.: Stocks/Bonds.

Although only a small fraction will ever develop the active form of tuberculosis (ATB) disease, chemoprophylaxis treatment in latent TB infected (LTBI) individuals is an effective strategy to control pathogen transmission. Characterizing immune responses in LTBI upon chemoprophylactic treatment is important to facilitate treatment monitoring, and thus improve TB control strategies. Here, we studied changes in the blood transcriptome in a cohort of 42 LTBI and 8 ATB participants who received anti-TB therapy. Based on the expression of previously published gene signatures of progression to ATB, we stratified the LTBI cohort in two groups and examined if individuals deemed to be at elevated risk of developing ATB before treatment (LTBI-Risk) differed from others (LTBI-Other). We found that LTBI-Risk and LTBI-Other groups were associated with two distinct transcriptomic treatment signatures, with the LTBI-Risk signature resembling that of treated ATB patients. Notably, overlapping genes between LTBI-Risk and ATB treatment signatures were associated with risk of progression to ATB and interferon (IFN) signaling, and were selectively downregulated upon treatment in the LTBI-Risk but not the LTBI-Other group. Our results suggest that transcriptomic reprogramming following treatment of LTBI is heterogeneous and can be used to distinguish LTBI-Risk individuals from the LTBI cohort at large.

Introduction Coccidioidomycosis is a fungal infection endemic in the southwestern United States (US). Primary pulmonary coccidioidomycosis (PPC) is a leading cause of community-acquired pneumonia (CAP) in this region, although its diagnosis is often delayed, leading to lag in antifungal treatment and subsequent morbidity. The impact of early empiric antifungal therapy as part of treatment for CAP in endemic areas on clinical outcomes is unknown. Methods Phase IV randomized, double-blind, placebo-controlled trial in individuals aged 18 years or older with CAP who met all eligibility criteria in Coccidioides endemic regions in the US. Eligible participants with CAP were randomized to receive either fluconazole (400 mg daily) or matching placebo for 42 days and were subsequently monitored for clinical resolution of their illness. Objectives The primary objective was to assess the clinical response of early empiric antifungal therapy with fluconazole through Day 22 in subjects with PPC who were adherent to the study intervention. Secondary objectives included: assessments of the impact of early empiric antifungal therapy with fluconazole through Day 22 and 43 in subjects with PPC regardless of adherence, comparisons of the clinical response and its individual components over time by treatment group in subjects with PPC, assessments of days lost from work or school, hospitalization, and all-cause mortality. Discussion This trial was halted early due to slow enrollment (72 participants in one year, 33 received fluconazole and 39 received placebo). Of those enrolled, eight (11%) met the study definition of PPC. The study design and challenges are discussed., Highlights • Clinical impact of early antifungal therapy for pneumonia in Coccidioides endemic regions is unknown. • We designed a phase IV trial in adults with community-acquired pneumonia in regions endemic for Coccidioides. • Trial was halted early due to slow enrollment and low prevalence of coccidioidomycosis in the enrollment population. • Lost to follow-up and treatment discontinuation were common in this trial.

Coccidioidomycosis is a fungal disease endemic to the southwestern United States, Mexico, and Central and South America. Prevalence rates are increasing steadily, and new endemic areas of Coccidioides are emerging. Standard treatment is often administered for months to decades, and intolerance to medications and treatment failures are common. No new treatments for coccidioidomycosis have been approved in the United States in nearly 40 years. On 5 August 2020, the US Food and Drug Administration convened experts in coccidioidomycosis from academia, industry, patient groups, and other government agencies to discuss the disease landscape and strategies to facilitate product development for treatment of coccidioidomycosis. This article summarizes the key topics concerning drug development for coccidioidomycosis presented by speakers and panelists during the workshop, such as unmet need, trial designs, endpoints, incentives, research and development support, and collaborations to facilitate antifungal drug development.

Background The lack of accurate and efficient diagnostic devices for extensively drug-resistant tuberculosis (XDR-TB) makes it a severe threat to global public health. A prospective clinical study in an intended-use cohort was designed to evaluate the Akonni Biosystems XDR-TB TruArray and lateral flow cell (XDR-LFC) to address this gap in tuberculosis diagnostics. Objective This paper presents the protocol for a study that aims to document the conceptualization and design of this evaluation method for early dissemination while data collection and analysis are ongoing. Methods The clinical study was conducted in three phases. The first phase was to observe changes in bacterial load and culture positivity in patient sputa over time and better understand the diversity of prospective clinical samples. The second phase was to prospectively collect clinical samples for sensitivity and specificity testing of the Akonni Biosystems XDR-LFC device. Lastly, the third phase was to explore the anti-TB drug concentrations in serum throughout the drug-resistant tuberculosis treatment. Results The methodology described includes the study design, laboratory sample handling, data collection, and the protection elements of human subjects of this clinical study to evaluate a potential new XDR-TB diagnostic device. A total of 664 participants were enrolled across the three phases. The implemented complex systems facilitated a thorough clinical data collection for an objective evaluation of the device. The study is closed to recruitment. The follow-up data collection and analysis are in progress. Conclusions This paper outlined a prospective cohort study protocol to evaluate a rapid XDR-TB detection device, which may be informative for other researchers with similar goals. International Registered Report Identifier (IRRID) DERR1-10.2196/26748

Conventional treatments to combat the tuberculosis (TB) epidemic are falling short, thus encouraging the search for novel antitubercular drugs acting on unexplored molecular targets. Several whole-cell phenotypic screenings have delivered bioactive compounds with potent antitubercular activity. However, their cellular target and mechanism of action remain largely unknown. Further evaluation of these compounds may include their screening in search for known antitubercular drug targets hits. Here, a collection of nearly 1400 mycobactericidal compounds was screened against Mycobacterium tuberculosis NaMN adenylyltransferase ( MtNadD), a key enzyme in the biogenesis of NAD cofactor that was recently validated as a new drug target for dormant and active tuberculosis. We found three chemotypes that efficiently inhibit MtNadD in the low micromolar range in vitro. SAR and cheminformatics studies of commercially available analogues point to a series of benzimidazolium derivatives, here named N2, with bactericidal activity on different mycobacteria, including M. abscessus, multidrug-resistant M. tuberculosis, and dormant M. smegmatis. The on-target activity was supported by the increased resistance of an M. smegmatis strain overexpressing the target and by a rapid decline in NAD(H) levels. A cocrystal structure of MtNadD with N2-8 inhibitor reveals that the binding of the inhibitor induced the formation of a new quaternary structure, a dimer-of-dimers where two copies of the inhibitor occupy symmetrical positions in the dimer interface, thus paving the way for the development of a new generation of selective MtNadD bioactive inhibitors. All these results strongly suggest that pharmacological inhibition of MtNadD is an effective strategy to combat dormant and resistant Mtb strains.

BACKGROUND The lack of accurate and efficient diagnostic devices for extensively drug-resistant tuberculosis (XDR-TB) makes it a severe threat to global public health. A prospective clinical study in an intended-use cohort was designed to evaluate the Akonni Biosystems XDR-TB TruArray and lateral flow cell (XDR-LFC) to address this gap in tuberculosis diagnostics. OBJECTIVE This paper presents the protocol for a study that aims to document the conceptualization and design of this evaluation method for early dissemination while data collection and analysis are ongoing. METHODS The clinical study was conducted in three phases. The first phase was to observe changes in bacterial load and culture positivity in patient sputa over time and better understand the diversity of prospective clinical samples. The second phase was to prospectively collect clinical samples for sensitivity and specificity testing of the Akonni Biosystems XDR-LFC device. Lastly, the third phase was to explore the anti-TB drug concentrations in serum throughout the drug-resistant tuberculosis treatment. RESULTS The methodology described includes the study design, laboratory sample handling, data collection, and the protection elements of human subjects of this clinical study to evaluate a potential new XDR-TB diagnostic device. A total of 664 participants were enrolled across the three phases. The implemented complex systems facilitated a thorough clinical data collection for an objective evaluation of the device. The study is closed to recruitment. The follow-up data collection and analysis are in progress. CONCLUSIONS This paper outlined a prospective cohort study protocol to evaluate a rapid XDR-TB detection device, which may be informative for other researchers with similar goals. INTERNATIONAL REGISTERED REPORT DERR1-10.2196/26748

Coccidioidal meningitis (CM) has high morbidity, and adjunctive measures to improve outcomes are needed. Using an established multicenter retrospective cohort study of CM (N = 221), we found that patients receiving adjunctive corticosteroids had a significant reduction in secondary cerebrovascular events (P = .0049). Those with CM-associated cerebrovascular events (8%) may benefit from short-term corticosteroids.

It is important to realize that guidelines cannot always account for individual variation among patients. They are not intended to supplant physician judgment with respect to particular patients or special clinical situations. Infectious Diseases Society of America considers adherence to these guidelines to be voluntary, with the ultimate determination regarding their application to be made by the physician in the light of each patient's individual circumstances.Coccidioidomycosis, also known as San Joaquin Valley fever, is a systemic infection endemic to parts of the southwestern United States and elsewhere in the Western Hemisphere. Residence in and recent travel to these areas are critical elements for the accurate recognition of patients who develop this infection. In this practice guideline, we have organized our recommendations to address actionable questions concerning the entire spectrum of clinical syndromes. These can range from initial pulmonary infection, which eventually resolves whether or not antifungal therapy is administered, to a variety of pulmonary and extrapulmonary complications. Additional recommendations address management of coccidioidomycosis occurring for special at-risk populations. Finally, preemptive management strategies are outlined in certain at-risk populations and after unintentional laboratory exposure.

Cross-resistance in rifamycins has been observed in rifampin (RIF)-resistant Mycobacterium tuberculosis complex isolates; some rpoB mutations do not confer broad in vitro rifamycin resistance. We examined 164 isolates, of which 102 were RIF-resistant, for differential resistance between RIF and rifabutin (RFB). A total of 42 unique single mutations or combinations of mutations were detected. The number of unique mutations identified exceeded that reported in any previous study. RFB and RIF MICs up to 8 μg/mL by MGIT 960 were studied; the cut-off values for susceptibility to RIF and RFB were 1 μg/mL and 0.5 μg/mL, respectively. We identified 31 isolates resistant to RIF but susceptible to RFB with the mutations D516V, D516F, 518 deletion, S522L, H526A, H526C, H526G, H526L, and two dual mutations (S522L + K527R and H526S + K527R). Clinical investigations using RFB to treat multidrug-resistant tuberculosis cases harboring those mutations are recommended.

Author(s): Seifert, M; Ajbani, K; Georghiou, SB; Catanzaro, D; Rodrigues, C; Crudu, V; Victor, TC; Garfein, RS; Catanzaro, A; Rodwell, TC | Abstract: ObjectiveTo evaluate the performance of a recently updated rapid molecular diagnostic test, GenoType® MTBDRplus version 2, designed to detect drug resistance in both acid-fast bacilli (AFB) smear-negative and -positive specimens.DesignSputum samples from 1128 patients at risk for multidrug-resistant tuberculosis (MDR-TB) were tested using MTBDRplus v2 and compared with reference standard MGIT™ 960™ drug susceptibility testing. The relationship of participant human immunodeficiency virus (HIV) status, diabetic status, previous treatment, and smear gradation to the likelihood of obtaining an interpretable result was assessed using logistic regression.ResultsThe sensitivity and specificity of MTBDRplus v2 for detecting MDR-TB, when compared to a reference standard, were respectively 96.0% (95%CI 93.5-97.6) and 99.2% (95%CI 97.0-99.9) in AFB smear-positive specimens and 82.8% (95%CI 63.5-93.5) and 98.3% (95%CI 89.9-99.9) in AFB smear-negative specimens. A dose-response relationship was observed between the proportion of interpretable test results and AFB smear bacterial load after adjusting for age, sex, body mass index, HIV status, previous treatment and diabetic status.ConclusionWhile MTBDRplus v2 performs well among both AFB smear-positive and -negative specimens, smear gradation appears to influence both the probability of obtaining an interpretable result and test sensitivity, indicating a significant association between bacillary load and test performance.

Background Although line-probe assays (LPAs) are promising, little research has been conducted to elucidate the true nature of indeterminate LPA results or assess the ability of these assays to perform on a wide range of clinical samples. Objective To evaluate the performance of the commercially available GenoType(®) MTBDRplus LPA against conventional BACTEC™ MGIT™ 960 culture and drug susceptibility testing (DST) among 308 pulmonary tuberculosis (PTB) and 32 extra-pulmonary TB samples. Results Invalid LPA results (defined as those with a missing Mycobacterium tuberculosis identification band) were obtained for 18 PTB samples, which were excluded from further analysis. The sensitivity and specificity of the MTBDRplus assay for multidrug-resistant TB, based upon the results obtained for the remaining 322 samples, was respectively 95.2% and 95.1%. Of 290 PTB samples, 40 (13.7%) were indeterminate on LPA (defined as the absence of both wild-type and corresponding mutation bands) for isoniazid (INH) and/or rifampicin (RMP), and were further evaluated by pyrosequencing (PSQ). Contrary to standard LPA interpretation, INH and RMP susceptibility were confirmed by both DST and PSQ in respectively 7.5% (3/40) and 27.5% (11/40) of indeterminate samples. Conclusion PSQ was found to be a valuable and rapid technique to resolve discrepancies in LPA test results that were not interpretable.

Objective/background The in vitro drug-susceptibility testing of Mycobacterium tuberculosis reports isolates as resistant or susceptible on the basis of single critical concentrations. It is evident that drug resistance in M. tuberculosis is quite heterogeneous, and involves low level, moderate level, and high level of drug-resistant phenotypes. Thus, the aim of our study was to correlate rrs ( X52917 ) and eis ( AF144099 ) promoter mutations, found in M. tuberculosis isolates, with corresponding minimum inhibitory concentrations of amikacin, kanamycin, and capreomycin. Methods Ninety M. tuberculosis clinical isolates were analyzed in this study. The minimum inhibitory concentrations were determined by MGIT 960 for 59 isolates with resistance-associated mutations in the rrs and eis promoter gene regions, and 31 isolates with wild-type sequences, as determined by the GenoType MTBDR sl (version 1) assay. Results The rrs A1401G mutation was identified in 48 isolates resistant to the second-line injectables. The eis promoter mutations C-14T ( n = 3), G-10C ( n = 3), G-10A ( n = 3), and C-12T ( n = 2) were found within 11 isolates with various resistance profiles to the second-line injectables. Thirty-one isolates had wild-type sequences for the rrs and eis promoter gene regions of interest, one of which was amikacin, kanamycin, and capreomycin resistant. The isolates with the rrs A1401G mutation had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of >40 mg/L, >20 mg/L, and 5–15 mg/L, respectively. The isolates with eis promoter mutations had amikacin, kanamycin, and capreomycin minimum inhibitory concentrations of 0.25–1.0 mg/L, 0.625–10 mg/L, and 0.625–2.5 mg/L, respectively. Conclusion This study provides a preliminary basis for the prediction of phenotypic-resistance levels to the second-line injectables based upon the presence of genetic mutations associated with amikacin, kanamycin, and capreomycin resistance. The results suggest that isolates with eis promoter mutations have consistently lower resistance levels to amikacin, kanamycin, and capreomycin than isolates with the rrs A1401G mutation.

Background Growth-based drug susceptibility testing (DST) is the reference standard for diagnosing drug-resistant tuberculosis (TB), but standard time to result (TTR) is typically ≥ 3 weeks. Rapid tests can reduce that TTR to days or hours, but accuracy may be lowered. In addition to the TTR and test accuracy, the cost of a diagnostic test may affect whether it is adopted in clinical settings. We examine the cost-effectiveness of rapid diagnostics for extremely drug-resistant TB (XDR-TB) in three different high-prevalence settings. Methods 1128 patients with confirmed TB were enrolled at clinics in Mumbai, India; Chisinau, Moldova; and Port Elizabeth, South Africa. Patient sputum samples underwent DST for first and second line TB drugs using 2 growth-based (MGIT, MODS) and 2 molecular (Pyrosequencing [PSQ], line-probe assays [LPA]) assays. TTR was the primary measure of effectiveness. Sensitivity and specificity were also evaluated. The cost to perform each test at each site was recorded and included test-specific materials, personnel, and equipment costs. Incremental cost-effectiveness ratios were calculated in terms of $/day saved. Sensitivity analyses examine the impact of batch size, equipment, and personnel costs. Results Our prior results indicated that the LPA and PSQ returned results in a little over 1 day. Mean cost per sample without equipment or overhead was $23, $28, $33, and $41 for the MODS, MGIT, PSQ, and LPA, respectively. For diagnosing XDR-TB, MODS was the most accurate, followed by PSQ, and LPA. MODS was quicker and less costly than MGIT. PSQ and LPA were considerably faster but cost more than MODS. Batch size and personnel costs were the main drivers of cost variation. Conclusions Multiple factors must be weighed when selecting a test for diagnosis of XDR-TB. Rapid tests can greatly improve the time required to diagnose drug-resistant TB, potentially improving treatment success, and preventing the spread of XDR-TB. Faster time to result must be weighed against the potential for reduced accuracy, and increased costs. Trial registration ClinicalTrials.gov Identifier: NCT02170441. Electronic supplementary material The online version of this article (10.1186/s12879-018-3013-0) contains supplementary material, which is available to authorized users.

Setting Rifampin (RIF)-resistant Mycobacterium tuberculosis (Mtb) is usually assumed to be resistant to all rifamycins. Increasing evidence indicates, however, that some rpoB mutations, detectable by rapid molecular diagnostics, confer resistance to RIF but not rifabutin (RFB), suggesting RFB may be effective for treatment of Mtb with these mutations.

Author(s): Garfein, RS; Catanzaro, DG; Rodwell, TC; Avalos, E; Jackson, RL; Kaping, J; Evasco, H; Rodrigues, C; Crudu, V; Lin, S-YG; Groessl, E; Hillery, N; Trollip, A; Ganiats, T; Victor, TC; Eisenach, K; Valafar, F; Channick, J; Qian, L; Catanzaro, A | Abstract: ObjectiveTo develop and evaluate rapid, molecular-based drug susceptibility testing (DST) for extensively drug-resistant tuberculosis (XDR-TB), we assembled a phenotypically and genotypically diverse collection of Mycobacterium tuberculosis isolates from patients evaluated for drug resistance in four high-burden countries.MethodsM. tuberculosis isolates from India (n = 111), Moldova (n = 90), the Philippines (n = 96), and South Africa (n = 103) were selected from existing regional and national repositories to maximize phenotypic diversity for resistance to isoniazid, rifampin (RMP), moxifloxacin, ofloxacin, amikacin, kanamycin, and capreomycin. MGIT™ 960 was performed on viable isolates in one laboratory using standardized procedures and drug concentrations. Genetic diversity within drug resistance phenotypes was assessed.ResultsNineteen distinct phenotypes were observed among 400 isolates with complete DST results. Diversity was greatest in the Philippines (14 phenotypes), and least in South Africa (9 phenotypes). Nearly all phenotypes included multiple genotypes. All sites provided isolates resistant to injectables but susceptible to fluoroquinolones. Many patients were taking drugs to which their disease was resistant.DiscussionDiverse phenotypes for XDR-TB-defining drugs, including resistance to fluoroquinolones and/or injectable drugs in RMP-susceptible isolates, indicate that RMP susceptibility does not ensure effectiveness of a standard four-drug regimen. Rapid, low-cost DST assays for first- and second-line drugs are thus needed.

Summary Background Prophylactic treatment of individuals with latent Mycobacterium tuberculosis infection is an essential component of tuberculosis control in some settings. In China, the prevalence of latent tuberculosis infection, and preventive interventions against this disease, have not been systematically studied. We aimed to assess the prevalence of latent tuberculosis and its associated risk factors in rural populations in China. Methods Between July 1, and Sept 30, 2013, we undertook a baseline survey of a population-based, multicentre, prospective cohort study of registered residents (≥5 years old) at four study sites in rural China. Eligible participants were identified by door-to-door survey with a household sampling design. We screened participants for active tuberculosis and history of tuberculosis then used a tuberculin skin test and an interferon-γ release assay (QuantiFERON [QFT]) to test for latent infection. We used odds ratios (ORs) and 95% CIs to assess variables associated with positivity of QFT and tuberculin skin tests. Findings 21 022 (90%) of 23 483 eligible participants completed a baseline survey. Age-standardised and sex-standardised rates of skin-test positivity (≥10 mm) ranged from 15% to 42%, and QFT positivity rates ranged from 13% to 20%. Rates of positivity for the tuberculin skin test and the QFT test were low in study participants younger than 20 years and gradually increased with age (p for trend Interpretation On the basis of findings showing that the performance of the tuberculin skin test might be affected by various factors including BCG vaccination and age, our results suggest that the prevalence of latent tuberculosis in China might be overestimated by skin tests compared with interferon-γ release assays. Funding The National Science and Technology Major Project of China, the Program for Changjiang Scholars and Innovative Research Team in University of China.

Reliable molecular diagnostics, which detect specific mutations associated with drug resistance, are promising technologies for the rapid identification and monitoring of drug resistance in Mycobacterium tuberculosis isolates. Pyrosequencing (PSQ) has the ability to detect mutations associated with first- and second-line anti-tuberculosis (TB) drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations. The aim of this project was to evaluate the performance of PSQ in predicting phenotypic drug resistance in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) clinical isolates from India, South Africa, Moldova, and the Philippines. A total of 187 archived isolates were run through a PSQ assay in order to identify M. tuberculosis (via the IS 6110 marker), and to detect mutations associated with M/XDR-TB within small stretches of nucleotides in selected loci. The molecular targets included katG , the inhA promoter and the ahpC-oxyR intergenic region for isoniazid (INH) resistance; the rpoB core region for rifampin (RIF) resistance; gyrA for fluoroquinolone (FQ) resistance; and rrs for amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance. PSQ data were compared to phenotypic mycobacterial growth indicator tube (MGIT) 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP. PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first- and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.

Pyrazinamide (PZA) is an important first-line drug in all existing and new tuberculosis (TB) treatment regimens. PZA-resistance in M. tuberculosis is increasing, especially among M/XDR cases. Noted issues with PZA Drug Susceptibility Testing (DST) have driven the search for alternative tests. This study provides a comprehensive assessment of PZA molecular diagnostics in M/XDR TB cases. A set of 296, mostly XDR, clinical M. tuberculosis isolates from four countries were subjected to DST for eight drugs, confirmatory Wayne’s assay, and whole-genome sequencing. Three genes implicated in PZA resistance, pncA, rpsA, and panD were investigated. Assuming all non-synonymous mutations cause resistance, we report 90% sensitivity and 65% specificity for a pncA-based molecular test. The addition of rpsA and panD potentially provides 2% increase in sensitivity. Molecular heterogeneity in pncA was associated with resistance and should be evaluated as a diagnostic tool. Mutations near the N-terminus and C-terminus of PZase were associated with East-Asian and Euro-American lineages, respectively. Finally, Euro-American isolates are most likely to have a wild-type PZase and escape molecular detection. Overall, the 8–10% resistance without markers may point to alternative mechanisms of resistance. Confirmatory mutagenesis may improve the disconcertingly low specificity but reduce sensitivity since not all mutations may cause resistance.

A new multinational study of the noncommercial MODS assay shows excellent performance in detecting M/XDR-tuberculosis http://ow.ly/FAEJ308OiOw

Treating 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.

The purpose of the present study was to correlate gyrA mutations found in Mycobacterium tuberculosis isolates using the GenoType(®) MTBDRsl assay with minimum inhibitory concentrations of the fluoroquinolone levofloxacin (LVX). Of 123 archived clinical M. tuberculosis isolates evaluated, 93 isolates had an Ala90Val, Ser91Pro, Asp94Ala, Asn/Tyr, Gly or His mutation and 30 were wild-type. Phenotypically, gyrA mutations Ala90Val, Ser91Pro or Asp94Ala showed a low level of resistance to LVX, while Asp94Asn/Tyr, Asp94Gly or Asp94His mutations had high-level resistance.

Objectives. We sought to compare prevalence and determinants of multidrug-resistant tuberculosis (MDR-TB) between tuberculosis patients in Baja California, Mexico, and Hispanic patients in California. Methods. Using data from Mexico’s National TB Drug Resistance Survey (2008–2009) and California Department of Public Health TB case registry (2004–2009), we assessed differences in MDR-TB prevalence comparing (1) Mexicans in Baja California, (2) Mexico-born Hispanics in California, (3) US-born Hispanics in California, and (4) California Hispanics born elsewhere. Results. MDR-TB prevalence was 2.1% in Baja California patients, 1.6% in Mexico-born California patients, 0.4% in US-born California patients, and 2.7% in Hispanic California patients born elsewhere. In multivariate analysis, previous antituberculosis treatment was associated with MDR-TB (odds ratio [OR] = 6.57; 95% confidence interval [CI] = 3.34, 12.96); Mexico-born TB patients in California (OR = 5.08; 95% CI = 1.19, 21.75) and those born elsewhere (OR = 7.69; 95% CI = 1.71, 34.67) had greater odds of MDR-TB compared with US-born patients (reference category). Conclusions. Hispanic patients born outside the US or Mexico were more likely to have MDR-TB than were those born within these countries. Possible explanations include different levels of exposure to resistant strains and inadequate treatment.

On June 13, 2012, a group of key stakeholders, leaders, and national experts on tuberculosis (TB), occupational health, and laboratory science met in Atlanta, Georgia, to focus national discussion on the higher than expected positive results occurring among low-risk, unexposed healthcare workers undergoing serial testing with interferon-γ release assays (IGRAs). The objectives of the meeting were to present the latest clinical and operational research findings on the topic, to discuss evaluation and treatment algorithms that are emerging in the absence of national guidance, and to develop a consensus on the action steps needed to assist programs and physicians in the interpretation of serial testing IGRA results. This report summarizes its proceedings.

Studies over the past several decades have dramatically increased our understanding of the immune response to Mycobacterium tuberculosis infection, and advances in proteomics and genomics have led to a new class of immune-diagnostic tests, termed interferon-γ (IFN-γ) release assays (IGRAs), which appear to obviate many of the problems encountered with the tuberculin skin test (TST). Worldwide, 2 IGRAs are currently commercially available. QuantiFERON-TB Gold In-Tube (Cellestis) is a third-generation product that uses an enzyme-linked immunosorbent assay to measure IFN-γ generated in whole blood stimulated with M. tuberculosis–specific antigens. T-Spot-TB (Oxford Immunotec) employs enzyme-linked immunosorbent spot technology to enumerate the number of purified lymphocytes that respond to M. tuberculosis–specific antigens by producing IFN-γ. These in vitro tests measure the host immune response to M. tuberculosis–specific antigens, which virtually eliminates false-positive cross reactions caused by bacillus Calmette-Guérin vaccination and/or exposure to environmental nontuberculous mycobacteria that plague the interpretation and accuracy of the tuberculin skin test (TST). The high specificity of IGRAs, together with sensitivity commensurate with or better than that of the TST, promises an accurate diagnosis and the ability to focus tuberculosis-control activities on those who are actually infected with M. tuberculosis. The Third Global Symposium was held over a 3-day period and was presented by the University of California, San Diego, Continuing Medical Education department; slides and sound recordings of each presentation are available at http://cme.ucsd.edu/igras/syllabus.html. A moderated discussion is also available at http://cme.ucsd.edu/igrasvideo. This document provides a summary of the key findings of the meeting, specifically focusing on the use of IGRAs in screening healthcare worker populations.

A new multi-national study of the non-commercial MODS assay shows excellent performance in detecting M/XDR-tuberculosis

Molecular diagnostic assays, with their ability to rapidly detect resistance-associated mutations in bacterial genes, are promising technologies to control the spread of drug-resistant tuberculosis (DR-TB). Sequencing assays provide detailed information for specific gene regions and can help diagnostic assay developers prioritize mutations for inclusion in their assays. We performed pyrosequencing of sevenMycobacterium tuberculosisgene regions (katG,inhA,ahpC,rpoB,gyrA,rrs, andeis) for 1,128 clinical specimens from India, Moldova, and South Africa. We determined the frequencies of each mutation among drug-resistant and -susceptible specimens based on phenotypic drug susceptibility testing results and examined mutation distributions by country. The most common mutation among isoniazid-resistant (INHr) specimens was thekatG315ACC mutation (87%). However, in the Eastern Cape, INHrspecimens had a lower frequency ofkatGmutations (44%) and higher frequencies ofinhA(47%) andahpC(10%) promoter mutations. The most common mutation among rifampin-resistant (RIFr) specimens was therpoB531TTG mutation (80%). The mutation was common in RIFrspecimens in Mumbai (83%) and Moldova (84%) but not the Eastern Cape (17%), where the 516GTC mutation appeared more frequently (57%). The most common mutation among fluoroquinolone-resistant specimens was thegyrA94GGC mutation (44%). Therrs1401G mutation was found in 84%, 84%, and 50% of amikacin-resistant, capreomycin-resistant, and kanamycin (KAN)-resistant (KANr) specimens, respectively. Theeispromoter mutation −12T was found in 26% of KANrand 4% of KAN-susceptible (KANs) specimens. Inclusion of theahpCandeispromoter gene regions was critical for optimal test sensitivity for the detection of INH resistance in the Eastern Cape and KAN resistance in Moldova. (This study has been registered atClinicalTrials.govunder registration number NCT02170441.)

Accurate identification of drug-resistant Mycobacterium tuberculosis is imperative for effective treatment and subsequent reduction in disease transmission. Line probe assays rapidly detect mutations associated with resistance and wild-type sequences associated with susceptibility. Examination of molecular-level performance is necessary for improved assay result interpretation and for continued diagnostic development. Using data collected from a large, multisite diagnostic study, probe hybridization results from line probe assays, MTBDR plus and MTBDR sl , were compared to those of sequencing, and the diagnostic performance of each individual mutation and wild-type probe was assessed. Line probe assay results classified as resistant due to the absence of wild-type probe hybridization were compared to those of sequencing to determine if novel mutations were inhibiting wild-type probe hybridization. The contribution of absent wild-type probe hybridization to the detection of drug resistance was assessed via comparison to a phenotypic reference standard. In our study, mutation probes demonstrated significantly higher specificities than wild-type probes and wild-type probes demonstrated marginally higher sensitivities than mutation probes, an ideal combination for detecting the presence of resistance conferring mutations while yielding the fewest number of false-positive results. The absence of wild-type probe hybridization without mutation probe hybridization was determined to be primarily the result of failure of mutation probe hybridization and not the result of novel or rare mutations. Compared to phenotypic culture-based drug susceptibility testing, the absence of wild-type probe hybridization without mutation probe hybridization significantly contributed to the detection of phenotypic rifampin and fluoroquinolone resistance with negligible increases in false-positive results.

Prospective population data on the incidence of tuberculosis (TB) infection has been sparsely reported in the global literature.A population-based prospective study was conducted in rural China to investigate the annual risk of TB infection, and its persistence using serial tuberculin skin tests (TSTs) and an interferon-γ release assay. In total, 13 580 eligible participants from four rural sites, identified as TST negative (The annual conversion rate of QFT among the study sites ranged between 2.1% and 4.9% (average 3.1%), and the incidence of TST conversion ranged between 6.0% and 31.1% (average 14.5%). During the second year's follow-up, infection persistence was investigated using 390 subjects with QFT conversions. Among them, 49.7% (164 out of 330) were found to be consistently QFT positive. Both the conversion and the persistence of QFT positivity were found to be significantly increased with increasing age.In conclusion, the annual TB infection rate was suggested to be ∼1.5% based on persistent positive results after QFT conversion in rural China. Therefore, infection control among those high-risk populations, including the elderly, should be prioritised for TB control in China.

Pregnancy is an established risk factor for the development of severe and disseminated coccidioidomycosis, particularly when infection is acquired during the later stages of gestation. Although recent studies suggest that the incidence of symptomatic coccidioidomycosis during pregnancy is decreasing and that outcome has improved, management is complicated by the observations that azole antifungal agents can be teratogenic when given to some women, particularly at high doses, early in pregnancy. This article summarizes the data on these issues and offers guidance on the management of coccidioidomycosis during pregnancy.

Background. Coccidioidomycosis can be difficult to treat with available therapies, particularly in patients with progressive or disseminated disease. Posaconazole is a new azole antifungal with potent activity against Coccidioides species, the causative agent of coccidioidomycosis. Methods. Twenty patients with chronic pulmonary or nonmeningeal disseminated coccidioidomycosis were enrolled in a multicenter trial to study the safety and tolerability of posaconazole therapy, with efficacy as a secondary end point. Patients received posaconazole (400 mg/day) in capsule formulation for up to 6 months. Safety was evaluated on the basis of the occurrence of adverse events. A satisfactory efficacy response was defined as a ≥50% reduction in the Mycoses Study Group score from baseline. Results. Seventeen (85%) of 20 patients had a satisfactory response to treatment. The median duration of treatment was 173 days. Paired baseline and end-of-treatment culture results for Coccidioides species were available for 4 patients, all of whom converted from being positive to being negative for Coccidioides species. Relapse was experienced by 3 of 9 patients who did not receive antifungal therapy during the follow-up period. In general, posaconazole therapy was well tolerated, with 12 of 20 patients reporting adverse events that were possibly or probably related to treatment. The most common adverse events were dry mouth (in 5 patients [25%]) and headache (in 3 patients [15%]). Conclusions. Courses of posaconazole therapy that were up to 6 months in duration were well tolerated in patients with coccidioidomycosis. Although this study was limited by the number of patients enrolled, it clearly demonstrates that posaconazole shows promise in the treatment of patients with coccidioidomycosis and warrants additional investigation in a full-scale clinical trial.

Diagnostic Criteria of Nontuberculous Mycobacterial Lung Disease Key Laboratory Features of NTM Health Careand Hygiene-associated Disease Prevention Prophylaxis and Treatment of NTM Disease Introduction Methods Taxonomy Epidemiology Pathogenesis Host Defense and Immune Defects Pulmonary Disease Body Morphotype Tumor Necrosis Factor Inhibition Laboratory Procedures Collection, Digestion, Decontamination, and Staining of Specimens Respiratory Specimens Body Fluids, Abscesses, and Tissues Blood Specimen Processing Smear Microscopy Culture Techniques Incubation of NTM Cultures NTM Identification Antimicrobial Susceptibility Testing for NTM Molecular Typing Methods of NTM Clinical Presentations and Diagnostic Criteria Pulmonary Disease Cystic Fibrosis Hypersensitivity-like Disease Transplant Recipients Disseminated Disease Lymphatic Disease Skin, Soft Tissue, and Bone Disease

Undetected and untreated, low-levels of drug resistant (DR) subpopulations in clinical Mycobacterium tuberculosis (Mtb) infections may lead to development of DR-tuberculosis, potentially resulting in treatment failure. Current phenotypic DR susceptibility testing has a theoretical potential for 1% sensitivity, is not quantitative, and requires several weeks to complete. The use of “single molecule-overlapping reads” (SMOR) analysis with next generation DNA sequencing for determination of ultra-rare target alleles in complex mixtures provides increased sensitivity over standard DNA sequencing. Ligation free amplicon sequencing with SMOR analysis enables the detection of resistant allele subpopulations at ≥0.1% of the total Mtb population in near real-time analysis. We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay’s performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples. SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations. This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.