Ivan Bastian

Mycobacterium tuberculosis Strains with Highly Discordant Rifampin Susceptibility Test Results

ABSTRACT The objectives of this study were to investigate the origin of highly discordant rifampin (rifampicin) (RMP) drug susceptibility test results obtained for Mycobacterium tuberculosis strains during proficiency testing. Nine Supra-National Tuberculosis Reference Laboratories tested the RMP susceptibilities of 19 selected M. tuberculosis strains, using standard culture-based methods. The strains were classified as definitely resistant (R) (n = 6) or susceptible (S) (n = 2) or probably resistant (PR) (n = 8) or susceptible (PS) (n = 3) based on rpoB mutations and treatment outcome. All methods yielded a susceptible result for the two S and three PS strains lacking an rpoB mutation and a resistant result for one R strain with a Ser531Leu mutation and one PR strain with a double mutation. Although the remaining 12 R and PR strains had rpoB mutations (four Asp516Tyr, three Leu511Pro, two Leu533Pro, one each His526Leu/Ser, and one Ile572Phe), they were all susceptible by the radiometric Bactec 460TB or Bactec 960 MGIT methods. In contrast, only one was susceptible by the proportion method on Löwenstein-Jensen medium and two on Middlebrook 7H10 agar. Low-level but probably clinically relevant RMP resistance linked to specific rpoB mutations is easily missed by standard growth-based methods, particularly the automated broth-based systems. Further studies are required to confirm these findings, to determine the frequency of these low-level-resistant isolates, and to identify technical improvements that may identify such strains.

L-arginine and vitamin D adjunctive therapies in pulmonary tuberculosis: a randomised, double-blind, placebo-controlled trial

Background Vitamin D (vitD) and L-arginine have important antimycobacterial effects in humans. Adjunctive therapy with these agents has the potential to improve outcomes in active tuberculosis (TB). Methods In a 4-arm randomised, double-blind, placebo-controlled factorial trial in adults with smear-positive pulmonary tuberculosis (PTB) in Timika, Indonesia, we tested the effect of oral adjunctive vitD 50,000 IU 4-weekly or matching placebo, and L-arginine 6.0 g daily or matching placebo, for 8 weeks, on proportions of participants with negative 4-week sputum culture, and on an 8-week clinical score (weight, FEV1, cough, sputum, haemoptysis). All participants with available endpoints were included in analyses according to the study arm to which they were originally assigned. Adults with new smear-positive PTB were eligible. The trial was registered at ClinicalTrials.gov NCT00677339. Results 200 participants were enrolled, less than the intended sample size: 50 received L-arginine + active vitD, 49 received L-arginine + placebo vit D, 51 received placebo L-arginine + active vitD and 50 received placebo L-arginine + placebo vitD. According to the factorial model, 99 people received arginine, 101 placebo arginine, 101 vitamin D, 99 placebo vitamin D. Results for the primary endpoints were available in 155 (4-week culture) and 167 (clinical score) participants. Sputum culture conversion was achieved by week 4 in 48/76 (63%) participants in the active L-arginine versus 48/79 (61%) in placebo L-arginine arms (risk difference −3%, 95% CI −19 to 13%), and in 44/75 (59%) in the active vitD versus 52/80 (65%) in the placebo vitD arms (risk difference 7%, 95% CI −9 to 22%). The mean clinical outcome score also did not differ between study arms. There were no effects of the interventions on adverse event rates including hypercalcaemia, or other secondary outcomes. Conclusion Neither vitD nor L-arginine supplementation, at the doses administered and with the power attained, affected TB outcomes. Registry ClinicalTrials.gov. Registry number: NCT00677339

Investigation of Spa Pools Associated with Lung Disorders Caused by Mycobacterium avium Complex in Immunocompetent Adults

ABSTRACT Three cases of Mycobacterium avium complex-related lung disorders were associated with two poorly maintained spa pools by genotypic investigations. Inadequate disinfection of the two spas had reduced the load of environmental bacteria to less than 1 CFU/ml but allowed levels of M. avium complex of 4.3 × 104 and 4.5 × 103 CFU/ml. Persistence of the disease-associated genotype was demonstrated in one spa pool for over 5 months until repeated treatments with greater than 10 mg of chlorine per liter for 1-h intervals eliminated M. avium complex from the spa pool. A fourth case of Mycobacterium avium complex-related lung disease was associated epidemiologically but not genotypically with another spa pool that had had no maintenance undertaken. This spa pool contained low numbers of mycobacteria by smear and was culture positive for M. avium complex, and the nonmycobacterial organism count was 5.2 × 106 CFU/ml. Public awareness about the proper maintenance of private (residential) spa pools must be promoted by health departments in partnership with spa pool retailers.

Evaluation of Mycobacteria Growth Indicator Tube for Direct and Indirect Drug Susceptibility Testing of Mycobacterium tuberculosis from Respiratory Specimens in a Siberian Prison Hospital

ABSTRACT The manual Mycobacteria Growth Indicator Tube (MGIT) method was evaluated for performing direct and indirect drug susceptibility testing (DST) of Mycobacterium tuberculosis for isoniazid and rifampin on 101 strongly smear-positive sputum specimens in a Siberian prison hospital. Using the indirect method of proportion (MOP) as the “gold standard,” the accuracies of isoniazid and rifampin susceptibility testing by the direct MGIT system were 97.0 and 94.1%, respectively. The accuracy of the indirect MGIT system was 98.0% for both drugs. The turnaround times from specimen processing to reporting of the DST results ranged between 4 and 23 (mean, 9.2) days by the direct MGIT method, 9 and 30 (mean, 15.3) days by the indirect MGIT method, and 26 and 101 (mean, 59.6) days by the indirect MOP. MGIT appears to be a reliable, rapid, and convenient method for performing direct and indirect DSTs in low-resource settings, but further studies are required to refine the direct DST protocol. Cost is the only factor prohibiting widespread implementation of MGIT.

Current thinking on the management of tuberculosis

High-income countries are moving toward tuberculosis (TB) elimination. Sophisticated diagnostic tests and effective treatment regimens are readily available. The range of available resources even makes effective treatment of multidrug-resistant tuberculosis (MDRTB) possible. The introduction of highly active antiretroviral therapy and specific TB control measures has reduced the incidence of HIV-associated TB disease. Unfortunately, the situation in low-income countries that carry 95% of the global TB burden is less positive. TB diagnosis still relies upon sputum smear microscopy. The management of MDRTB remains problematic though guidelines for DOTS-plus programs have been developed, and cheaper second-line drugs are becoming available. The HIV epidemic continues to confound TB control efforts, particularly in sub-Saharan Africa. The appropriate package of interventions for controlling HIV/TB disease remains undefined and unimplemented. The international community must provide the funding and technical support to address the alarming dichotomy in TB control that exists between rich and poor countries.

New World cutaneous leishmaniasis imported into Australia

Summary A case of cutaneous leishmaniasis in a traveller from Belize, Central America is reported. Leishmaniasis presents rarely in Australia and delays in diagnosis and treatment often occur. A high index of suspicion in a patient who has returned from an endemic region is required. Subsequent confirmation of a diagnosis of cutaneous leishmaniasis is best achieved by demonstration of the organism on skin biopsy, aspiration or smear. The histology is variable and depends on geographic, parasite species and host factors. Speciation of New World disease as either Leishmania braziliensis or Leishmania mexicana is important to determine the risk of later development of mucosal disease, which normally only occurs with L. braziliensis infection, and for optimal treatment. Several different modes of treatment have been suggested, but antimonials, such as sodium stibogluconate, remain the treatment of choice in New World cutaneous leishmaniasis.

Molecular diagnostics for tuberculosis

Summary The phenotypic methods of smear microscopy, culture and indirect drug susceptibility testing (DST) remain the ‘gold standard’ diagnostics for tuberculosis (TB) in 2015. However, this review demonstrates that genotypic methods are in the ascendancy. Current-generation nucleic acid amplification tests (NAATs) are important supplementary tests for the rapid direct detection of (multidrug-resistant) TB in specific clinical settings. Genotypic detection is already the preferred method of detecting rifampicin and pyrazinamide resistance. Next-generation NAATs able to detect about 10 colony forming units/mL of sputum could replace culture as the initial test for detecting TB. Whole genome sequencing could also plausibly replace phenotypic DST but much work is required in method standardisation, database development and elucidation of all resistance gene determinants. The challenge then will be to rollout these increasingly complex and expensive diagnostics in the low-income countries where TB is prevalent.

Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA

ABSTRACT Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium-M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera. Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera. We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico-predicted specificity for M. chimaera. Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera.

MYCOBACTERIUM PINNIPEDII TUBERCULOSIS IN A FREE-RANGING AUSTRALIAN FUR SEAL (ARCTOCEPHALUS PUSILLUS DORIFERUS) IN SOUTH AUSTRALIA

Abstract:  This report describes the first case in South Australia, Australia, of Mycobacterium pinnipedii tuberculosis in a free-ranging Australian fur seal (Arctocephalus pusillus doriferus). Severe pyogranulomatous pleuropneumonia with intrahistocytic acid-fast beaded filamentous bacilli was seen on histology. M. pinnipedii was confirmed by full 24-loci mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing. Spillover concerns for public health and cattle are discussed.

Culture-independent detection of nontuberculous mycobacteria in clinical respiratory samples

ABSTRACT Culture-based detection of nontuberculous Mycobacteria (NTM) in respiratory samples is time consuming and can be subject to overgrowth by nonmycobacterial bacteria. We describe a single-reaction TaqMan quantitative PCR assay for the direct detection of NTM species in clinical samples that is specific, sensitive, and robust.