Mycobacterium tuberculosis bloodstream infection prevalence, diagnosis, and mortality risk in seriously ill adults with HIV: a systematic review and meta-analysis of individual patient data

BACKGROUND: The clinical and epidemiological significance of HIV-associated Mycobacterium tuberculosis bloodstream infection (BSI) is incompletely understood. We hypothesised that M tuberculosis BSI prevalence has been underestimated, that it independently predicts death, and that sputum Xpert MTB/RIF has suboptimal diagnostic yield for M tuberculosis BSI. METHODS: We did a systematic review and individual patient data (IPD) meta-analysis of studies performing routine mycobacterial blood culture in a prospectively defined patient population of people with HIV aged 13 years or older. Studies were identified through searching PubMed and Scopus up to Nov 10, 2018, without language or date restrictions and through manual review of reference lists. Risk of bias in the included studies was assessed with an adapted QUADAS-2 framework. IPD were requested for all identified studies and subject to harmonised inclusion criteria: age 13 years or older, HIV positivity, available CD4 cell count, a valid mycobacterial blood culture result (excluding patients with missing data from lost or contaminated blood cultures), and meeting WHO definitions for suspected tuberculosis (presence of screening symptom). Predicted probabilities of M tuberculosis BSI from mixed-effects modelling were used to estimate prevalence. Estimates of diagnostic yield of sputum testing with Xpert (or culture if Xpert was unavailable) and of urine lipoarabinomannan (LAM) testing for M tuberculosis BSI were obtained by two-level random-effect meta-analysis. Estimates of mortality associated with M tuberculosis BSI were obtained by mixed-effect Cox proportional-hazard modelling and of effect of treatment delay on mortality by propensity-score analysis. This study is registered with PROSPERO, number 42016050022. FINDINGS:We identified 23 datasets for inclusion (20 published and three unpublished at time of search) and obtained IPD from 20, representing 96·2% of eligible IPD. Risk of bias for the included studies was assessed to be generally low except for on the patient selection domain, which was moderate in most studies. 5751 patients met harmonised IPD-level inclusion criteria. Technical factors such as number of blood cultures done, timing of blood cultures relative to blood sampling, and patient factors such as inpatient setting and CD4 cell count, explained significant heterogeneity between primary studies. The predicted probability of M tuberculosis BSI in hospital inpatients with HIV-associated tuberculosis, WHO danger signs, and a CD4 count of 76 cells per µL (the median for the cohort) was 45% (95% CI 38­52). The diagnostic yield of sputum in patients with M tuberculosis BSI was 77% (95% CI 63­87), increasing to 89% (80­94) when combined with urine LAM testing. Presence of M tuberculosis BSI compared with its absence in patients with HIV-associated tuberculosis increased risk of death before 30 days (adjusted hazard ratio 2·48, 95% CI 2·05­3·08) but not after 30 days (1·25, 0·84­2·49). In a propensity-score matched cohort of participants with HIV-associated tuberculosis (n=630), mortality increased in patients with M tuberculosis BSI who had a delay in anti-tuberculosis treatment of longer than 4 days compared with those who had no delay (odds ratio 3·15, 95% CI 1·16­8·84). INTERPRETATION:In critically ill adults with HIV-tuberculosis, M tuberculosis BSI is a frequent manifestation of tuberculosis and predicts mortality within 30 days. Improved diagnostic yield in patients with M tuberculosis BSI could be achieved through combined use of sputum Xpert and urine LAM. Anti-tuberculosis treatment delay might increase the risk of mortality in these patients

Mycobacterium tuberculosis bloodstream infection prevalence, diagnosis, and mortality risk in seriously ill adults with HIV: a systematic review and meta-analysis of individual patient data.

BACKGROUND: The clinical and epidemiological significance of HIV-associated Mycobacterium tuberculosis bloodstream infection (BSI) is incompletely understood. We hypothesised that M tuberculosis BSI prevalence has been underestimated, that it independently predicts death, and that sputum Xpert MTB/RIF has suboptimal diagnostic yield for M tuberculosis BSI. METHODS: We did a systematic review and individual patient data (IPD) meta-analysis of studies performing routine mycobacterial blood culture in a prospectively defined patient population of people with HIV aged 13 years or older. Studies were identified through searching PubMed and Scopus up to Nov 10, 2018, without language or date restrictions and through manual review of reference lists. Risk of bias in the included studies was assessed with an adapted QUADAS-2 framework. IPD were requested for all identified studies and subject to harmonised inclusion criteria: age 13 years or older, HIV positivity, available CD4 cell count, a valid mycobacterial blood culture result (excluding patients with missing data from lost or contaminated blood cultures), and meeting WHO definitions for suspected tuberculosis (presence of screening symptom). Predicted probabilities of M tuberculosis BSI from mixed-effects modelling were used to estimate prevalence. Estimates of diagnostic yield of sputum testing with Xpert (or culture if Xpert was unavailable) and of urine lipoarabinomannan (LAM) testing for M tuberculosis BSI were obtained by two-level random-effect meta-analysis. Estimates of mortality associated with M tuberculosis BSI were obtained by mixed-effect Cox proportional-hazard modelling and of effect of treatment delay on mortality by propensity-score analysis. This study is registered with PROSPERO, number 42016050022. FINDINGS: We identified 23 datasets for inclusion (20 published and three unpublished at time of search) and obtained IPD from 20, representing 96·2% of eligible IPD. Risk of bias for the included studies was assessed to be generally low except for on the patient selection domain, which was moderate in most studies. 5751 patients met harmonised IPD-level inclusion criteria. Technical factors such as number of blood cultures done, timing of blood cultures relative to blood sampling, and patient factors such as inpatient setting and CD4 cell count, explained significant heterogeneity between primary studies. The predicted probability of M tuberculosis BSI in hospital inpatients with HIV-associated tuberculosis, WHO danger signs, and a CD4 count of 76 cells per µL (the median for the cohort) was 45% (95% CI 38-52). The diagnostic yield of sputum in patients with M tuberculosis BSI was 77% (95% CI 63-87), increasing to 89% (80-94) when combined with urine LAM testing. Presence of M tuberculosis BSI compared with its absence in patients with HIV-associated tuberculosis increased risk of death before 30 days (adjusted hazard ratio 2·48, 95% CI 2·05-3·08) but not after 30 days (1·25, 0·84-2·49). In a propensity-score matched cohort of participants with HIV-associated tuberculosis (n=630), mortality increased in patients with M tuberculosis BSI who had a delay in anti-tuberculosis treatment of longer than 4 days compared with those who had no delay (odds ratio 3·15, 95% CI 1·16-8·84). INTERPRETATION: In critically ill adults with HIV-tuberculosis, M tuberculosis BSI is a frequent manifestation of tuberculosis and predicts mortality within 30 days. Improved diagnostic yield in patients with M tuberculosis BSI could be achieved through combined use of sputum Xpert and urine LAM. Anti-tuberculosis treatment delay might increase the risk of mortality in these patients. FUNDING: This study was supported by Wellcome fellowships 109105Z/15/A and 105165/Z/14/A.

Evaluation of 5 Novel protein biomarkers for the rapid diagnosis of pulmonary and extra-pulmonary tuberculosis: preliminary results.

Improved methods are required for the early and accurate diagnosis of tuberculosis, especially in the patients with smear-negative disease. Several biomarkers have been tried but most have shown poor sensitivity or specificity. In present study we aimed to evaluate the diagnostic utility of five novel antigens identified earlier by us. This is an initial study conducted on 250 subjects. The five recombinant antigens, named as rSS1 (Rv2145c), rSS2 (Rv0164), rSS3 (Rv1437), rSS4 (Rv1827) and rSS5 (Rv2970c), were expressed in pQE-30 expression vector, purified and their sero-diagnostic efficacy was evaluated in an unblinded manner using dot-blot and ELISA methods. The sensitivity and specificity of these novel antigens were compared with commercially available standard esat6 and 38 kDa antigens. Bacteriologically confirmed TB patients, non-TB disease controls and healthy individuals were included. which are based on novel antigen or novel technology, Area under curve (AUC) of the selected antigens were 0.98 (0.98-0.99) for rSS1, 0.88 (0.84-0.92) for rSS2, 0.88 (0.84-0.92) for rSS3, 0.95 (0.93-0.98) for rSS4 and 0.99 (0.98-1.0) for rSS5. Receiver operative characteristic (ROC) curve showed highly significant difference between TB and healthy subjects (p = <0.001). These initial findings, show that the recombinant antigens rSS1, rSS4 and rSS5 could be used as highly potential biomarkers for the serological diagnosis of active TB.

Comparative proteomic analysis of sequential isolates of Mycobacterium tuberculosis sensitive and resistant Beijing type from a patient with pulmonary tuberculosis.

AIM & OBJECTIVE: In India, tuberculosis (TB) is a foremost health problem, and the emergence of multidrug-resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (M. tuberculosis) has further complicated the situation. Although various mechanisms have been proposed to elucidate the emergence of resistance, our knowledge remains insufficient. The formation of a very complex network and drugs of proteins are countered by their efflux/modification or target over-expression/modification. The analysis of the over-expressed proteins and their qualitative and phenotypic evaluation before and after the development of drug-resistance may be the most appropriate tool to understand the mechanisms of the mechanism of development of drug-resistance. Most studies are performed on distinct strains. Therefore, the objective of this study was to compare the proteomic information of sequential isolates of M. tuberculosis Beijing type from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy. METHODS: In this study, a clinical isolate of M. tuberculosis was grown in Middlebrook 7H9 broth medium for 2weeks, and the cell lysate of isolates was prepared by sonication and centrifugation. We compared and analyzed the whole cell lysate proteins of M. tuberculosis sequential clinical isolate from a patient with pulmonary TB before and after the development of drug resistance using two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and bioinformatics tools. RESULTS: The genotypes of both isolates remained homologous, showing no re-infection. The first isolate (before treatment) was sensitive to all the first-line drugs, sequential isolate was found resistant to rifampicin (RIF) and isoniazid (INH) and developed mutations in rpoB, katG and inhA. The concentrations of 17 protein spots were found to be consistently over-expressed in RIF- and INH-resistant isolates. The most prominent and over-expressed proteins found during the development of drug resistance were wag31, Rv2714, GarA, SSB, FabG4, Probable lipase, Rv3924c, Rv3204A, Rv2031c, Rv3418c and GroES. The InterProScan and homology searches generated insights into the possible functions and essential domains of the proteins. Rv1827, Rv2626c, Rv2714, Rv2970c, Rv3208A, and Rv3881c showed significant in silico interaction with RIF and INH; thus, the over-expression in the drug-resistant isolates could be compensating the inhibited/modulated molecules. Other proteins, which are over-expressed but do not unveil good binding with drug, might be indirectly associated with RIF and INH. CONCLUSIONS: This proteomic study provides an understanding about the proteins that are over-expressed during the development of drug resistance. These over-expressed proteins, identified here, could prove useful as vaccine candidate, immunodiagnostic and possibly drug-resistant or chemotherapeutic markers in future.

Comparative proteomic analysis of sequential isolates of Mycobacterium tuberculosis from a patient with pulmonary tuberculosis turning from drug sensitive to multidrug resistant.

BACKGROUND & OBJECTIVES: Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). METHODS: In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The genotypes of all four isolates remained homologous, indicating no re-infection. The initial isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and rpoB. the intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF resistant isolates. The most prominent and overexpressed proteins found during the development of drug resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c. INTERPRETATION & CONCLUSIONS: This preliminary proteomic study provides an insight about the proteins that are upregulated during drug resistance development. These upregulated proteins, identified here, could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more studies are required to confirm these findings.

Targeted gene knockout and essentiality testing by homologous recombination.

This chapter provides an updated experimental protocol for generating allelic exchange mutants of mycobacteria by two-step selection using the p2NIL/pGOAL system. The types of mutants that can be generated using this approach are targeted gene knockouts marked with a drug resistance gene, unmarked deletion mutants, or strains in which a point mutation/s has been introduced into the target gene. A method for assessing the essentiality of a gene for mycobacterial growth by means of allelic exchange is also described. This method, which utilizes a merodiploid strain carrying a second copy of the gene of interest on an integration vector, allows the exploration by means of complement switching of structure-function relationships in proteins that are essential for mycobacterial growth.

Deciphering the sequential events during in vivo acquisition of drug resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) and the disease has remained a major health problem in most of the developing countries, particularly after the emergence of multidrug-resistant TB (MDR-TB). The MDR-TB is an intriguing subject and very little is known about the in vivo processes which take place during the acquisition of MDR. This study describes a unique case of pulmonary TB (PTB) from which four sequential isolates of MTB could be isolated while the patient was on anti-tubercular treatment. The first baseline isolate was sensitive to all drugs, but the subsequent three isolates acquired resistance to multiple drugs and finally the patient died after 27months post-diagnosis when his fourth isolate became resistant to isoniazid, rifampicin, ethambutol and kanamycin. All sequential cultures were identified as MTB using conventional and molecular methods, including 16s RNA sequencing and the spoligotyping. Spoligotyping followed by comparison with SITVITWEB database revealed that all the isolates belonged to the family of the Central Asian Strain Delhi (CAS1_Delhi, ST26) genotype, and no cross or mixed infections were observed. The drug resistance was further characterized at the molecular level by sequencing the target genes (katG, inhA, rpoB, embB, eis promoter region and rrs). The results revealed mutated alleles associated with resistance to the respective drugs. This unique case indicates that it is possible to isolate MTB during treatment if the strain is acquiring resistance. The data presented from four sequential isolates provides an insight into what sequential genetic and proteomic changes occur in the bacteria during the in vivo acquisition of MDR.

Vitamin B(12) metabolism in Mycobacterium tuberculosis.

Mycobacterium tuberculosis is included among a select group of bacteria possessing the capacity for de novo biosynthesis of vitamin B12, the largest and most complex natural organometallic cofactor. The bacillus is also able to scavenge B12 and related corrinoids utilizing an ATP-binding cassette-type protein that is distinct from the only known bacterial B12-specific transporter, BtuFCD. Consistent with the inferred requirement for vitamin B12 for metabolic function, the M. tuberculosis genome encodes two B12 riboswitches and three B12-dependent enzymes. Two of these enzymes have been shown to operate in methionine biosynthesis (MetH) and propionate utilization (MutAB), while the function of the putative nrdZ-encoded ribonucleotide reductase remains unknown. Taken together, these observations suggest that M. tuberculosis has the capacity to regulate core metabolic functions according to B12 availability - whether acquired via endogenous synthesis or through uptake from the host environment - and, therefore, imply that there is a role for vitamin B12 in pathogenesis, which remains poorly understood.

Co(II) and Cu(II) pyrophosphate complexes have selectivity and potency against Mycobacteria including Mycobacterium tuberculosis.

Tuberculosis (TB) causes up to 10 million incident cases worldwide per annum. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains are leading factors in the resurgence of TB cases and the need to produce new agents to combat such infection. Herein, we describe Co(II) and Cu(II) metal based complexes that feature the pyrophosphate ligand with notable selectivity and marked potency against Mycobacterium tuberculosis, including MDR strains. Such complexes are confirmed to be bacteriocidal and not affected by efflux inhibitors. Finally, while susceptibility to copper has recently been established for M. tuberculosis, the greater efficacy of cobalt observed herein is of considerable note and in line with the discovery of a copper metallothionein in M. tuberculosis.

A vitamin B12 transporter in Mycobacterium tuberculosis.

Vitamin B12-dependent enzymes function in core biochemical pathways in Mycobacterium tuberculosis, an obligate pathogen whose metabolism in vivo is poorly understood. Although M. tuberculosis can access vitamin B12 in vitro, it is uncertain whether the organism is able to scavenge B12 during host infection. This question is crucial to predictions of metabolic function, but its resolution is complicated by the absence in the M. tuberculosis genome of a direct homologue of BtuFCD, the only bacterial B12 transport system described to date. We applied genome-wide transposon mutagenesis to identify M. tuberculosis mutants defective in their ability to use exogenous B12. A small proportion of these mapped to Rv1314c, identifying the putative PduO-type ATP : co(I)rrinoid adenosyltransferase as essential for B12 assimilation. Most notably, however, insertions in Rv1819c dominated the mutant pool, revealing an unexpected function in B12 acquisition for an ATP-binding cassette (ABC)-type protein previously investigated as the mycobacterial BacA homologue. Moreover, targeted deletion of Rv1819c eliminated the ability of M. tuberculosis to transport B12 and related corrinoids in vitro. Our results establish an alternative to the canonical BtuCD-type system for B12 uptake in M. tuberculosis, and elucidate a role in B12 metabolism for an ABC protein implicated in chronic mycobacterial infection.

Poor performance of serological tests in the diagnosis of pulmonary tuberculosis: evidence from a contact tracing field study.

BACKGROUND: Delayed or missed diagnosis of TB continues to fuel the global TB epidemic, especially in resource limited settings. Use of serology for the diagnosis of tuberculosis, commonly used in India, is another factor. In the present study a commercially available serodiagnostic assay was assessed for its diagnostic value in combination with smear, culture and clinical manifestations. METHODOLOGY/PRINCIPAL FINDINGS: A total of 2300 subjects were recruited for the study, but 1041 subjects were excluded for various reasons. Thus 1259 subjects were included in the study of which 470 were pulmonary tuberculosis cases (440 of 470 were culture-positive) and 789 were their asymptomatic contacts. A house-to-house survey method was used. Blood samples were tested for IgM, IgA, and IgG antibodies using the Pathozyme Myco M (IgM), Myco A (IgA) and Myco G (IgG) enzyme immunoassay (EIA). Out of 470 PTB cases, BCG scar was positive in 82.34%. The Mantoux test and smear positivity rates in PTB cases were 94.3% (430/456), and 65.32% (307/470), respectively. Among the asymptomatic contacts, BCG scar was positive in 95.3% and Mantoux test was positive in 80.66% (442/548) contacts. No contact was found falsely smear positive. The sensitivity of IgM, IgA, and IgG EIA tests was 48.7%, 25.7% and 24.4%, respectively, while the specificity was 71.5%, 80.5%, 76.6%, respectively. Performance of EIAs was not affected by the previous BCG vaccination. However, prior BCG vaccination was statistically significantly (p = 0.005) associated with Mantoux test positivity in PTB cases but not in contacts (p = 0.127). The agreement between serology and Mantoux test was not significant. CONCLUSION: The commercial serological test evaluated showed poor sensitivity and specificity and suggests no utility for detection of pulmonary tuberculosis.

Mycobacterium avium subspecies Paratuberculosis and Crohn's regional ileitis: how strong is association?

Mycobacterium avium subsp. paratuberculosis (MAP) is a well-established etiological agent of Johne's disease in animals. In humans, similar clinical condition, first described by Crohn as regional ileitis in 1932, now known as Crohn's diseases (CD), has also been associated with this mycobacterial species. However, there are two schools of thoughts, one favoring MAP as its etiological agent while the second considers it as an immune-inflammatory condition triggered by an external factor. Onset of CD requires a series of events including predisposition of certain inherited genetic traits, associated environmental stimuli, and immune-inflammatory response. A combination of these factors probably leads to this disease. Recently, some human genes have also been identified which regulate ability to respond appropriately to the external factors. Added to these factors are concerns about the selection of clinical specimens and poor adherence to laboratory quality controls. The literature is full of contradictory findings, but there a lack of uniformity in the materials and methods used by many of these researchers. In this review, we provide our perspective under above circumstances and give our point of view which may open a platform for debate regarding the MAP as the etiological agent of human CD.

Urine as an adjunct specimen for the diagnosis of active pulmonary tuberculosis.

BACKGROUND: The diagnosis of pulmonary tuberculosis (PTB) is conventionally established by examination of three Ziehl-Neelsen stained smears; however, negative results do not preclude active TB. Since tubercle bacilli or their nucleic acids are also expected to be excreted through the kidneys, we assessed spot urine as a supplementary specimen for diagnosing PTB. METHODS: A total of 164 respiratory specimens (147 sputum, 15 bronchoalveolar lavage, and two gastric lavage) from 81 suspected PTB cases were prospectively collected and processed. A total of 112 non-TB controls were also included in the study. For three consecutive days, morning urine specimens were collected from all patients and controls, and were processed for culture by BACTEC MGIT 960 (mycobacteria growth indicator tube) and Lowenstein-Jensen methods and for PCR by amplifying a 441-bp fragment of the hsp65 gene (Mycobacterium genus-specific) and a 786-bp fragment of the cfp32 gene (TB complex-specific). RESULTS: Of the 81 patients suspected of having PTB, 46 (56.8%) were sputum culture-positive. Of these, 12 (26.1%) were also urine culture-positive for Mycobacterium tuberculosis. Of the 35 sputum culture-negative cases, three (8.6%) were urine culture-positive. The TB complex specific PCR (cfp32) was positive in 52.2% (24/46) of the bacteriologically-confirmed and 28.6% (10/35) of the bacteriologically-negative PTB patients. In none of the control subjects were urine culture or PCR found to be positive for M. tuberculosis. CONCLUSIONS: Specific PCR and culture examination of spot urine samples from suspected PTB patients significantly improved the detection rate of PTB and should be encouraged in resource-limited settings and where multiple pulmonary specimens are not feasible.

In-vitro antimycobacterial drug susceptibility testing of non-tubercular mycobacteria by tetrazolium microplate assay.

BACKGROUND: Non-tubercular mycobacteria (NTM) has not been given due attention till the recent epidemic of HIV. This has led to increasing interest of health care workers in their biology, epidemiology and drug resistance. However, timely detection and drug susceptibility profiling of NTM isolates are always difficult in resource poor settings like India. Furthermore, no standardized methodology or guidelines are available to reproduce the results with clinical concordance. OBJECTIVE: To find an alternative and rapid method for performing the drug susceptibility assay in a resource limited settings like India, we intended to evaluate the utility of Tetrazolium microplate assay (TEMA) in comparison with proportion method for reporting the drug resistance in clinical isolates of NTM. METHODS: A total of fifty-five NTM isolates were tested for susceptibility against Streptomycin, Rifampicin, Ethambutol, Ciprofloxacin, Ofloxacin, Azithromycin, and Clarithromycin by TEMA and the results were compared with agar proportion method (APM). RESULTS: Of the 55 isolates, 23 (41.8%) were sensitive to all the drugs and the remaining 32 (58.2%) were resistant to at least one drug. TEMA had very good concordance with APM except with minor discrepancies. Susceptibility results were obtained in the median of 5 to 9 days by TEMA. The NTM isolates were highly sensitive against Ofloxacin (98.18% sensitive) and Ciprofloxacin (90.09% sensitive). M. mucogenicum was sensitive only to Clarithromycin and resistant to all the other drugs tested. The concordance between TEMA and APM ranged between 96.4 - 100%. CONCLUSION: Tetrazolium Microplate Assay is a rapid and highly reproducible method. However, it must be performed only in tertiary level Mycobacteriology laboratories with proper bio-safety conditions.

High rate of extensively drug-resistant tuberculosis in Indian AIDS patients.

Fifty-four full-blown AIDS patients suspected of having HIV-tuberculosis co-infection were investigated for the prevalence of extensively drug-resistant (XDR) Mycobacterium tuberculosis. Out of the 54 patients, M. tuberculosis was isolated from 24 (44.4%). Twelve (50%) isolates of these had resistance to first-line drugs, whereas four (33.33%) were also resistant to second-line drugs. All four patients, in whom XDR M. tuberculosis was isolated, died within 2.6 months of diagnosis.

Novel method for clearing red blood cell debris from BacT/ALERT blood culture medium for improved microscopic and antimycobacterial drug susceptibility test results.

Even though automation in mycobacterial culture has immensely improved the detection of organisms, identification of species and antimycobacterial susceptibility testing from blood culture bottles remain cumbersome and error-prone due to the presence of intact red blood cells (RBCs). The removal or lysis of these RBCs and excessive protein from the blood components could theoretically help improve this process. The present study reports an effective method that uses ammonium chloride (NH(4)Cl) and Triton X-100 to lyse the RBCs in blood culture medium. The method was optimized by preparing various concentrations of NH(4)Cl and Triton X-100, and incubation conditions, leading to eight protocols. The lysis protocol with a concentration of 150 mM of NH(4)Cl, 0.5% Triton X-100, and 1% potassium bicarbonate, pH 7.0, and incubation at 37 degrees C for 15 min was found to be optimal. This method not only made the culture medium clear, the protein concentration decreased from 753.5+/-39.4 to 53.2+/-4.2 mg/mL in the M. tuberculosis-spiked culture medium and in the blood culture medium inoculated with the blood from tuberculosis patients. The method had no adverse effect on mycobacteria, and no depletion of M. tuberculosis colony-forming units was found. The lysate could be used for antimycobacterial susceptibility testing with no difficulty in setting the mycobacterial concentration of inoculum to 0.5 McFarland standards. Furthermore, this method had the added advantage in the microscopy and molecular methods for the speciation of Mycobacterium sp.

Nontuberculous mycobacterial infections in Indian AIDS patients detected by a novel set of ESAT-6 polymerase chain reaction primers.

Nontuberculous mycobacteria are often underdiagnosed due to lack of proper diagnostic facilities. To overcome this, we created a rapid PCR method for the species-specific diagnosis of Mycobacterium tuberculosis and its differentiation from other mycobacteria. A set of PCR primers targeting the gene encoding for early-secreted antigen-6 (ESAT-6) of the M. tuberculosis complex was designed and standardized on mycobacterial standard strains and on 75 recent isolates from AIDS patients and 70 isolates from HIV-negative patients seen at the hospital of the All India Institute of Medical Sciences, New Delhi, India. All 145 fresh mycobacterial isolates were identified using phenotypic methods and 16S rRNA PCR followed by sequencing of hypervariable region A. The ESAT-6 PCR detected all of the M. tuberculosis strains correctly (100% sensitivity), but none of the nontuberculous Mycobacterium spp. gave positive results (100% specific). Most nontuberculous mycobacteria were identified in patients with AIDS (24%) followed by those with tuberculous lymphadenitis (12.5%) and those with pulmonary tuberculosis whose treatment had failed (4.3%). The most common nontuberculous mycobacterial species isolated from AIDS patients was M. avium (6.6%), followed by M. fortuitum (5.7%), M. intracellulare and M. terrae (2.6% each). M. celatum, M. duvalii, M. austroafricanum, M. phlei and M. flavescence were also isolated from one patient each. The combination of genus-specific PCR primers with the novel ESAT-6 primer set could provide accurate and rapid diagnosis of mycobacteriosis.