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1.  Does Neutralization of Gastric Aspirates from Children with Suspected Intrathoracic Tuberculosis Affect Mycobacterial Yields on MGIT Culture? 
Journal of Clinical Microbiology  2013;51(6):1753-1756.
The microbiological confirmation of pulmonary tuberculosis in children relies on cultures of gastric aspirate (GA) specimens. Conventionally, GAs are neutralized to improve culture yields of mycobacteria. However, there are limited data to support this practice. To study the utility of neutralization of GAs with sodium bicarbonate in children with intrathoracic tuberculosis, a total of 116 children of either sex, aged 6 months to 14 years (median age, 120 months; interquartile range [IQR], 7 to 192 months), underwent gastric aspiration on 2 consecutive days. Gastric aspirates were divided into two aliquots, and only one aliquot was neutralized with 1% sodium bicarbonate. Both aliquots were processed for smear and culture examinations. Out of the 232 gastric aspirates, 12 (5.17%) were acid-fast bacilli (AFB) smear positive. There were no differences in smear positivity rates from samples with or without neutralization. The yield of Mycobacterium tuberculosis on a Bactec MGIT 960 culture system was significantly lower in the neutralized samples (16.3% [38/232]) than in the nonneutralized samples (21.5% [50/232]) (P = 0.023). There was no significant difference between the neutralized and the nonneutralized samples in time to detection using the MGIT 960 system (average, 24.6 days; IQR, 12 to 37 days) (P = 0.9). The contamination rates were significantly higher in the neutralized samples than in the nonneutralized samples (17.2% [40/232] versus 3.9% [9/232]) (P = 0.001). The agreement for positive mycobacterial culture between the two approaches was 66.5% (P = 0.001). Hence, we recommend that gastric aspirate samples not be neutralized with sodium bicarbonate prior to culture for M. tuberculosis.
doi:10.1128/JCM.00202-13
PMCID: PMC3716107  PMID: 23536406
2.  Two-Step Tuberculin Skin Testing in School-Going Adolescents with Initial 0-4 Millimeter Responses in a High Tuberculosis Prevalence Setting in South India 
PLoS ONE  2013;8(9):e71470.
Background
The utility of two-step tuberculin skin testing among adolescents in high tuberculosis prevalence settings is not well established.
Objectives
To determine the proportion and determinants of a 0-4 mm response to an initial standard tuberculin skin test (TST) and evaluating 'boosting' with repeat testing.
Methods
Adolescents between 11 and 18 years attending schools/colleges underwent a TST; those with a response of between 0–4 mm had a repeat TST 1-4 weeks later.
Results
Initial TST was done for 6608/6643 participants; 1257 (19%) developed a 0-4 mm response to the initial TST. Younger age and under-nutrition were more likely to be associated with a 0-4 mm response, while the presence of BCG (Bacillus Calmette Guerin) scar and higher socio-economic class were less likely to be associated with a 0-4 mm response. On repeat testing boosting was seen in 13.2% (145/1098; ≥ 6 mm over the initial test) while 4.3% showed boosting using a more conservative cutoff of a repeat TST ≥ 10 mm with an increment of at least 6 mm (47/1098). History of exposure to a tuberculosis (TB) case was associated with enhanced response.
Conclusion
The proportion of adolescents who demonstrated boosting on two-step TST testing in our study was relatively low. As a result repeat testing did not greatly alter the prevalence of TST positivity. However, the two-step TST helps identify individuals who can potentially boost their immune response to a second test, and thus, prevents them from being misclassified as those with newly acquired infection, or tuberculin converters. While two-step tuberculin skin testing may have a limited role in population- level TST surveys, it may be useful where serial tuberculin testing needs to be performed to distinguish those who show an enhanced response or boosters from those who indeed have a new infection, or converters.
doi:10.1371/journal.pone.0071470
PMCID: PMC3765300  PMID: 24039716
3.  Effect of Ascaris Lumbricoides specific IgE on tuberculin skin test responses in children in a high-burden setting: a cross-sectional community-based study 
BMC Infectious Diseases  2012;12:211.
Background
M.tuberculosis (M.tb) is associated with enhanced T helper cell type 1 (Th1) immune responses while helminth infection is associated with T helper cell type 2 (Th2) immune responses. Our aim was to investigate whether helminth infection could influence the ability to generate an appropriate Th1 immune response that is characterized by a positive tuberculin skin test (TST), in M.tb exposed children.
Methods
We completed a community-based, cross sectional household contact tracing study, using matched enrolment of HIV negative children with and without documented household M.tb exposure. We documented demographics, clinical characteristics, HIV status, M.tb exposure (using a standard contact score) and M.tb infection status (TST > = 10 mm). Ascaris lumbricoides-specific IgE was used as proxy for Ascaris infection/exposure.
Results
Of 271 children (median age 4 years (range: 4 months to 15 years)) enrolled, 65 participants (24%) were serum positive for Ascaris IgE. There were 168 (62%) children with a documented household tuberculosis contact and 107 (40%) were (TST) positive overall.
A positive TST was associated with increasing age (Odds Ratio (OR) =1.17, p < 0.001), increasing M.tb contact score (OR = 1.17, p < 0.001), previous tuberculosis treatment (OR = 4.8, p = 0.06) and previous isoniazid preventive treatment (OR = 3.16, p = 0.01). A visible bacillus Calmette-Guérin (BCG) scar was associated with reduced odds of being TST positive (OR = 0.42, p = 0.01).
Ascaris IgE was not associated with TST status in univariate analysis (OR = 0.9, p = 0.6), but multivariable logistic regression analysis suggested an inverse association between Ascaris IgE status and a positive TST (OR = 0.6, p = 0.08), when adjusted for age, and M.tb contact score. The addition of an age interaction term to the model suggested that the age effect was stronger among Ascaris IgE positive children; the effect of being Ascaris IgE positive significantly reduced the odds of being TST positive amongst younger children while this effect weakened with increasing age.
Conclusions
Our preliminary findings highlight a high prevalence of both Ascaris exposure/infection and M.tb infection in children in an urban setting. Helminth exposure/infection may reduce the immune response following M.tb exposure when controlling for epidemiological and clinical covariates. These findings might be relevant to the interpretation of immunological tests of M.tb infection in children.
doi:10.1186/1471-2334-12-211
PMCID: PMC3482567  PMID: 22966931
Tuberculosis; Helminth infection; Ascaris, M.tb infection; Immune polarization; Paediatric tuberculosis
4.  Baseline Predictors of Sputum Culture Conversion in Pulmonary Tuberculosis: Importance of Cavities, Smoking, Time to Detection and W-Beijing Genotype 
PLoS ONE  2012;7(1):e29588.
Background
Time to detection (TTD) on automated liquid mycobacterial cultures is an emerging biomarker of tuberculosis outcomes. The M. tuberculosis W-Beijing genotype is spreading globally, indicating a selective advantage. There is a paucity of data on the association between baseline TTD and W-Beijing genotype and tuberculosis outcomes.
Aim
To assess baseline predictors of failure of sputum culture conversion, within the first 2 months of antitubercular therapy, in participants with pulmonary tuberculosis.
Design
Between May 2005 and August 2008 we conducted a prospective cohort study of time to sputum culture conversion in ambulatory participants with first episodes of smear and culture positive pulmonary tuberculosis attending two primary care clinics in Cape Town, South Africa. Rifampicin resistance (diagnosed on phenotypic susceptibility testing) was an exclusion criterion. Sputum was collected weekly for 8 weeks for mycobacterial culture on liquid media (BACTEC MGIT 960). Due to missing data, multiple imputation was performed. Time to sputum culture conversion was analysed using a Cox-proportional hazards model. Bayesian model averaging determined the posterior effect probability for each variable.
Results
113 participants were enrolled (30.1% female, 10.5% HIV-infected, 44.2% W-Beijing genotype, and 89% cavities). On Kaplan Meier analysis 50.4% of participants underwent sputum culture conversion by 8 weeks. The following baseline factors were associated with slower sputum culture conversion: TTD (adjusted hazard ratio (aHR) = 1.11, 95% CI 1.02; 1.2), lung cavities (aHR = 0.13, 95% CI 0.02; 0.95), ever smoking (aHR = 0.32, 95% CI 0.1; 1.02) and the W-Beijing genotype (aHR = 0.51, 95% CI 0.25; 1.07). On Bayesian model averaging, posterior probability effects were strong for TTD, lung cavitation and smoking and moderate for W-Beijing genotype.
Conclusion
We found that baseline TTD, smoking, cavities and W-Beijing genotype were associated with delayed 2 month sputum culture. Larger studies are needed to confirm the relationship between the W-Beijing genotype and sputum culture conversion.
doi:10.1371/journal.pone.0029588
PMCID: PMC3251579  PMID: 22238625
6.  High Diversity of Mycobacterium tuberculosis Genotypes in South Africa and Preponderance of Mixed Infections among ST53 Isolates ▿  
Journal of Clinical Microbiology  2009;47(6):1848-1856.
The reemergence of tuberculosis (TB) has become a major health problem worldwide, especially in Asia and Africa. Failure to combat this disease due to nonadherence or inappropriate drug regimens has selected for the emergence of multiple-drug-resistant (MDR) TB. The development of new molecular genotyping techniques has revealed the presence of mixed Mycobacterium tuberculosis infections, which may accelerate the emergence of drug-resistant strains. There are some studies describing the local distribution of circulating strains in South Africa, but to date, reports describing the frequency and distribution of M. tuberculosis genotypes, and specifically MDR genotypes, across the different provinces are limited. Thus, 252 isolates (of which 109 were MDR) from eight of the nine provinces of South Africa were analyzed by spoligotyping. Spoligotyping showed 10 different lineages, and ST53 (11.1%) and ST1 (10.3%) were the most frequent genotypes. Of the 75 different spoligopatterns observed, 20 (7.9%) were previously unreported. Analysis of the mycobacterial interspersed repetitive units of variable-number tandem repeats of the ST53 and ST1 isolates revealed that ∼54% of the ST53 isolates were of mixed M. tuberculosis subpopulations. Drug resistance (defined as resistance to at least isoniazid and/or rifampin) could only be linked to a history of previous anti-TB treatment (adjusted odds ratio, 4.0; 95% confidence interval, 2.27 to 7.10; P = <0.0001). This study describes a high diversity of circulating genotypes in South Africa in addition to a high frequency of mixed M. tuberculosis subpopulations among the ST53 isolates. MDR TB in South Africa could not be attributed to the spread of any single lineage.
doi:10.1128/JCM.02167-08
PMCID: PMC2691085  PMID: 19386854
8.  Predominance of Mycobacterium tuberculosis EAI and Beijing Lineages in Yangon, Myanmar ▿  
Journal of Clinical Microbiology  2008;47(2):335-344.
Isolates of the Mycobacterium tuberculosis Beijing lineage are associated with high rates of transmission, hypervirulence and drug resistance. The Beijing lineage has been shown to dominate the tuberculosis (TB) epidemic in East Asia; however, the diversity and frequency of M. tuberculosis genotypes from Myanmar are unknown. We present the first comprehensive study describing the M. tuberculosis isolates circulating in Yangon, Myanmar. Thus, 310 isolates from pulmonary TB patients from Yangon, Myanmar, were genotyped by spoligotyping and IS6110-based restriction fragment length polymorphism analysis (IS6110 RFLP). The most frequent lineages observed were the East African-Indian (EAI; 48.4%; n = 150) and Beijing (31.9%; n = 99) lineages. Isolates belonging to the most frequent shared types (STs), ST1 (n = 98; Beijing), ST292 (n = 28; EAI), and ST89 (n = 11; EAI), had ≥75% similarity in their IS6110 patterns. Five of 11 Beijing isolates comprising five clusters with identical IS6110 RFLP patterns could be discriminated by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. Of the 150 EAI isolates, 40 isolates (26.7%) had only one IS6110 copy, and 17 of these isolates could be discriminated by MIRU-VNTR analysis. The findings from this study suggest that although there is a predominance of the ancient EAI lineage in Yangon, the TB epidemic in Yangon is driven by clonal expansion of the ST1 genotype. The Beijing lineage isolates (21.4%) were more likely (P = 0.009) than EAI lineage isolates to be multidrug resistant (MDR) (1.3%; odds ratio, 3.2, adjusted for the patients' history of exposure to anti-TB drugs), suggesting that the spread of MDR Beijing isolates is a major problem in Yangon.
doi:10.1128/JCM.01812-08
PMCID: PMC2643659  PMID: 19036933
9.  Pyrazinamide Resistance among South African Multidrug-Resistant Mycobacterium tuberculosis Isolates▿  
Journal of Clinical Microbiology  2008;46(10):3459-3464.
Pyrazinamide is important in tuberculosis treatment, as it is bactericidal to semidormant mycobacteria not killed by other antituberculosis drugs. Pyrazinamide is also one of the cornerstone drugs retained in the treatment of multidrug-resistant tuberculosis (MDR-TB). However, due to technical difficulties, routine drug susceptibility testing of Mycobacterium tuberculosis for pyrazinamide is, in many laboratories, not performed. The objective of our study was to generate information on pyrazinamide susceptibility among South African MDR and susceptible M. tuberculosis isolates from pulmonary tuberculosis patients. Seventy-one MDR and 59 fully susceptible M. tuberculosis isolates collected during the national surveillance study (2001 to 2002, by the Medical Research Council, South Africa) were examined for pyrazinamide susceptibility by the radiometric Bactec 460 TB system, pyrazinamidase activity (by Wayne's assay), and sequencing of the pncA gene. The frequency of pyrazinamide resistance (by the Bactec system) among the MDR M. tuberculosis isolates was 37 of 71 (52.1%) and 6 of 59 (10.2%) among fully sensitive isolates. A total of 25 unique mutations in the pncA gene were detected. The majority of these were point mutations that resulted in amino acid substitutions. Twenty-eight isolates had identical mutations in the pncA gene, but could be differentiated from each other by a combination of the spoligotype patterns and 12 mycobacterial interspersed repetitive-unit loci. A high proportion of South African MDR M. tuberculosis isolates were resistant to pyrazinamide, suggesting an evaluation of its role in patients treated previously for tuberculosis as well as its role in the treatment of MDR-TB.
doi:10.1128/JCM.00973-08
PMCID: PMC2566105  PMID: 18753350
10.  In-Depth Molecular Characterization of Mycobacterium tuberculosis from New Delhi – Predominance of Drug Resistant Isolates of the ‘Modern’ (TbD1−) Type 
PLoS ONE  2009;4(2):e4540.
Background
India has the highest estimated burden of tuberculosis in the world, accounting for 21% of all tuberculosis cases world-wide. However, due to lack of systematic analysis using multiple markers the available information on the genomic diversity of Mycobacterium tuberculosis in India is limited.
Methodology/Principal Findings
Thus, 65 M. tuberculosis isolates from New Delhi, India were analyzed by spoligotyping, MIRU-VNTR, large deletion PCR typing and single nucleotide polymorphism analysis (SNP). The Central Asian (CAS) 1 _DELHI sub-lineage was the most prevalent sub-lineage comprising 46.2% (n = 30) of all isolates, with shared-type (ST) 26 being the most dominant genotype comprising 24.6% (n = 16) of all isolates. Other sub-lineages observed were: East-African Indian (EAI)-5 (9.2%, n = 6), EAI6_BGD1 (6.2%, n = 4), EAI3_IND, CAS and T1 with 6.2% each (n = 4 each), Beijing (4.6%, n = 3), CAS2 (3.1%, n = 2), and X1 and X2 with 1 isolate each. Genotyping results from five isolates (7.7%) did not match any existing spoligopatterns, and one isolate, ST124, belonged to an undefined lineage. Twenty-six percent of the isolates belonged to the TbD1+ PGG1 genogroup. SNP analysis of the pncA gene revealed a CAS-lineage specific silent mutation, S65S, which was observed for all CAS-lineage isolates (except two ST26 isolates) and in 1 orphan. Mutations in the pncA gene, conferring resistance to pyrazinamide, were observed in 15.4% of all isolates. Collectively, mutations in the rpoB gene, the katG gene and in both rpoB and katG genes, conferring resistance to rifampicin and isoniazid, respectively, were more frequent in CAS1_DELHI isolates compared to non-CAS_DELHI isolates (OR: 3.1, CI95% [1.11, 8.70], P = 0.045). The increased frequency of drug-resistance could not be linked to the patients' history of previous anti-tuberculosis treatment (OR: 1.156, CI95% [0.40, 3.36], P = 0.79). Fifty-six percent of all new tuberculosis patients had mutations in either the katG gene or the rpoB gene, or in both katG and rpoB genes.
Conclusion
CAS1_DELHI isolates circulating in New Delhi, India have a high frequency of mutations in the rpoB and katG genes. A silent mutation (S65S) in the pncA gene can be used as a putative genetic marker for CAS-lineage isolates.
doi:10.1371/journal.pone.0004540
PMCID: PMC2641002  PMID: 19234602
11.  Genomic Diversity among Beijing and non-Beijing Mycobacterium tuberculosis Isolates from Myanmar 
PLoS ONE  2008;3(4):e1973.
Background
The Beijing family of Mycobacterium tuberculosis is dominant in countries in East Asia. Genomic polymorphisms are a source of diversity within the M. tuberculosis genome and may account for the variation of virulence among M. tuberculosis isolates. Till date there are no studies that have examined the genomic composition of M. tuberculosis isolates from the high TB-burden country, Myanmar.
Methodology/Principle Findings
Twenty-two M. tuberculosis isolates from Myanmar were screened on whole-genome arrays containing genes from M. tuberculosis H37Rv, M. tuberculosis CDC1551 and M. bovis AF22197. Screening identified 198 deletions or extra regions in the clinical isolates compared to H37Rv. Twenty-two regions differentiated between Beijing and non-Beijing isolates and were verified by PCR on an additional 40 isolates. Six regions (Rv0071-0074 [RD105], Rv1572-1576c [RD149], Rv1585c-1587c [RD149], MT1798-Rv1755c [RD152], Rv1761c [RD152] and Rv0279c) were deleted in Beijing isolates, of which 4 (Rv1572-1576c, Rv1585c-1587c, MT1798-Rv1755c and Rv1761c) were variably deleted among ST42 isolates, indicating a closer relationship between the Beijing and ST42 lineages. The TbD1 region, Mb1582-Mb1583 was deleted in Beijing and ST42 isolates. One M. bovis gene of unknown function, Mb3184c was present in all isolates, except 11 of 13 ST42 isolates. The CDC1551 gene, MT1360 coding for a putative adenylate cyclase, was present in all Beijing and ST42 isolates (except 1). The pks15/1 gene, coding for a putative virulence factor, was intact in all Beijing and non-Beijing isolates, except in ST42 and ST53 isolates.
Conclusion
This study describes previously unreported deletions/extra regions in Beijing and non-Beijing M. tuberculosis isolates. The modern and highly frequent ST42 lineage showed a closer relationship to the hypervirulent Beijing lineage than to the ancient non-Beijing lineages. The pks15/1 gene was disrupted only in modern non-Beijing isolates. This is the first report of an in-depth analysis on the genomic diversity of M. tuberculosis isolates from Myanmar.
doi:10.1371/journal.pone.0001973
PMCID: PMC2276860  PMID: 18398483
12.  Heterogeneity of Mycobacterium tuberculosis Isolates in Yangon, Myanmar 
Journal of Clinical Microbiology  2003;41(10):4907-4908.
doi:10.1128/JCM.41.10.4907-4908.2003
PMCID: PMC254380  PMID: 14532259
13.  Intestinal and Systemic Immune Responses to an Oral Cholera Toxoid B Subunit Whole-Cell Vaccine Administered during Zinc Supplementation  
Infection and Immunity  2003;71(7):3909-3913.
Zinc plays a critical role in the normal functioning of the immune system. We investigated whether zinc sulfate administered orally to adult zinc-replete volunteers modulates systemic and intestinal immune responses to an oral killed cholera toxoid B subunit (CTB) whole-cell cholera vaccine. The 30 participants were immunized twice, with a 17-day interval. The vaccinees in the intervention group ingested 45 mg of elemental zinc thrice daily for 9 days starting 2 days before each vaccine dose. The median serum anti-CTB immunoglobulin A (IgA) and IgG responses from day 0 to day 30, i.e. after two vaccine doses, were 13-fold lower (P value for identical distribution, <0.005) in the zinc-supplemented compared to the nonsupplemented vaccinees. The median serum vibriocidal responses from baseline to after one (day 0 to day 17) and two (day 0 to day 30) vaccine doses were at least sixfold (P = 0.033) and fourfold (P = 0.091) higher, while the median fecal anti-CTB IgA response after two doses was estimated to be fourfold higher (P = 0.084) in the zinc-supplemented vaccinees. These observations show that zinc reduces the antitoxin and may enhance the antibacterial responses in serum. Zinc may also improve the intestinal antitoxin immune response. Oral zinc administration has the potential to modify critical immune responses to antigens applied to mucosal surfaces.
doi:10.1128/IAI.71.7.3909-3913.2003
PMCID: PMC162035  PMID: 12819076
15.  Modulation of Transcriptional and Inflammatory Responses in Murine Macrophages by the Mycobacterium tuberculosis Mammalian Cell Entry (Mce) 1 Complex 
PLoS ONE  2011;6(10):e26295.
The outcome of many infections depends on the initial interactions between agent and host. Aiming at elucidating the effect of the M. tuberculosis Mce1 protein complex on host transcriptional and immunological responses to infection with M. tuberculosis, RNA from murine macrophages at 15, 30, 60 min, 4 and 10 hrs post-infection with M. tuberculosis H37Rv or Δ-mce1 H37Rv was analyzed by whole-genome microarrays and RT-QPCR. Immunological responses were measured using a 23-plex cytokine assay. Compared to uninfected controls, 524 versus 64 genes were up-regulated by 15 min post H37Rv- and Δ-mce1 H37Rv-infection, respectively. By 15 min post-H37Rv infection, a decline of 17 cytokines combined with up-regulation of Ccl24 (26.5-fold), Clec4a2 (23.2-fold) and Pparγ (10.5-fold) indicated an anti-inflammatory response initiated by IL-13. Down-regulation of Il13ra1 combined with up-regulation of Il12b (30.2-fold), suggested switch to a pro-inflammatory response by 4 hrs post H37Rv-infection. Whereas no significant change in cytokine concentration or transcription was observed during the first hour post Δ-mce1 H37Rv-infection, a significant decline of IL-1b, IL-9, IL-13, Eotaxin and GM-CSF combined with increased transcription of Il12b (25.1-fold) and Inb1 (17.9-fold) by 4 hrs, indicated a pro-inflammatory response. The balance between pro-and anti-inflammatory responses during the early stages of infection may have significant bearing on outcome.
doi:10.1371/journal.pone.0026295
PMCID: PMC3200323  PMID: 22039457

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