Ida Parwati

Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology

BackgroundThe Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database.ResultsThe fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network.ConclusionOur results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.

Possible underlying mechanisms for successful emergence of the Mycobacterium tuberculosis Beijing genotype strains

The wide geographic distribution of one clade of Mycobacterium tuberculosis, the Beijing genotype family, and its genetic homogeneity, suggests that strains belonging to this grouping might have a selective advantage over other M tuberculosis strains. This hypothesis was addressed by reviewing molecular-epidemiological, experimental, and clinical studies. Beijing strains represent about 50% of strains in east Asia and at least 13% of strains worldwide. Their emergence might be linked to escape from BCG vaccination, and to multidrug resistance, which is associated with the Beijing genotype in many areas. Different animal models have shown Beijing strains to be more virulent, and to cause more histopathological changes, higher outgrowth, and increased mortality. At a molecular level, Beijing strains have specific properties in terms of protein and lipid structures and their interaction with the human immune system. Finally, the Beijing genotype has been linked to polymorphisms in immune genes, suggesting the possibility of human-mycobacterial co-evolution. The emergence of the Beijing genotype family might represent an evolutionary response of M tuberculosis to vaccination or antibiotic treatment, with an important negative impact on tuberculosis control. More research is needed to further unravel the mechanisms underlying the emergence of M tuberculosis Beijing genotype strains, and examine the implications for future control strategies.

The Effect of Type 2 Diabetes Mellitus on the Presentation and Treatment Response of Pulmonary Tuberculosis

BACKGROUND Diabetes mellitus (DM) is a known risk factor for tuberculosis (TB), and with the increasing prevalence of type 2 DM in less developed regions, many patients with TB will have concomitant DM. Presently, little is known about the effect of DM on the clinical presentation and treatment outcome of TB. METHODS In an urban setting in Indonesia, 737 patients with pulmonary TB were screened for DM and were followed up prospectively during TB treatment. Clinical characteristics and outcome were compared between patients with TB who had DM and patients with TB who did not have DM. RESULTS DM was diagnosed in 14.8% of patients with TB and was associated with older age and a greater body weight. On presentation, diabetic patients with TB had more symptoms but had no evidence of more-severe TB. After 2 months, results of sputum microscopic examination was more often positive in diabetic patients (18.1% vs. 10.0%). After 6 months, 22.2% of cultured sputum specimens from diabetic patients were positive for Mycobacterium tuberculosis (adjusted odds ratio, 7.65; P=.004). CONCLUSION DM seems to have a negative effect on the outcome of TB treatment. The underlying mechanisms for the different response to treatment in diabetic patients with TB must be explored. Screening for DM and subsequent glycemic control may improve the outcome of TB treatment.

Pharmacokinetics and Tolerability of a Higher Rifampin Dose versus the Standard Dose in Pulmonary Tuberculosis Patients

ABSTRACT Rifampin is a key drug for tuberculosis (TB) treatment. The available data suggest that the currently applied 10-mg/kg of body weight dose of rifampin may be too low and that increasing the dose may shorten the treatment duration. A double-blind randomized phase II clinical trial was performed to investigate the effect of a higher dose of rifampin in terms of pharmacokinetics and tolerability. Fifty newly diagnosed adult Indonesian TB patients were randomized to receive a standard (450-mg, i.e., 10-mg/kg in Indonesian patients) or higher (600-mg) dose of rifampin in addition to other TB drugs. A full pharmacokinetic curve for rifampin, pyrazinamide, and ethambutol was recorded after 6 weeks of daily TB treatment. Tolerability was assessed during the 6-month treatment period. The geometric means of exposure to rifampin (area under the concentration-time curve from 0 to 24 h [AUC0-24]) were increased by 65% (P < 0.001) in the higher-dose group (79.7 mg·h/liter) compared to the standard-dose group (48.5 mg·h/liter). Maximum rifampin concentrations (Cmax) were 15.6 mg/liter versus 10.5 mg/liter (49% increase; P < 0.001). The percentage of patients for whom the rifampin Cmax was ≥8 mg/liter was 96% versus 79% (P = 0.094). The pharmacokinetics of pyrazinamide and ethambutol were similar in both groups. Mild (grade 1 or 2) hepatotoxicity was more common in the higher-dose group (46 versus 20%; P = 0.054), but no patient developed severe hepatotoxicity. Increasing the rifampin dose was associated with a more than dose-proportional increase in the mean AUC0-24 and Cmax of rifampin without affecting the incidence of serious adverse effects. Follow-up studies are warranted to assess whether high-dose rifampin may enable shortening of TB treatment.

Mycobacterium tuberculosis Beijing Genotype Is an Independent Risk Factor for Tuberculosis Treatment Failure in Indonesia

Animal studies have shown that the globally emerging Beijing genotype strains of Mycobacterium tuberculosis are more virulent than other strains. We examined whether Beijing strains increase treatment failure in a prospective cohort study in Indonesia. Among 818 tuberculosis cases, positive sputum culture results after 6 months of treatment were more common among patients infected with Beijing strains (33.4%) than among those infected with non-Beijing strains (relative risk, 1.94 [95% confidence interval, 1.26-3.00]), even after adjustment for differences in drug resistance. These data suggest that M. tuberculosis Beijing genotype strains have a higher capacity to withstand tuberculosis treatment, even in the absence of drug resistance.

Microevolution of Mycobacterium tuberculosis in a Tuberculosis Patient

ABSTRACT Five Mycobacterium tuberculosis isolates were obtained from three body sites from a Dutch patient. The isolates displayed a single genotype by 24-locus MIRU-VNTR typing (except for a single locus not amplified from one isolate) but were differentiated by small variations in IS6110 fingerprints, spoligotypes, 6 hypervariable MIRU-VNTR loci, and/or DiversiLab profiles, revealing patterns of microevolution in a clonal infection.

Polymorphisms in autophagy genes and susceptibility to tuberculosis.

Recent data suggest that autophagy is important for intracellular killing of Mycobacterium tuberculosis, and polymorphisms in the autophagy gene IRGM have been linked with susceptibility to tuberculosis (TB) among African-Americans, and with TB caused by particular M. tuberculosis genotypes in Ghana. We compared 22 polymorphisms of 14 autophagy genes between 1022 Indonesian TB patients and 952 matched controls, and between patients infected with different M. tuberculosis genotypes, as determined by spoligotyping. The same autophagy polymorphisms were studied in correlation with ex-vivo production of TNF, IL-1β, IL-6, IL-8, IFN-γ and IL-17 in healthy volunteers. No association was found between TB and polymorphisms in the genes ATG10, ATG16L2, ATG2B, ATG5, ATG9B, IRGM, LAMP1, LAMP3, P2RX7, WIPI1, MTOR and ATG4C. Associations were found between polymorphisms in LAMP1 (p = 0.02) and MTOR (p = 0.02) and infection with the successful M. tuberculosis Beijing genotype. The polymorphisms examined were not associated with M. tuberculosis induced cytokines, except for a polymorphism in ATG10, which was linked with IL-8 production (p = 0.04). All associations found lost statistical significance after correction for multiple testing. This first examination of a broad set of polymorphisms in autophagy genes fails to show a clear association with TB, with M. tuberculosis Beijing genotype infection or with ex-vivo pro-inflammatory cytokine production.

Comparison of real time IS6110-PCR, microscopy, and culture for diagnosis of tuberculous meningitis in a cohort of adult patients in Indonesia.

Background Bacteriological confirmation of tuberculous (TB) meningitis is difficult. Culture is slow and microscopy has insufficient sensitivity. We evaluated real time PCR targeting insertion sequence IS6110 among 230 consecutive adult patients with subacute meningitis in a referral hospital in Indonesia. Methods Cerebrospinal fluid (CSF) samples were examined using microscopy, solid and liquid culture, and real time IS6110-PCR with a fluorescence-labeled probe using DNA extracted from CSF. CSF samples from 40 non-infectious neurology patients were used as negative controls. IS6110-PCR results were linked with clinical and CSF characteristics. Results Most patients presented with subacute meningitis, after a median of 14 days of symptoms (range 7–30). After exclusion of cryptococcal and bacterial meningitis, 207 patients were classified as definite or probable TB meningitis; 17.9% with HIV infection. Among this group IS6110-PCR gave the highest positivity rate (68%, 95% CI 62–74%) compared with microscopy of ZN-stained slides (11%, 95% CI 7–15%), and mycobacterial culture using solid (36%, 95% CI 29–42%) and liquid (44%, 95% CI 37–51%) media. IS6110-PCR was positive in 92% of patients with culture-positive and 42% of patients with culture-negative probable TB meningitis. Among culture-negative patients, a positive PCR was associated with a history of TB treatment, a longer duration of illness, a higher CSF cell count and protein, and a lower CSF glucose. IS6110-PCR was negative in all CSF samples from non-meningitis control patients. Conclusions Real time IS6110-PCR is a quick, sensitive, and specific test for diagnosing of TB meningitis in this setting. Its performance in other (less-developed) settings needs further study.

The effect of HIV infection on adult meningitis in Indonesia: a prospective cohort study.

Objective:Indonesia has a concentrated but rapidly growing HIV epidemic. We examined the effect of HIV on causative organisms, clinical features and prognosis of adult meningitis. Design:A prospective cohort study. Methods:All adult patients at a referral hospital who underwent cerebrospinal fluid examination for suspected meningitis were examined for HIV and included in a prospective cohort study. Microbiological testing was done for common bacterial pathogens, mycobacteria and fungi. Patients were followed for at least 6 months, and logistic regression models were used to identify risk factors for mortality. Results:Among 185 patients who mostly presented with subacute meningitis, 60% were male and the median age was 30 years. HIV infection was present in 25% of the patients; almost two-thirds were newly confirmed, and all presented with severe immunosuppression (median CD4 cell count 13/μl, range 2–98). One-third of HIV-infected patients had cryptococcal meningitis whereas two-thirds suffered from tuberculosis. After 1 month, 41% of patients had died. HIV infection was strongly associated with 1-month mortality (adjusted odds ratio 12.15; 95% confidence interval 3.04–15.72) and death during extended follow-up (hazard ratio 2.48; 95% confidence interval 1.97–5.74). Conclusion:Although HIV is still uncommon in the general population in Indonesia, its prevalence among adult meningitis cases already seems high. Mycobacterium tuberculosis and Cryptococcus neoformans are the main causes of meningitis in this setting, and mortality is very high, especially in HIV-infected patients. Our data suggest that adult meningitis cases in Indonesia should be screened routinely for HIV infection. Further studies are needed to address the high mortality.

The diagnostic and prognostic value of dengue non-structural 1 antigen detection in a hyper-endemic region in indonesia

As dengue fever is undifferentiated from other febrile illnesses in the tropics and the clinical course is unpredictable, early diagnosis is important. Several commercial assays to detect dengue NS1 antigen have been developed; however, their performances vary and data is lacking from hyper-endemic areas where all four serotypes of dengue are equally represented. To assess the sensitivity of the Bio-Rad platelia Dengue NS1 antigen assay according to virus serotype, immune status, gender, and parameters of severe disease, acute sera from 220 individuals with confirmed dengue and 55 individuals with a non-dengue febrile illness were tested using the Bio-Rad platelia Dengue NS1 antigen assay. The overall sensitivity of the NS1 ELISA was 46.8% and the specificity was 100%. The sensitivity in primary infections was significantly higher than in secondary infections (100% vs. 35.7%). In secondary infections, the sensitivity of NS1 detection was highest in DENV-3 (47.1%), followed by DENV-1 (40.9%), DENV-2 (30%) and DENV-4 (27%) infections. NS1 was less frequently detected in sera with high titers of HI antibodies or in acute samples from patients whose pre-illness sera showed neutralizing antibodies to more than one serotype. The detection of NS1 was higher in females, severe cases, and individuals with lower platelet counts (<100,000/mm3). While the overall sensitivity of this NS1 ELISA is poor, our data suggest that in secondary infections, detection may be predictive of a more severe illness.