Histone acetylome-wide associations in immune cells from individuals with active Mycobacterium tuberculosis infection.

Host cell chromatin changes are thought to play an important role in the pathogenesis of infectious diseases. Here we describe a histone acetylome-wide association study (HAWAS) of an infectious disease, on the basis of genome-wide H3K27 acetylation profiling of peripheral blood granulocytes and monocytes from persons with active Mycobacterium tuberculosis (Mtb) infection and healthy controls. We detected >2,000 differentially acetylated loci in either cell type in a Singapore Chinese discovery cohort (n = 46), which were validated in a subsequent multi-ethnic Singapore cohort (n = 29), as well as a longitudinal cohort from South Africa (n = 26), thus demonstrating that HAWAS can be independently corroborated. Acetylation changes were correlated with differential gene expression. Differential acetylation was enriched near potassium channel genes, including KCNJ15, which modulates apoptosis and promotes Mtb clearance in vitro. We performed histone acetylation quantitative trait locus (haQTL) analysis on the dataset and identified 69 candidate causal variants for immune phenotypes among granulocyte haQTLs and 83 among monocyte haQTLs. Our study provides proof-of-principle for HAWAS to infer mechanisms of host response to pathogens.

Smear-positive pulmonary tuberculosis patients with previously abnormal chest radiographs: missed opportunities for early diagnosis.

INTRODUCTION: It is vital to diagnose and treat pulmonary tuberculosis (PTB) early, in the interests of the patient and public health. At the Singapore Tuberculosis Control Unit (TBCU), it was not uncommon to discover abnormal chest radiographs (CXRs) predating their PTB diagnosis by months to years in the electronic medical records (EMRs) of sputum acid-fast bacilli smear-positive patients. Our study explored this observation. METHODS: The EMRs of sputum smear-positive PTB patients treated at the TBCU from January to July 2014 were viewed for abnormal CXRs preceding their PTB diagnosis. Information in the EMRs pertaining to the indication for the CXRs, radiological reports (including whether the possibility of PTB was stated) and action taken was captured. RESULTS: Of the 254 sputum smear-positive patients, 108 had previous CXRs in their EMRs, of whom 39 (36.1%) had previous CXRs compatible with PTB. Most of these were performed in tertiary institutions and for reasons unrelated to PTB. No action was taken in response to these CXRs in 24 (61.5%) patients. 27 (69.2%) patients had abnormal CXRs dating back more than six months, with 12 (30.8%) dating back more than 30 months before the PTB diagnosis. Patients aged ≥ 55 years were significantly more likely to have previous CXRs that were compatible with PTB. CONCLUSION: PTB patients may be asymptomatic or have chronic indolent disease and remain undiagnosed for years. There is a need for vigilance within our healthcare system to seize opportunities for early diagnosis and treatment to prevent the spread of this infectious disease in Singapore.

Sub-clinical abnormalities detected by PET/MRI in household tuberculosis contacts.

BACKGROUND: The understanding of early events following TB exposure is limited by traditional tests that rely on detection of an immune response to infection, which is delayed, or on imaging tests with low sensitivity for early disease. We investigated for evidence of lung abnormalities in heavily exposed TB contacts using PET/MRI. METHODS: 30 household contacts of 20 index patients underwent clinical assessment, IGRA testing, chest x-ray and PET/MRI scan using 18-F-FDG. MRI images were examined by a radiology/nuclear medicine dual-qualified physician using a standardised report form, while PET/MRI images were examined independently by another radiology/nuclear medicine dual-qualified physician using a similar form. Standardised uptake value (SUV) was quantified for each abnormal lesion. RESULTS: IGRA was positive in 40%. PET/MRI scan was abnormal in 30%, predominantly FDG uptake in hilar or mediastinal lymph nodes and lung apices. We did not identify any relationship between PET/MRI findings and degree of exposure or IGRA status. CONCLUSION: PET-based imaging may provide important insights into the natural history following exposure to TB that may not be available from traditional tests of TB immune response or imaging. The clinical significance of the abnormalities is uncertain and merits further investigation in longitudinal studies.

Clofazimine drug susceptibility testing for Mycobacterium tuberculosis: the case of using the right diluent.

Accurate and reliable drug susceptibility testing (DST) is essential for the effective treatment and control of tuberculosis. With the increase in drug-resistant organisms, newer and less conventional antimicrobial agents are used for treatment. Recently, we found an unprecedented rise in the number of clofazimine-resistant Mycobacterium tuberculosis isolates in our laboratory. An investigation found that this phenomenon was due to a change in the method of drug preparation. We performed studies to assess the impact of water and dimethyl sulfoxide (DMSO) as a final diluent for clofazimine drug testing. Based on our findings, the use of DMSO as a solvent for M. tuberculosis DST was optimised using the BACTEC MGIT 960 platform.

Prevalence of and risk factors associated with latent tuberculosis in Singapore: A cross-sectional survey.

OBJECTIVES: This first cross-sectional survey on latent tuberculosis infection (LTBI) in Singapore was performed by utilizing the QuantiFERON Gold In-tube (QFT-GIT) assay to collect data on the prevalence of LTBI and to identify potential risk factors associated with LTBI. METHODS: Nationwide household addresses were selected randomly for enumeration, and Singaporeans or Permanent Residents aged 18-79 years were identified. One eligible member per household was selected using the Kish grid. Each participant answered a questionnaire assessing their medical history (including tuberculosis (TB)), socio-economic factors, and lifestyle factors. They also provided a blood specimen for the QFT-GIT assay. Participants with a positive QFT-GIT result were defined as having LTBI if they were asymptomatic. To identify independent risk factors, adjusted hazard ratios were obtained using the multivariable modified Breslow-Cox proportional hazards model. RESULTS: An overall QFT-GIT positivity rate of 12.7% was detected amongst 1682 Singapore residents. There was a wide variation in the positivity rate according to the participants' country of birth. Higher LTBI prevalence was also significantly associated with increasing age, lower educational and socio-economic status, and alcohol use. CONCLUSIONS: Given the high prevalence of LTBI amongst foreign-born residents from regional countries, similar studies should be conducted amongst migrants in Singapore to improve national guidelines on screening and preventive treatment against LTBI.

Investigation of a cluster of multi-drug resistant tuberculosis in a high-rise apartment block in Singapore.

OBJECTIVE: Between February 2012 and May 2016, six residents of an 11-storey apartment block were diagnosed with MDR-TB. Based on initial tests, all isolates had similar genotypic profiles, although there were no identifiable epidemiological transmission patterns between three cases. We present findings from the cluster investigation and results of a mass screening exercise. DESIGN: Free voluntary TB screening was offered to past and current residents of the apartment block, comprising an interview, Chest X-Ray, and Interferon Gamma Release Assay or Tuberculin skin test. Expected latent TB proportions were calculated using a reference population, and whole genome sequencing (WGS) was performed. RESULTS: The index case was involved in a separate gaming centre outbreak involving five patrons. 241 current (67.9% of 355 residents) and 18 past residents were screened. The latent TB proportion was 19.9%, which was at the higher end of the expected range. WGS confirmed relatedness of cases' MDR-TB isolates- eight of 10 isolates were genetically identical, while the remaining two were one Single Nucleotide Polymorphism apart. CONCLUSION: With WGS, TB clusters not apparent through regular activity-based contact tracing may be detected. Mass screening may help inform the extent of transmission, but is limited by participation and difficulties in interpretation.

Alcohol drinking and cigarette smoking in relation to risk of active tuberculosis: prospective cohort study.

INTRODUCTION: Heavy alcohol consumption increases the risk of active tuberculosis (TB). However, the relation between lower levels of alcohol intake and TB risk remains unclear. We aimed to evaluate the association between alcohol intake and risk of active TB and assess whether the associations were modified by smoking status, which is another risk factor for active TB. METHODS: The Singapore Chinese Health Study is a prospective cohort of 63 257 adults aged 45-74 years recruited from 1993 to 1998. Information on alcohol intake and smoking history was collected at recruitment. Active TB cases were identified via linkage with National TB Notification Registry. RESULTS: During a mean follow-up of 16.8 years, 1249 incident cases of active TB were identified. Among non-smokers, compared with total abstinence, participants who had monthly to weekly intake of alcohol had reduced TB risk (HR 0.70, 95% CI 0.55 to 0.89), but this reduction in risk with low-dose drinking was not observed among current smokers (HR 0.96, 95% CI 0.77 to 1.18; p for interaction=0.02). Comparatively, drinking 2+ drinks daily was associated with increased TB risk among current smokers (HR 1.51, 95% CI 1.11 to 2.05). This increased risk was not observed among non-smokers (HR 0.93, 95% CI 0.49 to 1.77) and the interaction between alcohol intake and smoking status was of borderline significance (p for interaction=0.08). In joint effect, compared with those who neither smoked nor drank, the risk of active TB increased from 1.82 (95% CI 1.57 to 2.10) in current smokers who were non-drinkers to 3.16 (95% CI 2.35 to 4.24) in current smokers who also drank 2+ drinks daily. CONCLUSION: While low intake of alcohol may protect against active TB among non-smokers, drinking 2+ drinks daily could act synergistically with smoking to increase the risk of active TB in current smokers.

Association and clinical utility of NAT2 in the prediction of isoniazid-induced liver injury in Singaporean patients.

BACKGROUND AND AIMS: Isoniazid (INH) is part of the first-line-therapy for tuberculosis (TB) but can cause drug-induced liver injury (DILI). Several candidate single nucleotide polymorphisms (SNPs) have been previously identified but the clinical utility of these SNPs in the prediction of INH-DILI remains uncertain. The aim of this study was to assess the association between selected candidate SNPs and the risk of INH-DILI and to assess the clinical validity of associated variants in a Singaporean population. METHODS: This was a case-control study where 24 INH-DILI cases and 79 controls were recruited from the TB control unit in a tertiary hospital. Logistic regression was used to test for the association between candidate SNPs and INH-DILI. NAT2 acetylator status was inferred from genotypes and tested for association with INH-DILI. Finally, clinical validity measures were estimated for significant variants. RESULTS: Two SNPs in NAT2 (rs1041983 and rs1495741) and NAT2 slow acetylators (SA) were significantly associated with INH-DILI (OR (95% CI) = 13.86 (4.30-44.70), 0.10 (0.03-0.33) and 9.98 (3.32-33.80), respectively). Based on an INH-DILI prevalence of 10%, the sensitivity, specificity, positive and negative predictive values of NAT2 SA were 75%, 78%, 28% and 97%, respectively. The population attributable fraction (PAF) and number needed to test (NNT) for NAT2 SA were estimated to be 0.67 and 4.08, respectively. A model with clinical and NAT2 acetylator status provided significantly better prediction for INH-DILI than a clinical model alone (area under receiver operating characteristic curve = 0.863 vs. 0.766, respectively, p = 0.027). CONCLUSIONS: We show the association between NAT2 SA and INH-DILI in a Singaporean population and demonstrated its clinical utility in the prediction of INH-DILI.

Tea Drinking and Its Association with Active Tuberculosis Incidence among Middle-Aged and Elderly Adults: The Singapore Chinese Health Study.

Experimental studies showed that tea polyphenols may inhibit growth of Mycobacterium tuberculosis. However, no prospective epidemiologic study has investigated tea drinking and the risk of active tuberculosis. We investigated this association in the Singapore Chinese Health Study, a prospective population-based cohort of 63,257 Chinese aged 45-74 years recruited between 1993 and 1998 in Singapore. Information on habitual drinking of tea (including black and green tea) and coffee was collected via structured questionnaires. Incident cases of active tuberculosis were identified via linkage with the nationwide tuberculosis registry up to 31 December 2014. Cox proportional hazard models were used to estimate the relation of tea and coffee consumption with tuberculosis risk. Over a mean 16.8 years of follow-up, we identified 1249 incident cases of active tuberculosis. Drinking either black or green tea was associated with a dose-dependent reduction in tuberculosis risk. Compared to non-drinkers, the hazard ratio (HR) (95% confidence interval (CI)) was 1.01 (0.85-1.21) in monthly tea drinkers, 0.84 (0.73-0.98) in weekly drinkers, and 0.82 (0.71-0.96) in daily drinkers (p for trend = 0.003). Coffee or caffeine intake was not significantly associated with tuberculosis risk. In conclusion, regular tea drinking was associated with a reduced risk of active tuberculosis.

Spatial dynamics of TB within a highly urbanised Asian metropolis using point patterns.

Singapore is a high-income country in a region with a high prevalence of tuberculosis. The Singapore Tuberculosis (TB) Elimination Program (STEP) was set up in 1997, and the better surveillance and clinical management practices initiated under STEP led to a decade-long decline in the incidence levels. However, incidence rates started to rise again since 2008. The reasons for this rise are unclear. This study involved a spatial analysis of the epidemiology of TB among Singapore residents. More than 30 000 cases reported during 1995-2011 and their residential addresses were analysed for spatial risk and spatial clustering, using spatial point pattern methodology. The principal factor responsible for the increasing resident TB incidence in Singapore is the changing age profile of the population. In particular the burgeoning population aged above 65 years accounts for the increase in reported cases. Singapore's population has one of the world's lowest fertility and mortality rates, and the elderly population is projected to grow substantially over the next few decades. Tuberculosis rates may therefore continue to rise even with static or improving case management and surveillance.

Ministry of Health Clinical Practice Guidelines: Prevention, Diagnosis and Management of Tuberculosis.

The Ministry of Health (MOH) has developed the clinical practice guidelines on Prevention, Diagnosis and Management of Tuberculosis to provide doctors and patients in Singapore with evidence-based treatment for tuberculosis. This article reproduces the introduction and executive summary (with recommendations from the guidelines) from the MOH clinical practice guidelines on Prevention, Diagnosis and Management of Tuberculosis, for the information of SMJ readers. The chapters and page numbers mentioned in the reproduced extract refer to the full text of the guidelines, which are available from the Ministry of Health website: http://www.moh.gov.sg/content/moh_web/healthprofessionalsportal/doctors/guidelines/cpg_medical.html. The recommendations should be used with reference to the full text of the guidelines. Following this article are multiple choice questions based on the full text of the guidelines.

First use of bedaquiline in a patient with XDR-TB in Singapore.

Drug-resistant tuberculosis (TB) continues to pose a threat to global control of TB: 3.5% of new and 20.5% of previously treated TB cases were estimated to have multidrug-resistant (MDR)-TB in 2013. Approximately 9% of patients with MDR-TB had extensively drug-resistant (XDR)-TB. A 30-year-old Vietnamese woman previously treated for TB in her home country presented with 5 months of cough and shortness of breath 1 year after migrating to Singapore. Xpert MTB/Rif testing showed rpoB gene mutation. Phenotypic drug susceptibility testing revealed XDR-TB. Second and third-line TB drugs were commenced. To strengthen the efficacy of her treatment regimen, the novel anti-TB drug bedaquiline was obtained for the patient on compassionate grounds. We report the first use in Singapore of bedaquiline for the treatment of XDR-TB.

Management of latent Mycobacterium tuberculosis infection: WHO guidelines for low tuberculosis burden countries.

Latent tuberculosis infection (LTBI) is characterised by the presence of immune responses to previously acquired Mycobacterium tuberculosis infection without clinical evidence of active tuberculosis (TB). Here we report evidence-based guidelines from the World Health Organization for a public health approach to the management of LTBI in high risk individuals in countries with high or middle upper income and TB incidence of <100 per 100 000 per year. The guidelines strongly recommend systematic testing and treatment of LTBI in people living with HIV, adult and child contacts of pulmonary TB cases, patients initiating anti-tumour necrosis factor treatment, patients receiving dialysis, patients preparing for organ or haematological transplantation, and patients with silicosis. In prisoners, healthcare workers, immigrants from high TB burden countries, homeless persons and illicit drug users, systematic testing and treatment of LTBI is conditionally recommended, according to TB epidemiology and resource availability. Either commercial interferon-gamma release assays or Mantoux tuberculin skin testing could be used to test for LTBI. Chest radiography should be performed before LTBI treatment to rule out active TB disease. Recommended treatment regimens for LTBI include: 6 or 9 month isoniazid; 12 week rifapentine plus isoniazid; 3-4 month isoniazid plus rifampicin; or 3-4 month rifampicin alone.

Outcome of a grocery voucher incentive scheme for low-income tuberculosis patients on directly observed therapy in Singapore.

INTRODUCTION: The 'DOT & Shop' scheme is sponsored by SATA CommHealth, a local non-governmental organisation. It was launched in July 2009, in collaboration with Singapore's Tuberculosis Control Unit (TBCU). Under this scheme, grocery vouchers are disbursed to low-income patients with tuberculosis (TB) at each clinic visit if they have been adherent to directly observed therapy (DOT). This study aimed to determine the effect of this incentive scheme on treatment completion rates and to report the characteristics of patients who were non-adherent to the scheme. METHODS: This descriptive study used data from the TBCU medical social worker database and the National TB Registry. RESULTS: From July 2009 to December 2012, a total of 883 TB patients were enrolled in the scheme. The overall treatment completion rates of the patients before (July 2006-June 2009) and after (July 2009-December 2012) the implementation of the scheme improved from 85.3% to 87.2% (p = 0.02). Patients under this scheme had a higher treatment completion rate (90.0%) than those not under this scheme (86.4%) (p < 0.01). It was found that the non-adherent patients were more likely to be of Malay ethnicity, younger and unemployed. CONCLUSION: We demonstrate the salutary effect of a non-governmental organisation-funded grocery voucher incentive scheme for low-income TB patients on DOT in Singapore.

Measurement of phenotype and absolute number of circulating heparin-binding hemagglutinin, ESAT-6 and CFP-10, and purified protein derivative antigen-specific CD4 T cells can discriminate active from latent tuberculosis infection.

The tuberculin skin test (TST) and interferon gamma (IFN-γ) release assays (IGRAs) are used as adjunctive tests for the evaluation of suspected cases of active tuberculosis (TB). However, a positive test does not differentiate latent from active TB. We investigated whether flow cytometric measurement of novel combinations of intracellular cytokines and surface makers on CD4 T cells could differentiate between active and latent TB after stimulation with Mycobacterium tuberculosis-specific proteins. Blood samples from 60 patients referred to the Singapore Tuberculosis Control Unit for evaluation for active TB or as TB contacts were stimulated with purified protein derivative (PPD), ESAT-6 and CFP-10, or heparin-binding hemagglutinin (HBHA). The CD4 T cell cytokine response (IFN-γ, interleukin-2 [IL-2], interleukin-17A [IL-17A], interleukin-22 [IL-22], granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor alpha [TNF-α]) and surface marker expression (CD27, CXCR3, and CD154) were then measured. We found that the proportion of PPD-specific CD4 T cells, defined as CD154(+) TNF-α(+) cells that were negative for CD27 and positive for GM-CSF, gave the strongest discrimination between subjects with latent and those with active TB (area under the receiver operator characteristic [ROC] curve of 0.9277; P < 0.0001). Also, the proportions and absolute numbers of HBHA-specific CD4 T cells were significantly higher in those with latent TB infection, particularly CD154(+) TNF-α(+) IFN-γ(+) IL-2(+) and CD154(+) TNF-α(+) CXCR3(+). Finally, we found that the ratio of ESAT-6- and CFP-10-responding to HBHA-responding CD4 T cells was significantly different between the two study populations. In conclusion, we found novel markers of M. tuberculosis-specific CD4 cells which differentiate between active and latent TB.

Time series analysis of demographic and temporal trends of tuberculosis in Singapore.

BACKGROUND: Singapore is an intermediate tuberculosis (TB) incidence country, with a recent rise in TB incidence from 2008, after a fall in incidence since 1998. This study identified population characteristics that were associated with the recent increase in TB cases, and built a predictive model of TB risk in Singapore. METHODS: Retrospective time series analysis was used to study TB notification data collected from 1995 to 2011 from the Singapore Tuberculosis Elimination Program (STEP) registry. A predictive model was developed based on the data collected from 1995 to 2010 and validated using the data collected in 2011. RESULTS: There was a significant difference in demographic characteristics between resident and non-resident TB cases. TB risk was higher in non-residents than in residents throughout the period. We found no significant association between demographic and macro-economic factors and annual incidence of TB with or without adjusting for the population-at-risk. Despite growing non-resident population, there was a significant decrease in the non-resident TB risk (p < 0.0001). However, there was no evidence of trend in the resident TB risk over this time period, though differences between different demographic groups were apparent with ethnic minorities experiencing higher incidence rates. CONCLUSION: The study found that despite an increasing size of non-resident population, TB risk among non-residents was decreasing at a rate of about 3% per year. There was an apparent seasonality in the TB reporting.

Molecular epidemiology of Mycobacterium tuberculosis complex in Singapore, 2006-2012.

BACKGROUND: Tuberculosis remains common in Singapore, increasing in incidence since 2008. We attempted to determine the molecular epidemiology of Mycobacterium tuberculosis complex (MTC) isolates locally, identifying major circulating genotypes and obtaining a glimpse of transmission dynamics. METHODOLOGY: Non-duplicate MTC isolates archived between 2006 and 2012 at the larger clinical tuberculosis laboratory in Singapore were sampled for spoligotyping and MIRU-VNTR typing, with case data obtained from the Singapore Tuberculosis Elimination Program registry database. Isolates between 2008 and 2012 were selected because of either multidrug-resistance or potential epidemiological linkage, whereas earlier isolates were randomly selected. Separate analyses were performed for the early (2006-2007) and later (2008-2012) study phases in view of potential selection bias. PRINCIPAL FINDINGS: A total of 1,612 MTC isolates were typed, constituting 13.1% of all culture-positive tuberculosis cases during this period. Multidrug-resistance was present in 91 (5.6%) isolates - higher than the national prevalence in view of selection bias. The majority of isolates belonged to the Beijing (45.8%) and EAI (22.8%) lineages. There were 347 (30.7%) and 133 (27.5%) cases clustered by combined spoligotyping and MIRU-VNTR typing from the earlier and later phases respectively. Patients within these clusters tended to be of Chinese ethnicity, Singapore resident, and have isolates belonging to the Beijing lineage. A review of prior contact investigation results for all patients with clustered isolates failed to reveal epidemiological links for the majority, suggesting either unknown transmission networks or inadequate specificity of the molecular typing methods in a country with a moderate incidence of tuberculosis. CONCLUSION: Our work demonstrates that Singapore has a large and heterogeneous distribution of MTC strains, and with possible cross-transmission over the past few years based on our molecular typing results. A universal MTC typing program coupled with enhanced contact investigations may be useful in further understanding the transmission dynamics of tuberculosis locally.