Identification of Significant Pathogenic Nontuberculous Mycobacteria Species from Presumptive TB Patients Using Partial hsp65 Gene Sequencing

Dyshelly Nurkartika Pascapurnama,1 Nabilla Ghina Zavitri,1 Raspati Cundarani Koesoemadinata,1 Adi Imam Cahyadi,2 Lidya Chaidir2,3 1Research Center for Care and Control of Infectious Diseases, Universitas Padjadjaran, Bandung, West Java, Indonesia; 2Department of Biomedical Sciences, Faculty of Medicine, Universitas Padjadjaran, Sumedang, West Java, Indonesia; 3Center for Translational Biomarker Research, Universitas Padjadjaran, Bandung, West Java, IndonesiaCorrespondence: Lidya Chaidir, Faculty of Medicine, Universitas Padjadjaran, Jln. Ir. Soekarno Km. 21 Jatinangor, Sumedang, 45363, West Java, Indonesia, Tel +62-22-8428812, Email [email protected]: To date, the diagnosis of nontuberculous mycobacteria (NTM) disease primarily relies on clinical symptoms and radiological features. Our objective was to apply a sequence-based analysis method by using partial gene sequencing of heat shock protein 65 (hsp65) to identify NTM species.Patients and Methods: A total of 32 stored isolates obtained from individuals suspected of having pulmonary NTM infection were subjected to solid Ogawa culture. Genomic DNA from each sample was extracted and used in a conventional polymerase chain reaction (PCR) targeting a specific region of hsp65 gene. Identified amplicons from the PCR were then subjected to targeted sequencing. Analysis of the obtained hsp65 sequence was performed using DNA Baser tool. The consensus sequences obtained were compared to references in the GenBank NCBI database to determine NTM species.Results: We identified several important NTM species which posses opportunistic characteristics. M. abscessus and M. chelonae are the most frequent NTM species identified in this study (40.63% and 18.75%, respectively). These two species have the potential to cause significant infections in human, ranging from opportunistic pulmonary infection to localized skin infection. Additionally, pathogenic NTM members of M. fortuitum group (MFG), M. avium, M. intracellulare, M. kansasii, and M. celatum were also found among all identified species.Conclusion: Sequence-based analysis is a promising method for identifying species of NTM. The hsp65 gene has a high discriminatory power to identify opportunistic pathogen NTM species in specimens in Indonesia. Consequently, hsp65 partial gene sequencing is considerable as an alternative and reliable approach for NTM speciation.Keywords: nontuberculous mycobacteria, species identification, hsp65 gene, sequence-based analysi

High tuberculosis incidence among people living with diabetes in Indonesia.

BACKGROUND: Data regarding the incidence of tuberculosis (TB) among people living with diabetes (PLWD) in TB-endemic settings are scarce. We examined TB incidence among PLWD in Indonesia who had previously been screened for latent TB infection (LTBI) and TB disease. METHODS: PLWD (≥18 y of age) in an urban setting were examined a mean 3.4 y after they had been screened for active TB and LTBI. Data on subsequent TB diagnosis were collected by interview and with chest X-ray, sputum smear and Mycobacterium tuberculosis culture. TB incidence rates were stratified for baseline LTBI status, as determined by the QuantiFERON interferon-gamma release assay (IGRA). RESULTS: Of 590 PLWD, 101 had died and 163 could not be contacted or refused. Among the 326 who were re-examined, 6 (1.8%; 95% confidence interval [CI] 0.7 to 4.0) reported being diagnosed already and a further 5 were diagnosed with active TB (1.5%; 95% CI 0.50 to 3.5). The TB incidence rate was 9.85 (95% CI 4.03 to 15.68) per 1000 person-years. TB incidence was higher among PLWD with baseline LTBI (17.13; 95% CI 5.25 to 29.00/1000 person-years) compared with those without LTBI (4.79; 95% CI -0.63 to 10.21), with an incidence rate ratio of 3.57 (95% CI 0.86 to 20.92; p=0.054). CONCLUSIONS: PLWD with LTBI in Indonesia and similar settings are likely to benefit from TB preventive therapy

Latent TB infection and pulmonary TB disease among patients with diabetes mellitus in Bandung, Indonesia.

Background: Screening and treatment of latent TB infection (LTBI) and TB disease could reduce diabetes mellitus (DM)-associated TB. We aimed to describe the prevalence of LTBI and pulmonary TB among patients with DM in a TB-endemic setting. Methods: Patients with DM attending a hospital and community centres in Bandung, Indonesia, underwent LTBI screening using interferon gamma release assay (IGRA). TB was investigated by sputum smear, culture and x-ray. TB contacts from a parallel study were age- and sex-matched to patients with DM to compare LTBI and TB disease prevalence. Results: Of 682 patients with DM screened, 651 (95.5%) were eligible. Among 'TB disease-free' patients, LTBI prevalence was 38.9% (206/530; 95% CI 34.7-43.2). Patients with DM were less likely to be IGRA positive than TB contacts (38.6%, 54/140; 95% CI 30.5-46.6 vs 68.6%, 96/140; 95% CI 60.9-72.3: p<0.001); but had a higher disease prevalence (4.9%, 8/164; 95% CI 1.6-8.2 vs 1.2%, 2/164; 95% CI -0.5 to 2.9: p=0.054). Patients with DM in crowded households had increased risk of LTBI (AOR 1.71; 95% CI 1.19-2.45). Conclusions: LTBI prevalence in patients with DM was lower than in household contacts, but patients with DM were more likely to have TB disease. Further studies should explore possible benefits of LTBI screening and preventive therapy in patients with DM in TB-endemic settings

Latent TB infection and pulmonary TB disease among patients with diabetes mellitus in Bandung, Indonesia.

Background: Screening and treatment of latent TB infection (LTBI) and TB disease could reduce diabetes mellitus (DM)-associated TB. We aimed to describe the prevalence of LTBI and pulmonary TB among patients with DM in a TB-endemic setting. Methods: Patients with DM attending a hospital and community centres in Bandung, Indonesia, underwent LTBI screening using interferon gamma release assay (IGRA). TB was investigated by sputum smear, culture and x-ray. TB contacts from a parallel study were age- and sex-matched to patients with DM to compare LTBI and TB disease prevalence. Results: Of 682 patients with DM screened, 651 (95.5%) were eligible. Among 'TB disease-free' patients, LTBI prevalence was 38.9% (206/530; 95% CI 34.7-43.2). Patients with DM were less likely to be IGRA positive than TB contacts (38.6%, 54/140; 95% CI 30.5-46.6 vs 68.6%, 96/140; 95% CI 60.9-72.3: p<0.001); but had a higher disease prevalence (4.9%, 8/164; 95% CI 1.6-8.2 vs 1.2%, 2/164; 95% CI -0.5 to 2.9: p=0.054). Patients with DM in crowded households had increased risk of LTBI (AOR 1.71; 95% CI 1.19-2.45). Conclusions: LTBI prevalence in patients with DM was lower than in household contacts, but patients with DM were more likely to have TB disease. Further studies should explore possible benefits of LTBI screening and preventive therapy in patients with DM in TB-endemic settings

Diabetes characteristics and long-term management needs in diabetic TB patients.

BACKGROUND: Diabetes mellitus (DM) is common among patients with TB. We assessed DM characteristics and long-term needs of DM-TB patients after completing TB treatment.METHODS: Newly diagnosed TB patients with DM were recruited for screening in a randomised clinical trial evaluating a simple algorithm to improve glycaemic control during TB treatment. DM characteristics, lifestyle and medication were compared before and after TB treatment and 6 months later. Risk of cardiovascular disease (CVD), albuminuria and neuropathy were assessed after TB treatment.RESULTS: Of 218 TB-DM patients identified, 170 (78%) were followed up. Half were males, the mean age was 53 years, 26.5% were newly diagnosed DM. High glycated haemoglobin at TB diagnosis (median 11.2%) decreased during TB treatment (to 7.4% with intensified management and 8.4% with standard care), but this effect was lost 6 months later (9.3%). Hypertension and dyslipidemia contributed to a high 10-year CVD risk (32.9% at month 6 and 35.5% at month 12). Neuropathy (33.8%) and albuminuria (61.3%) were common. After TB treatment, few patients used CVD-mitigating drugs.CONCLUSION: DM in TB-DM patients is characterised by poor glycaemic control, high CVD risk, and nephropathy. TB treatment provides opportunities for better DM management, but effort is needed to improve long-term care

The effect of a structured clinical algorithm on glycemic control in patients with combined tuberculosis and diabetes in Indonesia: a randomized trial.

AIMS: Diabetes mellitus (DM) is associated with worse tuberculosis (TB) treatment outcomes, especially among those with poor glycemic control. We examined whether a structured clinical algorithm could improve glycemic control in TB patients with DM. METHODS: In an open label randomized trial, TB-DM patients were randomized to scheduled counselling, glucose monitoring, and adjustment of medication using a structured clinical algorithm (intervention arm) or routine DM management (control arm), with glycated hemoglobin (HbA1c) at month 6 as the primary end point. RESULTS: We randomized 150 pulmonary TB-DM patients (92% culture positive, 51.3% male, mean age 53 years). Baseline mean HbA1c was 11.0% in the intervention arm (n=76) and 11.6% in the control arm (n=74). At 6 months, HbA1c had decreased more in the intervention arm compared with the control arm (a difference of 1.82% HbA1c, 95% CI 0.82-2.83, p<0.001). Five patients were hospitalized in the intervention arm and seven in the control arm. There was more hypoglycemia (35.0% vs 11.8%; p=0.002) in the intervention arm. Two deaths occurred in the intervention arm, one due to cardiorespiratory failure and one because of suspected septic shock and multiorgan failure. CONCLUSION: Regular monitoring and algorithmic adjustment of DM treatment led to improved glycemic control

Computer-assisted chest radiography reading for tuberculosis screening in people living with diabetes mellitus.

BACKGROUND: Diabetes mellitus is a significant risk factor for tuberculosis (TB). We evaluated the performance of computer-aided detection for tuberculosis (CAD4TB) in people living with diabetes mellitus (PLWD) in Indonesia. METHODS: PLWD underwent symptom screening and chest X-ray (CXR); sputum was examined in those with positive symptoms and/or CXR. Digital CXRs were scored using CAD4TB and analysed retrospectively using clinical and microbiological diagnosis as a reference. The area under the receiver operator curve (AUC) of CAD4TB scores was determined, and an optimal threshold score established. Agreement between CAD4TB and the radiologist's reading was determined. RESULTS: Among 346 included PLWD, seven (2.0%) had microbiologically confirmed and two (0.6%) had clinically diagnosed TB. The highest agreement of CAD4TB with radiologist reading was achieved using a threshold score of 70 (κ = 0.41, P < 0.001). The AUC for CAD4TB was 0.89 (95%CI 0.73-1.00). A threshold score of 65 for CAD4TB resulted in a sensitivity, specificity, positive predictive value and negative predictive value of respectively 88.9% (95%CI 51.8-99.7), 88.5% (95%CI 84.6-91.7), 17.0% (95%CI 7.6-30.8) and 99.6% (95%CI 98.2-100). With this threshold, 48 (13.9%) individuals needed microbiological examination and no microbiologically confirmed cases were missed. CONCLUSIONS: CAD4TB has potential as a triage tool for TB screening in PLWD, thereby significantly reducing the need for microbiological examination

Diabetes Mellitus Among Pulmonary Tuberculosis Patients From 4 Tuberculosis-endemic Countries: The TANDEM Study

Background Diabetes mellitus (DM) increases active tuberculosis (TB) risk and worsens TB outcomes, jeopardizing TB control especially in TB-endemic countries with rising DM prevalence rates. We assessed DM status and clinical correlates in TB patients across settings in Indonesia, Peru, Romania, and South Africa. Methods Age-adjusted DM prevalence was estimated using laboratory glycated hemoglobin (HbA1c) or fasting plasma glucose in TB patients. Detailed and standardized sociodemographic, anthropometric, and clinical measurements were made. Characteristics of TB patients with or without DM were compared using multilevel mixed-effect regression models with robust standard errors. Results Of 2185 TB patients (median age 36.6 years, 61.2% male, 3.8% human immunodeficiency virus–infected), 12.5% (267/2128) had DM, one third of whom were newly diagnosed. Age-standardized DM prevalence ranged from 10.9% (South Africa) to 19.7% (Indonesia). Median HbA1c in TB–DM patients ranged from 7.4% (Romania) to 11.3% (Indonesia). Compared to those without DM, TB–DM patients were older and had a higher body mass index (BMI) (P value < .05). Compared to those with newly diagnosed DM, TB patients with diagnosed DM had higher BMI and HbA1c, less severe TB, and more frequent comorbidities, DM complications, and hypertension (P value < .05). Conclusions We show that DM prevalence and clinical characteristics of TB–DM vary across settings. Diabetes is primarily known but untreated, hyperglycemia is often severe, and many patients with TB–DM have significant cardiovascular disease risk and severe TB. This underlines the need to improve strategies for better clinical management of combined TB and DM