Tween 80 Improves the Acid-Fast Bacilli Quantification in the Magnetic Nanoparticle-Based Colorimetric Biosensing Assay (NCBA)

Despite its reduced sensitivity, sputum smear microscopy (SSM) remains the main diagnostic test for detecting tuberculosis in many parts of the world. A new diagnostic technique, the magnetic nanoparticle-based colorimetric biosensing assay (NCBA) was optimized by evaluating different concentrations of glycan-functionalized magnetic nanoparticles (GMNP) and Tween 80 to improve the acid-fast bacilli (AFB) count. Comparative analysis was performed on 225 sputum smears: 30 with SSM, 107 with NCBA at different GMNP concentrations, and 88 with NCBA-Tween 80 at various concentrations and incubation times. AFB quantification was performed by adding the total number of AFB in all fields per smear and classified according to standard guidelines (scanty, 1+, 2+ and 3+). Smears by NCBA with low GMNP concentrations (≤1.5 mg/mL) showed higher AFB quantification compared to SSM. Cell enrichment of sputum samples by combining NCBA-GMNP, incubated with Tween 80 (5%) for three minutes, improved capture efficiency and increased AFB detection up to 445% over SSM. NCBA with Tween 80 offers the opportunity to improve TB diagnostics, mainly in paucibacillary cases. As this method provides biosafety with a simple and inexpensive methodology that obtains results in a short time, it might be considered as a point-of-care TB diagnostic method in regions where resources are limited.

Modern lineages of Mycobacterium tuberculosis were recently introduced in western India and demonstrate increased transmissibility

Background: Mycobacterium tuberculosis (Mtb) transmissibility may vary between lineages (or variants) and this may contribute to the slow decline of tuberculosis (TB) incidence. The objective of our study was to compare transmissibility across four major lineages (L1-4) of Mtb among participants from two cohort studies in Pune, India. Methods: We performed whole-genome sequencing (WGS) of Mtb sputum culture-positive isolates from participants in two prospective cohort studies of adults with pulmonary TB seeking care at public treatment centers in Pune, Maharashtra. We performed genotypic susceptibility prediction for both first- and second-line drugs using a previously validated random forest model. We used single nucleotide substitutions (SNS) and maximum likelihood estimation to build isolate phylogenies by lineage. We used Bayesian molecular dating to estimate ancestral node ages and compared tree characteristics using a two-sample Kolmogorov-Smirnov (KS) test. Results: Of the 642 isolates from distinct study participants that underwent WGS, 612 met sequence quality criteria. The median age of the 612 participants was 31 years (IQR 24.4-44.2), the majority were male (64.7%) and sputum smear-positive (83.3%), and 6.7% had co-infection with HIV. Most isolates belonged to L3 (44.6%). The majority (61.1%) of multidrug-resistant isolates (MDR, resistant to isoniazid and rifampin) belonged to L2 (P < 0.001 [Fisher's Exact]). There was no significant difference in host characteristics between participants infected with the four major lineages. In phylogenetic analysis, we measured shorter terminal branch lengths in the L2 tree compared to L1 and L3 trees indicating less time elapsing between transmission and sampling and higher transmissibility (median branch lengths: L2 - 3.3, L3 - 7.8, p <0.001). Branching times for L2 and L4 were more recent than L1 and L3 indicating recent introduction into the region (p < 0.01 [KS test]). Conclusion: Modern Mtb lineages (L2 and L4) were more recently introduced in western India, compared to older lineages (L1 and L3). L2 shows a higher frequency of drug-resistance and higher transmissibility. Our findings highlight the need for contact tracing around cases of TB due to L2, and heightened surveillance of TB antibiotic resistance in India.

Improved Diagnosis of Pulmonary Tuberculosis using Bleach (Sodium hypochlorite) Concentration Technique at Tertiary Care Hospital

Introduction: In the Revised National Tuberculosis Control Program (RNTCP), microscopic examination of sputum for acid-fast bacilli (AFB) is currently the backbone for the diagnosis of pulmonary tuberculosis. Studies have shown liquefaction and concentration of sputum by 5% sodium hypochlorite is useful in providing increased sensitivity and safety for the handling of specimens. Objective: To assess the utility of the 5% Sodium hypochlorite concentration method in increasing the sensitivity of smear microscopy for detection of AFB for diagnosis of pulmonary tuberculosis. Methods: The study included a total of 1000 sputum samples from 500 patients with suspected pulmonary tuberculosis. Direct smears were prepared from the sputum samples as per RNTCP guidelines. The remaining sputum was used for bleach concentration and smears prepared from the concentrated material. Both smears were stained by Ziehl-Neelsen staining and screened for acid-fast bacilli and graded according to the RNTCP guidelines. Results: A total of 158 samples (15.8%) from 89 patients were positive by a routine direct method whereas by concentration method 236 samples (23.6%) from 143 patients were found positive diagnosing additional 54 patients. The gain in sputum smear positivity of 7.8% over the routine method is highly significant (p=0.0000, χ2= 270) with a 10.8% increase in case detection. Conclusions: Improvement in the sensitivity of smears microscopy will be useful in case detection of tuberculosis especially in resource-poor countries. The increased positivity of microscopy by bleach method indicates that would prove useful if included in the RNTCP to improve case detection. Keywords: Concentration; smear positivity; sodium hypochlorite; tuberculosis.

Diagnostic Overview and Management of Adult Pulmonary Tuberculosis Patients Inpatient at RSU Royal Prima Medan in 2020

Tuberculosis is an inflammatory disease of the lung parenchyma caused by infection with Mycobacterium tuberculosis. This research aims to find out the description of the diagnosis and management of adult pulmonary TB patients hospitalized at RSU Royal Prima Medan in 2020. This study is a descriptive study. This study is based on medical record data on inpatients with a diagnosis of pulmonary TB at the Royal Prima General Hospital Medan in 2020. The youngest patient was 18 years old and the oldest was 82 years old. The main complaint was shortness of breath (53%), additional complaints were cough (48%), symmetrical physical examination of the thorax (91%), positive sputum smear examination (72%), combined medical management of pulmicort + fumadryl + paracetamol + levofloxacin + OAT category I (72%), the longest length of stay was 7-8 days (38%), the shortest was 11-12 days (1%). In conclusion, pulmonary TB occurs more in men in the 37- 45 year age group (22%), the most complaints are shortness of breath (53%), and the most widely used combination treatment is pulmicort + fumadryl + paracetamol + levofloxacin + drugs anti tuberculosis category I (72%).

Diagnostic Features and Mortality of Tuberculosis by TNF-alpha Inhibitor Use in the United States, 2010–2017

Background An elevated risk of tuberculosis disease (TB) in persons who have received tumor necrosis factor-alpha inhibitor medications (TNF-α inhibitors) has been reported for nearly two decades, but clinical diagnostic features and outcomes of TB in this population remain poorly described. Methods We analyzed national surveillance data for TB cases among persons aged 15 years and older reported in the United States during 2010–2017 and associated mortality data reported through 2019 to describe the clinical characteristics of those receiving TNF-α inhibitors. Results Of 70,129 TB cases analyzed, 504 (0.7%) of the patients had TNF-α inhibitor use reported at TB diagnosis. Patients with TNF-α inhibitor use at TB diagnosis were more likely than TB patients not receiving TNF-α inhibitors to have TB diagnosed in extrapulmonary sites in conjunction with pulmonary sites (28.8% vs 10.0%, P&lt;0.001). Patients receiving TNF-α inhibitors were less likely to have acid-fast bacilli (AFB) noted on sputum smear microscopy (25.6% vs 39.1%, P=0.04), and more likely to have drug-resistant disease (13.5% vs 10.0%, P&lt;0.001). TB-attributed deaths did not significantly differ between patients receiving and not receiving TNF-α inhibitors (adjusted odds ratio 1.46, 95% confidence interval 0.95-2.26). Conclusions Clinicians evaluating TNF-α inhibitor-treated patients should have a high index of suspicion for TB and be aware that extrapulmonary or sputum smear negative TB disease is more common in these patients. No significantly diminished survival of TB patients treated with TNF-α inhibitor therapy before TB diagnosis was noted.

Comparison between ziehl-neelsen staining and fluorescent staining of sputum samples to detect acid fast bacilli in suspected case of pulmonary tuberculosis at tertiary care hospital, Amreli, Gujarat

In India, Tuberculosis (TB) is one of the major community health problems.Pulmonary tuberculosis (PTB) is a respiratory disease. Causative organism for this is acid fast bacilli known as . It is the most ordinary disease affecting the lower socio-economic class in developing countries. Microbiological diagnosis is the heart for the effective treatment of pulmonary TB (PTB). The look forrapid and efficient method has resulted in several staining techniques. Objective of the study was to compare the results of ZN stain (RNTCP) with fluorescent stain by use of microscopy. The study was carried out in Microbiology Department, SMCGH, Amreli. 350 sputum samples (Spot and early morning sample) collected from 175 suspected case of the pulmonary tuberculosis. All 350 samples were processed by ZN stain and Fluorescent stain to detect acid fast bacilli. By use of microscope, the results of the stained smears were given according to RNTCP guideline.Out of 350 sputum smears, 52 (14.85%) and 61 (17.4%) were positive by ZN and FM staining respectively. Males are predominantly affected than females. Majority of the patients were in age above 50 years. Early morning samples were more reliable than spot samples for detection of acid fast bacilli for ZN stain, but not for fluorescent stain.Fluorescent staining with LED microscopy was more efficient than ZN staining for detection of acid fast bacilli from sputum smear.

Treatment Outcomes of the Standardized Shorter MDR-TB Regimen under Programmatic Setting: A Retrospective Study from a Tertiary Care Centre, India

Background: The shorter regimen was widely accepted and advocated for MDR-TB treatment compared tothe conventional longer regimen. Evaluating the performance of both regimens in a programmatic setting will help in tailoring the treatment regimen of MDR-TB. Objectives: 1. To estimate the duration of sputum smear conversion in the shorter MDR-TB regimen. 2. To compare the treatment outcomes of the shorter MDR-TB regimen with that of the longer conventional MDR regimen in a programmatic set up in India. 3. To estimate the adverse drug reactions in the shorter MDR-TB regimen. Methods: A retrospective cross-sectional study was conducted on 320 patients enrolled under programmatic management of drug resistant tuberculosis (PMDT) from April 2017 to May 2019 at a nodal DRTB center and a tertiary care hospital in India. Demographic and clinical characteristics of those who received a shorter MDR-TB regimen were recorded. Treatment outcomes of both regimens were recorded. Treatment success is defined as ‘disease cured and treatment completed’, whereas treatment failure was considered when the treatment was either terminated or changed due to lack of bacteriological conversion at the end of an extended intensive phase or culture reversion in the continuation phase. Results: The treatment success observed in the shorter MDR-TB regimen was 61.25%, which was significantly higher than the conventional longer regimen (p=0.0007). Treatment failures were higher with a shorter MDR-TB regimen (p=0.0001). Conclusion: Treatment success with the shorter MDR-TB regimen though higher than the conventional regimen, is still way behind the target treatment success rate. Improving treatment adherence remains pivotal for achieving end TB targets.

Clinico-radiological and bronchoscopic predictors of microbiological yield in sputum negative tuberculosis in Pakistan

To determine association of clinico-radiological factors and radiological activity with diagnostic yield in sputum-smear negative tuberculosis (TB). Prospective observational study in Military Hospital Rawalpindi from July to December 2018. Adult patients having no contraindications to bronchoscopy were included. HIV positive patients and those on anti-tuberculosis therapy for more than one week were excluded. High-Resolution Computed tomography (HRCT) findings were classified based on active and inactive tuberculosis features. Washings were sent for Acid-Fast Bacillus (AFB) smear, GeneXpert assay and cultures. Out of 215 patients, 42.3% (91) were diagnosed with microbiological or histological evidence of TB. On univariate analysis, cavitation (p-value <0.001), soft-tissue nodules (p-value 0.04), and endobronchial mucosal changes (p-value 0.02) were associated with culture positivity. Presence of cavitation (OR= 4.10; CI= 2.18,7.73; p-value<0.001) was the only independent predictor of microbiological yield. Diagnostic yield was 70%, 50%, 12.5% and 8.6% in patients with definitely active, probably active, indeterminate and inactive tuberculosis HRCT features respectively. Sensitivity, specificity, positive predictive value and negative predictive value of HRCT active TB were 95.38% (95% CI 87.10 -99.04), 48.00 % (95% CI 39.78 -56.30), 44.29% (95% CI 40.31 -48.33), 96.00 % (95%CI 88.70 -98.66) respectively. There was no significant association between age groups, smoking status and gender with diagnosis of tuberculosis in our study. Radiological activity and certain visualized bronchoscopic changes were associated with good diagnostic performance and can be used as predictive factors in diagnosis of active smear negative tuberculosis.

Use of rapid molecular tuberculosis diagnostics across Brazil’s incarcerated population

BackgroundIncarcerated individuals in Brazil are at high-risk of tuberculosis (TB), but their access to World Health Organization recommended diagnostics is poorly understood.MethodsWe conducted a retrospective cross-sectional study of newly diagnosed TB cases in Brazil’s notifiable disease registry, which includes information on diagnostic tests performed, from January 2015 through December 2018. We quantified reported use of TB diagnostics across incarcerated and non-incarcerated populations and tested for municipality-level factors associated with diagnostic usage among the incarcerated population with generalized linear regression.ResultsBetween 2015 and 2018, 258,014 individuals were newly diagnosed with TB, including 27,400 (10.6%) incarcerated individuals. Among these, 27.5% had an Xpert MTB/RIF test reported; 71.5% had sputum smear; 34.1% had culture; 70.9% had chest radiography. Xpert MTB/RIF use was greater among incarcerated than non-incarcerated individuals (36.2% vs 26.5%, p<0.001). However, we found spatial heterogeneity in state-level use of both Xpert MTB/RIF (range: 4.7-72.4% cases diagnosed) and chest radiography (range: 11.7-88.4%) in prisons. We identified seven municipalities with large incarcerated populations (>5000) with rates of Xpert MTB/RIF usage below the national average in incarcerated individuals.ConclusionPrioritizing expansion of rapid molecular diagnostics in prisons, particularly in regions with limited current usage of molecular diagnostics, will be an essential component of TB control.

Costs of TB services in India (No 1)

BACKGROUND: There is a dearth of economic analysis required to support increased investment in TB in India. This study estimates the costs of TB services from a health systems´ perspective to facilitate the efficient allocation of resources by India´s National Tuberculosis Elimination Programme.METHODS: Data were collected from a multi-stage, stratified random sample of 20 facilities delivering TB services in two purposively selected states in India as per Global Health Cost Consortium standards and using Value TB Data Collection Tool. Unit costs were estimated using the top-down (TD) and bottom-up (BU) methodology and are reported in 2018 US dollars.RESULTS: Cost of delivering 50 types of TB services and four interventions varied according to costing method. Key services included sputum smear microscopy, Xpert® MTB/RIF and X-ray with an average BU costs of respectively US$2.45, US$17.36 and US$2.85. Average BU cost for bacille Calmette-Guérin vaccination, passive case-finding, TB prevention in children under 5 years using isoniazid and first-line drug treatment in new pulmonary and extrapulmonary TB cases was respectively US$0.76, US$1.62, US$2.41, US$103 and US$98.CONCLUSION: The unit cost of TB services and outputs are now available to support investment decisions, as diagnosis algorithms are reviewed and prevention or treatment for TB are expanded or updated in India.