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.

Dynamics of Macrophage, T and B Cell Infiltration Within Pulmonary Granulomas Induced by Mycobacterium tuberculosis in Two Non-Human Primate Models of Aerosol Infection

Non-human primate models of Tuberculosis (TB) are one of the most commonly used within the experimental TB field because they closely mimic the whole spectrum of disease progression of human TB. However, the early cellular interactions of the pulmonary granuloma are still not well understood. The use of this model allows investigation into the early interactions of cells within pulmonary granulomas which cannot be undertaken in human samples. Pulmonary granulomas from rhesus and cynomolgus macaques from two timepoints post infection were categorised into categories 1 – 6 (early to late stage granulomas) and immunohistochemistry was used to identify CD68+ macrophages, CD3+ T cells and CD20+ B cells. Multinucleated giant cells and acid-fast bacilli were also quantified. At week four post infection, cynomolgus macaques were found to have more CD68+ cells than rhesus in all but category 1 granulomas. Cynomolgus also had a significantly higher percentage of CD20+ B cells in category 1 granulomas. At week twelve post infection, CD68+ cells were most abundant in category 4 and 5 granulomas in both species; however, there were no significant differences between them. CD3+ T cells and CD20+ B cells were significantly higher in the majority of granuloma categories in cynomolgus compared to rhesus. Multinucleated giant cells and acid-fast bacilli were most abundant in categories 5 and 6 at week 12 post challenge in both species. This study has identified the basic cellular composition and spatial distribution of immune cells within pulmonary granulomas in both rhesus and cynomolgus macaques over time. The data from this study will add to the knowledge already gained in this field and may inform future research on vaccines and therapeutics for TB.

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.

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.

Analysis of MTb Rapid Molecular Test Performance Towards Microscopical Acid Fast Bacilli Examination at Labuang Baji General Hospital

Tuberculosis (TB) is a global health problem, which is the third leading cause of death of all infectious diseases aroundthe world, included Indonesia. Acid Fast Bacilli (AFB) smear and rapid molecular assay for Mycobacterium tuberculosis (MTB)are the old and new examinations required for MTB laboratory diagnosis. This study aimed to compare the performance ofMTB rapid molecular assay and AFB smear in diagnosis and screening for TB patients. This observational retrospective studyused a cross-sectional approach, with a purposive sampling technique of 559 patients with suspected TB in Labuang BajiHospital, Makassar. This study was conducted from March 2019 to June 2019 by taking data from medical records fromJanuary 2018 to December 2018 at Labuang Baji Hospital, Makassar. Three hundred and forty-nine subjects were males(62.4%), and 210 subjects were females (37.6%). This study revealed sensitivity and specificity of 98.57% and 84.96%,respectively for MTB rapid molecular assay, and 68.65% and 99.44%, respectively for AFB smear, this shows that MTB rapidmolecular assay was superior to AFB smear in diagnosing TB patients.

Sputum Smear Non-conversion at the End of Intensive Phase of Tuberculosis Treatment at a Tertiary Center: A Descriptive Cross-sectional study

Introduction: Sputum non-conversion is smear positive tuberculosis despite anti-tubercular therapy. Various factors may lead to sputum non-conversion including resistance to anti-tubercular drugs, age, gender, disease severity, non-compliance, drugs unavailability etc. Little is known and studied about the contribution of these individual factors. Our study sought to determine the prevalence of sputum smear non-conversion in patients at the end of intensive phase of tuberculosis treatment visiting a tertiary-level institution in Nepal. Methods: A descriptive cross-sectional study was conducted among recorded data of patients undergoing sputum Acid Fast Bacilli staining at the end of intensive phase at National Tuberculosis Control Center from April 2018 to April 2020. The study was approved by Nepal Health Research Council (Registration no: 76012020 P). The convenient sampling method was adopted. The data were analyzed using Microsoft Excel. Point estimate at 95% Confidence Interval was calculated along with frequency and proportion for binary data. Results: Our study found that out of 830 samples that were tested by Acid Fast Bacilli stain at the end of intensive phase, 40 (4.82%) (3.37-6.28 at 95% Confidence Interval) were sputum smear non-converters. The mean age of sputum non-converters was 41.25±15.543 years. Conclusions: The study shows that a significant proportion of patients remain acid-fast stain positive despite the treatment. However, the proportion is low compared to other similar studies around the globe. This study provides program managers with evidence to support the development of more tailored tuberculosis care and need to conduct more intensive studies about various factors that may lead to non-conversion.

Autochthonous North American Leprosy: A Second Case in Canada

Autochthonous leprosy was reported in the Southern USA in 2011 and has comprised an average of 34% of new cases from 2015 to 2020 in that country. We report a similar case in a patient from Western Canada. A 50-year old male patient presented with a four-year history of a chronic rash. Pathology stains revealed acid-fast bacilli prompting specialist referral. Examination was suspicious for leprosy, which was confirmed on slit skin smears and molecular testing. The patient responded well to treatment. Genotypic testing mapped the organism to the 3I-2 SNP type, which is of European origin and is the type found in implicated armadillo species in North America.