Auramine-O Staining vs Ziehl Neelsen Staining: Advantages and Disadvantages

Background: Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is still a major public health concern around the world. Prompt detection of active tuberculosis cases helps in timely therapeutic intervention and reduces community transmission. Despite limited sensitivity, conventional microscopy is still used to diagnose pulmonary tuberculosis in high-burden nations such as India. This study, therefore, was aimed at assessing the diagnostic performance of microscopy by Ziehl Neelsen (ZN) and auramine (AO) staining in the diagnosis of pulmonary tuberculosis. Materials and methods: A prospective comparative study was done on the sputum samples of 2,395 adult patients from November 2018 to May 2020 suspected of having pulmonary tuberculosis visiting the Designated Microscopic Centre of SGT Medical College, Budhera, Gurugram. Each sample was subjected to ZN staining, and AO staining as per NTEP guidelines. Results: Out of the 2,395 samples studied, 161 (6.76%) and 224 (9.35%) were positive by ZN and AO staining methods respectively. Pauci-bacillary cases detected by AO were more than ZN staining. There were 63 more sputum samples detected by AO staining which were missed by ZN microscopy. Conclusion: When compared to conventional ZN staining, the auramine staining technique is more sensitive and takes less time to diagnose pulmonary tuberculosis

Quality of Same-Day Sputum Smears Microscopy and Presumptive Tuberculosis Patients Drop-out at Health Facilities of Addis Ababa, Ethiopia / 결핵

Comparative Evaluation of GENE-XPERT MTB/RIF with AFB Smear Microscopy Methods for the Diagnosis of Tuberculosis in Sputum Specimens of Suspected Pulmonary Tuberculosis Patients

Tuberculosis is a major public health problem worldwide and remains one of the most significant causes of death from an infectious agent. India contributes to 25.5% of the global new TB case detection. In recent times, emphasis has shifted from older phenotypically and biochemical methods of diagnosis to molecular methods such as GeneXpert MTB/RIF, a real time PCR that can detect MTB and rifampicin resistence simultaneously. Methods: This study aims at assessing the performance of acid fast bacilli (AFB) smear microscopy in comparison with GeneXpert MTB RIF, in the diagnosis of pulmonary tuberculosis. Study was a cross-sectional study carried out at the Department of Pulmonary Medicine, Muzaffarnagar Medical College, from January 2018 to April 2019. Result: The detection of MTB and rifampicin resistance using the Xpert MTB/RIF assay was assessed in 67 specimens from patients suspected of having pulmonary tuberculosis and compared with conventional smear microscopy. Out of these 67 sputum specimens, 38 samples were MTB positive by smear microscopy while 56 samples were MTB detected by Gene Xpert assay. Gene Xpert detected 18 additional tubercular cases and identify two cases of Rifampicin resistant MTB. Conclusion: Study show that there was no statistically significance in diagnostic value between GeneXpert and AFB smear microscopy in sputum samples however Gene Xpert MTB/RIF is useful method for rapid detection of MTB and Rifampicin resistance simultaneously.

Diagnostic Accuracy of Xpert MTB Compared to Smear Microscopy in Pulmonary vs Extrapulmonary Tuberculosis

Introduction: Mycobacterium tuberculosis is the mostsignificant infectious disease causing death worldwide.Inability to rapidly diagnose and treat the affected patientsleads to increased morbidity and mortality, developmentof secondary resistance. There is sparse data which showthe accuracy of diagnosis of tuberculosis by Gene Xpert inpatients. There is limited data on the usefulness of XpertMTB in the diagnosis of extrapulmonary TB. Study aimedto evaluate the diagnostic accuracy of XpertMTB comparedto smear microscopy in pulmonary and extra pulmonarytuberculosis.Material and methods: Prospective observational studyconducted from May 2017 to July 2017. The study wasconducted in Department of medicine, Department ofpulmonology at R.L.Jalappa hospital and research centre,Kolar. 139 tubercular suspects who were not human immunedeficiency virus (HIV) positive were included in the study.Result: Out of 139 patients 72 were males and 67 were females.99 pulmonary samples and 40extrapulmonary samples wereobtained. 93 pulmonary samples and 24 extrapulmonarysamples were culture positive. Smear microscopy has lowersensitivity in diagnosing pulmonary TB (46.2%) as well asextrapulmonary TB(16.6%).Conclusion: The Xpert MTB is a rapid, sensitive and areliable diagnostic test for TB than smear microscopy in bothpulmonary as well as extra pulmonary TB. The diagnosisof extrapulmonary TB should not solely depend on theresults of Xpert MTB and should be subjected to culture ofMycobacterium.

Clinical impact and cost analysis of the use of either the Xpert MTB Rif test or sputum smear microscopy in the diagnosis of pulmonary tuberculosis in Rio de Janeiro, Brazil

Abstract INTRODUCTION: The molecular test Xpert MTB/RIF (Xpert) has been recommended for use in the diagnosis of pulmonary tuberculosis (PTB); however, data on the cost of incorporating it under routine conditions in high-burden countries are scarce. The clinical impact and costs incurred in adopting the Xpert test in routine PTB diagnosis was evaluated in a prospective study conducted from November 2012 to November of 2013, in the City of Rio de Janeiro, Brazil. METHODS: The diagnostic and therapeutic cascade for TB treatment was evaluated using Xpert in the first stage (S1), and sputum smear microscopy (SSM) in the second stage (S2). The mean costs associated with each diagnostic test were calculated including equipment, human resources, supplies, and infrastructure. RESULTS: We included 232 subjects with probable TB (S1 = 87; S2 = 145). The sensitivities of Xpert and SSM were 91.7% (22/24) and 79.1% (34/43), respectively. The median time between triage and TB treatment initiation in S1 (n = 24) was 14.5 days (IQR 8-28.0) and in S2 (n = 43) it was 8 days [interquartile range (IQR) 6-12.0]. The estimated mean costs per examination in S1 and S2 were US$24.61 and US$6.98, respectively. CONCLUSIONS: Compared with SSM, Xpert test showed a greater sensitivity, but it also had a time delay with respect to treatment initiation and a higher mean cost per examination.

Microscopic detection of Mycobacterium tuberculosis in direct or processed sputum smears

Abstract INTRODUCTION: Microscopic identification of active pulmonary tuberculosis (PTB) from direct smears of sputum (DS) is widely used for detection, but has limited sensitivity. Here, we assessed the yield of acid-fast bacilli (AFB) detection in processed sputum smears (PSS). METHODS: Sputum samples were simultaneously analyzed by direct sputum smearing and after chemical treatment and spontaneous sedimentation. RESULTS: Of the 1,719 samples analyzed, 16.4% were positive for AFB in conventional DS and 21.4% in PSS, corresponding to a 30% increase in detection. CONCLUSIONS: Increased sensitivity from analyzing PSS and better safety protocols will contribute to improved detection and control of the disease.

Effectiveness of Tuberculosis Smear Microscopy in Laboratory Diagnosis of Mycobacterium tuberculosis in Resource Constrained Settings.

Aim: To determine the correlation of accuracy of direct smear microscopy compared with BACTEC MGIT 960. Design: The study prospectively compare direct smear microscopy with BACTEC MGIT 960 using the reference standard, Lowenstein Jensen culture. Place and Duration: The study was conducted in Zankli Medical Centre, Abuja, between November 2004 and July 2005. Methodology: 340 suspected patients for Mycobacterium tuberculosis referred from direct observation therapy clinics located in six different government owned health facilities were referred to our facility. These patients; male (192) and female (148) were between the age of 10 and 64 years old. Three sputa samples were collected over two consecutive days and direct smear microscopy and culture were performed on these samples. Results: When compared with the reference standard, BACTEC MGIT 960 has a sensitivity and specificity of 100.0% and 56.4% respectively, and a negative predictive value of 100.0%; indicating the proportion of AFB negative participants were actually not infected with M. tuberculosis when tested with BACTEC MGIT 960. The sensitivity of direct microscopy was significantly lower than BACTEC MGIT 960 (84.9% versus 100%, p<0.001) and the specificity was significantly higher (96.6% versus 56.4%, p<0.001). Conclusions: For the purpose of effectiveness of tuberculosis program in developing countries, direct smear microscopy may still be relevant in the diagnosis of Mycobacterium tuberculosis.

Same day sputum smear microscopy approach with modified ZN staining for the diagnosis of pulmonary tuberculosis in a microscopy centre at Rajahmundry.

Background: Sputum smear microscopy is the main‑stay in the diagnosis of pulmonary tuberculosis in many developing countries. To overcome the drop outs, same day diagnosis is ideal. Materials and Methods: In the current study, two spot sputum samples (SS2 approach) are collected within a gap of one hour (same day sputum smear microscopy) in addition to the standard spot morning (SM) approach. The smears were stained with standard Ziehl Neelsen (ZN) and modified ZN staining techniques. Results: Out of 1537 patients, sputum smear positivity (SSP) was 9.43% (146 patients) in SM approach with standard ZN staining. Smear positivity was increased to 9.8% (151 patients) with modified ZN staining. For SS2 approach, SSP was 9.37% (144 patients) and 9.8% (151 patients) with standard and modified ZN staining procedures, respectively. Conclusions: Diagnosis of lung tuberculosis is possible with two spot sputum samples with modified ZN staining.

Avaliação da qualidade das baciloscopias realizadas na Rede de Laboratórios de Tuberculose da Região Metropolitana da Baixada Santista/SP / Quality assessment of Sputum-Smear Microscopy performed by the Tuberculosis Laboratory Network of the Metropolitan Region of Baixada Santista/SP

O Programa Nacional de Controle da Tuberculose (TB) do Ministério da Saúde utiliza a baciloscopia de escarro como metodologia padrão no diagnóstico da TB pulmonar. Para garantir a qualidade do exame é recomendado o uso de Controle Externo de Qualidade (CEQ) da baciloscopia na rede de laboratórios de diagnóstico de TB. Neste estudo foi avaliada a qualidade das baciloscopias de escarro da rede de laboratórios locais, por meio de supervisão indireta, na Região Metropolitana da Baixada Santista/SP. Em nove laboratórios locais foram analisadas 5.691 baciloscopias, coradas pelo Ziehl-Neelsen. Tais amostras foram avaliadas nos seguintes itens: análise macroscópica (distensão) e microscópica (coloração) dos esfregaços e o índice baciloscópico (C%). Para avaliá-las, utilizou-se a metodologia padrão do Manual Nacional de Vigilância Laboratorial de TB e outras Micobactérias/MS de 2008. Utilizando-se metodologia recomendada, das 713 lâminas analisadas, 529 (74,2 %) foram satisfatórias na avaliação macroscópica e 631 (88,5 %), satisfatórias na avaliação microscópica. Na análise da releitura, uma unidade laboratorial apresentou dois resultados falsos positivos e C = 97,5 %. Embora os resultados encontrados tenham sido aceitáveis, sugere-se a realização de atividades contínuas do CEQ, com ênfase na aplicação de ações corretivas nas principais causas de erro no desenvolvimento da técnica de baciloscopia.

The Brazilian Ministry of Health national program for tuberculosis (TB) control (Programa Nacional deControle da Tuberculose) determines the sputum smear microscopy as the standard methodology for TBdiagnosis. In order to achieve diagnosis quality assurance it is recommended to set up an External QualityAssessment (EQA) scheme in the TB diagnosis laboratory network. This study aimed at evaluating thequality of sputum smear microscopy performed by the local laboratory network of the metropolitanarea of Baixada Santista, SP, by means of an indirect supervision. The 5,691 sputum samples stained withZiehl-Neelsen in nine local laboratories were evaluated. The applied EQA methodology was in accordancewith the National TB and Other AFB Laboratory Surveillance Manual – Ministry of Health, 2008. Theanalyzed parameters were: macroscopic (smear uniformity) and microscopic (staining) analyses of thesputum smears and bacilloscopic index (C%). Of 713 analyzed slides, 529 (74.2 %) were satisfactory onmacroscopic features and 631 (88.5 %) on microscopic evaluation. In the slides rechecking, one laboratoryshowed two false-positive results and C =97.5 %. Although the general results have been acceptable, acontinuous EQA activity should be carried out, emphasizing the use of correcting actions on the commonsources of error that may occur in performing the AFB microscopy technique.

Same day sputum smear microscopy approach for the diagnosis of pulmonary tuberculosis in a microscopy centre at Rajahmundry.

Background: Sputum smear microscopy is the initial and rapid diagnostic technique for tuberculosis. This requires two (spot and morning SM) sputum sample examinations over two days. Collection of two spot samples (SS2) on the same day would reduce the number of visits, time, money and early initiation of treatment. Methods: We evaluated same day approach (SS2) against standard (SM) for the diagnosis of pulmonary tuberculosis. Results: Out of 658 participants, same day approach could identify 62 cases, whereas standard approach could identify 64 cases. Both the approaches are equally effective (p>0.05). Conclusion: The diagnosis of pulmonary tuberculosis is possible in one day by examining two spot samples.

Proficiency panel testing - A reliable tool in external quality assessment of sputum smear microscopy services in Gujarat, India.

Objective: To assess the proficiency of Senior TB Laboratory Supervisors (STLSs) and district level Laboratory Technicians (LTs) in sputum smear microscopy. Method: Intermediate Reference Laboratory (IRL), Ahmedabad had manufactured and validated Proficiency Panel Testing slides from sputum samples, made On Site Evaluation (OSE) visits of District TB Centres (DTCs) in two rounds, and conducted Proficiency Panel Testing of STLSs & DTC-LTs from January 2005 to June 2009. Results: High level of concordance in Z-N smear grading was found between Microbiologist and district laboratory staff. DTC readers reported overall consistency level of more than 98% in Z-N grade agreement during both the IRL, EQA, OSE visits. The tendency to over-grade the panel slides was much higher (more than 22%) as compared to under-grade (less than 2%) them in “correct slides”. High False Positive (HFP) error was not observed in the present study. Conclusion: Laboratory supervisor’s proficiency can be quickly assessed by Proficiency Panel Testing, under multi-level quality assurance network system of sputum smear microscopy in public health programmes like the RNTCP. Proficiency Panel Testing is highly replicable and reproducible tool for quick and reliable assessment of proficiency of the staff and it can be made more effective by raising the proportion of lower grade positive slides in panel set of each reader. DTC readers’ overall agreement level of more than 98% in Z-N grade suggests high level of precision and excellent consistency during both the IRL, EQA, OSE rounds. It is concluded that even for a large network of sputum smear microscopy centres under public health programmes like the RNTCP in order to take corrective action, Proficiency Panel Testing can be effectively used for quick identification of suboptimal- technical performance of the supervisory staff.