Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients / Comparação de diferentes modalidades de diagnóstico para isolamento de Mycobacterium tuberculosis entre pacientes com suspeita de linfadenite tuberculosa

Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados para o isolamento completo do Mycobacterium tuberculosis.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients

Abstract Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

Resumo A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados para o isolamento completo do Mycobacterium tuberculosis.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients / Comparação de diferentes modalidades de diagnóstico para isolamento de Mycobacterium tuberculosis entre pacientes com suspeita de linfadenite tuberculosa

Abstract Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

Resumo A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado ​​para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados ​​para o isolamento completo do Mycobacterium tuberculosis.

Acid-fast bacteria as causative agents of skin and soft tissue infections: case presentations and literature review

ABSTRACT Acid-fast bacteria can be implicated in skin and soft tissue infections. Diagnostic identification can be challenging or not feasible by routine laboratory techniques, especially if there is no access to the Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technology. Here, we present two cases of skin and soft tissue infections caused by two different acid-fast bacteria, Nocardia brasiliensis and Mycobacterium marinum. They both grew on Löwenstein-Jensen medium, Sabouraud agar medium and blood agar medium. Both bacteria appeared acid-fast by Ziehl-Neelsen stain and Gram-positive by Gram stain. The identification was performed by MALDI-TOF MS and gene analysis. N. brasiliensis and nontuberculous mycobacterium M. marinum represent rare pathogens that cause severe skin and soft tissue infections. Failure to identify the causative agent and subsequent inappropriate or inadequate treatment may lead to severe complications or even disseminated disease, especially in immunocompromised individuals.

Gallbladder tuberculosis: A rare case report with review of literature

Gallbladder tuberculosis (TB) is a rare disease, and it might be challenging to distinguish it from gallbladder cancer on clinical history and radiology. It frequently mimics carcinoma in patients who initially appear with a gallbladder mass. Gallbladder TB is only identified after histology of the resected specimen since radiography lacks pathognomic characteristics. Here, we describe a unique case of gallbladder TB that was incidentally identified when a 49-year-old lady was being evaluated for suspected gallbladder cancer. Histology of the gallbladder exhibits necrotizing granulomatous inflammation with the presence of numerous pink-colored, curved, and beaded acid-fast bacilli that were recognized on the Ziehl-Neelsen stain.

Comparative Study of Conventional Ziehl Neelsen and Modified Bleach Method of Ziehl Neelsen with CBNAAT in the Diagnosis of Tuberculous Lymphadenitis

Tuberculosis is one of the major health problems in developing countries like India. Even though lungs are the commonly involved organs in Tuberculosis, extrapulmonary presentations are on the rise nowadays. Among the various types of presentations of extrapulmonary tuberculosis, lymphadenitis is the most common presentation. A spectrum of investigations is available for diagnosis, and molecular methods like CBNAAT and PCR analysis are highly reliable. But their disadvantage is their cost and requirement of trained personnel. FNA is the first line of investigation but is lacking in sensitivity and culture method is time consuming. So, Modified Ziehl Neelsen method with low cost and less time may be considered as an alternative. We wanted to evaluate the sensitivity of the Modified Bleach Ziehl Neelsen method and compare the Modified and Conventional Ziehl Neelsen Method in association with the CBNAAT in the diagnosis of TB lymphadenitis.METHODSDiagnostic validation study was conducted with 30 clinically suspected cases of tuberculosis. FNA was done and aspirated material was spread on 2 slides. Slides were stained with H & E stain and convention Ziehl Neelsen stain. Part of the aspirate material was centrifuged with 5 % sodium hypochlorite. Sediment was smeared on a slide and stained with Ziehl Neelsen stain. Remaining material was subjected to CBNAAT assay.RESULTSCorrelation shows that 9, 6, and 11 out of 30 cases were positive for tuberculosis in Conventional, Modified and CBNAAT methods respectively. Sensitivity was 81.81 %; specificity was 94.74%, positive predictive value was 90% and negative predictive value was 90% for Conventional Ziehl Neelsen Method and 54.55%, 94.73%, 85.71% and 78.26%, respectively for modified bleach method.CONCLUSIONSConventional method is found to be more sensitive than modified method. CBNAAT negative results do not rule out TB. So, they should be correlated with cytology and microbiological studies.

Identification of pulmonary paragonimiasis using Ziehl-Neelsen stain

Pulmonary paragonimiasis and tuberculosis are endemic in Asia, South America, and Africa. However, differential diagnosis among the diseases is difficult because they present with similar clinical symptoms and diagnostic features. Here, we report a case of pulmonary paragonimiasis that was identified using Ziehl-Neelsen stain after initially being assessed for pulmonary tuberculosis. Following anti-Paragonimus chemotherapy, the patient's symptoms, laboratory test results, and lung lesions improved. Thus, the identification of Paragonimus westermani using Ziehl-Neelsen stain can be considered in the diagnosis.

Tuberculosis of the central nervous system in cattle in Paraíba, Brazil / Tuberculose no sistema nervoso central de bovinos na Paraíba

This paper describes six cases of tuberculosis in the central nervous system (CNS) of cattle in the state of Paraíba in northeastern Brazil. We reviewed the autopsy reports of 851 bovine necropsies performed from 2003 to 2016. Seventy-three (8.6%) cattle were diagnosed with tuberculosis and six showed lesions in the CNS. Three cases affected cattle up to two-year-old and other three affected adults. Three cattle presented exclusively nervous signs, two had respiratory signs and weight loss and one did not present any clinical signs. At necropsy, five cattle had thickening of the leptomeninges of the cerebellum, pons, obex, spinal cord and cortex, mainly, in the region near the brain basilar Willis´ circle. Another animal, presented a single focal lesion in the cerebellum. Microscopically we observed moderate to severe granulomatous meningitis and encephalitis. Five cattle presented lesions in the lungs and mediastinal lymph nodes and three of them had disseminated lesions in other organs. In all cattle acid-fast bacilli were observed in the lesions and marked positive for immunohistochemistry with polyclonal antibody anti-Mycobacterium tuberculosis. It is concluded that bovine tuberculosis of central nervous system occurs sporadically in Paraíba, in cattle of different ages, most of them with disseminate lesions in other organs. The location of the lesions suggests that the agent invaded the brain by hematogenous route through the circle of Willis.(AU)

Descrevem-se seis casos de tuberculose no sistema nervoso central (SNC) em bovinos, no semiárido da Paraíba. Foram revisados os laudos de um total de 851 necropsias de bovinos realizadas no período de 2003 a 2016. Destes, 73 (8,6%) foram diagnosticados com tuberculose e seis apresentavam lesões no SNC. Três casos ocorreram em bovinos de até dois anos de idade e três em bovinos adultos. Três bovinos apresentaram exclusivamente sinais nervosos, dois tinham sinais respiratórios e perda de peso e um não apresentava nenhum sinal clínico. Macroscopicamente, em cinco bovinos, havia espessamento das leptomeninges do cerebelo, medula espinhal, ponte, obex, colículos e córtex, principalmente, na região basilar encefálica próxima ao polígono de Willis. Em apenas um bovino houve a presença de tubérculo único no cerebelo. Microscopicamente observou-se moderada a acentuada meningite e encefalite granulomatosa. Cinco bovinos apresentaram lesões pulmonares e nos gânglios mediastínicos e três deles tinham lesões disseminadas em outros órgãos. Em todos os bovinos foram encontrados bacilos ácido-álcool resistentes intralesionais e todos tiveram marcação positiva na técnica de imuno-histoquímica com anticorpo policlonal anti-Mycobacterium tuberculosis. Conclui-se que a tuberculose do sistema nervoso central de bovinos ocorre de forma esporádica na Paraíba, principalmente em bovinos com lesões disseminadas em outros órgãos. Sugere-se que a disseminação do agente ocorre pela via hematógena, possivelmente através do polígono de Willis.(AU)

A comparative study of Ziehl Neelsen, Gabbet‘s and Fluorescent staining methods to detect acid fast bacilli in sputum samples

Tuberculosis remains world's leading cause of death from a single infectious agent. Pulmonary tuberculosis (PTB) is the most common presentation of tuberculosis. Here we compare Ziehl-Neelsen (ZN), Gabbet's and Fluorescent staining (FS) methods to determine efficiency and cost effectiveness of diagnosing pulmonary tuberculosis from sputum samples. Three smears from 148 sputum samples of PTB cases were prepared and stained by the above methods. Of the samples examined, 71.6% and 53.4% and 85.8% cases were detected by Ziehl-Neelsen, Gabbet's and Fluorescence staining respectively. Fluorescent staining was found to be more reliable and cost effective method particularly when dealing with large sample loads than Ziehl-Neelsen and Gabbet's staining technique.

Detection of Cryptosporidium spp. in Diarrheic Immunocompetent Patients in Beni-Suef, Egypt: Insight into Epidemiology and Diagnosis

Cryptosporidium species is an important cause of gastrointestinal infections globally. This study aimed to shed light on its role in diarrheic immunocompetent patients in Beni-Suef, Egypt and to compare three diagnostic methods. Two hundred diarrheic patients, 37±16.8 year old, were enrolled. Stool samples were examined by light microscopy, using modified Ziehl-Neelsen stain (MZN) for Cryptosporidium spp. oocysts. Coproantigens were detected by sandwich ELISA. DNA molecular diagnosis was done by nested PCR. PCR yielded the highest detection rates (21.0%), compared to ELISA (12.5%) and MZN staining method (9.5%). The higher infection rates were in 20–40 year-old group, followed by 40–60 year-old. Association between epidemiologic factors was statistically not significant; positivity and gender, clinical manifestations, residence, source or water, or contact with animals. Cryptosporidiosis is an important enteric parasitic infection in Beni-Suef and PCR remains the gold standard for diagnosis.

Preliminary analysis of the value of Fite staining in the diagnosis of leprosy / 中华皮肤科杂志

Objective To evaluate the diagnostic value of Fite staining in leprosy histopathology.Methods Between 2013 and 2017,13 patients diagnosed with leprosy or suspected leprosy (high suspicion of leprosy based on clinical manifestations and hematoxylin-eosin staining,but negative acid-fast staining) in our department,were enrolled into this study.The histopathological sections were subjected to Fite staining,and the results were compared with those of acid-fast staining,so as to assess the value of Fite staining in the diagnosis of leprosy.Results Six patients with positive acid-fast staining still showed positive Fite staining.Among 7 patients with suspected leprosy and negative acid-fast staining,6 patients showed positive Fite staining with varying numbers of Mycobacterium leprae,and 1 showed negative Fite staining.Conclusion Fite staining can increase the detection rate of Mycobacterium leprae.

Real-time PCR assay for the diagnosis of pleural tuberculosis / Reacción en cadena de la polimerasa en tiempo real para el diagnóstico de tuberculosis pleural

Abstract Introduction: The diagnosis of pleural tuberculosis requires an invasive and time-consuming reference method. Polymerase chain reaction (PCR) is rapid, but validation in pleural tuberculosis is still weak. Objective: To establish the operating characteristics of real-time polymerase chain reaction (RT-PCR) hybridization probes for the diagnosis of pleural tuberculosis. Methods: The validity of the RT-PCR hybridization probes was evaluated compared to a composite reference method by a cross-sectional study at the Hospital Universitario de la Samaritana. 40 adults with lymphocytic pleural effusion were included. Pleural tuberculosis was confirmed (in 9 patients) if the patient had at least one of three tests using the positive reference method: Ziehl-Neelsen or Mycobacterium tuberculosis culture in fluid or pleural tissue, or pleural biopsy with granulomas. Pleural tuberculosis was ruled out (in 31 patients) if all three tests were negative. The operating characteristics of the RT-PCR, using the Mid-P Exact Test, were determined using the OpenEpi 2.3 Software (2009). Results: The RT-PCR hybridization probes showed a sensitivity of 66.7% (95% CI: 33.2%-90.7%) and a specificity of 93.5% (95% CI: 80.3%-98.9%). The PPV was 75.0% (95% CI: 38.8%-95.6%) and a NPV of 90.6% (95% CI: 76.6%-97.6%). Two false positives were found for the test, one with pleural mesothelioma and the other with chronic pleuritis with mesothelial hyperplasia. Conclusions: The RT-PCR hybridization probes had good specificity and acceptable sensitivity, but a negative value cannot rule out pleural tuberculosis.

Resumen Introducción: El diagnóstico de tuberculosis pleural requiere un método de referencia invasivo y demorado. La reacción en cadena de la polimerasa es rápida, pero su validación en tuberculosis pleural aún es débil. Objetivo: Establecer las características operativas de la reacción en cadena de la polimerasa en tiempo real (RT-PCR) sondas de hibridación para el diagnóstico de tuberculosis pleural. Métodos: Se evaluó la validez de la RT-PCR sondas de hibridación comparada con un método de referencia compuesto mediante un estudio transversal en el Hospital Universitario de la Samaritana. Se incluyeron 40 adultos con derrame pleural linfocitario. Tuberculosis pleural fue confirmada (en 9 pacientes) si el paciente tenía mínimo una de tres pruebas del método de referencia positiva: Ziehl-Neelsen o cultivo para Mycobacterium tuberculosis en líquido o tejido pleural, o biopsia pleural con granulomas; se descartó tuberculosis pleural (en 31 pacientes) si las tres pruebas eran negativas. Se determinaron las características operativas de la RT-PCR, mediante la Prueba Mid-P Exact, con el Software OpenEpi 2.3 (2009). Resultados: La RT-PCR sondas de hibridación mostró una sensibilidad del 66.7% (IC 95%: 33.2%-90.7%) y una especificidad del 93.5% (IC 95%: 80.3%-98.9%). El VPP fue de 75.0% (IC 95%: 38.8%-95.6%) y un VPN de 90.6% (IC 95%: 76.6%-97.6%). Se encontraron dos falsos positivos para la prueba, uno con mesotelioma pleural y otro con pleuritis crónica con hiperplasia mesotelial. Conclusiones: La RT-PCR sondas de hibridación tuvo una buena especificidad y una aceptable sensibilidad, pero un valor negativo no puede descartar tuberculosis pleural.

Acid-Fastness of Histoplasma in Surgical Pathology Practice

BACKGROUND: Histoplasmosis (HP) is diagnosed by visualizing intracellular microorganisms in biopsy and/or culture. Periodic-acid Schiff (PAS) and Gomori methenamine silver (GMS) staining methods are routinely used for identification. The acid-fast property of Histoplasma was identified decades ago, but acid-fast staining has not been practiced in current surgical pathology. Awareness of the acid-fast property of Histoplasma, which is due to mycolic acid in the cell wall, is important in distinguishing Histoplasma from other infective microorganisms. Here, we examined acid-fastness in previously diagnosed cases of Histoplasma using the Ziehl-Neelsen (ZN) stain and correlated those findings with other known fungal stains. METHODS: All cases diagnosed as HP were retrieved and reviewed along with ZN staining and other fungal stains. We also stained cases diagnosed with Cryptococcus and Leishmania as controls for comparison. RESULTS: A total of 54 patients ranging in age from 11 to 69 years were examined. The most common sites of infection were the skin, adrenal tissue, and respiratory tract. Of the total 43 tissue samples, 20 (46.5%) stained positive with the ZN stain. In viable cases, a significant proportion of microorganisms were positive while necrotic cases showed only rare ZN-positive yeasts. In comparison to PAS and GMS stains, there was a low burden of ZN-positive yeasts. Cryptococcus showed characteristic ZN staining and all cases of Leishmania were negative. CONCLUSIONS: Although the morphology of fungal organisms is the foundation of identification, surgical pathologists should be aware of the acid-fast property of fungi, particularly when there is the potential for confusion with other infective organisms.

Diseño del kit de tinción Ziehl Neelsen del Instituto nacional de Higiene "Rafael Rangel" / Design Kit Ziehl Neelsen the Instituto Nacional de Higiene "Rafael Rangel"

La tinción de Ziehl Neelsen (ZN), es una técnica de coloración de microorganismos para la identificación de patógenos, como Mycobacterium tuberculosis causante de la tuberculosis, que requiere de tres (03) soluciones: Carbol Fucsina Fenicada (Fucsina Básica), Azul de Metileno al 1% y Solución Decolorante, que se elaboran en la Sección de Reactivos y Colorantes del Instituto Nacional de Higiene "Rafael Rangel" y se emplean en el diagnóstico de tuberculosis. Esta investigación surgió con el propósito de comprobar el tiempo de caducidad y condiciones de almacenamiento de dichos productos y presentarlos en un estuche tipo kit para su distribución, en apoyo a la Red Nacional de Laboratorios de Salud Pública y comercialización con otros entes. Se realizó el ensayo de tres (3) lotes del kit de Ziehl Neelsen; con su respectiva contramuestra que fue evaluada en el análisis final. Se registraron los parámetros físicos de temperatura y humedad relativa bajo condiciones normales de almacenamiento en el laboratorio, con las muestras protegidas de la luz. Se evaluó la funcionalidad por medio de la tinción ZN observada bajo microscopio, de tres (03) muestras con ATCC 700686: M. peregrinum y ATCC 29213: S. aureus por lote; tomando en cuenta el exceso de colorante, y la definición de las coloraciones. Estas evaluaciones se realizaron durante dos (02) años encontrándose como resultado que física y funcionalmente los productos contentivos en el kit se mantenían estables, fijándose un tiempo de caducidad de dos (02) años.

Ziehl Neelsen (ZN) is a staining technique of microorganisms for the identification of pathogens as Mycobacterium tuberculosis, causative of tuberculosis, which requires three (03) solutions: Carbol Fuchsin combined with Phenol (Basic Fuchsin), Methylene Blue 1% and Bleaching solution, which are prepared in Section of Reagents and Coloring of the Instituto Nacional de Higiene "Rafael Rangel" and are used in the diagnosis of tuberculosis. This investigation was made with the purpose of checking the shelf life and storage conditions of these products and present them in a kit type container for distribution in support of the National Network of Public Health Laboratories and marketing with other entities. The analysis was performed in three (3) batches of the Ziehl Neelsen kit; with their respective counter sample that was evaluated in the final analysis. The physical parameters of temperature and relative humidity were recorded in the laboratory under normal storage conditions with samples protected from light. The functionality was evaluated through ZN staining being observed under a microscope three (03) samples with ATCC 700686: M. peregrinum and ATCC 29213: S. aureus by Batch; taking into consideration the excess dye, and the definition of the colors. These evaluations were conducted for two (02) years found as main result that physically and functionally the products in the kit were stable, and can set an expiration time of two years.

Role of fine needle aspiration cytology in diagnosing leprosy: in a tertiary care hospital.

Background: Leprosy, a chronic inflammatory granulomatous disease chiefly involving skin and peripheral nerves and occasionally other organ systems, caused by Mycobacterium leprae. It has tormented the human civilization through time immemorial. Leprosy remains a significant public health problem worldwide, especially in developing countries like India. The diagnosis of leprosy is not always easy because of long incubation period, over dependence of clinical expertise and a lack of rapid and simple diagnostic tool, patients remain undiagnosed for longer time. Fine needle aspiration (FNAC) technique is an inexpensive, rapid and accurate procedure for diagnosis of leprosy. We conducted a prospective study evaluating the ability of fine needle aspiration cytology in diagnosing and classifying leprosy lesions on Ridley-Jopling scale (R-J scale). The aim of this prospective study was to assess the usefulness of fine needle aspiration cytology in early diagnosis of leprosy, to identify specific cytological characteristics of diagnosis and to correlate the cytological smear findings with histopathology and to evaluate merits of relatively non-invasive procedure of FNAC over more invasive procedure - biopsy. Methods: The study is a hospital based prospective study carried out in the Department of Pathology and Department of Skin, Venereal Diseases, Leprosy, N.S.C.B. Medical College & Hospital, Jabalpur (M.P.) September 2010 to September 2013. Patients with new skin lesions were selected for the study. FNAC was performed and aspirates were evaluated for cytology using Hematoxylin and Eosin staining (H&E staining), Ziehl-Neelsen staining (ZN staining) and punch biopsy was collected. Results: Out of 50 cases, clinical and cytological correlation was seen in 88% tuberculoid leprosy, 93.7% of borderline tuberculoid, 33% of borderline lepromatous leprosy and 66% of lepromatous leprosy. While clinical with histopathological correlation revealed 100% specificity in tuberculoid leprosy, 100% in borderline tuberculoid, 66.6% in borderline lepromatous, 83.3% in lepromatous leprosy and 80% in indeterminate leprosy and 100% in histoid leprosy in our study. The overall cytodiagnostic accuracy has been 92% in present study. Conclusion: Our study demonstrates that the combination of FNAC and ZN staining for Acid Fast Bacilli (AFB) can provide a rapid diagnosis in majority of leprosy suspected cases. FNAC is a safe, simple, rapid, less-invasive, OPD procedure for early diagnosis and classification of leprosy cases.

Is universal sample processing methodology better than conventional techniques for detection of tuberculosis.

Background: The identification of infectious cases is a crucial first step for tuberculosis control programmes worldwide. It relies exclusively on the detection of acid‑fast bacilli in sputum by smear microscopy. Therefore, there is an urgent and definite need to improve the sensitivity of smear microscopy. Objective: The USP method was compared with the two most commonly used conventional methods of smear microscopy namely; direct smear microscopy and the microscopy by modified Petroff’s method. Materials and Methods: Two samples from each patient were taken from 197 patients of presumptive tuberculosis. One smear was made for direct Ziehl‑Neelsen staining and two smears were made after processing by two concentration methods i.e., modified Petroff’s and USP solution. LJ media were inoculated for culture after processing by both concentration methods. Results: Among 197 cases 93 were culture positive by either method. Out of 93 culture‑positive sample, 78.5% were direct smear positive, 89% were 4%NaOH smear positive and 96% were USP smear‑positive samples but difference in diagnostic accuracy of USP (96%) and modified Petroff method (93%) is not statistically significant (P > 0.01). Conclusion: The present study evaluated the smear microscopy by USP method with two conventional methods, direct microscopy and microscopy by modified Petroff’s method. The study concludes that although USP method is more sensitive than conventional methods, it is not feasible to include it in diagnosis of early tuberculosis within RNTCP.

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.

Evaluation on a modified Ziehl-Neelsen stainin the diagnosis of tuberculous meningitis / 中国神经精神疾病杂志

Objective Toevaluatea modified Ziehl-Neelsen(Z-N) stain in the diagnosis of tuberculous meningitis. Methods Cerebrospinal fluid specimens from 35 patients were stained by using the modified Ziehl-Neelsen staining. Re-sults The positive rate was 94.29% in 35 patients with tuberculous meningitisand the intracellular acid-fast bacilli was detected in 53.40%of all specimens. One case was stained positive in 15 patients with non-tuberculous meningitis. Con-clusion The modified Ziehl-Neelsen stain not only significantly improves the detection rates of tuberculous meningitisbut alsois able to identify intracellular M.tuberculosisin cerebrospinal fluidspecimen.Thus, the modified Z-N stain can be a convenient tool for diagnosing tuberculous meningitis.

Detection of Mycobacterium tuberculosis Complex in Lung Specimen of Slaughtered Cattle and Goats by a DNA Based multiplex Polymerase Chain Reaction and Ziehl-Neelsen Methods In Jos, Nigeria.

Aims: Determine percentage positive by the Ziehl-Neelsen (ZN) stain; detect 245 base pair fragment of the IS6110 gene for Mycobacterium tuberculosis complex using INS1 and INS2 primers and 500 base pair fragment of the RvD1Rv2031c gene for M. bovis using the JB21 and JB22 primers by a multiplex PCR (M-PCR);compare number of positive samples detected by ZN stain and M–PCR; determine prevalence rates of Mycobacterium tuberculosis complex between the two animal species studied and estimate the rates of detecting agents of tuberculosis using Ziehl-Neelsen (ZN) technique and a Multiplex- Polymerase Chain Reaction (M-PCR) in lung samples of slaughtered cattle and goats in the study area. Place and Duration of Study: Samples were analyzed at the Central Diagnostic Laboratory and Molecular Biology Departments of the National Veterinary Research Institute Vom. This work was carried out between July-October 2010. Study Design: Experimental. Methodology: Our PCR amplified the 245 base pair (bp) fragment which is specific for this group of Mycobacterium while ZN exploited the acid-fast nature of the organisms. We examined one hundred lung samples, of cattle and goats, fifty for each. Results: Out of the lung samples from cattle, 15 (30%) were positive by ZN while 9 (18%) were positive by PCR. Among the ZN negative samples from cattle, one was positive by M-PCR. All the 50 samples from goats were negative by the two diagnostic tools used in this study. Results obtained in this study showed 0% TB infection in goats and 18% in cattle. Conclusion: The study showed a high infection rate of tuberculosis among cattle sampled in the area of study, as such, preventive and curative measures have to be stepped up in controlling the zoonosis.

Improved diagnosis of tuberculosis in lymph node cytology by bleach method for detection of acid fast bacilli in comparison to conventional ziehl neelsen staining method.

Background: Extra pulmonary tuberculosis (TB) is prevalent in developing countries and its diagnosis is often delayed, thus increasing the morbidity and mortality. Bleach method is cost effective, sensitive and safe method for demonstration of Acid fast bacilli (AFB) and is very valuable in diagnosing a case of tuberculous lymphadenitis. This simple procedure would benefit the patients to receive an early and specific treatment. Aims & Objective: The aim of our study was early diagnosis of tuberculosis in lymph node Fine needle aspiration cytology (FNAC) by bleach method for detection of AFB in comparison to conventional Ziehl–Neelsen (ZN) method. Material and Methods: Total 115 cases clinically suspected as tuberculous lymphadenitis in one year duration were included in study. All the aspirates by FNAC were processed for routine cytology, ZN staining and bleach method. The significance of the bleach method over the ZN method was analyzed using the χ2 (chi-square) test. Results: Among the 115 aspirates, 59.13% (68/115) were indicative of TB on cytology, 27.83% (32/115) were positive for AFB on conventional ZN method and the smear positivity increased to 61.74% (71/115) on bleach method. Conclusion: The implementation of the bleach method clearly improves microscopic detection of AFB over ZN method. The bleach method can be easily performed and reduce chances of laboratory acquired infections.