Spatial distribution of Mycobacterium tuberculosis mRNA and secreted antigens in acid-fast negative human antemortem and resected tissue.

BACKGROUND: The ability to detect evidence of Mycobacterium tuberculosis (Mtb) infection within human tissues is critical to the study of Mtb physiology, tropism, and spatial distribution within TB lesions. The capacity of the widely-used Ziehl-Neelsen (ZN) staining method for identifying Mtb acid-fast bacilli (AFB) in tissue is highly variable, which can limit detection of Mtb bacilli for research and diagnostic purposes. Here, we sought to circumvent these limitations via detection of Mtb mRNA and secreted antigens in human tuberculous tissue. METHODS: We adapted RNAscope, an RNA in situ hybridisation (RISH) technique, to detect Mtb mRNA in ante- and postmortem human TB tissues and developed a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B). FINDINGS: We identified Mtb mRNA within intact and disintegrating bacilli as well as extrabacillary mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchiolar epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. INTERPRETATION: RNAscope and dual ZN/immunohistochemistry staining are well-suited for identifying subsets of intact Mtb and/or bacillary remnants in human tissue. RNAscope can identify Mtb mRNA in ZN-negative tissues from patients with TB and may have diagnostic potential in complex TB cases. FUNDING: Wellcome Leap Delta Tissue Program, Wellcome Strategic Core Award, the National Institutes of Health (NIH, USA), the Mary Heersink Institute for Global Health at UAB, the UAB Heersink School of Medicine.

Evaluation of Diagnostic Performance of Line Probe Assay on Smear-Negative Samples of Pulmonary Tuberculosis.

BACKGROUND: This study aims to compare the performance of line probe assay (LPA) on smear-negative samples with that of smear-positive samples for diagnosing pulmonary tuberculosis (PTB) and first-line drug sensitivity testing (FL DST). METHODS: A total of 196 sputum samples including both smear-positive (112) and negative (84) samples of patients suspected of PTB were subjected to LPA for TB detection and FL DST. TB culture followed by MPT 64 Ag was done and conventional FL DST was performed on all culture-positive isolates. Results of LPA on smear-negative were compared with smear-positive samples. RESULTS: The LPA confirmed the diagnosis of PTB in 104/112 smear-positive cases but in only 36/84 smear-negative cases. The assay had 47.36%, 72.72%, and 88.88% sensitivity and 86.96%, 95.23%, and 95.65% specificity in smear-negative cases compared to 89.09%, 95.83%, and 98.07% sensitivity and 100%, 98.36%, and 98.24% specificity in smear-positive cases for detecting Mycobacterium tuberculosis (MTB), rifampicin (RMP) resistance, and isoniazid (INH) resistance, respectively. CONCLUSION: LPA performance was better on smear-positive than smear-negative sputum samples. Further larger studies are needed to justify the use of LPA on smear-negative pulmonary samples for diagnosis.

Nocardia otitidiscaviarum causing pulmonary nocardiosis: a case report and its review of the literature.

Background: Infections caused by Nocardia spp. can occur in immunocompromised as well as immunocompetent individuals. Although nocardiosis is rare, it is being increasingly recognized owing to the rise in occurrence rate over the years. The documentation of pleural involvement in nocardiosis is rare in India. Case: We report a case of pulmonary nocardiosis in an immunocompromised individual caused by Nocardia otitidiscaviarum. Discussion: Pulmonary nocardiosis caused by Nocardia otitidiscaviarum may go unnoticed without clinical suspicion. Correct and timely identification is the key to proper patient management. Conclusion: Coordination between clinicians and microbiologists is necessary for early diagnosis and appropriate management of nocardiosis.

Isolated Unilateral Testicular Tuberculosis in a Young, Immunocompetent Patient: A Case Report.

Testicular or epididymal tuberculosis is a rare form of extrapulmonary tuberculosis. Extrapulmonary tuberculosis of any form is very difficult to diagnose by microscopy because it is usually paucibacillary. Therefore, molecular methods play a major role in the diagnosis of extrapulmonary tuberculosis. We present a rare case of unilateral testicular tuberculosis in a 23-year-old immunocompetent patient with no history of contact with a known tuberculosis case. He presented to us with swelling on his testis for one month and a discharging sinus in the left testis for 15 days, along with an intermittent fever for a week. A pus swab from the discharging sinus of the testis was sent to microbiology, where a cartridge-based nucleic acid amplification test (CBNAAT) was done, which detected Mycobacterium tuberculosis complex (MTBC), but resistance to rifampicin was not detected. A line probe assay was also done on the sample for first-line drugs, and no resistance was detected for rifampicin or isoniazid. The patient was started on first-line drugs in the intensive phase, and after the completion of two months of treatment, the patient's discharge stopped and he showed clinical improvement. Being a young patient, if he had not been diagnosed and treated as early as possible, it could have led to infertility. This again emphasizes the importance of molecular methods for the diagnosis of extrapulmonary tuberculosis.

A controlled evaluation of filter paper use during staining of sputum smears for tuberculosis microscopy.

Background: Some sputum smear microscopy protocols recommend placing filter paper over sputum smears during staining for Mycobacterium tuberculosis (TB) . We found no published evidence assessing whether this is beneficial. We aimed to evaluate the effect of filter paper on sputum smear microscopy results. Methods: Sputum samples were collected from 30 patients with confirmed pulmonary TB and 4 healthy control participants. From each sputum sample, six smears (204 smears in total) were prepared for staining with Ziehl-Neelsen (ZN), auramine or viability staining with fluorescein diacetate (FDA). Half of the slides subjected to each staining protocol were randomly selected to have Whatman grade 3 filter paper placed over the dried smears prior to stain application and removed prior to stain washing. The counts of acid-fast bacilli (AFB) and precipitates per 100 high-power microscopy fields of view, and the proportion of smear that appeared to have been washed away were recorded. Statistical analysis used a linear regression model adjusted by staining technique with a random effects term to correct for between-sample variability.   Results: The inclusion of filter paper in the staining protocol significantly decreased microscopy positivity independent of staining with ZN, auramine or FDA (p=0.01). Consistent with this finding, there were lower smear grades in slides stained using filter paper versus without (p=0.04), and filter paper use reduced AFB counts by 0.28 logarithms (95% confidence intervals, CI=0.018, 0.54, p=0.04) independent of staining technique. In all analyses, auramine was consistently more sensitive with higher AFB counts versus ZN (p=0.001), whereas FDA had lower sensitivity and lower AFB counts (p<0.0001). Filter paper use was not associated with the presence of any precipitate (p=0.5) or the probability of any smear washing away (p=0.6) during the staining process. Conclusions: Filter paper reduced the sensitivity of AFB microscopy and had no detectable beneficial effects so is not recommended.

Diagnostic Performance of Toluidine Blue Stain for Direct Wet Mount Detection of Cryptosporidium Oocysts: Qualitative and Quantitative Comparison to the Modified Ziehl-Neelsen Stain.

(1) Background: The wet mount staining technique is a simple, economical, and rapid method for detecting parasitic stages. The objective of the current study was to evaluate wet mount diagnostic stains for Cryptosporidium infection in human faecal samples and to compare the sensitivity and qualitative performance of toluidine blue (TolB) and modified Ziehl-Neelsen (mZN) stain. (2) Methods: The collection, purification, and molecular amplification of Cryptosporidium oocysts were performed. TolB, malachite green, trypan blue, and crystal violet were evaluated qualitatively to diagnose Cryptosporidium oocysts. The outperforming stain was compared to mZN using a quantitative evaluation and qualitative scoring system. (3) Results: The oocysts of Cryptosporidium parvum were effectively purified and utilized for spiking. TolB was the most effective diagnostic stain among wet mount stains for detecting Cryptosporidium oocysts. TolB outperformed mZN in terms of sensitivity, with 100% versus 79% at a concentration of 104 and 49% versus 23% at a concentration of 102. TolB had the most favourable qualitative stain characteristics against mZN; however, sample freshness and being a temporary stain were crucial considerations. (4) Conclusions: This study emphasizes that TolB, as a routine wet mount technique for screening Cryptosporidium infection, will provide a more sensitive and faster method than mZN staining.

The Use of Xpert MTB/RIF Ultra Testing for Early Diagnosis of Tuberculosis: A Retrospective Study from a Single-Center Database.

Tuberculosis (TB) is a multisystemic contagious disease produced by Mycobacterium tuberculosis complex bacteria (MTBC), with a prevalence of 65:100,000 inhabitants in Romania (six times higher than the European average). The diagnosis usually relies on the detection of MTBC in culture. Although this is a sensitive method of detection and remains the "gold standard", the results are obtained after several weeks. Nucleic acid amplification tests (NAATs), being a quick and sensitive method, represent progress in the diagnosis of TB. The aim of this study is to assess the assumption that NAAT using Xpert MTB/RIF is an efficient method of TB diagnosis and has the capacity to reduce false-positive results. Pathological samples from 862 patients with TB suspicion were tested using microscopic examination, molecular testing and bacterial culture. The results show that the Xpert MTB/RIF Ultra test has a sensitivity of 95% and a specificity of 96.4% compared with 54.8% sensitivity and 99.5% specificity for Ziehl-Neelsen stain microscopy, and an average of 30 days gained in the diagnosis of TB compared with bacterial culture. The implementation of molecular testing in TB laboratories leads to an important increase in early diagnostics of the disease and the prompter isolation and treatment of infected patients.

Pathological and molecular identification of Mycobacterium avium infection in a loft of domestic pigeons (Columba livia var. domestica) from India.

Mycobacterium avium is a zoonotic pathogen associated with a wide range of pulmonary and extrapulmonary manifestations in a range of host species like humans, animals, and birds. The disease is more common in the avian population, and opportunistic infections have been reported in immune-compromised or debilitated animals and humans. This study reports the pathological and molecular identification of Mycobacterium avium causing avian mycobacteriosis in a loft of domestic pigeons (Columba livia var. domestica). Out of 30 pigeons aged 2-3 years, ten adult racing pigeons revealed a severe chronic and debilitating disease followed by death. The clinical signs included chronic emaciation, dullness, ruffled feathers, lameness, and greenish, watery diarrhea. Post-mortem examination of birds revealed multifocal gray- to yellow-colored raised nodules in the liver parenchyma, spleen, lungs, intestines, bone marrow, and joints. Avian mycobacteriosis was suspected based on the tissue impression smears stained by Ziehl-Neelsen staining. Histopathological examination also revealed multifocal granulomatous lesions in affected organs, which is characteristic of avian mycobacteriosis. The PCR analysis based on 16S rRNA, IS1245, and IS901 regions suggested the presence of Mycobacterium avium infection belonging to either subspecies avium or sylvaticum. This is the first detailed report of avian mycobacteriosis in pigeons from India, warranting a strict surveillance program to identify the carrier status of these microorganisms in the pigeons, which may prove a fatal zoonotic infection in humans.

Comparison of Ziehl Neelsen microscopy and geneXpert for the diagnosis of pulmonary tuberculosis among pulmonary tuberculosis-suspected patients in northwest Ethiopia.

In low-income countries, the diagnosis of pulmonary tuberculosis (PTB) is most frequently achieved using Ziehl Neelsen (ZN) microscopy rather than the GeneXpert system. The performance of the former has not been evaluated against the latter in Ethiopia. A total of 180 PTB-suspected patients were enrolled in our study. Sputum specimens were tested both by ZN microscopy and geneXpert. The sensitivity, specificity, positive predictive value, and negative predictive value of ZN microscopy were 75%, 99.4%, 92.3%, and 97.6%, respectively. The Kappa value of agreement between the two diagnosis methods was 0.80. We concluded that there was a good agreement between ZN microscopy and the reference Xpert assay, which suggests that ZN microscopy is still a good diagnostic method in health care facilities that do not have the latter test.

Comparison of conventional diagnostic methods with molecular method for the diagnosis of pulmonary tuberculosis.

BACKGROUND: Tuberculosis remains one of the deadliest communicable diseases. Prompt diagnosis of active tuberculosis cases facilitates timely therapeutic intervention and minimizes the community transmission. Although conventional microscopy has low sensitivity, still it remains the corner stone for the diagnosis of pulmonary tuberculosis in high burden countries like India. On the other hand, Nucleic acid amplification techniques due to their rapidity and sensitivity, not only help in early diagnosis and management of tuberculosis but also curtail the transmission of the disease. This study therefore was aimed at assessing the diagnostic performance of Microscopy by Ziehl Neelsen (ZN) and Auramine Staining (AO) with Gene Xpert/CBNAAT (Cartridge based nucleic acid amplification test) in the diagnosis of Pulmonary Tuberculosis. METHODS: A prospective comparative study was done on the sputum samples of 1583 adult patients from November 2018 to May 2020 suspected of having pulmonary tuberculosis as per NTEP criteria visiting the Designated Microscopic Centre of SGT Medical College, Budhera, Gurugram. Each sample was subjected to ZN staining, AO staining, and was run on CBNAAT as per National Tuberculosis Elimination Program (NTEP) guidelines. The sensitivity, specificity, PPV and NPV and Area under the curve of ZN microscopy and Fluorescent Microscopy were calculated taking CBNAAT as reference in absence of culture. RESULTS: Out of the 1583 samples studied, 145 (9.15%) and 197 (12.44%) were positive by ZN and AO staining methods respectively. By CBNAAT 246 (15.54%) samples were positive for M. tuberculosis. AO was also able to detect more pauci-bacillary cases than ZN. While CBNAAT detected M. tuberculosis in 49 sputum samples which were missed by both methods of microscopy. On the other hand there were 9 samples which were positive for AFB by both the smear microscopy techniques but M. tuberculosis was not detected by CBNAAT, these were considered as Non-Tuberculous Mycobacteria. Seventeen samples were resistant to rifampicin. CONCLUSION: Auramine Staining technique is more sensitive and less time consuming for the diagnosis of pulmonary tuberculosis as compared to the conventional ZN Staining. CBNAAT can be a useful tool for early diagnosis of patients with high clinical suspicion of pulmonary tuberculosis and detecting rifampicin resistance.

Cryptosporidiosis in Yellow Nail Syndrome - A Rare Case Report.

BACKGROUND: Cryptosporidium species infection causes malabsorption and severe diarrhea in immunocompromised hosts. Association of Yellow Nail Syndrome (YNS) and Cryptosporidiosis is rare and has not been reported till date. Immunity can also be affected in this case of YNS is associated with autoimmune disorders. CASE PRESENTATION: Here, we describe a case of persistent diarrhea in an 8 month old YNS patient. Modified Ziehl-Neelsen staining and Saffranine-Methylene blue revealed oocyts of Cryptosporidium species. Following appropriate treatment, the patient's symptoms improved and the patient was discharged in a hemodynamically stable condition. DISCUSSION: Cryptosporidiosis is a significant cause of morbidity and mortality in immunocompromised patients. YNS per se as well as treatment including steroids leads to immunosuppression in individuals making them susceptible host for opportunistic infections like Cryptosporidiosis. CONCLUSION: Clinicians should be aware of the condition and screen for Cryptosporidiosis in any immunocompromised patients with diarrheal symptoms, as parasitic infection like this are opportunistic in them.

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.

Investigação da presença do bacilo Mycobacterium tuberculosis em amostras sugestivas de tuberculose oral / Investigation of the presence of Mycobacterium tuberculosis bacillus in samples suggestive of oral tuberculosis

A Tuberculose (TB) é um considerável problema de saúde pública mundial. Em 2021, de acordo com a Organização Mundial de Saúde (OMS) estimou-se que, no mundo, cerca de 10,6 milhões de pessoas desenvolveram TB e 1,4 milhão morreu devido à doença. Com isso, tornou-se a principal causa de morte por infecção em todo o mundo e uma das dez principais causas de morte em geral. A TB tem o pulmão como o principal sítio de acometimento, sendo denominada de TB Pulmonar (TBP). Porém pode ser diagnosticada em muitos órgãos do corpo de maneira Extrapulmonar (TBEP), sendo o linfonodo o local mais comum. Porém, o envolvimento pleural, neurológico, sinovial, pericárdico, abdominal, geniturinário e oral tem sido descrito, o que mostra a potencial capacidade de disseminação do Mycobacterium tuberculosis (MTB). A detecção do Bacilos Álcool-Ácido Resistentes (BAAR), geralmente ocorre pela observação das características microscópicas da morfologia dos tecidos, presença de granulomas com necrose caseosa, histiócitos epitelióides e células gigantes do tipo Langhans, associada à coloração para BAAR, pela técnica de Ziehl Neelsen (ZN). Ademais, investigação por imuno-histoquímica (IHQ), testes de amplificação de ácido nucleico pela Reação em Cadeia da Polimerase Hemianinhada (nested-PCR) e pelo sistema de detecção automatizado GeneXpert® MTB/RIF são métodos aplicados para o diagnóstico da infecção. Com isso, este estudo teve como objetivo investigar a presença do bacilo Mycobacterium tuberculosis em amostras orais em parafina que continham granulomas com necrose caseosa. Ao todo, como critério de inclusão, foram selecionadas biópsias que apresentaram granulomas com necrose caseosa, sugerindo o diagnóstico de TB. Foram excluídas aquelas que após a revisão das fichas e histológicas, não apresentavam os granulomas exibindo necrose caseosa e aquelas que foram de biópsias intraósseas. O M. tuberculosis foi procurado por meio da coloração de ZN, IHC, nested-PCR e ensaios GeneXpert® MTB/RIF. Foram então selecionadas nove amostras com granulomas com necrose caseosa. Houve predominância de indivíduos do sexo masculino (2,5:1), com idade média de 50 anos (±23,08; 19-89), sendo a língua o local anatômico mais afetado (n=4). O bacilo não foi identificado pela técnica de ZN em nenhuma amostra, e a coloração por IHC mostrou um padrão granular grosseiro, sugerindo M. tuberculosis, em três delas. Nested-PCR e os ensaios GeneXpert® MTB/RIF foram positivos em duas e três das amostras, respectivamente. Conclui-se que testes moleculares e IHC podem ser métodos auxiliares úteis para casos suspeitos de tuberculose.

Tuberculosis (TB) is a significant global public health issue. In 2021, according to the World Health Organization (WHO), it was estimated that approximately 10.6 million people developed TB worldwide, and 1.4 million died from the disease. Consequently, it became the leading cause of death due to infection worldwide and one of the top ten overall causes of death. TB primarily affects the lungs and is referred to as Pulmonary TB (PTB). However, it can be diagnosed in various organs of the body as Extrapulmonary TB (EPTB), with lymph nodes being the most common site of involvement. Moreover, pleural, neurological, synovial, pericardial, abdominal, genitourinary, and oral involvement have been described, demonstrating the potential for Mycobacterium tuberculosis (MTB) dissemination. The detection of Acid-Fast Bacilli (AFB) typically involves the observation of microscopic tissue characteristics, the presence of granulomas with caseous necrosis, epithelioid histiocytes, and Langhans giant cells, along with AFB staining using the Ziehl-Neelsen (ZN) technique. Furthermore, immunohistochemistry (IHC), nucleic acid amplification tests by Nested Polymerase Chain Reaction (nested-PCR), and the automated detection system GeneXpert® MTB/RIF are methods employed for diagnosing the infection. Therefore, the aim of this study was to investigate the presence of Mycobacterium tuberculosis in paraffin-embedded oral samples containing granulomas with caseous necrosis. Inclusion criteria were based on the selection of biopsies displaying granulomas with caseous necrosis, suggesting a diagnosis of TB. Biopsies without these features upon review of records and histological findings, as well as intraosseous biopsies, were excluded. M. tuberculosis was sought using ZN staining, IHC, nested-PCR, and GeneXpert® MTB/RIF assays. Nine samples with granulomas and caseous necrosis were selected. The majority of individuals were male (2.5:1 ratio), with an average age of 50 years (±23.08; range 19-89), and the tongue was the most affected anatomical site (n=4). AFB was not identified by the ZN technique in any of the samples, and IHC staining exhibited a coarse granular pattern, suggestive of M. tuberculosis, in three of them. Nested-PCR and GeneXpert® MTB/RIF assays yielded positive results in two and three of the samples, respectively. In conclusion, molecular tests and IHC can be valuable auxiliary methods for suspected cases of 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

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.

Fluorescein Diacetate Vital staining for detecting viability of acid-fast bacilli in sputum of pulmonary tuberculosis patients.

OBJECTIVE: To compare the performance of the Fluorescein diacetate (FDA) vital staining method with Ziehl-Neelsen staining method in detecting the viability of acid-fast bacilli using MGIT culture as "reference standard". METHODS: This was a single centre prospective observational study conducted from October 2015 to November 2016. Microbiologically confirmed ZN-Smear positive (3+) sputum specimens were obtained from 30 pulmonary tuberculosis patients taking anti-tuberculosis treatment at DOTS centre of NITRD, New Delhi. Patients were made available to collect the first baseline sputum sample before commencing treatment, and an early morning sputum sample was collected as per RNTCP guidelines. After starting treatment, sputum specimens were collected weekly in the first month and thereafter twice-weekly until 18th week. All sputum specimens from patients receiving anti-tuberculosis treatment were examined using Ziehl-Neelsen (ZN) smear microscopy, FDA vital staining, and MGIT culture. RESULT: Out of 360 follow up sputum specimens collected from 30 adult microbiologically confirmed ZN- Smear (3+) pulmonary tuberculosis patients, 146 were ZN-positive and 130 FDA-positive. Of 130 FDA-positive sputum samples, mycobacteria tuberculosis (MTB) growth was found in 116 sputum samples, of which 116 sputum specimens were positive for FDA. Additionally, 14 culture-negative specimens were FDA positive. No FDA-negative sputum samples were positive for MGIT culture. Among ZN positive specimens, FDA had 100% sensitivity and 85.3% specificity with an accuracy of 96.58% for the detection of viable mycobacteria. Among ZN negative sputum specimens, FDA had comparatively high specificity (95.7%). Using positive MGIT culture as a reference for viability, negative predictive value (NPV) and positive predictive value (PPV) from FDA vital staining method were found to be 100 and 89% respectively. CONCLUSION: FDA staining is a simple and rapid tool for identifying viable MTB bacilli. Because of its excellent NPV and encouraging specificity, FDA staining is useful to identify patients with non-viable bacilli (FDA negative) among retreatment cases at diagnosis and patients on anti-tuberculosis treatment for both drug-sensitive and drug-resistant tuberculosis for follow up for the response of treatment.

Application of Ziehl Neelsen Staining Method for Taenia spp. Eggs Differentiation.

Background: Three of Taenia species, named Taenia saginata, T. solium and T. asiatica can be found in Indonesia, but only T. solium can lead to neurocysticercosis. The morphology of those 3 Taenia spp. egg is indistinguishable by standard parasitology procedure. We aimed to use Ziehl Neelsen staining for differentiation of eggs of T. saginata and T. solium. Methods: As many as 40 containers of stool samples from the positive helminthiasis patients in Jakarta, Indonesia were collected during the year 2018. From each container, 10 slides prepared for staining with Kato-Katz technique as the preliminary examination. From those stool samples with positive taeniasis, we then once again made 10 slides/container for Ziehl Neelsen staining. Results: The first 400 slides stained with Kato-Katz technique as preliminary test were all positive for Taenia spp. The second 400 slides, we got 244 slides that gave good results as we could distinguished between the eggs of T. saginata and T. solium, meanwhile the remainder 156 slides gave unconfirmed results. From those 244 slides, 154 slides showed T. saginata eggs with magenta colored and 90 slides showed T. solium eggs with blue/purple colored. The eggs of T. solium slightly smaller if compared to Taenia saginata and had round shape, meanwhile T. saginata eggs were oval in shape. Conclusion: Ziehl Neelsen staining method can be used as an alternative parasitological method to differentiate the eggs of T. saginata and T. solium.

Effect of 80% ethanol or 10% formalin fixation, freezing at -20 °C and staining on Myxobolus (Myxosporea) spores to be deposited in parasitological collections.

The preparation of myxosporeans for the description of myxospores and their preservation as type material in parasitological collections show great variations. Most frequently, formalin and ethanol are used for fixation and Giemsa solution for staining spores. In this work, authors studied the effect of 80% ethanol and 10% formalin fixation, freezing at -20 °C and staining on the size and transparency of two Myxobolus species of cyprinid fishes, M. bramae and M. bliccae spore, and recommended a new method for the deposition of type material to parasitological collections in museums. The studies have commended that fresh spores from mature plasmodia are the best material for measuring the size and studying the inner structures, the number of polar tubules in polar capsules and the morphological characters of the intercapsular appendix. The obtained quantitative data suggest that cryo- and chemical preservation do not have a notable negative effect on spores compared to fresh samples but they decrease the transparency of spores. Staining the spores with Ziehl-Neelsen has proved to be a useful method for studying the fine structure without size reduction, while Giemsa staining induced a shrinkage of spores so it seems to be not ideal for description of a new species. When treating spores of Myxobolus spp. with Lugol's solution, iodinophilous vacuoles in the sporoplasm were not recognised but visualisation of the coils of polar tubules was enhanced. As a type material for newly described species, authors suggest phototypes and spores fixed in 80% ethanol to be deposited into collections, as this preservation method is suitable for subsequent research, such as re-measurements and molecular analysis.

The Re-Identification of Previously Unidentifiable Clinical Non-Tuberculous Mycobacterial Isolates Shows Great Species Diversity and the Presence of Other Acid-Fast Genera.

Non-tuberculous mycobacteria that cannot be identified at the species level represent a challenge for clinical laboratories, as proper species assignment is key to implementing successful treatments or epidemiological studies. We re-identified forty-eight isolates of Ziehl-Neelsen (ZN)-staining-positive "acid-fast bacilli" (AFB), which were isolated in a clinical laboratory and previously identified as Mycobacterium species but were unidentifiable at the species level with the hsp65 PCR restriction fragment length polymorphism analysis (PRA). As most isolates also could not be identified confidently via 16S, hsp65, or rpoB DNA sequencing and a nBLAST search analysis, we employed a phylogenetic method for their identification using the sequences of the 16S rDNA, which resulted in the identification of most AFB and a Mycobacterium species diversity not found before in our laboratory. Most were rare species with only a few clinical reports. Moreover, although selected with the ZN staining as AFB, not all isolates belonged to the genus Mycobacterium, and we report for the first time in Latin America the isolation of Nocardia puris, Tsukamurella pulmosis, and Gordonia sputi from sputum samples of symptomatic patients. We conclude that ZN staining does not differentiate between the genus Mycobacterium and other genera of AFB. Moreover, there is a need for a simple and more accurate tree-based identification method for mycobacterial species. For this purpose, and in development in our lab, is a web-based identification system using a phylogenetic analysis (including all AFB genera) based on 16S rDNA sequences (and in the future multigene datasets) and the closest relatives.