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Objective: To identify the clinical, laboratory, and radiological markers that could predict a positive GeneXpert result in patients suspected of tuberculous meningitis. Methods: In this prospective, observational study, patients with tuberculous meningitis were systematically evaluated. Various clinical, laboratory [including cerebrospinal fluid (CSF) microscopy, culture, and GeneXpert], and neuroimaging factors were examined. All participants were administered anti-tuberculous treatment and corticosteroids. A six-month follow-up was done to evaluate the outcome. Results: Of the 116 patients studied, 54 (46.6%) tested positive for GeneXpert, while 62 (53.4%) were negative. Third cranial nerve involvement (OR 3.71, 95% CI 1.052-13.09, P=0.04) and the presence of basal exudates on neuroimaging (OR 5.22, 95% CI 2.03-13.42, P=0.001) emerged as independent predictive factors for a positive GeneXpert result. A positive CSF GeneXpert result (P=0.002) and drug resistance (P=0.004) were significantly linked to adverse outcomes. Additionally, a high score on the Medical Research Council stage III (OR 5.64, 95% CI 1.18-26.87, P=0.03) and elevated CSF cell counts (OR 1.002, 95% CI 1.00-1.001, P=0.03) were identified as independent predictors of poor prognosis. Conclusions: Third cranial nerve involvement and the presence of basal exudates were significant indicators of a positive GeneXpert result. MRC stage III disability and elevated CSF cell counts predicted poor outcomes.

Objective: To identify the clinical, laboratory, and radiological markers that could predict a positive GeneXpert result in patients suspected of tuberculous meningitis. Methods: In this prospective, observational study, patients with tuberculous meningitis were systematically evaluated. Various clinical, laboratory [including cerebrospinal fluid (CSF) microscopy, culture, and GeneXpert], and neuroimaging factors were examined. All participants were administered anti-tuberculous treatment and corticosteroids. A six-month follow-up was done to evaluate the outcome. Results: Of the 116 patients studied, 54 (46.6%) tested positive for GeneXpert, while 62 (53.4%) were negative. Third cranial nerve involvement (OR 3.71, 95% CI 1.052-13.09, P =0.04) and the presence of basal exudates on neuroimaging (OR 5.22, 95% CI 2.03-13.42, P =0.001) emerged as independent predictive factors for a positive GeneXpert result. A positive CSF GeneXpert result (P =0.002) and drug resistance (P =0.004) were significantly linked to adverse outcomes. Additionally, a high score on the Medical Research Council stage III (OR 5.64, 95% CI 1.18-26.87, P =0.03) and elevated CSF cell counts (OR 1.002, 95% CI 1.00-1.001, P =0.03) were identified as independent predictors of poor prognosis. Conclusions: Third cranial nerve involvement and the presence of basal exudates were significant indicators of a positive GeneXpert result. MRC stage III disability and elevated CSF cell counts predicted poor outcomes. [ABSTRACT FROM AUTHOR]

Background Culture of Mycobacterium tuberculosis (MTB) using egg-based solid media like Lowenstein Jensen (LJ) is the gold standard for tuberculosis diagnosis but requires extended incubation time. Rapid diagnostic tests, such as Ziehl-Neelsen (ZN) staining and the MPT64 antigen rapid test, are essential for early detection. Objective This study aimed to evaluate the effectiveness of ZN staining and the MPT64 rapid test in detecting MTB and Mycobacterium other than tuberculosis (MOTT) during different culture times. Methods Using a cross-sectional design, 110 culture-positive samples were analyzed from Hasan Sadikin Hospital Bandung over two months. Specimens were cultured on LJ media for eight weeks, with weekly observation of colony growth. ZN staining and MPT64 tests were performed on growing colonies. Results Less than four weeks culture, 61 samples (55.5%) were culture-negative, 45 (40.9%) were positive for MOTT, and the remainder were contaminated. In more than four weeks culture, 48 samples (43.6%) were positive for MTB, 45 samples (40.9%) were positive for MOTT, and 13 (11.8%) were culture-negative, and the remaining were contaminated. ZN-positive and MPT64-negative results indicated MOTT in less than four weeks culture, while ZN-positive and MPT64-positive results indicated MTB in more than four weeks culture. Conclusion While ZN staining was positive for both MTB and MOTT colonies, the MPT64 rapid antigen test was specific for MTB, supporting its use in confirming MTB detection alongside culture methods.

Sputum microscopy is the most important tool for the detection of Mycobacterium tuberculosis in peripheral laboratories. Sodium hypochlorite concentration technique prior to sputum microscopy may improve the detection of AFB bacilli over direct Ziehl Neelsen’s staining method. A prospective study was conducted at Kasturba Medical College, Manipal. Consecutive sputum samples were collected for 3 months. ZN stained smears were made directly of fresh specimens and of specimens that were processed with 3.5% sodium hypochlorite (NaOCl), from each patient. Then, ZN stained smears were observed under light microscopy. A total of 239 samples were collected. The yield of positive smears with ZN staining after NaOCl centrifugation was higher than that with microscopy after direct ZN stain. The percentage increase yield of ZN staining after NaOCl centrifugation over direct ZN staining was 3.3% (8/239). Bleach centrifugation can help in increasing the yield of positivity in sputum smear.

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.

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. [ABSTRACT FROM AUTHOR]

Sputum microscopy is the most important tool for the detection of Mycobacterium tuberculosis in peripheral laboratories. Sodium hypochlorite concentration technique prior to sputum microscopy may improve the detection of AFB bacilli over direct Ziehl Neelsen’s staining method. A prospective study was conducted at Kasturba Medical College, Manipal. Consecutive sputum samples were collected for 3 months. ZN stained smears were made directly of fresh specimens and of specimens that were processed with 3.5% sodium hypochlorite (NaOCl), from each patient. Then, ZN stained smears were observed under light microscopy. A total of 239 samples were collected. The yield of positive smears with ZN staining after NaOCl centrifugation was higher than that with microscopy after direct ZN stain. The percentage increase yield of ZN staining after NaOCl centrifugation over direct ZN staining was 3.3% (8/239). Bleach centrifugation can help in increasing the yield of positivity in sputum smear. [ABSTRACT FROM AUTHOR]

Daisuke Fukushi,1 Keigo Murakami,2 Yuji Watanabe,3 Norihiko Sugimoto,4 Hirotsugu Uehara,4 Masafumi Seki5 1Division of Gastroenterology, Tohoku Medical and Pharmaceutical University Hospital, Sendai City, Miyagi, Japan; 2Division of Pathology, Tohoku Medical and Pharmaceutical University Hospital, Sendai City, Miyagi, Japan; 3Laboratory for Clinical Microbiology, Tohoku Medical and Pharmaceutical University Hospital, Sendai City, Miyagi, Japan; 4Fuso Pharmaceutical Industry, Osaka City, Osaka, Japan; 5Division of Infectious Diseases and Infection Control, Tohoku Medical and Pharmaceutical University Hospital, Sendai City, Miyagi, JapanCorrespondence: Masafumi Seki, Division of Infectious Diseases and Infection Control, Tohoku Medical and Pharmaceutical University, 1-15-1 Fukumuro, Miyagino-ku, Sendai City, Miyagi, 983-8536, Japan, Tel +81-22-983-1221, Fax +81-22-290-8959, Email m-seki@tohoku-mpu.ac.jp; seki@hosp.tohoku-mpu.ac.jpBackground: Detection of Mycobacterium avium complex (MAC) in tissue is essential for the diagnosis of MAC infections when the Mycobacterium is not isolated from sputum. However, detection of MAC in paraffin-embedded sections has not been established.Methods: We encountered two patients with suspected MAC infections after surgery: patient 1 had a pulmonary nodule that was initially suspected to be lung cancer and was excised under video-assisted thoracoscopic surgery (VATS). Patient 2, who was under treatment with steroids and anti-IL-6 inhibitors for rheumatoid arthritis, was suspected to have disseminated ileocecal cancer with metastasis to the lung and skin. In both cases, we postoperatively detected MAC genes in paraffin-embedded tissue sections using the novel mycobacterial nucleic acid identification test, ie tissue-direct polymerase chain reaction (tdPCR)-based nucleic acid lateral flow immunoassay (NALFIA). Both patients showed granulomatous lesions with hematoxylin-eosin staining, and mycobacteria by Ziehl–Neelsen staining in tissue sections from the lung and skin, respectively, although MAC were not isolated from the sections. MAC genes were finally detected by tdPCR-NALFIA in both cases.Conclusion: Although Ziehl–Neelsen staining and culture tests are the gold standard in identifying causative mycobacteria, the rapid results of tdPCR-NALFIA performed simultaneously with sputum and/or tissue culture may make it an important auxiliary diagnostic tool for identifying mycobacterial infection, leading to improvement in the management of MAC patients.Keywords: Mycobacterium avium complex, MAC, Ziehl–Neelsen staining, rapid diagnosis

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. [Display omitted] • The effect of fixation, freezing at −20 C and staining on myxospores was studied. • Model materials were myxospores of Myxobolus bramae and M. bliccae. • No changes in size were observed on frozen at −20 C and 80% ethanol- or 10% formalin-fixed spores. • A shrinkage of spores was noticed when staining spores with Giemsa solution. • Phototypes and 80% ethanol-fixed spores are recommended for collections as type material. [ABSTRACT FROM AUTHOR]

Aim: This study aimed to determine the prevalence of microbiologically confirmed female genital tuberculosis (FGTB) infection in patients attending a tertiary care hospital in North India., Materials and Methods: A total of 623 endometrial biopsy samples were processed in the mycobacteriology laboratory from the outpatient and inpatient gynecology departments between May 2022 and February 2024. Ziehl-Neelsen (ZN) smear was performed on all samples. Each sample was subsequently subjected to cartridge-based nucleic acid amplification test (CBNAAT) Xpert Mycobacterium tuberculosis (MTB)/rifampicin (RIF) Ultra (Cepheid, Sunnyvale, California), and LJ culture irrespective of the ZN smear result and demographic details of the patients were recorded simultaneously., Results: Of the 623 endometrial biopsy samples tested, 17 (2.73%) samples were positive for MTB by CBNAAT Ultra. The disease was most commonly observed in the reproductive age group between 21 and 35 years. ZN smear and culture were negative for all the samples received for endometrial TB. Of these 17 positive samples, one (5.9 %) was MTB detected (medium), four (23.5 %) (low), two (11.8 %) (very low), and 10 (58.8 %) (trace). RIF resistance was detected in one (5.9 %) of these 17 samples. However, the 10 samples that were detected as MTB trace and RIF indeterminate ideally need to be processed further by culture or by line probe assay to determine the drug resistance., Conclusion: Genital TB is characterized by a low bacterial load. Therefore, a highly sensitive and specific molecular test is essential for confirming the diagnosis microbiologically. Our study found that Xpert MTB/RIF Ultra is particularly valuable because it detected 58.8% of cases as "TRACE," which might be missed by other testing methods. Early diagnosis using this test can significantly impact patient management, especially in cases related to infertility. This study effectively highlights the role of GeneXpert Ultra in documenting extra-pulmonary TB, including genital TB., Competing Interests: Human subjects: Consent for treatment and open access publication was obtained or waived by all participants in this study. Institutional Ethics Committee of ESIC Medical College & Hospital (ESIC MC&H), Faridabad issued approval 34 X/11/13/2023- IEC/DHR/92. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Gunasekaran et al.)

Background: Extrapulmonary tuberculosis (EPTB) is still a major health problem in developing countries. Diagnosis of EPTB is difficult due to paucity of acid-fast bacilli in clinical specimens. Objectives: To study the diagnostic role of cartridge-based nucleic acid amplification test (CBNAAT) and rifampicin resistance in EPTB on fine-needle aspiration samples. Materials and Methods: It is a prospective study comprising 79 patients of clinically suspected EPTB cases for 1 year duration. All patients underwent fine-needle aspiration cytology (FNAC) with Ziehl–Neelsen (ZN) staining followed by CBNAAT for confirmation of tuberculosis (TB) and to detect rifampicin resistance. Data have been collected and analyzed regarding sensitivity, specificity, positive predictive value, and negative predictive value of FNAC in comparison with CBNAAT. Results: Out of 79 patients, 47 (59.49%) patients were male and cervical lymph node was the most common site of aspiration (42, 52.16%). Granuloma with necrosis (38, 48.10%) followed by necrosis only (14, 17.72%) were the common findings on FNAC. Out of 79 suspected EPTB patients, CBNAAT confirmed 62 patients as having TB. Specificity of FNAC with ZN staining was 100.0%; however, sensitivity was low (43.55%) compared to CBNAAT. Only three cases were detected as rifampicin resistant. Conclusion: Compared to FNAC, CBNAAT has very good sensitivity and specificity in the diagnosis of EPTB. It has also as added advantage of detecting rifampicin resistance at the same time. CBNAAT should be used for diagnosis of EPTB wherever the facility is available.

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Background: Extrapulmonary tuberculosis (EPTB) is still a major health problem in developing countries. Diagnosis of EPTB is difficult due to paucity of acid-fast bacilli in clinical specimens. Objectives: To study the diagnostic role of cartridge-based nucleic acid amplification test (CBNAAT) and rifampicin resistance in EPTB on fine-needle aspiration samples. Materials and Methods: It is a prospective study comprising 79 patients of clinically suspected EPTB cases for 1 year duration. All patients underwent fine-needle aspiration cytology (FNAC) with Ziehl–Neelsen (ZN) staining followed by CBNAAT for confirmation of tuberculosis (TB) and to detect rifampicin resistance. Data have been collected and analyzed regarding sensitivity, specificity, positive predictive value, and negative predictive value of FNAC in comparison with CBNAAT. Results: Out of 79 patients, 47 (59.49%) patients were male and cervical lymph node was the most common site of aspiration (42, 52.16%). Granuloma with necrosis (38, 48.10%) followed by necrosis only (14, 17.72%) were the common findings on FNAC. Out of 79 suspected EPTB patients, CBNAAT confirmed 62 patients as having TB. Specificity of FNAC with ZN staining was 100.0%; however, sensitivity was low (43.55%) compared to CBNAAT. Only three cases were detected as rifampicin resistant. Conclusion: Compared to FNAC, CBNAAT has very good sensitivity and specificity in the diagnosis of EPTB. It has also as added advantage of detecting rifampicin resistance at the same time. CBNAAT should be used for diagnosis of EPTB wherever the facility is available. [ABSTRACT FROM AUTHOR]

Background Tuberculosis (TB) remains a significant public health challenge, particularly in developing countries, where delayed diagnosis contributes to ongoing transmission. Ziehl-Neelsen (ZN) smear microscopy, commonly used for TB diagnosis, has limitations in sensitivity, especially in cases of extrapulmonary tuberculosis (ETB). The GeneXpert Mycobacterium tuberculosis (MTB)/rifampicin (RIF) assay, a molecular diagnostic tool, offers rapid and accurate detection of MTB and RIF resistance. This study aimed to compare the diagnostic efficacy of GeneXpert MTB/RIF with ZN staining in detecting pulmonary tuberculosis (PTB) and ETB. Methods A prospective study was conducted over two years (April 2022 to April 2024) at a tertiary care teaching hospital in India. A total of 319 clinical samples from patients with suspected PTB and ETB were analyzed. Samples underwent ZN staining and GeneXpert MTB/RIF assay. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated using mycobacterial culture as the gold standard. The chi-square test was employed to compare diagnostic accuracy, with a p-value of <0.05 considered statistically significant. Results Of the 319 samples, ZN staining was positive in 18.2% of cases, while GeneXpert was positive in 21.6%. GeneXpert demonstrated a perfect sensitivity of 100% and a specificity of 98.81%, compared to ZN staining's sensitivity of 84.85% and specificity of 99.21%. GeneXpert showed superior performance in detecting TB in both pulmonary and extrapulmonary samples, with a statistically significant difference (p<0.001). Additionally, GeneXpert identified six cases of RIF resistance. Conclusion The GeneXpert MTB/RIF assay outperforms ZN staining in diagnosing TB, offering higher sensitivity and comparable specificity. Its ability to detect RIF resistance adds significant clinical value. Despite its cost, the integration of GeneXpert into routine diagnostic workflows, particularly in high TB prevalence areas, is recommended to enhance early detection and treatment, thereby reducing TB transmission., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Mahaveer Institute of Medical Sciences and Research issued approval MIMSR/IEC/2022/3/008. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Gupta et al.)

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., (© 2022 The Authors.)