Lowenstein-Jensen Research Articles

Evaluation of Colorimetric Nitrate Reductase Assay in Liquid Medium for Detection of Resistance to First-line Antitubercular Drugs.

On a global scale, India holds the distinction of having the greatest number of tuberculosis (TB) cases caused by Mycobacterium tuberculosis (MTB) complex. The study aimed at evaluating the sensitivity, specificity, accuracy, cost, rapidity, and feasibility of the performance of the colorimetric nitrate reductase-based antibiotic susceptibility (CONRAS) test against the indirect proportion method (IPM) on Lowenstein-Jensen media as the gold standard. A comparative cross-sectional study was performed on 51 MTB isolates. Fresh subcultures were used for drug susceptibility testing by IPM on the Lowenstein-Jensen medium and the CONRAS method in liquid medium. Quality control for drug susceptibility testing was done using a known sensitive strain of MTB (H37Rv) and strains resistant to both isoniazid (INH) and rifampicin (RIF) - multidrug-resistant (MDR), mono-resistant to RIF, streptomycin (STM), and ethambutol (EMB). Statistical analysis was performed using MedCalc software (Version 20.027). CONRAS, carried out in microfuge tubes, was cost-efficient and easy to perform/interpret with most results being available in 10 days compared to 42 days in the case of IPM. The sensitivity, specificity, and accuracy of RIF and INH were 100%, 97.37%, and 98.04 and 93.33%, 97.59%, and 96.08%, respectively, which translates into an almost perfect agreement between the two methods as indicated by κ value of 0.905 and 0.949, respectively, for the two drugs. The performance of CONRAS was less satisfactory for STM and EMB when compared to IPM. CONRAS may serve as a useful test for the detection of MDR-TB because of its accuracy, low cost, ease of performance/interpretation, and rapidity when compared to IPM on LJ medium. It does not involve the use of expensive reagents and equipment, as is the case with molecular methods like GeneXpert and line probe assay, making it a suitable option for the detection of MDR-TB in resource-poor settings.

Prevalence of Rifampicin resistance tuberculosis among presumptive tuberculosis patients in Egypt-2021: a national health facility-based survey

BackgroundThe magnitude of MDR-TB cases was noticeable in Egypt. However, the last national survey was 11-years ago. The current survey was conducted to determine the prevalence of rifampicin resistance among sputum smear-positive pulmonary tuberculosis patients in Egypt.MethodsA national health facility-based cross-sectional study was conducted in 14 randomly selected governorates in Egypt between August 2020 and September 2021. All presumptive TB cases, either new or previously treated according to WHO definitions, with no gender, age, or nationality limitations, and provided informed consent were included in the study. Each patient completed a case report form (CRF). The CRF included socio-demographic and clinical data. Sputum samples were collected according to standard techniques and cultured on Lowenstein-Jensen (L-J) medium. Gene X-pert test was carried out first on the samples for simultaneous identification of MTB and rifampicin resistance. The prevalence of RR was calculated using crude, cluster, and weighted methods. Factors associated with RR were analyzed by bivariate and multivariate techniques.ResultsAmong the total 849 presumptive TB patients enrolled in the study, 710 (83.6%) patients were subjected to Gene X-pert testing (MTB/RIF). The crude prevalence of RR was 3.32% (95% CI: 1.89–4.76%) among the new cases and 9.46% (95% CI: 2.63–16.29%) among the retreated cases with an overall estimate of 3.99%; (95% CI: 2.51–5.47%). By cluster analysis the overall prevalence of RR was 5.01% (95% CI: 2.90–7.13). Factors associated with the prevalence of RR were co-morbidity with bronchial asthma, drug abuse and history of contact with a family member with TB.ConclusionThe prevalence of RR among either new or retreated cases TB patients was lower than the previous Egyptian rates in 2010–2012. The strongest predictor associated with RR was comorbidity with bronchial asthma.

Drug Resistance Tests for Mycobacterium Tuberculosis and Compatibility between Tests; From Past to Today

Objective: The gold standard for pulmonary tuberculosis diagnosis is the demonstration of Mycobacterium tuberculosis bacilli. Drug susceptibility test results of the bacilli obtained are crucial in tuberculosis treatment management. The various tests used to determine drug susceptibility must yield the same result. In the present study, we aimed to evaluate the compatibility of drug susceptibility test results obtained by Löwenstein-Jensen (L-J) and BACTEC 460TB methods from the past to the present. Material and Methods: Sputum results from 79 patients suspected of multidrug-resistant pulmonary tuberculosis (MDR-TB) clinically, radiologically, and bacteriologically were evaluated for isoniazid, rifampicin, ethambutol, and streptomycin between June 1997 and June 1998. Culture and drug sensitivity tests on the L-J medium were conducted at the Heybeliada Chest Diseases and Thoracic Surgery Center bacteriology laboratory, while culture and drug sensitivity tests with the BACTEC 460 TB system were performed at another center. The results were assessed for compatibility using the Kappa (κ) test, a tool for comparing two independent parameters. Results: All drug sensitivity tests for isoniazid, rifampicin, ethambutol, and streptomycin were collectively evaluated. It was determined that 263 (83.3%) of 316 drug sensitivity tests yielded concordant results, while 53 (16.7%) produced discordant results. The drug sensitivity tests using L-J and BACTEC 460TB methods indicated compatibility only for streptomycin (κ = 0.715). In contrast, they yielded different results for isoniazid (κ = 0.585), ethambutol (κ = 0.552), and rifampicin (κ = 0.507). Streptomycin exhibited compatibility, while isoniazid, ethambutol, and rifampicin showed incompatibility between the L-J and BACTEC 460TB methods. Conclusion: Drug sensitivity tests are pivotal in tuberculosis treatment management. While literature suggests compatibility between L-J and BACTEC 460TB methods, our study revealed incompatibility. Evaluation of drug sensitivities may lead to confusing results. Current practices involving studies in the same laboratory and genetic testing contribute to faster and more accurate outcomes in managing drug-resistant tuberculosis. Genetic tests and reference laboratories remain crucial for antituberculosis drug sensitivity, emphasizing their continued importance.

Comparison of Lowenstein Jensen Media and Ogawa Media Usage for Viability Test of BCG Vaccine Pasteur P11732 and Russian (Moscow) - 384 sub-strains.

The BCG vaccines on the market have employed a Mycobacterium bovis (M. bovis) sub-strains derived from the initial strain. To date, there has been no recommendation regarding the sub-strains with the highest effectiveness when administered to humans. Because it remains the standard for Tuberculosis treatment, the quality of the BCG vaccine must be verified. The viability test is one of the parameters for BCG vaccine quality control. The culture method has become the gold standard for viability testing with various testing media. The present study aimed to evaluate the performance of Lowenstein Jensen (LJ) and Ogawa media for the viability test of Pasteur 1173P2 and Russian (Moscow) - 384 sub-strains of M. bovis in the BCG vaccine. The number of culturable particles of each sub-strain in the BCG vaccine was estimated and statistically evaluated using the t-test. The colonies of the Pasteur 1173P2 have characteristics; tended to clump on both mediums with tiny, rough, and pale yellow/cream colors. Although the colony character of the Russian (Moscow) - 384 generally has similar feature, it did not cluster and had a smooth texture. In terms of growth rate, LJ and Ogawa media performed similarly for Pasteur 1173P2 and Russian (Moscow) - 384 sub-strains. Maximum growth is reached by the fifth week. The culturable particles of Pasteur P1173P2 sub-strains did not differ between mediums. Whereas the growth of the Russian (Moscow) - 384 sub-strains was statistically better on Ogawa media. The results of this study reveal that the performance of the media used for determining the number of culturable particles is based on the sub-strains of M. bovis present in the BCG vaccine.

Rapid detection of drug-resistant Mycobacterium tuberculosis by Modified MODS assay suitable for resource-poor settings.

Cross contamination and biosafety are concerns with the microscopic observation drug susceptibility assay. To address these issues, we modified the MODS technique in the current study. Two hundred and seventy-five samples were processed on LJ media and drug susceptibility was performed by the Indirect agar proportion method. A modified MODS test was done in tissue culture bottles. GenoType MTBDRplus assay was performed to detect the resistance and mutational pattern associated with the resistances. Sensitivity, specificity, positive predictive value, and negative predictive value for the detection of tuberculosis by modified MODS were 97.44%, 80.00%, 97.44%, and 80.00% respectively. The perfect agreement was seen between modified MODS and the Indirect agar proportion method for drug susceptibility testing of isoniazid (kappa = 0.923) and rifampicin (kappa = 1). The contamination rate, cost and TAT for modified MODS were less as compared to the solid media. In the case of MDR-TB isolates S531L (66.66%) was the most prevalent mutation in the rpoB gene followed by S315T2 mutation (58.33%) and T8C (41.66%) in katG and inhA gene respectively. In hetero-resistant strains, C-15T mutation (37.50%) was the most common followed by A-16G (12.50%) in the inhA gene. In INH mono-resistant strains only two mutations were observed i.e., S-315T1(50%) and C-15T (50%) in the katG and inhA genes respectively. Modified MODS proved to be cost-effective and user-friendly, with minimal risk to the handler and no cross-contamination between samples were observed. Hence, it can be used in low-income countries for early detection of tuberculosis and its resistance.

Disseminated Tuberculosis Presenting Section as Transverse Myelitis with Bone Marrow Involvement: A Rare Case of Pyrexia of Unknown Origin

In India, both pulmonary and Extrapulmonary Tuberculosis (TB) are prevalent. Central Nervous System TB (CNS-TB) is rare, with spinal intramedullary TB (SIMT) presenting as Longitudinally Extensive Transverse Myelitis (LETM) being even rarer. Diagnosing LETM often involves considering alternative aetiologies, frequently overlooking TB, particularly in patients without primary pulmonary TB. A 31-year-old male presented with high-grade fever, weight loss, and right hypochondriac pain. Initial investigations revealed pancytopenia and hepatosplenomegaly. Bone marrow biopsy showed granulomas and Acid-Fast Bacilli (AFB), but there was no evidence of Mycobacterium tuberculosis on the Cartridge-based Nucleic Acid Amplification Test (CBNAAT), leading to a presumptive diagnosis of disseminated TB. The patient was started on Antitubercular Therapy (ATT) but was non compliant with his medication, resulting in worsening symptoms, including paraparesis and urinary bladder involvement. Further investigations revealed LETM and ring-enhancing lesions in the brain. Despite negative CSF CBNAAT tests, empirical ATT with corticosteroids was resumed, and Mycobacterium tuberculosis was later confirmed via Lowenstein-Jensen (LJ) culture of the bone marrow. This case underscores the diagnostic challenges of disseminated TB with LETM, highlighting the importance of a high clinical suspicion and thorough evaluation, especially in endemic regions.

Acı Su Koşullarında Avrupa Deniz Levreklerinde (Dicentrarchus labrax) Mycobacterium marinum Enfeksiyonu

Mycobacteriosis is a well-known zoonotic disease that affects both fish and human. The pathogen, Mycobacterium marinum, cause systemic and lethal disease in fish and can be observed in farmed and aquarium species. In this study, M.marinum infection was detected in European sea bass (Dicentrarchus labrax) with common clinical and pathological findings in brackish water conditions (3.92 ‰). Granulomas in the enlarged spleen and liver were observed. The pathogen was isolated on Löwenstein-Jensen (L-J) media and the molecular identification verified the causative agent as M.marinum. Several granulomas were detected in histopathological examination. European sea bass has one of the highest production numbers in Türkiye, and this study aims to present mycobacteriosis in detail with clinical and pathological effects on European sea bass from brackish water conditions.

2584. Predictors of Clinically Significant Infection in Patients with Mycobacterium simiae Isolated from Respiratory Samples: a Retrospective Study from a Tertiary Care Center in Lebanon

Abstract Background Mycobacterium simiae is a rare slow-growing, non-tuberculous mycobacterium (NTM). It is predominant in Middle Eastern countries like Israel, Iran and Lebanon and is the most isolated NTM spp. at our institution (40%). Little is known about its reservoirs, pathogenesis, risk factors and indications for treatment. Methods We retrospectively studied patients with respiratory cultures showing M. simiae at the American University of Beirut Medical Center (AUBMC) over 19 years. We used the American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA) criteria to define NTM lung disease. Samples were cultured at AUBMC using Lowenstein-Jensen media and the BacT/ALERT system. NTM isolates were referred for speciation and susceptibility testing at Mayo Clinic, MN, USA. Results We identified 72 isolates from 66 patients. Most were obtained from sputum samples (57.6%), the rest from bronchoalveolar lavages (BAL). Acid-fast bacilli smear was positive in 27% of samples with no difference between sputum and BAL. The median age of patients was 64 years and 51.5% were females. Bronchiectasis (34.5%), chronic obstructive pulmonary disease (27.3%) and immunosuppression (33.3%) were common (Table 1). As per the ATS/IDSA criteria, 67.9% of patients had NTM lung disease. The most common symptoms were cough (83.3%) and sputum production (74.5%). All patients except 2 had abnormal findings on CT imaging like nodules (58.5%), tree in bud aspect (45.3%) and bronchiectasis (24.5%). Among those, 55.6% had abnormal CT findings preceding the M. simiae isolation. Bronchiectasis was more common in those who satisfied the ATS/IDSA criteria versus those who did not (38.9% vs 20.0%). Otherwise, we found no significant differences between both groups (Table 2). Treatment was given to 88.9% of patients who satisfied the NTM lung disease and 42.1% of those who did not (p< 0.001) with a median duration of 12 months. Of those with available follow-up, relapse was reported in 24% of cases. Characteristics of patients with M. simiae isolated from respiratory samples. BMI, body mass index, COPD, chronic obstructive pulmonary disease, AFB, acid-fast bacilli. *Median(Interquartile range) **Composite variable including hematologic malignancy, solid organ malignancy, hematopoietic transplantation, solid organ transplant, chronic steroids treatment, and active chemotherapy. Clinical and radiological differences between patients with M. simiae lung disease and those colonized according to the ATS/IDSA criteria. BMI, body mass index, COPD, chronic obstructive pulmonary disease. *Median (Interquartile range) Conclusion M. simiae is a significant cause of NTM lung disease at our institution with a high rate of relapse. Further research is required to understand its geographic restriction, predisposing factors, and identify patients at risk for clinically significant infection. Disclosures Souha S Kanj, MD, Gilead: Advisor/Consultant|Menarini: Advisor/Consultant|MSD: Advisor/Consultant|Pfizer: Advisor/Consultant

Effects of sputum bacillary load and age on GeneXpert and traditional methods in pulmonary tuberculosis: a 4-year retrospective comparative study

BackgroundThe purpose of this study was to evaluate the diagnostic value of the GeneXpert® MTB/RIF (Xpert®), Auramine O staining method, and Lowenstein-Jensen medium for bacteriologically confirmed pulmonary tuberculosis and explore the effects of the sputum bacillary load (SBL) and qRT‒PCR threshold cycle (Ct) value on the detection methods.MethodsWe retrospectively analysed the results in the Department of Infectious Disease for 49 months. The χ2 test was used to compare the performances of each method, receiver operating characteristic curve analysis was used to determine the optimal cut-off values, and the factors associated with a false-negative result from Xpert® were analysed by logistic regression.ResultsSimultaneous analysis of 980 sputum specimens showed that the positive detection rate of Xpert® did not increase with increasing SBL, and there were differences between the three when SBL ≤ 1 + (all P < 0.05). There was a good negative correlation between the Ct value and the SBL (P < 0.0001). Age was an independent risk factor for false-negative Xpert® results (P = 0.029), and when Ct < 16, the diagnostic sensitivity and specificity were both 100.00%. The optimal cut-off Ct values for resegmentation based on the drug resistance classification were < 18.6, 18.6–34.1, and > 34.1 cycles.ConclusionsXpert® was not affected by SBL but it was by age, and it is more advantageous when SBL ≤ 1 + . The results regarding rifampicin resistance were reliable, and the novel Ct segmentation was a practical and more clinically meaningful classification method for diagnosing rifampicin resistance. These findings will help improve physicians’ ability to accurately diagnose TB.

Interaction between mesenchymal stromal cells and tuberculous mycobacteria in vitro

The objective: in an in vitro experiment, we compared phagocytic parameters of mesenchymal stromal cells (MSCs) and macrophages to tuberculous mycobacteria, assessed the ability of MSCs and macrophages to lyse mycobacteria or maintain their intracellular growth, their effect on formation of phenotypic drug resistance of mycobacteria, as well as the effect of tuberculous mycobacteria on the type of MSCs cell death.Subjects and Methods. Balb/c male mice, aged 6 to 8 weeks, were used in the experiment. Bone marrow MSCs were obtained from femurs and tibias by further cultivation, peritoneal macrophages were elicited with 4% alpha-glucan. The intracellular content of mycobacteria was counted using a confocal microscope with x 400 magnification. Susceptibility of mycobacteria to isoniazid and development of phenotypic drug resistance after culturing MSCs and macrophages with MTB on Lowenstein–Jensen medium was assessed by counting CFU. In 5 days after the infection, the number of apoptotic and necrotic MSCs and macrophages was determined by a flow cytometer.Results. On Day 1, the total number of phagocytosed MTB, as well as the number of phagocytic-active macrophages, exceeds the corresponding figures for MSCs more than twice. MSCs phagocytize tuberculous mycobacteria in a smaller amount, but MTB reproduces in them more actively: the number of CFU after 7 days of cell cultivation with MTB exceeded the corresponding parameter by almost 50 times after 24 hours of cultivation. In cultures of infected MSCs cultivated for 7 days, regardless of the presence of isoniazid, there was a rapid growth of tuberculous mycobacteria. On Day 5 after infection of macrophage culture with tuberculous mycobacteria, the number of necrotic cells was 2.7 times greater than that of uninfected necrotic macrophages, but the number of apototic cells in these groups differed slightly. In the culture of MSCs, there were 8.5 times more infected nectrotic cells versus uninfected necrotic MSCs, and the number of necrotic MSCs was 4.5 times higher than the number of MSCs with apoptosis, while in the culture of infected macrophages, the number of necrotic cells was the same as number of apoptotic cells. Unlike macrophages, treatment of MSCs with isoniazid did not inhibit the intracellular proliferation of MTB.Conclusion. MSCs have the ability to phagocytose mycobacteria, but they do it less actively than macrophages and, unlike macrophages, they are not able to restrain the reproduction of tuberculous mycobacteria. Mycobacteria have phenotypic drug resistance in MSCs. In MSCs, when infected with tuberculous mycobacteria, there is a pronounced shift towards necrosis in the type of cell death, which can lead to dissemination of MTB and development of local destructive changes.

The acetylation of pknH is linked to the ethambutol resistance of Mycobacterium tuberculosis.

EmbR, a substrate of pknH in Mycobacterium tuberculosis (Mtb), is related to the ethambutol (EMB) resistance. This study aimed to investigate the relationship between acetylation of pknH and the resistance of EMB mono-resistant Mtb. The EMB mono-resistant Mtb strain was constructed based on the MYCOTB and the Löwenstein-Jensen (LJ) proportion method. The growth kinetics was used to evaluate the bacterial growth. Escherichia coli, as the host of Mtb, was used for cloning and protein purification. Moreover, the immunoprecipitation was performed along with western blot to evaluate the EmbR phosphorylation and pknH acetylation. Each independent experiment was conducted in triplicate. EMB mono-resistant Mtb strain was successfully constructed according to the results of MIC values of 14 anti-Mtb drugs. The EMB resistant (ER) Mtb strain showed faster growth than the wild-type (WT) Mtb strain, and the difference was statistically significant. Moreover, pknH robustly phosphorylates EmbR, and pknH and acetylated pknH protein levels were downregulated in ER strain. The acetylation of pknH may reduce the phosphorylation of EmbR to inhibit the growth of Mtb strain. Enhancing the acetylation of pknH may be a promising method to inhibit the EMB resistance against Mtb.

Phenotypic and genotypic drug susceptibility patterns of Mycobacterium tuberculosis isolates from pulmonary tuberculosis patients in Central and Southern Ethiopia.

The persistence of tuberculosis (TB) infection in some patients after treatment has highlighted the importance of drug susceptibility testing (DST). This study aimed to determine the drug susceptibility patterns of Mycobacterium tuberculosis (M. tuberculosis) isolates from pulmonary TB (PTB) patients in Central and Southern Ethiopia. A health institution-based cross-sectional study was conducted between July 2021 and April 2022. Sputum samples were collected from newly diagnosed smear microscopy and/or Xpert MTB/RIF-positive PTB patients. The samples were processed and cultivated in Lowenstein-Jensen (LJ) pyruvate and glycerol medium. M. tuberculosis isolates were identified using polymerase chain reaction (PCR) based region of difference 9 (RD9) deletion typing. Phenotypic DST patterns of the isolates were characterized using the BACTEC MGIT™ 960 instrument with SIRE kit. Isoniazid (INH) and Rifampicin (RIF) resistant M. tuberculosis isolates were identified using the GenoType® MTBDRplus assay. Sputum samples were collected from 350 PTB patients, 315 (90%) of which were culture-positive, and phenotypic and genotypic DST were determined for 266 and 261 isolates, respectively. Due to invalid results and missing data, 6% (16/266) of the isolates were excluded, while 94% (250/266) were included in the paired analysis. According to the findings, 14.4% (36/250) of the isolates tested positive for resistance to at least one anti-TB drug. Gene mutations were observed only in the rpoB and katG gene loci, indicating RIF and high-level INH resistance. The GenoType® MTBDRplus assay has a sensitivity of 42% and a specificity of 100% in detecting INH-resistant M. tuberculosis isolates, with a kappa value of 0.56 (95%CI: 0.36-0.76) compared to the BACTEC MGIT™ DST. The overall discordance between the two methods was 5.6% (14/250) for INH alone and 0% for RIF resistance and MDR-TB (resistance to both INH and RIF) detection. This study reveals a higher prevalence of phenotypic and genotypic discordant INH-resistant M. tuberculosis isolates in the study area. The use of whole-genome sequencing (WGS) is essential for gaining a comprehensive understanding of these discrepancies within INH-resistant M. tuberculosis strains.

Сложность диагностики туберкулеза в учреждениях общей лечебной сети

Aim. The aim of the study is to analyze the reasons for the erroneous diagnostics of TB in the general health care institutions and identify ways to improve diagnostics. Materials and methods. 147 medical cards of inpatients who were in the Donetsk Republican Clinical Tuberculosis Hospital, for whom active TB was excluded were analyzed. Groups were identified: 89 HIV-negative (group 1) and 58 HIV-infected (group 2). Laboratory, radiation and instrumental examinations, sputum examination by microscopy, molecular genetic analysis GeenXpertMBT/Rif, culture on Middlbrook medium and Lowenstein-Jensen medium were performed. Statistical analysis was performed using the MedStat package. Results and discussions. Hospitalization in the anti-tuberculosis hospital was considered justified in 24.7% of HIV-negative and 34.5% of HIV-infected patients due to the similarity of the clinical and radiological picture of the disease, as well as false-positive results of sputum examination before hospitalization (in 49.1% of examined HIV-negative patients). These patients required in-depth examination, and in 18.0% and 10.3%, respectively, test therapy with anti-tuberculosis drugs. Most often, pneumonia and oncological pathology were mistaken for pulmonary tuberculosis: 31.5% and 25.8% in the 1st group and 48.4% and 25.8% in the 2nd group, in both groups the verification of tumor processes was the most difficult: out of 31 cancer patients, 17 (54.8%) people received anti-tuberculosis treatment, of 7 fatal cases, in 6 the diagnosis was verified by autopsy. Development of secondary forms of pulmonary tuberculosis is not accompanied by hilar lymphadenopathy in immunocompetent patients: in group 1 enlarged lymph nodes were observed in 29 (32.6%) patients, of whom oncological pathology was revealed in 11 (37.9%), sarcoidosis – in 8 (27.6%), pneumonia – in 6 (20.7%), interstitial lung diseases (ILD) – in 2 (6.9%). Of 31 HIV-infected patients with suspected pulmonary tuberculosis, 8 (25.8%) were diagnosed with Pneumocystis pneumonia, requiring treatment outside the TB dispensary. The reasons for the erroneous diagnostics of tuberculous meningitis in HIV-infected people were insufficient coverage of neuroimaging, genetic molecular testing of cerebrospinal fluid and serodiagnosis of blood (cerebrospinal fluid). An important counterargument against the tuberculous etiology of CNS damage is the absence of active tuberculosis of the lungs and/or other organs: 77.7% of patients with suspected TM had no fresh changes in the lungs. Conclusion. Improving the quality of sputum microscopy, carrying out, if indicated, antibacterial (antifungal) treatment, diagnostic bronchoscopy, CT/MRT, consultations with specialists can reduce the frequency of erroneous diagnosis of tuberculosis and avoid hospitalization in the anti-tuberculosis infectious diseases department.

Role of GeneXpert in Diagnosis of Smear Negative Pulmonary Tuberculosis

Abstract Background Pulmonary TB considered as a global health problem with specific concern in developing countries that need rapid methods in diagnosis with accurate results. Objective To evaluate the diagnostic yield of GeneXpert assay in diagnosis of TB infection in patients with negative sputum smear. Methods A total 346 cases with suspect clinical and radiological finding for TB infection was subjected to direct sputum analysis under microscope to find acid fast bacilli (AFB); the negative smear then subjected to GeneXpet assay analysis and sputum culture on LJ media. Results All cases with negative GeneXpert results, had negative culture, while 45 cases of 50 that had positive GeneXpert assay finding showed positive culture. The diagnostic yield of test was excellent (94.7%). The sensitivity, specificity, PPV and NPV were 100, 89.8, 90 and 100 respectively. Conclusion The performance of GeneXpert assay in diagnosing TB among smear negative patients was reasonable, and may fast the decision of treatment.

RETROSPECTIVE STUDY EVALUATING THE EFFICACY OF LJ MEDIUM ALONG WITH PENICILLIN TO IMPROVE CULTURE YIELD OF MYCOBACTERIUM TUBERCULOSIS IN PATIENTS

This study evaluated the efficacy of penicillin in improving the culture yield of M. Tuberculosis in cases of pulmonary tuberculosis. A retrospective study was conducted in the Department of Pathology, MMDC, Multan. A total of 350 suspected pulmonary tuberculosis patients were selected for the study. An early morning sputum specimen was collected. NAOH citrate and N-acetyl-L-cysteine were used for digesting, decontaminating, and homogenizing the sputum. The samples were processed on Lowenstein Jensen media and then Lowenstein Jensen media with penicillin. The cultures were then examined after 2 months. On LJ media, 100 (28.5%) suspected cases were positive, and 80 (22.8%) cases were contaminated. On LJ media with penicillin, 145 (41.4%) cases were positive, and 15 (4.3%) were contaminated. In addition, 30 (8.5%) were 1+ grading, 15 (4.3%) were 2+ grading on both media, 95 (27.1%) were 3+ grading on LJ media, and 50 (14.2%) were 3+ grading on LJ media with penicillin. Contamination in Mycobacterium tuberculosis is reduced by adding penicillin to the LJ media, and isolation-positive cultures are enhanced.

Comparative Diagnostic of Cervical Tuberculous Lymphadenitis: PCR is a Fast, Efficient, and Improved Diagnostic Approach.

The study included 100 clinically suspected cases of TBLN. Fine needle aspirate (FNA) samples were processed for cytology staining and cultured on LJ & BACTEC 12B media. The biochemical tests were performed to identify the isolates at the species level. Additionally, for PCR, DNA was extracted and used for the diagnosis and identification of mycobacterial species. Patients ranged from 2 to 45 years with a mean age of 24.96 ± 9.10 years. Out of 100 patients, 73% had clinical symptoms of weight loss, followed by fever (72%), anorexia (66%), and night sweats (58%). 24% of patients were found to be smear-positive after Ziehl-Neelsen (ZN) staining and statistically highly significant with PCR. On LJ medium 34% and on BACTEC radiometric 45% of samples were smearing positive. Overall, 48% of cases were PCR-positive for TBLN. When compared with culture, the sensitivity and specificity of PCR were 93.75% and 100%, respectively, which are higher than cytology. The true positive predictive value (PPV) and negative predictive value (NPV) were 83.3% and 61.5%, respectively. This study suggests that PCR is a rapid, sensitive, and specific tool for correct diagnosis of TBLN cases as compared to staining and culture which lead to the early and proper management of mycobacterial diseases.

Effect of Stenotrophomonas maltophilia on Tuberculosis.

Tuberculosis (TB) is an important infectious disease suffered by many countries, including China. In this stage, accurate diagnosis and treatment are key measures for the prevention and control of TB. Stenotrophomonas maltophilia is a global emerging Gram-negative, multidrug-resistant (MDR) organism characterized by its high contribution to the increase in crude mortality rates. By single cell preparation and strain identification, we isolated S. maltophilia from stored cultures of Mycobacterium tuberculosis (Mtb). We found that S. maltophilia could not be removed from sputum by alkali treatment or inhibited by antibiotic mixture added to MGIT 960 indicator tubes. When co-cultured with Mtb on a Löwenstein-Jensen (L-J) slant, it could inhibit the growth of Mtb and liquefy the medium. More seriously, it was resistant to 10 of the 12 anti-TB drugs, including isoniazid and rifampin, and made the mixed samples display multidrug-resistant Mtb (MDR-TB) results in the drug sensitivity test, which might change a treatment regimen and increase disease burden. Following, we conducted a small-scale surveillance which showed that the isolation rate of S. maltophilia in TB patients was 6.74%, but these patients had no special characteristics and the presence of S. maltophilia was hidden. The effect of S. maltophilus on TB and its mechanism are unclear and require more attention. IMPORTANCE China is a high-burden country for tuberculosis (TB), multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB), and HIV-associated TB. Increasing the positive rate of culture and the accuracy of antibiotic susceptibility testing (AST) are important for diagnosis, treatment, and control of TB. In our study, we found that the isolation rate of Stenotrophomonas maltophilia in TB patients was not neglectable and that this bacterium affects the isolation and AST results of TB. Due to a lack of relevant research, the impact of S. maltophilia on the course and outcome of TB is unclear. However, the characteristics of S. maltophilia that increase disease mortality require attention. Therefore, in the clinical testing of TB, in addition to mycobacteria, it is recommended to increase the detection of co-infected bacteria and improve the awareness of TB clinicians of these bacteria.

Diagnostic Approach to Extrapulmonary Tuberculosis by Cartridge-based Nucleic Acid Amplification Test.

Tuberculosis (TB) is a highly infectious disease causing millions of cases worldwide. Though pulmonary TB is the most common form of infection, extrapulmonary cases are also very rampant and are responsible for a large number of cases. But the diagnosis of extrapulmonary cases is quite difficult because of varied manifestations and the paucibacillary nature of the infection. Cartridge-based nucleic acid amplification test (CBNAAT) is a simple, rapid test that is very efficient in the early diagnosis of these extrapulmonary cases [extrapulmonary TB (EPTB)]. A study was done to establish the usefulness of CBNAAT in the early diagnosis of EPTB cases. A comparative study was conducted in a rural tertiary care hospital in West Bengal, India, for 8 months (July 2021-February 2022). Samples were collected from different sites like pleural fluid, lymph nodes, cerebrospinal fluid (CSF), pus, ascitic fluid, and tissue aspirate and subjected to both CBNAAT and smear staining and examination under a fluorescent microscope. Positive samples were cultured, examined, and compared. From 593 samples collected from different sites in suspected cases of EPTB-52 samples were positive by CBNAAT, and six cases showed rifampicin resistance (RIF resistant). Smear staining of the samples by auramine-rhodamine stains and examined under the fluorescent microscope for acid-fast bacilli identifying 33 samples; the rest were negative. Slides showing acid-fast bacilli were cultured on Lowenstein-Jensen media. Cartridge-based nucleic acid amplification test (CBNAAT) is a very useful assay for the early diagnosis of extrapulmonary cases as it can accurately identify false negative samples by smear microscopy.

The Effect of Inoculum Size on Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis.

Phenotypic drug susceptibility testing (DST) requires a standardized amount of inoculum to produce reproducible susceptibility results. The most critical step in the application of DST in Mycobacterium tuberculosis isolates is the preparation of the bacterial inoculum. In this study, the effect of bacterial inoculum prepared in various McFarland turbidities on primary antituberculosis drug susceptibility of M. tuberculosis strains was investigated. Five standard ATCC strains (ATCC 27294 [H37Rv], ATCC 35822 [izoniazid-resistant], ATCC 35838 [rifampicin-resistant], ATCC 35820 [streptomycin-resistant], ATCC 35837 [ethambutol-resistant]) were tested. Inoculums of McFarland standard of 0.5, 1, 2, 3, and 1:100 dilutions of 1 McFarland standard of each strain were used. The effect of inoculum size on DST results was determined by the proportion method in Lowenstein-Jensen (LJ) medium and nitrate reductase assay (NRA) in the LJ medium. In both test methods, the increase in inoculum size did not affect the DST results of the strains. On the contrary, DST results were obtained more rapidly as a result of the use of dense inoculum. DST results obtained in all McFarland turbidities were found to be 100% compatible with the recommended amount of inoculum, 1:100 dilution of 1 McFarland standard (inoculum size of gold standard method). In conclusion, the use of a high amount of inoculum did not change the drug susceptibility profile of tuberculosis bacilli. Minimizing manipulations during the inoculum preparation phase of susceptibility testing, this outcome will decrease the need for equipment and make the test application easier, particularly in developing countries. IMPORTANCE During DST application, it can be challenging to evenly homogenize TB cell clumps with lipid-rich cell walls. These experiments must be carried out under Biosafety Level-3 (BSL-3) laboratory conditions with personal protective equipment and taking safety precautions because the procedures applied at this stage cause the formation of bacillus-laden aerosols and carry a serious risk of transmission. Considering this situation, this stage is important given that it is not possible to establish a BSL-3 laboratory in poor and developing countries. Reducing the manipulations to be applied during the preparation of bacterial turbidity will minimize the risk of aerosol formation. Perhaps there will be no need to do these steps for susceptibility tests in these countries or even in developed countries.

Effective Use of Penicillin to Improve Culture Yield for Mycobacterium Tuberculosis

Background: The aim of this study to evaluate the Mycobacterium tuberculosis (MTC) sputum culture contamination and recovery between Lowenstein-Jensenmedium (LJ) contains Pencillin+LJ and free Pencillin LJ. Study design: This was a cross sectional study conducted at Khawaja Safdar Medical College, Sialkot /Allama Iqbal Memorial Hospital Sialkot for the duration of six months from September 2022 to February 2023. Methods: A total number of the participants was 140. The male and female gender were both included who suffered pulmonary tuberculosis (PTB). The sample n=140 were analyzed for culture and contamination. The sputum samples were taken in the early morning in the falcon tube and digested, decontaminated, and homogenized by Modified Petroff method. The specimen of sputum was processed on addition of Penicillin+ LJ media, and incubation was carried out at 37°C. After eight weeks, cultures were examined. The data were analyzed by SPSS 21 software. Results: The male 64.2% show greater number of cases as compared to female 36%. The suspected cases of PTB was compared with Pencillin+ LJ media and LJ media alone. 21.4% of the samples were positive mycobacterium (MTB), 79% were contaminated on LJ media while 93% were positive (MTB), and 7.1% of the samples were contaminated on LJ media without penicillin. This difference was statistically significant (p&lt;0.005**). The grade 2+/3+ show 50% recovery by the treatment of pencillin+LJ on positive sample and 47% contaminated sample P&lt;0.001** while graded scanty/+1 show recovery was 38%; p=0.001** with pencillin+LJ then 37% contaminated sample; p=0.002**. The difference in recovery between treatment with penicillin and without pencillin LJ was statistically significant; p&lt;0.005. Conclusion: The contamination rate was reduced by 47% by penicillin+LJ media. Penicillin offered great alternatives for decreasing contamination in LJ alone. Penicillin should be further used as a medium supplement to enhance recovery for LJ media. Keywords: Culture media, Mycobacterium, Penicillin