Mycobacterium Tuberculosis Culture Research Articles

Addition of computed tomography chest increases the diagnosis rate in patients with suspected intestinal tuberculosis.

Background/AimsIntestinal tuberculosis (ITB) is difficult to diagnose due to poor sensitivity of definitive diagnostic tests. ITB may be associated with concomitant pulmonary tuberculosis (PTB) which may remain undetected on chest X-ray. We assessed the role of contrast enhanced computed tomography (CECT) chest in detecting the prevalence of active PTB, and increasing the diagnostic yield in patients with suspected ITB.MethodsConsecutive treatment naïve patients with suspected ITB (n=200) who underwent CECT chest (n=88) and had follow-up duration>1 year were recruited in this retrospective study (February 2016 to October 2018). ITB was diagnosed in the presence of caseating granuloma, positive acid fast stain or culture for Mycobacterium tuberculosis on biopsy, presence of necrotic lymph nodes (LNs) on CT enterography or positive response to anti-tubercular therapy. Evidence of active tuberculosis on CECT-chest was defined as presence of centrilobular nodules with or without consolidation/miliary nodules/thick-walled cavity/enlarged necrotic mediastinal LNs.ResultsSixty-five of eighty-eight patients (mean age, 33.8±12.8 years; 47.7% of females) were finally diagnosed as ITB (4-caseating granuloma on biopsy, 12-necrotic LNs on CT enterography, 1-both, and 48-response to anti-tubercular therapy) and 23 were diagnosed as Crohn’s disease. Findings of active TB on CECT chest with or without necrotic abdominal LNs were demonstrated in 5 and 20 patients, respectively. No patient with Crohn’s disease had necrotic abdominal LNs or active PTB. Addition of CECT chest in the diagnostic algorithm improved the sensitivity of ITB diagnosis from 26.2% to 56.9%.ConclusionsAddition of CECT chest significantly improves the sensitivity for definite diagnosis in a patient with suspected ITB.

Genital footprints of extragenital tuberculosis in infertile women: Comparison of various diagnostic modalities

BackgroundGenital tuberculosis (TB) continues to remain an important cause of infertility in women, especially in developing countries. It is mostly consequent to a primary infection elsewhere in the body. The diagnosis is challenging, considering its paucibacillary nature. Although there are many studies on association of genital tuberculosis with infertility, there is paucity of literature on impact of extragenital tuberculosis on fertility of women through involvement of female reproductive organs. The various diagnostic modalities available have limitations and quest is ongoing for the best diagnostic test. MethodThis was a prospective observational study conducted at the infertility clinic of a tertiary care health facility where 60 infertile women with either tubal factor or unexplained infertility with or without past history of extragenital tuberculosis were enrolled as study subjects or controls respectively. Mantoux test was performed in all women and diagnostic laparo-hysteroscopy was performed in all women to look for any evidence of uterine and/or tubal damage. The peritoneal fluid was sent for GeneXpert and Liquid culture for mycobacterium tuberculosis. Results of Mantoux test, GeneXpert and liquid culture were compared with the laparohysteroscopic findings. ResultOf the thirty infertile women in the study group, 27/30 (90%) had a history of pulmonary tuberculosis and 3/30 (10%) had history of tubercular cervical lymphadenopathy. It was observed that Mantoux test was positive (induration >10 mm) in 27/30 (90%) of women in the study group as compared to only 4/30 (13.3%) controls. Abnormal hysteroscopic findings were documented in 26.6% (8/30) study group women as compared to 6.6% (2/30) women in the control group. Similarly, 60% (18/30) of women in the study group had abnormal laparoscopic findings compared to 33% (10/30) in the control group. Seven out of thirty (23.3%) women were positive for GeneXpert in the study group compared to only 1/30 (3.3%) in the control group. Similarly, liquid culture was positive in 6/30 (20%) of women in the study group as compared to 1/30 (3.3%) in the control group. All the above differences were statistically significant. We observed that the sensitivity of Mantoux test (75.8%) stand alone was higher than the other tests combined (50%). However, specificity and positive predictive value (PPV) increases markedly (up to 100%) to when all the three tests are combined. ConclusionThe authors conclude that all women presenting with infertility should be screened for a past history of tuberculosis and actively worked up for genital tuberculosis in case the history is positive. The various available tests (Mantoux test, GeneXpert and liquid culture) have their limitations for the diagnosis of genital tuberculosis. Thus an approach of early resort to laparohysteroscopy in suspected patients is desirable so that definitive management may be instituted timely and promptly.

Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates

Rapid detection of tuberculosis (TB) infection is paramount to curb further transmission. The gold standard for this remains mycobacterial culture, however emerging evidence confirms the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens. These bacteria do not grow under standard culture conditions and require the presence of culture filtrate (CF), from axenic cultures of Mycobacterium tuberculosis (Mtb), to emerge. It has been hypothesized that molecules such as resuscitation promoting factors (Rpfs), fatty acids and cyclic-AMP (cAMP) present in CF are responsible for the growth stimulatory activity. Herein, we tested the ability of CF from the non-pathogenic bacterium Mycobacterium smegmatis (Msm) to stimulate the growth of DCTB, as this organism provides a more tractable source of CF. We also interrogated the role of Mtb Rpfs in stimulation of DCTB by creating recombinant strains of Msm that express Mtb rpf genes in various combinations. CF derived from this panel of strains was tested on sputum from individuals with drug susceptible TB prior to treatment. CF from wild type Msm did not enable detection of DCTB in a manner akin to Mtb CF preparations and whilst the addition of RpfABMtb and RpfABCDEMtb to an Msm mutant devoid of its native rpfs did improve detection of DCTB compared to the no CF control, it was not statistically different to the empty vector control. To further investigate the role of Rpfs, we compared the growth stimulatory activity of CF from Mtb, with and without Rpfs and found these to be equivalent. Next, we tested chemically diverse fatty acids and cAMP for growth stimulation and whilst some selective stimulatory effect was observed, this was not significantly higher than the media control and not comparable to CF. Together, these data indicate that the growth stimulatory effect observed with Mtb CF is most likely the result of a combination of factors. Future work aimed at identifying the nature of these growth stimulatory molecules may facilitate improvement of culture-based diagnostics for TB.

Cross-Contamination versus Outbreak: Pre-XDR Mycobacterial Strains Confirmed by Whole-Genome Sequencing.

Whole-genome sequencing (WGS) is promising for the quality control of laboratory facilities for Mycobacterium tuberculosis (MTB) strains. We describe the clinical and laboratory characteristics of false positive versus true positive MTB cultures based on WGS, which were experienced in a real clinical setting. Strain harvest and DNA extraction from seven isolates from pre-extensive drug-resistant (pre-XDR) TB patients transferred to the Korea University Ansan Hospital were performed, and epidemiologic links and clinical information, including the phenotypic drug susceptibility test (pDST), were investigated. WGS was performed using Ion GeneStudio with an ION530tm chip (average sequencing depth, ~100-fold). In the phylogenetic tree, identical and different strains were distributed separately. Five of the seven isolates were identical; the remaining two isolates differed from the others. The images of the referred pre-XDR-TB patients with false positive MTB that were analyzed were of regions close to old TB scars. Further, the results of WGS gene mutation analysis for ethambutol, streptomycin, and fluoroquinolone resistance in all six patients were not concordant with the pDST results. WGS and clinical information were useful in differentiating laboratory cross-contamination from true positive TB, thereby avoiding the unnecessary treatment of false positive patients and delay in treating true positive TB patients, with reliable genotypic drug resistance results.

Application of video-assisted thoracoscopic lung resections for differential diagnosis of lung diseases

The objective of the study: to determine the place and effectiveness of video-assisted thoracoscopic lung resection (VATS resections) in the differential diagnostic procedure for examination of patients with chest diseases.Subjects and methods. In 2017-2019, 1,190 patients with suspected respiratory tuberculosis were examined in the differential diagnostic department of TB Dispensary, the Ministry of Health of the Kabardino-Balkarian Republic; 106 (8.9%) needed VATS since it was impossible to verify their diagnosis despite various biopsies. The age of 106 patients varied from 18 to 80 years old, there were 66 men (62.0%), and 40 women (38.0%). The preoperative diagnosis was as follows: single or multiple lung lesions – 83 (76.9%) cases, disseminated lung disease of unknown etiology or interstitial lung disease of unspecified etiology – 25 (23.1%) cases.In all cases, the surgical specimens were sent for comprehensive tests, including histological tests, microbiological tests (fluorescent microscopy with Ziehl-Nielsen staining, culture for Mycobacterium tuberculosis (MTB) by Bactec, and culture for secondary flora and fungi), and molecular genetic tests to detect MTB DNA.Results. The diagnostic efficiency when using VATS resections, histological and microbiological testing of surgical specimens made 98.1% (95% CI 93.38-99.48; Wilson's method). Among the diagnosed diseases, pulmonary tuberculosis was confirmed in 47/106 (44.3%) patients, cancer – 37/106 (34.9%), the rest of patients were diagnosed with some other disorders including orphan diseases.

Diagnostic value of Xpert MTB/RIF assay on pleural tissue obtained via closed pleural biopsy among persons with presumptive tuberculous pleuritis.

Background Tuberculous pleuritis (TBP) is a common extrapulmonary tuberculosis that contributes to the tuberculosis burden. Xpert MTB/RIF assay is a promising method for rapid diagnosis of TBP. The diagnostic value of Xpert MTB/RIF assay in pleural tissue obtained via closed pleural biopsy among sputum acid-fast bacilli (AFB) smear-negative persons is not well studied. Objectives To evaluate the diagnostic value of Xpert MTB/RIF assay on diagnosis of TB in pleural tissue obtained via blind closed pleural biopsy. Methods Closed pleural biopsy using Cope needle was performed on adult patients who presented with lymphocyte predominant exudative pleural effusion. Xpert MTB/RIF assay was performed in parallel to pathology and mycobacterial culture of the pleural tissue specimen to determine its sensitivity and specificity. Final clinical diagnosis of TBP was determined by improvement in 2-months follow-up of anti-tuberculous treatment. Results A total of 33 patients were included in the study. The median (interquartile range (IQR)) age was 27 (25 - 42) years. The sensitivity and specificity of Xpert MTB/RIF assay was 30% and 100% compared with Mycobacterium tuberculosis culture as the gold standard, and 20% and 95.7% compared with histopathology as the gold standard. Conclusion Xpert MTB/RIF assay in pleural tissue obtained by closed pleural biopsy did not increase diagnostic yield, but it shortens time for diagnosis compared with conventional methods.

Immunological Aspects of Diagnosis and Management of Childhood Tuberculosis.

The diagnosis of tuberculosis (TB) in children is difficult because of the low sensitivity and specificity of traditional microbiology techniques in this age group. Whereas in adults the culture of Mycobacterium tuberculosis (M. tuberculosis), the gold standard test, detects 80% of positive cases, it only detects around 30–40% of cases in children. The new methods based on the immune response to M. tuberculosis infection could be affected by many factors. It is necessary to evaluate the medical record, clinical features, presence of drug-resistant M. tuberculosis strains, comorbidities, and BCG vaccination history for the diagnosis in children. There is no ideal biomarker for all TB cases in children. A new strategy based on personalized diagnosis could be used to evaluate specific molecules produced by the host immune response and make therapeutic decisions in each child, thereby changing standard immunological signatures to personalized signatures in TB. In this way, immune diagnosis, prognosis, and the use of potential immunomodulators as adjunct TB treatments will meet personalized treatment.

Gene Xpert/MTB RIF assay for spinal tuberculosis- sensitivity, specificity and clinical utility

BackgroundXpert MTB/RIF assay is a rapid automated molecular test with excellent reported sensitivity, specificity for diagnosis of pulmonary and extrapulmonary Mycobacterium tuberculosis (MTB) infections. However, the clinical utility and accuracy in STB is not well established. A study was conducted to report on the sensitivity, specificity and clinical utility of the Xpert MTB/RIF assay in spinal tuberculosis (STB). MethodsA retrospective review of medical records was performed for 136 patients that underwent spinal biopsy for suspected spondylodiscitis. Reports for acid fast bacilli (AFB) smear, gram stain, pyogenic culture, MTB culture, histopathology, Xpert MTB/RIF assay, and drug sensitivity testing were reviewed. ‘Reference standard for diagnosis of STB’ was based on positive histopathology and/or MTB culture evidence and was considered as MTB positive. Any samples returning a positive pyogenic or fungal culture were considered as MTB negative. The sensitivity, specificity for Xpert MTB/RIF was assessed against the reference standard. ResultsA total of 125 patients were considered for final analysis, 86 patients met the criteria for ‘Reference standard for diagnosis of TB spine’ (MTB positive). This includes nine patients that were MGIT culture only positive; 45 that were histopathology only positive and 32 were both culture and histopathology positive. There were 39 culture proven (pyogenic-37 and fungal-2) patients included in MTB negative group. The 86 MTB positive patients, included 53 (61.6%) tissue samples and 33 (38.4%) pus samples. The overall analysis showed a 65.1% sensitivity, 100% specificity, 100% PPV and 56.5% NPV for the Xpert MTB/RIF. ConclusionsGene Xpert MTB/RIF showed excellent specificity and was accurate in the identification of drug resistance. The sensitivity was 65% and sampling techniques using pus samples rather than tissue samples could be a possible reason for lower sensitivity.

Application value of metagenomic next-generation sequencing (mNGS) in the diagnosis of different types of tuberculosis

Objective: To investigate the diagnostic value of metagenomic next generation sequencing(mNGS)for different types of tuberculosis(TB). Methods: A retrospective analysis was performed on suspected TB patients admitted to Shanghai Pulmonary Hospital from January 1, 2019 to December 31, 2019. The patients underwent mNGS test, acid-fast staining smear, Mycobacterium tuberculosis (MTB) culture and Xpert MTB/RIF(Xpert). A total of 205 TB suspects were finally included, including 120 males and 85 females, with a median age of 34 (16, 70) years. There were 165 cases of TB, including 92 males and 73 females, with a median age of 30 (12, 67) years. There were 40 non-TB patients, 28 males and 12 females, with a median age of 44.6 (24, 78) years, including 28 cases of infectious diseases and 12 cases of malignant tumors. With the final diagnosis as the reference standard, the diagnostic efficiency of mNGS in different types of tuberculosis (including pulmonary tuberculosis and extrapulmonary tuberculosis) and differences in diagnostic sensitivities between mNGS and other detection methods were calculated, respectively. P<0.05 was considered to be statistically different. Results: With clinical diagnosis as the reference standard, the sensitivity of mNGS to diagnose TB in this study was 60% (99/165, 95%CI: 0.53-0.67), and the specificity was 100% (40/40, 95%CI 0.90-1.00). The positive predictive value was 100% (99/99, 95%CI: 0.95-1.00), and the negative predictive value was 37.74% (40/106, 95%CI: 0.29-0.48). The sensitivity of mNGS in TB was 60% (99/165), higher than that of smear (10.9%, 18/165), Xpert(43.64%, 72/165) and culture (23.03%, 38/165). All the differences were statistically significant (P=0.00). The sensitivity of mNGS in pulmonary TB was 59%(59/100), which was significantly higher than that of smear (15%, 15/100) and culture (26%, 26/100), all P<0.001, but the difference was not statistically significant compared with Xpert (52%,52/100, P=0.09). In the extrapulmonary tuberculosis (EPTB), the sensitivity of mNGS was 61.54% (40/65), higher than that of acid fast staining(4.62%,3/65), Xpert (30.77%, 20/65)and culture (18.45%, 12/65). All the differences were statistically significant (all P<0.001). Conclusion: The mNGS was rapid and efficient in detection of MTB, and was superior to other traditional diagnostic methods for TB, especially for EPTB.

Whole Cell Active Inhibitors of Mycobacterial Lipoamide Dehydrogenase Afford Selectivity over the Human Enzyme through Tight Binding Interactions.

Tuberculosis remains a leading cause of death from a single bacterial infection worldwide. Efforts to develop new treatment options call for expansion into an unexplored target space to expand the drug pipeline and bypass resistance to current antibiotics. Lipoamide dehydrogenase is a metabolic and antioxidant enzyme critical for mycobacterial growth and survival in mice. Sulfonamide analogs were previously identified as potent and selective inhibitors of mycobacterial lipoamide dehydrogenase in vitro but lacked activity against whole mycobacteria. Here we present the development of analogs with improved permeability, potency, and selectivity, which inhibit the growth of Mycobacterium tuberculosis in axenic culture on carbohydrates and within mouse primary macrophages. They increase intrabacterial pyruvate levels, supporting their on-target activity within mycobacteria. Distinct modalities of binding between the mycobacterial and human enzymes contribute to improved potency and hence selectivity through induced-fit tight binding interactions within the mycobacterial but not human enzyme, as indicated by kinetic analysis and crystallography.

Diagnostic Accuracy of Gene Xpert on Bronchoscopy Washings in Patients with Suspected Pulmonary Tuberculosis

Objective: The objective of this study was to determine the diagnostic accuracy of GeneXpert assay of bronchial washing in detecting pulmonary tuberculosis in suspected TB patients with no sputum production taking culture of bronchial washing as gold standard. Methods: This is cross sectional study. Data was collected through structured questionnaire. Patients had bronchoscopy according to standard protocol. Their bronchial washings were sent for culture and GeneXpert assay for Mycobacterium Tuberculosis. Data analysis was done using SPSS 20 version. Results: From one hundred and seventy patients the age was observed as 16-60 years. AFB was detected by GeneXpert assay in 45.3% patients. AFB was detected by culture report in 47.6% patients. The sensitivity of GeneXpert was observed as 87.65%, specificity was 93.26%, and diagnostic accuracy was calculated as 90.59%. By using chi-square t-test, there was significant association found between GeneXpert and Culture report in both gender in all age groups with any duration of symptoms with p-value = 0.000. Conclusion: The GeneXpert assay was highly effective for detecting pulmonary tuberculosis in suspected TB patients who do not have sputum production. Key Words: GeneXpert Assay, Culture Report, Bronchial Washing, Pulmonary Tuberculosis How to Cite: Waseem A. K, Manzoor N, Javaid A, Musharaf M.S, Sohail N, Majeed N. Diagnostic accuracy of gene xpert on bronchoscopy washings in patients with suspected pulmonary tuberculosis. Esculapio.2020;16(04):33-36.

Value of next-generation sequencing in early diagnosis of patients with tuberculous meningitis

ObjectivesTo assess the value of next-generation sequencing (NGS) technology in early diagnosis of patients with tuberculous meningitis (TBM). Methods56 patients with clinically suspected TBM who came to Shandong Provincial Chest Hospital from February 2, 2018 to August 2, 2018 were prospectively included, and the clinical diagnosis and treatment outcomes were followed up. NGS was performed for the cerebrospinal fluid specimens submitted for test on the BGISEQ-100 platform of Tianjin Huada Gene Research Institute and the obtained pathogen sequences were compared with the pathogen data to get the final results. The NGS results were positive for detecting the unique matching sequence of the Mycobacterium tuberculosis (MTB) complex and negative for no unique matching sequence. Patients confirmed with TBM should have at least one of the following four items: cerebrospinal fluid MTB culture positive, smear positive, Xpert MTB/RIF test positive, or MTB nucleic acid polymerase chain reaction (PCR) test positive; clinically diagnosed patients were those with clinically suspected TBM and effective anti-tuberculosis treatment; non-TBM patients were those with other pathogenic basis or clinical exclusion of TBM. The sensitivity and specificity of NGS in early diagnosis of TBM were analyzed. Results22 patients were confirmed with TBM, of which 13 were positive for Xpert MTB/RIF test, 6 were positive for cerebrospinal fluid MTB culture, 5 were positive for MTB nucleic acid PCR test, 12 patients were clinically diagnosed with TBM, and there were 16 cases of non-TBM patients. Among confirmed and clinically diagnosed patients, 20 cases of MTB complex were detected by NGS technology, with a sensitivity of 58.8% (20/34) and specificity of 100% (16/16). Among confirmed patients, the sensitivity of NGS was 63.6% (14/22). Of the 50 specimens that were simultaneously subjected to traditional methods, Xpert MTB/RIF test and NGS, the specificity of the three methods was 100% (16/16) based on clinical diagnosis, and the sensitivity was 29.4% (10/34), 38.2% (13/34), and 58.8% (20/34) respectively. The difference of sensitivity between the first two detection methods and NGS was statistically significant (McNemar test, p = 0.013, x2 = 5.786 and p = 0.065, x2 = 3.273). The sensitivity of traditional methods combined with NGS was as high as 82.4% (28/34). ConclusionsNGS technology could rapidly detect the MTB complex in cerebrospinal fluid with significant sensitivity and specificity, which could be used as an early diagnosis index of TBM. NGS combined with MTB culture could increase the detection rate.

The diagnosis of tuberculous meningitis in adults and adolescents: protocol for a systematic review and individual patient data meta-analysis to inform a multivariable prediction model

Background: Tuberculous meningitis (TBM) is the most lethal and disabling form of tuberculosis. Delayed diagnosis and treatment, which is a risk factor for poor outcome, is caused in part by lack of availability of diagnostic tests that are both rapid and accurate. Several attempts have been made to develop clinical scoring systems to fill this gap, but none have performed sufficiently well to be broadly implemented. We aim to identify and validate a set of clinical predictors that accurately classify TBM using individual patient data (IPD) from published studies. Methods: We will perform a systematic review and obtain IPD from studies published from the year 1990 which undertook diagnostic testing for TBM in adolescents or adults using at least one of, microscopy for acid-fast bacilli, commercial nucleic acid amplification test for Mycobacterium tuberculosis or mycobacterial culture of cerebrospinal fluid. Clinical data that have previously been shown to be associated with TBM, and can inform the final diagnosis, will be requested. The data-set will be divided into training and test/validation data-sets for model building. A predictive logistic model will be built using a training set with patients with definite TBM and no TBM. Should it be warranted, factor analysis may be employed, depending on evidence for multicollinearity or the case for including latent variables in the model. Discussion: We will systematically identify and extract key clinical parameters associated with TBM from published studies and use a ‘big data’ approach to develop and validate a clinical prediction model with enhanced generalisability. The final model will be made available through a smartphone application. Further work will be external validation of the model and test of efficacy in a randomised controlled trial.

Clinical outcomes and molecular characterization of drug-resistant tuberculosis in pre- and extensively drug-resistant disease based on line probe assays

Multidrug-resistant tuberculosis (MDR-TB) represents a significant impact in transmission, outcome, and health costs. The World Health Organization recommends implementation of rapid diagnostic methods for multidrug-resistance detection. This study was performed to evaluate the frequency of pre- and extensively drug resistant tuberculosis (pre-XDR-TB and XDR-TB) among MDR-TB patients, the pattern of resistance mutations for fluoroquinolones and the clinical outcome. Adult patients followed at a Brazilian regional reference center for TB, from January 2013 to June 2019 were included. Stored Mycobacterium tuberculosis (Mtb) cultures were recovered, the DNA was extracted, and the susceptibility test was performed using the line probe assay for second line antimycobacterial drugs, Genotype MTBDRsl version 2.0 (Hain Lifescience, CmbH, Germany). Among 33 MDR-TB included patients, we diagnosed XDR-TB or pre-XDR in five (15%) cases. Of these, mutations related to fluoroquinolones resistance were observed in four Mtb isolates, including one who had no phenotypic resistance profile. In two other patients with phenotypic resistance to ofloxacin, genotypic resistance was not found. Case fatality rate was 60% in pre/XDR-TB group, compared to 3.6% in the remaining of patients. This study observed few cases of pre-XDR and XDR-TB among a MDR-TB cohort. Phenotypic and genotypic assays presented good agreement. Clinical outcome was more favorable for patients with susceptibility to fluoroquinolones and injectable drugs.

Microbiological diagnosis of tuberculosis in Russia in 2020:achievements and challenges

SummaryMonitoring the status of microbiological diagnostics of tuberculosis is of major national and international importance for achieving tuberculosis control objectives. Objective. To study and characterize the current state, major trends and current trends in the development of microbiological diagnostics of TB in progress. Materials and methods. The study covered health care organiza-tions of the executive authorities in the field of health services of the subjects of the Russian Federation. The data of industrial and single time statistical observation, as well as answers to special enquiries were studied. Results and discussion. Detection of TB patients by means of sputum microscopy is insufficient, amount-ing to 14.9% in new cases and 10.8% in recurrent ones. The increase in the share of pulmonary TB patients with a positive sputum microscopy up to 35.1% can be as-sociated with a worsening of active detection due to a new coronavirus infection. The share of newly-detected pulmonary TB patients with a positive culture for Myco-bacterium tuberculosis has also increased to 52.8%, but this is below the target (54.0%) and will not reach the target by 2024 (60.0%). The decrease is seen at both preanalytical and analytical phases, mainly in selected regions. The coverage by rapid methods was 87.4%, including WHO-recommended was 58.0%, which is be-low the target (90%). The coverage by drug sensitivity testing (DST) was 97.1% (target 100%). The coverage by DST of patients with resistance to rifampicin and fluoro-quinolones was 93.5%, the coverage by DST of patients with resistance to linezolid was 35.0%, to bedaquiline — 8.5%, to two drugs — 6.6%. Conclusion. It is advisable to intensify the targeted supervision by specialists in microbiological diagnostics of TB with low target values, paying attention to participation in quality assessment programmes, ensuring the possibility of DST to bedaqui-line and linezolid.

Female genital tuberculosis in Pakistan - A retrospective review of 10-year laboratory data and analysis of 32 cases.

Female genital tuberculosis (FGTB) is an underobserved clinical entity owing to diagnostic challenges stemming from difficulty of obtaining diagnostic specimens and paucibacillary nature of the disease. Yet, FGTB is a cause of infertility, pelvic pain, or menstrual irregularities in high-burden countries. To assess laboratory and microbiology diagnostic utilization for FGTB in Pakistan, we have collected data from 2007 to 2016 to inform the need for improved laboratory diagnostics. The objectives of this study were to determine the proportion of FGTB as culture-confirmed extrapulmonary tuberculosis (EPTB) and to describe the characteristics of women with culture-confirmed FGTB in a nationwide laboratory network in Pakistan. A retrospective database was established by accessing laboratory archives and analyzed by sex and source to determine extrapulmonary cases among women. Data were checked for quality, and after removing patient identifiers and duplicate samples, frequencies were calculated in MS Excel. Clinical characteristics of patients were derived from a linked hospital database for those patients who were diagnosed and managed at the affiliated university hospital in Karachi, Pakistan. Over 10 years, 410,748 mycobacterial cultures were received from multiple geographic sites throughout Pakistan and processed at the study laboratory. The overall mean culture positivity rate was 5.9% ± 3.5%, while the mean culture positivity rate among females was 2.8% ± 0.8%. Among female culture-confirmed tuberculosis cases, the pulmonary-to-EPTB ratio of infection was 5. Over 10 years, a total of 32 FGTB cases were reported on the basis of positive cultures for Mycobacterium tuberculosis; 3 (9.4%) were rifampin resistant. FGTB currently constitutes a small but significant proportion of culture-confirmed EPTB. A fewer number of laboratory requisitions suggest the need to increase awareness and testing. The advent of high-sensitivity molecular testing on extrapulmonary specimens has the potential to improve diagnostic accuracy and improved detection of FGTB cases in high-burden regions.

Comparison of N-acetyl-L-cysteine-sodium hydroxide and Modified Petroff's Decontamination Methods for Mycobacterium tuberculosis Culture

Indonesia is one of 22 countries with a high incidence of TB in the world, particularly related to TB-HIV and MDR-TB cases. Contamination of normal flora from nasopharyngeal tract is the main problem to isolate Mycobacterium tuberculosis (MTB) from sputum. It is needed a safe solution to decontaminate without killing MTB bacilli. N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) is compared with modified Petroff’s which is widely used in laboratories, to get better outcome. Of the 110 sputum samples were collected from suspected cases of Pulmonary TB. Decontamination using 2% NALC-NaOH and Modifed Petroff (4% NaOH) was performed before AFB smear and culture on LJ medium. Each group was assessed for contamination and culture positive rate. The positive culture was validated using chromatography test for detected antigen MPT-64 and PNB. Result of this study showed that NALC-NaOH and modified Petroff’s methods did not significantly affect positivity rate of Acid-Fast Bacillus (AFB) smear, 71% and 66% respectively. Contamination on culture was significantly (p=0,034) higher in samples treated with NALC-NaOH (21%) compared to Modified Petroff methods(13%). Proportion of positive culture on samples treated with NALC-NaOH was lower than Modified Petroff, 65% and 70% respectively with p value=1. Conclusion of this study showed that sputum decontamination using Modified Petroff methods is still more effective than NALC-NaOH to increase positivity rate of MTB culture. However, the two methods were not significantly different to get positivity result on the microscopic examination of AFB.

Single Nucleotide Polymorphism in the rpoB Mycobacterium tuberculosis gene from Papua-Indonesia and Its Impact on Rifampicin Resistance: A Whole-Genome Sequencing Analysis

In the antibiotic era, Tuberculosis (TB) drugs resistance especially Rifampicin (RIF) is highly reported around the world. Resistance of RIF is caused by the mutation of genes that associated with RIF receptor. The aims of this study are detecting the Single Nucleotide Polymorphism of Rifampicin resistant genes using Whole Genome Sequencing (WGS) and analysing the profile of protein changing caused by SNP. Twenty Mycobacterium tuberculosis culture samples were passed on WGS procedure and 19 samples were adequate to further bioinformatics analysis. Single Nucleotide Polymorphisms Analysis was done using TBprofiler. Based on TBProfiler, seventeen samples were resistant to rifampicin. The mutations that cause the resistance are S450L, D435Y, H445Y, 430P, Q432K. Other Single Nucleotide Polymorphisms H835R, V534M and R224C were also found. The H835R mutants are present together with the S450L, V534M with S450L mutants, and R224C with Q432K mutants. Native protein for RNA Polymerase Subunit β used was the result of separation from the crystal structure of Mycobacterium tuberculosis H37Rv RNA polymerase (PDB: 5UHB). Binding affinity RIF to RNA Polymerase Subunit β calculated using AutoDock vina. Construction of mutant 3D structures using FoldX5. From the analysis, it was found that seventeen samples were resistant to rifampicin and two samples did not contain SNP which could cause resistance to rifampicin.

Ex Juvantibus Diagnosis of Recurrent Granulomatous Flexor Tenosynovitis: Insidious Mycobacterium Tuberculosis

Objective We report a case of recurrent painful and disabling granulomatous flexor tenosynovitis of the right wrist in 50-year-old female patient. Summary of background data The proper diagnosis was made ex juvantibus after repeated cultivation of Mycobacteria yielded negative results and local disease control was surgically unsuccessful. Methods It was then decided to start at first with oral clarithromycin at 500 mg, 2 times daily, during 6-month period throughout which there was no sign of local recurrence. However, synovectomy had to be performed 3 times in total during the period of 2 years. Although the noninfectious causes were excluded, and infectious agent was not to be determined, the decision was made to administer full anti-tuberculosis medication therapy. At about the same time, we were finally able to obtain a positive Mycobacterium tuberculosis culture after a sudden onset of right axillary lymphadenitis, but only two and half years from the occurrence of first symptoms related to right wrist tenosynovitis.

Development of technology for longterm storage of Mycobacterium tuberculosis cultures

Objective. To develop a technology for freezing and long-term storage of mycobacteria cultures and implement into a microbiological laboratory routine practice. Materials and Methods. For comparison between different condition, the media for freezing mycobacteria containing 0.9% NaCl or Middlebrook’s 7H9 medium with the addition of glycerol at various concentrations: 0%, 4.0%, 20.0%, 50.0% (by final volume) was evaluated. To freeze suspensions of cultures, two methods were used: one – using a cryo-freezer (cooling rate of 1°C/min), the second – direct placement in a deep freeze at a temperature of -80°C. Suspension of a museum strain of Mycobacterium tuberculosis H37Rv was used for preparing suspension. The efficiency of the chosen storage method was assessed by the proportion of viable cells obtained after thawing the suspensions (total number of cells according to quantitative PCR data/number of CFU in Lowenstein-Jensen medium). Results. After storage under various conditions, from 1.2% to 16.9% of mycobacterial cells remained viable. The maximum percentage of viable cells of 16.9% (95% CI 8.6–42.4%) reached when stored in a freezer at a temperature of -80°C without using a cryo-freezer in a medium consisting of 0.9% NaCl with glycerol (20.0% by final volume). However, no statistically significant differences in viable cells were found in the medium containing 0.9% NaCl solution with the addition of 4.0%, 50.0% glycerol, as well as Middlebrook’s 7H9 medium with and without glycerol. Conclusions. A technology for long-term storage of mycobacterial cultures has been developed, based on freezing a cell suspension at a temperature of -80°C in a medium consisting of a 0.9% NaCl solution with the glycerol (20.0% volume fraction), which provides a high degree of cell viability preservation, is lowcost and can be easily integrated into the routine work of a microbiological laboratory.