Lowenstein-Jensen Research Articles

Second-Line Drug Susceptibility Testing of Mycobacterium tuberculosis Isolates from Patients in Bayelsa State, Nigeria

Aim: To determine the second-line drug susceptibility testing of Mycobacterium tuberculosis isolates from patients in Bayelsa State, Nigeria.
 Study Design: A cross sectional study was carried out in this research.
 Place and Duration of Study: Directly Observed Treatment Short-course (DOTS) Centers across Bayelsa State, between March 2020 and November 2021.
 Methodology: Ethical approval was obtained from Bayelsa State Ministry of Health, Yenagoa, Nigeria. Informations such as age, sex and residential address was obtained with the help of a questionnaire. A total of 100 sputum sample was collected from 100 patients across all the Local Government Areas. Sputum sample decontamination and homogenization was done using the Sodium Hydroxide/N- Acetyl -L- Cysteine Citrate Solution. Sputum samples were cultured on solid Lowenstein Jensen (LJ) Media. All growths on LJ media were confirmed with Ziehl Neelsen staining and Standard Bioline antigen test. Drug susceptibility test was carried out after bacterial DNA was extracted and amplified using Line Probe Assay (MTBDRplus assay ver 2).
 Results: Out of 100 patients, 15 had confirmed growth of Mycobacterium tuberculosis. All isolate had no form of mutation on gyr gene, meaning 100% of isolates were susceptibility to flouroquinolones. There were also no mutation detected on rrs gene therefore all strains are also susceptible to Kanamycin, Amikasin and capreomycin. Out of the 15 isolates 14 had no mutation on the eis gene while 1% had mutation of WT2 and MUT1.
 Conclusion: A good percentage of the isolates are susceptible to second line drugs, therefore cases of extensive drug resistance is not common in Bayelsa State, Nigeria.

IMMUNE RESPONSE UPON THE ADMINISTRATION OF RECOMBINANT PROTEIN ANTIBODIES Ag-38 KDa Mycobacterium tuberculosis AND RIFAMPICIN EX-VIVO.

Background:Development a granuloma model resembling latent tuberculosis in vitro is needed with a fast and efficient time to be used as an effective therapy. This study aimed to form efficient granulomas, increase cellular immunity and humoral immunity, and evaluate growth on media using recombinant protein antibody Ag38kDa, Rifampicin, and a combination of both. Peripheral Blood Mononuclear Cell (PBMC) in vitro is derived from a healthy individual separated from monocytes and lymphocytes.Materials and methods:Monocytes are matured into macrophages and then combined macrophages and lymphocytes to the Roswell Park Memorial Institute (RPMI) medium. Flow cytometry analysis was used to count the number of cells, and cytokine levels were measured using ELISA. The result from the treatment was planted on the Lowenstein-Jensen medium.Results:Granulomas-like aggregates was formed after one-day post-infection with Mycobacterium tuberculosis (M.tb). A significant increase in immune response occurred in the number of macrophages, Th1, and Tregs in the combination group compared to the Mtb infection group. The number of Th2 and Th17 cells in the combination group was compared with the control but not significantly. TNF-α cytokine levels increased in the combination group compared to Mtb infection, while in IL-4, we found between all groups, there was no significant difference. Bacterial colonies on culture in the Lowenstein-Jensen medium were only seen in positive controls.Conclusion:Our study concluded that administration of a combination between Ag38kDa recombinant antibody and rifampicin could inhibit granuloma formation and enhance immune response.

Stool-based Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis in children at a teaching and referral hospital in Southwest Ethiopia.

Diagnosis of tuberculosis (TB) in children is challenging mainly due to the difficulty of obtaining respiratory specimen and lack of sensitive diagnostic tests. The objective of this study was to evaluate the diagnostic performance of Xpert MTB/RIF (Xpert here after) for the diagnosis of pulmonary TB (PTB) from stool specimen in children. A cross-sectional study was conducted among consecutively recruited children (less than 15 years old) with presumptive PTB at Jimma Medical Center, Ethiopia. One pulmonary specimen (expectorated sputum or gastric aspirate) was collected from each participant and tested for TB by Xpert and Lowenstein-Jensen (LJ) culture. In addition, one stool specimen per child was collected and tested by Xpert after a single step, centrifuge-free stool processing method adapted from KNCV TB Foundation. Diagnostic performance of Xpert was calculated with reference to LJ culture and to a composite reference standards (CRS) comprising of confirmed TB (positive by Xpert and/or culture) and unconfirmed TB (clinical diagnosis with improvement after anti-TB treatment). A total of 178 children were enrolled; 152 of whom had complete microbiological results. Overall, TB was diagnosed in 13.2% (20/152) of the children with presumptive TB. Of these, only ten had microbiologically confirmed TB (positive Xpert and/or culture) and the remaining ten were clinically diagnosed with positive response to anti-TB treatment and were classified as unconfirmed TB. Stool Xpert had sensitivity of 100% (95%CI: 66.4-100) and specificity of 99.3% (95%CI: 96.2-100) compared to culture; however, the sensitivity was decreased to 50% (95%CI: 27.2-72.8) when compared to CRS. The Xpert on gastric aspirate had sensitivity of 77.8% (95%CI: 40-97.2) compared to culture and 40% (95%CI: 19.1-64) compared to CRS. The sensitivity of Xpert for stool sample is comparable to that for gastric aspirate. Stool sample is a potential alternative to pulmonary specimen in the diagnosis of pulmonary TB in children using Xpert.

Comparison of Conventional Methods with Newer Diagnostic Modalities to Detect Genital Tuberculosis in Infertile Women.

Genital tuberculosis is one of the leading causes of female infertility. Paucibacillary nature of the disease in the female genital system often makes its diagnosis difficult. No single test has been able to accurately diagnose genital tuberculosis. In this study we aim to compare conventional diagnostic tests for tuberculosis like Acid Fast Bacilli (AFB) Staining, Lowenstein Jensen (LJ) Culture and Histopathology with newer tests like PCR, MGIT 960, GeneXpert. This study included 67 infertile women from Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi. They were subjected to detailed history and routine investigations, namely Haemogram, ESR, Mantoux test, Chest X-ray and pelvic ultrasound to look for the findings of tuberculosis. A premenstrual endometrial aspirate was taken and was subjected to the AFB Staining, LJ Culture, Histopathology, PCR, MGIT 960, Gene Xpert, and the test results were compared. 35.8% (24/67) of women were diagnosed with genital tuberculosis using the diagnostic criteria. With culture as the gold standard, the positivity of genital TB was 19.4% (13/67). Majority of infertile patients with low index of suspicion clinically were positive for genital tuberculosis. Therefore, all the patients of infertility should be routinely evaluated for genital tuberculosis. PCR and MGIT 960 have shown promising results in the newer methods. LJ culture and histopathology are still the most reliable and available diagnostic methods. The usefulness of AFB Staining and GeneXpert remains questionable.

Spoligotyping of Mycobacterium tuberculosis isolates from Pulmonary Tuberculosis patients from North Karnataka, India.

Tuberculosis (TB) is a leading cause of morbidity and mortality in low income countries. Multi-drug resistant (MDR-TB) is seen as the reason for many TB outbreaks globally and is also a threat to control programmes. India accounts for 27% TB cases worldwide. Our study was undertaken to understand the outbreaks related to MTB. All the sputum samples were subjected to microscopy and smear positive samples were cultured on Lowenstein-Jensen (L-J) media. Identification was carried by biochemical analysis. A total of 57 isolates were subjected to Drug Susceptibility testing (DST) and spoligotyping, where eleven MDR-TB isolates were confirmed, of which ten were SIT1/Beijing and one SIT53/T1. Spoligotyping results showed that the predominant lineage in this region was SIT1/Beijing followed by SIT124/U and the strains which did not match spoligodatabase were named as orphans. In this study, MDR-TB was associated with SIT1/Beijing and mono resistance belonged to CAS1_DEL.

Diagnostic performance of GeneXpert MTB/RIF in detecting MTB in smear-negative presumptive TB patients

BackgroundTuberculosis (TB) is a leading cause of morbidity and mortality worldwide. Control of TB is lingering by the lack of diagnostic tests that are simple, rapid, yet accurate. Thus, smear-negative pulmonary TB often misses the diagnosis. The study evaluated the performance of GeneXpert MTB/RIF assay for the detection of Mycobacterium tuberculosis (MTB).MethodsThe study was carried out from June to December 2016 in Nepal Tuberculosis Center, Bhaktapur, Nepal. A total of 173 sputum samples were collected and processed by microscopy [Auramine-O staining and Ziehl–Neelsen (ZN) staining], followed by GeneXpert MTB/RIF assay and culture in Lowenstein-Jensen (LJ) medium.ResultsOf 173 sputum samples, 162 (93.6%) were smear-negative. Of 162 smear-negative sputum samples, 35 (21.6%) were confirmed to have MTB by culture, and 31 (19.1%) by GeneXpert MTB/RIF assay. Of 31 GeneXpert-positive samples, 25 (80.6%) were susceptible, 4 (12.9%) were resistant, and 2 (6.45%) were intermediate to rifampicin. The sensitivity, specificity, positive predictive value, and negative predictive value of GeneXpert MTB/RIF assay for smear-negative sputum samples were 74.3%, 96.6%, 86.7%, and 92%, respectively. The GeneXpert MTB/RIF has a substantial diagnostic agreement of 90.91% with culture (Cohen’s Kappa coefficient = 0.73).ConclusionThe diagnostic performance of GeneXpert MTB/RIF assay was almost on par with culture, and thus can be relied upon for MTB detection in smear-negative sputum samples.

Integration of cytopathology with molecular tests to improve the lab diagnosis for TBLN suspected patients.

BackgroundTuberculosis lymphadenitis (TBLN) diagnosis is often challenging in most resource poor settings. Often cytopathologic diagnosis of TBLN suspected patients is inconclusive impeding timely clinical management of TBLN suspected patients, further exposing suspected patients either for unnecessary use of antibiotics or empirical treatment. This may lead to inappropriate treatment outcome or more suffering of suspected patients from the disease. In this study, an integrated diagnostic approach has been evaluated to elucidate its utility in the identification of TBLN suspected patients.MethodsA cross-sectional study was conducted on 96 clinically diagnosed TBLN suspected patients, where fine needle aspirate (FNA) samples were collected at the time of diagnosis. FNA cytology, Ziehl-Neelsen (ZN), Auramine O (AO) staining, GeneXpert MTB/RIF and Real time PCR (RT-PCR) were performed on concentrated FNA samples. Considering culture as a gold standard, the sensitivity, specificity, positive and negative predictive values were calculated. Cohen’s Kappa value was used to measure interrater variability and level of agreement and a P-value of <0.05 was considered as statistically significant.ResultOut of the 96 FNA sample, 12 (12.5%) were identified to have Mycobacterium tuberculosis (Mtb) using ZN staining, 27 (28.1%) using AO staining, 51 (53.2%) using FNAC, 43 (44.7%) using GeneXpert MTB/RIF, 51 (53.1%) using Real time PCR (RT-PCR) and 36 (37.5%) using Lowenstein-Jensen (LJ) culture. Compared to LJ culture, the sensitivities of GeneXpert MTB/RIF, RT-PCR, and FNAC were 91.7%, 97.2%, and 97.2%, respectively and the specificities were 83.3%, 73.3%, and 68.3%, respectively. GeneXpert MTB/RIF and RT-PCR when combined with FNAC detected 61 (63.5%) cases as having Mtb, and the sensitivity and specificity was 100% and 58.3%, respectively.ConclusionFNA cytology and RT-PCR detected more TBLN cases compared to other Mtb detection tools and the detection sensitivity even improved when FNA cytology was combined with GeneXpert MTB/RIF, performed on concentrated FNA sample, suggesting the combined tests as an alternative approach for improved diagnosis of TBLN.

Spoligotyping of Clinical Isolates of Mycobacterium tuberculosis Complex Species in the Oromia Region of Ethiopia.

BackgroundTuberculosis (TB) is a leading cause of morbidity and mortality in Ethiopia. Investigation of the Mycobacterium tuberculosis complex (MTBC) species circulating in the Ethiopian population would contribute to the efforts made to control TB in the country. Therefore, this study was conducted to investigate the MTBC species and spoligo patterns in the Oromia region (central) of Ethiopia.MethodsA cross-sectional study design was used to recruit 450 smear positive pulmonary TB (PTB) cases from the Oromia region between September 2017 and August 2018. Mycobacteria were isolated from sputum samples on the Lowenstein Jensen (LJ) medium. Molecular identification of the isolates was performed by spoligotyping. The results of spoligotyping were transferred into a query box in the SITVIT2 database and Run TB-Lineage in the TB Insight website for the identification of spoligo international type (SIT) number and linages of the isolates, respectively. Statistical Product and Service Solutions (SPSS) 20 was applied for statistical analysis.ResultsThree hundred and fifteen isolates were grouped under 181 different spoligotype patterns. The most dominantly isolated spoligotype pattern was SIT149 and it consisted of 23 isolates. The majority of the isolates were grouped under Euro-American (EA), East-African-Indian (EAI), and Indo-Oceanic (IO) lineages. These lineages consisted of 79.4, 9.8, and 9.8% of the isolates, respectively. One hundred and sixty-five of the isolates were classified under 31 clustered spoligotypes whereas the remaining 150 were singleton types. Furthermore, 91.1% of the total isolates were classified as orphan types. Clustering of spoligotypes was associated (p < 0.001) with EAI lineage.ConclusionSIT149 and EA lineage were predominantly isolated from the Oromia region substantiating the findings of the similar studies conducted in other regions of Ethiopia. The observation of significant number of singleton and orphan spoligotypes warrants for additional genetic typing of the isolates using method(s) with a better discriminatory power than spoligotyping.

Accuracy of Xpert MTB/RIF assay in diagnosis of tuberculosis a comparison to culture

Culture method is the most sensitive and specific method for the detection of Mycobacterium tuberculosis. Despite this, cultures are highly prone to contamination and the process can still take several days and does require expensive equipment, strict biosafety practices and well trained technical staff .Among the currently available Nucleic Acid Amplification Tests (NAAT), the Xpert MTB/RIF Assay (CB-NAAT), the LINE Probe Assay (LPA) and the Loop-Mediated Isothermal Amplification (LAMP) are endorsed by WHO for in vitro diagnosis of TB. The present study was carried out to assess the performance of CB-NAAT (Cepheid GeneXpert®) system for the diagnosis of MTB in both pulmonary and extra pulmonary specimens, within the demographic area of south Kashmir, India. Materials and methods: In this study Pulmonary &amp; extra pulmonary specimens received at the Microbiology laboratory, from the new clinically suspected tuberculosis patients were used. The samples received at the laboratory were divided into three portions, one part each was used for AFB direct smear preparation, CB-NAAT and culture, respectively. A smear was reported positive if either the direct or the concentrated smear showed the presence of AFB. The concentrates were cultured on LJ media and incubated for up to 8 weeks.

A Report on Drug Resistance Patterns of Mycobacterium tuberculosis Isolates in Northern Iran.

There are limited data on the Mycobacterium tuberculosis (MTB) drug resistance in regions located at the proximity of the Caspian Sea. We aimed to assess the drug resistance patterns of the MTB isolates to anti-tuberculosis drugs in patients from four northern provinces of Iran between April 2013 and March 2019. Drug susceptibility testing (DST) was performed by culturing MTB isolates on the Lowenstein-Jensen medium using the proportion method. Out of 963 MTB isolates, 927 (96.3%) were recovered from Iranian cases and 36 (3.7%) were from Afghan immigrants. Based on DST, 59 (6.1%) showed any drug resistance pattern, while 18 patients (1.9%) were multidrug-resistant (MDR) or rifampicin-resistant (RR). Resistance to streptomycin (STR), isoniazid (INZ), rifampicin (RIN), and ethambutol (ETL) was reported in 33 (3.4%), 28 (2.9%), 18 (1.9%), and 12 isolates (1.2%), respectively. The rate of MDR/RR in four northern provinces of Iran was in line with previous reports from the World Health Organization. Due to proximity to the former Soviet Union, which had a high rate of MDR/RR isolates, the establishment of cross-border tuberculosis (TB) control strategies is recommended to reduce the possibility of MDR-TB transmission. Moreover, DST for all TB cases is recommended as an effective diagnostic tool for optimal monitoring and control of drug resistance in these areas. Future studies with a molecular epidemiology approach will be needed to evaluate the transmission dynamics of MTB in these regions.

Detection of Mycobacterium tuberculosis Rifampicin Resistance Conferred by Borderline rpoB Mutations: Xpert MTB/RIF is Superior to Phenotypic Drug Susceptibility Testing

ObjectiveTo compare the ability of detection of borderline rifampicin resistance in Mycobacterium tuberculosis between molecular assay and phenotypic drug susceptibility tests.MethodsFifty-seven isolates with His445Leu, Asp435Tyr, Leu452Pro, Leu430Pro, His445Asn, Ile491Phe, and His445Ser mutations in rpoB gene identified by whole-genome sequencing conferring borderline rifampicin resistance were included. Molecular-based Xpert MTB/RIF, phenotypic Löwenstein–Jensen (L-J) medium-based drug susceptibility test (DST) with a critical concentration of 40.0μg/mL and minimal inhibitory concentration (MIC) assay were performed to detect borderline rifampicin resistance.ResultsWhen using Xpert MTB/RIF, 48/57 (84.2%) isolates exhibited resistance to rifampicin. 25/57 (43.9%) and 33/57 (57.9%) isolates showed rifampicin resistance by L-J medium-based DST with 4 and 6 weeks of incubation, respectively. 30/57 (52.6%) and 40/57 (70.2%) strains were resistant to rifampicin by MIC method at cutoff values of 1.0 and 0.5μg/mL, respectively. The detection rate of rifampicin resistance of Xpert MTB/RIF was significantly higher than that of phenotypic methods (p < 0.001). Of the 57 isolates with borderline rpoB mutations, 5 (8.8%) had MICs of 0.25 or 0.12μg/mL, 22 (38.6%) had MICs of 0.5μg/mL or 1.0μg/mL, and 30 (52.6%) other isolates showed MICs ≥2.0μg/mL.ConclusionMolecular-based Xpert MTB/RIF showed superior ability to detect borderline rifampicin resistance over phenotypic DST methods. Extending the incubation time of L-J DST or lowering the cutoff value of the MIC method can improve borderline rifampicin resistance detection.

Rapid Detection of Mycobacterium tuberculosis in Pleural Fluid Using Resuscitation-Promoting Factor-Based Thin Layer Agar Culture Method.

BackgroundPleural tuberculous is difficult to diagnose. Culture is still considered the gold standard, especially in resource-limited settings where quick, cheap, and easy techniques are needed. The aim of the study was to evaluate resuscitation-promoting factors (Rpfs)-based thin layer agar (TLA) culture method for quick detection of Mycobacterium tuberculosis in pleural fluid.MethodsPatients with suspected pleural TB were enrolled prospectively in our hospital, pleural fluid of all patients were collected, stained with Ziehl–Neelsen for acid-fast bacilli (AFB), cultured on Rpfs-TLA, TLA, and Löwenstein–Jensen (LJ) medium, and identified according to recommended procedures.ResultsA total of 137 suspected pleural TB were enrolled and categorized, including 103 pleural TB (49 confirmed and 54 probable pleural TB) and 34 non-TBP patients. The sensitivity of Rpfs-TLA for total pleural TB was 43.7% (34.5∼53.3%), higher than that of TLA 29.1% (21.2∼38.5%) and LJ 26.2% (18.7∼35.5%) (p < 0.01), and all specificity was 100% in the diagnosis of pleural TB. Median time to detection of a positive culture was 11.8 days (95% CI 10.4∼13.4) for Rpfs-TLA, 21.0 days (95% CI 19.1∼22.9) for TLA, and 30.5 days (95% CI 28.5∼32.5) for LJ (p < 0.001).ConclusionRpfs-TLA is an accurate, rapid, cheap, and easy culture method, which makes it promising for use in clinical laboratories.

Subcutaneous Nocardiosis in a child with T- cell Immunodeficiency: A case report

Nocardia are weakly gram-positive, filamentous bacteria found worldwide in soils. Infection with Nocardia is rare inimmunocompetent patientsbut may leadto severe disease in immunocompromised patients. Reported cases in paediatric age group are few, and the literature is limited. We present a case of Nocardia isolated from pus from a skin lesion of eleven year old immunocompromised male child. Pus from Skin lesion on Ziehl-Neelsen (ZN) stain showed Acid Fast, Branching filamentous Bacteria. Pus culture on Lowenstein-Jensen (LJ) media showed glabrous, tough and waxycolonies. It was confirmed to be Nocardia species on Gram stain and modified ZN Stain. No organism was isolated on culture from Blood, urine and Cerebrospinal fluid (CSF). High clinical suspicion is required for diagnosis of slow growing bacteria such as Nocardia as they can be easily be missed on routine bacteriological culture.

Non-tuberculous mycobacteria, not Mycobacterium bovis, are a significant cause of TB-like lesions observed in slaughtered cattle in Ghana

ObjectivesThe aim was to isolate and identify the species of mycobacteria causing tuberculous-like (TB-like) lesions in cattle in Ghana. MethodsBetween 2019 and 2020, 68 bovine tissue samples with TB-like lesions, identified during post slaughter examination, were obtained from four major abattoirs close to border towns in Ghana. The samples were cultured on Lowenstein–Jensen medium. Isolated bacteria were characterized by Ziehl–Neelsen staining and observation for acid-fast bacilli (AFB) under a microscope. DNA was extracted from AFB-positive isolates, and mycobacterial speciation was performed by line probe assay using GenoType Mycobacterium CM and also with mycobacterial 16S rRNA gene amplification and sequencing. ResultsNo Mycobacterium bovis was identified; however 53 bacterial isolates were obtained, of which 41 were non-tuberculous mycobacteria (NTM) strains and 12 were gram-positive bacteria. The predominant NTM species was M. fortuitum (43.9%, 18/41), with the rest being M. novocastrense, M. terrae, M. flavescens, M. holsaticum, M. cosmeticum, M. virginiense, M. intracellulare, M. mageritense, M. minnesotensis, M. duvalii, M. lehmannii, and M. koreense. ConclusionsIn cattle, NTM contribute significantly to lesions observed during slaughter examination and may be an important cause of zoonotic tuberculosis. A One Health surveillance of NTM in Ghana would provide insights into their clinical significance.

Performance of WHO-Endorsed Rapid Tests for Detection of Susceptibility to First-Line Drugs in Patients with Pulmonary Tuberculosis in Bangladesh.

The fast and accurate detection of susceptibility in drugs is a major challenge for a successful tuberculosis (TB) control programme. This study evaluated the performance of WHO-endorsed rapid diagnostic tools, such as BACTEC MGIT 960 SIRE (MGIT SIRE), GenoType MTBDRplus (MTBDRplus) and Xpert MTB/RIF (Xpert), for detecting susceptibility to first-line anti-TB drugs among pulmonary TB patients in Bangladesh. A total of 825 sputum samples with results from drug susceptibility testing (DST) against first-line anti-TB drugs in the MGIT SIRE, MTBDRplus and Xpert assays were evaluated and compared with the gold standard proportion susceptibility method of the Lowenstein–Jensen (LJ) medium. The overall sensitivities of MGIT SIRE were 97.6%, 90.0%, 61.3% and 44.9%, while specificities were 89.9%, 94.5%, 91.3% and 92.2% for detection of susceptibility to isoniazid (INH), rifampicin (RIF), streptomycin (STR) and ethambutol (EMB), respectively. For MTBDRplus, the sensitivities were 88.0% and 88.7%, and the specificities were 97.4% and 97.8% for the detection of susceptibility to INH and RIF, respectively. Xpert demonstrated a sensitivity and specificity of 94.8% and 99.5%, respectively, for the detection of RIF susceptibility. All tests performed significantly better in retreated TB patients compared with primary TB cases. For detection of RIF and INH susceptibility, all three assays showed almost perfect agreement with the LJ method, although MGIT SIRE exhibited low agreement for STR and EMB. Considering the high performance, shorter turnaround time and ease of use, molecular-based approaches Xpert and MTBDRplus can be widely implemented throughout the country for the rapid detection of drug-resistant TB.

Epidemiology of Avian Tuberculosis in Selected Districts of Oromia Region, Ethiopia.

Avian tuberculosis is a growing public health concern and a significant impediment to socioeconomic development, especially in developing countries, where the risk of infection is high. The disease is predominantly caused by Mycobacterium avium belonging to serotypes 1, 2, 3, and 6 (genotypes IS901 and IS1245) and Mycobacterium genavense. It mostly occurs in older birds and immunocompromised individuals due to the greater opportunity for infection with age and host immunity. A cross-sectional study anticipated to generate epidemiological information on avian tuberculosis was carried out from November 2016 to June 2017 at highland areas of Gerar Jarso, Ada'a (midland), and Boset (lowland) districts of Oromia region, Ethiopia. Two hundred seventy-three village chickens comprising local breeds, exotic breeds, and crossbreeds of both sexes were used in the study. Single intradermal avian tuberculin test, postmortem inspection of positive reactors, mycobacteriological culturing, and histopathological examination were used to assess information on the epidemiology of the disease. Subsequently, avian tuberculin test revealed an overall apparent prevalence of 11.4% (31/273) and a specific prevalence of 6.8% (6/88) in the highland, 13.4% (13/97) in the midland, and 13.6% (12/88) in the lowland study districts. Besides, it signified a higher odd of exposure in crossbreeds and females as compared to locals and males. In addition, greater odd of exposure was observed in chickens at mid- and low altitudes as compared to those at the highland. Moreover, 40.9% (9/22) of positive reactor chickens sacrificed for necropsy showed gross pathological lesions. Similarly, histopathological examination revealed a granuloma characterized by central necrosis and peripheral mononuclear lymphocytes. Nevertheless, only 0.02% (2/120) of the cultured tissues had shown colonial growth up to 12 weeks of incubation, and both were seen on sodium pyruvate-enriched Lowenstein–Jensen medium slants. Generally, the study revealed an overall increment of the apparent prevalence of avian tuberculosis with decreasing altitude. Besides, it signified a relative breed and sex variation in the risk of acquiring the disease, with crossbreeds and female chickens having higher odds of exposure.

Bovine mastitis caused by rapid-growth environmental mycobacteria

Rapid-growth mycobacteria were isolated from two cases of cow mastitis with similar clinical appearance and within a narrow time frame. Mycobacteria were isolated on blood esculine agar. The isolated mycobacteria were Gram stained, Ziehl-Nielsen stained and tested for growth at 25°C, 37°C and 42°C, iron uptake, growth on Löwenstein-Jensen (LJ) agar with and without 5% NaCl, arylsulphatase (3 days), tween 80 hydrolysis, tellurite reduction, nitrate reductase and niacin synthesis. Molecular identification was performed using the Mycobacteria GenoType CM and AS tests (Hain Diagnostika, Nehren, Germany). One isolate was additionally sequenced for the hsp65, rpoB, 16S rRNA gene sequence and transcribed spacer sequence (ITS) DNA. Susceptibility testing of isolates was performed on the Sensititre Rapmycol plate (TREK Diagnostic Systems Ltd.) for trimethoprim/sulfamethoxasole, linezolid, ciprofloxacin, imipenem, moxifloxacin, cefepime, cefoxitin, amoxicillin / clavulanic acid, amikacin, ceftriaxone, doxycycline, minocycline, tigecycline, tobramycine and clarythromycine. Gram-positive acid-resistant rods were observed in stained smears. Both strains grew at 25°C, 37°C and 42°C on LJ medium, and on LJ medium containing 5 % NaCl. The conventional biochemical tests for iron uptake, arylsulphatase (3 days), Tween 80 hydrolysis, tellurite reduction and nitrate reductase were positive, while the niacin test was negative. Both isolates were identified by the GenoType Mycobacterium CM as Mycobacterium fortuitum II/ Mycobacterium mageritense, while application of the GenoType Mycobacterium AS kit identified both isolates as belonging to the species Mycobacterium smegmatis. Analysis of the isolate sequences (strain DS) for 16S ribosomal RNA confirmed a 100% identical result with Mycobacterium smegmatis strain INHR2. According to the CLSI criteria, both strains were sensitive to sulfametoxazole/trimethoprim, linezolid, doxicycline, amikacin and tobramycin. The strains differed in their sensitivity to cefoxitim, and both strains were resistant to clarithromycin. There was a strong difference between the isolates in sensitivity toward cefoxitime and tigecycline.

A Comparative Study of Cartridge-based Nucleic Acid Amplification Test and ZiehlÂ-Neelsen Stain with Culture on LowensteinÂ-Jensen Media as Gold Standard for the Diagnosis of Pulmonary Tuberculosis

A Comparative Study of Cartridge-based Nucleic Acid Amplification Test and ZiehlÂ-Neelsen Stain with Culture on LowensteinÂ-Jensen Media as Gold Standard for the Diagnosis of Pulmonary Tuberculosis

ПОРІВНЯННЯ СИСТЕМ GENEXPERT MTB/RIF І GENOTYPE ЗІ СТРИПАМИ MTBDRPLUS ДЛЯ ВИЯВЛЕННЯ МУТАЦІЙ, ПОВ’ЯЗАНИХ З РЕЗИСТЕНТНІСТЮ M. TUBERCULOSIS ДО РИФАМПІЦИНУ ПРИ ТУБЕРКУЛЬОЗІ

COMPARISON OF GENEXPERT MTB/RIF AND GENOTYPE SYSTEMS WITH MTBDRPLUS STRIPS FOR DETECTION OF MUTATIONS, ASSOCIATED WITH M. TUBERCULOSIS RESISTANCE TO RIFAMPICIN IN TUBERCULOSIS A. A. Zhurilo, A. I. Barbova, Yu. A. Cherednik, P. S. Trofi mova, S. V. Mironchenko, O. V. Pavlova, A. V. Chernov, L. M. Sladkova Summary The aim was to analyze the level of compliance of two molecular genetic methods GeneXpert MTB/RIF and GenoTypeDRplus in determining the drug resistance of M. tuberculosis to rifampicin when detecting mutations in the RRDR region associated with drug resistance. Object and methods. We studied strains of M. tuberculosis with resistance to rifampicin, which was detected by any of the studied molecular genetic methods. 96 sputum samples were taken. Sputum smears were examined for the presence of acid-fast bacteria by microscopy after staining with the Ziehl-Neelsen method. The material was inoculated into Middlebrook 7H9 broth and on Lowenstein-Jensen medium. The liquid medium was incubated in the BACTEC MGIT system. An immunochromatographic test was used to identify the strains. The drug susceptibility test of M. tuberculosis to rifampicin was performed using the BACTEC MGIT system. The GeneXpert MTB/RIF test was performed according to the manufacturer’s instructions. The GenoTypeDRplus assay was performed on decontaminated and concentrated sputum samples. The process was carried out in three stages: DNA extraction from the processed sputum sample; amplification of the RRDR region by PCR; hybridization of the PCR product to specific oligonucleotide probes immobilized on the test strip. For sequencing, M. tuberculosis DNA isolation was performed using the QIAamp® DNA Mini Kit. The DNA concentration was measured on a Denovix Quantus spectrophotometer. Targeted panel amplification was performed using the Deeplex Myc-TB kit. Amplicon purification was performed using Agencourt AMPure XP magnetic beads. Quantitative analysis of the purified amplification products was performed using a Qubit fluorometer. The M. tuberculosis DNA library was prepared for sample sequencing using the Nextera XT DNA library preparation kit. The library used 5.0 μl of input DNA at a concentration of 0.2 ng/μl. Sequencing was performed on MiSeq equipment with the library normalization and denaturation protocol. Results and discussion. The GeneXpert MTB/RIF and MTBDRplus systems target the same 81 bp rifampicin resistance domain. (RRDR) subunits of bacterial RNA polymerase (rpoB) for mutation detection using DNA probes, i.e. there is a correspondence of probes to each other and an expected similarity of probe binding. We analyzed all sputum samples using GeneXpert MTB/RIF and GenoType MTBDRplus and phenotypic BACCTEC MGIT methods. The level of agreement between two molecular genetic methods for the detection of rifampicin-associated mutations in the RRDR region has been established. The RRDR 81bp region of the rpoB gene of mismatched cases was studied by sequencing. GeneXpert MTB/RIF and GenoType DRplus matched the phenotypic method in 92.7% and 89.6% of cases of M. tuberculosis resistance, respectively. Complete agreement between the results of GeneXpert MTB/RIF and GenoTypeMTBDRplus was observed in 92.7% of cases. GeneXpert MTB/RIF and GenoType DRplus showed a similar pattern of binding failure of wild type probes (WT-probes) when scanning the 81 bp region (RRDRdomain), which leads to stability diagnostics through probe failure software. Sequencing of the RRDR region of “mismatched” strains showed that GeneXpert probes detected seven “mismatched” cases correctly, and GenoTypeDRplus was erroneous in all cases. GeneXpert has demonstrated greater accuracy in R-resistance detection for mismatched isolates compared to GenoTypeDRplus. GeneXpert MTB/RIF has a number of other benefits over GenoTypeDRplus. GeneXpert MTB/RIF is relatively easier to implement, biosafety requirements are minimal, study times are shorter, and the study process is more automated, resulting in less human error and more reproducible results. Given these facts and the results of GeneXpert MTB/RIF found in the study, it is recommended that GeneXpert MTB/RIF be used to detect MDR-TB. Conclusions. Sequencing of the 81bp RRDR region of mismatched M. tuberculosis strains showed that GeneXpert MTB/RIF performed more accurately than GenoTypeDRplus in detecting mutations associated with rifampicin resistance. GeneXpert MTB/RIF is relatively easier to perform, biosafety requirements are minimal, time to study is shorter, and the study process is more automated, resulting in less human error and greater reproducibility of results, so it is reasonable to use it for the detection of multidrug-resistant tuberculosis. The Deeplex Myc-TB analytical solution delivers a wealth of insightful information from antimycobacterial drug resistance markers and speeds up data analysis with easy-to-use software. Key words: tuberculosis, Mycobacterium tuberculosis, drug resistance, GeneXpert MTB/RIF and GenoTypeDRplus molecular genetic systems, BACTEC MGIT phenotypic system, sequencing. Ukr. Pulmonol. J

Investigation of species distribution of nontuberculosis mycobacteria isolated from sputum samples in patients with suspected pulmonary tuberculosis.

Rapid and accurate identification of mycobacteria is important for the species-specific treatment of the disease. The aim of this study was the identification at the species level of 34 nontuberculous mycobacteria strains isolated from respiratory tract samples and 14 reference strains as by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Isolates derived from clinical specimens were subcultured in the Lowenstein-Jensen medium. Deoxyribonucleic acid isolation was carried out using the boiling method. PCR amplification was performed using primers specific to the hsp65 gene region. The PCR products were digested BstEII and HaEIII enzymes. All samples were studied comparatively by two different centers. In our study, the most common species were found to be Mycobacterium intracellulare in 23.52% (8/34). The performance of the PCR-RFLP method in detecting mycobacteria was found to be 82.35%. The PCR-RFLP method is a rapid, cheap, and practical method for the identification of mycobacteria.