Hain Lifescience Research Articles

Genetic Determinants of Clarithromycin and Fluoroquinolones Resistance in Helicobacter pylori in Serbia.

Stomach infections by Helicobacter pylori can cause acute or chronic gastritis, peptic ulcers, and gastric cancer. The rise in antibiotic resistance is a significant health issue highlighted by the World Health Organization. The increasing number of treatment failures underscores the necessity for antibiotic susceptibility testing (AST). The study aimed to investigate the current prevalence and resistance to fluoroquinolones and clarithromycin with their detected mutations. Stomach biopsies from symptomatic patients were subjected to molecular testing by GenoType Helico DR kit (Hain Lifescience GmbH, Nehren, Germany). Positive findings on the presence of H. pylori were detected in 42.4% of symptomatic patients, with the significant majority of patients (69%) having previously failed treatments. The resistance rates to fluoroquinolones and clarithromycin were 53.9% and 58.5%, respectively, with significantly higher rates in secondary resistant strains. The main resistance markers in fluoroquinolones and clarithromycin were N87K (27.4%) and A2147G (78.6%), respectively. Hetero-resistance or mixed genotypes were detected in over 20% of tested patients. During the study period, a significant increase in trends in both fluoroquinolones and clarithromycin resistance rates was observed. Results indicate the need for the implementation of the latest Maastricht VI Consensus recommendations for both AST whenever possible and the use of tailored guided therapy options due to high resistance rates and possible treatment failures. The GenoType Helico DR kit is a useful tool for AST, especially in cases of mixed H. pylori genotypes.

Drug-resistant Mycobacterium tuberculosis among Nepalese patients at a tuberculosis referral center.

Multidrug-resistant tuberculosis (MDR-TB), characterized by isoniazid and rifampicin resistance, is caused by chromosomal mutations that restrict treatment options and complicate tuberculosis management. This study sought to investigate the prevalence of pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) tuberculosis, as well as mutation pattern, in Nepalese patients with MDR/rifampicin-resistant (RR)-TB strains. A cross-sectional study was conducted on MDR/RR-TB patients at the German Nepal Tuberculosis Project from June 2017 to June 2018. The MTBDRsl line probe assay identified pre-XDR-TB and XDR-TB. Pre-XDR-TB included MDR/RR-TB with resistance to any fluoroquinolone (FLQ), while XDR-TB included MDR/RR-TB with resistance to any FLQ and at least one additional group A drug. Mutation status was determined by comparing bands on reaction zones [gyrA and gyrB for FLQ resistance, rrs for SILD resistance, and eis for low-level kanamycin resistance, according to the GenoType MTBDRsl VER 2.0, Hain Lifescience GmbH, Nehren, Germany definition of pre-XDR and XDR] to the evaluation sheet. SPSS version 17.0 was used for data analysis. Out of a total of 171 patients with MDR/RR-TB, 160 had (93.57%) had MTBC, of whom 57 (35.63%) had pre-XDR-TB and 10 (6.25%) had XDR-TB. Among the pre-XDR-TB strains, 56 (98.25%) were FLQ resistant, while 1 (1.75%) was SLID resistant. The most frequent mutations were found at codons MUT3C (57.14%, 32/56) and MUT1 (23.21%, 13/56) of the gyrA gene. One patient had SLID resistant genotype at the MUT1 codon of the rrs gene (100%, 1/1). XDR-TB mutation bands were mostly detected on MUT1 (30%, 3/10) of the gyrA and rrs, MUT3C (30%, 3/10) of the gyrA, and MUT1 (30%, 3/10) of the rrs. Pre-XDR-TB had a significantly higher likelihood than XDR-TB, with different specific mutation bands present in gyrA and rrs genes.

Dual-centre evaluation of the FluoroType MTBDR version 2 assay for detection of Mycobacterium tuberculosis complex and resistance-conferring mutations in pulmonary and extrapulmonary samples from Denmark, Germany and Sierra Leone

ObjectivesWe evaluated the ability of FluoroType MTBDR version 2 (FTv2; Hain Lifescience), a second-step real-time PCR assay, to simultaneously detect Mycobacterium tuberculosis complex (MTBC) DNA and mutations conferring resistance to rifampicin (RIF) and isoniazid (INH), in pulmonary and extrapulmonary samples from patients and compared them with corresponding cultures. MethodsFTv2 MTBC was evaluated on 1815 and 432 samples from Denmark (DK) and Germany (DE), respectively. RIF and INH resistance mutations were assessed in the German samples and 110 samples from Sierra Leone and subsequently compared to phenotypic antimicrobial susceptibility testing and a composite reference DNA (CRD) based on the GenoType MTBDR line-probe assay and Sanger sequencing or whole-genome sequencing. ResultsOf the 584 (557 smear-negative) Danish and 277 (85 smear-negative) German sputum samples, 42 (16) and 246 (54) were culture positive, and 44 (18) and 222 (35) were FTv2 positive, providing an FTv2 sensitivity and specificity of 0.86 (0.63) and 0.98 (DK), 0.90 (0.65) and 1.00 (DE), respectively. The count, sensitivities, and specificities for all pulmonary samples were 1434, 0.79, and 0.99 (DK) and 347, 0.86, and 1.00 (DE), respectively; for extrapulmonary samples, 381, 0.33, 0.99 (DK) and 83, 0.50, and 1.00 (DE). The valid count, sensitivity, and specificity compared with CRD for detecting resistance mutations were RIF 355, 0.99, 0.96, and INH 340, 1.00, and 0.98, respectively. DiscussionFTv2 reliably detects MTBC DNA in pulmonary and extrapulmonary samples and detects resistance mutations for INH and RIF resistance in inhA promoter, katG, and rpoB genes.

Retrospective and prospective evaluation of the FluoroType®-Mycobacteria VER 1.0 assay for the identification of mycobacteria from cultures in a French center.

Rapid, reliable identification of mycobacteria from positive cultures is essential for patient management, particularly for the differential diagnosis of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) species. The aim of the present study was to evaluate a new "In-Vitro-Diagnostic"-certified PCR kit, FluoroType®-Mycobacteria VER1.0 (Hain Lifescience GmbH) for NTM and MTBC identification from cultures. Mycobacteria identification isolated from positive cultures during routine practice at the Lyon university hospital mycobacteria laboratory obtained by hsp65 amplification/sequencing were compared retrospectively and prospectively to those obtained by and the FluoroType®-Mycobacteria VER1.0kit. The overall agreement between hsp65 amplification/sequencing and the FluoroType®-Mycobacteria VER1.0kit was 88.4% (84/95); 91.2% (52/57) for the retrospective period and 84.2% (32/38) for the prospective period. There were 9 (9.5%) minor discrepancies (species in the FluoroType®-Mycobacteria VER1.0database and identified at genus level): 4 during the retrospective period, 5 during the prospective period; and 2 (2.1%) major discrepancies (species in the FluoroType®-Mycobacteria VER1.0database and identified incorrectly to species level): 1 during the retrospective period (M. kumamotonense identified as M. abscessus subsp massiliense by the kit) and 1 during the prospective period (M. chimaera identified as M. smegmatis by the kit). Including concordant results at genus level and minor discrepancies, 17.9% (17/95) of strains were identified as Mycobacterium sp. by the FluoroType®-Mycobacteria-VER1.0kit. The good performance of the FluoroType®-Mycobacteria-VER1.0kit with few major discrepancies could enable its use for first-line identification of positive mycobacteria cultures. However, an alternative identification method at least for reference laboratories is needed owing to the non-negligible proportion of NTM strains were identified at genus level.

Mutational analysis in drug resistant Mycobacterium tuberculosis in Western Uttar Pradesh

BackgroundMulti-drug resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes. This study was carried out with the aim to determine the pattern of drug resistance against Mycobacterium tuberculosis towards first line ATT (anti-tubercular treatment) in sputum smear–positive patients using Line Probe Assay (LPA). MethodsA cross sectional prospective study was carried out in a tertiary care Hospital of Meerut. A total of 898 sputum samples (on spot and early morning) collected from 449 suspected pulmonary tuberculosis patients as per RNTCP guidelines were screened by microscopy. Decontamination was done by N-acetyl-l-cysteine and sodium hydroxide. Then smear positive samples were subjected to 1st line drug susceptibility testing (DST) using LPA GenoType® MTBDRplus (HAIN Life Science) assay, a molecular method which allows rapid detection of Rifampicin (Rif) and Isoniazid (INH) resistance. ResultsThe overall burden of MDR TB in this geographical area was 7.9 %. Mono-resistance with Rif alone was around 2.8 %. However, the mono-resistance with INH (inhA gene) and INH (katG gene) was 2.8 % and 1.1 % respectively. Drug resistance of Rif was due to mutations in rpoB gene while resistances to INH were more commonly due to mutation in inhA gene followed by katG gene. TB was more commonly seen in the age group of 30–59 years (43.8 %) and predominantly in males. ConclusionTuberculosis positivity rate is high in Western Uttar Pradesh. Burden of MDR TB in Western Uttar Pradesh was similar to National data. Line probe assay can be used as a primary method to diagnose multi drug resistant TB as done in present study which can help in earlier initiation of correct therapy.

Description of Mycobacterium pinniadriaticum sp. nov., isolated from a noble pen shell (Pinna nobilis) population in Croatia.

Shortly before the mass mortality event of the noble pen shell (Pinna nobilis) population in the south-eastern Adriatic coast, two rapidly growing Mycobacterium strains CVI_P3T (DSM 114013 T, ATCC TSD-295 T) and CVI_P4 were obtained from the organs of individual mollusks during the regular health status monitoring. The strains were identified as members of the genus Mycobacterium using basic phenotypic characteristics, genus-specific PCR assays targeting the hsp65 and 16S rRNA genes and the commercial hybridization kit GenoType Mycobacterium CM (Hain Lifescience, Germany). MALDI-TOF mass spectrometry did not provide reliable identification using the Bruker Biotyper Database. Genome-wide phylogeny and average nucleotide identity (ANI) values confirmed that the studied strains are clearly differentiated from their closest phylogenetic relative Mycobacterium aromaticivorans and other validly published Mycobacterium species (ANI ≤ 85.0%). The type strain CVI_P3T was further characterized by a polyphasic approach using both phenotypic and genotypic methods. Based on the phenotypic, chemotaxonomic and phylogenetic results, we conclude that strains CVI_P3T and CVI_P4 represent a novel species, for which the name Mycobacterium pinniadriaticum sp. nov. is proposed.

Pattern and characteristics of mutations conferring resistance to second line drugs in Mycobacterium tuberculosis isolates of pulmonary and extrapulmonary TB samples

Background & objectivesThe purpose of present study is to analyse the distribution and pattern of genetic mutations in PRE-XDR-TB and extensive drug resistant Mycobacterium tuberculosis (XDR-TB) using second-line line probe assay and to compare them with different parameters. MethodSputum, Lymph node aspirate and cold accesses from patients with rifampicin resistant Tuberculosis were subjected to first line and second line Probe Assay (Genotype MTBDRsl by Hain Life Science, Germany) to assess additional drug resistance to fluroquinolones (Levofloxacin & Moxifloxacin) and Aminoglycosides (Amikacin, Ofloxacin and Kanamycin). The genetic mutation pattern was analysed and compared with demographic, clinical and other parameters. ResultsThe final study population included 123 fluoroquinolone resistant isolates including 14 isolates with additional second line aminoglycosides drug resistance. The most frequent mutation observed among Gyr A drug resistance mutation was D94G (Gyr A MUT3C, 50/123,40%) corresponding to high level resistance to levofloxacin and moxifloxacin. The most frequent wild type mutant among Gyr A gene locus was WT 3 (85/123,69%). The most common mutation among second line aminoglycoside resistant isolates was at eis WT2 (7/14,50%) followed by rrs MUT 2 (4/14,29%). ConclusionsGyrA MUT3C (Asp94Gly) was the most common mutation in Gyr A gene locus in M. tuberculosis causing high level levofloxacin and moxifloxacin resistance. Patients with Asp94Gly mutation was significantly associated with underweight body mass index (p = 0.026). This study also observed that history of anti-tuberculosis therapy is a risk factor for FQ drug resistance mutations (p < 0.001).

Molecular Identification of Non-tuberculous Mycobacteria in Suspected Tuberculosis Cases in Central India.

Introduction Mycobacterium tuberculosis complex (MTBC), the primary cause of tuberculosis (TB), must be accurately identified to implement effective patient management and control strategies. Non-tuberculous mycobacteria (NTM) in suspected TB cases can result in erroneous diagnoses and needless treatment. Objective The study aimed to identify NTM in patients suspected of TB at a tertiary care hospital in central India using molecular methods. Methods This prospective study enrolled 400 suspected pulmonary and extra-pulmonary TB patients. Patients between the age of two to 90 years, of either gender, new and previously treated cases, Culture positive, patients with immune-compromised status, patients not responding to ATT, HIV positive and negative, and willing to give consent were included in the study. Liquid culture via the Mycobacterial growth indicator tube (MGIT) system was used to culture mycobacteria from clinical samples. The SD Bioline Ag MPT64 Test (Standard Diagnostics, South Korea) and in-house multiplex-PCR (mPCR) were used to differentiate between Mycobacterium tuberculosis complex and NTM species for the molecular identification of NTM GenoType® Mycobacterium Common Mycobacteria (CM) assay kit (HAIN Life Science, Nehren, Germany) was used following the manufacturer's protocol. Results Only 59/400 (14.7%) of the samples produced a positive result in MGIT culture, indicating the presence of mycobacteria, and 85.25% of the remaining 341 samples were negative for mycobacterial growth. Further investigation of these 59 cultures with mPCR and SD Bioline Ag MPT64 test showed that 12 (20.33%) cultures were determined to be NTM, while the remaining 47 (79.67%) were identified as MTBC. Genotype characterization with GenoType® mycobacterium CM assay kit revealed that five of the 12 NTM isolates (41.67%) showed patterns that were consistent with Mycobacterium (M.) fortuitum, three (25%) showed patterns that were consistent with M. abscessus, and four (33.33%) showed patterns that were consistent with M. tuberculosis. Conclusion These resultsemphasizethe value of molecular methods for precisely identifying mycobacterial species, particularly in suspected TB cases. The high prevalence of NTM in positive cultures emphasizes the significance of differentiating between MTBC and NTM to prevent misdiagnosis and ensure proper care. Understanding the epidemiology and clinical significance of these organisms in central India is made possible by the identification of particular NTM species.

A Case of Mycobacterium abscessus Identified by Suspicious Gram Staining in the Blood Culture of a Patient with Chronic Renal Failure

Mycobacterium abscessus (M.abscessus), which is from the group of non-tuberculosis mycobacteria and is widely found in the natural environment, has been reported with increasing frequency as the causative agent of various infections; especially in the lower respiratory tract and in immuncompromised people. In this report, a case of M.abscessus, which developed tubular adenoma, pancytopenia and sepsis on the basis of chronic renal failure (CRF) was diagnosed by suspecting the causative agent in the Gram stain examination prepared from blood culture, was presented. A 49-year-old patient with CRF, who had complaints of weight loss, weakness, and loss of appetite for the last six months, admitted to the emergency department with a 7-8-day history of severe diarrhea and fever. Besides other tests, as the white blood cell count was 1.6 x 103/µl, neutrophil count was 80.6%, hemoglobin was 9.3 g/ dl and the platelet value was 36 x 103/µl in the blood samples, the patient was first taken into internal medicine service and then to the intensive care unit with a preliminary diagnosis of hypotension and sepsis. Meropenem and teicoplanin were started with the preliminary diagnosis of peritonitis in the internal medicine service. In addition to other tests, on the fifth day of antibiotic treatment, two consecutive sets of blood cultures were taken and sent to the microbiology laboratory. A positive signal was obtained from two aerobic blood culture samples at 42 and 45 hours of incubation in the BacT/Alert device. No bacteria were observed in the Gram staining of these samples and Erhlich Ziehl Neelsen (EZN) staining was performed because the structures considered as dye residues were noted as a result of the examination. Acid-fast bacteria were observed in the EZN-stained slide examination, and a panic report was given to the clinician. The patient died shortly after the notification was made in the evening hours. On culture plates inoculated after a positive signal, at the end of two days of aerobic incubation at 37 °C, small smooth S colonies grew on chocolate and sheep blood agar. Growing bacteria were detected as positive by EZN staining and identified as M.abscessus with 99.9% confidence by MALDI-TOF MS. After the bacterium was named as M.abscessus, the isolates were sent to the tuberculosis central laboratory of Süreyyapaşa Chest Diseases and Thoracic Surgery Hospital for molecular typing. After DNA extraction from the growing colonies and polymerase chain reaction (PCR), they were typed using the GenoType NTM-DR (Hain Lifescience GmbH, Germany) kit and identified as M.abscessus, consistent with the MALDITOF MS result. After the species level identification, the erm, rrl (clarithromycin, azithromycin), and rrs (kanamycin, amikacin, and gentamicin) genes were investigated in the isolate, and it was determined that the bacteria were resistant to macrolides and sensitive to aminoglycosides. In the clinic, it should be noted that, non-tuberculous mycobacteria may play a role as an agent in immunocompromised people. On the other hand, it should be considered that non-tuberculosis bacteria may be the causative agent, with gram-positive bacilli appearing as stain residues or pale staining in Gram stains made from samples of such patients. As in this case, if the agent is seen as dye residue in blood culture Gram staining samples, it may be life-saving to suspect the agent and to report the result to the clinician accurately and quickly after EZN staining.

Evaluation and clinical impact of MALDI Biotyper Mycobacteria Library v6.0 for identification of nontuberculous mycobacteria by MALDI‐TOF mass spectrometry

Major challenges in the identification of non-tuberculous mycobacteria (NTM) by MALDI-TOF MS include protein extraction protocol and updating of the NTM database. The aim of this study was to evaluate MALDI Biotyper Mycobacteria Library v6.0 (Bruker Daltonics GmbH, Bremen, Germany) for identification of clinical NTM isolates and its impact on clinical management. NTM isolates cultivated from clinical samples in 101 patients were identified simultaneously by PCR-reverse hybridization (Hain Lifescience GmbH, Nehren, Germany) as a routinely used reference molecular method and using MALDI Biotyper Microflex LT/SH after protein extraction. Each isolate was applied to eight spots, and mean scores were used in analysis. MALDI-TOF MS obtained correct identification to the species level for 95 (94.06%) NTM isolates. The majority of correctly identified isolates (92/95; 96.84%) were identified with high-confidence score of ≥1.80 and only 3.16% (3/95) with a score of <1.80. Mean value ± SD of RGM NTM isolates (2.127 ± 0.172) was statistically significant higher in comparison to SGM NTM isolates (2.027 ± 0.142) with a p value of 0.007. In comparison to PCR-reverse hybridization, discordant identification results by MALDI-TOF MS were found in six (6/101; 5.94%) NTM isolates for which clinical data were analyzed. We demonstrated a high confidence NTM identifications using Mycobacterium Library v 6.0 on routine clinical isolates. This is the first study that analyzed MALDI-TOF MS identification results of NTM isolates in the context of clinical data, and it showed that MALDI-TOF MS with its updated databases could help clarify the epidemiology, clinical characteristics, and course of infections caused by less frequent NTM species.

Fluoroquinolone resistance mutations among Mycobacterium tuberculosis and their interconnection with treatment outcome.

Fluoroquinolone (FQ) antibiotics are among the most potent second-line antitubercular drugs these days. The aim of the study was to analyze the frequency and pattern of genetic mutation in preextensive (pre-XDR) and extensively drug-resistant Mycobacterium tuberculosis using second-line line probe assay (LPA) and to compare drug-resistant mutations with different treatment outcomes. Sputum, lymph node aspirate, and cold accesses from patients with rifampicin-resistant Tuberculosis (TB) were subjected to first-line and second-line LPA (Genotype MTBDRsl by Hain Life Science, Germany) to assess additional drug resistance to fluoroquinolones (levofloxacin and moxifloxacin). Final treatment outcomes as per the National TB Elimination Program were assessed and compared with the mutation profile. One hundred and fifty subjects were observed to have mutations associated with resistance to FQs and constituted the final study population. The most frequent mutation observed among GyrA drug resistance mutation was D94G (Gyr A MUT3C, 44/150, 66%) corresponding to high-level resistance to levofloxacin and moxifloxacin. The same mutation was associated with poor treatment outcome as died or treatment failure (odds ratio 2.50, relative risk 1.67, P = 0.043). The most common hetero-resistance mutation pattern observed in GyrA gene was wild type plus Asp94Gly mutation in 24.6% of isolates. GyrA MUT3C hybridization corresponding to single-point mutation of aspartic acid to glycine at codon 94 constitutes the most common mutation in GyrA gene locus in M. tuberculosis with significant association with treatment outcome as died compared to those with treatment outcome as cured.

MTBDRplus and MTBDRsl for simultaneous diagnosis of gastrointestinal tuberculosis and detection of first-line and second-line drug resistance.

Emergence of drug resistance, especially to second-line drugs, hampers tuberculosis elimination efforts. The present study aimed to evaluate MTBDRplus and MTBDRsl assays for detecting first-line and second-line drug resistance, respectively, in gastrointestinal tuberculosis (GITB). Thirty ileocecal biopsy specimens, processed in the Department of Microbiology between 2012 and 2022, that showed growth of Mycobacterium tuberculosis on culture were included in the study. DNA, extracted from culture, was subjected to MTBDRplus and MTBDRsl (Hain Lifescience GmbH, Nehren, Germany), following manufacturer's instructions. Their performance was compared against phenotypic drug susceptibility testing (pDST) and gene sequencing. Out of the 30 specimens, 4 (13.33%) were mono-isoniazid resistant, 4 (13.33%) were multidrug resistant (MDR), 2 (6.67%) were pre-extensively drug resistant (pre-XDR), and 2 (6.67%) were mono-fluoroquinolone resistant. The results were 100% concordant with pDST and gene sequencing. In the wake of growing drug resistance in all forms of extrapulmonary tuberculosis, including GITB, MTBDRplus and MTBDRsl are reliable tools for screening of resistance to both first-line and second-line drugs.

ЭТИОЛОГИЧЕСКАЯ ВЕРИФИКАЦИЯ МИКОБАКТЕРИОЗА ЛЕГКИХ ПРИ ИССЛЕДОВАНИИ ОПЕРАЦИОННОГО МАТЕРИАЛА У ПАЦИЕНТОВ С ДИАГНОЗОМ А16.0 – РЕАЛЬНАЯ ПРАКТИКА

Objective: to evaluate effectiveness of etiological verification of pulmonary mycobacteriosis (PM) in patients with diagnosed “pulmonary TB without bacteriological or histological confirmation” (А16.0) as per ICD-10 and species diversity of nontuberculous mycobacteria (NTM) in resection samples from the studied cohort. Materials and methods.We analyzed test results from 28 patients with initially diagnosed “pulmonary TB without bacteriological or histological confirmation” (А16.0) as per ICD-10 and verified PM according to resection sample studies. The microbiological study (at least two sputum samples, one bronchobiopsy sample, and one resection sample) included luminescent microscopy (LUM), extraction of M. tuberculosis DNA or NTM DNA by polymerase chain reaction (PCR), culture growth on liquid media in the ВАСТЕС MGIT 960 system, identification of mycobacteria by molecular genetic methods: real-time PCR for M. tuberculosis DNA detection (Syntol, Russia) and DNA-strip technology (GenoType Mycobacterium CM/AS Hain Lifescience). Results. The analysis of referral diagnoses established that in more than half of patients with diagnosed “pulmonary TB without bacteriological or histological confirmation” (А16.0) changes in the lungs had been referred to tuberculoma (64.3%) or infiltrative TB (21.4%); 28.6% of patients had received TB treatment as per chemotherapy regimen no. 3, and 17.8% of patients – broad-spectrum antibiotics. TB treatment duration varied from 4 to 28 months. Atypical lung resections were performed in most cases (75%). NTM DNA detection in resection samples was the most informative method of etiological verification of PMB (100% of cases). NTM culture growth was observed in 17.9% of cases. Only slowly-growing NTM were represented in the studied cohort as etiologically significant. Conclusion. The diagnosis verification is based on data obtained by microbiological and/or histological, cytological studies. Studies of resection samples from TB suspects significantly improve diagnostic effectiveness.

A glimpse into the genotype and clinical importance of non tuberculous mycobacteria among pulmonary tuberculosis patients: The case of Ethiopia.

Laboratory identification of nontuberculous mycobacteria (NTM) species is not regularly performed while, they have a public health importance with a prevalence of more than 5% among pulmonary tuberculosis (PTB) patients in Ethiopia. Hence, this study aimed to identify the NTM species and their clinical significance among PTB patients. A retrospective study was conducted at the Ethiopian Public Health Institution’s (EPHI’s) national TB referral laboratory. Stored NTM isolates were genotyped using GenoType Mycobacterium CM/AS kit (Hain Life science, Germany). Data pertinent to the study was extracted from the EPHI’s database and patients’ medical records. Between January 2 & December 28 of 2017, a total of 3,834 samples were processed from 698 TB patients of whom 50% were female. Among 3,317 samples with mycobacterial culture results 7.3% were NTM and majority of them were identified from smear negative TB patients. M. simiae was the /predominant NTM among the genotyped isolates. All the studied NTM species were not clinically important however, considering the similarity of clinical and radiologic findings between NTM and MTBC infected patients, integrating NTM species identification in the routine TB laboratory diagnosis may augment clinicians’ decision particularly in DR-TB patients. Additional similar prospective study with a larger sample size is recommended. Moreover, urgent improvements on patients’ record keeping practice are required in the studied hospitals.

Peculiarities of the chemo­drug resistance of M. tuberculosis to anti­tuberculous medications among children and adolescents from multidrug­resistant tuberculous focies

Objective — to study the peculiarities of the resistance of M. tuberculosis (MTB) to antimycobacterial drugs (AMBD) among children and adolescents living in multidrug-resistant tuber­culous focies.&#x0D; Materials and methods. 246 children were examined, 145 of them had drug-resistant tuberculosis (TB), 101 children — susceptible form of the specific process, and 102 adult patients who became the source of the disease. Microbiological study in children, adolescents and adults included: detection of MTB in sputum by smear microscopy, seeding on Levenstein—Jensen medium, typing of isolated MTB on BACTEC MGIT 960, determination of drug susceptibility test of MTB strains to AMBD I and II lines, and molecular genetic testing of sputum, including the Gene Xpert MTB/RIF method and the Hain Lifescience linear probe analysis.&#x0D; Results and discussion. Researches show that children under one year of age (29.8 %) and up to 4 years of age (73.1 %) are the most vulnerable to TB in the multidrug-resistant tuberculous focies. Study of the resistance profile of MTB in multidrug-resistant foci was found almost complete coincidence of the profile of the resistance of MTB in children with a source of infection. In particular, children from contact compared with sick adolescents are in 3.5 times more likely to be resistant to the combination of HRS, in 2.7 times — to HR, in 1.8 times — to HRE, in 2.7 times less often — to HRESZ, in 1.6 times — to HRES. In adolescents resistance is most often found to the combination of HRSE (66.1 %), HRESZ (13.6 %), in contrast to the source of TB infection and children. Extensive drug resistance is 2.8 times more common in adolescents than in children (14.1 % vs. 5.0 %, p &gt; 0.01).&#x0D; Conclusions. Researches have shown a high risk of MDR-TB of lungs among children (42.7 %) from multidrug-resistant tuberculous focies. This indicates the need to improve the methods of obtaining respiratory samples to determine the susceptibility/resistance of MTB to AMBD. In the centers of multidrug-resistant tuberculosis infection, more than a third of children (35.8 %) were not vaccinated in the maternity hospital and no chemoprophylaxis was prescribed for contact persons.

Agreement between CBNAAT, liquid culture and line probe assay for detection of Mycobacterium tuberculosis and anti-tubercular drug resistance in extrapulmonary samples

PurposeCartridge based nucleic acid amplification test (CBNAAT) has been endorsed by the WHO as the screening test for diagnosing extrapulmonary tuberculosis (EPTB). In the present study we report the agreement between CBNAAT (Xpert MTB/RIF), liquid culture (LC) and line probe assay (LPA) for diagnosis of Mycobacterium tuberculosis and detection of drug resistance among EPTB cases. MethodsThe EP samples were subjected to CBNAAT (Xpert MTB/RIF, Cepheid, USA) and wherever possible, to LC (MGIT 960, Becton Dickinson, USA) followed sequentially by first line and second line-LPA (FL-LPA, SL-LPA, Hain Lifescience, Germany) on the isolates. ResultsTotal 566/4080 (13.9%) EP samples were detected positive for M. tuberculosis on CBNAAT. Aspirates from lymph nodes were most often positive (11/30; 36.6%), followed by pus (240/873; 27.5%) and CSF samples (166/104; 15.8%). The detection of M. tuberculosis was more in adults than children except in tissue biopsy samples. Rifampicin resistance was also higher among adults except CSF in which resistance was more in children. Total 185 of 566 (32.7%) CBNAAT positive and 770 of 3510 (21.9%) CBNAAT negative samples could be cultured of which 110/185 (59.4%) and 33/770 (4.3%) respectively turned positive. FL-LPA and SL-LPA of 143 culture isolates showed that 27 isolates had drug resistance, of which 3 (2.1%) were XDR, 11 (7.7%) were Pre-XDR (FQ) and 13 (9.1%) were MDR. Of these 27 resistant isolates, 12 were negative by CBNAAT and two were mislabeled as Rifampicin sensitive or indeterminate based on the unique RpoB gene mutation patterns on LPA. The positive and negative agreements between LC and CBNAAT for detection of M. tuberculosis were 67.1% and 92.7% respectively and between LPA and CBNAAT for rifampicin resistance detection were 98.9% and 92.9% respectively. ConclusionsFor EPTB, CBNAAT should be accompanied with LC wherever possible irrespective of the CBNAAT result.

Molecular characterisation of second-line drug resistance among drug resistant tuberculosis patients tested in Uganda: a two and a half-year\u2019s review

BackgroundSecond-line drug resistance (SLD) among tuberculosis (TB) patients is a serious emerging challenge towards global control of the disease. We characterized SLD-resistance conferring-mutations among TB patients with rifampicin and/or isoniazid (RIF and/or INH) drug-resistance tested at the Uganda National TB Reference Laboratory (NTRL) between June 2017 and December 2019.MethodsThis was a descriptive cross-sectional secondary data analysis of 20,508 M. tuberculosis isolates of new and previously treated patients’ resistant to RIF and/or INH. DNA strips with valid results to characterise the SLD resistance using the commercial Line Probe Assay Genotype MTBDRsl Version 2.0 Assay (Hain Life Science, Nehren, Germany) were reviewed. Data were analysed with STATAv15 using cross-tabulation for frequency and proportions of known resistance-conferring mutations to injectable agents (IA) and fluoroquinolones (FQ).ResultsAmong the eligible participants, 12,993/20,508 (63.4%) were male and median (IQR) age 32 (24–43). A total of 576/20,508 (2.8%) of the M. tuberculosis isolates from participants had resistance to RIF and/or INH. These included; 102/576 (17.7%) single drug-resistant and 474/576 (82.3%) multidrug-resistant (MDR) strains. Only 102 patients had test results for FQ of whom 70/102 (68.6%) and 01/102 (0.98%) had resistance-conferring mutations in the gyrA locus and gyrB locus respectively. Among patients with FQ resistance, gyrAD94G 42.6% (30.0–55.9) and gyrA A90V 41.1% (28.6–54.3) mutations were most observed. Only one mutation, E540D was detected in the gyrB locus. A total of 26 patients had resistance-conferring mutations to IA in whom, 20/26 77.0% (56.4–91.0) had A1401G mutation in the rrs gene locus.ConclusionsOur study reveals a high proportion of mutations known to confer high-level fluoroquinolone drug-resistance among patients with rifampicin and/or isoniazid drug resistance. Utilizing routinely generated laboratory data from existing molecular diagnostic methods may aid real-time surveillance of emerging tuberculosis drug-resistance in resource-limited settings.

Patients with non-tuberculous mycobacteriosis in clinical practice

Incidence and prevalence of non-tuberculous mycobacteriosis (NTM) are recorded in many countries, but the rates vary. There are no official statistics on the prevalence and incidence of NTMs in the Russian Federation at the moment. The regulatory and expert clinical database for NTMs has not yet been developed. The aim of thе article is to share the experience of the Federal State Scientific Institution “Central Research Institute of Tuberculosis” (FSSI “CRIT”) in working with patients with NTMB over the past 7 years. Methods. The study included 190 patients with a confirmed diagnosis of NTMB who were treated at the FSSI “CRIT” in 2011 – 2018. Before the diagnosis of NTMB, all patients were followed for a long time by various specialists (pulmonologists, general practitioners) for various chronic respiratory diseases. NTMs were isolated from the diagnostic material by the culture method. The material was inoculated into a liquid medium in an automated BACTEC MGIT 960 system and into Levenshtein – Jensen dense medium until the culture growth was obtained. The HTM species was determined using DNA strips GenoType® Mycobacterium CM by Hain Lifescience (Germany). Results. More than half of the examined patients were registered with a phthisiatrician for various clinical forms of pulmonary tuberculosis (disseminated, fibro-cavernous, focal). In 85.6% of cases, no MBT DNA was found in the sputum and skin tests with recombinant tuberculosis antigen (Diaskintest®) were negative. Nevertheless, the changes in the lungs detected on the chest CT and even a single detection of acid-fast microorganisms (AFB) without typing led to an erroneous diagnosis of tuberculosis. In some patients (6.8%), a combination of pulmonary tuberculosis and NTMB was diagnosed. Bronchiectasis and COPD were the most common chronic nonspecific lung diseases. 66.7% of the patients had centrilobular foci characterized by the involvement of not only the bronchial wall but also the pulmonary vessels. NTMB resistance to a wide range of antibacterial drugs was determined in 118 patients (out of 190). The prolonged use of chemotherapy drugs revealed a wide range of drug intolerance. Side effects were handled in various ways: drug replacement (with consideration to the sensitivity), temporary withdrawal, and drug discontinuation. Conclusion. This work reflects the long-term experience of the FSSI “CRIT” staff with patients with NTMs, which can be used in creating national recommendations on NTMs.

Nontuberculous Mycobacteria in Portugal: Trends from the last decade

Introduction and objectivesNontuberculous mycobacteria (NTM) are opportunistic human pathogens found in the environment. The transmission seems to be associated with inhalation of aerosol droplets, ingestion or trauma events. Recent studies indicate that NTM disease is increasing worldwide, however, the true clinical impact of NTM infections is difficult to determine due to challenges in discriminating between disease and colonization as they are ubiquitous in the environment. In addition, understanding the epidemiology of NTM is difficult and has not yet been established. In this work, we used a country NTM representative collection from the National Reference Laboratory for Tuberculosis (NRL-TB) of the National Institute of Health (INSA), to characterize the circulation trends of NTM species in Portugal and the most affected regions, contributing to a better understanding of the NTM epidemiology. Material and methodsWe conducted a nationwide retrospective study where all individuals with positive NTM cultures at the NRL-TB of the INSA from 2014 to December 2020 were included. Positive cultures were identified using GenoType Mycobacterium CM/AS® (Hain Lifescience) according to manufacturer's instructions, or hsp65 DNA sequencing as previously described. Social-demographic data from patients were also analyzed and patients classified into 3 groups according only to microbiological data, “definite NTM disease”, “NTM colonization” and, “possible NTM disease”. ResultsIn the period 2014-2020, the NRL-TB performed 50397 cultures. Among these, 1118 cultures were NTM positive retrieved from 944. Most of our cases were in patients whose mean age was 64±15.9 years, and no significant differences between gender was observed, although more frequent in male patients. Overall, from the 944 cases, we were able to identified 93 “definite NTM disease” cases and 79 “possible NTM disease”. Mycobacterium avium complex (MAC) (40,8%), Mycobacterium abscessus-chelonae complex (MABC) (9,6%) and Mycobacterium fortuitum (6,3%) were responsible for most of the infections. The geographical distribution of NTM cases varied significantly and was possible to observe that was independent of population density. The region were most cases occurred was Lisbon Metropolitan Area (31,9%), followed by North (25,3%) and Centre (24,4%), however North region has the highest number of “definite NTM disease” cases (n=33). ConclusionsThis is the first national wide epidemiological study on this subject, contributing to a better understanding of NTM dynamics in Portugal. MAC was the NTM species responsible for the majority of infections and, LMA the region with the highest number of cases. It was also possible to conclude that the number of NTM isolates is independent of the demography of the region.

Accuracy of GenoQuick MTB test in detection of Mycobacterium tuberculosis in sputum from TB presumptive patients in Uganda.

Objective:The objective of the study was to determine the diagnostic performance of the GenoQuick MTB test on heated sputum against the conventional Lowenstein–Jensen Mycobacterium tuberculosis culture as the reference method for tuberculosis diagnosis.Introduction:Fast, reliable, and easy-to-use tests for tuberculosis diagnosis are essential to achieving the Sustainable Development Goal of diagnosing and treating 90% of tuberculosis patients by 2030. We evaluated the diagnostic performance of the GenoQuick MTB, a polymerase chain reaction–lateral flow test, in Uganda, a resource-constrained, high tuberculosis- and HIV-burden setting.Methods:Fresh sputum samples from presumptive tuberculosis patients at Mulago Hospital were tested for M. tuberculosis using smear microscopy, GenoQuick MTB test, and Lowenstein–Jensen culture. For the GenoQuick MTB test, mycobacterial DNA was extracted by heating sputum at 95°C for 30 min while DNA amplification and detection were done following the manufacturer’s protocol (Hain Lifescience, Nehren, Germany). Sensitivity, specificity, and kappa agreements were calculated against Lowenstein–Jensen M. tuberculosis culture as a reference test using STATA V12.Results:Of the 86 tested samples, 30.2% had culture-confirmed pulmonary tuberculosis. Overall, sensitivity was higher for GenoQuick MTB (81%, 95% confidence interval: 60%−93%) than for smear microscopy (69%, 95% confidence interval: 48%−86%). Among people living with HIV, sensitivity was identical for GenoQuick MTB and smear tests (75%, 95% confidence interval: 42%−95%). Contrastingly, smear had a higher overall specificity (98%, 95% confidence interval: 91%−100%) than for GenoQuick MTB (92%, 95% confidence interval: 81%−97%). A similar trend of specificity was observed among the people living with HIV for smear microscopy (100%, 95% CI: 87%−100%) and for GenoQuick MTB (96%, 95% confidence interval: 81%−100%).Conclusion:The GenoQuick MTB test could be a potential tuberculosis diagnostic test given its higher sensitivity. Evaluation of this test in larger studies is recommended.