Drug susceptibility testing of Mycobacterium tuberculosis using next generation sequencing and Mykrobe software

Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis. Drug susceptibility testing is performed by phenotypic and molecular tests. Commonly used for phenotypic drug susceptibility testing is the automated BACTEC system in a liquid culture medium. Drug susceptibility by line probe molecular tests was introduced almost 15 years ago. Recently whole genome sequencing (WGS) analysis of M. tuberculosis strains demonstrated that genotyping of drug-resistance could be accurately performed. Several software tools were developed.Our study aimed to perform whole-genome sequencing on phenotypically confirmed multi-drug resistant (MDR) M. tuberculosis strains, to identify drug-resistant mutations and to compare whole-genome sequencing profiles with line probe assay and phenotypic results.Materials and methods. We performed analysis on 34 MDR M. tuberculosis Bulgarian strains. Phenotypic drug susceptibility testing was performed on the BACTEC system. For molecular testing of drug susceptibility to first- and second-line tuberculostatics, we applied line probe assay Geno Type MTBDR plus v.1.0 и Geno Type MTBDR sl v.1.0. Sequencing was performed on MiSeq. Generated FASTQ files were analyzed for known drugresistant mutations with the software platform Mykrobe v.0.8.1.Results. All three methods — phenotypic analysis using the BACTEC system, genetic analysis of strains applying the Geno Type test and Mykrobe software gave comparable sensitivity/resistance results for the studied strains. All phenotypically proven rifampicin and isoniazid-resistant strains were 100% confirmed using Mykrobe software. The C-15T mutation is a marker for isoniazid resistance in strains of the SIT41 spoligotype. We observed a 75% (21/28) agreement between BACTEC and Mykrobe for ethambutol resistance. Phenotypically, 87% (n = 27) of the strains are resistant to streptomycin, but only 59% (n = 19) are proven by Mykrobe software. Comparing phenotypic and genotypic resistance to ofloxacin, amikacin and kanamycin, we observed 100% coincidence of results.Conclusions. Whole-genome sequencing approach is relatively expensive and laborious but useful for detailed analysis such as epidemiological genotyping and molecular drug susceptibility testing.

Frequency and Antimicrobial Susceptibility Pattern of Gram-Negative Bacilli Isolated From Urine Specimens at a Tertiary Care Setting

OBJECTIVES: To find out the frequency and pattern of conventional antibiotic susceptibility of gram-negative bacilli cultured from urine specimens of patients at a tertiary care setting. METHODOLOGY: This study was conducted at the Microbiology Department of Combined Military Hospital Multan from June 2016 to May 2017. The data in this retrospective descriptive study was collected from urine culture records of the Microbiology Department, CMH Multan. Only those urine specimens who revealed positive gram-negative bacilli cultures were included in the study. Drug susceptibility patterns of these isolates were recorded against routinely used antibiotics (e.g. Nitrofurantoin, Imipenem, Sulbactum-cefoperazone, Gentamicin and Ciprofloxacin) and evaluated accordingly. RESULTS: A total of 1703 urine specimens were submitted for culture and antibiotics susceptibility testing during the period of study. A total of 128 specimens showed growth of gram-negative rods. Imipenem (95% sensitivity), Sulbactam- Cefoperazone (88% sensitivity) and Nitrofurantoin (87% sensitivity) were highly effective antibiotics against the cultured gram-negative bacilli in the study. CONCLUSION: This study showed that E. coli is the commonest cause of urinary tract infection (UTIs), followed by Klebsiella and Enterobacter species among gram-negative bacilli in our set up. In-vitro efficacy of Imipenem, Sulbactam- Cefoperazone and Nitrofurantoin was found to be the highest against these gram-negative bacilli as compared to other antimicrobials. On the contrary, in-vitro efficacy of ciprofloxacin and gentamycin was found to be extremely low.

Clinical Characteristics and Antimicrobial Susceptibility of Mycobacterium intracellulare and Mycobacterium abscessus Pulmonary Diseases: A Retrospective Study

The incidence of nontuberculous mycobacteria (NTM) diseases is increasing every year. The present study was performed to investigate the clinical characteristics, CT findings, and drug susceptibility test (DST) results of patients diagnosed with M. intracellulare or M. abscessus nontuberculous mycobacterial pulmonary disease (NTMPD). This retrospective study included patients diagnosed with NTMPD due to M. intracellulare or M. abscessus for the first time at Anhui Chest Hospital between 01/2019 and 12/2021. The patients were grouped as M. intracellulare-NTMPD group or M. abscessus-NTMPD group. Clinical features, imaging data and DST data, were collected. Patients with M. intracellulare infection had a higher rate of acid-fast smears (66.1% vs. 45.2%, P = 0.032 ) and a higher rate of cavitation based on pulmonary imaging (49.6% vs. 19.4%, P = 0.002 ) than patients with M. abscessus infection, but both groups had negative TB-RNA and GeneXpert results, with no other characteristics significant differences. The results of DST showed that M. intracellulare had high susceptibility rate to moxifloxacin (95.9%), amikacin (90.1%), clarithromycin (91.7%), and rifabutin (90.1%). M. abscessus had the highest susceptibility rate to amikacin (71.0%) and clarithromycin (71.0%). The clinical features of M. intracellulare pneumopathy and M. abscessus pneumopathy are highly similar. It may be easily misdiagnosed, and therefore, early strain identification is necessary. M. intracellulare has a high susceptibility rate to moxifloxacin, amikacin, clarithromycin, and rifabutin, while M. abscessus has the highest susceptibility rate to amikacin and clarithromycin. This study provides an important clinical basis for improving the management of NTMPD.

Reverse dot blot hybridization assay— An expeditious diagnostic tool for drug resistant TB

Tuberculosis (TB) is one of the leading infectious diseases that is highly transmissible. It is reported to cause approximately 10 million infections and 1.4 million deaths globally in the year of 2019.¹ Prompt detection and treatment of TB are essential to intercept the proliferation of the disease. In the case of drug- resistant TB, failure to detect the resistance of anti TB drugs may give rise to extensively drug- resistant tuberculosis (XDR-TB). Thus, the rapid detection of drug resistance is vital;, however, the current standard drug susceptibility tests (DST) may take up to 12 weeks raising an alarming concern.² A study was done in China by Li Wan et al, on the accuracy of the reverse dot blot hybridization assay (RDBH) for rapidly detecting the resistance of four anti TB drugs (rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB)) in mycobacterium tuberculosis (MTB) isolates.³ Continuous...

Multidrug Resistance and Serotype Distribution of Salmonella Enterica Isolated from Homemade Recipe Fermented Ground Pork (Nham) in Northeastern Thailand

Salmonellosis is caused by a thousand serotypes of Salmonella enterica. The sour taste inherent to Nham leads people believe that this fermented ground pork dish is safe from pathogenic microorganisms. The aim of this study was to evaluate the prevalence, serotype, drug susceptibility, and antimicrobial resistance (AMR) genes of Salmonella spp. in homemade recipes of Nham. There were 52 samples from different Nham makers in 3 northeastern provinces of Thailand collected between August and November 2019. Further, 30 Salmonella isolates (57.7 %) and 14 different serovars were identified: S. Rissen (23.3 %) was the most prevalent, followed by S. Typhimurium (16.7 %), S. Give and S. Virchow (10 % each), and S. Agona and S. Kouka (6.7 % each). All isolates carried AMR genes but 7 (23.3 %) were antibiotic susceptible and 23 (76.7 %) borne a resistance phenotype. The Salmonella isolates were resistant to tetracycline (63 %), sulfamethoxazole/trimethoprim (36.7 %), streptomycin (33.3 %), nalidixic acid (30 %), cefotaxime (16.7 %), and enrofloxacin (3.3 %). Among the 23 AMR genes in our analysis, there were gyrB (100 %), tetA (93.3 %), aadA (93.3 %), sul1, sul2, sul3 (23.3 - 33.3 %), dfrA12 (16.7 %), qnrS, (6.7 %), and mcr6 (6.7 %). Two strains had the mcr6 gene but were susceptible to colistin. Our findings suggest that naturally occurring lactic acid bacteria in the Nham products are insufficient to inhibit Salmonella contamination of this pork-based food. HIGHLIGHTS Highly presence of Salmonella in fermented ground pork (Nham) All identified Salmonella isolates in the Nham have AMR genes A few Salmonella isolates carry AMR genes but are antibiotic susceptible Two Salmonella isolates contain the mcr6 gene but are susceptible to colistin

Change in Quality of Tuberculosis (TB) Care since National Quality Assessment Program of TB Healthcare Service

Purpose: This study aims to examine the quality of tuberculosis (TB) care after the 1^st to 3^rd national quality assessment (QA) program for TB healthcare service in Korea was conducted.Methods: We analyzed Health Insurance Review & Assessment Service (HIRA) claims data of new TB patients during the period of January to June from 2018-2020. The new TB patients were defined as TB patients reported to Korea Centers for Disease Control and Prevention Agency (KCDA). The unit of analysis was the patient. Chi-square tests were used to analyze the differences in indicator value according to the types of medical facilities. The QA indicators of TB care were divided into 3 areas consisting of the following 7 quality indicators: 4 indicators of diagnosis test (the rate of acid-fast bacilli smear, the rate of acid-fast bacilli culture, the rate of Mycobacterium tuberculosis-polymerase chain reaction, drug susceptibility test), 1 compliance of treatment guideline, and 2 indicators of care management of TB patients (encounter rate, day of therapy).Results: The QA program for TB care was conducted among 8,246 patients from 534 facilities in 2020. The value of the 7 quality indicators was shown to increase as a result of the QA program. The indicators of the diagnostic test were all higher than 95%, with the exception of the drug susceptibility test which was 84.8%. Both indicators for care management of TB patients were 88.5%.Conclusion: The quality of TB care has been improving with the implementation of the QA program. In order to continue to improve the quality of TB care, it will be necessary to disclose the results of the QA program in medical facilities in the future.

Diagnosis of tuberculosis among COVID-19 suspected cases in Ghana

Background Tuberculosis (TB) and COVID-19 pandemics are both diseases of public health threat globally. Both diseases are caused by pathogens that infect mainly the respiratory system, and are involved in airborne transmission; they also share some clinical signs and symptoms. We, therefore, took advantage of collected sputum samples at the early stage of COVID-19 outbreak in Ghana to conduct differential diagnoses of long-standing endemic respiratory illness, particularly tuberculosis. Methodology Sputum samples collected through the enhanced national surveys from suspected COVID-19 patients and contact tracing cases were analyzed for TB. The sputum samples were processed using Cepheid’s GeneXpert MTB/RIF assay in pools of 4 samples to determine the presence of Mycobacterium tuberculosis complex. Positive pools were then decoupled and analyzed individually. Details of positive TB samples were forwarded to the NTP for appropriate case management. Results Seven-hundred and seventy-four sputum samples were analyzed for Mycobacterium tuberculosis in both suspected COVID-19 cases (679/774, 87.7%) and their contacts (95/774, 12.3%). A total of 111 (14.3%) were diagnosed with SARS CoV-2 infection and six (0.8%) out of the 774 individuals tested positive for pulmonary tuberculosis: five (83.3%) males and one female (16.7%). Drug susceptibility analysis identified 1 (16.7%) rifampicin-resistant tuberculosis case. Out of the six TB positive cases, 2 (33.3%) tested positive for COVID-19 indicating a coinfection. Stratifying by demography, three out of the six (50%) were from the Ayawaso West District. All positive cases received appropriate treatment at the respective sub-district according to the national guidelines. Conclusion Our findings highlight the need for differential diagnosis among COVID-19 suspected cases and regular active TB surveillance in TB endemic settings.

Diagnostic accuracy of Truenat Tuberculosis and Rifampicin-Resistance assays in Addis Ababa, Ethiopia

Background Rapid and sensitive Tuberculosis (TB) diagnosis closer to patients is a key global TB control priority. Truenat assays (MTB, MTB Plus, and MTB-RIF Dx) are new TB molecular diagnostic tools for the detection of TB and Rifampicin (RIF)-resistance from sputum samples. The diagnostic accuracy of the assays is needed prior to implementation in clinical use in Ethiopia. This study aimed to determine the sensitivity and specificity of Truenat assays; and aimed to compare the assays to the Xpert MTB/RIF assay. Methods A prospective evaluation study was conducted among 200 presumptive TB patients in microscopy centers in Addis Ababa, Ethiopia from May 2019 to December 2020. Culture (Solid and Liquid methods) and phenotypic (liquid method) drug susceptibility testing (DST) were used as a reference standard. Results Of 200 adult participants, culture confirmed TB cases were 25 (12.5%), and only one isolate was resistant to RIF by phenotypic DST. The sensitivity of Truenat MTB was 88.0% [95% CI 70.1, 95.8], while 91.7 [95% CI 74.2, 97.7] for Truenat MTB Plus at the microscopy centers. The specificity of Truenat MTB was 97.2% [95% CI 93.1, 98.9], while for Truenat MTB Plus was 97.2% [95% CI 93.0, 99.0]. The sensitivity of Truenat MTB was 90.5% while for MTB Plus, 100% compared to the Xpert MTB/RIF assay. Conclusion Truenat assays were found to have high diagnostic accuracy. The assays have the potential to be used as a point of care (POC) TB diagnostic tests.