Drug resistance for Mycobacterium tuberculosis testing in China

Kadri Altraja^*

Department of Mycobacteriolog, University of Tartu; South Africa

Abstract

Background Despite the fact that Mycobacterium TB phenotypic drug susceptibility testing (DST) can take up to 6-8 weeks, nothing is known about how drug susceptibility is changed over this time [1]. Methods Using 359 patients with pulmonary tuberculosis who had baseline DST results from a Mtb isolate collected at the time of TB diagnosis and follow-up DST results from a Mtb isolate collected when baseline DST results were available between 2013 and 2018, we conducted a prospective cohort study to examine the development of drug resistance during turnaround time [2]. The distinction between acquired drug resistance, exogenous reinfection, and mixed infection was determined using whole-genome sequencing [3]. 116 (32.3%) of the participants in the study developed DR to four first-line medications during the TAT for DST. 21 pairings of the 116 pairs of isolates included in the WGS were categorised with changes in single nucleotide polymorphisms smaller than were classed as acquired drug resistance [4]. Four couples were identified as having mixed infections because they showed small variations in linked genotypes and had intermediate SNP differences [5]. High SNP differences in the remaining 91 pairings were indicative of exogenous reinfection [6].

Keywords

Additional drug resistance • Turnaround time • Drug-susceptibility testing

Introduction

The emergence of drug-resistant Mtb isolates during TAT for DST was greatly aided by the external reinfection of drug-resistant strains, emphasising the necessity of both quick DST procedures and enhanced infection control [7]. Both middle-income and low-income nations continue to struggle with the serious public health issue of tuberculosis [8]. The turnaround time for Mycobacterium TB drug-susceptibility testing, measured as the interval from the time sputum is collected for culture to the time results are available, typically takes around Reinfection, in which a patient contracts a drug-resistant strain that is markedly different from the strain that initially infected them, may result in the acquisition of increased medication resistance [9]. The development of drug resistance during treatment can also be caused by acquired drug resistance linked to mutations of a drug-resistant gene as well as mixed infection with drugsusceptible and drug-resistant strains within the same patient [10]. TB therapy and management may be hampered by inaccurate categorization in the absence of genetic techniques to identify these occurrences. With the advent of whole genome sequencing, it is now feasible to discriminate between a mixed infection of many Mtb strains and an exogenous infection with a genetically distinct Mtb strain in the same sputum material. Thus, we took action [11]. To examine the shifting drug-susceptibility patterns in serial sputum culture-positive Mtb isolates during TAT for DST and to examine how the acquisition of further drug resistance happened, a prospective cohort study with three study sites in China was conducted [12]. For the initial diagnosis of TB, baseline sputum samples were sent for smear microscopy or the WHO-recommended fast molecular techniques (TB-LAMP, Eiken; Hain Lifescience, Germany); after the diagnosis was confirmed with PTB, the patients began the normal course of therapy [13]. The duplicate baseline sputum samples were delivered to the local TB reference labs on the same day for phenotypic DST on BACTEC MGITTM and culture [14]. When baseline DST became available, a fresh sample of sputum was usually taken, and it was submitted for culture and phenotypic DST [15]. All isolates' phenotypic DST four first-line medications: pyrazinamide, isoniazid, and rifampicin Patients with pulmonary TB who received first-line medications as part of routine therapy from 2013 to 2018 and had sputum culture confirmation were included. Sputum culture-confirmed TB patients having a baseline DST result of an M.

Discussion

Tuberculosis isolate obtained at the time of TB diagnosis and a follow-up DST result of an M. tuberculosis isolate obtained when the baseline DST result was available met the inclusion criteria. The Furan University public health school authorised the study. After TB was diagnosed using smear microscopy or a quick diagnostic test approved by the WHO, all individuals with the disease got first-line medications as routine therapy. The recommended standard treatment plan for newly treated TB patients is provided by the WHO. The previous guidelines of an extended 8-month retreatment regimen, monthlong intense phase, and month-long maintenance term of Sputum samples were taken for the study at the following times: when TB was diagnosed and when baseline DST results were available. The TB registry was used to gather baseline data on patients with culture-confirmed TB. This data included sociodemographic factors such as age, sex, place of residence, clinical information about newly or previously treated patients, disease severity on chest X-rays, comorbidities, bacterial diagnosis, and drug susceptibility. It also included dates of baseline sputum culture submission and baseline phenotypic DST completion. It also included minimum inhibitory concentration levels and first-line DST TAT for DST was determined to be the time between the collection of baseline sputum for culture and the availability of the baseline DST result. Based on bacteriological diagnosis and medication susceptibility, follow-up data was gathered, including sputum culture results and phenotypic DST findings of follow-up isolates. In order to detect potential exogenous reinfection and mixed infections with acquired drug resistance, we included baseline and follow-up isolates with added drug resistance during genotyping in this investigation. In order to distinguish between exogenous reinfection, mixed infection, and acquired drug resistance produced by mutations linked with drug-resistant genes, 116 pairs of patients who had developed resistance to four first-line medications were further compared using whole-genome sequencing. For each purified DNA sample, we created a 300-basepair paired-end library in accordance with the Illumina paired-end protocol. A maximum likelihood method was used to determine the observed frequency for each SNP. SNPs with frequencies above 95% were regarded as fixed mutations considering the lowest sequencing depth included in our research, whereas those with frequencies between 5% and 95% were defined as genetically significant. separate strains. While significant difference in pairs was defined as the discovery of a secondary drug-resistant strain with a difference when comparing the baseline isolate with the follow-up isolate and a developed resistance to any first-line drugs, acquired drug resistance was defined as SNPs difference by comparing the genotypes of baseline and follow-up isolates. In the trial sites, a total of patients received a bacteriological diagnosis of proven pulmonary TB during the course of the year-long study period. These were removed because they had negative sputum cultures and culture-positive samples that had failed baseline phenotypic tests. In addition, we eliminated patients who had completed their TB treatment in less than a month, were lost to follow-up, or had been moved before the data were available. A total of patients from the enrolled patients were whenever baseline DST values were available, sputum culture came out negative.

Finally, the final study comprised 359 individuals with a Each patient's follow-up sputum sample was obtained and cultured after the DST results of the baseline isolates were available. Of them, 359 patients including 77 with drug-susceptible isolates, 112 with monodrug resistant isolates, patients with PDR isolates, and 131 with MDRTB at the time of TB diagnosis—had positive sputum cultures, and follow-up DST was carried out. The first and subsequent drug resistance profiles when follow-up DST data were available, two isolates with EMB-resistance at TB diagnosis were reclassified as RIF-resistance and four isolates with EMB-resistance at TB diagnosis were reclassified as susceptible TB. Additionally, at the time of TB diagnosis, 11 MDR isolates had either PZA resistance or EMB resistance. Based on the subsequent drug-resistant profiles, susceptibility and EMB-susceptibility were determined. Additionally, 28 Mtb isolates lost their resistance to PZA or EMB during the TAT for DST. One baseline isolate and a follow-up isolate were collected at the time the baseline phenotypic DST findings were available, making 116 pairs of Mtb isolates with the development of further drug resistance in total. The WGS data of the 116 follow-up Mtb isolates revealed isolates as having acquired drug resistance with SNPs differences ranging from 3.4% isolates as mixed infection, which corresponds to a combination of majority and minority genotype. The other isolates displayed genetically distinct strains with SNPs differences ranging from EMB-resistant strains and pncA in PZA-resistant strains. Among Further analysis of the WGS data revealed indications of mixed infection among three non-MDR-TB pairs and one MDR-TB pair, which corresponded to a mixture of the majority and minority genotype among the baseline collected isolates, in the patients with genetically unique strains. The follow-up isolate and the minority genotype were closely linked, according to phylogenetic reconstruction. Furthermore, PZA heteroresistance Patients were divided into exogenous reinfection of a drug resistant strain among those with strains that acquired increased drug resistance.

Conclusion

Eight isolates from patients with exogenous reinfection mapped closely to other isolates, and WGS-based resistance mutations among the serially tested isolates were compatible with phenotypic DST, showing inter-individual transmission. Two MDR-TB patients and four non-MDR-TB patients at a healthcare institution suggested probable transmission, according to a subsequent epidemiological analysis, whereas during the course of the therapy, patients in non-MDR couples lived next to one other. A combined strategy focusing on quick detection of active tuberculosis illness and drug-resistant TB, followed by quick commencement of appropriate therapy, is necessary to reduce the transmission risk of drug-resistant Mtb strains. Additionally, by stepping up molecular DST, drug-resistant TB may be quickly recognised and treated appropriately, making patients much less contagious to others. Another conclusion from our study was that mixed infections were challenging to identify with culture-based DST and that using inadequate antibiotic dosages might favour the development of resistant bacteria. Four instances were reported, suggesting mixed infection with a drug-resistant strain, where an unsuspected sub-population of drug-resistant strains was discovered at first DST diagnosis.

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