Development of a rapid detection method for the macrolide resistance gene in Mycobacterium avium using the amplification refractory mutation system-loop-mediated isothermal amplification method.

Macrolide antibiotics such as clarithromycin (CLR) and azithromycin are the key drugs used in multidrug therapy for Mycobacterium avium complex (MAC) diseases. For these antibacterial drugs, drug susceptibility has been correlated with clinical response in MAC diseases. We have previously demonstrated the correlation between drug susceptibility and mutations in the 23S rRNA gene, which confers resistance to macrolides. Herein, we developed a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) technique to identify mutations in the 23S rRNA gene of M. avium. We examined the applicability of the ARMS-LAMP method to genomic DNA extracted from six genotypes of M. avium clinical isolates. The M. avium isolates were classified into 21 CLR-resistant and 9 CLR-susceptible strains based on the results of drug susceptibility tests; the 23S rRNA genes of these strains were sequenced and analyzed using the ARMS-LAMP method. Sequence analysis revealed that the 9 CLR-sensitive strains were wild-type strains, whereas the 21 CLR-resistant strains comprised 20 mutant-type strains and one wild-type strain. Using ARMS-LAMP, no amplification from genomic DNAs of the 10 wild-type strains was observed using the mutant-type mismatch primer sets (MTPSs); however, amplification from the 20 mutant-type strain DNAs was observed using the MTPSs. The rapid detection method developed by us integrates ARMS-LAMP with a real-time turbidimeter, which can help determine drug resistance in a few hours. In conclusion, ARMS-LAMP might be a new clinically beneficial technology for rapid detection of mutations.IMPORTANCEMultidrug therapy for pulmonary Mycobacterium avium complex disease is centered on the macrolide antibiotics clarithromycin and azithromycin, and resistance to macrolides is an important prognosticator for clinical aggravation. Therefore, it is important to develop a quick and easy method for detecting resistance to macrolides. Drug resistance is known to be correlated with mutations in macrolide resistance genes. We developed a rapid detection method using amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene, which is a macrolide resistance gene. Furthermore, we examined the applicability of this method using M. avium clinical isolates. The rapid method developed by us for detection of the macrolide resistance gene by integrating ARMS-LAMP and a real-time turbidimeter can help in detection of drug resistance within a few hours. Since this method does not require expensive equipment or special techniques and shows high analytical speed, it would be very useful in clinical practice.

Tackling Nontuberculous Mycobacteria by Repurposable Drugs and Potential Leads from Natural Products.

Nontuberculous Mycobacteria (NTM) refer to bacteria other than all Mycobacterium species that do not cause tuberculosis or leprosy, excluding the species of the Mycobacterium tu-berculosis complex, M. leprae and M. lepromatosis. NTM are ubiquitous and present in soils and natural waters. NTM can survive in a wide range of environmental conditions. The direct inocu-lum of the NTM from water or other materials is most likely a source of infections. NTMs are re-sponsible for several illnesses, including pulmonary alveolar proteinosis, cystic fibrosis, bronchi-ectasis, chronic obstructive pneumoconiosis, and pulmonary disease. Recent reports suggest that NTM species have become insensitive to sterilizing agents, antiseptics, and disinfectants. The ef-ficacy of existing anti-NTM regimens is diminishing and has been compromised due to drug re-sistance. New and recurring cases of multidrug-resistant NTM strains are increasing. Thus, there is an urgent need for ant-NTM regimens with novel modes of action. This review sheds light on the mode of antimicrobial resistance in the NTM species. Then, we discussed the repurposable drugs (antibiotics) that have shown new indications (activity against NTM strains) that could be developed for treating NTM infections. Also, we have summarised recently identified natural leads acting against NTM, which have the potential for treating NTM-associated infections.

Novel mutations found in Mycobacterium leprae DNA repair gene nth from central India.

INTRODUCTION: The importance of DNA repair enzymes in maintaining genomic integrity is highlighted by the hypothesis that DNA damage by reactive oxygen/nitrogen species produced inside the host cell is essential for the mutagenesis process. Endonuclease III (Nth), formamidopyrimide (Fpg) and endonuclease VIII (Nei) DNA glycosylases are essential components of the bacterial base excision repair process. Mycobacterium leprae lost both fpg/nei genes during the reductive evolution event and only has the nth (ML2301) gene. This study aims to characterize the mutations in the nth gene of M. leprae strains and explore its correlation with drug-resistance. METHOD: A total of 91 M. leprae positive DNA samples extracted from skin biopsy samples of newly diagnosed leprosy patients from NSCB Hospital Jabalpur were assessed for the nth gene as well as drug resistance-associated loci of the rpoB, gyrA and folP1 genes through PCR followed by Sanger sequencing. RESULTS: Of these 91 patients, a total of two insertion frameshift mutations, two synonymous and seven nonsynonymous mutations were found in nth in seven samples. Sixteen samples were found to be resistant to ofloxacin and one was found to be dapsone resistant as per the known DRDR mutations. No mutations were found in the rpoB region. Interestingly, none of the nth mutations were identified in the drug-resistant associated samples. CONCLUSION: The in-silico structural analysis of the non-synonymous mutations in the Nth predicted five of them were to be deleterious. Our results suggest that the mutations in the nth gene may be potential markers for phylogenetic and epidemiological studies.

High frequency of ofloxacin resistance patterns of Mycobacterium leprae from India: An indication to revisit second line anti-leprosy treatment regimen.

OBJECTIVES: Drug resistance in leprosy is an emerging concern, leading to treatment failures, recurrences, and potential spread of resistant Mycobacterium leprae in the community. In this study, we aimed to assess drug resistance prevalence and patterns amongst leprosy patients at a tertiary care referral hospital in India. METHODS: Mutations in drug resistance determining regions for dapsone, rifampicin, and ofloxacin of the M. leprae genome in DNA extracted from skin biopsies of 136 leprosy patients (treatment-naive = 67, with persistent skin lesions = 35, with recurrence = 34) were analysed by polymerase chain reaction followed by Sanger sequencing. Wild-type strain (Thai-53) was used as a reference strain. RESULTS: Resistance mutations were identified in a total of 23 patients, constituting 16.9% of the cohort. Within this subset of 23 cases, resistance to ofloxacin was observed in 17 individuals (12.5%), while resistance to both dapsone and rifampicin was detected in three patients each (2.2% for both). The occurrence of ofloxacin resistance showed minimal disparity between recurrent and treatment-naive cases, at 17.6% and 16.4%, respectively. Dapsone resistance emerged in two treatment-naive cases and one case with persistent skin lesions. Notably, none of the treatment-naive cases or those with recurrence/relapse exhibited rifampicin resistance. Subsequently, no statistically significant correlation was identified between other clinical variables and the presence of antimicrobial resistance. CONCLUSIONS: The occurrence of resistance to the current multidrug therapy regimen (specifically dapsone and rifampicin) and to ofloxacin, a secondary antileprosy medication in M. leprae, represents a concerning scenario. This calls for an expansion towards bactericidal drug options and the establishment of robust surveillance for drug resistance in countries burdened with high leprosy rates. Moreover, the introduction of stringent antimicrobial stewardship initiatives is imperative. As a single centre study, it represents a limited, cross-sectional view of the real situation in the field.

Specialized active leprosy search strategies in an endemic area of the Brazilian Amazon identifies a hypermutated Mycobacterium leprae strain causing primary drug resistance.

Introduction: Leprosy, an infectious disease caused by Mycobacterium leprae, remains a public health concern in endemic countries, particularly in Brazil. In this study, we conducted an active surveillance campaign in the hyperendemic city of Castanhal in the northeastern part of the state of Pará using clinical signs and symptoms combined with serological and molecular tools to diagnose new cases and to identify drug resistance of circulating M. leprae strains and their distribution in the community. Methods: During an active surveillance of one week, we enrolled 318 individuals using three different strategies to enroll subjects for this study: (i) an active survey of previously treated cases from 2006 to 2016 found in the Brazil National Notifiable Disease Information System database (n = 23) and their healthy household contacts (HHC) (n = 57); (ii) an active survey of school children (SC) from two primary public schools in low-income neighborhoods (n = 178), followed by visits to the houses of these newly diagnosed SC (n = 7) to examine their HHC (n = 34) where we diagnosed additional new cases (n = 6); (iii) and those people who spontaneously presented themselves to our team or the local health center with clinical signs and/or symptoms of leprosy (n = 6) with subsequent follow-up of their HHC when the case was confirmed (n = 20) where we diagnosed two additional cases (n = 2). Individuals received a dermato-neurological examination, 5 ml of peripheral blood was collected to assess the anti-PGL-I titer by ELISA and intradermal earlobe skin scrapings were taken from HHC and cases for amplification of the M. leprae RLEP region by qPCR. Results: Anti-PGL-I positivity was highest in the new leprosy case group (52%) followed by the treated group (40.9%), HHC (40%) and lowest in SC (24.6%). RLEP qPCR from SSS was performed on 124 individuals, 22 in treated cases, 24 in newly diagnosed leprosy cases, and 78 in HHC. We detected 29.0% (36/124) positivity overall in this sample set. The positivity in treated cases was 31.8% (7/22), while in newly diagnosed leprosy cases the number of positives were higher, 45.8% (11/23) and lower in HHC at 23.7% (18/76). Whole genome sequencing of M. leprae from biopsies of three infected individuals from one extended family revealed a hypermutated M. leprae strain in an unusual case of primary drug resistance while the other two strains were drug sensitive. Discussion: This study represents the extent of leprosy in an active surveillance campaign during a single week in the city of Castanhal, a city that we have previously surveyed several times during the past ten years. Our results indicate the continuing high transmission of leprosy that includes fairly high rates of new cases detected in children indicating recent spread by multiple foci of infection in the community. An unusual case of a hypermutated M. leprae strain in a case of primary drug resistance was discovered. It also revealed a high hidden prevalence of overt disease and subclinical infection that remains a challenge for correct clinical diagnosis by signs and symptoms that may be aided using adjunct laboratory tests, such as RLEP qPCR and anti-PGL-I serology.

Effectiveness and safety of multidrug therapy containing clofazimine for paucibacillary leprosy and clarithromycin for rifampicin-resistant leprosy: a systematic review and meta-analysis.

Introduction: The present study aimed to evaluate leprosy cure and relapse rates as primary outcomes related to two additional strategies for leprosy treatment: clofazimine for paucibacillary (PB) leprosy patients and clarithromycin for patients with rifampicin-resistant leprosy. Methods: We conducted two systematic reviews (protocols CRD42022308272 and CRD42022308260). We searched the PubMed, EMBASE, Web of Science, Scopus, LILACS, Virtual Health Library and Cochrane Library databases, registers of clinical trial databases and gray literature. We included clinical trials evaluating the addition of clofazimine to PB leprosy treatment and the use of clarithromycin for treating patients with rifampicin-resistant leprosy. Risk of bias (RoB) in randomized clinical trials was assessed by the RoB 2 tool and that in non-randomized clinical trials was assessed by the ROBINS-I tool; and the certainty of the evidence was assessed by the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system. A meta-analysis of dichotomous outcomes was performed. Results: For clofazimine, four studies were included. Cure and relapse rates were not different with the addition of clofazimine to PB leprosy treatment and demonstrated very low certainty of evidence. For clarithromycin, six studies were included. Considerable heterogeneity resulted from the difference between comparators, and studies showed no difference in the assessed outcomes with the addition of clarithromycin to rifampicin-resistant leprosy treatment. Mild adverse events were reported for both drugs but did not significantly impact treatment. Discussion: The effectiveness of both drugs still needs to be determined. Adding clofazimine to PB leprosy treatment may reduce the repercussions of an incorrect operational classification with no apparent relevant side effects. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022308272; https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022308260, identifier: CRD42022308272; CRD42022308260.

Inhibitory effect of natural compounds on Dihydropteroate synthase of Mycobacterium leprae: Molecular dynamic study.

Leprosy is a chronic infectious disease caused by a bacillus, Mycobacterium leprae. According to official data from 139 countries in the 6 WHO Regions, there were 127558 new leprosy cases worldwide in 2020. Leprosy mainly affects the skin, the peripheral nerves, mucosa of the upper respiratory tract, and the eyes. If this disease is left untreated, can harm the skin, nerves, limbs, eyes, and skin permanently. The disease is curable with multidrug therapy. Over a period of time Mycobacterium leprae has become resistant to these drugs. Therefore, new therapeutic molecules are warranted. This study was aimed to carry out the in-silico analysis to determine the inhibitory effect of natural compounds on Dihydropteroate synthase (DHPS) of Mycobacterium leprae. The DHPS is a key enzyme in the folate biosynthesis pathway in M. leprae and acts as a competitive inhibitor of PABA. The 3D structure of DHPS protein was modeled using homology modeling and was validated. Molecular docking and simulation along with other in-silico methods were employed to determine the inhibitory effect of ligand molecules towards DHPS target protein. Results revealed ZINC03830554 molecule as a potential inhibitor of DHPS. Binding experiments and bioassays utilizing this strong inhibitor molecule against purified DHPS protein are necessary to validate these early findings.Communicated by Ramaswamy H. Sarma.

Drug Resistance (Dapsone, Rifampicin, Ofloxacin) and Resistance-Related Gene Mutation Features in Leprosy Patients: A Systematic Review and Meta-Analysis.

Dapsone (DDS), Rifampicin (RIF) and Ofloxacin (OFL) are drugs recommended by the World Health Organization (WHO) for the treatment of leprosy. In the context of leprosy, resistance to these drugs occurs mainly due to mutations in the target genes (Folp1, RpoB and GyrA). It is important to monitor antimicrobial resistance in patients with leprosy. Therefore, we performed a meta-analysis of drug resistance in Mycobacterium leprae and the mutational profile of the target genes. In this paper, we limited the study period to May 2022 and searched PubMed, Web of Science (WOS), Scopus, and Embase databases for identified studies. Two independent reviewers extracted the study data. Mutation and drug-resistance rates were estimated in Stata 16.0. The results demonstrated that the drug-resistance rate was 10.18% (95% CI: 7.85-12.51). Subgroup analysis showed the highest resistance rate was in the Western Pacific region (17.05%, 95% CI:1.80 to 13.78), and it was higher after 2009 than before [(11.39%, 7.46-15.33) vs. 6.59% (3.66-9.53)]. We can conclude that the rate among new cases (7.25%, 95% CI: 4.65-9.84) was lower than the relapsed (14.26%, 95 CI%: 9.82-18.71). Mutation rates of Folp1, RpoB and GyrA were 4.40% (95% CI: 3.02-5.77), 3.66% (95% CI: 2.41-4.90) and 1.28% (95% CI: 0.87-1.71) respectively, while the rate for polygenes mutation was 1.73% (0.83-2.63). For further analysis, we used 368 drug-resistant strains as research subjects and found that codons (Ser, Pro, Ala) on RpoB, Folp1 and GyrA are the most common mutation sites in the determining region (DRDR). In addition, the most common substitution patterns of Folp1, RpoB, and GyrA are Pro→Leu, Ser→Leu, and Ala→Val. This study found that a higher proportion of patients has developed resistance to these drugs, and the rate has increased since 2009, which continue to pose a challenge to clinicians. In addition, the amino acid alterations in the sequence of the DRDR regions and the substitution patterns mentioned in the study also provide new ideas for clinical treatment options.

Risk factors for dapsone resistance in leprosy patients: a systematic meta-analysis.

OBJECTIVES: Dapsone is one of the important drugs in the treatment of leprosy. The present study aims to evaluate the resistance of Mycobacterium leprae isolates to dapsone, in turn assisting in implementing better control strategies for leprosy elimination. METHODS: A systematic literature search was conducted in PubMed, Embase, Medline, and Web of Science. Two independent reviewers selected the literature according to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), extracted data, and evaluated the risk of bias. Drug resistance data were pooled using the random-effects model. Subgroup analysis was performed based on across sampling time, region, study population (treatment status, relapses status), and sample size. RESULTS: A total of 30 studies were included. The results of meta-analysis showed that the dapsone resistance rate of leprosy patients after treatment was 8% (95% confidence interval [CI], 6%-10%). Compared to the rates of primary resistance of new cases without treatment therapy (pooled incidence, 4% [95% CI, 2%-5%]), treatment cases (13% [95% CI 9%-16%]) had secondary resistance, and relapse cases (26% [95% CI, 18%-33%]) had drug resistance. In addition, the drug resistance rate of monotherapy was significantly increased than that of relapsed patients treated with diamino-diphenylsulfone monotherapy. Subgroup analysis showed that the patients in the Western Pacific have the highest dapsone resistance, and the resistance to dapsone was slightly lower after 2005. For sample size, the rate in the group under 100 samples was significantly higher than in the other. CONCLUSION: Dapsone resistance is closely related to leprosy relapse and long-term drug use. Dapsone monotherapy is one of important reasons for drug resistance in relapsed cases. Drug resistance varies among different populations and regions of the world.

Ofloxacin resistance in multibacillary new leprosy cases from Purulia, West Bengal: a threat to effective secondary line treatment for rifampicin-resistant leprosy cases.

OBJECTIVES: Purulia is one of the high-endemic districts for leprosy in West Bengal (the eastern part of India). The annual new case detection rate (ANCDR) of leprosy in West Bengal is 6.04/100000 (DGHS 2019-20). Our earlier report provided evidence of secondary drug resistance in relapse cases of leprosy. The aim of the current study was to observe primary drug resistance patterns for dapsone, rifampicin, and ofloxacin amongst new leprosy patients from Purulia, West Bengal in order to better understand the emergence of primary resistance to these drugs. METHODS: In the present study, slit-skin smear samples were collected from 145 newly diagnosed leprosy cases from The Leprosy Mission (TLM) Purulia hospital between 2017 and 2018. DNA was extracted from these samples and the Mycobacterium leprae genome was analyzed for genes associated with drug resistance by polymerase chain reaction (PCR), followed by Sanger sequencing. Wild-type strain (Thai-53) and mouse footpad-derived drug-resistant strain (Z-4) were used as reference strains. RESULTS: Of 145 cases, 25 cases showed mutations in genes associated with resistance to rifampicin, dapsone, and ofloxacin (as described by the World Health Organization, rpoB, folP, and gyrA, respectively) through Sanger sequencing. Of these 25 cases, 16 cases showed mutations in ofloxacin, two cases showed mutations in combinations of ofloxacin and rifampicin, four cases showed a mutation only in rifampicin, one case showed mutations in combinations of rifampicin and dapsone, and two cases showed mutations only in dapsone. CONCLUSION: Results from this study indicated the emergence of resistance to antileprosy drugs in new cases of leprosy. As ofloxacin is the alternate drug for the treatment of rifampicin-resistant cases, the emergence of new cases with resistance to ofloxacin indicates that ofloxacin-resistant M. leprae strains are actively circulating in this endemic region (i.e., Purulia, West Bengal), posing challenges for the effective treatment of rifampicin-resistant cases.

Proportion and trend of primary resistance among Multidrug resistant Tuberculosis patients in Ethiopia.

Background: Evidence based information on the proportion & trend of primary resistance among multidrug resistance (MDR) TB patients is important for designing effective strategies in the control of the disease. Methods: A retrospective record review of 348 MDR/RR-TB patients treated at All African Leprosy Rehabilitation & Training (ALERT) Center from January 2014- December 2018. Categorical variables were compared using Chi-square/Fisher exact test as appropriate. Trend analysis was done using chi-square & linear regression. Logistic regression analysis was done to determine the factors associated with primary MDR/RR TB. Adjusted Odds Ratio (AOR) with 95% CI and p value < 5% were used to report factors associated. Result: Proportion of primary resistance among MDR/RR TB patients was 25.9% with 95% CI 21.3-30.3%. The proportion increased form 9.7% in 2014 to 43.4% in 2018 at a yearly increasing rate of 9.27%. Contact history to TB patient & year of diagnosis 2017 and 2018 were significantly associated with primary resistance AOR (95% CI) & p value 4.15(1.75-9.84) p = 0.001, 3.87(1.44-10.39) p = 0.007, 3.43(1.20-9.84) p = 0.022 respectively. Conclusion: The study revealed a high proportion of primary resistance among MDR/RR TB during the study period with a linearly increasing fashion thus a need for due attention in the efforts to control MDR TB.

Laboratory perspectives for Leprosy: Diagnostic, prognostic and predictive tools.

The diagnosis of leprosy poses several challenges. The bacillary load, serology, and tissue response are determined by the host immune status, which make individual tests unsuitable across the spectrum. The sensitivity of tests for identifying paucibacillary cases remains limited, on the other hand, many tests lack specificity in differentiating contacts from diseased cases. Nonetheless, a plethora of laboratory tests have been added to the armamentarium of the clinicians dealing with leprosy. In the current review, we critically analyze the tests available for diagnosis, prognostication, and prediction of treatment response in leprosy. We discuss in brief the conventional tests available and detail the newer serologic and molecular tests added over the past few years with an attempt to suggest the pros and cons of each, and the tests best fit for each clinical scenario. Slit skin smears and skin or nerve biopsies are primarily performed to exclude clinical mimics, confirm a diagnosis, and immunologically subtype the case. Antibody titres of phenolic glycolipid-1 and its synthetic variants can be measured in serum and saliva and provide noninvasive means to detect leprosy with good specificity. Conventional, quantitative, real-time, and other variants of PCR can detect M. leprae DNA and have been used to effect in blood, tissue, and urine samples. T helper I and II cytokine signatures can be used to differentiate the subtypes of leprosy. Newer machine learning algorithms use combinations of these tests to predict the development of leprosy in contacts. Tests to detect treatment response, antimicrobial drug resistance, and predict the onset of reactions in leprosy can be used to advantage. We compare the characteristics of these tests and suggest an algorithm for leprosy diagnosis optimally utilizing them in various clinical settings.

Drug resistance in leprosy: An update following 70years of chemotherapy.

Leprosy is one of the oldest infectious diseases, reported for more than 2000years. Leprosy elimination goal as a public health problem set by the World Health Organization, aiming for a global prevalence rate<1 patient in a population of 10,000, was achieved in 2000 mainly thanks to the worldwide use of leprosy drugs starting in the 1980s and their access at no cost for patients since 1995. However, around 200,000 new cases are still reported each year, particularly in India, Brazil, and Indonesia. As with other bacteria of medical interest, antimicrobial resistance is observed in Mycobacterium leprae strains in several parts of the world, despite multidrug therapy being the recommended standard leprosy treatment to avoid resistance selection since 1982. Therefore, identifying and monitoring resistance is necessary. We provide an overview of the historical facts that led to the current drug resistance situation, the antibiotics effective against M. leprae, their mechanisms of action and resistance, and resistance detection methods. We also discuss therapeutic management of the resistant cases, new genes with potential roles in drug resistance and bacterial adaptation, new drugs under investigation, and the risk for resistance selection with the chemoprophylaxis measures.

Antimicrobial Resistance among Leprosy Patients in Brazil: Real-World Data Based on the National Surveillance Plan.

Brazil ranks second among countries for new cases and first for relapse cases of leprosy worldwide. The Mycobacterium leprae Resistance Surveillance Plan was established. We aimed to present the results of a 2-year follow-up of the National Surveillance Plan in Brazil. A cross-sectional study of leprosy cases was performed to investigate antimicrobial resistance (AMR) in Brazil from October 2018 to September 2020. Molecular screening targeting genes related to dapsone (folP1), rifampin (rpoB), and ofloxacin resistance (gyrA) was performed. During the referral period, 63,520 active leprosy patients were registered in Brazil, and 1,183 fulfilled the inclusion criteria for molecular AMR investigation. In total, only 16 (1.4%) patients had genetic polymorphisms associated with AMR. Of these, 8 (50%) had cases of leprosy relapse, 7 (43.8%) had cases of suspected therapeutic failure with standard treatment, and 1 (6.2%) was a case of new leprosy presentation. M. leprae strains with AMR-associated mutations were found for all three genes screened. Isolates from two patients showed simultaneous resistance to dapsone and rifampin, indicating multidrug resistance (MDR). No significant relationship between clinical variables and the presence of AMR was identified. Our study revealed a low frequency of AMR in Brazil. Isolates were resistant mainly to dapsone, and a very low number of isolates were resistant to rifampin, the main bactericidal agent for leprosy, or presented MDR, reinforcing the importance of the standard World Health Organization multidrug therapy. The greater frequency of AMR among relapsed patients supports the need to constantly monitor this group.

Application of Droplet Digital PCR to Detection of Mycobacterium tuberculosis and Mycobacterium leprae Infections: A Narrative Review.

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB) infection, which has seriously endangered human health for many years. With the emergence of multidrug-resistant and extensively drug-resistant MTB, the prevention and treatment of TB has become a pressing need. Early diagnosis, drug resistance monitoring, and control of disease transmission are critical aspects in the prevention and treatment of TB. However, the currently available diagnostic technologies and drug sensitivity tests are time consuming, and thus, it is difficult to achieve the goal of early diagnosis and detection drug sensitivity, which results in limited control of disease transmission. The development of molecular testing technology has gradually achieved the vision of rapid and accurate diagnosis of TB. Droplet digital PCR (ddPCR) is an excellent nucleic acid quantification method with high sensitivity and no need for a calibration curve. Herein, we review the application of ddPCR in TB diagnosis and drug resistance detection and transmission monitoring.

Prevalence and antimicrobial susceptibility level of typhoid fever in Ethiopia: A systematic review and meta-analysis.

Typhoid fever continues to be a health challenge in low-and middle-income countries where access to clean water and sanitation infrastructure is scarce. The non-confirmatory diagnostic method continues to hinder effective diagnosis and treatment, ensuring in a high antimicrobial resistance. This systematic review and meta-analysis aimed to estimate the pooled prevalence and antimicrobial susceptibility level of typhoid fever in Ethiopia. The review was designed based on the condition-context-population review approach. Fifteen eligible articles were identified from PubMed, Google Scholar, and Science Direct databases. Risk of bias and quality of studies were assessed using the Joanna Briggs Institute's appraisal criteria. Heterogeneity was assessed using Cochran's Q test and I2 statistics. The review protocol was registered in PROSPERO (registration number CRD42021224478). The estimated pooled prevalence of typhoid fever from blood and stool culture diagnosis was 3% (95% CI: 2%-4%, p < 0.01) (I2  = 82.25) and Widal test examination 33% (95% CI: 22%-44%) (I2  = 99.14). The sub-group analyses identified a lower detection of typhoid fever of 2% (95% CI: 1%-3%) among febrile patients compared to typhoid suspected cases of 6% (95% CI: 2%-9%). The stool culture test identified was twofold higher, value of 4% (95% CI: 2%-7%) salmonella S. Typhi infection than blood culture test of 2% (95% CI: 1%-4%). The antimicrobial susceptibility of salmonella S. Typhi for antibiotics was 94%, 80% and 65% for ceftriaxone, ciprofloxacin, and gentamycin respectively. Low susceptibility of salmonella S. Typhi isolates against nalidixic acid 22% (95% CI: 2%-46%) and chloramphenicol 11% (95% CI: 2%-20%) were observed. The diagnosis of typhoid fever was under or overestimated depending on the diagnostic modality. The Widal test which identified as nonreliable has long been used in Ethiopia for the diagnosis of salmonella S. Typhi causing high diagnosis uncertainties. Antimicrobial susceptibility of salmonella S. Typhi was low for most nationally recommended antibiotics. Ethiopian Food and Drug Authority must strengthen its continued monitoring and enhanced national antimicrobial surveillance system using the best available state-of-the-art technology and or tools to inform the rising resistance of salmonella S. Typhi towards the prescription of standard antibiotics. Finally, it is crucial to develop an evidence-based clinical decision-making support system for the diagnosis, empiric treatment and prevention of antimicrobial resistance.

Drug resistance in leprosy: an update following 70 years of chemotherapy

Leprosy is one of the oldest infectious diseases, reported for more than 2000 years. Leprosy elimination goal as a public health problem set by the World Health Organization, aiming for a global prevalence rate < 1 patient in a population of 10,000, was achieved in 2000 mainly thanks to the worldwide use of leprosy drugs starting in the 1980s and their access at no cost for patients since 1995. However, around 200,000 new cases are still reported each year, particularly in India, Brazil, and Indonesia. As with other bacteria of medical interest, antimicrobial resistance is observed in Mycobacterium leprae strains in several parts of the world, despite multidrug therapy being the recommended standard leprosy treatment to avoid resistance selection since 1982. Therefore, identifying and monitoring resistance is necessary. We provide an overview of the historical facts that led to the current drug resistance situation, the antibiotics effective against M. leprae, their mechanisms of action and resistance, and resistance detection methods. We also discuss therapeutic management of the resistant cases, new genes with potential roles in drug resistance and bacterial adaptation, new drugs under investigation, and the risk for resistance selection with the chemoprophylaxis measures.

Antimicrobial resistance among leprosy patients in Brazil: real-world data based on the National Surveillance plan

Brazil ranks second among countries for new cases and first for relapse cases of leprosy worldwide. The Mycobacterium leprae Resistance Surveillance Plan was established. We aimed to present the results of a 2-year follow-up of the National Surveillance Plan in Brazil. A cross-sectional study of leprosy cases was performed to investigate antimicrobial resistance (AMR) in Brazil from October 2018 to September 2020. Molecular screening targeting genes related to dapsone (folP1), rifampin (rpoB), and ofloxacin resistance (gyrA) was performed. During the referral period, 63,520 active leprosy patients were registered in Brazil, and 1,183 fulfilled the inclusion criteria for molecular AMR investigation. In total, only 16 (1.4%) patients had genetic polymorphisms associated with AMR. Of these, 8 (50%) had cases of leprosy relapse, 7 (43.8%) had cases of suspected therapeutic failure with standard treatment, and 1 (6.2%) was a case of new leprosy presentation. M. leprae strains with AMR-associated mutations were found for all three genes screened. Isolates from two patients showed simultaneous resistance to dapsone and rifampin, indicating multidrug resistance (MDR). No significant relation ship between clinical variables and the presence of AMR was identified. Our study revealed a low frequency of AMR in Brazil. Isolates were resistant mainly to dapsone, and a very low number of isolates were resistant to rifampin, the main bactericidal agent for leprosy, or presented MDR, reinforcing the importance of the standard World Health Organization multidrug therapy. The greater frequency of AMR among relapsed patients supports the need to constantly monitor this group

Mycobacterium leprae: aspectos da resistência aos fármacos na poliquimioterapia / Drug resistance in multidrug therapy for Mycobacterium leprae

Introdução: O diagnóstico da hanseníase possui números significativos que causam preocupação à saúde pública. Os casos de resistência medicamentosa nessa doença se iniciaram em meados dos anos 60 e diante do problema, a Organização Mundial da Saúde instituiu em 1981 a poliquimioterapia, associação dos antibióticos rifampicina, dapsona e clofazimina, tratamento atual de escolha. A resistência aos fármacos na hanseníase é reportada pela literatura, desvelando um obstáculo à sua eliminação. Apresentamos nessa revisão os principais aspectos da resistência medicamentosa no tratamento para hanseníase e seus impactos. Metodologia: Revisão sistemática sobre os aspectos da resistência medicamentosa utilizando a pesquisa exploratória como metodologia de abordagem. Foram pesquisados os termos resistência medicamentosa, hanseníase, recidiva, alterações genéticas e os operadores booleanos "and" e "or" na busca. Resultados e discussão: A dificuldade de tomar a medicação corretamente foi um dos principais fatores que acarretaram resistência do bacilo Mycobacterium leprae aos fármacos. Homens de países norte e sul-americanos e asiáticos foram os mais atingidos por episódios de resistência. A resistência medicamentosa é uma das principais causas de recidivas em hanseníase. O principal fármaco causador de resistência medicamentosa descrito nos trabalhos foi a dapsona (46,6%) e a maioria das alterações genéticas encontradas estão no gene rpoB; 23,2% dos registros relatados foram de resistência secundária aos fármacos e, também, sete casos de resistência múltipla a esses medicamentos. Conclusão: Os principais aspectos da resistência medicamentosa na hanseníase são os equívocos ao ingerir os medicamentos e as alterações genéticas na bactéria. Os impactos causados estão na dificuldade de refazer o tratamento, a possibilidade de nova transmissão e o aparecimento de sintomas mais graves.

Introduction: The diagnosis of leprosy has significant numbers causing public health concern. Reports of drug resistance in this disease begun in the mid-1960s and due to this problem, the World Health Organization instituted a multidrug therapy with rifampicin, dapsone, and clofazimine antibiotic association in 1981, which is currently the first-choice treatment for leprosy. Cases of drug resistance have been reported in literature, revealing an obstacle to the eradication of the disease. This paper has the purpose of presenting the key aspects and impacts of drug resistance in the treatment for leprosy. Methods: Systematic review of the drug resistance aspects using exploratory research as an approach methodology. The authors searched the terms drug resistance, leprosy, recurrence, genetic alterations, and the Boolean operators "and" and "or" between them. Results and discussion: The difficulty in taking the medication correctly was one of the key factors that led to drug resistance for Mycobacterium leprae. Men from North and South American, as well as from Asian countries, were the most affected by episodes of resistance. Drug resistance is one of the main causes of leprosy recurrences. Dapsone was the most frequently identified drug resistance in the studies (46.6%), while most of the genetic alterations were found in the rpoB gene; 23.2% of the cases were from secondary resistance episodes, and seven cases of multiple resistance were reported. Conclusion: The misconceptions when taking the treatment and the Mycobacterium leprae genetic alterations have been described as the key aspects of drugs resistance in leprosy and the impacts caused are the difficulty in redoing the treatment, the possibility of new transmission, and the appearance of more severe symptoms.

Drug Resistance in Nontuberculous Mycobacteria: Mechanisms and Models.

The genus Mycobacteria comprises a multitude of species known to cause serious disease in humans, including Mycobacterium tuberculosis and M. leprae, the responsible agents for tuberculosis and leprosy, respectively. In addition, there is a worldwide spike in the number of infections caused by a mixed group of species such as the M. avium, M. abscessus and M. ulcerans complexes, collectively called nontuberculous mycobacteria (NTMs). The situation is forecasted to worsen because, like tuberculosis, NTMs either naturally possess or are developing high resistance against conventional antibiotics. It is, therefore, important to implement and develop models that allow us to effectively examine the fundamental questions of NTM virulence, as well as to apply them for the discovery of new and improved therapies. This literature review will focus on the known molecular mechanisms behind drug resistance in NTM and the current models that may be used to test new effective antimicrobial therapies.