Molecular Detection Of Drug Resistance Research Articles

Molecular detection of drug resistance in clinical specimens of leprosy patients from a tertiary care leprosy centre in South India

Background Leprosy is one of the neglected tropical diseases (NTD) experiencing relative stagnancy in the global number of new cases. Objective This study aims to find the proportion of antimicrobial resistance in the clinical specimens of leprosy patients attending a tertiary care leprosy Institute in South India. Materials and methods Skin biopsy specimens (31 Nos) were tested by Polymerase chain reaction (PCR) for identification of M. leprae, and PCR followed by DNA sequencing for antimicrobial resistance (AMR) detection. Results All 31 specimens tested for M. leprae identification were positive. Resistance to dapsone was 6.5%, while rifampicin and ofloxacin showed 100% sensitivity. Conclusion This study warrants the need for a vigilant surveillance mechanism to monitor the trend of antimicrobial resistance in leprosy.

Mycobacterium simiae Isolates Subtypes and Molecular Drug Susceptibility in Iran

Background: Despite the clinical and epidemiological importance of Mycobacterium simiae worldwide, including in Iran, there is no clear and effective treatment regimen for M. simiae and its different subtypes. Objectives: Concerning the superiority of molecular approaches, this study aims to identify the common M. simiae subtypes submitted to the National Reference Tuberculosis (TB) Laboratory of Iran and study the presence of drug resistance by molecular detection methods. Methods: We included sputum samples with M. simiae confirmation submitted to the National Reference TB Laboratory of Iran from May 2014 to May 2016. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used for drug susceptibility testing (DST). Results: Among 7200 TB suspected patients, a total of 60 M. simiae cases belonging to subtype I were identified. All the included clinical isolates met the American Thoracic Society (ATS) and Infectious Diseases Society of America (IDSA) diagnostic criteria and were considered the disease’s causative pathogen. Males (58.33%), elderly (68.54%), and patients with a history of TB (51.42%) were shown to be more prone to infection with the disease. All clinical isolates of M. simiae were resistant to rifampin (RIF) and isoniazid (INH). Amikacin/kanamycin (AMK/KAN) and ciprofloxacin (CIP) susceptibility was found to be 91.66% and 88.33%, respectively. Conclusions: Subtype I was exclusively identified among M. simiae patients in Iran. Molecular detection of drug resistance suggests that amikacin/kanamycin and ciprofloxacin could be used to treat patients infected with M. simiae subtype I.

Study on PVL, blaOXA-23 and blaOXA-51 Genes in Drug Resistant <i>Staphylococcus aureus</i> Causing Surgical-Sites and Traumatic Wounds Infections, Sudan

Background: The characteristics of Staphylococcus aureus that made it the most important cause of wound infections are environmental spread antimicrobials resistance and virulence. Absence of molecular detection of drug resistance and virulence factors in many developing countries limits the epidemiological information. This study conducted to identify PVL virulence gene, and blaOXA-23 and blaOXA-51 drug resistance genes of Staphylococcus aureus isolated from surgical-sites infections (SSIs) and traumatic wounds. Methods: A cross-sectional study was conducted from 2019 to 2021, in which 70 cefepime resistant Staphylococcus aureus were used, the strains were isolated from patients of SSIs and traumatic wounds admitted to the department of General Surgery in Wad Medani Teaching Hospital. Mannitol salt agar was used for primary culture followed by biochemical identification and Kirby Bauer susceptibility testing. Single and multiplex PCR protocols performed for bacterial confirmation and target genes detection. Results: Staphylococcus aureus strains from SSIs constituted 56% (39/70) from which 41% (16/39) possessed PVL gene while 42% (13/31) of wound infections strains were positive for PVL gene. Presence of PVL gene was significantly associated with resistance to meropenem (P. value 0.023) and ceftriaxone (P. value 0.037). blaOXA-23 was significantly detected with resistance to meropenem, augmentin and ceftriaxone. While blaOXA-51 was significantly identified among Staphylococcus aureus strains that showed resistance to meropenem and ciprofloxacin. Conclusion: This is the first study in Sudan that identified blaOXA-23 and blaOXA-51 in Staphylococcus aureus and correlated them to resistance to commonly used antimicrobials. Meropenem resistant Staphylococcus aureus were significantly positive for PVL, blaOXA-23 and baOXA-51 genes.

Profiling and identification of novel rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis clinical isolates from Pakistan

IntroductionRifampicin (RIF) is one of the most effective anti-tuberculosis first-line drugs prescribed along with isoniazid. However, the emergence of RIF resistance Mycobacterium tuberculosis (MTB) isolates is a major issue towards tuberculosis (TB) control program in high MDR TB-burdened countries including Pakistan. Molecular data behind phenotypic resistance is essential for better management of RIF resistance which has been linked with mutations in rpoB gene. Since molecular studies on RIF resistance is limited in Pakistan, the current study was aimed to investigate the molecular data of mutations in rpoB gene behind phenotypic RIF resistance isolates in Pakistan. MethodA total of 322 phenotypically RIF-resistant isolates were randomly selected from National TB Reference Laboratory, Pakistan for sequencing while 380 RIF resistance whole-genome sequencing (WGS) of Pakistani isolates (BioProject PRJEB25972), were also analyzed for rpoB mutations. ResultAmong the 702 RIF resistance samples, 675 (96.1%) isolates harbored mutations in rpoB in which 663 (94.4%) were detected within the Rifampicin Resistance Determining Region (RRDR) also known as a mutation hot spot region, including three novel. Among these mutations, 657 (97.3%) were substitutions including 603 (89.3%) single nucleotide polymorphism, 49 (7.25%) double and five (0.8%) triple. About 94.4% of Phenotypic RIF resistance strains, exhibited mutations in RRDR, which were also detectable by GeneXpert. ConclusionMutations in the RRDR region of rpoB is a major mechanism of RIF resistance in MTB circulating isolates in Pakistan. Molecular detection of drug resistance is a faster and better approach than phenotypic drug susceptibility testing to reduce the time for transmission of RIF resistance strains in population. Such insights will inform the deployment of anti-TB drug regimens and disease control tools and strategies in high burden settings, such as Pakistan.

801. Emergence of Multi-Drug Resistance Tuberculosis During the Treatment Course of Pan-Susceptible TB: A Case Series

BackgroundSuccessful treatment of tuberculosis (TB) requires monitoring for clinical, radiographic, and microbiologic improvement. Even after negative cultures are obtained, there should be continued monitoring of sputa. If cultures become positive during treatment of drug susceptible TB (DS-TB), there should be concern for multi-drug-resistant tuberculosis (MDR-TB). We present two cases diagnosed with DS-TB who developed MDR-TB during treatment. Case Report: Case 1 is a 33-year-old male who was incarcerated in Peru. During incarceration in 2008, three of his cellmates had MDR-TB and he was diagnosed with DS-TB and treated with directly observed therapy (DOT) for 7 months. In Texas in 2015 he was diagnosed with DS-TB and was initiated on rifampin, isoniazid, pyrazinamide, and ethambutol (RIPE). Five months into DOT, his sputa became culture positive with molecular detection of drug resistance (MDDR) and drug susceptibility testing (DST) revealing resistance to all of RIPE. Repeat MDDR and DST of the 2015 isolate showed no resistance. Genotyping of the two isolates were identical by mycobacterial interspersed repetitive units (MIRU) and spoligotyping. However, whole genome sequencing showed two different isolates. Case 2 is a 63-year-old female diagnosed with DS-TB in Saipan and started on RIPE in April 2017. She was on DOT until July when she moved to Texas and was lost to follow-up until September. She claims adherence with rifampin and isoniazid during this time. All sputa collected between diagnosis and September were smear and culture negative. Six months into therapy, she had sputa that was culture positive with MDDR and DST showing MDR-TB. Her isolates from Saipan and Texas were sent for genotyping. The MIRU and spoligotyping showed two different isolates.ConclusionThese cases show the importance of following cultures throughout treatment. Traditionally, MDR-TB is thought to be due to poor adherence. However, in high prevalence areas, heterogeneous infection with two different strains is an important consideration for the cause of MDR-TB. Concomitant infection of DS and MDR-TB can occur with MDR-TB not being detected until far into therapy. These cases represent heterogeneous exogenous infection of DS and MDR-TB—only discovered after meticulous culture monitoring.Disclosures All authors: No reported disclosures.

770. Multi-Drug-Resistant Tuberculosis Cases in Arkansas in 2017: A Tale of Two Threats

BackgroundMulti-drug-resistant tuberculosis (MDR-TB) is a threat to TB elimination strategies worldwide. From 1998 to 2016, six cases of MDR-TB were reported in Arkansas. In 2017 alone, three cases were detected. We sought to describe the characteristics of these cases to inform our MDR-TB prevention strategy in AR.MethodsThe surveillance database identified three MDR-TB cases in 2017. A detailed review was done to define the demographics, clinical presentation, and laboratory reports relating to drug susceptibility testing (DST), including molecular detection of drug resistance (MDDR). A search was done in the Genotyping database for genotype patterns of the patient isolates.ResultsAll three cases were born outside the United States and developed active disease after arrival in AR. Case 1, age 52, was born in the Marshall Islands, arrived in 2016, and had a history of Type 2 diabetes. He developed MDR-TB in February 2017. Case 2, age 42, was born in Mexico, arrived over 20 years ago, and was HIV positive. He developed TB in July 2016 with a pan-sensitive organism and completed an intermittent treatment regimen. A second TB episode with matching genotype but different drug sensitivities occurred in April 2017, less than 4 months after treatment completion, and was considered treatment failure. Case 3, age 56, was born in the Philippines, arrived in 1990, and was reportedly treated for latent TB infection in 1993 with 6 months of isoniazid. She visited the Philippines April–May 2017 and developed MDR-TB in October 2017. Her isolate was in cluster with a case in Oklahoma who came from Mexico in 2006 and was admitted in an AR hospital with a pan-sensitive organism. There are no epidemiological links between the two cases; only one isolate in each case. Because both isolates were identified in AR State TB laboratory, a complex contamination has been considered with no definite resolution at this time.ConclusionMDR-TB, due to both primary and secondary drug resistance, remains a threat in AR. Cooperation and communication between all levels of healthcare are crucial to avoid delayed diagnosis of MDR-TB. Timely DST via technologies like GeneXpert and MDDR service at CDC is critical. Consultation from Centers of Excellence is vital in the treatment of MDR-TB complicated by diabetes and HIV. Whole-genome sequencing could provide clarity in the cluster with discordant DST patterns.Disclosures All authors: No reported disclosures.

Detection of Apparent Cell-free M. tuberculosis DNA from Plasma

New diagnostics are needed to improve clinicians’ ability to detect tuberculosis (TB) disease in key populations such as children and persons living with HIV and to rapidly detect drug resistance. Circulating cell-free DNA (ccfDNA) in plasma is a diagnostic target in new obstetric and oncologic applications, but its utility for diagnosing TB is not known. Here we show that Mycobacterium tuberculosis complex DNA can be detected in plasma of persons with sputum smear-positive TB, even in the absence of mycobacteremia. Among 40 participants with bacteriologically-confirmed smear-positive TB disease who had plasma tested by quantitative PCR (qPCR), 18/40 (45%) had a positive result on at least one triplicate reaction. Our results suggest that plasma DNA may be a useful target for improving clinicians’ ability to diagnose TB. We anticipate these findings to be the starting point for optimized methods of TB ccfDNA testing and sequence-based diagnostic applications such as molecular detection of drug resistance.

The Use of a Shared Services Model for Mycobacteriology Testing: Lessons Learned.

Public health laboratories (PHLs) provide essential services in the diagnosis and surveillance of diseases of public health concern, such as tuberculosis. Maintaining access to high-quality laboratory testing is critical to continued disease detection and decline of tuberculosis cases in the United States. We investigated the practical experience of sharing tuberculosis testing services between PHLs through the Shared Services Project. The Shared Services Project was a 9-month-long project funded through the Association of Public Health Laboratories and the Centers for Disease Control and Prevention during 2012-2013 as a one-time funding opportunity to consortiums of PHLs that proposed collaborative approaches to sharing tuberculosis laboratory services. Submitting PHLs maintained testing while simultaneously sending specimens to reference laboratories to compare turnaround times. During the 9-month project period, 107 Mycobacterium tuberculosis complex submissions for growth-based drug susceptibility testing and molecular detection of drug resistance testing occurred among the 3 consortiums. The median transit time for all submissions was 1.0 day. Overall, median drug susceptibility testing turnaround time (date of receipt in submitting laboratory to result) for parallel testing performed in house by submitting laboratories was 31.0 days; it was 43.0 days for reference laboratories. The median turnaround time for molecular detection of drug resistance results was 1.0 day (mean = 2.8; range, 0-14) from specimen receipt at the reference laboratories. The shared services model holds promise for specialized tuberculosis testing. Sharing of services requires a balance among quality, timeliness, efficiency, communication, and fiscal costs.

Molecular detection of drug resistance to ofloxacin and kanamycin in Mycobacterium tuberculosis by using multiplex allele-specific PCR

Drug resistance in tuberculosis (TB) is the biggest global health challenge as it hinders the tuberculosis control program and makes the disease more worsen. Molecular methods interrupt the spread of drug resistance by facilitating the appropriate anti- tuberculosis therapy at correct time through rapid diagnosis of multi drug resistant (MDR) and extensively drug resistant tuberculosis (XDR-TB). In this study we standardized and evaluated the diagnostic utility of multiplex allele specific PCR (MAS-PCR) targeting gyrA D94G and rrs A1401G mutations for detection of resistance against two key drugs (ofloxacin and kanamycin) of second line anti tuberculosis treatment. MAS-PCR assays targeting gyrA D94G and rrs A1401G for ofloxacin (OFL) and kanamycin (KAN) resistance respectively were carried out on 150 multidrug resistant isolates of Mycobacterium tuberculosis. The results were compared with phenotypic drug susceptibility test against ofloxacin and kanamycin by using proportion method on MGIT 960. Of 150 MDR isolates 50 were resistant to both ofloxacin and kanamycin, 36 were resistant to ofloxacin only, 8 were resistant to kanamycin only and 56 were susceptible to both the drugs. MAS-PCR correctly identified gyrA D94G and rrs A1401G mutations in phenotypically resistant isolates with a specificity of 100%. The sensitivity of MAS-PCR was 88.66%, 93.55% and 86% for OFL, KAN and XDR-TB respectively. There was no mutation detected at gyrA D94G region of 12.86% (11 of 86) OFL resistant isolates while 6.89% (4 of 58) of KAN resistant isolates did not carry rrs A1401G substitution. MAS-PCR proves to be a rapid tool for detection of drug resistance which could also be used as an initial marker for screening of XDR-TB.

Mycobacteria.

The immunocompromised host is at increased risk of Mycobacterium tuberculosis complex and nontuberculous mycobacteria infection. Although Mycobacterium tuberculosis complex is a significant mycobacterial pathogen, nontuberculous mycobacteria causes substantial disease in those with suppressed immune responses. Mycobacterial infections can cause significant morbidity and mortality in this patient population, and rapid identification and susceptibility testing of the mycobacterial species is paramount to patient management and outcomes. Mycobacterial diagnostics has undergone some significant advances in the last two decades with immunodiagnostics (interferon gamma release assay), microscopy (light-emitting diode), culture (automated broth-based systems), identification (direct PCR, sequencing and matrix-assisted laser-desorption ionization-time of flight mass spectrometry) and susceptibility testing (molecular detection of drug resistance from direct specimens or positive cultures). Employing the most rapid and sensitive methods in the mycobacterial laboratory will have a tremendous impact on patient care and, in the case of Mycobacterium tuberculosis complex, in the control of tuberculosis.

Molecular and Growth-Based Drug Susceptibility Testing of Mycobacterium tuberculosis Complex for Ethambutol Resistance in the United States.

Ethambutol (EMB) is used as a part of drug regimens for treatment of tuberculosis (TB). Susceptibility of Mycobacterium tuberculosis complex (MTBC) isolates to EMB can be discerned by DNA sequencing to detect mutations in the embB gene associated with resistance. US Public Health Laboratories (PHL) primarily use growth-based drug susceptibility test (DST) methods to determine EMB resistance. The Centers for Disease Control and Prevention (CDC) provides a service for molecular detection of drug resistance (MDDR) by DNA sequencing and concurrent growth-based DST using agar proportion. PHL and CDC test results were compared for 211 MTBC samples submitted to CDC from September 2009 through February 2011. Concordance between growth-based DST results from PHL and CDC was 88.2%. A growth-based comparison of 39 samples, where an embB mutation associated with EMB resistance was detected, revealed a higher percentage of EMB resistance by CDC (84.6%) than by PHL (59.0%) which was significant (P value = 0.002). Discordance between all growth-based test results from PHL and CDC was also significant (P value = 0.003). Most discordance was linked to false susceptibility using the BACTEC™ MGIT™ 960 (MGIT) growth-based system. Our analysis supports coalescing growth-based and molecular results for an informed interpretation of potential EMB resistance.

Concordance between Molecular and Phenotypic Testing of Mycobacterium tuberculosis Complex Isolates for Resistance to Rifampin and Isoniazid in the United States

Multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis complex (MTBC) are defined by resistance to at least rifampin (RMP) and isoniazid (INH). Rapid and accurate detection of multidrug resistance is essential for effective treatment and interruption of disease transmission of tuberculosis (TB). Overdiagnosis of MDR TB may result in treatment with second-line drugs that are more costly, less effective, and more poorly tolerated than first-line drugs. CDC offers rapid confirmation of MDR TB by the molecular detection of drug resistance (MDDR) for mutations associated with resistance to RMP and INH along with analysis for resistance to other first-line and second-line drugs. Simultaneously, CDC does growth-based phenotypic drug susceptibility testing (DST) by the indirect agar proportion method for a panel of first-line and second-line antituberculosis drugs. We reviewed discordance between molecular and phenotypic DST for INH and RMP for 285 isolates submitted as MTBC to CDC from September 2009 to February 2011. We compared CDC's results with those from the submitting public health laboratories (PHL). Concordances between molecular and phenotypic testing at CDC were 97.4% for RMP and 92.5% for INH resistance. Concordances between CDC's molecular testing and PHL DST results were 93.9% for RMP and 90.0% for INH. Overall concordance between CDC molecular and PHL DST results was 91.7% for RMP and INH collectively. Discordance was primarily attributable to the absence of known INH resistance mutations in isolates found to be INH resistant by DST and detection of mutations associated with low-level RMP resistance in isolates that were RMP susceptible by phenotypic DST. Both molecular and phenotypic test results should be considered for the diagnosis of MDR TB.

Molecular detection of drug resistance in Mycobacterium leprae

Molecular detection of drug resistance in Mycobacterium leprae

Laboratory diagnostic aspects of drug resistant tuberculosis.

Multi-drug resistant strains of Mycobacterium tuberculosis (MDR-TB) are present world wide, and in many areas constitute a serious threat to the efficacy of TB control programs. The most effective strategies to limit further spread of MDR-TB are rapid detection of drug resistance followed by prompt and effective therapy. Routine laboratory diagnosis of drug resistance in TB requires a viable, pure culture of M. tuberculosis. Use of liquid media has decreased the turn around time for susceptibility test results however, because of the slow growth of M. tuberculosis these assays can still take 10 to 14 days. Alternatively, an increased understanding of the molecular basis for resistance to the antituberculosis drugs can greatly contribute to further decreasing turn around time. Based on this information, more precise and rapid molecular testing can be developed and lead to more appropriate and timely treatment regimens. In this review, we discuss methods for, and problems encountered in, performing TB drug susceptibility assays. Descriptions of routine protocols will be followed by recent developments in molecular detection of drug resistance.

Advances in Molecular Diagnosis of Tuberculosis.

Acccurate and early diagnosis of tuberculosis is important for its effective management. Several methods for the diagnosis of tuberculosis are available which include tuberculin test, radiological examination and other imaging methods and sputum smear microscopy. Characteristic histopathology is a very important approach but there could be problems to get representative specimen and non specific features could be a problem. The immunology is often not conclusive as antibodies and delayed type hypersensitivity response persist long after the subsidence of sub-clinical or clinical disease. Sputum smear microscopy has both the problems of sensitivity and specificity. Culture is more sensitive and is presently the yardstick for diagnosis, but the time required and frequent negative results in paucibacillary specimens are important limitations. During the last decade, major advances in understanding the genetic structure of mycobacteria have been made. Based on this newer knowledge about the specific gene sequences, several gene probes/gene amplification systems for tuberculosis have been developed [1, 2]. These molecular tools and methods can be used for the confirmation of identity of isolates, direct detection of gene sequences from the clinical specimens and also molecular detection of drug resistance.

Prediction of Drug Resistance in M. tuberculosis: Molecular Mechanisms, Tools, and Applications

Management of Tuberculosis is complicated by the emergence of drug resistant strains of Mycobacterium tuberculosis and this poses a threat to the success of Tuberculosis control programmes. Drug susceptibility testing by culture is time-consuming and technically difficult. It is known that resistance to drugs is due to a number of genomic mutations in specific genes of M. tuberculosis. These mutations in combination with molecular techniques can be used as markers for drug resistance, since drug susceptible isolates lack the corresponding gene mutations. This review focuses on molecular mechanisms, methods and applications as a possible new diagnostic tool for the early molecular detection of drug resistance in M. tuberculosis.

Application of molecular methods for detection and transmission analysis of mycobacterium tuberculosis drug resistance in patients attending a reference hospital in Italy.

A molecular analysis of drug-resistant isolates of Mycobacterium tuberculosis was done in a population with a high prevalence of human immunodeficiency virus infection. Seventy-one consecutive isolates were tested for genotypic resistance to isoniazid, rifampicin, streptomycin, and ethambutol by polymerase chain reaction-single strand conformation polymorphism analysis and automated sequencing of target regions. Phenotypic and genotypic resistance to isoniazid, rifampicin, streptomycin, and ethambutol were detected in 23.4%, 11.2%, 7%, and 5.6% of isolates and in 87%, 88%, 40%, and 100% of resistant isolates, respectively. Specificity was 100% for all target regions. When rpoB, katG, and ahpC mutation analysis were combined, 86% of resistant isolates to any drug were identified. No mutations in inhA were found in isoniazid-resistant isolates. Molecular detection of drug resistance, particularly for isoniazid and rifampicin, may represent a sensitive and very specific technique. The strategy of selecting rpoB, katG, and ahpC to quickly identify most resistant isolates, with a relevant saving of resources, is warranted.