Rapid Detection Of Resistance Research Articles

Molecular tools of antibiotic resistance for Helicobacter pylori: an overview in Latin America

Antibiotic resistance in the treatment of H. pylori infection is the principal reason for the failure of eradication regimens using the triple therapy. We reviewed the mechanisms of H. pylori antibiotic resistance and assessed the molecular tools commonly used to detect antibiotic resistance in Latin America. Most commonly reported molecular techniques were PCR and sequencing, as well as its variants PCR-RFLP and qPCR to detect the genes and mutations involved in the resistance to clarithromycin (CLR), amoxicillin (AMX), levofloxacin (LVX), tetracycline (TET), and metronidazole (MTZ). The mutation-associated resistance rates varied from 2.2% to 83.3% for CLA and 12% to 100% for LVX in 7 and 4 countries, respectively, whereas for MTZ the resistance frequency was between 50% to 100% in 4 countries. For TET and AMX, frequency of the resistance was in the range of 0% to 27% (5 and 3 countries, respectively). Molecular tools can be used for the rapid detection of resistance to CLR, LVX, and TET, whereas genotypic analysis is not appropriate to detect resistance to AMX and MTZ due to genomic changes found in the rdxA and pbp1A genes. The revision of the different molecular methods showed that qPCR and RFLP are the best tools to detect the resistance of H. pylori. Few molecular tools have been developed in Latin America to detect H. pylori resistance to antibiotics. More studies would be needed to understand better efficient strategies to prevent further emergence of antimicrobial resistance.

Agreement between Phenotypically Detected Linezolid Resistance and Mutations in rrl and rplC Genes of Mycobacterium tuberculosis Isolates Using Nanopore Sequencing.

Phenotypic drug susceptibility testing (DST) is considered the gold standard for detecting linezolid (LZD) resistance in Mycobacterium tuberculosis (MTB), but it is time-consuming. Nanopore sequencing offers a potentially faster alternative approach. This study evaluated the agreement between phenotypically detected LZD resistance and mutations in the rrl and rplC genes of MTB isolates using nanopore sequencing. Consecutive drug-resistant MTB isolates from pulmonary samples collected in 2021 underwent liquid culture (LC) DST for LZD. All resistant isolates and an equal number of susceptible isolates were subjected to targeted sequencing of the rrl and rplC genes using nanopore technology. Sequencing identified a C154R mutation in the rplC gene in only one LZD-resistant isolate. No mutations were detected in the rrl gene. The agreement between sequencing and LC-DST for detecting LZD resistance was poor (Cohen's kappa: 0.03571, 95% confidence interval [CI]: -0.034-0.105). Additionally, no significant association was found between LZD resistance and clinical or microbiological outcomes at 6-month follow-up. This study revealed a considerable discrepancy between phenotypic and genotypic detection of LZD resistance in MTB. Further research is needed to better understand the genetic mechanisms underlying LZD resistance and to develop reliable molecular diagnostics for rapid resistance detection.

Evaluation of Whole Genome Sequencing-Based Predictions of Antimicrobial Resistance to TB First Line Agents: A Lesson from 5 Years of Data.

Phenotypic susceptibility testing of the Mycobacterium tuberculosis complex (MTBC) isolate requires culture growth, which can delay rapid detection of resistant cases. Whole genome sequencing (WGS) and data analysis pipelines can assist in predicting resistance to antimicrobials used in the treatment of tuberculosis (TB). This study compared phenotypic susceptibility testing results and WGS-based predictions of antimicrobial resistance (AMR) to four first-line antimicrobials-isoniazid, rifampin, ethambutol, and pyrazinamide-for MTBC isolates tested between the years 2018-2022. For this 5-year retrospective analysis, the WGS sensitivity for predicting resistance for isoniazid, rifampin, ethambutol, and pyrazinamide using Mykrobe was 86.7%, 100.0%, 100.0%, and 47.8%, respectively, and the specificity was 99.4%, 99.5%, 98.7%, and 99.9%, respectively. The predictive values improved slightly using Mykrobe corrections applied using TB Profiler, i.e., the WGS sensitivity for isoniazid, rifampin, ethambutol, and pyrazinamide was 92.31%, 100%, 100%, and 57.78%, respectively, and the specificity was 99.63%. 99.45%, 98.93%, and 99.93%, respectively. The utilization of WGS-based testing addresses concerns regarding test turnaround time and enables analysis for MTBC member identification, antimicrobial resistance prediction, detection of mixed cultures, and strain genotyping, all through a single laboratory test. WGS enables rapid resistance detection compared to traditional phenotypic susceptibility testing methods using the WHO TB mutation catalog, providing an insight into lesser-known mutations, which should be added to prediction databases as high-confidence mutations are recognized. The WGS-based methods can support TB elimination efforts in Canada and globally by ensuring the early start of appropriate treatment, rapidly limiting the spread of TB outbreaks.

Rapid resistance detection is reliable for prompt adaptation of isoniazid resistant tuberculosis management

ObjectivesAppropriate tuberculosis (TB) management requires anti-TB drugs resistance detection. We assessed the performance of rapid resistance detection assays and their impact on treatment adaptation, focusing on isoniazid resistant (Hr) TB. MethodsFrom 2016 to 2022, all TB cases enrolled in 3 hospitals were reviewed for phenotypic drug susceptibility testing (p-DST) and genotypic DST (g-DST) performed by rapid molecular testing, and next generation sequencing (NGS). Clinical characteristics, treatment and outcome were collected for Hr-TB patients. The concordance between g-DST and p-DST results, and delay between treatment initiation and results of g-DST and p-DST were respectively recorded to assess the contribution of DST results on Hr-TB management. ResultsAmong 654 TB cases enrolled, 29 were Hr-TB. Concordance between g-DST by rapid molecular methods and p-DST was 76.9 %, whilst concordance between NGS-based g-DST and p-DST was 98.7 %. Rapid resistance detection significantly fastened Hr-TB treatment adaptation (median delay between g-DST results and treatment modification was 6 days). It consisted in fluoroquinolone implementation for 17/23 patients; outcome was favourable except for 2 patients who died before DST reporting. ConclusionRapid resistance detection fastened treatment adaptation. Also, NGS-based g-DST showed almost perfect concordance with p-DST, thus providing rapid and safe culture-free DST alternative.

Development of a rapid detection assay for acetolactate synthase inhibitors resistance in three Amaranthus weed species through loop-mediated isothermal amplification.

The early detection of herbicide resistance in weeds is a key factor to avoid herbicide waste and improve agriculture sustainability. The present study aimed to develop and validate an allele-specific loop-mediated isothermal amplification (AS-LAMP) assay for the quick on-site detection of the resistance-endowing point mutation Trp-574-Leu in the acetolactate synthase (ALS) gene in three widely diffused Amaranthus weed species: Amaranthus retroflexus, Amaranthus hybridus and Amaranthus tuberculatus. The AS-LAMP protocol was developed on wild-type and ALS-mutant plants of the three species and revealed that the amplification approach with only the primer set specific for the mutant allele (574-Leu) was the most promising. The validation and estimation of the AS-LAMP performance evaluated by comparing the results with those of the molecular marker (cleaved amplified polymorphic sequences) indicated that, although the sensitivity and specificity were relatively high in all species (overall 100 and > 65%, respectively), precision was high for A. hybridus L. and A. retroflexus L. (75 and 79%, respectively), but quite low for A. tuberculatus (Moq.) J. D. Sauer (59%). The LAMP assay was also effective on crude genomic DNA extraction, allowing the quick detection of mutant plants in field situation (on site resistance detection). The proposed AS-LAMP method has proven to be a promising technique for rapid detection of resistance as a result of Trp-574-Leu on the two monoecious weedy Amaranthus species but resulted less effective in the genetically variable dioecious species A. tuberculatus. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

TaqMan-MGB PCR Method for Rapid Detection of QoI Fungicide Resistance in Chinese Populations of Plasmopara viticola.

Grape downy mildew caused by Plasmopara viticola is one of the most devastating diseases of grapevine worldwide. Quinone outside inhibitor (QoI) fungicides are commonly used for the control of the pathogen in grape fields across China. However, their recurrent use could lead to the emergence of resistance against these compounds. Based on the most common mutation in resistant isolates, a glycine to alanine substitution at amino acid position 143 (G143A) in the cytochrome b protein, a TaqMan-MGB PCR was developed for the rapid detection of resistance to the QoI fungicide azoxystrobin in P. viticola. Specificity and sensitivity of this method showed it could specifically detect the point mutations linked with QoI resistance in P. viticola, and the detection limit was 0.2 pg. It could also quantify the resistance allele even in isolate mixtures containing as little as 5% QoI-resistant P. viticola strains. With this method, a large P. viticola population (n = 2,373) was screened, and QoI-resistant isolates were identified for the first time in China. The average frequencies of the resistant genotype from eight major-grapevine regions were up to 66%. Taken together, the results not only provide a novel tool for the rapid distinction and quantification of the QoI-resistant allele in P. viticola but also provide important references for fungicide selection and application, which will facilitate resistance management of grape downy mildew and improve grape production systems in Chinese vineyards.

Rapid test to detect insecticide resistance in field populations of Spodoptera frugiperda (Lepidoptera: Noctuidae).

Introduction: The development of insecticide resistance in Spodoptera frugiperda populations is a serious threat to the crop industry. Given the spread of invasive resistant populations, prospective monitoring should be accelerated, and the development of diagnostic tools for rapid and accurate assessments of insecticide resistance is essential. Methods: First, the discriminating dose and diagnostic time of the kit were determined by the glass vial method based on a susceptible strain. Then, pests that were collected from field populations were used to determine their susceptibility to seven insecticides by using the diagnostic kit. Finally, the accuracy of the kit was verified based on correlation analyses and the likelihood of insecticide control failure was assessed. Results: Here, we describe a diagnostic kit that enables the rapid detection of resistance to chlorpyrifos, bifenthrin, deltamethrin, lambda-cyhalothrin, phoxim, chlorantraniliprole and chlorfenapyr within 1-2h in S. frugiperda at diagnostic doses of 0.98, 0.84, 0.38, 1.64, 0.0082, 1.75 and 0.65μg/cm2, respectively. The linear equation between mortalities under diagnostic doses and actual resistance ratios measured by the diet-overlay bioassay was determined. The high correlation indicates that the insecticide resistance levels diagnosed by the kit were consistent with the results of the diet-overlay bioassay. Moreover, we found a significant negative correlation between diagnostic mortality and the likelihood of control failure for bifenthrin (r = -0.899, p = 0.001), deltamethrin (r = -0.737, p = 0.024) and lambda-cyhalothrin (r = -0.871, p = 0.002). Discussion: The insecticide resistance diagnostic kit for S. frugiperda is a user-friendly tool (portable, short detection time). Its excellent performance qualifies the kit as a reliable screening tool for identifying effective insecticides in sustainable resistance management.

Detection of azole-resistant Aspergillus fumigatus in the environment from air, plant debris, compost, and soil.

Aspergillus fumigatus is a ubiquitous fungus, a saprophyte of plants, and an opportunistic pathogen of humans. Azole fungicides are used in agriculture to control plant pathogens, and azoles are also used as a first line of treatment for aspergillosis. The continued exposure of A. fumigatus to azoles in the environment has likely led to azole resistance in the clinic where infections result in high levels of mortality. Pan-azole resistance in environmental isolates is most often associated with tandem-repeat (TR) mutations containing 34 or 46 nucleotides in the cyp51A gene. Because the rapid detection of resistance is important for public health, PCR-based techniques have been developed to detect TR mutations in clinical samples. We are interested in identifying agricultural environments conducive to resistance development, but environmental surveillance of resistance has focused on labor-intensive isolation of the fungus followed by screening for resistance. Our goal was to develop assays for the rapid detection of pan-azole-resistant A. fumigatus directly from air, plants, compost, and soil samples. To accomplish this, we optimized DNA extractions for air filters, soil, compost, and plant debris and standardized two nested-PCR assays targeting the TR mutations. Sensitivity and specificity of the assays were tested using A. fumigatus DNA from wild type and TR-based resistant isolates and with soil and air filters spiked with conidia of the same isolates. The nested-PCR assays were sensitive to 5 fg and specific to A. fumigatus without cross-reaction with DNA from other soil microorganisms. Environmental samples from agricultural settings in Georgia, USA were sampled and tested. The TR46 allele was recovered from 30% of samples, including air, soil and plant debris samples from compost, hibiscus and hemp. These assays allow rapid surveillance of resistant isolates directly from environmental samples improving our identification of hotspots of azole-resistant A. fumigatus.

Clinical utility of target-based next-generation sequencing for drug-resistant TB.

BACKGROUND: In high TB burden countries, access to drug susceptibility testing is a major bottleneck. Targeted next-generation sequencing (tNGS) is a promising technology for rapid resistance detection. This study assessed the role of tNGS for the diagnosis of drug-resistant TB (DR-TB).METHODS: A total of 161 samples from bacteriologically confirmed TB cases were subjected to tNGS using the Deeplex® Myc-TB kit and sequenced using the MiSeq platform. These samples were also processed for conventional phenotypic DST (pDST) using 13 drugs on Mycobacteria Growth Indicator Tube and line-probe assays (MTBDRplus and MTBDRsl).RESULTS: There were 146 DR-TB and 15 drug-susceptible TB (DS-TB) samples. About 70% of patients with DR-TB had no previous TB treatment history. Overall, 88.2% had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB), 58.5% pre-extensively drug-resistant TB (pre-XDR-TB) and 9.2% had XDR-TB as defined by the WHO (2020). Around 8% (n = 13) of samples were non-culturable; however, identified 8 were resistant to first and second-line drugs using tNGS. Resistance frequency was similar across methods, with discordance in drugs less reliable using pDST or with limited mutational representation within databases. Sensitivities were aligned with literature reports for most drugs. We observed 10% heteroresistance, while 75% of strains were of Lineages 2 and 3.CONCLUSIONS: Programme data supported tNGS in the diagnosis of DR-TB for early treatment using individualised regimens.

Evaluation of the BL-RED (Beta-Lactamase Rapid Electrochemical Detection) test for the rapid detection of resistance to third-generation cephalosporins in Enterobacteriaceae

The sensitivity of NG-test CTX-M Multi assay and BL-RED test incubated 10 min for the detection of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae was 80.6% and 90.3% respectively. Using an extended 60 min incubation with the BL-RED test, its sensitivity was increased to 100% and 60.9% for ESBL-producing and cephalosporinase-overexpressing Enterobacteriaceae respectively.

First insights into the phylogenetic diversity of Mycobacterium tuberculosis in Kuwait and evaluation of REBA MTB-MDR assay for rapid detection of MDR-TB

Early detection of Mycobacterium tuberculosis (Mtb) in clinical specimens, its susceptibility to anti-TB drugs and disruption of infection transmission to new hosts are essential components for global tuberculosis (TB) control efforts. This study investigated major Mtb genotypes circulating in Kuwait and evaluated the performance of REBA MTB-MDR (REBA) test in comparison to GenoType MTBDRplus (gMTBDR+) assay for rapid detection of resistance of Mtb to isoniazid and rifampicin (MDR-TB). M. tuberculosis isolates (n = 256) originating predominantly from expatriate patients during a 6-month period were tested by spoligotyping and a dendrogram was created by UPGMA using MIRU-VNTRplus software. Phenotypic drug susceptibility testing (DST) was performed by MGIT 960 system. Genotypic DST for isoniazid and rifampicin was done by REBA and gMTBDR+ assays. Spoligotyping assigned 188 (73.4%) isolates to specific spoligotype international type (SIT) while 68 isolates exhibited orphan patterns. All major M. tuberculosis lineages were detected and EAI, CAS and Beijing families were predominant. Phylogenetic tree showed 131 patterns with 105 isolates exhibiting a unique pattern while 151 isolates clustered in 26 patterns. Fifteen isolates were resistant to one/more drugs. REBA and gMTBDR+ detected isoniazid resistance in 11/12 and 10/12 and rifampicin resistance in 4/5 and 4/5 resistant isolates, respectively. The diversity of SIT patterns are highly suggestive of infection of most expatriate patients with unique Mtb strains, likely acquired in their native countries before their arrival in Kuwait. Both, REBA and gMTBDR+ assays performed similarly for detection of resistance of Mtb to isoniazid and rifampicin for rapid detection of MDR-TB.

Evaluation of xpert Mycobacterium tuberculosis rifampicin for tuberculosis diagnosis in a reference laboratory.

The Xpert Mycobacterium tuberculosis Rifampicin (MTB-RIF) is a technological innovation that presents precision and speed in the diagnosis of tuberculosis (TB). The study aimed to evaluate the performance of Xpert MTB/RIF in the TB diagnosis and compare its results with those of culture, species identification, Antimicrobial Susceptibility Testing (AST), and rpoB gene sequencing of discordant results in AST, used for the diagnosis of TB in a reference laboratory. Cross-sectional descriptive study of pulmonary and extrapulmonary samples requesting Xpert MTB/RIF and culture for TB diagnosis from 2015 to 2019 at Adolfo Lutz Institute-São Paulo/Brazil. The analysis was performed with Epi-Info 7.2.1, presenting the distribution of frequencies and, for comparative analyses, Pearson's Chi-square test and Fisher's exact test were used, considering P ≤ 0.05 as statistically significant. For variables agreement, the Kappa method was used. A total of 1575 pulmonary and extrapulmonary samples were analyzed using Xpert MTB/RIF and culture, of which 293 were positive for the MTB Complex in both methodologies with a sensitivity of 94.55%; specificity of 95.97%; accuracy 95.69%; positive predictive value 85.53%; negative predictive value 98.59%, substantial agreement by Kappa 0.87, and detection sensitivity even at lower levels of bacillary load (P < 0.05). The Xpert MTB/RIF and AST showed concordant results (P < 0.05). The study brings forward that the Xpert MTB/RIF shows substantial agreement with the results of culture and AST, contributing to the diagnosis of TB and the rapid detection of resistance. The sequencing of resistant cultures in Xpert MTB/RIF and sensitivity in AST identified the H526N mutation, characterized by a low level of resistance to RIF.

Antibiotic resistance acquisition versus primary transmission in the presentation of extensively drug-resistant tuberculosis.

Mycobacterium tuberculosis is the leading cause of mortality worldwide due to a single bacterial pathogen. Of concern is the negative impact that the COVID-19 pandemic has had on the control of tuberculosis (TB) including drug-resistant forms of the disease. Antimicrobial resistance increases the likelihood of worsened outcomes in TB patients including treatment failure and death. Multidrug-resistant (MDR) strains, resistant to first-line drugs isoniazid and rifampin, and extensively drug-resistant (XDR) strains with further resistance to second-line drugs (SLD), threaten control programs designed to lower TB incidence and end the disease as a public health challenge by 2030, in accordance with UN Sustainable Development Goals. Tackling TB requires an understanding of the pathways through which drug resistance emerges. Here, the roles of acquired resistance mutation, and primary transmission, are examined with regard to XDR-TB. It is apparent that XDR-TB can emerge from MDR-TB through a small number of additional resistance mutations that occur in patients undergoing drug treatment. Rapid detection of resistance, to first-line drugs and SLD, at the initiation of and during treatment, and prompt adjustment of regimens are required to ensure treatment success in these patients. Primary transmission is predicted to make an increasing contribution to the XDR-TB caseload in the future. Much work is required to improve the implementation of the World Health Organization-recommended infection control practices and block onward transmission of XDR-TB patients to contacts including health-care workers. Finally, limiting background resistance to fluoroquinolones in pre-XDR strains of M. tuberculosis will necessitate better antimicrobial stewardship in the broader use of this drug class.

An insecticide phenotypic resistance diagnostic kit for cotton aphid Aphis gossypii Glover (Hemiptera Aphididae).

Aphis gossypii is a notorious pest worldwide, and evidence of resistance of A. gossypii to various insecticides has been documented. Diagnostic tools for the rapid and accurate assessment of insecticide resistance are urgently needed to implement effective pest control and insecticide resistance management strategies. Using this diagnostic kit based on the glass vial bioassay, detection results can be obtained in 3h and the values of 897.86, 133.57, 12 037.45, 2849.26, 19 457.33 and 215.60 ng/cm2 were finally identified as the actual diagnostic doses of imidacloprid, acetamiprid, thiamethoxam, nitenpyram, dinotefuran and sulfoxaflor, respectively. The regression equation between mortalities under diagnostic doses and actual resistance ratios tested by the leaf-dipping method were conducted in different strains of A. gossypii, and the diagnostic mortality of A. gossypii was negatively correlated with the resistance ratio to imidacloprid (r=-0.986, P=0.002), acetamiprid (r=-0.964, P=0.008), thiamethoxam (r=-0.930, P=0.022), nitenpyram (r=-0.950, P=0.013), dinotefuran (r=-0.976, P=0.004) and sulfoxaflor (r=-0.937, P=0.019). Moreover, four A. gossypii field populations were selected to apply the diagnostic kit in the field. A diagnostic kit based on the glass vial bioassay for the rapid detection of resistance to imidacloprid, acetamiprid, thiamethoxam, nitenpyram, dinotefuran and sulfoxaflor in A. gossypii was developed. The insecticide diagnostic kit for A. gossypii can be a useful screening tool to determine effective insecticides quickly and accurately. © 2022 Society of Chemical Industry.

Performance evaluation of the FAST™ System and the FAST-PBC Prep™ cartridges for speeded-up positive blood culture testing.

ObjectivesAs time to appropriate antimicrobial therapy is major to reduce sepsis mortality, there is great interest in the development of tools for direct identification (ID) and antimicrobial susceptibility testing (AST) of positive blood cultures (PBC). Very recently, the FAST™ System (Qvella) has been developed to isolate and concentrate microorganisms directly from PBCs, resulting in the recovery of a Liquid Colony™ (LC) within 30 min. The LC can be used as equivalent of an overnight subcultured colony for downstream testing. We aimed to evaluate the performances of the FAST™ System and FAST-PBC Prep™ cartridges by testing the resulting LC for direct ID, AST and rapid resistance detection.Materials and methodsProspectively, FAST™ System testing was carried out on each patient’s first PBC with a monomicrobial Gram-stain result. In the second arm of the study, FAST™ System testing was carried out on blood cultures spiked with multidrug-resistant bacteria. Downstream testing using the LC included MALDI-TOF MS ID with the Bruker Biotyper® smart system, rapid resistance detection testing including the Abbott Diagnostics Clearview™ PBP2a SA Culture Colony Test (PBP2a) and the Bio-Rad βLACTA™ Test (βLT). AST was performed using the Becton Dickinson Phoenix™ System or by Bio-Rad disk diffusion using filter paper disk following EUCAST 2020 breakpoint criteria.ResultsFAST™ System testing was completed on 198 prospective PBCs and 80 spiked blood cultures. After exclusion of polymicrobial blood cultures, performance evaluation compared with standard of care results was carried out on 266 PBCs. Concordant, erroneous and no ID results included 238/266 (89.5%), 1/266 (0.4%), 27/266 (10.2%) PBCs, respectively. Sensitivity and specificity for PBP2a were 100% (10/10) and 75% (15/20), respectively. Sensitivity and specificity for βLT were 95.8% (23/24) and 100% (42/42), respectively. Categorical agreement for all 160 tested strains was 98% (2299/2346) with 1.2% (8/657) very major errors and 0.7% (10/1347) major errors.ConclusionFAST™ System testing is a reliable approach for direct downstream testing of PBCs including MALDI-TOF MS ID, BD Phoenix™ and Bio-Rad disk diffusion AST as well as rapid resistance testing assays. Next steps include optimal integration of the FAST™ System in the PBC workflow with a view toward clinical studies.

Performance of the GenoType MTBDRsl in a programmatic setting, South Africa.

BACKGROUND: The GenoType MTBDRsl v2 is a molecular test designed for the rapid detection of resistance to second-line anti-TB drugs in Mycobacterium tuberculosis complex (MTBC).OBJECTIVE: To assess the use of MTBDRsl in a programmatic setting and to describe the resistance patterns in a high HIV-TB-endemic area in South Africa.METHODS: We performed a retrospective data analysis of all MTBDRsl results in patients with newly diagnosed rifampicin-resistant TB (RR-TB). We compared its performance on direct testing of smear-positive and smear-negative specimens. Results were examined to observe the detected resistance-conferring mutations.RESULTS: Of 1873 RR-TB/multidrug-resistant TB (MDR-TB), 37.4% were smear-negative and 62.5% were smear-positive. Among smear-negative specimens, the MTBDRsl showed an inconclusive rate of 61.2%, while the inconclusive rate from smear-positive specimens was 6.6%. The most common mutation observed in case of fluoroquinolone resistance occurred at the gyrA gene, codon 90 (A90V) (61/158, 38.6%), and the most common mutation in injectable aminoglycoside resistance occurred in the rrs region, A1401G (71/108, 65.7%).CONCLUSION: In HIV-TB-prevalent settings, routine use of the MTBDRsl is more effective when performed directly on smear-positive specimens. In view of currently used injectable-free regimens, this test requires revision.

Implementation of a New Protocol for Direct Identification from Urine in the Routine Microbiological Diagnosis.

Background: The direct identification of uropathogens from urine samples, in combination with the rapid detection of resistance, would allow early adjustment of empirical antimicrobial treatment. Methods: Two hundred and ninety-eight urine samples processed between 1 June and 31 December 2020, selected with flow cytometry, with direct identification by MALDI-TOF mass spectrometry, and rapid detection of extended-spectrum beta-lactamase (ESBL) and carbapenemases-producing strains by lateral flow were analyzed. Results: The positive predictive value of the direct identification of the 86 samples that met the flow cytometry criterion (>5000 bacteria/µL) was 96.4%. Reliable direct identification was obtained in 14 of the 27 (51.8%) urinary source bacteraemias. There was 100% agreement between the lateral flow and antibiogram in the detection of ESBL and carbapenemases. Conclusion: the protocol for the direct identification and rapid detection of ESBL and carbapenemases-producing strains from urine samples is a reliable and useful tool.

Drug resistant TB – latest developments in epidemiology, diagnostics and management

AimThe aim of this review is to inform the reader on the latest developments in epidemiology, diagnostics and management. EpidemiologyDrug-resistant Tuberculosis (DR-TB) continues to be a current global health threat, and is defined by higher morbidity and mortality, sequelae, higher cost and complexity. The WHO classifies drug-resistant TB into 5 categories: isoniazid-resistant TB, rifampicin resistant (RR)-TB and MDR-TB, (TB resistant to isoniazid and rifampicin), pre-extensively drug-resistant TB (pre-XDR-TB) which is MDR-TB with resistance to a fluoroquinolone and finally XDR-TB that is TB resistant to rifampicin, plus any fluoroquinolone, plus at least one further priority A drug (bedaquiline or linezolid). Of 500,000 estimated new cases of RR-TB in 2020, only 157 903 cases are notified. Only about a third of cases are detected and treated annually. DiagnosticsRecently newer rapid diagnostic methods like the GeneXpert, whole genome sequencing and Myc-TB offer solutions for rapid detection of resistance. TreatmentThe availability of new TB drugs and shorter treatment regimens have been recommended for the management of DR-TB. ConclusionDespite advances in diagnostics and treatments we still have to find and treat two thirds of the drug resistant cases that go undetected and therefore go untreated each year. Control of TB and elimination will only occur if cases are detected, diagnosed and treated promptly.

Antimicrobial Susceptibility Testing: A Comprehensive Review of Currently Used Methods.

Antimicrobial resistance (AMR) has emerged as a major threat to public health globally. Accurate and rapid detection of resistance to antimicrobial drugs, and subsequent appropriate antimicrobial treatment, combined with antimicrobial stewardship, are essential for controlling the emergence and spread of AMR. This article reviews common antimicrobial susceptibility testing (AST) methods and relevant issues concerning the advantages and disadvantages of each method. Although accurate, classic technologies used in clinical microbiology to profile antimicrobial susceptibility are time-consuming and relatively expensive. As a result, physicians often prescribe empirical antimicrobial therapies and broad-spectrum antibiotics. Although recently developed AST systems have shown advantages over traditional methods in terms of testing speed and the potential for providing a deeper insight into resistance mechanisms, extensive validation is required to translate these methodologies to clinical practice. With a continuous increase in antimicrobial resistance, additional efforts are needed to develop innovative, rapid, accurate, and portable diagnostic tools for AST. The wide implementation of novel devices would enable the identification of the optimal treatment approaches and the surveillance of antibiotic resistance in health, agriculture, and the environment, allowing monitoring and better tackling the emergence of AMR.

Direct detection of resistance to fluoroquinolones/SLIDs in sputum specimen by GenoType MTBDRsl v.2.0 assay A study from Eastern Uttar Pradesh, India

BackgroundAccording to World Health Organization (WHO), drug-resistant tuberculosis (DR-TB) is a major contributor to antimicrobial resistance globally and continues to be a public health threat. Annually, about half a million people fall ill with DR-TB globally. The gradual increase in resistance to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs), poses a serious threat to effective TB control and adequate patient management. Therefore, WHO suggests the use of GenoType MTBDRsl v.2.0 assay for detection of multiple mutations associated with FQs and SLIDs. Hence, the study was conducted to determine the prevalence of resistance to FQs and SLIDs by comparing direct GenoType MTBDRsl v.2.0 assay with phenotypic drug susceptibility testing (DST).MethodsThe study was conducted on 1320 smear positive sputum samples from a total of 2536 RR-TB, confirmed by GeneXpert MTB/RIF. The smear positive specimens were decontaminated, and DNA extraction was performed. Furthermore, the extracted DNA was used for GenoType MTBDRsl v.2.0 assay. While 20% of the decontaminated specimens were inoculated in Mycobacterium growth indicator tube (MGIT) for drug susceptibility testing (DST).ResultsOut of 1320 smear positive sputum samples, 1178 were identified as Mycobacterium tuberculosis complex (MTBC) and remaining were negative by GenoType MTBDRsl v.2.0 assay. Of the 1178 MTBC positive, 26.6% were sensitive to both FQs and SLIDs, whereas 57.3% were only FQs resistant and 15.9% were resistant to both FQs and SLIDs. Further DST of 225 isolates by liquid culture showed that 17% were sensitive to both FQs and SLIDs, 61.3% were only FQs resistant and 21.3% were resistant to both. The specificity for FQs and SLIDs was 92.31% and 100% whereas sensitivity was 100% respectively by GenoType MTBDRsl v.2.0 assay in direct sputum samples.ConclusionsOur study clearly suggests that GenoType MTBDRsl v.2.0 assay is a reliable test for the rapid detection of resistance to second-line drugs after confirmation by GeneXpert MTB/RIF assay for RR-TB. Though, high rate FQ (ofloxacin) resistance was seen in our setting, moxifloxacin could be used as treatment option owing to very low resistance.