Identification Of Resistance Mutations Research Articles

Evolution of Laboratory Diagnosis of Tuberculosis.

Tuberculosis (TB) is an infectious disease of global public health importance caused by the Mycobacterium tuberculosis complex. Despite advances in diagnosis and treatment, this disease has worsened with the emergence of multidrug-resistant strains of tuberculosis. We aim to present and review the history, progress, and future directions in the diagnosis of tuberculosis by evaluating the current methods of laboratory diagnosis of tuberculosis, with a special emphasis on microscopic examination and cultivation on solid and liquid media, as well as an approach to molecular assays. The microscopic method, although widely used, has its limitations, and the use and evaluation of other techniques are essential for a complete and accurate diagnosis. Bacterial cultures, both in solid and liquid media, are essential methods in the diagnosis of TB. Culture on a solid medium provides specificity and accuracy, while culture on a liquid medium brings rapidity and increased sensitivity. Molecular tests such as LPA and Xpert MTB/RIF have been found to offer significant benefits in the rapid and accurate diagnosis of TB, including drug-resistant forms. These tests allow the identification of resistance mutations and provide essential information for choosing the right treatment. We conclude that combined diagnostic methods, using several techniques and approaches, provide the best result in the laboratory diagnosis of TB. Improving the quality and accessibility of tests, as well as the implementation of advanced technologies, is essential to help improve the sensitivity, efficiency, and accuracy of TB diagnosis.

Clinical utility of next generation sequencing (NGS) on circulating tumor DNA (ctDNA) in patients (pts) with pancreatic cancer (PC).

4154 Background: ctDNA provides opportunities for identifying targeted therapies, a source to conduct NGS when tissue acquisition is infeasible, detection of minimal residual disease (MRD), and identification of resistance mutations. There are limited large data sets to inform clinical utility and limited correlation of NGS of ctDNA and tumor in PC. Herein, we evaluate the ctDNA detection rate in multiple cohorts of PC and report the ctDNA-tissue genotype concordance in PC at Memorial Sloan Kettering (MSK). Methods: Pts with PC at MSK who had ctDNA prospectively collected using the MSK-ACCESS 129 gene ctDNA NGS assay, were identified. ctDNA detection was defined as the identification of a mutation, copy number (no.) alteration or structural variant. Tissue-based NGS using MSK-IMPACT gene assay was performed for pts with adequate tissue, and matched with ctDNA by date. Clinical, pathologic and outcome data were abstracted. Data are summarized with descriptive statistics. Overall survival (OS) estimated by Kaplan-Meier method from date of ctDNA draw to death/last follow up. Results: From 08/2019 to 07/2022, N= 414 pts with PC and ≥ 1 ctDNA sample included. Median age 69 years (range 29-92), female N=206 (50%). Stage at ctDNA collection: Stage I-III N= 203 (49%); Stage IV N= 211 (51%). ctDNA detection rate by no. of involved organs; 0 organs (MRD), N=7/30 (23%); 1-2 organs, N=158/270 (58.5%); 3-4 organs, N=82/102 (80.4%); >5 organs, 12/12 (100%). CtDNA detection rate by site of metastasis; Liver only, 75/99 (76%); peritoneum only, 15/25 (60%); lung only, 10/22 (45%). In pts with detected ctDNA median CA 19-9 was 494 U/mL (range 0, 247674) vs 100 U/mL (range 1, 22360) in pts with undetected ctDNA. In pts with detected ctDNA median CEA was 7 ng/mL (range 1, 2020) vs 4 ng/mL (range 1, 99) in pts with undetected ctDNA. In stage IV untreated pts, median OS 13 months(m) (95% CI; 7.3, 16) and 10 m (95% CI; 5.6, -) for ctDNA detected vs undetected, respectively. ctDNA detection rates by stage, and concordance between ctDNA and tissue-based NGS for common driver mutations KRAS, TP53, CDKN2A and SMAD4 in N=131 matched pairs are summarized in the table. Conclusions: ctDNA detection rates are high (89%) in pts with untreated stage IV PC, with high concordance between ctDNA and tissue-based NGS (87% - 95%). In untreated stage I-III PC, detection and concordance rates are lower (<50%). Detection rates are associated with disease burden, site of metastasis, CA 19-9/CEA levels. ctDNA is a promising tool in detection of somatic alterations in PC, particularly in stage IV disease, and is a complementary adjunct to tumor-based NGS. [Table: see text]

The value of defining molecular resistance in patients with progressive EGFR and ALK-driven lung cancer in a public system.

3126 Background: Repeat molecular profiling, except to detect EGFR T790M, is not routinely performed in Canadian patients with lung cancer progressing on EGFR tyrosine kinase inhibitors (TKIs). We performed genomic profiling on post-progression biopsies in patients with stage IV non-small cell lung cancer (NSCLC) and known EGFR/ ALK aberrations treated with TKIs to identify resistance mechanisms, evaluate options for subsequent treatment, and to assess clinical trial eligibility and costs. Methods: From Feb 2018-Aug 2020, post-progression tumour biopsies from consenting patients at a major cancer centre underwent genomic profiling (ThermoFisher OCA v3.0 including hotspots, fusions, and copy number variations in 161 cancer-associated genes). Outcomes of interest were the identification of resistance mutations, actionable targets, clinical trial eligibility (per clinicaltrials.gov), and costs. Results: Thirty-two patients consented to the study. Most, 84% (n = 27), had successful testing completed while 16% (n = 5) had insufficient tissue. Median age of the cohort was 56 yrs, 59% (n = 16) were female, 74% (n = 20) were never-smokers, 81% (n = 22) had ECOG performance status 0-1, and 67% (n = 18) were Asian. The majority, 81% (n = 22) had EGFR mutated NSCLC, and had progressed on EGFR TKIs (15 with previously identified T790M had progressed on osimertinib), and 19% (n = 5) had ALK fusions. Patients had received a median of 2 prior lines of targeted therapy prior to re-biopsy (IQR 1.5,3). One patient had evidence of small cell transformation and associated TP53 and RB1 mutations, 11% (n = 3) had acquired EGFR C797S mutations, and 11% (n = 3) had acquired ALK resistance point mutations (G1202R n = 2, I1171N n = 1). Genomic profiling identified additional actionable targets in 19% of patients (n = 5: MET exon 14 skip mutation n = 1, MET amplification n = 2, BRAF V600E n = 2). Overall, 33% (n = 9) patients had management-changing resistance mechanisms identified (small cell transformation n = 1, actionable targets n = 5, ALK inhibitor resistance = 3). New clinical trial options based on genomic profiling results were identified for 67% (n = 18) of patients. Incremental costs for repeat genomic profiling were approximately $880 CAD per case. Conclusions: Molecular profiling upon development of resistance to targeted therapy in our cohort revealed actionable resistance mechanisms for over a third of patients and clinical trial options for 67%. These incremental benefits for patients highlight the importance of routine molecular profiling in the setting of acquired TKI resistance in lung cancer.

Acaricide resistance status and identification of resistance mutations in populations of the two-spotted spider mite Tetranychus urticae from Ethiopia.

The intensive use of pesticides is a common practice for the management of the two-spotted spider mite, Tetranychus urticae, in greenhouses and field farms of Ethiopia. However, incidence of resistance and possible resistance mechanisms in T. urticae populations from Ethiopia have not yet been studied. Here, we assessed the toxicity of various acaricides-bifenazate, abamectin, emamectin benzoate, profenofos, fenbutatin oxide, fenpyroximate, amitraz and chlorfenapyr-on T. urticae populations sampled from six flower greenhouse farms, three strawberry greenhouse farms, one field-grown vegetable farm and two wild populations. In parallel, all populations were screened for known target-site mutations. All tested populations were fully susceptible to bifenazate, abamectin, emamectin benzoate and profenofos, but resistant against fenbutatin oxide and fenpyroximate. Four populations showed considerable levels of resistance against amitraz and one population was resistant to chlorfenapyr. Several target-site mutations were identified in the tested populations, including G119S, A201S, T280A, G328A and F331W/C/Y in acetylcholinesterase and the F1538I and L1024V mutation in the voltage-gated sodium channel. The F1538I mutation was found in eight out of 12 populations, whereas the L1024V mutation was only found in two populations. The H92R mutation in the PSST subunit of complex I and the I1017F mutation in chitin synthase 1 was detected in half of the tested populations. The G326E and I321T mutations in the glutamate-gated chloride channel 3 were also detected, but more rarely, whereas mitochondrial cytochrome b mutations were not detected. The current study revealed multiple resistance patterns in Ethiopian T. urticae populations and together with the wide presence of target-site mutations, calls for the wise use of acaricides in the management of T. urticae in Ethiopia.

Generation of a protective murine monoclonal antibody against the stem of influenza hemagglutinins from group 1 viruses and identification of resistance mutations against it.

Vaccines that elicit broadly cross-neutralizing antibodies, including antibodies that target the conserved stem of hemagglutinin (HA), are being developed as a strategy for next-generation influenza vaccines that protect against influenza across multiple years. However, efficient induction of cross-neutralizing antibodies remains a challenge, and potential escape mutations have not been well characterized. Here we elicited cross-neutralizing antibodies by immunizing animals with the hemagglutinins from H5 and H9 subtype influenza A viruses that are sensitive to neutralization by stem antibodies. We further isolated and characterized an HA stem monoclonal antibody 4C2 that broadly neutralizes group 1 influenza viruses and identified HA mutations that reduced sensitivity to stem antibodies. Our results offer insights for next-generation influenza vaccine strategies for inducing cross-neutralizing antibodies.

Review of current diagnostic methods and advances in Helicobacter pylori diagnostics in the era of next generation sequencing.

Helicobacter pylori (H. pylori) infection is highly prevalent in the human population and may lead to severe gastrointestinal pathology including gastric and duodenal ulcers, mucosa associated tissue lymphoma and gastric adenocarcinoma. In recent years, an alarming increase in antimicrobial resistance and subsequently failing empiric H. pylori eradication therapies have been noted worldwide, also in many European countries. Therefore, rapid and accurate determination of H. pylori’s antibiotic susceptibility prior to the administration of eradication regimens becomes ever more important. Traditionally, detection of H.pylori and its antimicrobial resistance is done by culture and phenotypic drug susceptibility testing that are cumbersome with a long turn-around-time. Recent advances in diagnostics provide new tools, like real-time polymerase chain reaction (PCR) and line probe assays, to diagnose H. pylori infection and antimicrobial resistance to certain antibiotics, directly from clinical specimens. Moreover, high-throughput whole genome sequencing technologies allow the rapid analysis of the pathogen’s genome, thereby allowing identification of resistance mutations and associated antibiotic resistance. In the first part of this review, we will give an overview on currently available diagnostic methods for detection of H. pylori and its drug resistance and their implementation in H. pylori management. The second part of the review focusses on the use of next generation sequencing technology in H. pylori research. To this end, we conducted a literature search for original research articles in English using the terms “Helicobacter”, “transcriptomic”, “transcriptome”, “next generation sequencing” and “whole genome sequencing”. This review is aimed to bridge the gap between current diagnostic practice (histology, rapid urease test, H. pylori culture, PCR and line probe assays) and new sequencing technologies and their potential implementation in diagnostic laboratory settings in order to complement the currently recommended H. pylori management guidelines and subsequently improve public health.

Abstract 3654: Identification of resistance mutations to crizotinib through circulating tumor cell (CTC) analysis in ALK-rearranged non-small-cell lung cancer (NSCLC)

Abstract Background Non-invasive methods including CTCs are crucial to develop for the implementation of precision medicine in the treatment of NSCLC. We recently reported how CTCs with abnormal ALK FISH-patterns monitored on treatment can stratify patients at risk of early-resistance to crizotinib (Pailler et al., Cancer Res, 2017). ALK-rearranged NSCLC patients treated with crizotinib develop resistance which manifest by the upregulation of bypass signaling pathways in two-thirds of cases. Here, we evaluated whether individual CTCs could identify resistance mutations to crizotinib and represent tumor diversity compared to single-site tumor-biopsies. Methods Individual CTCs were isolated from seven ALK-rearranged patients at resistance to crizotinib. Matched tumor-biopsies were available for three. Two isolation strategies were used: (i) ISET-filtration, immunofluorescence (Hoescht/CD45/epithelial-markers/mesenchymal-marker), scanning, laser-microdissection; (ii) RosetteSep, immunofluorescence (Hoescht/CD45/ALK/cytokeratins), cell-sorting. A process including Ampli1 whole-genome amplification, quality controls, multiplex PCR with two panels (Ampli1 CHPCustomBeta cancer panel developed by Silicon Biosystems and a home-made panel targeting all ALK mutations) and Ion Torrent next-generation sequencing (NGS) was established. Ten to 17 pools with 1-5 CTCs and one CD45+ cell pool were analyzed for each patient. A bioinformatic workflow was developed including determination of allele drop-out (ADO), false-positive rate (FPR) and positive predicted value (PPV). Results By comparing germline variants present in constitutional and CD45+ pool DNA, we calculated for each sample the statistics of mean ADO, FPR and PPV which were respectively 19%, 2.6x10-4 and 69%. When variants were present into two pools, the PPV and FPR were 96% and 2.39 10-5 respectively. In the three patients with an available tumor-biopsy, a limited number of shared mutations between CTC and matched tumor-biopsies were identified. In contrast, CTC-private mutations (exclusively present in CTCs and not in matched biopsies) present in at least two CTC pools were identified including in FLT3, SMAD4, KIT, PI3KCA, EGFR, KRAS, PDGFRA genes among which several were COSMIC mutations. A higher degree of mutational diversity was observed in CTCs compared to tumor-biopsies. Conclusion Using a rigorously qualified workflow, we reported for the first time the identification of resistance mutations in individually isolated CTCs of ALK-rearranged patients. In accordance with published series from tumor-biopsies, our CTC results highlighted the role of bypass signaling pathways in resistance to crizotinib. Our study provides important perspectives on using CTCs for precision medicine and informing on tumor heterogeneity in NSCLC patients. Citation Format: Emma Pailler, Vincent Faugeroux, Marianne Oulhen, Claudio Forcato, Mélanie Laporte, Yann Lecluse, Ludovic Lacroix, Maud NgoCamus, Claudio Nicotra, Jordi Remon, Laura Mezquita, David Planchard, Jean-Charles Soria, Nicolò Manaresi, Benjamin Besse, Françoise Farace. Identification of resistance mutations to crizotinib through circulating tumor cell (CTC) analysis in ALK-rearranged non-small-cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3654.

Genetic polymorphism of thymidine kinase (TK) and DNA polymerase (pol) of clinical varicella-zoster virus (VZV) isolates collected over three decades

BackgroundGenotypic resistance testing of varicella-zoster virus (VZV) strains to antivirals is of high relevance in immunocompromised patients with VZV reactivations unresponsive to therapy. However, the knowledge on mutations associated with natural gene polymorphism or resistance is limited. ObjectivesTo examine the genotype of the thymidine kinase (TK) and DNA polymerase (pol) of unselected clinical VZV isolates collected between 1984 and 2014 and to verify the phenotype related to novel amino acid (aa) substitutions. Study designThe TK and DNA pol genes of 169 VZV isolates were analyzed by amplification and sequencing. Sequences were compared to that of the reference strain Dumas. The phenotype to acyclovir and other antivirals was examined in isolates with novel aa substitutions using modified plaque reduction assay. ResultsIn the TK of four strains, four different aa substitutions were detected, apart from the known change S288L that was present in all strains compared to Dumas. All four substitutions have hitherto not been described in the literature and were phenotypically classified as natural gene polymorphisms although two out of them (S51L, K186R) were localized in conserved gene centers. The DNA pol of 34 isolates exhibited 19 different substitutions, 14 out of them were novel, and two (R753K, V777I) were within conserved gene regions. Again, these changes were characterized as natural gene polymorphisms. ConclusionsNon-synonymous mutations in VZV TK or DNA pol conferring natural gene polymorphism are rare events. Nevertheless, the phenotypic characterization of 18 novel polymorphisms can help to provide a better identification of resistance mutations.

Abstract 5886: High-throughput mutagenesis reveals novel mechanisms of drug resistance in the proto-oncogene Src kinase

Abstract Kinase inhibitors are integral for the treatment of certain cancers, but the emergence of drug resistance limits their long-term effectiveness. Resistance can arise through multiple routes, including via mutations in the kinase that render it insensitive to drug. Although some resistance mutations for a few inhibitors are well characterized, most remain uncharacterized or even unidentified, particularly in new or emerging kinase targets. Ideally, we could characterize every possible resistance mutation that could arise during treatment with each inhibitor. The resulting comprehensive data could improve clinical decision-making for patients with drug-resistant tumors. However, current experimental methods for discovering resistance mutations cannot achieve the required scale, preventing us from realizing this goal. We developed a method for parallel assessment of the degree of inhibitor resistance of nearly all possible single mutants of the promising drug target Src kinase. We generated a comprehensive single mutant library of Src and subjected it to selection for Src activity either alone or in the presence of the inhibitor dasatinib. Then, we measured the change in frequency of each mutant during selection by deep sequencing. Using this data, we classified the impact of each Src mutant, both in terms of activity and resistance to dasatinib. Our method identified dozens of previously uncharacterized dasatinib-resistance mutations in Src. As expected, some of these novel resistance mutations cluster near the ATP-binding cleft of the catalytic domain. However, a surprisingly large number occur at residues distal to dasatinib’s binding site. Biochemical validations of a subset of these newly identified resistance mutants are ongoing. Although dasatinib is not currently approved for Src inhibition, these results could be useful to direct treatment for those patients currently enrolled in dasatinib clinical trials. Our results demonstrate the power of our method for the characterization of resistance mutations in drug targets. In contrast with previous low-throughput and retrospective methods, our approach enables the comprehensive and prospective identification of resistance mutations. The approach can reveal resistance mechanisms, even before a drug is clinically approved. Importantly, our method allows for the rapid profiling of other Src inhibitors and potentially the discovery of inhibitor-specific resistance mutations. Finally, we are using our data to explore hypotheses regarding the structure, function, or regulation of Src kinase. Note: This abstract was not presented at the meeting. Citation Format: Ethan Ahler, Sujata Chakraborty, Linglan Fan, Dustin J. Maly, Douglas M. Fowler. High-throughput mutagenesis reveals novel mechanisms of drug resistance in the proto-oncogene Src kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5886. doi:10.1158/1538-7445.AM2017-5886

The Emerging Role of "Liquid Biopsies," Circulating Tumor Cells, and Circulating Cell-Free Tumor DNA in Lung Cancer Diagnosis and Identification of Resistance Mutations.

Therapeutic advances in the treatment of lung cancer are in part due to a more complete understanding of its genomic portrait. The serial monitoring of tumor genotypes, which are instable and prone to changes under selective pressure, is becoming increasingly needed. Although tumor biopsies remain the reference standard for the diagnosis and genotyping of lung cancer, they are invasive and not always feasible. The "liquid biopsies" have the potential to overcome many of these hurdles, allowing a rapid and accurate identification of de novo and resistant genetic alterations and a real-time monitoring of treatment responses. In this review, we provide insights into new liquid diagnostic platforms and discuss the role of circulating tumor cells and circulating tumor DNA in the diagnosis and identification of resistance mutations in lung cancer.

Molecular characterization of hepatitis C virus for determination of subtypes and detection of resistance mutations to protease inhibitors in a group of intravenous drug users co-infected with HIV.

Modifications in therapeutic regimens for the treatment of hepatitis C virus (HCV) have been observed since the approval of viral protease inhibitors (PI), and the selection of natural drug-resistant variants has been also reported. Thus, it becomes crucial to be aware of consequences of new therapeutic approaches and make available tools for monitoring the infection. The study aimed to apply an "in-house" method for amplification and sequencing of the NS3 region which is the target of PI, and allowing simultaneously the classification of viral subtypes and identification of resistance mutations. Forty-seven samples collected from HIV injecting drug users and drug naive for HCV protease inhibitors were tested for anti-HCV antibodies, 93.6% of them had a positive result and in 70.5% was determined HCV active infection. High frequency of subtype 1a (46.2%), followed by an equal proportion of subtypes 3a, 4a, and 4d (15.4%) was obtained. Two potential recombinants, RF1_2k/1b (3.8%) and 2q/2k (3.8%) were identified. Substitutions V36L/P, T54A, I72L/N/T/V, Q80K/G, S122R/T, D168Q, and I170L/V were observed in 65.4% of the samples. The T54A and Q80K mutations, and the combination V36L + T54A were also identified. Polymorphisms were observed exclusively associated with specific genotypes, particularly, I72L and D168Q with genotype 3, and S122T with genotype 4. The V36L substitution was identified in 92.8% of sequences of non-genotype 1 denoting that this amino acid substitution is a natural polymorphism associated with non-genotype 1 strains. Although no major PI resistance mutations were detected, a more extensive study is needed to evaluate the impact of mutations identified in efficacy of PI treatment.

Antiviral efficacy of adefovir dipivoxil in the treatment of chronic hepatitis B subjects from Indian subcontinent

Adefovir is one of the therapeutic options for the treatment of chronic hepatitis B. A total of 30 adefovir-experienced subjects with the median treatment duration of 12 (interquartile range (IQR) 6-18) months were studied. Virological response was measured by hepatitis B virus deoxyribonucleic acid (HBV DNA) levels. HBV reverse transcriptase (rt) domains were sequenced for the identification of resistance mutations. Among the 30 subjects, two (7%) showed virological response and 19 (63%) were non-responders. The virological response for the remaining nine (30%) subjects was not determined. On sequence analysis, two subjects were identified with rtI169L and rtA181V mutation after 9 months and 18 months of adefovir treatment, respectively. Though the frequencies of adefovir resistance mutations are low, a large majority of subjects showed non-response. Therefore, adefovir in the management of HBV should be used judiciously.

Genomic insights into the fate of colistin resistance and Acinetobacter baumannii during patient treatment

Bacterial whole-genome sequencing (WGS) of human pathogens has provided unprecedented insights into the evolution of antibiotic resistance. Most studies have focused on identification of resistance mutations, leaving one to speculate on the fate of these mutants once the antibiotic selective pressure is removed. We performed WGS on longitudinal isolates of Acinetobacter baumannii from patients undergoing colistin treatment, and upon subsequent drug withdrawal. In each of the four patients, colistin resistance evolved via mutations at the pmr locus. Upon colistin withdrawal, an ancestral susceptible strain outcompeted resistant isolates in three of the four cases. In the final case, resistance was also lost, but by a compensatory inactivating mutation in the transcriptional regulator of the pmr locus. Notably, this inactivating mutation reduced the probability of reacquiring colistin resistance when subsequently challenged in vitro. On face value, these results supported an in vivo fitness cost preventing the evolution of stable colistin resistance. However, more careful analysis of WGS data identified genomic evidence for stable colistin resistance undetected by clinical microbiological assays. Transcriptional studies validated this genomic hypothesis, showing increased pmr expression of the initial isolate. Moreover, altering the environmental growth conditions of the clinical assay recapitulated the classification as colistin resistant. Additional targeted sequencing revealed that this isolate evolved undetected in a patient undergoing colistin treatment, and was then transmitted to other hospitalized patients, further demonstrating its stability in the absence of colistin. This study provides a unique window into mutational pathways taken in response to antibiotic pressure in vivo, and demonstrates the potential for genome sequence data to predict resistance phenotypes.

Magnitude of Gene Mutations Conferring Drug Resistance in Mycobacterium Tuberculosis Isolates from Lymph Node Aspirates in Ethiopia

Objective: Resistance to drugs is due to particular genomic mutations in the specific genes of Mycobacterium tuberculosis. Timely genetic characterization will allow identification of resistance mutations that will optimize an effective antibiotic treatment regimen. We determine the magnitude of gene mutations conferring resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among tuberculosis (TB) lymphadenitis patients.Methods: A cross sectional prospective study was conducted among 226 M.tuberculosis isolates from culture positive lymph node aspirates collected from TB lymphadenitis patients between April 2012 and May 2012. Detection of mutations conferring resistance to drugs was carried out using GenoType® MTBDRplus and GenoType® MTBDRsl assay.Results: Out of the 226 strains, mutations conferring resistance to INH, RMP, multidrug resistance tuberculosis (MDR-TB) and EMB were 8, 3, 2 and 2 isolates, respectively. There was no isolated strain that showed mutation in the inhA promoter region gene. All INH resistant strains had mutations in the katG gene at codon 315 with amino acid change of S315T1. Among rifampicin resistant strains, two isolates displayed mutations at codon 531 in the rpoB gene with amino acid change of S531L and one isolate was by omission of wild type probes at Q513L. According to mutations associated with ethambutol resistance, all of the isolates had mutations in the embB gene with aminoacid change of M306I. All isolates resistant to INH, RMP and MDR using BacT/AlerT 3D system were correctly identified by GenoType® MTBDRplus assay.Conclusion: We observed mutations conferring resistance to INH at S315T1 of the katG gene, RMP at S531L and Q513L in the rpoB genes and EMB at M306I of the embB gene. In the absence of conventional drug susceptibility testing, the effort to develop easy, rapid and cost effective molecular assays for drug resistance TB monitoring is definitely desirable and the GenoType® MTBDRplus assay was found to be a useful method for diagnosis of resistance to INH, RMP and MDR from lymph node aspirates. Further molecular cluster analysis to determine transmission dynamics of mutated strain is required.

Genotypic resistance testing in HIV-infected pregnant women in an urban setting.

This study examines the utility of HIV genotypic resistance testing (GT) in pregnant women at their initial pregnancy evaluation. A retrospective medical record review of 50 consecutive HIV-infected pregnant women in whom GT was obtained in the Bronx, New York was conducted. Twenty-eight (56%) were antiretroviral experienced, including 12 on antiretroviral therapy (ART) at time of GT. Major mutations were found in 11 (24%) of 45 amplifiable GTs. Major resistance mutations were identified against nucleoside reverse transcriptase inhibitors (NRTIs) in six (13%) patients; against non-nucleoside reverse transcriptase inhibitors (NNRTIs) in eight (18%) patients, and against protease inhibitors in two (4%) patients. Duration of ART exposure was significantly associated with identification of resistance mutations by GT for NRTIs and NNRTIs (P < or =0.05). Results of this study indicate that GT at presentation may have implications on the initial choice of ART in up to one-quarter of HIV-infected pregnant women, especially with current or prior antiretroviral use.

Molecular based detection for drug resistance in mycobacterium tuberculosis

Multi- drug resistant tuberculosis continues to be a serious problem, particularly among some developing countries. Early detection of drug resistance in clinical M. tuberculosis isolates is crucial for appropriate treatment and to prevent the development of further resistance. Compared to conventional methods using solid media, the introduction of manual and automated methods (BACTEC or MB/BacT) for susceptibility testing in liquid media has resulted from 4 to 6 weeks to 3 to 15 days. The identification of resistance mutations, e.g., the genetic basis for RIF resistance, enables the development of molecular test that allows the detection of resistant strains within 1 day. One approach is the use of molecular analysis to detect mutations that are associated with resistance to drugs including INH and RIF. In the case of INH, mutations of the katG, inhA, kasA, and ahpC genes are responsible for the majority of INH-resistant M. tuberculosis, whereas mutations of rpoB are responsible for RIF-resistant M. tuberculosis. (Med J Indones 2003; 12: 259-65)

Examination of single and multiple mutations involved in resistance to quinolones in Staphylococcus aureus by a combination of PCR and denaturing high-performance liquid chromatography (DHPLC).

Detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that has been used to detect mutations in human DNA. We report on the first study to use this technique as a tool to detect mutations in genes encoding antibiotic resistance in bacteria. Three methicillin-sensitive and three methicillin-resistant clinical Staphylococcus aureus isolates, susceptible to ciprofloxacin (MIC <or= 0.4 mg/L), were used to derive mutants resistant to ciprofloxacin, levofloxacin, sparfloxacin, trovafloxacin and moxifloxacin. Genomic DNA from each strain was subjected to PCR amplification of 225-500 bp regions spanning the quinolone resistance determining regions of the gyrA, gyrB, grlA and grlB genes. Following DNA sequencing of these amplicons and identification of resistance mutations, DHPLC was undertaken to correlate the distinctive chromatogram with DNA sequence. The mutations detected by DHPLC resulted in the following amino acid substitutions: Ser-84-->Leu, Ser-112-->Pro, Glu-88-->Lys in GyrA, Glu-84-->Val, Ser-80-->Phe in GrlA, Pro-456-->Ser in GyrB and Glu-422-->Asp, Pro-451-->Ser, Asp-432-->Gly in GrlB. Mutations could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This system facilitates the detection of resistance alleles, providing a rapid (5 min per sample), economic (96 sample per run) and reliable technique for characterizing antibiotic resistance in bacteria.