Drug Resistance Screening Research Articles

The role of cytomegalovirus infection and disease in pediatric bone marrow transplant recipients in Mexico City in the context of viral drug resistance

We aimed to identify those pediatric patients undergoing ABMT with CMV EOD who developed GCV resistance. Forty-seven patients were analyzed following ABMT. Prospective post-transplant CMV monitoring was performed weekly for the detection of viral leukocyte DNAaemia, viral plasma DNAaemia, and viral DNAuria by PCR. Plasma DNAaemia was confirmed from whole blood by the detection of CMV pp67 late mRNA using NASBA technology. In the cases of persistence of viral DNA in plasma, and positive viral RNA detection in blood, CMV drug resistance screening by comprehensive PCR-based RFLP and sequencing of the viral UL97 gene were performed retrospectively. Thirty of the 47 (63.82%) patients showed active CMV infection with 27/30 (74.4%) patients belonging to the D+R+ group and 25/30 with proven viral replication. In total, 2/30 (6.6%) children developed CMV pneumonia proven by immunohistochemistry. Screening of the viral UL97 gene revealed in one of these two cases (1/30, 3.3%) the simultaneous presence of two point mutations in codon 460 (M460V, M460I) conferring GCV resistance. The CMV seroprevalence (81%) and the incidence of active infection (63.8%) in Mexican children undergoing ABMT are very high.

DNA Suspension Arrays: Silencing Discrete Artifacts for High-Sensitivity Applications

Detection of low frequency single nucleotide polymorphisms (SNPs) has important implications in early screening for tumorgenesis, genetic disorders and pathogen drug resistance. Nucleic acid arrays are a powerful tool for genome-scale SNP analysis, but detection of low-frequency SNPs in a mixed population on an array is problematic. We demonstrate a model assay for HIV-1 drug resistance mutations, wherein ligase discrimination products are collected on a suspension array. In developing this system, we discovered that signal from multiple polymorphisms was obscured by two discrete hybridization artifacts. Specifically: 1) tethering of unligated probes on the template DNA elicited false signal and 2) unpredictable probe secondary structures impaired probe capture and suppressed legitimate signal from the array. Two sets of oligonucleotides were used to disrupt these structures; one to displace unligated reporter labels from the bead-bound species and another to occupy sequences which interfered with array hybridization. This artifact silencing system resulted in a mean 21-fold increased sensitivity for 29 minority variants of 17 codons in our model assay for mutations most commonly associated with HIV-1 drug resistance. Furthermore, since the artifacts we characterized are not unique to our system, their specific inhibition might improve the quality of data from solid-state microarrays as well as from the growing number of multiple analyte suspension arrays relying on sequence-specific nucleic acid target capture.

AP24163 Inhibits the Gatekeeper Mutant of BCR‐ABL and Suppresses In vitro Resistance

Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance in patients with chronic myelogenous leukaemia. The second generation BCR/ABL inhibitors nilotinib and dasatinib effectively inhibit most imatinib resistance variants, but are ineffective against the gatekeeper mutant, T315I. Gatekeeper mutation activates the kinase by stabilizing the hydrophobic spine. Here, we describe that the rationally designed compound AP24163 can inhibit native and gatekeeper mutants of the BCR/ABL kinase. Structural modelling suggests that AP24163 affects the flexibility of the P-loop and destabilizes the active conformation by disrupting the hydrophobic spine. In vitro screening for drug resistance identified clones with compound mutations involving both the P-loop and T315I. Our studies provide structural insights for the design of inhibitors against the gatekeeper mutant and suggest that up-front combination therapy may be required to prevent the emergence of compound-resistant mutations.

Primary HIV-1 Drug Resistance and Polymorphic Patterns Among Injecting Drug Users (IDUs) in Chennai, Southern India

India has 1.1 million injecting drug users (IDUs) with an HIV prevalence rate as high as 64%. Drug resistance screening before therapy is beneficial to the individual. Here, we have studied primary drug resistance among IDUs in Chennai. Specimens (n = 55) collected between March 2005 and April 2006 were subjected to genotyping assay. The mutations for the drug resistance were interpreted using the Stanford HIV Drug Resistance Database. M41LM (1.8%), K65KN (1.8%), and G73GS (2.7%) were found to be associated with low-level resistance to zidovudine (ZDV), stavudine (d4T), abacavir (ABC), didanosine (ddI), emtricitabine (FTC), tenofovir (TDF), and saquinavir (SQV) in each specimen. The rate of polymorphisms is significantly different from universally established subtype ''C''-specific polymorphisms (P < .0001). The presence of drug resistance mutations, though minimal, is alarming as it could spread easily between IDUs and from them to their sexual partners. The genetic variation is of importance in vaccine design.

An HIV-1 integrase genotype assay for the detection of drug resistance mutations

The integrase inhibitors (e.g. Raltegravir) are a new class of antiretroviral drugs that have recently become available for the treatment of patients with multi-drug resistant HIV-1 within Australia. The emergence of mutations that confer resistance to the integrase inhibitors has been observed in vivo; however, no commercial genotyping assay is currently available to screen for resistance to these drugs. The HIV-1 integrase gene was amplified from plasma-derived HIV-1 viral RNA via reverse transcription-polymerase chain reaction and genotype determined via population DNA sequencing. Drug resistance mutations and polymorphisms were detected using the Stanford University online HIV database. Assay sensitivity and reproducibility were determined using clinical and laboratory-derived samples. Our in-house assay was capable of genotyping the integrase gene from all samples tested (n = 30) of HIV-1 subtypes B, C, D, F, CFR01_AE and CRF02_AG and can amplify the integrase region from plasma samples containing as few as 50 HIV RNA copies/mL. The assay is highly reproducible (average nucleotide concordance = 99.6%, n = 4) and is capable of detecting resistance-associated mutations. This assay is suitable for routine drug resistance screening of plasma samples from HIV-infected patients receiving integrase inhibitor antiretroviral drugs and also serves as a useful research tool.

Characterization of rpo B Gene for Detection of Rifampicin Drug Resistance by SSCP and Sequence Analysis

Background: Due to the emergence of multidrug resistant TB (MDR-TB) in recent time, the rapid detection of resistance to first line antituberculosis drug such as Rifampicin (which is generally considered a marker for determination of MDR strains) was felt worldwide. Methods: Rifampicin susceptibility of 22 rifampicin resistant and 11 rifampicin susceptible Mycobacterium tuberculosis strains was analyzed by PCR-Single Strand Conformation Polymorphism(SSCP) and validated by DNA sequencing within the 157 bp region of the rpo B gene(Ala500 to Val550) for checking its utility as a rapid screening test for determination of MDR drug resistance. Results: RMP resistance was detected successfully by PCR-SSCP in 20/22(90.90%) of the RMP- resistant strain. 2 RMPr strains showed identical PCR-SSCP pattern with wild type H37Rv. There were seen seven different mutation in the amplified region of rpo B gene of 20 RMPr: codon 513(CAA→CCA), 516(GAC→GTC), 507 (GGC→GAC), 526(CAC→GAC, TAC), 531(TCG→TTG, TGG), 522(TCG→TGG), 533(GTG→CCG). Thus this study demonstrated the high specificity(100%) and sensitivity(90.90%) of PCR-SSCP method for detection of mutation in rpo B gene; 77.27% of RMPr strain showed a single mutation and 9.09% had no mutation. Conclusion: Our data support the common notion that rifampicin resistance genotypes are generally present in codons 516, 526, and 531, most frequently found in M. tuberculosis population regardless of geographic origin and can be rapidly detected by PCR-SSCP making it as a useful tool in primary screening of drug resistance.

Inhibition of BCR/ABL-T315I by Dismantling the Hydrophobic Spine.

Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance (IMR) in patients with chronic myelogenous leukemia (CML). The second generation BCR/ABL inhibitors, Nilotinib and Dasatinib, are effective in inhibiting essentially all IMR variants, but not the gatekeeper mutant T315I. Substitution of a bulky hydrophobic residue for the gatekeeper threonine not only causes steric blockade to the inhibitor but also stabilizes the active kinase conformation through a network of hydrophobic connections dubbed the hydrophobic-spine. In this study we describe the molecular mechanisms employed by the gatekeeper mutation to stabilize the active conformation, and demonstrate that these structural components can be targeted by the small molecule inhibitor compound #14, which efficiently inhibits native and gatekeeper mutant forms of the BCR/ABL kinase. Structural modeling and mutagenesis of residues constituting the spine suggests that compound #14 inhibits the kinase by disrupting the hydrophobic spine. Screening for drug resistance in vitro selected for clones having compound mutations involving both the P-loop and gatekeeper residues. Our studies provide structural guidance for the design of inhibitors against the gatekeeper mutant, and suggest that combination therapy may be required to prevent the emergence of compound resistance mutations.

Characterization ofrpoBmutations associated with rifampin resistance inMycobacterium tuberculosisfrom eastern China

The aim of this study was to investigate the features of rpoB gene mutations associated with Rifampin (RIF) resistance in Mycobacterium tuberculosis (M. tuberculosis) in eastern China. The mutations of rpoB gene in 56 clinical isolates of M. tuberculosis resisted to one to four first-line drugs (rifampin, isonicotinyl hydrazide, ethambutol and streptomycin) were analysed by polymerase chain reaction single strand conformation polymorphism analysis (PCR-SSCP) and DNA sequencing. The results of PCR-SSCP showed 52 isolates were positive (existing rpoB mutation) including 47 isolates resisted to RIF. Subsequent results of DNA sequencing showed that 54 isolates had rpoB gene mutation including 49 isolates resisted to RIF. The most frequently mutated sites were at codons 526 (73.2%), 513 (10.7%) and 531 (3.5%). The rpoB codon 526 was the most frequently mutated site of RIF-resistant M. tuberculosis strains in eastern China and its frequency is significantly higher (P < 0.0001) compared with that in other areas of China and in other geographic regions worldwide. Our results reveal that geographic variation is responsible for rpoB mutations in M. tuberculosis and the resulting information will be helpful to improve a novel rapid molecular drug resistance screening approach for MDR TB.

To investigate the clinical diagnostic value of four mycobacteria tuberculosis detection methods

目的 探讨转录酶主导基因TB-RNA扩增法、噬菌体裂解法、3D培养法和涂片法4种实验室检测方法对结核病临床诊断的应用价值.方法 用以上4种方法对临床每份送检标本同时进行同步检测.其中痰液110份、胸腔积液54份、咽拭子37份、支气管冲洗液31份、脑脊髓液13份、尿液12份、颈部淋巴液8份和其他体液(包括腹腔积液、血液、心包积液、脓性分泌物和大便等)20份以及妇科标本(包括白带、经血等)6份等共计291份.最后对上述4种方法检测结果进行评估.结果 291份送检标本中,转录酶主导基因TB-RNA扩增法的总阳性率为37.1%,噬菌体裂解法为28.9%,3D培养法为27.5%和涂片法为10.3%;4种方法检测的敏感度、特异度分别依次为:54.3%和100%、41.7%和98.9%、31.7%和83.5%以及14.6%和98.9%.结论 转录酶主导基因TB-RNA扩增法对结核病临床诊断有较高的应用价值;涂片法阳性率低但其成本低廉且能在实验室中广泛开展;噬菌体裂解法的阳性率略高于培养法;而培养法阳性率远低于转录酶主导基因TB-RNA扩增法和略低于噬菌体裂解法,且需时较长,但培养出的分枝杆菌可做药物敏感性试验和菌型鉴定。

Genotypic Analysis of Multidrug-Resistant Mycobacterium tuberculosis Isolates Recovered From Central China

DNA sequencing analysis was used to investigate genetic alterations in the rpoB, katG, and inhA regulatory region and embB in 66 Mycobacterium tuberculosis isolates recovered from Central China. Of the 36 multidrug-resistant isolates, 33 (92%) had mutations in the amplified region of rpoB. The most frequent mutation (58%, 19/36) was S531L (TCG-->TTG). At least one mutation was found in the katG and inhA regulatory region in 83% (30/36) of the multidrug-resistant isolates, and mutations at katG codon 315 were identified in 78% (28/36). Alterations at embB306 may not confer resistance to EMB, and embB306 mutants were more frequently accompanied by rpoB mutations (100%, 16/16) than by katG 315 mutations (75%, 12/16). Our results show that geographic variation in the molecular genetic mechanism is responsible for drug resistance in multidrug-resistant M. tuberculosis. This observation will facilitate the development of a rapid molecular drug resistance screening approach for drug-resistant M. tuberculosis.

Gastrointestinal stromal tumor (GIST) pathogenesis, familial GIST, and animal models

Once a poorly understood pathologic entity, gastrointestinal stromal tumor (GIST) has emerged in recent years as a distinct oncologic-molecular paradigm that is now a leading model for kinase-targeted therapies in Oncology. Most GISTs are KIT-expressing and KIT-signaling driven mesenchymal tumors, many of which have KIT-activating mutations. A small subset of GIST show activating mutations in PDGFRA, encoding for a related member of the type III receptor tyrosine kinase family. The revelation of KIT expression as a diagnostic signature of GIST has not only revolutionized the pathologic criteria in classifying GIST, but also shed light onto the histogenesis of these tumors. The similarities in KIT immunoreactivity and ultrastructural appearance between GISTs and the intestinal pacemaker, the interstitial cells of Cajal (ICC), suggested that GISTs derive from or differentiate toward the ICC lineage. KIT plays a significant role in proliferation, survival, and differentiation of hematopoietic stem cells, mast cells, melanocytes, and interstitial cells of Cajal; and activating KIT mutations have been identified in tumors affecting most of these cell lineages. The observation that KIT mutations may be inherited, as seen in familial GIST syndrome, was used to develop murine models harboring a germline gain-of-function mutation, as a model for studying of KIT oncogenic mechanisms. These murine models of human GIST promise to become powerful preclinical tools in elucidating oncogenic signaling mechanisms by using genetic approaches and targeted pharmacological intervention. As true animal models of human cancer, they provide superior information compared with the more commonly used xenografts and transgenic mouse models. This review summarizes the recent knowledge on the central role of KIT oncogenic activation and subsequent signal transduction in the pathogenesis of GIST. In addition, we provide an updated discussion on familial GIST syndrome in relationship to genotype-phenotype characteristics. A succinct description of the available murine models of human GIST is presented and their applicability in further understanding of the KIT oncogenic signaling, drug screening, and mechanisms of drug resistance is discussed. This is a US government work. There are no restrictions on its use.

Mycobacteriophage and their application to disease control

The resurgence of tuberculosis and emergence of drug resistant disease has stimulated fresh research into mycobacteriophage. Studies are currently underway to develop phage-based tools for therapeutic and diagnostic use. Previous attempts at mycobacteriophage therapy in experimentally infected animals were not successful and alternative strategies of phage delivery that enable killing of intracellular bacteria are required. Replication of mycobacteriophage provides a simple means of detecting viable bacteria and good progress has been made towards the development of new phage-based diagnostic tools. When screening isolates for resistance to the major antituberculosis drug rifampicin phage-based tests have been shown to have high sensitivity. For the diagnosis of pulmonary tuberculosis evaluation studies indicate that current phage tests are not as sensitive as traditional culture methods. Further trials are needed to determine whether they might have a role in the detection of smear negative tuberculosis. A second generation of phage tests are under development following the construction of luciferase reporter phage. Preliminary data suggests they may offer rapid detection of mycobacteria and simple screening for drug resistance. The potential of mycobacteriophage to detect and treat other mycobacterial diseases remains largely unexplored. 2. INTRODUCTION

Analysis for a limited number of gene codons can predict drug resistance of Mycobacterium tuberculosis in a high-incidence community.

Correct and rapid diagnosis is essential in the management of multidrug-resistant tuberculosis (MDR-TB). In this population-based study of 61 patients with drug-resistant tuberculosis, we evaluated the frequency of mutations and compared the performance of genotypic (mutation analysis by dot blot hybridization) and phenotypic (indirect proportion method) drug resistance tests. Three selected codons (rpoB531, rpoB526, and katG315) allowed identification of 90% of MDR-TB cases. Ninety percent of rifampin, streptomycin, and ethambutol resistance and 75% of isoniazid resistance were detected by screening for six codons: rpoB531, rpoB526, rrs-513, rpsL43, embB306, and katG315. The performance (reproducibility, sensitivity, and specificity) of the genotypic method was superior to that of the routine phenotypic method, with the exception of sensitivity for isoniazid resistance. A commercialized molecular genetic test for a limited number of target loci might be a good alternative for a drug resistance screening test in the context of an MDR "DOTS-plus" strategy.

Transmission and prevalence of HIV resistance among treatment-naive subjects.

Recent reports demonstrate the transmission of drug-resistant HIV-1 variants to newly infected individuals, although estimates of the prevalence of drug resistance among populations from different geographic regions are highly varied. The interpretation and comparison of available study results are confounded by the lack of consensus regarding the nomenclature and reporting of antiviral resistance. This report re-evaluates previously presented and published data using uniform criteria for genotypic and phenotypic drug resistance. Treatment-inexperienced and recently or chronically infected populations are reviewed. The prevalence of transmitted drug resistance ranges from 1% to 11% among recently infected persons using these criteria. Programmes to monitor and characterize drug resistance among newly infected persons and their source partners are essential to evaluate the selection processes that influence the transmission of certain genetic variants of HIV. Temporal trends in the prevalence of transmitted drug resistance among diverse populations are necessary to evaluate the potential need for selective and generalized drug resistance screening programmes among newly infected, treatment-naive patients.

Diagnosis and treatment of cytomegalovirus infection.

Cytomegalovirus (CMV) infection remains a major cause of morbidity and mortality in recipients of an allogeneic stem cell transplant (SCT). Due to the broad application of antiviral prophylaxis and preemptive therapy, a decrease in early-onset and a subsequent increase in late-onset CMV disease has been observed. New data on the latency state and reactivation of CMV have been presented, the role of T-cell responses in the control of CMV further substantiated, and viral immune escape mechanisms described in more detail. Sensitive diagnostic assays using nucleic acid amplification and hybridization techniques have been commercialized and will allow standardization of CMV diagnostics in antiviral drug trials. Quantification of the viral load will be increasingly considered for initiation and, in patients with persistence of high viral titers despite antiviral therapy, screening for antiviral drug resistance. Clinical data are emerging to show that, apart from ganciclovir, foscarnet can be given safely even after allogeneic SCT. Additional drugs like lobucavir and cidofovir have been used for specific indications. Interactions of immunosuppressive drugs and antiviral compounds of clinical relevance have been described. Thus, therapeutic drug monitoring and targeted antiviral drug dosing will become standard practice in antiviral treatment strategies in patients following allogeneic SCT.

A simplified assay for screening of drug resistance of cell-associated cytomegalovirus strains.

Conventional phenotypic drug resistance determination of cell-free clinical human cytomegalovirus (HCMV) isolates is usually very laborious and may take 8-12 weeks, since serially passages of slowly growing viral isolates in tissue cultures are required to obtain a sufficient viral titer for an appropriate inoculum. Rapid screening of a large number of samples would therefore only be possible if simplified, less work-intensive methods are employed. The aim of this work was to develop an assay which speeds up the whole procedure of phenotypic drug resistance determination. Steps of the classical plaque reduction assay should be simplified or omitted, but on the other hand, the assay should be reliable and reproducible. Twenty-six clinical HCMV isolates from 20 immunocompromised patients (ten pre-treatment and 16 post-treatment with ganciclovir) were tested for drug susceptibility with the simplified plaque reduction assay. Most isolates were tested at least twice in independent assays. Virus titration could be avoided by using four different doses of cell-associated virus from the secondary culture for coculture susceptibility testing. Drug susceptibility values were determined by plaque titration and Probit analysis. All clinical HCMV isolates tested showed a mean ganciclovir ID50 value of 1.98 microM, (range 0.2-12.2; median 0.95) and a mean foscarnet ID50 value of 92.4 microM (range 35.7-181; median 81). All except one isolate were classified ganciclovir sensitive when compared to ID50 values of two ganciclovir resistant control stains (53.7 +/- 6.4 and 12.7 +/- 0.9 microM) and the sensitive laboratory strain Towne (2.1 +/- 0.8 microM). Repeated tests of individual isolates were reproducible, although the infectivity of the inoculum has not been determined prior of the assay. The mean time which elapsed between receipt of the clinical specimen and read-out of the assay was circa 4 weeks. Phenotypic resistance testing of HCMV isolates following to this protocol drastically reduces expenditure of time and work. The assay allows reliably the discrimination of HCMV isolates as drug resistant or sensitive according to the recent classification criteria of the AIDS Clinical Trials Group (ACTG). The simple handling and uncomplicated calibration of this assay facilitates the screening of large specimen numbers and renders drug susceptibility determination of HCMV more accessible to diagnostic routine use.

Initial screening for antituberculous drug resistance at an inpatient facility in Leon, Nicaragua.

Antituberculous (anti-TB) drug resistance has become a major tuberculosis control issue in the United States, where this situation has closely paralleled the current acquired immunodeficiency syndrome epidemic associated with human immunodeficiency virus type-1 (HIV-1) infections. In less developed countries, especially those like Nicaragua with an apparently low prevalence of known HIV-1 infections, less is known about the epidemiology of antituberculous drug resistance. To understand the potential extent of this problem in Nicaragua, we conducted a cross-sectional prevalence study at Nicaragua's only inpatient tuberculosis treatment facility, located in Leon, Nicaragua. A radiometric method was used during recovery, purification, and drug susceptibility testing of clinical Mycobacterium tuberculosis isolates. Resistance to at least one of the major anti-TB medications was found in 15 (40.5%) of 37 sputum isolates, of which seven (19%) were resistant to either isoniazid alone, or to isoniazid plus another agent other than rifampin. Five were resistant to at least isoniazid and rifampin (i.e., 13.5% demonstrated multidrug resistance). Two isolates were resistant to pyrazinamide alone, and one was resistant to streptomycin alone. These initial results suggest that anti-TB drug resistance is a defined problem for tuberculosis control programs in Nicaragua, a problem that is largely related to individual noncompliance, lack of extensive drug susceptibility testing facilities, and a general unavailability of expensive anti-TB medications for re-treatment. Ongoing surveillance for drug resistance, using the methodology presented here, might assist Nicaraguan public health officials in their tuberculosis control programs.