Analysis of HCV resistance mutations during combination therapy with protease inhibitor boceprevir and PEG-IFN α-2b using TaqMan mismatch amplification mutation assay

2008 ◽  
Vol 153 (2) ◽  
pp. 156-162 ◽  
Author(s):  
Stephanie Curry ◽  
Ping Qiu ◽  
Xiao Tong
Keyword(s):  
Combination Therapy ◽  
Protease Inhibitor ◽  
Resistance Mutations ◽  
Mutation Assay ◽  
Mismatch Amplification Mutation Assay

scholarly journals Ciprofloxacin Resistance in Campylobacter jejuniIsolates: Detection of gyrA Resistance Mutations by Mismatch Amplification Mutation Assay PCR and DNA Sequence Analysis

1999 ◽  
Vol 37 (10) ◽  
pp. 3276-3280 ◽  
Author(s):  
Gerald Zirnstein ◽  
Yu Li ◽  
Bala Swaminathan ◽  
Frederick Angulo
Keyword(s):  
Sequence Analysis ◽  
Dna Sequence ◽  
Campylobacter Jejuni ◽  
Dna Sequence Analysis ◽  
Quinolone Resistance ◽  
Resistance Mutations ◽  
Ciprofloxacin Resistance ◽  
Mutation Assay ◽  
Mismatch Amplification Mutation Assay

The gyrA quinolone resistance determining region was sequenced from 13 ciprofloxacin-resistant and 20 ciprofloxacin-susceptible Campylobacter jejuni isolates. All isolates resistant to ciprofloxacin had Thr-86-to-Ile mutations, a mutation frequently associated with the acquisition of resistance to fluoroquinolones. A mismatch amplification mutation assay (MAMA) PCR protocol was developed that detects this gyrA mutation in quinolone-resistant isolates. The MAMA PCR provides a means for routine detection of the gyrA mutation without the need for sequencing the gyrA gene.

Analysis of Ciprofloxacin-Resistant Salmonella Strains from Swine, Chicken, and Their Carcasses in Taiwan and Detection of parC Resistance Mutations by a Mismatch Amplification Mutation Assay PCR

2009 ◽  
Vol 72 (1) ◽  
pp. 14-20 ◽  
Author(s):  
CHENG-CHUNG LIN ◽  
TER-HSIN CHEN ◽  
YU-CHIH WANG ◽  
CHAO-CHIN CHANG ◽  
SHIH-LING HSUAN ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Fast Method ◽  
Resistance Mutations ◽  
Specific Pcr ◽  
Pcr Products ◽  
High Level ◽  
Mutation Assay ◽  
Pcr Targeting ◽  
Level Resistance ◽  
Mismatch Amplification Mutation Assay

One hundred thirty-three Salmonella strains isolated from the viscera of swine, chicken, and carcasses of swine and chicken in Taiwan from 2004 to 2006 were identified to serotype level and analyzed for their susceptibility to ciprofloxacin. In total, 76 (57%) strains of the Salmonella isolates exhibited high-level resistance to ciprofloxacin, having MICs ranging from 16 to 64 μg/ml. DNA sequence analysis revealed that mutations in the quinolone resistance–determining regions of gyrA (Ser83Phe, Asp87Gly or Asp87Asn), parC (Ser80Arg, or Ser80Ile or Glu84Lys), and parE (Ser458Pro) genes were associated with the Salmonella strains that demonstrated resistance to ciprofloxacin. A mismatch amplification mutation assay (MAMA)–PCR was developed to identify mutations in parC at codons 80 and 84. Specific PCR products were only recovered from ciprofloxacin-resistant Salmonella strains but not from the susceptible strains. MAMA-PCR targeting the mutations in parC correlated with what DNA sequencing revealed. In conclusion, monitoring ciprofloxacin-resistant Salmonella from animal sources should be performed on a regular basis. MAMA-PCR targeting parC could provide a fast method for those laboratories interested in quickly characterizing the resistance profile and with little access to DNA sequencing facilities.

scholarly journals Evaluation of reverse transcriptase and protease inhibitors in two-drug combinations against human immunodeficiency virus replication.

1996 ◽  
Vol 40 (6) ◽  
pp. 1346-1351 ◽  
Author(s):  
C A Deminie ◽  
C M Bechtold ◽  
D Stock ◽  
M Alam ◽  
F Djang ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Combination Therapy ◽  
Protease Inhibitor ◽  
Reverse Transcriptase ◽  
Protease Inhibitors ◽  
Nucleoside Analogs ◽  
Drug Combinations ◽  
Hiv Protease ◽  
Human Immunodeficiency Virus Replication ◽  
Immunodeficiency Virus

Current treatments for human immunodeficiency virus (HIV) include both reverse transcriptase and protease inhibitors. Results from in vitro and clinical studies suggest that combination therapy can be more effective than single drugs in reducing viral burden. To evaluate compounds for combination therapy, stavudine (d4T), didanosine (ddI), or BMS-186,318, an HIV protease inhibitor, were combined with other clinically relevant compounds and tested in a T-cell line (CEM-SS) that was infected with HIV-RF or in peripheral blood mononuclear cells infected with a clinical HIV isolate. The combined drug effects were analyzed by the methods described by Chou and Talalay (Adv. Enzyme Regul. 22:27-55, 1984) as well as by Prichard et al. (Antimicrob. Agents Chemother. 37:540-545, 1993). The results showed that combining two nucleoside analogs (d4T-ddI, d4T-zidovudine [AZT], and d4T-zalcitabine [ddC]), two HIV protease inhibitors (BMS-186,318-saquinavir, BMS-186,318-SC-52151, and BMS-186,318-MK-639) or a reverse transcriptase and a protease inhibitor (BMS-186,318-d4T, BMS-186,318-ddI, BMS-186,318-AZT, d4T-saquinavir, d4T-MK-639, and ddI-MK-639) yielded additive to synergistic antiviral effects. In general, analysis of data by either method gave consistent results. In addition, combined antiviral treatments involving nucleoside analogs gave slightly different outcomes in the two cell types, presumably because of a difference in phosphorylation patterns. Importantly, no strong antagonism was observed with the drug combinations studied. These data should provide useful information for the design of clinical trials of combined chemotherapy.

scholarly journals Protease Inhibitor Resistance Analysis in the MONARK Trial Comparing First-Line Lopinavir-Ritonavir Monotherapy to Lopinavir-Ritonavir plus Zidovudine and Lamivudine Triple Therapy

2009 ◽  
Vol 53 (7) ◽  
pp. 2934-2939 ◽  
Author(s):  
Constance Delaugerre ◽  
Philippe Flandre ◽  
Marie Laure Chaix ◽  
Jade Ghosn ◽  
François Raffi ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Triple Therapy ◽  
Virological Failure ◽  
Therapy Group ◽  
Resistance Mutation ◽  
Resistance Testing ◽  
Initial Screening ◽  
Resistance Mutations ◽  
Cd4 Cell Count ◽  
Hiv 1

ABSTRACT The MONARK study was a pilot randomized trial comparing the safety and efficacy of lopinavir-ritonavir (LPV/r) monotherapy to those of LPV/r-zidovudine-lamivudine triple therapy for antiretroviral-naïve human immunodeficiency virus type 1 (HIV-1)-infected patients. Resistance testing was performed at the time of initial screening and at the time of virological failure (defined to include low-level viremia with >50 and <400 HIV-1 virus RNA copies/ml of plasma). Changes from the baseline sequences, including mutations noted on the 2008 International AIDS Society—USA list of resistance-associated protease mutations, were considered. Drug resistance testing was performed for 38 patients (5 of 53 on triple therapy and 33 of 83 on monotherapy). By week 96 (W96), virus samples from 18 of 33 patients in the monotherapy arm showed changes from baseline sequences, and 5 of these patients had viruses with major protease inhibitor (PI) resistance-associated mutations (M46I at W40, L76V at W48, M46I and L76V at W48, L10F and V82A at W72, and L76V at W84). Data on virus phenotypes detected at the time of initial screening and the time of virological failure were available for four patients in whom major PI resistance mutations developed, and these data revealed a mean increase of 2.2-fold (range, 0.75- to 4.6-fold) in the LPV 50% inhibitory concentration. All three patients in whom the L76V PI resistance mutation developed were infected with HIV-1 subtype CRF02_AG. In the triple-therapy group, no major PI resistance mutation was selected among the three patients with protease changes by W48. No association between the baseline CD4 cell count and the viral load, the W4 and final viral loads, or the final LPV trough concentration and the emergence of a major PI resistance mutation was found. Major PI resistance-associated mutations were detected in 5 (6%) of 83 patients treated with LPV/r monotherapy, suggesting that LPV/r monotherapy is an inappropriate first option. The mutation L76V may be considered in further studies of lopinavir resistance.

scholarly journals Combination Treatment with Hepatitis C Virus Protease and NS5A Inhibitors Is Effective against Recombinant Genotype 1a, 2a, and 3a Viruses

2012 ◽  
Vol 57 (3) ◽  
pp. 1291-1303 ◽  
Author(s):  
Judith M. Gottwein ◽  
Sanne B. Jensen ◽  
Yi-Ping Li ◽  
Lubna Ghanem ◽  
Troels K. H. Scheel ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Combination Therapy ◽  
Combination Treatment ◽  
Resistance Mutations ◽  
Adaptive Mutations ◽  
Genotype 1A ◽  
Drug Concentrations ◽  
Low Concentrations ◽  
Ns3 Protease

ABSTRACTWith the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi-FL) recombinants relying only on the JFH1 NS3 helicase, NS5B, and the 3′ untranslated region. With identified adaptive mutations, semi-FL recombinants of genotypes(isolates) 1a(TN) and 3a(S52) produced supernatant infectivity titers of ∼4 log10focus-forming units/ml in Huh7.5 cells. Genotype 1a(TN) adaptive mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed the higher efficacy of the NS3 protease inhibitor asunaprevir (BMS-650032) and the NS5A inhibitor daclatasvir (BMS-790052) against 1a(TN and H77) than 3a(S52) viruses. Asunaprevir had intermediate efficacy against previously developed 2a recombinants J6/JFH1 and J6cc. Daclatasvir had intermediate efficacy against J6/JFH1, while low sensitivity was confirmed against J6cc. Using a cross-titration scheme, infected cultures were treated until viral escape or on-treatment virologic suppression occurred. Compared to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations. Inhibitors showed synergism at drug concentrations reportedin vivo. In summary, semi-FL HCV recombinants, including the most advanced reported genotype 3a infectious culture system, permitted genotype-specific analysis of combination treatment in the context of the complete viral life cycle. Despite differential sensitivity to lead compound NS3 protease and NS5A inhibitors, genotype 1a, 2a, and 3a viruses were suppressed by combination treatment with relatively low concentrations.

scholarly journals Development of a mismatch amplification mutation assay to correctly serotype isolates of Streptococcus suis serotypes 1, 2, 1/2, and 14

2020 ◽  
Vol 32 (3) ◽  
pp. 490-494 ◽  
Author(s):  
Sonia Lacouture ◽  
Masatoshi Okura ◽  
Daisuke Takamatsu ◽  
Lorelei Corsaut ◽  
Marcelo Gottschalk
Keyword(s):  
Capsular Polysaccharide ◽  
Streptococcus Suis ◽  
Zoonotic Potential ◽  
Nucleotide Polymorphism ◽  
Emerging Pathogen ◽  
Single Nucleotide ◽  
Routine Identification ◽  
Serotype 1 ◽  
Mutation Assay ◽  
Mismatch Amplification Mutation Assay

Streptococcus suis is one of the most important bacterial swine pathogens worldwide and is an emerging pathogen in humans. There are 29 serotypes, and serotyping, which is based on the antigenicity of the capsular polysaccharide (CPS) or on its coding genes, is often part of routine identification and provides further information regarding S. suis virulence and zoonotic potential. Serotypes 2 and 14 possess high zoonotic potential, and serotype 1/2 is the serotype most frequently isolated from diseased pigs in North America. PCR has replaced antibody-based techniques to perform serotyping. However, traditional PCR is not able to differentiate serotype 2 from 1/2 and serotype 1 from 14, given that the only difference in the cps loci of those serotype pairs is a nonsynonymous single-nucleotide polymorphism. We developed a mismatch amplification mutation assay (MAMA)-PCR that was able to correctly serotype 148 isolates previously known to be serotypes 1, 2, 1/2, or 14. This technique will be highly useful in animal and human health laboratories performing PCR serotyping of S. suis isolates.

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