Background: Access to antiretroviral therapy for low-income countries has expanded during recent years. More than 9.7 million people living with HIV/AIDS in low and middle income countries were receiving antiretroviral therapy at the end of 2012. As the coverage of antiretroviral therapy continues to grow in resourcelimited settings, emergence and transmission of HIV drug resistance is inevitable; this could potentially restrict future therapeutic options and increase treatment cost by requiring more expensive antiretroviral regimens. Appropriate selection of successful antiretroviral regimen for HIV infected patients initiating therapy mainly relies on drug resistance testing, moreover the increasing number of individuals on first-line antiretroviral treatment requires closed virological monitoring to ensure timely and appropriate switch to second-line regimens soon after failure, to sustain the effectiveness of standard first and second-line treatment combinations. In this context, drug resistance testing is gradually becoming an indispensable tool for the surveillance and prevention of emerging resistant viral HIV strains as well as for individual patient monitoring. Genotyping assays are currently the reference methods to detect HIV drug resistance (HIVDR) mutations; however their use is limited in resource-limited settings because of their high cost. Several laboratories in developing countries have developed and validated highly sensitive in-house HIV genotyping assays comparable to FDA-approved assays to reduce the cost and to overcome subtype specificities, but drug resistance testing is still not affordable for the majority of patients in need. Objectives: In the present work, we aim to ascertain HIV drug resistance patterns in different antiretroviral treatment contexts (naïve, first and second-line) and determine viral susceptibility to recently developed drugs [DRV/r, integrase inhibitors (INI), CCR5 antagonists] for potential third line antiretroviral treatment options in Cameroon, using in-house genotyping assays. Furthermore we aim to develop and validate a low cost PCR-based point mutation assay namely the Amplification Refractory Mutation System PCR (ARMS-PCR) to detect the most frequent HIV-1 drug resistance mutations (M184V, T215Y/F, K103N and Y181C) affecting first line regimens in Cameroon as an alternative to sequencing-based assays. Material and methods: Firstly, 66 patients in Cameroon were evaluated by sequencing the protease-reverse transcriptase, integrase and envelope-V3 loop to determine their susceptibility to potential third line drugs in Cameroon. Secondly, 15 samples with different biological characteristics were selected to develop an ARMSPCR assay to detect the most common HIVDR mutations (M184V, T215Y/F, K103N and Y181C) in Cameroon. Finally the ARMS-PCR assay was used to investigate the mutation profile of 75 HIV infected patients and to determine its performance. Discordant results between ARMS-PCR and sequencing were further investigated with Trugene® HIV genotyping assay, a FDA-approved commercial test for resistance testing. Results: In the PR-RT region, the rate of HIVDR-associated mutations increased significantly when moving from ART-naïve (6.7%: 1/15) to first-line (79.2%: 19/24) and to second-line (100%: 23/23) ART-experienced patients. Only accessory mutations to INI were found, and similarly distributed across sub-set of patients with different ART history. An overall rate of 37.9% (22/58) viral tropism switch from CCR5 to CXCR4 was observed, ranging from 26.7% (4/15) CXCR4 among ARTnaïve patients, to 47.4% (9/19) among first-line ART-experienced patients, to 37.5% (9/24) among second-line ART-experienced patients. The ARMS-PCR assay was able to identify the most common HIV-1 drug resistance mutations tested with high sensitivity (70-96.8%), high specificity (90.6-100%), good positive predictive value (77.7-100%) and good negative predictive value (95.4-97.6) compared to sequencing. 46.6% (35/75), 20% (15/75), 28% (21/75) and 12% (9/75) patients were identified with M184V, T215Y/F, K103N and Y181C drug resistance mutations respectively. Conclusion: HIV drug resistance mutations increase with long-term antiretroviral treatment exposure; integrase inhibitors are suitable for patients failing second-line therapy in resource-limited settings with multi drug resistance mutation while CCR5 antagonists require tropism testing. ARMS-PCR assay has a good performance compared to sequencing for the identification of the most common drug resistance mutations. Its simplicity, affordability and cost-effectiveness make it a pragmatic approach to monitor HIV-1 infected patients in resource-limited settings.

(2014). Characterization of HIV-1 drug resistance mutations in Cameroon: utility of cost-effective tools in resource-limited settings.

Characterization of HIV-1 drug resistance mutations in Cameroon: utility of cost-effective tools in resource-limited settings

NANFACK, AUBIN JOSEPH
2014-01-01

Abstract

Background: Access to antiretroviral therapy for low-income countries has expanded during recent years. More than 9.7 million people living with HIV/AIDS in low and middle income countries were receiving antiretroviral therapy at the end of 2012. As the coverage of antiretroviral therapy continues to grow in resourcelimited settings, emergence and transmission of HIV drug resistance is inevitable; this could potentially restrict future therapeutic options and increase treatment cost by requiring more expensive antiretroviral regimens. Appropriate selection of successful antiretroviral regimen for HIV infected patients initiating therapy mainly relies on drug resistance testing, moreover the increasing number of individuals on first-line antiretroviral treatment requires closed virological monitoring to ensure timely and appropriate switch to second-line regimens soon after failure, to sustain the effectiveness of standard first and second-line treatment combinations. In this context, drug resistance testing is gradually becoming an indispensable tool for the surveillance and prevention of emerging resistant viral HIV strains as well as for individual patient monitoring. Genotyping assays are currently the reference methods to detect HIV drug resistance (HIVDR) mutations; however their use is limited in resource-limited settings because of their high cost. Several laboratories in developing countries have developed and validated highly sensitive in-house HIV genotyping assays comparable to FDA-approved assays to reduce the cost and to overcome subtype specificities, but drug resistance testing is still not affordable for the majority of patients in need. Objectives: In the present work, we aim to ascertain HIV drug resistance patterns in different antiretroviral treatment contexts (naïve, first and second-line) and determine viral susceptibility to recently developed drugs [DRV/r, integrase inhibitors (INI), CCR5 antagonists] for potential third line antiretroviral treatment options in Cameroon, using in-house genotyping assays. Furthermore we aim to develop and validate a low cost PCR-based point mutation assay namely the Amplification Refractory Mutation System PCR (ARMS-PCR) to detect the most frequent HIV-1 drug resistance mutations (M184V, T215Y/F, K103N and Y181C) affecting first line regimens in Cameroon as an alternative to sequencing-based assays. Material and methods: Firstly, 66 patients in Cameroon were evaluated by sequencing the protease-reverse transcriptase, integrase and envelope-V3 loop to determine their susceptibility to potential third line drugs in Cameroon. Secondly, 15 samples with different biological characteristics were selected to develop an ARMSPCR assay to detect the most common HIVDR mutations (M184V, T215Y/F, K103N and Y181C) in Cameroon. Finally the ARMS-PCR assay was used to investigate the mutation profile of 75 HIV infected patients and to determine its performance. Discordant results between ARMS-PCR and sequencing were further investigated with Trugene® HIV genotyping assay, a FDA-approved commercial test for resistance testing. Results: In the PR-RT region, the rate of HIVDR-associated mutations increased significantly when moving from ART-naïve (6.7%: 1/15) to first-line (79.2%: 19/24) and to second-line (100%: 23/23) ART-experienced patients. Only accessory mutations to INI were found, and similarly distributed across sub-set of patients with different ART history. An overall rate of 37.9% (22/58) viral tropism switch from CCR5 to CXCR4 was observed, ranging from 26.7% (4/15) CXCR4 among ARTnaïve patients, to 47.4% (9/19) among first-line ART-experienced patients, to 37.5% (9/24) among second-line ART-experienced patients. The ARMS-PCR assay was able to identify the most common HIV-1 drug resistance mutations tested with high sensitivity (70-96.8%), high specificity (90.6-100%), good positive predictive value (77.7-100%) and good negative predictive value (95.4-97.6) compared to sequencing. 46.6% (35/75), 20% (15/75), 28% (21/75) and 12% (9/75) patients were identified with M184V, T215Y/F, K103N and Y181C drug resistance mutations respectively. Conclusion: HIV drug resistance mutations increase with long-term antiretroviral treatment exposure; integrase inhibitors are suitable for patients failing second-line therapy in resource-limited settings with multi drug resistance mutation while CCR5 antagonists require tropism testing. ARMS-PCR assay has a good performance compared to sequencing for the identification of the most common drug resistance mutations. Its simplicity, affordability and cost-effectiveness make it a pragmatic approach to monitor HIV-1 infected patients in resource-limited settings.
2014/2015
Immunologia e biotecnologie applicate
27.
Settore MED/17 - Malattie Infettive
English
Tesi di dottorato
(2014). Characterization of HIV-1 drug resistance mutations in Cameroon: utility of cost-effective tools in resource-limited settings.
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