Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission.
Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-sp...
| Main Authors: | , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2015-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4607442?pdf=render |
| _version_ | 1818953102905049088 |
|---|---|
| author | Andrea Hauser Claudia Kuecherer Andrea Kunz Piotr Wojtek Dabrowski Aleksandar Radonić Andreas Nitsche Stefanie Theuring Norbert Bannert Julius Sewangi Paulina Mbezi Festo Dugange Gundel Harms Karolin Meixenberger |
| author_facet | Andrea Hauser Claudia Kuecherer Andrea Kunz Piotr Wojtek Dabrowski Aleksandar Radonić Andreas Nitsche Stefanie Theuring Norbert Bannert Julius Sewangi Paulina Mbezi Festo Dugange Gundel Harms Karolin Meixenberger |
| author_sort | Andrea Hauser |
| collection | DOAJ |
| description | Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT).Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System.Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage.Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies. |
| first_indexed | 2024-12-20T10:00:56Z |
| format | Article |
| id | doaj.art-09a6524b36564a0e8ae7dd8b65d120f6 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-20T10:00:56Z |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-09a6524b36564a0e8ae7dd8b65d120f62022-12-21T19:44:20ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-011010e014080910.1371/journal.pone.0140809Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission.Andrea HauserClaudia KuechererAndrea KunzPiotr Wojtek DabrowskiAleksandar RadonićAndreas NitscheStefanie TheuringNorbert BannertJulius SewangiPaulina MbeziFesto DugangeGundel HarmsKarolin MeixenbergerPregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT).Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System.Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage.Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.http://europepmc.org/articles/PMC4607442?pdf=render |
| spellingShingle | Andrea Hauser Claudia Kuecherer Andrea Kunz Piotr Wojtek Dabrowski Aleksandar Radonić Andreas Nitsche Stefanie Theuring Norbert Bannert Julius Sewangi Paulina Mbezi Festo Dugange Gundel Harms Karolin Meixenberger Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission. PLoS ONE |
| title | Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission. |
| title_full | Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission. |
| title_fullStr | Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission. |
| title_full_unstemmed | Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission. |
| title_short | Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission. |
| title_sort | comparison of 454 ultra deep sequencing and allele specific real time pcr with regard to the detection of emerging drug resistant minor hiv 1 variants after antiretroviral prophylaxis for vertical transmission |
| url | http://europepmc.org/articles/PMC4607442?pdf=render |
| work_keys_str_mv | AT andreahauser comparisonof454ultradeepsequencingandallelespecificrealtimepcrwithregardtothedetectionofemergingdrugresistantminorhiv1variantsafterantiretroviralprophylaxisforverticaltransmission AT claudiakuecherer comparisonof454ultradeepsequencingandallelespecificrealtimepcrwithregardtothedetectionofemergingdrugresistantminorhiv1variantsafterantiretroviralprophylaxisforverticaltransmission AT andreakunz comparisonof454ultradeepsequencingandallelespecificrealtimepcrwithregardtothedetectionofemergingdrugresistantminorhiv1variantsafterantiretroviralprophylaxisforverticaltransmission AT piotrwojtekdabrowski comparisonof454ultradeepsequencingandallelespecificrealtimepcrwithregardtothedetectionofemergingdrugresistantminorhiv1variantsafterantiretroviralprophylaxisforverticaltransmission AT aleksandarradonic comparisonof454ultradeepsequencingandallelespecificrealtimepcrwithregardtothedetectionofemergingdrugresistantminorhiv1variantsafterantiretroviralprophylaxisforverticaltransmission AT andreasnitsche comparisonof454ultradeepsequencingandallelespecificrealtimepcrwithregardtothedetectionofemergingdrugresistantminorhiv1variantsafterantiretroviralprophylaxisforverticaltransmission AT stefanietheuring comparisonof454ultradeepsequencingandallelespecificrealtimepcrwithregardtothedetectionofemergingdrugresistantminorhiv1variantsafterantiretroviralprophylaxisforverticaltransmission AT norbertbannert comparisonof454ultradeepsequencingandallelespecificrealtimepcrwithregardtothedetectionofemergingdrugresistantminorhiv1variantsafterantiretroviralprophylaxisforverticaltransmission AT juliussewangi comparisonof454ultradeepsequencingandallelespecificrealtimepcrwithregardtothedetectionofemergingdrugresistantminorhiv1variantsafterantiretroviralprophylaxisforverticaltransmission AT paulinambezi comparisonof454ultradeepsequencingandallelespecificrealtimepcrwithregardtothedetectionofemergingdrugresistantminorhiv1variantsafterantiretroviralprophylaxisforverticaltransmission AT festodugange comparisonof454ultradeepsequencingandallelespecificrealtimepcrwithregardtothedetectionofemergingdrugresistantminorhiv1variantsafterantiretroviralprophylaxisforverticaltransmission AT gundelharms comparisonof454ultradeepsequencingandallelespecificrealtimepcrwithregardtothedetectionofemergingdrugresistantminorhiv1variantsafterantiretroviralprophylaxisforverticaltransmission AT karolinmeixenberger comparisonof454ultradeepsequencingandallelespecificrealtimepcrwithregardtothedetectionofemergingdrugresistantminorhiv1variantsafterantiretroviralprophylaxisforverticaltransmission |