Surveillance technology for HIV-1 subtype C in Ethiopia: An env-based NASBA molecular beacon assay to discriminate between subcluster C and C′

https://doi.org/10.1016/j.jviromet.2005.05.028Get rights and content

Abstract

Forty-nine samples with known C2V3 sequences were used for the evaluation of an env-based molecular beacon assay to distinguish between the two genetic subclusters C and C′ which characterize the HIV-1 epidemic in Ethiopia. Two subcluster C and two subcluster C′ beacons targeting two different loci in the C2V3 region were developed. Using a three beacon-based (2C and 1C′ = C prime), isothermal amplification assay, concordance with DNA sequencing was achieved for 43 (87.8%) samples. Sensitivity was 81.8% and specificity 97.4% for subcluster C beacons. For the subcluster C′ beacon, a sensitivity of 97% and a specificity of 87.5% was achieved. Five samples were ambiguous by sequencing of which two samples were subcluster C′ by the beacon assay and one subcluster C. Two of the samples remained ambiguous with different beacon-pair combinations as well. From samples with a clear C or C′ phylogeny by sequencing, three were undetected by the first-line beacon genotyping assay. Genotype ambiguity was resolved in the three samples using beacon pair combinations restricted to each targeted locus. The beacons were evaluated further in a panel including all HIV-1 subtypes. Four of five subtype C isolates were identified correctly, and no cross-reactivity was observed with other subtypes.

Introduction

Global tracking of the genetic variability of human immunodeficiency virus-type 1 is important from both epidemiological and treatment perspectives. The identification of HIV-1 clades or subtypes (Robertson et al., 2000) relies on sequence analysis of the env, gag or pol genes (Myers et al., 1994, Pasquier et al., 2001, Njouom et al., 2003). Sequencing remains the gold standard but other subtyping technologies that are less costly, offering ease of performance and which are therefore more practical to adopt for large population-based screening in developing countries have undergone extensive evaluation (Barin et al., 1996, Luigi et al., 1995, Luo et al., 1998).

HIV-1 subtype C is estimated to account for at least 56% of all global infections (Esparza and Bhamarapravati, 2000). The HIV-1 epidemic in Ethiopia is overwhelmingly subtype C, since the start of the epidemic over two decades ago (Abebe et al., 2001a, Abebe et al., 2001b). Sequence analysis has demonstrated the presence of a distinct subcluster (C prime or C′) within the main subtype C group viruses in Ethiopia (Abebe et al., 2000), which is increasing in prevalence over time in surveyed population groups. The functional relevance of two distinct genetic subclusters of HIV-1 subtype C in Ethiopia remains to be investigated. The development of a rapid, sensitive and highly specific assay to discriminate between the two genetic subclusters of HIV-1 subtype C circulating in Ethiopia based on the gag gene has previously been reported (Ayele et al., 2004). In the present study, the development of an env (C2V3) gene-based assay utilizing a similar real-time NASBA format to discriminate between subcluster C and C′ is described. Genotyping of C2V3 is of interest since there is some indication that a C′ envelope gene may confer a selective transmission advantage as compared to C (Pollakis et al., 2003). This is inferred from the observation that among recombinant isolates C′ predominates in the envelope gene, and therefore the continued evaluation of the C/C′ ratio would be a good indicator of the evolution dynamics in the two regions of the genome.

The two regions (gag and env) were targeted for development of assays to discriminate between the subclusters as these contain the principal immunodominant regions which are important to consider in vaccine design (Van Baalen et al., 1996, Neurath and Strick, 1990). The env-based assay in combination with the gag-based test is expected to aid in surveillance efforts that would in addition, greatly aid in identifying functional differences between C and C′ viruses in Ethiopia. Application of this tool will enable detailed analysis of differences, if any, in disease progression patterns over the long term in individuals infected with one or the other subcluster, which might be ascribed to the intrinsic biological properties of those viruses.

Section snippets

Sample selection

Plasma samples from 49 HIV-seropositive individuals enrolled in the Ethio-Netherlands AIDS Research Project (ENARP) cohort to study the natural history of HIV infection in Ethiopia were selected. Details of the cohort are as described elsewhere (Sahlu et al., 1998).

HIV-1 extraction and amplification

HIV-1 RNA was isolated from 200 μl each of plasma using the silica-based Boom extraction technique for nucleic acids (Boom et al., 1990). Viral load was determined for all individuals using the NucliSens HIV-1 QT assay (bioMérieux,

Results

During assay development various beacon combinations from both loci were tested and genotyping results coming closest to those generated by DNA sequencing were found using the two C beacons (MB146 and MB148) in combination with one C′ beacon located in the downstream region of the PCR amplified fragment (MB147) (data not shown). Hence, this triple beacon combination (MB146/147/148) was adopted as the first-line screening assay for genotyping all 49 samples in the present study. The method was

Discussion

This study describes the performance of a novel env-based genotyping assay having a high sensitivity and specificity to distinguish between the two main subclusters of HIV-1 subtype C circulating in Ethiopia. The assay is tailor-made to the Ethiopian situation where HIV-1 subtype C remains fairly homogeneous, indicating perhaps a more recent epidemic compared to places, like South Africa where multiple lineages of HIV-1 subtype C have been reported (Gordon et al., 2003).

Concordance in results

Acknowledgements

This study is a part of the Ethio-Netherlands AIDS Research Project (ENARP), a collaborative effort of the Ethiopian Health and Nutrition Research Institute (EHNRI, Addis Ababa, Ethiopia), the Amsterdam Municipal Health Service (GG/GD), the Academic Medical Center of the University of Amsterdam (AMC), and the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB, Amsterdam, the Netherlands). Financial support of ENARP is from the Netherlands Ministry of Foreign Affairs

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