An Improved Algorithm for Determining HIV Type 1 Subtypes in a Primary Laboratory in Uganda
Pontiano KaleebuDavid YirrellNeil FrenchFred LyagobaAlleluiah RutebemberwaR. Cheingsong‐PopovCharles F. GilksBenon BiryahwahoJonathan WeberJimmy Whitworth
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Abstract:
A pilot study was undertaken with the objective of developing a simple, economical, and efficient algorithm through which to subtype HIV-1 in a large epidemiological cohort study in Uganda. A peptide enzyme immunoassay (PEIA) employing both V3 and gp41 regions and a heteroduplex mobility assay (HM A) were evaluated in comparison with DNA sequencing. Of 146 samples selected, 115 (79%) were successfully sequenced. Taking sequence data as the "gold standard" other assays were compared with these data. The HMA correctly identified 95 (83%) of the samples, and only 1 sample was wrongly identified. The V3 PEIA alone and in combination with gp41 peptides correctly identified 76 and 78% of the samples, respectively; however, the number of wrongly identified samples was four times less with the combination compared with V3 peptides alone (4 versus 16%). The sensitivity, specificity, and positive and negative predictive values for serotype A and D samples were greater for the combination than V3 peptides alone. We have described a new algorithm to segregate subtypes A and D. This algorithm uses the two peptide assays followed by HMA and then DNA sequencing for untypable samples, giving an accuracy of 95% at a cost of 37 and 21% for consumables compared with subtyping all the samples by HMA or DNA sequencing, respectively. This proposed approach is suitable for epidemiological studies in Uganda and other regions with a predominance of A and D subtypes.Keywords:
Subtyping
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Abstract The purpose of subtyping is to differentiate bacterial isolates beyond the classification of species or subspecies. Subtyping methods can be grouped into two broad categories based on the cellular components targeted: (1) phenotypic subtyping methods that differentiate isolates by the enzymes, proteins, or other metabolites expressed by the cell, and (2) molecular subtyping methods that discriminate isolates based on interrogation of nucleic acid sequences. The two major types of molecular subtyping methods include band-based methods based on fragment pattern data or DNA fingerprints, and methods that generate DNA sequence data. Molecular subtyping methods have shown that Listeria monocytogenes isolates can be classified into four genetic lineages or divisions. Although band-based molecular subtyping methods continue to serve as the gold standard for routine molecular subtyping of most clinically important foodborne pathogens, including L. monocytogenes, the explosion of recently completed and ongoing DNA sequencing projects, and thus available DNA sequence data, have stimulated efforts to develop highly discriminatory and high-throughput DNA sequence-based subtyping methods for L. monocytogenes. L. monocytogenes represents one of the most highly sequenced human pathogens; more than 20 genome sequences are currently available for this organism. This review provides an overview of the concepts behind subtyping and discusses the application of molecular subtyping methods, with an emphasis on DNA sequence-based subtyping methods to characterize L. monocytogenes.
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DNA fragments of HIV-1 env gene were amplified by nested PCR from uncultured peripheral blood mononuclear cells (PBMCs) obtained from 24 HIV-1 infected individuals. The PCR products were separated by melting and annealing with denatured PCR product prepared from reference plasimd of representative subtypes. Heteroduplex were then formed between the single-stranded DNA from the two sources and were analysed on polyacrylamide gels. The results from heteroduplex mobility assay (HMA) were compared with HIV-1 subtype results determined by DNA sequencing. With advantages of high speed, low cost and high specificity, HMA is a reliable screening method for HIV-1 subtyping.
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Consistent subtyping is employed in some gradual type systems to validate type conversions. The original definition by Siek and Taha serves as a guideline for designing gradual type systems with subtyping. Polymorphic types à la System F also induce a subtyping relation that relates polymorphic types to their instantiations. However Siek and Taha's definition is not adequate for polymorphic subtyping. The first goal of this paper is to propose a generalization of consistent subtyping that is adequate for polymorphic subtyping, and subsumes the original definition by Siek and Taha. The new definition of consistent subtyping provides novel insights with respect to previous polymorphic gradual type systems, which did not employ consistent subtyping. The second goal of this paper is to present a gradually typed calculus for implicit (higher-rank) polymorphism that uses our new notion of consistent subtyping. We develop both declarative and (bidirectional) algorithmic versions for the type system. We prove that the new calculus satisfies all static aspects of the refined criteria for gradual typing, which are mechanically formalized using the Coq proof assistant.
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ABSTRACT The gag -based heteroduplex mobility assay ( gag -HMA) was evaluated for its ease and reliability in subtyping circulating recombinant forms (CRFs) of human immunodeficiency virus type 1 (HIV-1) in Côte d'Ivoire. One hundred thirty-two plasma samples were analyzed blindly for HIV-1 subtypes by sequencing the pol gene and by gag -HMA. DNA sequencing was used as the “gold standard.” Of the 132 samples sequenced, 108 (82%) were CRF02_AG, 14 (11%) were pure subtype A, 5 (4%) were subtype G, 3 (2%) were subtype D, 1 was CRF01_AE, and 1 was subtype H. The gag -HMA correctly classified 126 (95.5%) of the samples. Of the 108 samples that were classified as CRF02_AG by DNA sequencing, 107 (99%) were correctly identified by gag -HMA, resulting in a positive predictive value of 96.4%. The gag -HMA seems to be a valuable tool for understanding the molecular epidemiology of HIV-1 CRF02_AG in Côte d'Ivoire and West Africa, which could be important for developing and evaluating AIDS vaccines, although DNA sequencing remains necessary for accurate molecular epidemiology.
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Nowadays, there are various HIV-1 subtyping techniques, among them full-length sequencing is the most accurate technique to characterize HIV viral genomes and that will reveal the subtype and any recombinant structure of the genome. Although sequencing remains the most informative method, it is technically challenging, time consuming and expensive for use as a screening tool for large-scale surveillance, especially in developing countries. To circumvent this problem, easier and faster techniques are needed for routine subtyping. One of the techniques the Heteroduplex Mobility Assay (HMA) has been introduced by UNAIDS in several developing countries as a tool for monitoring subtypes distribution. HMA has also been used to rapidly characterize HIV-1 strains as part of the WHO sponsored vaccine evaluation programmed to identify subtypes and to investigate the heterogeneity of strains in the different parts of the world including South East Asia (SEA) countries such as Thailand, etc. In my study, the Heteroduplex Mobility Assay (HMA) was started to utilize for the first time in Myanmar to determine the circulating HIV-1 subtypes in our country. Moreover, it serves as a useful tool to determine the prevalence and distribution of HIV-1 subtypes and segregation of these subtypes among the different risk groups in Yangon (Lower Myanmar). Forty plasma samples were collected from 3 different risk groups: 10 from heterosexual male, 10 from heterosexual female, 12 from commercial sex workers (CSWs) and 8 from injecting drug users (IDUs) during 2000–2003. The viral RNA from plasma samples of 40 Western-blott confirmed HIV-infected individuals were extracted. These purified viral RNAs were then amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and subsequently by nested (second round) PCR with the primers for gag (p24) region. The reference plasmid DNAs were also amplified by nested PCR with the primers for gag (p24) region. Out of 40 samples, only 22 samples were amplified by gag (p24) PCR allowing subsequent subtype determination. HIV-1 subtype of 22 samples were determined by HIV -1 Gag (p24) HMA. Of the 22 samples, 9 (40.9%) were subtype C, 5 (22.7%) were subtype B' (Thai B cluster within Subtype B), 2 (9.1%) were subtype E (CRF 01-AE), 2 (9.1%) were subtype A and 4 (18.2%) were untypable. Sequencing is normally recommended for these untypeable samples. From this study, HIV-1 gag subtypes C was newly discovered in Yangon (Lower Myanmar) samples for the first time, but previously found in Mandalay (Central Myanmar) since 2000. Subtype C was most commonly found (9 out of 22) in our study and was more predominant in the heterosexual group. In fact, this study is the pioneer ones in Myanmar to apply the HMA technique in our own laboratory setting. The gag subtype A and E was detected only among persons presumably infected sexually and the subtype B' and C were found in both IDUs and persons with sexual exposure. If possible, further confirmation of these subtypes by sequencing should be done. Out of 40 samples non-amplifiable 18 samples were subsequently amplified with second primer set for gag (p 17) region. Only 4 samples amplified but not classified into subtypes due to the limitation of facilities (lack of the corresponding reference plasmids for gag (p 17) region). So, transfer of HIV-1 gag/env HMA technology to the developing countries contributes to more accurate estimations of the prevalence of HIV-1 subtypes and intersubtype recombinants and segregation of these subtypes in different risk groups in those parts of the world where the technology for sequencing is not readily available.
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Small-ruminant lentiviruses (SRLVs) display a high genetic diversity and are currently classified into five genotypes and an increasing number of subtypes. The co-circulation of subtypes in restricted geographical regions, combined with the occurrence of cross-species infection, suggests the need for development of a large-scale screening methodology for rapid monitoring of the prevalence of the various genetic subtypes and their genetic evolution. Here, a heteroduplex mobility assay (HMA) was developed for the rapid identification of group B subtypes. The assay was validated for both the p14 nucleocapsid-coding region of the gag gene and the V1-V2 region of the env gene using a panel of reference standards and was applied to the genetic subtyping of SRLV field isolates from five mixed flocks in France. Subtyping of 75 blood samples using the env HMA revealed a preferential distribution of subtypes B1 and B2 in sheep and goats, despite direct evidence for interspecies transmission of both subtypes. Adding the gag HMA to the env HMA provided evidence for dual infection and putative recombination between subtypes B1 and B2 in five goats, and between groups A and B in one sheep. Phylogenetic analysis revealed that 100 % (23/23) and 96.7 % (30/31) of samples were correctly classified using the gag and env HMAs, respectively. These results indicate that dual infection and recombination may be a significant source of new variation in SRLV and provide a useful tool for the rapid genetic subtyping of SRLV isolates, which could be relevant for the development of more accurate diagnosis of prevalent SRLV strains in different countries.
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The gp120 region of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene exhibits a high level of genetic heterogeneity across the group M subtypes. The heteroduplex mobility assay (HMA) has successfully been used to assign subtype classifications, but C2V5 primers often fail to amplify African strains. We developed an env gp41-based HMA for which the target sequence is amplified with highly conserved gp41 primers, known to efficiently amplify nucleic acids from HIV-1 group M, N, and O viruses. By using gp41 from a new panel of reference strains, the subtype assignments made by our modified HMA were concordant with those obtained by sequencing and phylogenetic analysis of 34 field strains from 10 countries representing subtypes A to G. Testing of field strains from Nigeria further demonstrated the utility of this modified assay. Of 28 samples, all could be amplified with gp41 primers but only 17 (60.7%) could be amplified with the standard C2V5 primers. Therefore, gp41-based HMA can be a useful tool for the rapid monitoring of prevalent subtypes in countries with divergent strains of circulating HIV-1.
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Double-stranded heteroduplex molecules that form between a mutant and wild-type DNA strand are often distinguished from homoduplex molecules upon gel electrophoresis. This method, heteroduplex analysis (HA), can be performed rapidly without radioisotopes or specialized equipment. Modifications and enhancements of the HA method have been developed that increase the sensitivity of detection of single-base pair alterations. © 1995 Wiley-Liss, Inc.1 This article is a US Government work and, as such, is in the public domain in the United States of America.
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