Identification of a new HIV-1 circulating recombinant form (CRF159_01103) derived from CRF103_01B and CRF01_AE in Hebei Province, China.

Recombinant HIV-1 genomes identified in three or more epidemiological unrelated individuals are defined as circulating recombinant forms (CRFs). CRFs can further recombine with other pure subtypes or recombinants to produce secondary recombinants. In this study, a new HIV-1 intersubtype CRF, designated CRF159_01103, isolated from three men who have sex with men with no epidemiological linkage, was identified in Baoding city, Hebei Province, China. CRF159_01103 was derived from CRF103_01B and CRF01_AE. Bayesian molecular clock analysis was performed on the CRF01-AE and CRF103_01B regions of CRF159_01103. The time of origin of CRF159_01103 was predicted to be 2018-2019, indicating that it is a recent recombinant virus. The emergence of CRF159_01103 has increased the complexity of the HIV-1 epidemic in Hebei Province.

Identification of a HIV-1 circulating BF1 recombinant form (CRF75_BF1) of Brazilian origin that also circulates in Southwestern Europe.

Introduction: The high recombinogenic potential of HIV-1 has resulted in the generation of countless unique recombinant forms (URFs) and around 120 reported circulating recombinant forms (CRFs). Here we identify through analyses of near full-length genomes (NFLG) a new HIV-1 CRF derived from subtypes B and F1. Methods: HIV-1 protease-reverse transcriptase (Pr-RT) sequences were obtained by RT-PCR amplification from plasma RNA. Near full-length genome sequences were obtained after amplification by RT-PCR in 5 overlapping fragments. Phylogenetic sequence analyses were performed via maximum likelihood. Mosaic structures were analyzed by bootscanning and phylogenetic analyses of genome segments. Temporal and geographical estimations of clade emergence were performed with a Bayesian coalescent method. Results: Through phylogenetic analyses of HIV-1 Pr-RT sequences obtained by us from samples collected in Spain and downloaded from databases, we identified a BF1 recombinant cluster segregating from previously reported CRFs comprising 52 viruses, most from Brazil (n = 26), Spain (n = 11), and Italy (n = 9). The analyses of NFLG genomes of 4 viruses of the cluster, 2 from Spain and 2 from Italy, allowed to identify a new CRF, designated CRF75_BF1, which exhibits a complex mosaic structure with 20 breakpoints. All 4 patients harboring CRF75_BF1 viruses studied by us had CD4+ T-cell lymphocyte counts below 220/mm3 less than one year after diagnosis, a proportion significantly higher (p = 0.0074) than the 29% found in other patients studied in Spain by us during the same period. The origin of the clade comprising CRF75_BF1 and related viruses was estimated around 1984 in Brazil, with subsequent introduction of CRF75_BF1 in Italy around 1992, and migration from Italy to Spain around 1999. Conclusion: A new HIV-1 CRF, designated CRF75_BF1, has been identified. CRF75_BF1 is the 6th CRF of South American origin initially identified in Western Europe, reflecting the increasing relationship of South American and European HIV-1 epidemics. The finding of low CD4+ T-cell lymphocyte counts early after diagnosis in patients harboring CRF75_BF1 viruses warrants further investigation on the virulence of this variant.

Characterization of two novel HIV-1 second-generation recombinants (CRF01_AE/CRF07_BC) identified in Hebei Province, China.

Introduction: The unique recombinant forms (URFs) of HIV-1 consist of a mixture of subtypes, and each URF has a unique breakpoint. In this study, we identified the near fulllength genome (NFLG) sequences of two novel HIV-1 URFs (Sample ID: BDD034A and BDL060) isolated during HIV-1 molecular surveillance in 2022 in Baoding city, Hebei Province, China. Methods: The two sequences were aligned with subtype reference sequences and CRFs from China using MAFFT v7.0, and the alignments were adjusted manually using BioEdit (v7.2.5.0). Phylogenetic and subregion trees were constructed using MEGA11 with the neighbor-joining (N-J) method. Recombination breakpoints were identified by SimPlot (v3.5.1) based on Bootscan analyses. Results: Recombinant breakpoint analysis revealed that the NFLGs of BDD034A and BDL060 were composed of CRF01_AE and CRF07_BC, containing seven segments, respectively. For BDD034A, three CRF01_AE fragments were inserted into the CRF07_BC main framework, whereas for BDL060, three CRF07_BC fragments were inserted into the CRF01_AE main framework. Discussion: The emergence of the CRF01_AE/CRF07_BC recombinant strains indicates that HIV-1 co-infection is common. The increasing genetic complexity of the HIV-1 epidemic in China warrants continued investigation.

Comprehensive Genetic Characterization of Four Novel HIV-1 Circulating Recombinant Forms (CRF129_56G, CRF130_A1B, CRF131_A1B, and CRF138_cpx): Insights from Molecular Epidemiology in Cyprus.

Molecular investigations of the HIV-1 pol region (2253-5250 in the HXB2 genome) were conducted on sequences obtained from 331 individuals infected with HIV-1 in Cyprus between 2017 and 2021. This study unveiled four distinct HIV-1 putative transmission clusters, encompassing 19 previously unidentified HIV-1 recombinants. These recombinants, each comprising eight, three, four, and four sequences, respectively, did not align with previously established Circulating Recombinant Forms (CRFs). To characterize these novel HIV-1 recombinants, near-full-length genome sequences were successfully obtained for 16 of the 19 recombinants (790-8795 in the HXB2 genome) using an in-house-developed RT-PCR assay. Phylogenetic analyses, employing MEGAX and Cluster-Picker, along with confirmatory neighbor-joining tree analyses of subregions, were conducted to identify distinct clusters and determine subtypes. The uniqueness of the HIV-1 recombinants was evident in their exclusive clustering within generated maximum likelihood trees. Recombination analyses highlighted the distinct chimeric nature of these recombinants, with consistent mosaic patterns observed across all sequences within each of the four putative transmission clusters. Conclusive genetic characterization identified four novel HIV-1 CRFs: CRF129_56G, CRF130_A1B, CRF131_A1B, and CRF138_cpx. CRF129_56G exhibited two recombination breakpoints and three fragments of subtypes CRF56_cpx and G. Both CRF130_A1B and CRF131_A1B featured seven recombination breakpoints and eight fragments of subtypes A1 and B. CRF138_cpx displayed five recombination breakpoints and six fragments of subtypes CRF22_01A1 and F2, along with an unclassified fragment. Additional BLAST analyses identified a Unique Recombinant Form (URF) of CRF138_cpx with three additional recombination sites, involving subtype F2, a fragment of unknown subtype origin, and CRF138_cpx. Post-identification, all putative transmission clusters remained active, with CRF130_A1B, CRF131_A1B, and CRF138_cpx clusters exhibiting further growth. Furthermore, international connections were identified through BLAST analyses, linking one sequence from the USA to the CRF130_A1B strain, and three sequences from Belgium and Cameroon to the CRF138_cpx strain. This study contributes valuable insights into the dynamic landscape of HIV-1 diversity and transmission patterns, emphasizing the need for ongoing molecular surveillance and global collaboration in tracking emerging viral variants.

HIV-1 Circulating Recombinant Forms (CRFs) and Unique Recombinant Forms (URFs) in Israel, 2010-2018.

Monitoring HIV-1 circulating recombinant forms (CRFs) and unique recombinant forms (URFs) is important for disease surveillance. Recombination may affect prevention efforts and interfere with the diagnosis and treatment of HIV-1 infection. Here, we characterized the epidemiology of HIV-1 CRFs and URFs in Israel. Partial pol sequences from treatment naïve patients diagnosed in 2010−2018 were assessed using the recombinant identification program (RIP), the recombinant detection program (RDP5), and using the maximum-likelihood phylogenetic method, using 410 reference sequences obtained from the Los Alamos database. CRFs and URFs were identified in 11% (213/1940) of all sequenced cases. The median age at diagnosis was 38 (30−47) years, 61% originated from Israel, and 82% were male. The most common were CRF02_AG (30.5%), CRF01_AE (16.9%), and the more complex forms CRF01_AE/CRF02_AG/A3 (10.8%) and B/F1 (7%). A significant increase in their overall proportion was observed in recent years (8.1% in 2010−2012, 20.3% in 2016−2018, p < 0.001). This increase was most prominent in individuals carrying CRF02_AG (2.5% in 2010−2015, 9.8% in 2016−2018, p < 0.001). Men who have sex with men (MSM) was the most common risk group; however, those infected with the secondary recombinant CRF02_AG/A6 were mainly injecting drug users (IDUs). The most common resistance mutations were K103N (5/213, 2.3%) and E138A (18/213, 8.5%) in the reverse transcriptase. Only E138A was more frequent in the recombinants compared with the classic subtypes and was significantly associated with a specific secondary CRF, CRF02_AG/A4. We concluded that CRFs and URFs were mainly detected in Israeli-born MSM and that an increase in the overall proportion of such HIV-1 sequences could be observed in more recent years.

Characterization of a novel HIV-1 circulating recombinant form, CRF91_cpx, comprising CRF02_AG, G, J, and U, mostly among men who have sex with men.

Prospective molecular studies of HIV-1 pol region (2253-5250 in HXB2 genome) sequences from sequenced samples of 269 HIV-1-infected patients in Cyprus (2017-2021) revealed a transmission cluster of 14 unknown HIV-1 recombinants that were not classified as previously established CRFs. The earliest recombinant was collected in September 2017, and the transmission cluster continued to grow until November 2020. Near full-length HIV-1 genome sequences of the 11 of the 14 recombinants were successfully obtained (790-8795 in HXB2 genome) and aligned against a reference dataset of HIV-1 subtypes and CRFs. We employed MEGAX for maximum-likelihood tree construction (GTR model, 1000 bootstrap replicates), Cluster-Picker for phylogenetic clustering analysis (genetic distance ≤0.045, bootstrap support value ≥70%), and REGA-3.0 for subtype determination. Bootscan and similarity plot analyses (sliding window of 400 nucleotides overlapped by 40 nucleotides) were conducted using SimPlot-v3.5.1, and subregion confirmatory neighbour-joining tree analyses were conducted using MEGAX (Kimura two-parameter model, 1000 bootstrap replicates, ≥70% bootstrap-support value). Exclusive clustering of the HIV-1 recombinants revealed their uniqueness. The recombination analyses illustrated the same unique mosaic pattern with six putative intersubtype recombination breakpoints, seven fragments of subtypes CRF02_AG, G, J and an unclassified fragment. We conclusively characterized the mosaic structure of the novel HIV-1 CRF, named CRF91_cpx, by the Los Alamos HIV Sequence Database. Additionally, we identified a URF of CRF91_cpx with two additional recombination sites, generated by a recombination event between subtype B and CRF91_cpx. Since the identification of CRF91_cpx, two additional patient samples have been entered into the CRF91_cpx transmission cluster, demonstrating active growth.

Viruses Previously Identified in Brazil as Belonging to HIV-1 CRF72_BF1 Represent Two Closely Related Circulating Recombinant Forms, One of Which, Designated CRF122_BF1, Is Also Circulating in Spain.

Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Those derived from recombination between subtype B and subsubtype F1, with 18 reported, most of them of South American origin, are among the most diverse. In this study, we identified a HIV-1 BF1 recombinant cluster that is expanding in Spain, transmitted mainly via heterosexual contact, which, analyzed in near full-length genomes in four viruses, exhibited a coincident BF1 mosaic structure, with 12 breakpoints, that fully coincided with that of two viruses (10BR_MG003 and 10BR_MG005) from Brazil, previously classified as CRF72_BF1. The three remaining Brazilian viruses (10BR_MG002, 10BR_MG004, and 10BR_MG008) previously identified as CRF72_BF1 exhibited mosaic structures highly similar, but not identical, to that of the Spanish viruses and to 10BR_MG003 and 10BR_MG005, with discrepant subtypes in two short genome segments, located in pol and gp120env. Based on these results, we propose that the five viruses from Brazil previously identified as CRF72_BF1 actually belong to two closely related CRFs, one comprising 10BR_MG002, 10BR_MG004, and 10BR_MG008, which keep their CRF72_BF1 designation, and the other, designated CRF122_BF1, comprising 10BR_MG003, 10BR_MG005, and the viruses of the identified Spanish cluster. Three other BF1 recombinant genomes, two from Brazil and one from Italy, previously identified as unique recombinant forms, were classified as CRF72_BF1. CRF122_BF1, but not CRF72_BF1, was associated with protease L89M substitution, which was reported to contribute to antiretroviral drug resistance. Phylodynamic analyses estimate the emergence of CRF122_BF1 in Brazil around 1987. Given their close phylogenetic relationship and similar structures, the grouping of CRF72_BF1 and CRF122_BF1 in a CRF family is proposed.

Identification of two novel HIV-1 circulating recombinant forms of CRF111_01C and CRF116_0108 in southwestern Yunnan, China.

Yunnan, the region hardest hit by HIV/AIDS in China, is also an area with the most abundant HIV-1 genetic diversity. A large number of novel HIV-1 circulating recombinant forms (CRFs) and unique recombinants were identified among injection drug users in Yunnan; however, few were found among sexual contacts. Here, we obtained 15 near full-length genome sequences (NFLGs) from HIV-1 seropositive heterosexual contacts in Yunnan who received antiretroviral therapy during the period from 2014 to 2016. Phylogenetic analysis showed that six NFLGs belonged to CRF01_AE (n = 3) and CRF106_cpx (n = 3), and the other nine sequences were novel inter-subtype recombinants. Of the recombinants, two novel CRFs (CRF111_01 C (n = 4) and CRF116_0108 (n = 4)) and one CRF106_cpx variant (n = 1) were identified. CRF111_01 C had a CRF01_AE backbone with seven subtype C fragments inserted into the gag, pol, vif, env, nef and 3'LTR regions. CRF116_0108 had a CRF08_BC backbone with a CRF01_AE fragment inserted into the pol, tat, rev, vif, vpr, vpu and env regions. Phylogeographic analyses estimated that CRF111_01 C and CRF116_0108 originated approximately 1995.7-1998.6 and 1991.7-1993.7, respectively. These identifications of two novel HIV-1 CRFs highlighted the importance of continuous surveillance in heterosexual contacts and other high-risk groups in this region and the surrounding regions.

Molecular Epidemiology of HIV-1 Virus in Egypt: A Major Change in the Circulating Subtypes.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) is characterized by high genetic diversity due to its high mutation and recombination rates. Although, there is an increasing prevalence of Circulating Recombinant Forms (CRFs) worldwide, subtype B is still recognized as the predominant subtype in the Middle East and North Africa (MENA) region. There is a limited sampling of HIV in this region due to its low prevalence. The main purpose of this study is to provide a summary of the current status of the resident HIV subtypes and their distribution among Egyptian patients. METHODOLOGY: Forty-five HIV-1 patients were included in this study. Partial pol gene covering the protease (PR) and Reverse Transcriptase (RT) was successfully amplified in 21 HIV patients using nested PCR of cDNA of the viral genomic RNA, then sequenced. The sequence data were used for viral HIV-1 subtyping by 5 online subtyping tools: NCBI viral genotyping tool, Stanford University HIV database (HIVDB) subtyping program, REGA tool, Context-Based Modeling for Expeditious Typing (COMET) tool, and Recombinant Identification Program (RIP) tool. The final subtype assignment was based on molecular phylogenetic analysis. RESULTS: Unexpectedly, non-B subtypes are dominating, with the most common circulating one is CRF02_AG (57.1%) followed by subtype B (14.3%), subtype BG recombinant (9.5%), CRF35_ AD (9.5%), subtype A1 and CRF06_cpx (4.8% each). CONCLUSION: To the best of our knowledge, this is the first study to tackle HIV-1 subtyping among the group of HIV-1 patients in Egypt. CRF02_AG is the most prevalent subtype in Egypt.

Molecular Epidemiological Analysis of the Origin and Transmission Dynamics of the HIV-1 CRF01_AE Sub-Epidemic in Bulgaria.

HIV-1 subtype CRF01_AE is the second most predominant strain in Bulgaria, yet little is known about the molecular epidemiology of its origin and transmissibility. We used a phylodynamics approach to better understand this sub-epidemic by analyzing 270 HIV-1 polymerase (pol) sequences collected from persons diagnosed with HIV/AIDS between 1995 and 2019. Using network analyses at a 1.5% genetic distance threshold (d), we found a large 154-member outbreak cluster composed mostly of persons who inject drugs (PWID) that were predominantly men. At d = 0.5%, which was used to identify more recent transmission, the large cluster dissociated into three clusters of 18, 12, and 7 members, respectively, five dyads, and 107 singletons. Phylogenetic analysis of the Bulgarian sequences with publicly available global sequences showed that CRF01_AE likely originated from multiple Asian countries, with Vietnam as the likely source of the outbreak cluster between 1988 and 1990. Our findings indicate that CRF01_AE was introduced into Bulgaria multiple times since 1988, and infections then rapidly spread among PWID locally with bridging to other risk groups and countries. CRF01_AE continues to spread in Bulgaria as evidenced by the more recent large clusters identified at d = 0.5%, highlighting the importance of public health prevention efforts in the PWID communities.

Non-B variants of HIV-1 in San Francisco, California.

The HIV-1 epidemic in the US has historically been dominated by subtype B. HIV subtype diversity has not been extensively examined in most US cities to determine whether non-B variants have become established, as has been observed in many other global regions. We describe the diversity of non-B variants and present evidence of local transmission of non-B HIV in San Francisco. Viral sequences collected from patients between 2000 and 2016 were matched to the San Francisco HIV/AIDS case registry. HIV subtype was determined using COMET. Phylogenies were reconstructed using the pol region of subtypes A, C, D, G, CRF01_AE, CRF02_AG, and CRF07_BC, with reference sequences from the LANL HIV database. Associations of non-B subtypes and circulating recombinant forms (CRFs) with patient characteristics were assessed using multivariable logistic regression. Out of 11,381 sequences, 10,669 were from 7235 registry cases, of which 141 (2%) had non-B subtypes and CRFs and 72 (1%) had unique recombinant forms. CRF01_AE (0.8%) and subtype C (0.5%) were the most prevalent non-B forms. The frequency of non-B subtypes and CRFs increased in San Francisco during years 2000-2016. Out of 146 transmission events involving non-B study sequences, 18% indicated local transmission within the study population and 74% appeared to be inward migration of the virus. Compared to 7016 cases with only subtype B, 141 cases with non-B sequences were more likely to be of non-US country of birth (aOR = 11.02; p < 0.001), of Asian/Pacific-Islander race/ethnicity (aOR = 3.17; p < 0.001), and diagnosed after 2009 (aOR = 4.81; p < 0.001). Results suggest that most non-B infections were likely acquired outside the US and that local transmission of non-B forms has occurred but so far has not produced extensive transmission networks. Thus, non-B variants were not widely established in San Francisco, an observation that differs from cities worldwide with more diverse epidemics.

Diverse Large HIV-1 Non-subtype B Clusters Are Spreading Among Men Who Have Sex With Men in Spain.

In Western Europe, the HIV-1 epidemic among men who have sex with men (MSM) is dominated by subtype B. However, recently, other genetic forms have been reported to circulate in this population, as evidenced by their grouping in clusters predominantly comprising European individuals. Here we describe four large HIV-1 non-subtype B clusters spreading among MSM in Spain. Samples were collected in 9 regions. A pol fragment was amplified from plasma RNA or blood-extracted DNA. Phylogenetic analyses were performed via maximum likelihood, including database sequences of the same genetic forms as the identified clusters. Times and locations of the most recent common ancestors (MRCA) of clusters were estimated with a Bayesian method. Five large non-subtype B clusters associated with MSM were identified. The largest one, of F1 subtype, was reported previously. The other four were of CRF02_AG (CRF02_1; n = 115) and subtypes A1 (A1_1; n = 66), F1 (F1_3; n = 36), and C (C_7; n = 17). Most individuals belonging to them had been diagnosed of HIV-1 infection in the last 10 years. Each cluster comprised viruses from 3 to 8 Spanish regions and also comprised or was related to viruses from other countries: CRF02_1 comprised a Japanese subcluster and viruses from 8 other countries from Western Europe, Asia, and South America; A1_1 comprised viruses from Portugal, United Kingom, and United States, and was related to the A1 strain circulating in Greece, Albania and Cyprus; F1_3 was related to viruses from Romania; and C_7 comprised viruses from Portugal and was related to a virus from Mozambique. A subcluster within CRF02_1 was associated with heterosexual transmission. Near full-length genomes of each cluster were of uniform genetic form. Times of MRCAs of CRF02_1, A1_1, F1_3, and C_7 were estimated around 1986, 1989, 2013, and 1983, respectively. MRCA locations for CRF02_1 and A1_1 were uncertain (however initial expansions in Spain in Madrid and Vigo, respectively, were estimated) and were most probable in Bilbao, Spain, for F1_3 and Portugal for C_7. These results show that the HIV-1 epidemic among MSM in Spain is becoming increasingly diverse through the expansion of diverse non-subtype B clusters, comprising or related to viruses circulating in other countries.

Diversity of HIV-1 in Aquitaine, Southwestern France, 2012-2016.

We have estimated the prevalence of the different viral subtypes between January 2012 and December 2016 in HIV-1-infected patients of the Aquitaine region (southwest part of France) who had a routine HIV-1 genotype resistance testing (GRT) centralized at the Bordeaux University Hospital. GRT was performed on viral RNA (1,784 samples) before treatment initiation or at failure, whereas proviral DNA was used as template (1,420 samples) in the event of a treatment switch in patients with viral load below 50 copies/mL. Pol and integrase sequences were obtained; subtypes, circulating recombinant forms (CRFs), and unique recombinant forms (URFs) were assigned by combining the results of SCUEAL, REGA, COMET, and HIV BLAST. Globally, subtype B was predominant with 71.7%, whereas non-B subtypes accounted for 28.3%. Within the non-B viruses, CRF02_AG was the most prominent (11.6%) followed by non-B non-URF (13.5%), A, CRF01_AE, G, CRF06_cpx, F, C, D, H, J, and finally URF (3.2%). The analysis of the two compartments separately showed that RNA exhibits higher percentages of non-B viruses than DNA. This study reveals a high degree of diversity of HIV-1 non-B subtype strains in Aquitaine, with an increasing prevalence of CRF02_AG and URF in the population investigated for viral RNA, that is, including more recently detected HIV-1-infected patients. Future studies should attempt to identify the transmission clusters while paying special attention to URF, since they seem to be increasing in the population and could potentially host CRF.

Comparative Evaluation of Subtyping Tools for Surveillance of Newly Emerging HIV-1 Strains.

HIV-1 non-B subtypes/circulating recombinant forms (CRFs) are increasing worldwide. Since subtype identification can be clinically relevant, we assessed the added value in HIV-1 subtyping using updated molecular phylogeny (Mphy) and the performance of routinely used automated tools. Updated Mphy (2015 updated reference sequences), used as a gold standard, was performed to subtype 13,116 HIV-1 protease/reverse transcriptase sequences and then compared with previous Mphy (reference sequences until 2014) and with COMET, REGA, SCUEAL, and Stanford subtyping tools. Updated Mphy classified subtype B as the most prevalent (73.4%), followed by CRF02_AG (7.9%), C (4.6%), F1 (3.4%), A1 (2.2%), G (1.6%), CRF12_BF (1.2%), and other subtypes (5.7%). A 2.3% proportion of sequences were reassigned as different subtypes or CRFs because of misclassification by previous Mphy. Overall, the tool most concordant with updated Mphy was Stanford-v8.1 (95.4%), followed by COMET (93.8%), REGA-v3 (92.5%), Stanford-old (91.1%), and SCUEAL (85.9%). All the tools had a high sensitivity (≥98.0%) and specificity (≥95.7%) for subtype B. Regarding non-B subtypes, Stanford-v8.1 was the best tool for C, D, and F subtypes and for CRFs 01, 02, 06, 11, and 36 (sensitivity, ≥92.6%; specificity, ≥99.1%). A1 and G subtypes were better classified by COMET (92.3%) and REGA-v3 (98.6%), respectively. Our findings confirm Mphy as the gold standard for accurate HIV-1 subtyping, although Stanford-v8.1, occasionally combined with COMET or REGA-v3, represents an effective subtyping approach in clinical settings. Periodic updating of HIV-1 reference sequences is fundamental to improving subtype characterization in the context of an effective epidemiological surveillance of non-B strains.

Evaluation of the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test as an alternative to Western blot for confirmation of HIV infection.

BACKGROUND: In the United States, a new HIV diagnostic algorithm has been proposed that uses an HIV-1/HIV-2 antibody differentiation immunoassay instead of Western blot or immunofluoresence for confirmatory testing. OBJECTIVES: To evaluate the Multispot HIV-1/HIV-2 Rapid Test (Multispot) as an alternative to Western blot analysis for confirmation of HIV infection. STUDY DESIGN: A series of 205 serum and plasma specimens positive for HIV-1 or HIV-2 were used to compare the performance of Multispot to a standard HIV-1 Western blot. Positive samples included 63 specimens from patients>18 months of age, 33 proficiency survey specimens, and 109 specimens from nine commercial seroconversion and performance panels. In addition, 63 specimens from 51 HIV-exposed, uninfected children≤18 months of age in various stages of seroreversion and 192 HIV-negative samples were tested. Specimens were initially screened using a 4th generation HIV Ag/Ab Combo assay. RESULTS: Multispot readily discriminated between individuals with HIV-1 or HIV-2 infection and those who were uninfected. Of the 205 samples repeatedly reactive by the 4th generation screening assay, infection status was correctly confirmed by Multispot in 83.9% (172/205) compared to 68.8% (141/205) for Western blot. Multispot detected HIV-1 earlier in 27.6% of low-titer antibody specimens called indeterminate by Western blot, and effectively reduced the number of indeterminate results in seroreverting HIV-1 exposed, uninfected infants and for HIV-2 infections misinterpreted as indeterminate or positive by HIV-1 Western blot. CONCLUSIONS: Multispot offers speed and simplicity over Western blot and has an excellent performance for differentiation and confirmation of antibodies to HIV-1 and HIV-2.

Automated subtyping of HIV-1 genetic sequences for clinical and surveillance purposes: performance evaluation of the new REGA version 3 and seven other tools.

BACKGROUND: To investigate differences in pathogenesis, diagnosis and resistance pathways between HIV-1 subtypes, an accurate subtyping tool for large datasets is needed. We aimed to evaluate the performance of automated subtyping tools to classify the different subtypes and circulating recombinant forms using pol, the most sequenced region in clinical practice. We also present the upgraded version 3 of the Rega HIV subtyping tool (REGAv3). METHODOLOGY: HIV-1 pol sequences (PR+RT) for 4674 patients retrieved from the Portuguese HIV Drug Resistance Database, and 1872 pol sequences trimmed from full-length genomes retrieved from the Los Alamos database were classified with statistical-based tools such as COMET, jpHMM and STAR; similarity-based tools such as NCBI and Stanford; and phylogenetic-based tools such as REGA version 2 (REGAv2), REGAv3, and SCUEAL. The performance of these tools, for pol, and for PR and RT separately, was compared in terms of reproducibility, sensitivity and specificity with respect to the gold standard which was manual phylogenetic analysis of the pol region. RESULTS: The sensitivity and specificity for subtypes B and C was more than 96% for seven tools, but was variable for other subtypes such as A, D, F and G. With regard to the most common circulating recombinant forms (CRFs), the sensitivity and specificity for CRF01_AE was ~99% with statistical-based tools, with phylogenetic-based tools and with Stanford, one of the similarity based tools. CRF02_AG was correctly identified for more than 96% by COMET, REGAv3, Stanford and STAR. All the tools reached a specificity of more than 97% for most of the subtypes and the two main CRFs (CRF01_AE and CRF02_AG). Other CRFs were identified only by COMET, REGAv2, REGAv3, and SCUEAL and with variable sensitivity. When analyzing sequences for PR and RT separately, the performance for PR was generally lower and variable between the tools. Similarity and statistical-based tools were 100% reproducible, but this was lower for phylogenetic-based tools such as REGA (~99%) and SCUEAL (~96%). CONCLUSIONS: REGAv3 had an improved performance for subtype B and CRF02_AG compared to REGAv2 and is now able to also identify all epidemiologically relevant CRFs. In general the best performing tools, in alphabetical order, were COMET, jpHMM, REGAv3, and SCUEAL when analyzing pure subtypes in the pol region, and COMET and REGAv3 when analyzing most of the CRFs. Based on this study, we recommend to confirm subtyping with 2 well performing tools, and be cautious with the interpretation of short sequences.