Evidence for Onward Transmission of HIV-1 Non-B Subtype Strains in the United Kingdom

An increasing proportion of new HIV diagnoses in the United Kingdom and other European countries are attributable to non-B subtype infections, mainly among black Africans with infections heterosexually acquired in sub-Saharan Africa. We examined whether there was evidence for onward transmission of non-B subtypes within an ethnically diverse HIV-1-infected cohort in South London. Three hundred eighty-four HIV-1-infected patients attending Kings College Hospital were subtyped using an in-house enzyme-linked immunoassay and env sequencing. Epidemiologic data were obtained from medical chart review and the patients' physician and were used to establish the most likely source and country of infection. Overall, 344 patients (154 black African, 148 white UK-born, and 42 black Caribbean) had an identifiable subtype. The prevalence of non-B subtypes among the black African, white, and black Caribbean patients was 96.8%, 14.2%, and 31%, respectively. Most non-B subtype infections were identified in black Africans (149 of 183 cases) and were mainly acquired in sub-Saharan Africa, but 22.9% (42 of 183 cases) of all non-B infections were probably acquired in the United Kingdom. Among the 21 white UK-born patients infected with a non-B subtype, 15 probably acquired the infection in the United Kingdom and only 6 of these patients reported a source sexual partner from an HIV endemic area. All 13 black Caribbean patients with a non-B infection most likely acquired their infection in the United Kingdom, most of whom (8 of 13 patients) were probably infected by a partner from an HIV endemic area. Potential acquisition of HIV infection in the United Kingdom was lowest among black African patients with a non-B infection, and most of these infections were probably acquired from a partner originating from an HIV endemic area. This study provides the first evidence for onward transmission of non-B subtypes in the United Kingdom, particularly among the black Caribbean population.

The recent epidemiology of HIV-1 infection in the United Kingdom and most Western European countries with large immigrant communities has been characterized by increasing genetic diversity and a marked increase in non-B subtype genetic strains among newly diagnosed cases.^1-9 For example, in the United Kingdom, the number of new diagnoses attributable to heterosexually acquired infection has risen almost 4-fold since 1996,¹⁰ and three quarters of these infections in 2003 were among black Africans from sub-Saharan Africa, who now constitute the second largest group affected by HIV/AIDS in the United Kingdom. The number of new HIV diagnoses in the United Kingdom among persons from the Caribbean as well as among those born in the United Kingdom to parents of Caribbean origin has also increased more than 6-fold since 1995.¹⁰

It has been assumed that most non-B subtype infections in the United Kingdom and Western Europe are linked to migration from or, to a lesser extent, travel to areas of high HIV-1 prevalence, mainly sub-Saharan Africa and Southeast Asia, where non-B genetic forms predominate. To date, there has been no evidence for onward propagation of non-B subtypes in the United Kingdom and only limited data to suggest UK acquisition of infection among black Africans living in the United Kingdom. Information on the origin of these infections is limited, however, because supporting epidemiologic data are frequently lacking. Based on reported HIV diagnoses between 1995 and 2003, 9% of all heterosexually infected individuals probably acquired their infection in the United Kingdom,¹⁰ and these diagnoses have risen from 158 in 1999 to 341 in 2003. In addition, 4% of 1567 black African HIV-infected patients diagnosed in 2002 reported that they may have acquired their infection in the United Kingdom (K. Sinka, personal communication, Communicable Disease Surveillance Center, Health Protection Agency). To date, no study has been undertaken to establish whether there is onward propagation of non-B subtypes within the United Kingdom or acquisition of subtype B infection among black Africans living in the United Kingdom.

An insight into the possible source or geographic origin of infection may be gained from knowledge of the distribution of subtypes in different HIV-1-infected populations. Given the increasingly diverse and dynamic nature of the HIV epidemic, however, epidemiologic and molecular data may be required before making inferences on the probable source of an individual's HIV strain. For example, in Western Europe, where the HIV-1 epidemic has historically been dominated by subtype B viral strains, the presence of a non-B subtype infection would generally suggest that the person had acquired the infection overseas or from an individual who had himself or herself acquired the infection in an HIV endemic country. Given the increasing diversity of HIV strains cocirculating in Europe, however, the possibility of acquiring a non-B strain in the United Kingdom from a UK-born source cannot be excluded.¹¹ Similarly, the predominance of A, C, and D subtypes as well as circulating recombinant forms (CRFs) and mosaic viruses in Africa and the relative scarcity of subtype B viral strains¹ would suggest that the presence of B subtype infections among black Africans in the United Kingdom may arise because of acquisition from a non-African UK source. Finally, although the subtype distribution in the Caribbean basin is dynamic and complex, non-B subtype infections still account for less than 20% of infections in this region.¹ As such, the presence of non-B infection in black Caribbean patients, together with supporting epidemiologic data, may suggest other sources of transmission. The main objective of this study was to characterize the genetic diversity and determine the probable source of non-B subtype infection in a large, ethnically diverse, HIV-1-infected cohort in South London.

METHODS

Study Population

The King's College Hospital HIV clinic is based in the inner London boroughs of Lambeth, Southwark, and Lewisham, which, in addition to a large migrant population from sub-Saharan Africa and a significant black Caribbean community, have the highest rate of new HIV infections in the United Kingdom.¹² We undertook a cross-sectional study on the distribution of HIV-1 subtypes in 384 HIV-1-infected patients. This was based on an approximately 50% random sample of all black African, white UK-born, and black Caribbean patients who had attended the HIV clinic on at least 1 occasion over a 1-year period between May 1999 and May 2000.¹³

Data Collection

The local HIV clinic database was used to obtain demographic (ethnic origin, country of birth, gender, HIV transmission risk group, and age at HIV diagnosis), clinical, and laboratory data (clinical stage at presentation, CD4 cell count, and viral load within 3 months of HIV diagnosis). The most likely source and country of HIV infection was determined by 2 researchers (IA and PJE) for each patient based on a review of key epidemiologic data obtained from the medical records or, when this information was not available or poorly documented, from the patient's physician. These variables included country of birth; duration of residence in the United Kingdom; and the ethnicity, country of birth, and HIV status of any sexual partners preceding HIV diagnosis, if known. A detailed history of HIV risk behaviors, sexual partners, and likely source of infection of the patient is obtained routinely at the initial post-HIV test counseling visit by a trained health advisor for all patients. Age, stage of disease, and CD4 cell count at HIV diagnosis, together with dates of previous negative HIV test results, were also used as proxy data for estimating likely duration of infection. For example, when the diagnosis was made in the country of origin before entering the United Kingdom, this was considered the country of acquisition. In contrast, if an individual had a previous documented negative test result in the United Kingdom with no subsequent reported exposure abroad, this was considered to represent probable UK acquisition.

Patients were categorized into 3 ethnic groups-white UK-born (white patients born in the United Kingdom and Ireland only), black African, and black Caribbean-according to their self-reported ethnicity on the clinic registration form and country of birth. Those who described themselves as black other on the registration form were categorized as black African if at least 1 parent was born in sub-Saharan Africa and as black Caribbean if at least 1 parent was born in the Caribbean.

Laboratory Methods

HIV-1 serotyping was performed on the first available plasma sample after HIV diagnosis from all 384 patients using an in-house enzyme-linked immunoassay (EIA) directed against peptide antigens representative of the V3 region of the outer envelope glycoprotein gp120¹⁴ and was used to discriminate between B and non-B subtypes.¹⁵Env sequencing was performed to assign a subtype to samples identified as non-B using EIA (n = 90: black African [n = 67], white [n = 12], and black Caribbean [n = 11]) and in samples with mixed or nonreactivity (n = 57) on serology. In addition, env sequencing was performed to validate subtype B infections in 21 of 26 black Africans and 16 of 23 black Caribbeans with an EIA-determined subtype B infection. One white patient with subtype B on EIA was also subtyped.

Env sequencing was performed at the Dulwich Health Protection Agency (n = 155) or the Sexually Transmitted and Blood-Borne Virus Laboratory, Health Protection Agency (HPA), Colindale, London (n = 30). Samples processed at Dulwich HPA were extracted using QiAamp Viral RNA Mini Kits (Qiagen Ltd, Crawley, UK) according to the manufacturer's instructions. Amplification and cycle sequencing reactions were carried out as previously described.¹⁶ Sequencing reactions (1.5 μL) were run on a Visible Genetics sequencing system under standard conditions using version 3.1.6 software. Samples handled at the HPA, Colindale, were extracted using the method of Boom et al,¹⁷ and amplification was performed as previously described.^11,18-20 Sequencing was performed using a CEQ 2000 XL DNA Analysis System capillary sequencer (Beckman Coulter, High Wycombe, UK) according to the manufacturer's instructions.

Samples from which sufficient sequence data could be obtained were assigned a subtype by phylogenetic analysis of env gene alignments. HIV-1 reference sequences were obtained from the Los Alamos HIV sequence database (http://www.hiv.lanl.gov/). Alignments of the study sequences (where at least 240 base pairs [bp] of unambiguous sequence were available), representative sequences of group M subtypes (A-K), the most common CRFs, and groups O and N were generated using the latest version of CLUSTAL W (http://www.ebi.ac.uk/clustalw/) within Bioedit v4.8.5. The parameters of the optimal model of evolution were estimated using Modeltest v3.0 within the phylogenetic analysis package Phylogenetic Analysis Using Parsimony (Paup*), and neighbor-joining trees (bootstrapped × 1000) were generated. The tree topology was used to assign subtypes based on a high level of bootstrap support (>70) for each subtype or CRF structure. When a phylogenetically determined subtype was available, this was used to assign a definitive subtype. Two further subtyping methods were applied as a validation of the phylogenetic analysis results and to enable a subtype to be assigned to samples for which phylogenetic analysis was not possible: the National Center for Biotechnology Information (NCBI) Retrovirus Genotyping Tool (http://www.ncbi.nlm.nih.gov/retroviruses/subtype/subtype.html), with a window size of 100 bp and an increment step of 50 bp, and the Basic Local Alignment Search Tool (BLAST 2.0) search tool at Los Alamos database (http://hiv-web.lanl.gov/content/hiv-db/basic_blast.html). In summary, 164 samples were assigned a subtype by env sequencing; of these, phylogenetic analysis was used to assign subtype in 115 patient samples and the Los Alamos method was used in 49 patient samples.

Algorithm for Assignment of HIV-1 Subtype

We developed a final algorithm to incorporate serology and genotypic subtype.¹³ When an HIV-1 subtype was available by genotyping (n = 164), this was used to assign a subtype for the analysis. To provide a consistent subtyping strategy, only genotypic subtypes assigned by genotyping the env V3 loop region are presented in this study. An EIA-defined subtype B infection was assigned in white (n = 109) and black Caribbean (n = 9) patients for whom no genotype data were available. Because serology is unreliable for discriminating between B and non-B subtype in low subtype B prevalence populations such as black Africans,¹⁵ only subtype B infections in black Africans that had been confirmed by sequencing were included in the analysis, and 8 serologically determined subtype B infections for which no sequence was obtained were excluded. Sixty-two patients with non-B subtype infection on EIA for whom a sequence was not obtained were classified as non-B subtype. Patients for whom a subtype could not be assigned by either method were excluded (n = 32).

Statistical Methods

We compared the demographic, clinical, and laboratory characteristics at HIV diagnosis among black African, white UK-born, and black Caribbean patients infected with B versus non-B subtypes using χ² tests for categoric variables and Kruskal-Wallis or Wilcoxon rank-sum tests for continuous variables. Data were analyzed using Stata 7.5 (Stata Corporation, College Station, TX).

RESULTS

Subtype Distribution Among Ethnic Groups

Using our subtyping algorithm, it was possible to assign a subtype in 89.6% (344 of 384) of patients. Non-B subtypes accounted for 96.8% (149 of 154) of infections among black Africans, 31% (13 of 42) of infections among black Caribbeans, and 14.2% (21 of 148) of infections among whites. Figure 1 shows the distribution of non-B subtypes as determined by the algorithm according to ethnic group.

Figure 1

Black African Patients

Non-B subtypes accounted for 96.8% (149 of 154) infections among black Africans, comprising 30 each with subtypes A and C; 21 with subtype D; 13 with CRF02_AG; a single infection with each of subtypes F2, G, and CRF01_AE; 2 with mosaic forms (A/G and C/D); and 50 with unspecified non-B subtypes based on EIA (see Fig. 1A). Compared with those with non-B infection, the 5 patients with subtype B were more likely to have been born in the United Kingdom (40% vs. 6.7%; P = 0.01). There was also a trend for these patients to present with more advanced disease (AIDS diagnosis at HIV diagnosis: 40% vs. 24.2%) and a low CD4 cell count (81 × 10⁶/L vs. 201 × 10⁶/L) (see Table 1), but these differences were not statistically significant.

Table 1

Of the 5 black African patients with subtype B infection confirmed by genotyping, 3 were heterosexual women born in sub-Saharan Africa (Congo, Ivory Coast, and Nigeria) and 2 were UK-born (1 homosexual man and 1 heterosexual man). Three of the 5 patients are likely to have acquired their B subtype infection in the United Kingdom. One was an African-born woman resident in the United Kingdom for 10 years who presented with a probable HIV seroconversion illness during a relationship with an African partner in the United Kingdom. The other 2 patients had spent their childhood in Nigeria but only reported sexual partners since their arrival in the United Kingdom, although the ethnicity of the source partner was unknown.

Of 149 black Africans with non-B subtypes, 3 who were born in the United Kingdom had acquired their infection through vertical transmission from their mothers and 14 (9.4%) gave a history suggestive of HIV acquisition in the United Kingdom. Nine of these were heterosexual women (3 UK-born and 6 born in sub-Saharan Africa), and 5 were heterosexual men (4 born in sub-Saharan Africa and 1 born in the United Kingdom). In 7 patients, this was based on the observation that they had all had a previous negative HIV test result since they had been living in the United Kingdom; 3 subsequently had sexual partners from sub-Saharan Africa in the United Kingdom, 1 had a sexual partner from the Caribbean, and the ethnicity of their sexual partners in the United Kingdom was unknown for the remaining 3. A further 7 patients were born in the United Kingdom (n = 4) or were long-term residents (n = 3) and had sexual partners in the United Kingdom (5 from sub-Saharan Africa, 1 from the Caribbean, and 1 UK-born partner of unknown ethnicity). Therefore, we determined that 65.7% (98 of 149) of patients had acquired their infection before their arrival in the United Kingdom; but in a further 22.8% (34 of 149 patients), it was not possible to be definitive as to whether their non-B HIV infection was acquired in the United Kingdom or Africa.

White Patients

Twenty-one (14.2%) white UK-born patients were infected with non-B subtypes (4 with subtype D, 2 each with subtype A and C, 1 each with CRF01_AE and CRF02_AG, and 11 with non-B subtype on EIA; see Fig. 1B). Patients infected with a non-B subtype were less likely to be homosexual than those with B subtype (61.9% vs. 81.8%; P = 0.01; see Table 1).

Of the 21 patients with non-B subtype infection, 3 were heterosexual women, 3 were heterosexual men, 2 were male injecting drug users (IDUs), and the remaining 13 were homosexual men. Overall, 15 of these 21 patients probably acquired their non-B infection from a sexual partner in the United Kingdom, and in 6, the source sexual partner was from an HIV endemic area. We determined that all 3 heterosexual women probably acquired their non-B subtype HIV infection from sexual partners in the United Kingdom (these were an IDU from Spain, a Jamaican man, and a UK-born black African man). Of the 3 heterosexual men, 2 acquired their infection from sexual partners in Africa (Kenya and Botswana) and 1 from a known HIV-positive black African partner in the United Kingdom. Both male IDUs were infected in the United Kingdom, probably through needle sharing, although 1 also had a known HIV-infected sexual partner. Of the 13 homosexual men, more than half probably acquired their infection in the United Kingdom (n = 9): 7 from UK-born partners, 1 from a Southern European partner, and 1 from a known HIV-positive white South African partner. Of note, however, 5 of these 9 non-B infections were based on serology alone, and there were insufficient remaining samples to verify by sequencing and phylogenetic analyses. In only 2 cases was infection probably acquired during travel overseas from partners in Southeast Asia (Thailand and Malaysia).

Black Caribbean Patients

Nearly a third (13 of 42) of black Caribbean patients were infected with non-B subtypes, comprising 5 with subtype C; 3 with subtype A; 1 each with subtype D, F, CRF01_AE, and CRF02_AG; and 1 with non-B subtype on EIA (see Fig. 1C). Subtype B infection was more likely to be homosexually acquired (non-B vs. B in heterosexuals: 84.6% vs. 37.9%, B vs. non-B in homosexuals: 58.6% vs. 15.4%; P = 0.01). Of note, black Caribbeans had an intermediate CD4 cell count at presentation between whites and black Africans (283 vs. 358 vs. 192 × 10⁶/L; Kruskall-Wallis test, P < 0.001; see Table 1).

Of the 13 black Caribbeans with non-B subtypes, 8 were born in the United Kingdom (5 heterosexual women and 3 heterosexual men) and 5 in Jamaica, of whom 2 were heterosexual women, 2 were heterosexual men, and 1 was a homosexual man. Of the 5 born in Jamaica, 3 had been resident in the United Kingdom for more than 10 years at the time of their HIV diagnosis and believed they had acquired HIV in the United Kingdom; 1 woman had been resident in the United Kingdom since the age of 14 years, and another who had had a negative HIV test result a year earlier reported a recent partner of unknown ethnicity in the United Kingdom. Therefore, all black Caribbeans infected with non-B subtype (n = 13) are likely to have acquired their infection in the United Kingdom: of these, 8 had partners from sub-Saharan Africa now resident in the United Kingdom (Nigeria, Zambia, Ghana, Zimbabwe, Angola, and Uganda), 2 had partners from Jamaica, and there was no information available on their sexual partners for 4. We also assessed the likely source and country of acquisition for the black Caribbean patients infected with B subtype (n = 29). Of these, 58.6% (17 of 29 patients) were homosexual men (9 UK-born, 6 born in the Caribbean, and 2 born in France), 8 were heterosexual women (6 UK-born and 2 born in the Caribbean), 3 were heterosexual men (1 UK-born and 2 born in the Caribbean), and 1 was an IDU (UK-born). Overall, 13 patients were probably infected in the United Kingdom and Europe, 5 were probably infected in the Caribbean, and it was not possible to determine whether the remaining 11 were infected in the Caribbean or the United Kingdom.

Correlation Between Serotyping and Genotyping

The results obtained from serotyping and genotyping in our black African and black Caribbean populations were compared. Among black African patients with an EIA-defined subtype B infection, 80.7% (21 of 26 patients) were sequenced (in 5 patients, there was an insufficient sample). Only 1 infection (4.7%) was confirmed as subtype B. Of the remainder, there were 10 subtype D infections, 4 were subtype A, 2 were CRF02_AG, and 1 was subtype C. Sequence data could not be obtained from the remaining 3 samples. Of the samples from black Africans with non-B subtypes that were sequenced, 87.9% (58 of 66 samples) were confirmed as non-B subtypes, 3 were found to be subtype B, and 5 could not be sequenced. Similarly, sequencing of black Caribbean patients whose viruses were identified as non-B subtypes by EIA confirmed 63.6% (7 of 11 infections) to be non-B subtypes and 3 to be B subtype, and the sequence could not be obtained on one sample. Among black Caribbean patients identified with a subtype B infection by EIA, sequencing confirmed 75% (12 of 16 infections) to be subtype B, 1 to be subtype A, and 1 to be subtype D. Sequence data could not be obtained for 2 patients.

DISCUSSION

Our findings represent the first documented evidence using molecular and epidemiologic data for onward transmission of non-B HIV-1 strains within the United Kingdom. Although most (81.4%) of the non-B subtype infections in our study cohort were among black Africans (149 of 183 infections) and are likely to have been acquired in sub-Saharan Africa, we identified that approximately 23% (42 of 183 infections) of all non-B infections were probably acquired in the United Kingdom. Concerns have been raised for several years about the potential for onward sexual propagation of non-B virus strains in Western Europe; however, to date, there have been only isolated reports among IDUs in Finland²¹ and Spain⁸ as well as 5 cases in New York.²² Two studies conducted in the late 1990s, the first from Amsterdam²³ and another from Belgium,²⁴ found no evidence for spread of non-B subtypes beyond those persons with an epidemiologic link with an HIV endemic region, despite the presence of multiple HIV subtypes among individuals with heterosexually acquired infection in Amsterdam.

We found that most non-B infections in our white and black Caribbean patients but only 9.4% of non-B infections in our black Africans were acquired in the United Kingdom. In almost all the black Caribbean and African patients with non-B infection acquired in the United Kingdom, this was the result of sexual contact with persons originating from an HIV endemic area. In contrast, although nearly three quarters (15 of 21 patients) of white UK-born patients with non-B subtype infection are likely to have acquired their infection in the United Kingdom or Europe, only 6 reported probable acquisition of infection directly from a person originating from an HIV endemic area. The remainder reported their source sexual partners as being mainly UK-born white or from Europe but living in the United Kingdom or as IDUs. Only 4 individuals had sexual exposure abroad in regions of the world where non-B subtypes predominate. All 7 patients who reported probable acquisition of non-B infection from other white UK-born partners were homosexual men, although in several cases, there was some uncertainty about the definitive source of infection and the non-B subtype was not confirmed on sequencing. Overall, 11% of our 117 white homosexual male patients were infected with non-B subtypes, which is substantially higher than the 0.84% of 355 samples from homosexual or bisexual men in the United Kingdom reported previously.² In our study, however, this finding was based only on a serologic subtype in a significant proportion. This finding needs to be confirmed with sequencing in a nationally representative sample of HIV-1-infected homosexual men but suggests a propensity for increasing genetic diversity within the homosexual population in the United Kingdom.

More than half of subtype B infections among the 29 black Caribbeans were identified in homosexual or bisexual men, and most were probably infected in the United Kingdom or elsewhere in Europe. All the black Caribbean patients with non-B subtypes reported that they probably acquired their HIV infection in the United Kingdom. Most of these non-B infections were likely to have been acquired from an African sexual partner. In part, this may explain the higher prevalence of non-B subtypes observed in our black Caribbean patients (31%) compared with the prevalence reported from surveys in the Caribbean (19.3%).¹

Among black Africans infected with non-B subtypes who probably acquired their infection in the United Kingdom, most infections were likely acquired from African partners, which is consistent with data from 2 other studies (K. Sinka, personal communication, Communicable Disease Surveillance Center 24). Four percent of 1567 black Africans with newly diagnosed HIV infection in the United Kingdom reported that they might have acquired their infection in the United Kingdom (K. Sinka, personal communication, Communicable Disease Surveillance Center). Similarly, a case note review of 275 HIV-infected black Africans in South London suggested that between 5% and 10% of infections were probably acquired in the United Kingdom, and in a further 20% to 30% of infections, it was not possible to decide whether the infection was more likely to have been acquired in Africa or the United Kingdom.²⁵

The low prevalence of subtype B infection among black Africans observed in this study is consistent with the low prevalence (0.19%-4.03%) of subtype B infection reported in sub-Saharan Africa.¹ It is noteworthy that our 5 black African patients with B subtype infection presented with more advanced HIV disease than those with non-B subtypes. The significance of this observation is unclear, given the small sample size, and warrants confirmation in a larger study. Although several studies have failed to demonstrate a difference in the rate of disease progression between B and non-B subtypes,^13,26,27 such a comparison has not been undertaken previously among black Africans because of the rarity of the B subtype in sub-Saharan Africa.

Sexual mixing patterns are thought to play an important role in the potential for heterosexual spread in host countries. A study among migrant groups in Amsterdam found that disassortative sexual mixing occurred frequently, demonstrating that a bridge exists for the spread of HIV and sexually transmitted diseases between ethnic groups.²⁸ In our study, most white and black Caribbean heterosexuals infected with non-B subtypes reported probable infection by partners of black African or Caribbean ethnicity. In contrast, none of the homosexual men reported black African partners (1 had a white partner from South Africa, and 2 had partners from Southeast Asia). Although this finding may reflect incomplete reporting of all potentially HIV-infected sexual partners and the numbers are limited, our data suggest that disassortative sexual mixing with African or other non-white UK partners may be occurring, particularly among the black Caribbean and white heterosexual population.

It was possible to assign a subtype to nearly 90% of samples using the combination algorithm applying EIA and genotyping in this study. When it was not possible to assign a subtype by EIA or amplification was not possible using polymerase chain reaction (PCR), this could most likely be attributed to sample deterioration as a result of freeze-thawing, small sample volume, and/or low viral load because of antiretroviral therapy, which may all contribute to degradation in sample quality and a reduced viral load. The genetic diversity observed is not thought to be a likely cause of amplification failure, because the PCR and sequencing primers have previously been shown to detect all group M subtypes.¹¹ The EIA and genotyping approach targeted the region of env encompassing the V3 loop, thus providing good confidence in the comparability of results between the 2 techniques. In addition, although genotyping was performed at 2 different sites, all env sequences were included in a single alignment and bootstrap values of >70% were observed for each subtype cluster.

Our results show a greater level of concordance between the Los Alamos subtyping tool and phylogenetic analysis; in 13 discrepant results between all 3 methods, 9 were concordant between the Los Alamos and phylogenetic methods. Similar results were reported in a subset of these samples (n = 100), where an overall agreement of 89% between all 3 genotypic subtyping methods was found; however, 92% of results were concordant between the Los Alamos and phylogenetic methods.¹⁶ The discrepancies found using the BLAST subtyping tool could not be resolved by altering the window size or the increment step. Overall differences between the Web-based and phylogenetic gold standard subtyping methods are likely to be attributable to the different computational methods each employs. The discordant results between the 2 Web-based genotyping methods may be attributable to differences in the comparative algorithms and reference sequences used by the programs. The BLAST subtyping tool employs a set of 117 reference subtypes representing the 14 HIV-1 subtypes and genotypes and 16 CRFs,²⁹ whereas the Los Alamos tool compares the query sequence with all the sequences in the database (>100,000 sequences).

Serotyping has been shown to be of good specificity for differentiating subtype B from non-B infections in populations predominantly infected with B subtype.¹⁵ It is less reliable in areas of high HIV-1 genetic heterogeneity, however, may not differentiate between the major non-B subtypes, and cannot detect recombinant forms.³⁰ In our comparison of serotyping and genotyping in black African patients, we found that serotyping was extremely poor in discriminating between B and non-B subtypes. Indeed, it was only possible to confirm 1 sample as subtype B using env sequencing among those with an EIA-defined subtype B. In contrast, there was a good correlation between serologic and genotypically determined subtype in the black Caribbean population. Phylogenetic studies have shown that subtypes B and D are most closely related to each other.³¹ Most of the samples identified as B on serology were among Ugandans, and half of these were subsequently confirmed as subtype D, which has been shown to account for 38% of HIV-1 infections in Uganda.³² Poor specificity in differentiating between subtypes B and D by EIA is likely to be a significant factor in limiting the use of serotyping among black Africans, particularly in areas with a high proportion of subtype D infections.

There are several other caveats to our methodologic approach and interpretation of findings. We did not have the benefit of standardized prospective data collection, and the designation of likely country and source of infection was based, in part, on variables such as duration of residence in the United Kingdom, country of birth of sexual partners, and travel history before an HIV diagnosis, which were often poorly documented in the medical records. As a result, our findings may represent an underestimation of the number of infections acquired through overseas travel. There may also have been a reporting bias, with sexual partners from areas perceived as high risk, such as sub-Saharan Africa, being preferentially reported and sexual exposures during overseas travel being underreported. Sources of infection were generally assigned using reported sexual partners together with estimated duration of infection based on the CD4 cell count at HIV diagnosis. Nevertheless, in those patients with non-B infection and no clear documentation on their likely source sexual partners, we cannot exclude the possibility that they also acquired their infection in or from a person originating from an HIV endemic area.

Overall, the relatively high proportion of non-B subtypes observed most probably reflects the high proportion of black Africans (approximately 45%) in our local HIV population in South London. The proportion of non-B subtypes among black Africans in this study is similar to that observed in a previous UK unlinked anonymous HIV surveillance program.¹¹ A similarly high proportion of black Africans with a heterosexually acquired non-B infection (15%-80%) has also been reported from several Western European countries,³³ and it is likely that onward transmission of non-B subtypes is now also occurring in these countries. The high proportion of CRFs among UK-born HIV-infected heterosexuals in a recent survey in the United Kingdom is also consistent with sexual mixing between migrants and UK-born individuals.¹¹

In summary, our findings demonstrate a substantial diversity of HIV-1 subtypes among our HIV-infected population in South London, with evidence for ongoing low-level transmission of non-B subtype infection in the United Kingdom, mainly among black Caribbean and UK-born white individuals. We observed evidence of second-generation transmission of non-B subtype infection to white UK-born individuals from persons with no apparent epidemiologic link to an HIV endemic area. Non-B subtype infections acquired as a result of overseas travel represented a small proportion of infections in our patient cohort, although this may be an underestimation, because a full travel history with sexual partnerships over the decade before HIV diagnosis was usually not available. This increasing genetic diversity has implications for clinical management, particularly in the detection of resistance, and monitoring of virologic response to therapy.^34-37 This is likely to be further complicated by the potential emergence of novel genetic forms through recombination with locally circulating B subtype viruses, as has occurred in Argentina,³⁸ Cuba,³⁹ and Spain,⁸ where a substantial proportion of recent infections with non-B subtype or intersubtype recombinant viruses has been reported. With increasing migration and international travel, the diversity of HIV-1 subtypes in Western Europe and the potential for onward second-generation transmission should continue to increase. A program of continuous molecular epidemiologic surveillance is required to monitor the impact of non-B strains on the epidemic in the United Kingdom and other European countries more systematically by tracking the level of propagation of imported non-B subtype viruses or of locally generated recombinants. This is going to require the establishment of prospective data collection with subtyping in a sample of newly infected individuals of different ethnicity and transmission groups at sentinel sites with linked epidemiologic questionnaires.