JAIDS Journal of Acquired Immune Deficiency Syndromes:
Epidemiology and Social Science
Infection With HIV Type 1 Group M Non-B Subtypes in Individuals Living in New York City
Achkar, Jacqueline M. MD*; Burda, Sherri T.†; Konings, Frank A. J.‡; Urbanski, Mateusz M.†; Williams, Constance A. U.†; Seifen, Dorothee§; Kahirimbanyi, Martha N. MD‖; Vogler, Mary MD*; Parta, Mark MD*; Lupatkin, Helene C. MD*; Zolla-Pazner, Susan PhD†§; Nyambi, Phillipe N. PhD†§
From the *Department of Medicine, New York University School of Medicine, New York, NY, the †Department of Pathology, New York University School of Medicine, New York, NY, the ‡Department of Microbiology, New York University School of Medicine, New York, NY, the §Research Enhancement Award Program, Veterans Affairs Medical Center of the New York Harbor Healthcare System, New York, NY, and the ‖African Services Committee, New York, NY.
Received for publication October 15, 2003; accepted March 2, 2004.
Supported in part by National Institutes of Health grants AI 07382, AI 47053, HL 59725, and AI 36085, by a grant from the New York University Institute of Urban Health and Global Development, and by research funds from the Department of Veterans Affairs.
Presented in part at the AIDS Vaccine 2003 Conference, September 2003, New York, NY (abstract 461), and the 41st Annual Meeting of the Infectious Disease Society of America, October 2003, San Diego, CA (abstract 1148).
Reprints: Jacqueline M. Achkar, Department of Medicine, New York University School of Medicine, c/o VA Medical Center, 423 East 23rd Street, Room 18124N, New York, NY 10010 (e-mail: Jacqueline.Achkar@med.nyu.edu).
Objective: To document infection with HIV type 1 (HIV-1) group M non-B subtypes in individuals living in New York City.
Design: From October 1999 through April 2003, HIV-1–seropositive individuals were selected from 3 clinics in New York City based on having risk factors for infection with HIV-1 non-B subtypes.
Methods: HIV-1 RNA was extracted from plasma samples, and partial gag, pol, or env genes were amplified by PCR analysis. The infecting HIV-1 group M subtype was determined based on results of either heteroduplex mobility assay or sequencing and phylogenetic analysis.
Results: Ninety-seven subjects were enrolled in the study. Of the 97 subjects, 91 (94%) were selected based on having emigrated from a non-European country, while 6 (6%) were native United States citizens. Subtypes were successfully determined in 53 (55%) of the 97 plasma samples tested. The subtypes in 2 plasma samples were unclassifiable. HIV-1 infections were classified as those due to the following group M subtypes: A (n = 4; 7%), B (n = 12; 22%), C (n = 8; 15%), F (n = 2; 4%), CRF01_AE–like (n = 7; 13%), CRF02_AG–like (n = 19; 34%), an intersubtype recombinant form Ggag/Aenv (n = 1; 2%), and unclassifiable viruses (n = 2; 4%).
Conclusion: This study reveals infection with a broad variety of HIV-1 group M subtypes mostly in the immigrant population of New York City as well as how several non-B subtypes are being introduced into the United States.
Molecular epidemiological studies have identified 3 phylogenetically distinct groups of HIV type 1 (HIV-1): group M (major), group O (outlier), and group N (new or non-M, non-O). 1–3 Isolates of group M cause most infections worldwide, whereas viruses of groups O and N are linked to Central Africa and cause only few HIV-1 infections. 4,5 Nine subtypes designated A–D, F–H, J, and K are recognized within group M (http://www.hiv.lanl.gov). In addition, 15 circulating recombinant forms (CRFs), each of which has a mosaic structure of ≥2 parental subtypes, has been fully sequenced, and has been identified in 3 epidemiologically unrelated individuals, are recognized as of today (http://www.hiv.lanl.gov).
Much of our understanding of HIV-1 disease derives from studies in the developed world where HIV-1 infection is predominantly due to subtype B. However, on a global scale, non-B subtypes cause most infections. The largest proportion of HIV-1 infections in the year 2000 was due to subtype C strains (47.2%). Subtype A together with CRF02_AG viruses were estimated to be the second leading cause of the pandemic (27%), followed by subtype B strains (12.3%). 4 Subtype C viruses cause most infections in southern Africa, Ethiopia, and India, whereas subtype A viruses are predominant in areas of central and East Africa. Subtype B is the main genetic form in western and central Europe, the Americas, and Australia. 4 The same analysis also confirmed an increasing role of CRFs in the pandemic. In fact, throughout all of West Africa and in parts of central Africa the most prevalent genetic form is the recombinant virus CRF02_AG. 4
Infection with HIV-1 non-B subtypes in the Western world is commonly linked to travel to or immigration from areas with a high occurrence of non-B strains. The prevalence of infection due to non-B subtypes in some HIV clinics of metropolitan areas in western Europe is well documented and ranges from 3% to 50%, mostly depending on the proportion of immigrants attending those clinics. 6–9 Information about the prevalence of HIV-1 non-B subtypes in certain areas of the United States is sparse. 10,11 Most reports describe only small numbers of non-B subtype–infected individuals. 12–16 However, non-B strains that are being introduced into the United States population can be expected to spread. Knowledge about these strains is important because subtypes might differ in pathogenicity 17,18 and the virus variability is recognized as a potential problem for the diagnosis and treatment of HIV-1 infection as well as HIV-1 vaccine design. 11,15,19–24
New York City is one of the main places of residence for immigrants and travelers from regions with a high prevalence of HIV-1 non-B subtypes. Because immigration and travel from these regions is one of the main risk factors for non-B subtype infection, this study was designed to document the variety of non-B subtypes infecting individuals who live in New York City and have such risk factors.
STUDY SUBJECTS, MATERIALS, AND METHODS
From October 1999 through April 2003, HIV-1–seropositive individuals were selected from 3 clinics in New York City (Bellevue Hospital, St. Vincent's Hospital, and African Services Committee). Subjects were enrolled based on risk factors for non-B subtype infection. These risk factors included emigration from a non-European country, having a history of HIV risk practices in regions where non-B subtypes are prevalent or with subjects originating from those regions, or having an unexplained low or undetectable HIV-1 viral load (VL) in spite of a low CD4 cell count. The hospital-based HIV clinics from which the subjects were selected are in Manhattan and serve a heterogeneous population of both immigrants and native United States citizens. The African Services Committee is located in Harlem in uptown Manhattan and provides health, social, and legal services to HIV-infected African immigrants living in New York City. HIV-1 seropositivity was confirmed in all study subjects via ELISA and Western blot tests. After explaining the purpose of the study to all subjects, written consent was obtained before blood samples were obtained and clinical and personal data were recorded.
VL was determined using the Bayer HIV-1 RNA 3.0 Assay (bDNA; Bayer Corporation, Tarrytown, NY) according to the manufacturer's protocol. The lowest value reported for different HIV-1 subtypes by the assay is 75 copies/mL.
HIV-1 Subtype Analysis
First, plasma samples were tested either by the hetero-duplex mobility assay (HMA) or by sequencing followed by phylogenetic analysis for the infecting HIV-1 group M subtype. Viruses exhibiting an indeterminate HMA pattern were subtyped by sequencing and phylogenetic analysis.
RNA Extraction and RT-PCR Analysis
RNA extraction was performed on 100 μL of plasma as previously described by Boom et al. 25 One-tube RT-PCR analysis (Access RT-PCR System; Promega, Madison, MI) was performed according to the manufacturer's recommendations for amplification of gag, pol, or env.
HIV-1 gag, pol, and env RT-PCR Analysis
HIV-1 gag RT-PCR analysis was performed as described by Heyndrickx et al. 26 The amplified 460-nt gag gene fragment covered the p24–p7 region. Amplification of the ~300-nt protease region was performed as described by Zhong et al. 27 An ~1260-nt fragment of the pol gene, covering the PR and part of the RT region, was amplified. The forward primer NYUPOL6 in combination with the 2 reverse primers NYU-POL11 (5′-TGGCTCTTGGTAAATTTGATAT-3′; location 3563–3584) and NYUPOL12 (5′-GTTAATTGTTTTACAT-CATTAGTGT-3′; location 3631–3655) were designed and used in the first-round PCR assay, while primers NYUPOL7 27 and NYU POL4 (5′-TCAGTGTTACTATATCTGTTAGTG-GTTTG-3′, location 3407–3435) were used in the nested PCR assay as described by Zhong et al. 27 The primer locations were based on the HXB2 numbering engine (http://www.hiv.lanl.gov). HIV-1 env RT-PCR analysis was performed as described by Delwart et al. 28 The amplified 666-nt env gene fragment covered the V3 through V5 coding domains.
HMAs for env and gag genes were performed as described by Delwart et al 28,29 and Heyndrickx et al, 26 respectively, using kits obtained through the NIH Reagent Repository (Bethesda, MD). A result of subtype A in env was distinguished further by HMA of gag. To better differentiate among subtypes A, CRF01_AE, and CRF02_AG in gag, the amount of urea used in the gel was increased from 20% to 30%, and references for subtypes A, CRF02_AG, and CRF01_AE supplied in the kit were used. 26
Samples with indeterminate HMA results for gag or env and samples with PCR products of the pol region were sequenced and phylogenetically analyzed. RNA extraction and PCR amplification were performed as described above. PCR products were purified from agarose gels by using the QIAQuick Spin kit (QIAGEN, Inc., Valencia, CA) according to the manufacturer's recommendations. DNA sequencing was performed using an automated DNA sequencer (373XL; Applied Biosystems, Foster City, CA) at the sequencing service center of New York University School of Medicine. Subsequently, the sequences were aligned with previously reported HIV-1 strains of various subtypes from the HIV databases at http://hiv-web.lanl.gov. Multiple alignments were performed automatically by CLUSTAL X 30 with minor manual adjustments and gap stripping where necessary. For subtype determination, phylogenetic analysis of the aligned sequences was performed by the neighbor-joining method of TREECON (TREECON for Windows, version 1.3b). 31 The distance calculation was performed by the Kimura 2-parameter method. The statistical robustness of the neighbor-joining tree and reliability of the branching patterns were confirmed by bootstrapping (1000 replicates).
Sequences that could not be classified by phylogenetic analysis were further analyzed by bootstrap plotting (window, 500 bp; step, 40 bp; 1000 bootstraps) using SimPlot 3.2 beta software. 32
The sequences reported here have been deposited in GenBank under accession numbers AY524144–AY524180.
A total of 97 HIV-1–seropositive individuals were enrolled in the study. Ninety-one subjects were identified on the basis of emigration from a non-European country. Of these 91 subjects, 53 had emigrated from sub-Saharan Africa (35 from West and central Africa; 18 from East and southern Africa), 22 from Asia, and 16 from Latin America and the Caribbean Islands. Six study subjects were native United States citizens; 4 were enrolled based on having had partners from regions where non-B subtype viruses are prevalent, and 2 were enrolled because they had unexplained discordant CD4 cell count/VL results. The mean age ± SD of the study participants was 39 ± 9 years, with 62 males (64%) and 35 females (36%). Heterosexual contact was the main self-reported mode of infection in 81 individuals (84%). Fifty-nine subjects (61%) were receiving highly active antiretroviral treatment, and 43 (44%) had an undetectable VL (<75 copies/mL) at the time of enrollment. To calculate the mean and median VLs, all values of <75 copies/mL were taken as 75 copies/mL. The mean VL ± SD was 21,863 ± 73,430 copies/mL (median, 113 copies/mL). The mean CD4 cell count ± SD was 321 ± 213/mm3 (median, 307/mm3). For the immigrants providing information on their length of stay in the United States, the mean length of stay in the United States ± SD was 5.9 ± 6.0 years.
HIV-1 Subtype Analysis
Viral RNA extraction and PCR analysis of partial gag, pol or env were successful in 55 of the 97 plasma samples tested. No PCR amplification product was obtained for 42 plasma samples. HMA was used to determine 18 subtypes (3 in env; 15 in gag), and sequencing and phylogenetic analysis provided subtype identification for 37 (2 in gag; 34 in pol; 1 in env).
Analysis of the 55 samples with genomic viral RNA products revealed 4 group M subtypes, 2 CRFs, 1 intersubtype recombinant, and 2 unclassifiable subtypes. These included subtypes A (n = 4; 7%), B (n = 12; 22%), C (n = 8; 15%), and F (n = 2; 4%), CRF01_AE–like (n = 7; 13%) and CRF02_AG–like (n = 19; 34%) viruses, an intersubtype recombinant form Ggag/Aenv (n = 1; 2%), and unclassifiable viruses (n = 2; 4%). These subtype designations refer only to the specific gene fragment analyzed. Table 1 lists the genetic subtypes in the respective genomic regions analyzed for all 55 samples, and Figure 1A shows the phylogenetic tree for viruses sequenced in the protease region of pol. There is a lack of support for the cluster at several nodes in our phylogenetic tree (Fig. 1A), mostly because all protease sequences were run together to show 1 tree. To avoid the influences on the bootstrap value, phylogenetic analysis was performed separately for each sequence. We added asterisks at the nodes of the phylogenetic tree for protease sequences, indicating the relevant decreased bootstrap value when sequences were run together (eg, from range 53%–76% to 26% for CRF02_AG). Protease sequences with bootstrap values of <70% (9 of 14 CRF02_AG–like strains) were further investigated by performing an HIV blast at the HIV sequence database (http://www.hiv.lanl.gov). All of these sequences showed high similarity (>97%) with at least the first 30 CRF02_AG strains in the database. Our results show that most sequences of the protease region can be subtyped (29 [88%] of 33) as previously described, 33–35 although a few (4 [12%] of 33) were unclassifiable.
In 3 (02USNYU2913, 02USNYU2767, and 02USNYU3104) of the 4 samples that were unclassifiable, a larger fragment of pol (1260 bp) was successfully amplified. Figure 1B shows the phylogenetic analysis for these sequences after gap stripping and demonstrates how the sequences of samples 02USNYU2913 and 02USNYU2767 cluster with subtype F but are distinct from reference strains of sub-subtypes F1 and F2. Sample 02USNYU3104 remained unclassifiable. SimPlot analysis 32 of this sequence revealed that the first 500 bp clustered significantly with reference subtype J, while the remaining 700 bp did not cluster significantly with any of the other reference subtypes (data not shown), leaving sample 02USNYU3104 unclassifiable. Attempts to amplify gag, a larger fragment of pol or env in sample 02USNYU3062 with an undetectable VL of <75 copies/mL, were unsuccessful. This sample is marked as U (unclassifiable) on Figure 1A. Phylogenetic analysis of partial gag (samples 02USNYU2819 and 99USNYU2063) or env (sample 02USNYU2775) sequences showed classification into group M subtypes (Table 1; phylogenetic data not shown). The lack of detection of viral RNA in 42 plasma samples could have been due to the following reasons: first, 39 (93%) of 42 plasma samples were from individuals receiving highly active antiretroviral treatment with a bDNA VL of <75 copies/mL, and peripheral blood mononuclear cells were not available; second, in the remaining 3 samples, aberrant viral strains may have been missed due to the lack of primer specificity despite using universal primer sets for HIV-1 group M strains. 26–28 Further analysis is ongoing to determine whether these patients are infected with aberrant viruses. Such results will be reported elsewhere.
HIV-1 Subtypes According to Travel History and Geographic Origin of Study Subjects
All the following results refer to the 55 samples for which subtype identity was obtained. Results from the different subtyping approaches are included in Table 1, and characteristics of the subjects are tabulated according to region and country of origin, sex, age, mode of transmission, and length of stay in the United States.
Individuals from sub-Saharan Africa formed the largest group, with 35 of 55 subjects originating from 16 different countries. Most sub-Saharan Africans (27 of 35) had emigrated from West and central Africa. We found CRF02_AG–like viruses to be the infecting strain in 17 (63%) of these 27 subjects, with the remainder being infected with subtypes A (1), B (3), C (2), and F (2), an intersubtype recombinant form Ggag/Aenv (1), and an unclassifiable virus (1) (Table 1). These findings are in accordance with the high diversity of group M subtypes originating from this region, with CRF02_AG accounting for 70%–90% in parts of this region. 4,36,37
In the 8 subjects from southern and East African immigrants, subtype C viruses were the predominant infecting strain in 6, and subtype A viruses were found in the other 2 (Table 1). Again, our findings are in accordance with the high prevalence of subtype C viruses in southern Africa and Ethiopia and subtype A viruses in East Africa. 4,38
CRF01_AE–like viruses were the infecting strain in 7 of 8 Asian immigrants (of whom 6 were Chinese and 1 was Nepalese). Before arrival in the United States, the Chinese immigrants transited through Burma or Thailand for a 6- to 9-month period during which they engaged in high-risk encounters with local female commercial sex workers; they believed that this was the source of their HIV infection.
In the 8 Caribbean and Latin American immigrants, subtype B viruses were the infecting strain in 6, while 2 were infected with non-B subtype viruses (Table 1). HIV infection in the Caribbean is predominantly due to subtype B, while the most common subtypes found in Latin America are B and F or recombinants between these 2 subtypes. 4,39 Thus, these individuals could have been infected in their countries of origin or in the United States. One Haitian immigrant, infected with a CRF02_AG–like virus (sample 02USNYU2825;Table 1 and Fig. 1A) worked for several years in Ivory Coast before immigrating to the United States. One Jamaican immigrant was infected with an unclassifiable subtype (sample 02USNYU3104;Table 1 and Fig. 1B).
Of the 4 United States citizens, 2 were infected with subtype B viruses, while the other 2 who did not have a history of traveling outside the United States, taking intravenous drugs, or receiving blood products were infected with a non-B subtype virus (Table 1). One individual, who was infected with a subtype A virus, gave a history of multiple homosexual contacts and did not know the source of his infection. The other individual infected with a CRF02_AG–like virus thought she had acquired her infection through heterosexual contact with her partner, an immigrant from Barbados whose travel history was unknown to her.
Overall, we determined that 43 (78%) of 55 individuals in our study population were infected with non-B subtype viruses. Of these 43 non-B subtype viruses, 41 (95%) were found in the immigrant population of New York City, while 2 (5%) were found in native United States citizens who had no travel history.
This study is among the first to demonstrate the broad diversity of group M subtypes in HIV-1–infected individuals in the United States and the first to demonstrate the extent of this diversity in individuals living in New York City. The high number (43) of individuals infected with non-B subtype viruses in this study was based on enrolling only subjects with risk factors for non-B subtype infection, most of whom were immigrants from regions where non-B subtypes prevail.
To our knowledge, there have been only 2 reports concerning HIV non-B subtype infections in New York City residents 14,15: Jenny-Avital and Beatrice 15 described 13 HIV-1–seropositive individuals, mostly immigrants, who were infected with non-B subtypes. However, diagnosis was based on a serologic assay allowing them only to report subtypes as non-B. Weidle et al 14 found that 3 of 252 United States residents with long-term HIV infection at the Bronx–Lebanon Hospital Center in New York City were infected with non-B subtypes; the strains were identified as subtypes A and F and an inter-subtype recombinant form Fpol/Benv. Our analysis revealed 4 group M subtypes, 2 CRFs, 1 intersubtype recombinant form, and 2 unclassifiable viruses in our study population with risk factors for non-B subtype infection. Although subtype determination can refer only to the HIV-1 gene fragments analyzed, we were able to identify subtypes A (n = 4; 7%), B (n = 12; 22%), C (n = 8; 15%), and F (n = 2; 4%), CRF01_AE–like (n = 7; 13%) and CRF02_AG–like (n = 19; 34%) viruses, an inter-subtype recombinant form Ggag/Aenv (n = 1; 2%), and unclassifiable viruses (n = 2; 4%). The predominance of CRF02_AG–like viruses infecting 19 of 55 individuals was because 27 of 55 study subjects were immigrants from West and central Africa where CRF02_AG accounts for up to 70%–90% of HIV-1 subtypes in certain areas. 4,36,37
Overall, there was a strong association between the identified HIV-1 non-B subtypes and the prevalence of these subtypes in the regions from which the study subjects originated. However, some subjects, like the 6 Chinese immigrants infected with CRF01_AE–like viruses, were probably infected during their long journey before arrival in the United States. Although CRF01_AE can be found in certain provinces of China, 27,40 most infections in China are due to subtype B and BC recombinant forms. 41,42 CRF01_AE, on the other hand, caused >60% of infections in the year 2000 in Southeast Asia and is the most common heterosexually transmitted subtype in Thailand. 4,43 Our subtype results together with the high-risk behavior in that region and their prolonged stay in that area suggest that these Chinese immigrants were probably infected in Southeast Asia. Another example of the relevance of travel history in HIV-1–seropositive immigrants is the Haitian immigrant (sample 02USNYU2825) who was infected with a CRF02_AG–like virus that he probably acquired while living in West Africa. These cases are similar to the first described cases of non-B subtype infections in native United States citizens who acquired non-B viruses during overseas employment and introduced them into the United States upon their return. 12
In our study, 2 native United States citizens without travel history were infected with HIV-1 non-B subtypes. To our knowledge, only a few cases of domestic transmission of non-B subtypes in the United States have been reported, most with a link to regions of endemicity. 14,16 In a recently reported study, Delwart et al 44 analyzed blood samples from HIV-infected blood donors collected throughout the United States between 1997 and 2000 and found that 2% (7 of 312) of the HIV-seropositive blood donors were infected with non-B subtype strains. Four of these 7 non-B subtype infections were likely acquired after sex with an HIV-seropositive African in the United States or abroad, while the remaining 3 infections were found in United States born individuals who did not report sex with someone from Africa.
Knowledge about non-B subtypes that are newly introduced into the United States is important for several reasons. First, it implies that continued monitoring of HIV genetic diversity throughout the United States is needed to determine whether non-B subtypes are increasingly detected and transmitted. Second, the emergence of non-B subtypes in a region where predominantly B subtypes are found will confound efforts to protect against infection with subtype-specific vaccines. Third, subtypes might differ in pathogenicity, 17,18 and fourth, virus variability is a potential problem for the diagnosis and treatment of HIV-1 infection. 11,15,19–24
Although differences regarding transmission and disease progression have been described in persons infected with different HIV-1 non-B subtypes living in African settings, 17,18,45,46 Alaeus et al 47 found no differences in disease progression between native Swedes infected with subtype B and Africans infected with various non-B subtypes living in Sweden.
Information on HIV-1 VL and drug resistance–associated mutations has significant implications for treatment strategies. Subtype variation can have a major influence on VL quantification by different methods. 15,19,20 For example, the RT-PCR–based assay Amplicor HIV-1 MONITOR test version 1.0 (Roche Diagnostics, Branchburg, NJ) has lead to falsely low VL measurements for certain non-B viruses compared with the new Amplicor HIV-1 MONITOR test version 1.5 (Roche Diagnostics). 15,19,20 In our study, we used the bDNA test because it detects HIV-1 by hybridization to multiple sequences derived from multiple subtypes (A–F), resulting in accurate VL quantification for B and most non-B strains. 19,20
Most drug susceptibility studies of HIV-1 have involved subtype B strains. However, the prevalence of non-B strains continues to increase in industrialized countries, and antiretroviral therapy has recently become available in certain developing countries where non-B subtypes predominate. HIV-1 genetic diversity has been shown to be a major obstacle for certain antiretroviral drug resistance hybridization-based assays, 21 and information on the impact of viral diversity on natural susceptibility to antiretroviral drugs is limited and mostly based on in vitro data. 23,24 Major mutations linked to resistance to nonnucleoside reverse transcriptase inhibitors were detected in group O viruses, 23 and primary resistance mutations to fusion inhibitors and polymorphism in the gp41 sequences of HIV-1 group M non-B subtypes and recombinants have been described recently. 48 In spite of many minor mutations, no major mutations associated with resistance to nucleoside reverse transcriptase inhibitors or protease inhibitors were detected in group M non-B viruses isolated from treatment-naive patients. 11,34,35,49 Even though proteases from subtypes C and A exhibit a higher biochemical fitness in the presence of widely prescribed protease inhibitors, 24 no significant differences in immunologic and virological responses to highly active antiretroviral treatment have been shown in patients infected with non-B versus B subtypes to date. 50,51
In conclusion, this study documents the broad variety of HIV-1 group M subtypes currently circulating in New York City. Although, at present, this HIV-1 molecular diversity is to a large extent determined by the structure of the immigrant population in New York City, these newly introduced viruses might spread in the United States. Clinicians should strongly consider subtype analysis for HIV-1–positive individuals with risk factors for non-B subtype infection, because the result might change their clinical management.
The authors thank the clinicians from St. Vincent's Hospital for their collaboration and referral of patients for subtype analysis and Felicité Nduku from the African Services Committee and Sumon Chin from the Chinese-American Planning Council for their help in obtaining personal and clinical data from study subjects. They also thank Anne Dwyer, Jeanne O'Leary, Diana Vargas, and Xiaohong Wang for their help in determining CD4 cell counts and VLs for the study subjects.
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