High HIV Type 1 Prevalence and Wide Genetic Diversitywith Dominance of Recombinant Strains But Low Levelof Antiretroviral Drug-Resistance Mutations in Untreated

Patients in Northeast Gabon, Central Africa

Armel Mintsa-Ndong,1 Melanie Caron,1,2 Jean-Christophe Plantier,3 Maria Makuwa,1

Simon Le Hello,1 Valerie Courgnaud,4 Pierre Roques,1,2,5 and Mirdad Kazanji1,2,6

Abstract

The northeast of Gabon, central Africa is characterized by high population density and a high rate of immi-gration from the surrounding countries. To determine the prevalence, circulating subtypes, and antiretroviralresistance mutations of HIV-1, 810 blood samples were collected from the general population of the two maincities (Oyem and Makokou) of this region. Of these, 61 (7.5%) were found to be positive for HIV-1. Analysis ofthe env (gp120), pol, and gag (p24) sequences as well as phylogenetic analyses showed at least eight different virallineages. The most prevalent strains were CRF02 recombinants, followed by subtypes A, D, and C. The re-maining strains were found to be F, J, G, and also, for the first time in Gabon, the recombinant form CRF11cpx.Analysis of antiretroviral drug-resistance mutations in protease and reverse transcriptase from this untreatedpopulation showed a low level of specific mutations. These mutations were associated with subtype poly-morphism rather than with resistance to antiretroviral drugs. The wide diversity and the emergence of re-combinant strains are in accordance with the rapid spread of new HIV strains in the population and, thus, thedynamic evolution of the epidemic.

Introduction

Today, about 70% of all HIV-1 infections worldwide

are found in sub-Saharan Africa (www.unaids.org). InGabon, a central Africa country, the prevalence of HIV-1 in-fection in 2006 was estimated to be less than 8%,1 but noprevalence studies have been published. Although socialconditions are improving and HIV-1 prevention programs arebeing introduced in Gabon, the actual HIV prevalence is dif-ficult to determine and might be underestimated, particularlyin areas with a high population density and a high rate ofimmigration from surrounding countries with high HIVprevalence.2–5

The distribution of HIV-1 subtypes differs from one conti-nent to another, but the widest genetic diversity has been foundin Africa.6 The geographic distribution of subtypes, however,

undergoes constant change. The wide genetic diversity of HIV-1 described in westcentral Africa, where pure subtypes, manyunclassified strains, and complex recombinants have beenfound, suggests that the HIV-1 pandemic originated there.7

It has been reported previously that HIV diversity is maximalin central Africa.5,7–9 A study in Gabon showed an increasedprevalence of HIV-1 and high strain diversity.10 Makuwa et al.11

investigated HIV-1 diversity among infected persons in the twomain cities of Gabon and observed a high proportion (49%) ofsubtype A. Several studies showed wide diversity and thepresence of recombinant strains such as CRF02 and CRF MAL-like.12 Similar findings were made in neighboring countries, suchas Cameroon13 and Congo,14 where highly diverse HIV-1 groupsand subtypes were found. Recently, Caron et al.15 conducted anepidemiological survey among manganese miners in Gabon toevaluate the prevalence of HIV-1 infection and examined the

1Unite de Retrovirologie, Centre International de Recherches Medicales de Franceville, Franceville, Gabon.2Service de Cooperation et d’Action Culturelle, French Embassy, Libreville, Gabon.3Hopital Charles Nicolle, Centre Hospitalier Universitaire de Rouen, Rouen, France.4Institut de Genetique Moleculaire CNRS Unite Mixte de Recherches 5535, Universite Montpellier II, Montpellier, France.5Service d’Immuno-virologie, Unite Mixte de Recherches E1, Pais XI, Centre d’Etudes Nucleaires, Fontenay aux Roses, France.6Reseau International des Instituts Pasteur, Institut Pasteur, Paris, France.

AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 25, Number 4, 2009ª Mary Ann Liebert, Inc.DOI: 10.1089=aid.2008.0223

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diversity of HIV-1 strains by characterizing polymorphism of thepol gene in order to observe drug resistance-associated muta-tions. They found that the majority of the strains studied wererelated to CRF02_AG (57%), followed by subtype A (9%) andCRF01_AE or subtype B (4%).

This rapid evolution of HIV strain diversity underscoresthe need for continuous monitoring of the molecular epide-miology of HIV-1 in Gabon and other central African coun-tries. There is clear evidence that the efficiency of diagnosticassays and antiretroviral (ARV) drugs differs for HIV-1 vari-ants.16 Furthermore, the introduction of new or geneticallyappropriate ARV treatment in sub-Saharan countries willrequire such data and would be beneficial.

In Gabon, except in the main cities (Libreville, the capital,Franceville, and Port-Gentil), few patients have had access toHIV-1 treatment,17 and there is no medical structure in thecountry with the capacity to treat sexually transmitted dis-eases. Two studies on drug resistance have, however, beenconducted in the country. A study in Libreville showed a highprevalence of the main mutations that induce resistance tonucleoside analogue reverse transcriptase (RT) inhibitors inHIV-1-infected and treated individuals.17 The second study,conducted among manganese miners in Gabon by ourgroup,15 showed that most of the mutations detected in the polgene corresponded to subtype polymorphisms, with no spe-cific resistance to ARV drugs.

Nothing is known about the prevalence of HIV-1=2 innortheast Gabon, an area characterized by a high populationdensity and a high rate of immigration from Equatorial Gui-nea, Congo, and Cameroon. Thus, the aim of this study was toevaluate the prevalences of HIV-1 and HIV-2 and then tocharacterize the HIV strains circulating in the general popu-lations of the two main cities in the northeast region, Oyemand Makokou. We also describe polymorphisms of the polgene, particularly at the protease (P) and RT coding regions, inorder to identify native drug resistance mutations, whichmight affect ARV treatment of this untreated population.

Materials and Methods

Area and population

Gabon occupies 270,000 km2 on the Gulf of Guinea near theEquator and between Cameroon, Equatorial Guinea, andCongo, with tropical forest covering 75% of the territory. Ourstudy was conducted in the main cities (Oyem and Makokou)of the northwest of the country (Fig. 1). We enrolled 810persons aged 13–60 years into an HIV epidemiological survey,with 417 sampled in Makokou and 393 in Oyem by clustersampling on the basis of the population distribution. Thestudy was approved by local and French ethics committees,and informed consent was obtained from each person before ablood sample was taken by locally trained health care staff.All the sampled persons were symptomless; the survey wasanonymous, but age and geographic origin were recorded.

Serological studies

All plasma samples were tested for the presence of anti-bodies to HIV-1=HIV-2 with ELISA assay (Genscreen HIV1þ 2, Bio-Rad, Marne la Coquette, France), and with a rapidtest (Determine HIV-1=2, Abbott, USA). The antibody patternwas confirmed by Western blot (New Lav Blot1, Bio-Rad,Marne la Coquette, France).

Molecular studies

DNA was extracted by the phenol chloroform method.Polymerase chain reactions (PCRs) were performed for the envgene (gp120) with the outer primer ED5=ED12 or ED3=ED14for the first round and ES7=ES8 for nested PCR; for the gaggene (p24), the outer primer was G60=G01, with H1gag1584=gag12V for nested PCR, as described previously.18

To study mutations in the P and RT coding regions, the polgene was amplified by the consensus PCR method of theNational Agency for AIDS Research (ANRS), France (HIV-1PCR and Sequencing Procedures, ANRS AC-11 ResistanceGroup, www.hivfrenchresistance.org).19,20,21

The reference strains from GenBank (http:==hiv-web.land.gov) were aligned with the newly obtained Gabonesesequences by ClustalW 1.8.22 Phylogenetic trees were con-structed from nucleotide alignments by the neighbor-joiningmethod with the MEGA3 package (only bootstrapping valueshigher than 60% were considered significant).23

Genotypic resistance to ARV drugs was defined accordingto the 2007 Stanford Resistance Surveillance list of criticalmutations.24 HIV strain genotyping was performed by partialpol sequence analysis with the HIV Drug Resistance Databaseof Stanford University (http:==hivdb.stanford.edu), the latestversion of the ANRS (www.hivfrenchresistance.org) and the2007 IAS (www.iasusa.org) algorithms.

Results

HIV-1 prevalence

Of the 810 samples, 61 [7.5%; 95% confidence interval (CI),5.71–9.35] were HIV positive. Of these, 26 (6.6%) originatedfrom Oyem and 35 (8.4%) from Makokou. Western blot andthe ELISA method indicated no HIV-2 or HIV-1 group O or N

FIG. 1. Map of Gabon with the two main cities and prov-inces in the northeast of the country in which the study wasconducted.

412 MINTSA-NDONG ET AL.

in our population. Of the 61 positive samples, 33 (8.5%) werefrom women and 28 (6.6%) from men, with no significantdifference by sex. As seen in Table 1, the HIV-1 seroprevalencewas 11.6% for persons aged 25–36 years and 11.0% for thoseaged 37–48 years. The percentages were significantly lower( p¼ 0.0001) for persons aged 13–24 years (3.8%) and for thoseover 49 years (5.5%).

Characterization of circulating HIV-1 strains

To evaluate the strains circulating in this area of centralAfrica, a 600-bp fragment in the env gene (gp120) from 25positive samples was amplified and then sequenced. Phylo-genetic analysis showed wide genetic diversity and thepresence of subtypes A1, A2, C, D, F, G, J, and the recombi-nant form CRF02_AG (Fig. 2A). To evaluate the presence ofrecombinant strains, a 450-bp fragment in the gag gene from16 samples was amplified and sequenced. Sequence andphylogenetic analysis of gag fragments showed that most(11=16) belonged to the recombinant subtypes CRF02_AGand CRF11cpx (Fig. 2B). The remaining strains belonged tosubtypes A, D, C, and F.

Characterization of drug-resistance mutations

A fragment of 1050 bp in the pol gene (Protease-RT) from 16samples was also amplified. The sequence data for the P andRT coding regions are shown in Tables 2 and 3, respectively.In the P region, high polymorphism was found (Table 2); butall the observed mutations corresponded to minor drug re-sistance. Some common polymorphic mutations such asL10V=I found in patients GA.Mkk24, GA.Mkk248, andGA.Mkk332 as well as E35G, seen for patient GA.Mkk332, area nonpolymorphic mutation that could be weakly associatedwith a decreased virological response to some protease in-hibitors. The most frequent polymorphic mutations in the RTregion are shown in Table 3. No mutation known to be as-sociated with nucleoside or nonnucleoside RT inhibitor drugresistance was detected at the relevant positions. K103R, seenfor patient GA.Mkk85, is a minor mutation that alone has noeffect on susceptibility to nonnucleoside RT inhibitors.

Discussion

We present the first data on the seroepidemiology, geneticdiversity, and ARV resistance mutations of the strains circu-

lating in the general population of northeast Gabon. In con-trast to other central African countries, such as Cameroon andthe Central African Republic, few data are available on HIVprevalence and genetic diversity in Gabon. In 1994, Delaportet al. reported the results of a serological survey performed inLibreville showing an HIV prevalence of 2.2%.10 UNAIDSreported that the estimated prevalence among persons aged15–49 years in 2006 communicated by the Gabonese Gov-ernment was 7.9%1; however, no published data are available.The latter estimate was made on the basis of two rapid sero-logical tests, without Western blot confirmation. Thus, ourseroprevalence study provides the first rates obtained bystatistically valid methods based on laboratory tests withconfirmation by Western blot, showing a seroprevalence ofHIV of 7.5% in the two main cities in the north of the country.These two cities are characterized by extensive immigrationfrom the surrounding countries, although the rate is higherthan that in Cameroon (6.7%) in the same year.25 Further-more, this prevalence is also higher than that found in severalother sub-Saharan African countries, such as Mali (1.9%) andCote d’Ivoire (7%).2 On the basis of the few data publishedpreviously, the prevalence of HIV-1 in Gabon appears to beincreasing with time, from 2.2% in 199410 and 4.3% in 1998.26

Curiously, the HIV prevalence was higher in the generalpopulation of the northeast than in manganese miners(2.9%).15 The difference might be due to differences in thesamples collected in the two surveys. In our previous study inminers, 80% were men, whereas the population presented inthis study was more representative of the general populationincluding 52% of men and 48% of women. The prevalence ofHIV in men in the northeast (6.6%) is, however, still higherthan that in the miners. The low prevalence in the minersmight be due to a certain extent to a ‘‘healthy worker’’ effect,as men selected to work in the mines have a higher educa-tional level and better understanding of HIV transmissionthan other populations living in the surrounding areas. Fur-thermore, preventive measures and educational programs todecrease the spread of HIV are frequently conducted inmining companies in Gabon. This could also explain in partthe low prevalence in miners compared to other populationgroups.

Our results indicate wide viral diversity in Gabon andshow that at least eight different viral lineages are circulating,corroborating previous studies showing high prevalences ofrecombinant strains in central Africa.11,12,27 We thus showed

Table 1. Prevalence of Antibodies to HIV-1 in the General Population of Northeast Gabon

by Sex and Age Group

HIV antibodies Crude

Variable No. positive=no. tested % 95% CI Odds ratio 95% CI

SexMen 28=423 6.6 4.3–9.0 0.76 0.45–1.28Women 33=387 8.5 5.8–11.3 1.31 0.78–2.21

Age range (years)13–24 14=361 3.8 1.9–5.9 0.35 0.19–0.6525–36 30=258 11.6 7.7–15.5 2.21 1.31–3.7437–48 13=118 11.0 5.4–16.7 1.66 0.87–3.1749–60 4=73 5.5 0.3–10.7 0.69 0.24–1.96

All 61=810 7.5 5.7–9.4

HIV-1 PREVALENCE AND DIVERSITY IN GABON 413

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10CD.T Z 96.96T Z B F 061 (AF289548)D CD.83.ELI (K03454)

GA.Mkk356(AM903423)GA.Mkk227(AM903410)D T Z 01.A 280 (AY 253311)

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A 2.CY 94.94CY 01741 (AF286237)A 1 UG 98.98U G57136 (AF484509)A 1 K E.94 Q2317 (AF004885)

A 1.UG 92.92U G037 (U51190)11CP X .GR.GR17 (AF179368)

GA.Mkk222(AM903406)19CP X C U 99.CU 38 (AY588970)

13CP X .CM 96.1849 (AF460972)

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J.S E .93 S E 7887 (AF082394)J.S E .94 S E 7022 (AF0823935)

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GA.Mkk209(AM903416)

GA.Mkk308(AM903414)GA.Mkk192(AM903405)GA.Mkk27(AM903408)GA.Oye080(AM903390)

GA.Mkk106(AM903403)GA.Mkk235(AM903413)

GA.Mkk116(AM903404)GA.Mkk248(AM903411)

GA.Mkk85(AM903409)GA.Mkk274(AM903418)

GA.Oye298(AM903395)GA.Mkk351(AM903407)

GA.Mkk169(AM903417)GA.Mkk93(AM903419)

GA.Mkk363(AM903397)

GA.Mkk108(AM903421)

FIG. 2. Phylogenetic tree of the HIV-1 env-gp120 region (A) and gag gene (B). The Gabonese samples (GA.Mkk andGA.Oye, in bold) were aligned and compared with the corresponding sequence of isolates from the database of all subtypesof HIV-1. Brackets on the right indicate the subtype clusters. Phylogenetic relationships were reconstructed by the distanceneighbor-joining method and rooted with HIV-1 groups O. The bootstrap values (percentage of 1000 replicates) supportingparticular subtypes are indicated next to the node.

414 MINTSA-NDONG ET AL.

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GA.Mkk227(AM903448)

GA.Mkk116(AM903444)

GA.Mkk24(AM903450)

GA.Mkk248(AM903451)

GA.Mkk222(AM903446)GA.Mkk106(AM903441)GA.Mkk274(AM903457)

GA.Mkk115(AM903443)GA.Mkk108(AM903442)

GA.Mkk85(AM903453)GA.Mkk235(AM903449)

GA.Mkk192(AM903445)

GA.Mkk351(AM903452)GA.Mkk169(AM903456)GA.Mkk93(AM903455)

FIG. 2. (Continued).

Table 2. Amino Acid Polymorphism in HIV Protease from Isolates Obtained from Northeast

Gabon, Central Africa

Subtypes No. strains L10 T12 I13 K14 I15 G16 Q18 L19 K20 E35 M36 N37 R41

CRF02_AG 8 2A 7V 5R 1E 1HQ 1I 8I 2D 8I 2D 7K1A 1ED 1KR

CRF01_AE 1 1V 1K 1V 1D 1I 1KD 2 1R 1V 1I 1D 1I 2KJ 2 1I 1R 1IV 1A 1R 1G 2I 2D 1K

1EF 1 1V 1V 1IT 1IT 1EG 1Q 1R 1D 1I

Subtypes K45 R57 D60 Q61 L63 I64 E65 C67 H69 K70 I72 V77 L89 I93

CRF02_AG 1H 1HY 2V 1EK 8K 1G 8M1KR

CRF01_AE 1K 1R 1I 1MD 1KR 1P 1L 1HQ 1IJ 1K 1E 1E 1I 2E 2K 1V 1I 1I 1L

1N 1G 1T 1MF 1K 1V 1M

Table 3. Amino Acid Polymorphism in HIV Reverse Transcriptase from Isolates Obtained

from Northeast Gabon, Central Africa

Subtypes No. strains E6 K11 K20 T27 V35 E36 T39 E40 K43 S48 K49 V60 T84 G93 K103 K104 D121

CRF02_AG 8 1K 2R 1S 6T 4D 1A 1KR 3R 4I 1AT 1R 1T2K 1AT

CRF01_AE 2 1T 2T 1K 1D 1R 1DY1N

D 2 1R 1F 1R 1I 1R 1Y1Y

J 1 1D 1TF 1 1TK 1 1T 1K

Subtypes E122 D123 I135 T139 G141 I142 S162 T165 E169 R172 K173 Q174 D177 I178 D186 G196 T200

CRF02_AG 4V 1I 1GR 1V 7A 1IT 1A 5T 6K 8E 1M 8A2L 1IT 1A 1E 1V

1AT 1EQ1E

CRF01_AE 1G 1T 1T 1D 1A 1K 2E 1E1EK 1S

D 1E 1T 1N 1C 1K 1E 1R 2E 1L 1AJ 1S 1A 1K 1E 1G 1IF 1DE 1F 1I 1K 1EK 1C 1T 1R 1E 1M

1A

Subtypes I202 E204 Q207 R211 W229 T240 P243 I244 V245 E248

CRF02_AG 1V 5E 4K 1L 1AT 3Q 1D1D 1E1G 1M1N

CRF01_AE 1A 1S 1P 1D1N 1Q

D 1K 2E 1K 1C1T

J 1V 1E 1L 1LF 1D 1K 1Q 1PK 1K 1A

416 MINTSA-NDONG ET AL.

that HIV-seropositive persons in Gabon are infected withsubtypes G, D, J, and C, and for the first time we identified theCRF11-cpx strain, which has been reported previously inneighboring countries.17 We also confirmed that CRF02_AG,encountered widely in west Africa,28 is now present in centralAfrica. It is still the most prevalent subtype in Gabon as re-cently described in Gabonese miners.15 The wide genetic di-versity of circulating strains reported in other parts of centralAfrica, such as in the Democratic Republic of the Congo,29 ismirrored by the high degree of HIV-1 heterogeneity in Gabon,as many of our strains clustered with the IbNG-like strain(CRF02_AG), similarly but later than in Cameroon.30

In this study, we also analyzed potential resistance muta-tions of HIV-1 to ARV drugs in this untreated semiruralpopulation of clearly identified origin. Previously, Pandreaet al. showed minor mutations associated with viral resistanceto ARV drugs in a limited number of strains, with no samplesof clear Gabonese origin.12 Vergne et al. showed the presenceof major mutations inducing resistance to ARV drugs in alimited number of treated patents (11=19) in Libreville after 17months of treatment, which may reflect inappropriate druguse.31 Recently, we showed minor mutations not associatedwith ARV drug resistance but rather with subtype polymor-phism in a closed miner group.15

In the present study, many of the observed mutations wererelated to subtype polymorphism rather than to resistance.L10, the minor resistance mutation to protease inhibitors, canbe associated with resistance to each protease inhibitor whenpresent with other mutations and occurs commonly in 5–10%of untreated persons. E35, the other protease inhibitor minormutation observed, is a nonpolymorphic mutation that isslightly more common in viruses from protease inhibitor-treated (particularly nelfinavir-treated) persons a than un-treated persons. It was weakly associated with a decreasedvirological response to tipranavir in resistance trials.24,32

The nonnucleoside RT inhibitor resistance mutation ob-served (K103R) occurs in 1–2% of untreated persons and byitself has no effect on nonnucleoside RT inhibitor suscepti-bility; however, the combination of K103R and V179D re-duces intermediate resistance to nevirapine, delavirdine, andefavirenz.33

In studies conducted in the countries surrounding Gabon(Cameroon, the Democratic Republic of the Congo, and theCentral African Republic), a similar result was obtained: theprevalence of drug resistance-associated mutations was verylow.29,34,35

In conclusion, the high seroprevalence (7.5%) and the widegenetic diversity of HIV-1 in Gabon show that the features ofthe epidemic in this country are now similar to those in otherwestcentral African countries. The large number of cocircu-lating HIV-1 subtypes and the wide intrasubtype diversity,together with a high prevalence of recombinant viruses, areconsistent with an old epidemic in central Africa, which isprobably the epicenter of the emergence of HIV-1 group M.36

The low level of drug resistance mutations in the P and RTcoding regions is consistent with an ARV-naive population, asthese drugs were introduced into Gabon relatively recently(less than 5 years in the northeast). It would be informative torepeat the study in order to evaluate the impact of a greateravailability of ARVs on the incidence of HIV-1 drug resis-tance. Moreover, the increasing diversity of HIV-1 in Gabon,with potential consequences for molecular diagnosis and

follow-up of infected patients, calls for regular studies of thedynamics of HIV-1 in this country. Our data indicate the needfor a regional evaluation and for cooperation among thesecountries to address the HIV epidemic, which clearly crossesnational borders.

Sequence Data

The GenBank accession numbers for the sequences re-ported in this study are AM903389 to AM903398, AM903395to AM903398, AM903403 to AM903421, and AM903423 forthe env-gp120 region, AM903424 to AM903440 for the pol, P,and RT coding regions, and AM903441 to AM903457 for thegag polyprotein region.

Acknowledgments

Armel Mintsa-Ndong is the recipient of a fellowship fromthe European Community. This study was funded by theAgence Nationale pour la Recherche sur le Sida, France. TheCentre de Recherches Medicales de Franceville, Gabon, isfunded by the Gabonese Government, Total-Gabon, and theFrench Foreign Ministry. Pierre Roques and Mirdad Kazanjicontributed equally to this work.

Disclosure Statement

No competing financial interests exist.

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Address reprint requests to:Mirdad Kazanji

Laboratoire de RetrovirologieCIRMF, BP 769

Franceville, Gabon

E-mail: m.kazanji@cirmf.org

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