HIV genotyping among female sex workers in the State of Santa Catarina Genotipagem do HIV em mulheres profissionais do sexo no Estado de Santa Catarina

The objective of this study was to investigate the frequency of HIV infection among female sex workers in the port area of Imbituba (State of Santa Catarina), and to identify the viral subtype and its susceptibility to antiretroviral medications. Ninety women were interviewed between December 2003 and February 2004. Six (6.7%) were HIV-positive. Genotyping for HIV, performed on four samples, detected subtype C in three of them, which is predominant in Africa and Asia, and subtype B in one of them, which is prevalent in Brazil, USA and Europe. The results suggest that the Port of Imbituba may be one of the gateways for HIV-1 subtype C to enter Brazil, and for its dissemination to the rest of the country and the Mercosul area, along the highway BR-101. This points towards the need for preventive work to reduce the introduction and dissemination of HIV subtype C in Brazil. Key-words: HIV. Sex workers. Subtype C. Brazil. RESUMO O objetivo deste estudo foi verificar a freqüência da infecção pelo HIV em profissionais do sexo, atuantes em Imbituba (SC), identificar o subtipo viral e a suscetibilidade do vírus aos medicamentos antiretrovirais. De dezembro de 2003 a fevereiro de 2004, foram entrevistadas 90 mulheres, profissionais do sexo, e a freqüência de HIV nessa população foi de 6,7%. O teste de genotipagem para o HIV, realizado em quatro amostras, detectou em três delas o subtipo C, que é predominante na África e Ásia, e em uma o subtipo B, prevalente no Brasil, EUA e Europa. Os resultados sugerem que o Porto de Imbituba pode ser uma das portas de entrada para do HIV-1 subtipo C no Brasil, e a partir dessa localidade ocorrer sua disseminação para o restante do País e países do Mercosul pela rodovia BR-101. Isto aponta para a necessidade de trabalhos de prevenção, com a finalidade de reduzir a introdução, transmissão e disseminação do HIV subtipo C. Palavras-chaves: HIV. Profissionais do sexo. Subtipo C. Brasil. 1. Universidade do Sul de Santa Catarina, Tubarão, SC. 2. Instituto de Infectologia Emílio Ribas, Secretaria de Estado da Saúde de São Paulo, São Paulo, SP. 3. Laboratório de Retrovirus, Instituto Adolfo Lutz, Secretaria de Estado da Saúde de São Paulo, São Paulo, SP. Esse trabalho foi aprovado pelo Comitê de Ética e Pesquisa da Unisul em 13 de novembro de 2003 sob o código 03.024.4.06.III. Address to: Dra. Fabiana Schuelter Trevisol. Av. �os�� Ac�cio �oreira ���, �airro Dehon, ���0��0�0 Tubarão, SC, �rasil. Tel: �� 4� 3621�3000; Fax: �� 4� 3622�1�0� e�mail: fabiana.trevisol@unisul.br Recebido para publicação em 24/11/2006 Aceito em 23/�/200� The human immunodeficiency virus (HIV) is an emerging pathogen with great genetic polymorphism. Two types of virus (HIV�1 and HIV�2) have been identified as etiological agents for AIDS. HIV�2 is concentrated on the African continent, with a small number of cases in Europe and the United States. HIV�1 has spread worldwide. In order to identify the different subtypes of HIV�1, it has been divided into three large groups: � (�ajor), O (Outlier), and N (non�� and non�O) 14 . At least 10 subtypes derived from the � group have already been detected, and these have been classified with the letters from A to �. These variations generally occur in the env and gag regions of the virus. Studies conducted around the world have determined the distribution of the viral subtypes according to their predominant geographic localization: subtype A predominates in central Africa; �, in the United States, Europe, South America and Thailand; C, in India, southern Africa and �razil ; D, in central Africa; E, in Thailand and the Central African Republic; F, in �razil, Romania and the Democratic Republic of the Congo (DRC; former Zaire); G, in DRC, Gabon and Taiwan; G, in DRC, Gabon and Taiwan; H, in DRC and Gabon. Up to �une 2006, 433,06� cases had been notified in �razil, and the number of studies on the viral genotypes of HIV is still small. A study conducted among 10� patients who underwent HIV genotyping tests in Porto Alegre (State of Rio Grande do Sul), from 1��4 through 1���, found prevalences of ��% for subtype

The human immunodeficiency virus (HIV) is an emerging pathogen with great genetic polymorphism.Two types of virus (HIV�1 and HIV�2) have been identified as etiological agents for AIDS.HIV�2 is concentrated on the African continent, with a small number of cases in Europe and the United States.HIV�1 has spread worldwide.In order to identify the different subtypes of HIV�1, it has been divided into three large groups: � (�ajor), O (Outlier), and N (non�� and non�O) � 14 1� .
At least 10 subtypes derived from the � group have already been detected, and these have been classified with the letters from A to �.These variations generally occur in the env and gag regions of the virus 1� .Studies conducted around the world have determined the distribution of the viral subtypes according to their predominant geographic localization: subtype A predominates in central Africa; �, in the United States, Europe, South America and Thailand; C, in India, southern Africa and �razil � 13 ; D, in central Africa; E, in Thailand and the Central African Republic; F, in �razil, Romania and the Democratic Republic of the Congo (DRC; former Zaire); G, in DRC, Gabon and Taiwan; G, in DRC, Gabon and Taiwan; H, in DRC and Gabon � .
Up to �une 2006, 433,06� cases had been notified 12 in �razil, and the number of studies on the viral genotypes of HIV is still small.A study conducted among 10� patients who underwent HIV genotyping tests in Porto Alegre (State of Rio Grande do Sul), from 1��4 through 1���, found prevalences of ��% for subtype � and 22% for subtype C � .A study performed in Rio de �aneiro from 2002 to 2003, among �4� HIV�infected patients undergoing antiretroviral treatment, found subtype C in only 0.4% of the sample 4 .Knowledge of the distribution of these HIV genotypes is essential for better understanding of HIV epidemiology, pathogenesis and response to antiviral treatments, and with regard to the appearance of resistance to medications, development of vaccines, and updating of diagnostic methods.

MATERIAl And METHOdS
�etween December 2003 and February 2004, 13� women were contacted.They were all sex workers at the port City of Imbituba (State of Santa Catarina; SC), and were registered with the non�governmental organization Indústria da Solidariedade (ISO).The Research Ethics Committee of the University of Southern Santa Catarina (Unisul) had approved the study.However, only �0 of these women accepted participation.After they agreed to sign the informed consent statement, blood samples were collected for anti�HIV antibody detection.The HIV�1 serological test was performed at the Clinical Laboratory of Unisul using the Abbott® HIV kit, �.E.I.A. in the automated Axsym System and the Roche® immunoassay HIV kit, with ELISA methodology in automated Cobas Core.The positive or undetermined results were confirmed by indirect immunofluorescence or Western blot analysis, as a confirmatory test for HIV 11 .At the test stage, a second sample was collected in the case of positive or undetermined results for HIV, for each assay.The confirmatory tests were performed at the Public Health Reference Laboratory (LACEN), in Florianópolis (SC).
The women whose biological samples showed reactivity in the tests were invited to give a new blood sample, which was analyzed using the �D® Vacutainer system with EDTA.The tubes that contained the biological samples were centrifuged for 1� minutes at 2,�00rpm, and the plasma obtained was frozen at -20°C until the time of performing the genotyping and detection of pol gene mutations, which are associated with the sensitivity to antiretroviral tests.These tests were performed at the Virology Service of the Adolfo Lutz Institute, São Paulo State Department of Health, and followed the procedures described below: Molecular analysis.HIV�1 RNA was extracted from �00ul of plasma using Trizol LS reagent (Invitrogen, USA).Reverse transcription was performed with 200U of SuperScript II, (Invitrogen, USA), 300ng of random hexamer primers (Invitrogen, USA), 0.�m� of deoxynucleotide triphosphate, �.�U of RNase Inhibitor (Invitrogen, USA) and 10m� of dithiothreitol, in a final volume of 20µl at 42°C for �0 min in 1x reaction buffer.Polymerase chain reaction (PCR) of the target sequences was performed using a nested protocol to amplify protease (PR) and reverse transcriptase (RT) genes.The relative average positions of sequences to HX�2 were: PR (2,240�2,�4�) and RT (2,664 -3,214) (Los Alamos National Laboratory � Sequence Locator Tool).For de�amplification of the pol gene, the first PCR allowed an amplicon product of 1.2Kb to be obtained.The nested PCR allowed the coding genes for PR and RT to be obtained.The samples with negative amplification were recovered using alternative primers or a commercial kit (Viroseq 2.1; Abbott).Nested PCR products were purified using the Concert T� Rapid PCR purification system (Invitrogen, USA) and quantified on agarose 2% gel with low DNA �ass Ladder T� (Invitrogen, USA).Sequencing of both the PR and RT was performed using fluorescent dideoxynucleotides (ddNTPs) from the A�I Prism �ig Dye T� Terminator (Applied �iosystems Inc., Foster City, CA, USA), resolved on 3,100 automatic sequencer (Applied �iosystems Inc., Foster City, CA, USA).
The genetic sequencing of PR and RT allowed genetic information to be obtained for genotype analysis.The analysis and editing of the genetic sequences were performed using the Sequence Navigator software, in which forward and reverse sequences formed a consensus file in FASTA format.The molecular data were subjected to genotype resistance analysis using the Stanford HIV Drug Resistance Database website (hivdb.stanford.edu),which produces a report on susceptibility to antiretroviral drugs.After alignment using the Clustal X program against a reference set from Los Alamos Laboratory (http://www.ncbi.nih.gov/projects/genotyping/formpage.cgi) and manual correction using the �ioEdit software, neighbor joining (N�) trees � were constructed under HKY �� distance matrices, using PAUP*4.10b10for PR and/or RT.HIV�1 group O was used as the outgroup.�ootstrap values (1,000 replicates) above �0% were considered significant.

RESUlTS
The mean age of the �0 female participants in the study was 2� ± � years, ranging from 1� to 60.The data showed that �� (��.6%) were white, �6 (�4.4%) had children (with reports of previous maternal breastfeeding), the educational level was low (incomplete elementary school) and the socioeconomic classification was low, predominantly class D, according to the �razilian criteria � .Furthermore, only four of the women were born in Imbituba, and the women stated that they were frequently moving from one city to another, and from state to state (Table 1).
With regard to sexual practices and risky behavior, a statistical association was found between the number of clients attended per day and HIV infection (p = 0.00�).There were also associations between HIV infection and sexual relations without frequent use of condoms (p = 0.01�), and between HIV infection and the use of inhaled illicit drugs (p = 0.0�3) (Table 2).
Six (6.�%) women, out of all the women who took part in this study, presented reactivity in the anti�HIV test.Among these six, five agreed to undergo a new blood sample collection for the HIV�1 genotyping test.In four samples it was possible to obtain amplified genetic material.HIV�1 subtype C was found in three of the samples and HIV�1 subtype � in the remaining sample.The mutations confering resistance to antiretroviral drugs indicated that three samples were susceptible to protease inhibitors, nucleosides and non�nucleoside  analogs.One sample presented low or intermediary resistance to protease inhibitors, high resistance to non�nucleoside analogs and lamivudine, low resistance to abacavir, and susceptibility to zidovudine and stavudine Table 3.

dISCUSSIOn
Out of the �0 women interviewed in this study, six were infected with HIV, which corresponds to a seroprevalence rate of 6.�%.This seroprevalence rate is similar to those found in other studies among sex workers in �razil, such as the one carried out in Paranagu� (State of Paran�; PR) which reported an HIV infection rate of 4.�% among 132 women 1 .A similar study performed by the STD/AIDS reference center in Vitória (State of Espírito Santo; ES), revealed an infection rate of �.6% among 140 women 16 .Another study conducted among 4� street sex workers in Santos (State of São Paulo; SP), presented an infection rate of 11.1% 2 .Therefore, the results found in this study are similar to those found by other researchers in different regions of �razil.In 1��6, the HIV infection rate among sex workers was approximately 1�% in �razil, according to the �inistry of Health.The last survey by the �inistry of Health, in cooperation with �rasília University in 2002, revealed that the HIV infection rate was 6% among sex workers in different regions of �razil (n = 3,000), which is similar to that found in this study.The prevalence rate among sex workers in �razil was lower than that found in Canada (1�%), China (10%) and Thailand (1�%), and is higher than that found in India (�%) and Argentina (4%) 10 .

Table 3 -Profile of susceptibility to HIV-1 found in female sex workers surveyed between December 2003 and February 2004 in Imbituba (SC).
In the present study, HIV�1 subtype C was found in three (��%) of the four samples in which HIV was recovered.This subtype is usually found in Africa, along with subtype D. HIV�1 subtype � is usually found in the Americas and Europe in approximately �0% of the infected individuals, including �razil.HIV�1 F is associated with blood transmission through the use of injected illicit drugs 6 1� .In a study performed among 10� AIDS patients at the Hospital da Fundação, University of Rio Grande, in the State of Rio Grande do Sul, in 2002, subtype C was found in ��% of them, subtype � was found in 32% and subtype F in 3%, which differs from other regions in �razil 1� .A study carried out in 2001 among �� users of illicit injected drugs in the cities of Santos and São Paulo, in the State of São Paulo, showed subtype � in ��% of the infected individuals, F in �.6% and C in 2.4%, in �3 out of the �� samples 1� .�oth of the above two studies discuss the possibility that the appearance of subtype C may have occurred later than that of subtype �, thus explaining the distribution mentioned above 20 .A study performed in �rasilia among 1� infected individuals found subtype � prevalence in 1� (��.�%) of the samples 3 .Another study performed among 1�0 sex workers in Addis Ababa, Ethiopia, found serum reactivity in 4�% (6� samples) in the anti�HIV test, such that HIV�1 subtype C was present in 66 (��.�%) women, and subtype D in one woman � .
In the present study, it can be suggested that the introduction of subtype C must have occurred as a result of the arrival of foreign ships.According to the �aritime Traffic Sector of the Port �anagement Department, there is great variation in the movements in the port, in relation to the origin and destination of foreign ships.The routes of both the �razilian and foreign ships change all the time, as does the kind of cargo transported.According to an assessment made within that department, the first semester of 2003 registered ships that had stopped at ports in Saudi Arabia, Argentina, Nigeria, Chile, Poland, United States, Italy, Ukraine, Yemen and �razil.Highway �R 101 goes through the City of Imbituba, an important road that connects �razil and other �ercosul countries.Thus, infection by HIV subtype C can disseminate to other regions both through the port and along the �R 101 highway.The sex workers in that city have sex with sailors and truck drivers.The City of Imbituba may be a critical site in �razil for the transmission and dissemination of distinct viral stocks in that region, in �razil and in the other �ercosul countries.Furthermore, it was found that 36.�% of the studied women were transitory workers in the region, which shows that this population is in constant movement from one city to another.Since they do not have a fixed residence, they migrate frequently to other port cities, such as Itajaí, Camboriú and São Francisco do Sul, in Santa Catarina, and Paranagu�, in Paran�.�oreover, even those who stated that they had a fixed residence in Imbituba said that they also worked in other prostitution areas, particularly in Tubarão, Florianópolis and �alne�rio Camboriú, and returned occasionally to their home cities, especially when few ships were in the docks.
Another situation that must be taken into consideration in relation to the transmission and dissemination of subtype C is that Imbituba is also a place that attracts many tourists from other regions of �razil and from abroad, due to its many beaches, traditional fairs and festivities like carnival and the Shrimp Fair.On those occasions there is increased use of alcohol and illicit drugs and an increase in sexual encounters, thus increasing the risk of contamination of the sex workers and these tourists, as well as disseminating HIV.
Considering that the predominant HIV type found in this study was HIV�1 subtype C, rather than subtype �, which predominates in the rest of �razil, governmental and non�governmental programs should pay more attention to the Port of Imbituba, while concentrating their efforts on better examination of the viral dynamics in this location, as well as developing preventive action.
Further studies on genotyping are recommended for this region, including a greater number of volunteers who are not necessarily sex workers.
delavirdine susceptible susceptible susceptible high�level resistance efavirenz susceptible susceptible susceptible high�level resistance nevirapine susceptible susceptible susceptible high�level resistance *Using antiretroviral drug therapy.

Table 1 -Demographic and socioeconomic characteristics of the female sex workers surveyed in Imbituba (State of Santa Catarina; SC), from December 2003 to February 2004.
*The �razilian economic classification criteria categorize the economic classes (A1, A2, �1, �2, C, D, and E) based on the family's income by combining the purchasing power with the educational background.