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RICARDO DA SILVA DE SOUZA
AVALIAÇÃO DA EPIDEMIA DO HIV-1
SUBTIPO C NO SUL DO BRASIL
Tese apresentada á Universidade Federal de São Paulo - Escola Paulista de
Medicina - Disciplina de Doenças Infecciosas e Parasitárias - Laboratório de
Retrovirologia, para obtenção do Título de Doutor em Doenças Infecciosas e
Parasitárias
São Paulo
2008
1
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da Silva de Souza, Ricardo
AVALIAÇÃO DA EPIDEMIA DO HIV-1 SUBTIPO C NO SUL DO
BRASIL/ Ricardo da Silva de Souza – São Paulo, 2008.
Tese (Doutorado) – Universidade Federal de São Paulo. Escola
Paulista de Medicina. Programa de Pós-Graduação da Disciplina
de Doenças Infecciosas e Parasitárias.
Evaluation of HIV-1 the subtype C epidemic in south Brazil .
1. acute infection, 2. HIV genetic diversity, 3. resistance mutations
2
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Dedicatória
A minha mãe, Maria Tereza da Silva de Souza, que sempre me apoiou em
todos os momentos da minha vida.
A meu falecido pai, Gilberto David de Souza, por sempre apoiar e orientar as
minhas escolhas.
A minha esposa, Carla Tessari de Souza, por compreender e apoiar a minha
busca por mais conhecimento, mesmo tendo sofrido com a minha ausência. Por
me amar incondicionalmente e me fazer orgulhoso da nossa família.
A minha irmã Maria Rosane da Silva de Souza, por compartilhar os meus
anseios e história de vida, mesmo que distante.
Ao professor e mentor Dr. Ricardo Sobhie Diaz.
3
“
A filosofia de uma pessoa não é melhor expressa em palavras; ela é
expressa pelas escolhas que a pessoa faz. A longo prazo, moldamos
nossas vidas e moldamos a nós mesmos. O processo nunca termina
até que morramos. E, as escolhas que fizemos são, no final das
contas, nossa própria responsabilidade.”
Eleanor Roosevelt
4
Agradecimentos
Esta tese representa a concretização de um esforço de diversas
instituições, profissionais, amigos e familiares. Gostaria de agradecer
especialmente as seguintes pessoas:
Ao meu orientador, Prof. Dr. Ricardo Sobhie Diaz, por compartilhar o
seu conhecimento e por sua paciência e bom humor.
Aos Prof(s). Dr. Christopher Pilcher e Kim Page-Shafer, por me
receberem no Center for AIDS Prevention Studies (CAPS) da
Universidade da Califórnia em San Francisco (EUA) para a análise dos
dados compilados, contribuindo assim para minha formação científica.
A Prof(s). Dr(s). Gail Shor Posner e Charles Mitchell, diretores do
programa Fogarty da Universidade de Miami (EUA), por me apoiarem
financeiramente, por me receberem em Miami e contribuírem
tecnicamente na análise dos dados referentes aos métodos
laboratoriais deste estudo.
Aos colegas do Laboratório de Retrovirologia que me receberam de
braços abertos e que sem eles os resultados das genotipagens não
estariam disponíveis. Em especial a Cecília Sucupira por facilitar minha
estada em São Paulo e pela importante contribuição técnica com a
realização da análise da diversidade genética e resistência genotípica
das amostras obtidas.
A Antonio Charlys da Costa, secretário da pós-graduação, pela
prestatividade e compreensão nas questões referentes ao andamento
do curso e nas questões burocráticas deste trabalho. E principalmente
pela amizade mesmo que distante.
5
A Lilian Amaral Inocencio, coordenadora da Unidade de Laboratório do
Programa Nacional de DST/AIDS do Ministério da Saúde, pelo apoio
institucional ao projeto e por sua amizade e confiança.
A equipe do Laboratório de Pesquisa em HIV/AIDS da Universidade de
Caxias do Sul pelo apoio operacional na condução do projeto que
originou esta tese.
As equipes dos sítios de pesquisa afiliados por compreenderem a
importância deste trabalho e ativamente participarem de sua
realização.
Ao Instituto Nacional da Saúde dos EUA (NIH) pelo apoio e suporte
financeiro para a realização deste projeto.
A Deus, por me abençoar neste caminho.
6
Sumário
INTRODUÇÃO...................................................................................................... 8
OBJETIVOS........................................................................................................ 14
ARTIGO 1 - DETECTION OF ACUTE HIV INFECTION USING A FOURTH-
GENERATION EIA SCREENING ASSAY IN SOUTHERN BRAZIL ................. 15
ARTIGO 2 - DIFFERENCES IN TRANSMITTED DRUG RESISTANCE AND
TRANSMISSION EFFICIENCY BETWEEN HIV-1 SUBTYPES IN SOUTHERN
BRAZIL............................................................................................................... 34
CONCLUSÕES................................................................................................... 60
REFERÊNCIAS BIBLIOGRÁFICAS .................................................................. 62
7
INTRODUÇÃO
A epidemia causada pelo Vírus da Imunodeficiência Humana do Tipo 1 (HIV-1)
está sofrendo grandes transformações tanto a nível epidemiológico como a nível
molecular. No Brasil, o subtipo mais freqüente de HIV-1 é o subtipo B, mas, nos
últimos dez anos, outros subtipos tais como F, C, e, as Formas Recombinantes
Circulantes (CRFs) tem sido descritas [1,2]. Recentemente as CRFs 28 e 29,
recombinantes entre clades B e F, foram caracterizadas e descritas no estado de
Sâo Paulo [3] e a CRF 31, recombinante entre clades B e C, foi descrita na
Região Sul [4].
O HIV-1 subtipo C foi inicialmente identificado no Brasil em Porto Alegre, RS no
início dos anos 90. Desde então, a prevalência do subtipo C na Região Sul
cresceu de 3% em 1994, para 22% em 2000, atingiu 37% no ano de 2004 e
chegou a 39% em 2007 [5]. Especula-se que o HIV-1C seja mais virulento a
nível populacional que o HIV-1B [6]. Embora este fenômeno já tenha sido
observado na África do Sul [7] e Índia [8] não há estudos conhecidos avaliando
os fatores virológicos associados ao crescimento do subtipo C no sul do Brasil.
Um maior conhecimento das características moleculares do HIV circulante e dos
mecanismos da resposta imunológica na nossa população é fundamental para a
compreensão da epidemia, desenvolvimento de produtos vacinais eficazes e
para identificação de novas estratégias de prevenção e tratamento. Entretanto,
para um melhor entendimento do crescimento do subtipo C se faz necessário
estudar as características virais durante a fase aguda e recente, isto é as fases
iniciais da infecção, quando o vírus ainda não se adaptou a resposta imune do
hospedeiro.
Aproximadamente, 35 milhões de pessoas estão infectadas pelo HIV [9],
praticamente todas passaram por um período de infecção aguda (IAH). Embora
dezenas de milhares destes indivíduos tenham apresentado sintomas
compatíveis com a síndrome retroviral aguda apenas poucos milhares de
pacientes com IAH foram diangnosticados até hoje. Historicamente, a
dificuldade para identificar pacientes com IAH levou a uma falta de conhecimento
sobre sua história natural e consequentemente incertezas sobre a necessidade e
8
o tratamento ideal [10]. Entretanto, novas tecnologias laboratoriais foram
introduzidas, as quais tornaram o reconhecimento da IAH na rotina clínica uma
realidade.
Infecção aguda pelo HIV ou infecção primária é o primeiro estágio da doença
causada pelo HIV. Nesta fase, o RNA do HIV ou carga viral pode ser detectado
no sangue antes do aparecimento de anticorpos específicos. A infecção primária
pode ser diferenciada da infecção recente (quando a detecção de anticorpos é
realizada por meio da utilização de ensaios imunoenzimáticos (EIE) comerciais,
mas a concentração ou a avidez dos anticorpos ainda é reduzida); da infecção
estabelecida, ou crônica, quando a resposta mediada por anticorpos está
completamente desenvolvida. Até 66% dos pacientes com IAH apresentam uma
síndrome clínica inespecífica, autolimitada, semelhante à mononucleose,
chamada de síndrome retroviral aguda [11-14]. Estudos sugerem que o período
de incubação da IAH varia de 5 a 31 dias, contados a partir da exposição até o
aparecimento dos sintomas, e possui uma duração média de 14 dias [11, 1-19].
Uma das principais características da síndrome retroviral aguda inclui o pico de
viremia do HIV (tipicamente em milhões de cópias de RNA do HIV por mL de
sangue) seguida, uma ou duas semanas mais tarde, pelo aparecimento de
anticorpos ou soroconversão [19,20] e, um ou dois meses mais tarde, por uma
diminuição expressiva da viremia plasmática [21]. O diagnóstico da IAH é
dificultado pela ausência de anticorpos durante as primeiras semanas da
infecção. Por isto, a avaliação laboratorial deve incluir testes que detectam
ácidos nucléicos ou antígenos do HIV, como o antígeno p24.
Estudos que avaliaram os testes necessários para o diagnóstico da IAH [15,16]
indicam o uso de testes suplementares para detecção do antígeno p24 ou testes
de amplificação dos ácidos nucléicos (TAANs) apenas em situações em que haja
suspeita clínica de IAH. Tais situações podem não ser tão incomuns como
previamente assumido. Rosenberg e colaboradores [22] observaram que 1.0%
dos pacientes com testes negativos para mononucleose infecciosa pelo vírus
Epstein-Barr (VEB) apresentavam sorologia consistente com infecção aguda
pelo HIV. Pincus e colaboradores [23] observaram, em um serviço de urgência
9
da cidade de Boston (EUA), que 1.0% dos pacientes com “sintomas virais”
apresentavam IAH, mesmo na ausência de suspeita clínica.
Em estudo de revisão de 30 casos bem sucedidos de diagnóstico de IAH no
estado da Carolina do Norte (EUA), Weintrob e colaboradores [24,25] relataram
que a maioria dos pacientes necessitou retornar ao médico pelo menos três
vezes até que IAH fosse considerada uma explicação para as queixas
inespecíficas de síndrome retroviral aguda.
Devido às dificuldades associadas ao reconhecimento clínico da IAH, as
pesquisas na área tem focado na adaptação de algoritmos de testagem que
permitam o uso de ensaios moleculares ou testes para detecção do antígeno
p24 na rotina de triagem do HIV como testes suplementares.
Nos ensaios moleculares disponíveis atualmente, o RNA do HIV ou carga viral,
pode ser detectado com precisão em amostras de sangue periférico alguns dias
após a infecção. A estratégia ideal compreende retestar as amostras anti-HIV
negativas com metodologias moleculares. Este procedimento detectaria a maior
parte dos casos de IAH nos indivíduos testados. No entanto esta estratégia é de
alto custo e também eleva as chances de resultados falso-positivos [16].
Estratégias de testagem em grupo ou “pools” foram utilizadas, inicialmente, pelos
bancos de sangue como uma solução para o elevado custo dos ensaios
moleculares. Esta estratégia é baseada na criação de grupos ou pools de
amostras de vários indivíduos. Na triagem de grupos de amostras, o grupo que
apresentar resultado positivo no ensaio molecular deverá ser desmembrado e
suas amostras testadas individualmente. Os algoritmos de testagem em grupo
reduzem o número de amostras individuais que necessitam testagem molecular,
reduzindo o custo e, quando feita em níveis, também diminuem o potencial de
resultados falso-positivos, aumentando a acurácia do processo de testagem.
A aplicação da testagem em pools na triagem para o HIV foi primeiramente
sugerida por Quinn, et al [26] e utilizada, prospectivamente, no programa público
do estado da Carolina do Norte (EUA), na população de indivíduos que
buscavam testagem para HIV no início do ano de 2002 [27]. Nos primeiros 12
meses do programa de testagem suplementar com testes moleculares em pools
10
de amostras anti-HIV negativas um número adicional de 23 casos (4% das
infecções identificadas no estado) apresentavam infecção aguda.
Resultados similares foram obtidos em diversas populações urbanas que
buscam testagem para HIV: São Francisco [25], Los Angeles [28], Atlanta [29] e
Seattle [30] nos Estados Unidos e em Johannesberg [31], na Àfrica do Sul. Estes
estudos indicam que a testagem de pools de amostras com TAANs pode ser
uma alternativa promissora, entretanto, existem limitações nesta estratégia. Os
TAANs são técnica e operacionalmente complexos, necessitam nível avançado
de infra-estrutura laboratorial, de programas de qualidade e requerem que as
amostras de sangue total sejam processadas previamente à realização do
ensaio. Assim sendo, a estratégia de testagem de pools de amostras com
TAANs pode não ser viável em áreas com limitada infra-estrutura laboratorial, ou
situações onde a flebotomia convencional e o processamento de amostras
possam ser problemáticos.
Em situações em que a testagem em pools seguida de TAAN não seja viável,
uma abordagem alternativa para melhorar a acurácia (valor preditivo positivo) e
as questões de custo ligadas aos TAANs ou testes de antígenos virais é
direcionar a testagem para populações com reconhecida alta prevalência de IAH.
Estudos recentes avaliaram prospectivamente estratégias alternativas de triagem
da IAH em áreas endêmicas como na Índia [13], Sul do Brasil [32], África do Sul
[31] e Maláui [22, 33]. Os pesquisadores identificaram entre dois e três casos de
IAH por 1.000 indivíduos nas suas respectivas populações de testagem. É
importante salientar, que dois estudos [28,33] utilizaram ensaios do tipo enzime
linked immunosorbent assay (ELISA) que detectam somente antígeno p24 para
identificação de casos agudos e outro estudo [32] utilizou ELISA de quarta
geração, ou seja, que detecta simultaneamente antígeno p24 e anticorpo anti-
HIV. Esses estudos sugerem que a detecção de IAH, em larga escala, com
métodos não-moleculares são viáveis mesmo em áreas do mundo com limitada
infra-estrutura laboratorial.
11
Estudos para avaliar o desempenho de metodologias laboratoriais alternativas
para a identificação de casos de IAH em áreas com alta prevalência do HIV são
muito importantes para entendermos os mecanismos de transmissão do HIV e
sua relação com a diversidade genética.
A resistência a drogas anti-retrovirais pode ser dividida em duas categorias:
transmitida ou primária, que ocorre quando um vírus que apresenta mutações
associadas a resistência é transmitido para um indivíduo virgem de tratamento;
adquirida ou secundária, que ocorre em indíviduos após um período de
tratamento anti-retroviral. Embora os dois tipos sejam preocupantes, a
resistência primária ao HIV tem o potencial de rapidamente reverter o eficácia do
tratamento anti-retroviral a nível populacional.
Os testes para detecção de resistência genotípica, comumente denominados de
genotipagem, se tornaram uma ferramenta importante no monitoramento de
pacientes infectados pelo HIV sob terapia anti-retroviral como também em
situações para orientar a terapêutica inicial. Esse teste identifica as posições das
mutações e/ou polimorfismos no genoma do HIV associados à resistência aos
anti-retrovirais. Com o aumento global da diversidade genética do HIV surge a
necessidade de vigilância sistemática da resistencia primária e a caracterização
das mutações ou vias mutacionais de resistência dos subtipos circulantes em
nossa população com o objetivo de garantir o sucesso dos programas de
tratamento anti-retroviral.
Casos de transmissão de vírus resistentes tem sido relatados nos últimos dez
anos [34]. Estudos recentes sugerem que a prevalência da resistência primária
no Brasil está sofrendo mudanças importantes com características regionais. Em
estudo recente realizado no estado da Bahia, Pedroso [35] e colaboradores
observaram uma prevalência de resistência primária de 18.9%. Sucupira [36] e
colegas observaram uma prevalência de 36% em Santos. Por outro lado, estudo
realizado no Rio de janeiro, em 2007, reportou prevalência de apenas 1.4% [37].
Estudos que tenham a capacidade de integrar novos conhecimentos sobre
metodologias laboratoriais eficazes, adequadas a nossa realidade e, novos
12
conhecimentos sobre a biologia da transmissão são vitais para o entendimento
da epidemia e sua erradicação.
Este trabalho faz parte de uma iniciativa do Laboratório de Pesquisa em
HIV/AIDS da Universidade de Caxias do Sul (UCS), do Laboratório de
Retrovirologia do da Disciplina de Doenças Infecciosas e Parasitárias da Escola
Paulista de Medicina e de seus colaboradores em resposta a chamada de
projetos de pesquisas International Research on Infectious Diseases (IRID) do
Instituto Nacional da Saúde (NIH) do governo norte-americano. O Laboratório de
Pesquisa em HIV/AIDS da UCS recebeu aprovação de financiamento do projeto
com titulo em inglês de “Evaluation of HIV Subtype C Epidemic in South Brazil” e
recebeu aprovação da Comissão Nacional de Ética em Pesquisa (CONEP) para
este projeto.
13
OBJETIVOS
1. Avaliar o desempenho de um ensaio imunoenzimático de quarta geração
para detectar infecção aguda pelo HIV.
2. Determinar a prevalência da resistência primária e descrever as
características genotípicas do HIV-1 subtipo B e C entre os indivíduos
virgens de tratamento que freqüentam Centros de Testagem e
Aconselhamento Anônimos da Região Sul do Brasil.
14
Artigo 1
Detection of Acute HIV Infection Using a Fourth-Generation
EIA Screening Assay in Southern Brazil
Ricardo S. de Souza
,1,6
, Robert W. Ryder
2
, Susan A. Fiscus
3
, Ada Cachafeiro
3
,
M. Kerkau
3
, Luciene Scherer
4
, R.D. Sperhacke
1
,Simone Castro
5
,
,
Ricardo
Sobhie Diaz
6,
and Christopher D. Pilcher
7
1
Laboratório de Pesquisa em HIV/AIDS, Universidade de Caxias do Sul, Caxias
do Sul, Brazil;
2
Center for Internacional Health and Development, Boston
University School of Public Health, Boston MA;
3
Center for AIDS Research,
University of North Carolina at Chapel Hill, Chapel Hill, United States,
4
Fundação
Estadual de Pesquisa e Produção em Saúde, Porto Alegre, Brazil,
5
Laboratório
Central do Município de Porto Alegre, Porto Alegre, Brazil,
6
Laboratório de
Retrovirologia, Universidade Federal de Sâo Paulo, Sao Paulo, Brazil and
7
HIV/AIDS Division, Universiity of California-San Francisco, San Francisco,
United States.
Corresponding author:
Ricardo S. de Souza,
Universidade de Caxias do Sul
Laboratório de Pesquisa em HIV/AIDS
Rua Francisco Getúlio Vargas 1130, Bloco S, Sala 315
95070-560 Caxias do Sul, RS – Brazil
Running head: Detection of acute HIV infection
15
The study was presented in part at the
XVI International AIDS Conference,
Toronto, Canada, August 13-18, 2006,
Oral abstract session TUAB0201.
This research was funded by the following grants from the National Institutes
of Health: R03 AI64037, 2 D43 TW000017, R01 MH068686 and UNC CFAR
grant.
None of the authors have any commercial or other association that might
pose a conflict of interest.
16
Abstract
Detecting acute HIV infections is an important HIV prevention goal. ‘Fourth-
generation’ enzyme immunoassays (EIAs) detect both HIV antibody and HIV
antigens. We compared fourth-generation EIA, antibody-only third generation and
HIV RNA assay performance using 933 specimens collected consecutively from
HIV testing clients in south Brazil. The fourth-generation assay detected 183 HIV
infections (subtype B and C) with 100% sensitivity and 99.8% specificity. The
antibody testing algorithm failed to identify three cases of acute HIV infection
(1.6% of confirmed cases). These results suggest that fourth-generation EIA
based screening may have sufficient accuracy to facilitate acute HIV based
prevention strategies in resource limited, high prevalence settings with diverse
HIV subtypes.
Key Words: Acute HIV infection, Fourth-generation EIA, pooled HIV RNA, HIV
screening.
17
Introduction
Acute HIV infections (i.e., those occurring in the previous 1-3 months, when
standard HIV antibody tests remain negative) have been estimated to account for
as many as half of all HIV transmission in some populations [1]. Recent studies
demonstrating that antibody negative acute cases may represent between 4% to
39% of infected patients presenting for HIV testing have further highlighted the
potential importance of targeted treatment, surveillance and prevention activities
focusing on acute infection [2-4]. To date, most experience with testing for recent
HIV infection has relied on “detuned”, less-sensitive antibody assays calibrated to
identify antibody positive individuals with recent seroconversion on the basis of
low titer and/or low affinity antibodies [5]. Another used method, the BED capture
enzyme immunoassay is based on the increasing proportion of anti-HIV1 IgG in
total IgG following seroconversion [6]. However, such assays diagnose new
infections with a 1-2 month delay and do not identify acute infection.
Techniques for real-time diagnosis of acute HIV at HIV testing, adding screening
tests for HIV antigens (e.g., p24) or nucleic acids (HIV RNA) to antibody testing
algorithms have been explored [7-9]. The gold standard for AHI (acute HIV
infection) diagnosis remains HIV RNA testing, which is highly sensitive and can
be made both efficient and accurate through the use of pooling strategies [10].
However, HIV p24 antigen assays are more rapid, less expensive and less
technically demanding [11], and have been used with some success to identify
AHI cases in high incidence HIV testing populations, achieving up to 75%
sensitivity when compared to a reference standard including HIV RNA [12]. For
HIV screening, it is important to emphasize that p24 or HIV RNA are
supplemental tests: specimens are first classified as antibody negative, positive
or indeterminate using a classical screening/confirmatory antibody testing
algorithm. If antibody negative or indeterminate, they can then be screened for
nucleic acid or antigen. This strategy increases window period case identification
but increases the cost of screening for AHI and the turnaround time of results.
Fourth-generation HIV enzyme immunoassays (EIA) were developed by the
blood screening industry in the 1990s to detect both antigen and antibody
18
simultaneously [13]. Although they have found limited use in blood screening,
they have several potential advantages for HIV testing in the clinical setting.
They are highly sensitive for detection of HIV during the window period of AHI:
testing on seroconversion panels suggests that commercially available fourth-
generation assays can detect AHI within 3 days after these infections are first
detectable by HIV RNA testing [14]. They can also be performed easily with
several hours’ turnaround time using equipment available in many HIV
laboratories in the developing world. Interestingly, the clinical performance of
these assays has not been well studied.
In this study we evaluated the ability of a fourth-generation EIA to detect acute
HIV infection in comparison to HIV RNA, in an HIV testing population in southern
Brazil, an area with mixed subtypes and a recognized increase of the HIV-1 clade
C [15].
19
Subjects, Materials and Methods
We conducted a retrospective, cross-sectional, diagnostic performance
evaluation involving plasma samples submitted for analysis to the Vila dos
Comerciários (VCC) Health Clinic (Porto Alegre, Brazil) and anonymized for
completion of performance evaluation. Individuals seeking routine anonymous
HIV voluntary counseling and testing at VCC were screened for HIV. Clients with
inconsistent results on this testing were counseled as HIV indeterminate and had
confirmatory testing performed. All clients completed a routine questionnaire
detailing demographics and HIV risk factors at the time of specimen collection.
Consecutive samples were collected from patients that had received routine HIV
testing from the VCC voluntary and counseling testing center between January
and March 2004. Initial HIV testing was performed by the VCC laboratory. All
specimens having negative or indeterminate specimens results were anonymized
by the VCC lab, associated with a study code and submitted to the Virology
Laboratory at the University of North Carolina at Chapel Hill, USA (UNC) for
additional HIV RNA screening. Antibody negative or indeterminate specimens
from subjects of all ages and genders were transferred. For purposes of data
analysis, the VCC laboratory provided the study team with initial HIV testing
results and limited additional information (gender, age, and follow-up confirmatory
HIV test results) for all clients screened during the study period.
HIV testing by the VCC laboratory was carried out using a fourth-generation HIV
EIA (Genscreen Plus HIVAg-Ab; Biorad, Hercules, CA, USA), which detects HIV
p24 antigen and anti-HIV. This assay has been in use at the VCC laboratory
since 2003. For specimens that were positive on the fourth-generation HIV test,
the VCC laboratory performed immediate confirmatory anti-HIV testing using
three different anti-HIV tests in parallel: the Axsym HIV1/2 gO MEIA (Abbott
Laboratories, IL, USA), a third generation EIA; the indirect immunofluorescence
assay (IFA) from Biomanguinhos (Fiocruz, Rio de Janeiro, Brazil); and the
Determine HIV rapid test (Abbott, Abbott Park, IL, USA). Western blot (WB) (HIV
Blot 2.2, Genelabs Diagnostics, Singapore) was performed to help resolve cases
with inconsistent or low-signal positive antibody testing results, and was
20
interpreted according to Centers for Disease Control and Prevention (CDC)
criteria [16].
Specimens found to be negative using the fourth-generation EIA were screened
for HIV RNA at the virology laboratory at UNC using the Roche assay (Amplicor
Monitor HIV RNA version 1.5; Pleasanton, CA, USA) and a 1:12 robotic minipool
strategy after the technique of Busch and colleagues [17]. Specimens that were
positive on the fourth-generation EIA but negative or indeterminate on one or
more follow-up anti-HIV test were submitted for individual HIV RNA testing if
specimen remnants were available. Following all HIV RNA testing, RNA positive
specimens were then submitted for population sequencing of pol for preliminary
HIV subtype analysis (ViroSeq™ v2.0, Celera Diagnostics, Alameda, California,
USA).
HIV infection status was determined using all available data. HIV negative status
was defined by having consistent negative results on all testing. Acute HIV
infection (AHI) was defined by having one or more negative antibody screening
tests results, with infection status supported by a positive HIV RNA result or
documented seroconversion. Established HIV Infection was defined by having
consistently positive results on confirmatory antibody testing.
Diagnostic performance of the fourth generation EIA and routine antibody EIA
screening assays were assessed using a reference standard that included all
detectable HIV infections, according to the above definitions (rather than only
antibody-positive infections). Sensitivity, specificity and positive and negative
predictive values were calculated with 95 percent confidence intervals. Analyses
were conducted using SAS (version 8.2; SAS Institute).
All study procedures were approved by the human subjects review boards of the
University of Caxias do Sul and the University of North Carolina School of
Medicine.
21
Results
In total, 933 consecutive specimens were received by the VCC laboratory during
the study period. The majority of those seeking testing were men (61%) between
the ages of 5 and 64, with a mean age of 31.9 years. Females comprised 39% of
the group with a mean age of 32.5 years. The study’s HIV screening procedures
are illustrated in Figure 1. Of 933 specimens screened, 932 were fully evaluable
and 181 (19.4%) of these were confirmed as HIV infected by the complete
algorithm. One specimen (with results suggesting probable, but unconfirmed
acute HIV infection) was not able to be classified according to our study
definitions, and was excluded from the primary analysis.
All confirmed HIV infections were positive on the fourth-generation EIA; none of
the 750 confirmed fourth-generation negative specimens tested positive for HIV
RNA.
Five fourth-generation EIA positive specimens had negative or inconsistent
results on confirmatory antibody testing (see Table 1). Of these 5 possible acute
cases, 3 (A,B,C) were repeatedly HIV RNA positive meeting the study definition
of acute HIV infection. A fourth possible acute case (D) had no remnant
specimen available for confirmatory RNA testing and no documentation of
seroconversion and was unclassifiable. The final case (E) had insufficient
sample but did follow-up and failed to seroconvert by any assay 30 days after the
first test, and was classified as HIV negative.
Two of the three RNA positive acute cases were sequenced. One was found to
be HIV subtype B and the other a recombinant CB.
The overall prevalence of acute HIV infection in the study population was 0.32%
(3/932), representing 1.6% of all confirmed HIV infections. Considering all
evaluable specimens, the sensitivity (Se) of the fourth-generation EIA for HIV
infection in this study was 100.0% (95% confidence interval: 98.3,100.0) and the
estimated specificity (Sp) was 99.8% (99.2,100.0). Positive predictive value
(PPV) was 99.4% (96.9,100.0) and negative predictive value (NPV) was 100.0 %
(99.6,100.0).
22
The sensitivity of antibody assays for confirmed HIV infection varied
(immunoflourescence, 98.3%; third generation EIA, 99.4%, rapid test 100%);
several false-positive antibody test results were noted, but the specificity, PPV
and NPV of these assays were not evaluable in this study.
23
Discussion
In a clinic setting with high HIV prevalence in Southern Brazil, the fourth-
generation EIA assay accurately identified 1.6% of HIV cases that were missed
by standard antibody tests and showed a high specificity (99.8%). The proportion
of acute (antibody-negative) cases found in this testing population was in accord
with findings of other studies in high risk populations that have used HIV RNA-
based testing [12,9]. We were unable to demonstrate an advantage to including
HIV RNA screening over and above the fourth-generation EIA for detection of
acute HIV infections. These results suggest a simple, low-tech alternative to HIV
RNA screening for accurate identification of acute HIV infections in resource-
constrained settings with a high burden of HIV infections.
We demonstrated the assay’s high sensitivity and specificity in a population with
mixed HIV subtypes: in a separate report working with the same specimens,
Rodrigues and colleagues [21],documented that 32% of the HIV infections in this
study were of subtype B, 58% were subtype C and 3% were F recombinants.
High sensitivity across HIV subtypes is likely typical of the current generation of
such tests [18]. The limits of detection of modern fourth-generation assays range
from 3 to 10 pg of p24 Ag/ml, comparable to those for p24 Ag-only assays [19],
and they accordingly reduce the seroconversion window period to around 2
weeks, within 3 days of (non-pooled) HIV RNA assays [20]. High specificity
(99.8%) of our study’s screening assay has been reported previously [18] and is
superior to the reported specificities of many antibody screening tests in current
use.
The method used in this study confers some potential advantages in closing the
window period for HIV infection. First, the fourth-generation screening assay is
typically used as a primary screening test, and supplemental (antibody-only)
testing is only performed on the small number of samples that screen positive by
the fourth-generation test. By contrast, combined antibody/RNA or antibody/p24
screening algorithms demand that both antibody-negative and antibody-positive
results be confirmed with some form of supplemental testing before results can
be reported. Thus, using the fourth-generation assay ‘up front’ dramatically
24
reduces the number of specimens to be retested. This potential advantage may
be of less impact in testing populations with very low HIV prevalence, where
specimen pooling/RNA algorithms can be highly efficient [7]. In high prevalence
settings, however, specimen pooling/RNA algorithms are unlikely to approach the
testing efficiency of fourth-generation EIA-based testing [10].
Second, fourth-generation EIAs are relatively simple, suitable for testing sizable
numbers of samples, and easily adapted to automated platforms. Their list price
is similar to that of third-generation assays, making their use feasible in resource-
limited settings.
Third, some new fourth-generation assays [14] read detection of antigen and
antibody as separate results—allowing probable acute and 4established cases to
be distinguished in one single simple assay. This could dramatically reduce the
turnaround time for identification and confirmation of suspected acute cases.
Fourth, nucleic acid based test may be potentially more problematic than EIA
based tests in places were more genetically diverse HIV circulates, since nucleic
acid tests sensitivity relies in the efficient annealing of primers and probes.
If the performance and cost-effectiveness of fourth-generation EIAs are borne out
in larger studies, the results of this study suggest that the expanded use of fourth-
generation assays by public health programs could help create important
opportunities for acute HIV-centered prevention without the need for complex
specimen pooling and nucleic acid amplification. Projecting the use of the fourth-
generation EIA in the Brazilian epidemic by applying the number of HIV tests
performed in the country and the reported prevalence of HIV infection, we
estimate that as many as 2,500 (acute) cases presenting for testing in Brazil have
been missed from 2000 to 2005.
We recognize that the small number of samples constitutes a limitation of the
present study. However, methods to reliably identify acute HIV infection are
urgently needed to develop strategies focusing on reducing HIV transmission by
individuals with acute HIV infection. Our results suggest that simple, fourth-
generation EIA screening can detect acute HIV infections with sufficient accuracy
25
to make acute HIV based prevention strategies feasible in high HIV prevalence,
resource limited settings with diverse circulating HIV subtypes.
26
Acknowledgements
Members of the AMPLIAR study team include: Carla de Souza, Leonardo da
Motta, Machiline Paganella, Gisele Preusller, Daniela Costa, Nemora Barcellos,
Vania Debastiani. The authors thank Dr. Gail Shor-Posner, Dr. Toye Brewer and
Dr. Charles Mitchell for revision of the manuscript, Carla de Souza for
administrative and regulatory support, and Ada Cachafeiro for technical expertise
in the performance of the Roche Amplicor assay.
27
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45(7):2274-7.
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Performance evaluation of three automated human immunodeficiency
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19. Ly, T. D., Laperche S., Brennan C., Vallari A., et al. Evaluation of the
sensitivity and specificity of six HIV combined p24 antigen and antibody
assays. J Virol. Methods 2004; 122:185-194.
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combined p24 antigen and antibody assays replace p24 antigen specific
assays?
J Virol Methods. 2007 Jul;143(1):86-94
21. Rodrigues R, Scherer LC, Oliveira CM, Franco HM, Sperhacke RD, Ferreira
JL, Castro SM, Stella IM, Brigido LF.
Low prevalence of primary antiretroviral
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116 (1-2):201-7.
30
Figure and Tables Legends
Figure 1: Flow chart of the Screening Procedures for Combined HIV antigen and
antibody (fourth-generation) and antibody-only EIAs in a Testing Site in Southern
Brazil.
Figure Sublegend:
4th Gen EIA: Fourth generation enzyme immunoassay (EIA)
3rd Gen EIA: Third generation enzyme immunoassay (EIA)
IFA: Indirect immunofluorescence assay
RT: Rapid test
WB: Western Blot
QNS: Quantity not sufficient
Table 1: Classification of AHI According to Test Results and Follow up
Information.
Table Sublegend:
ID: Specimen identification
4th Gen EIA: Fourth generation enzyme immunoassay (EIA)
3rd Gen EIA: Third generation enzyme immunoassay (EIA)
IFA: Indirect immunofluorescence assay
RT: Rapid test
WB: Western Blot
ND: Not done
Indet: Indeterminate
QNS: Quantity not sufficient
31
Figure 1.
IFA, 3
rd
Gen EIA, RT, WB
178 Antibody
Positive
Established HIV
Infection
5 Antibody
Negative
RNA 1:1
3 RNA Positive
Acute HIV
Infection
2 QNS
Follow-up
RNA 1:12
0 RNA
Positive
750 RNA
Negative
HIV Negative
933
4
th
-Generation EIA Screening
183
4
th
Gen Positive
750
4
th
Gen Negative
1 No Follow-up
Unconfirmed
Acute HIV
Infection
1 Failed to
Seroconvert
HIV Negative
32
Table 1.
ID
4
th
Gen
EIA
3
rd
Gen
EIA
IFA RT WB
HIV
RNA
Subtype
Sero-
conversion
Acute
HIV
A + + - + + + CB Yes Yes
B + - - + ND + QNS
No
information
Yes
C + + + + Indet + B Yes Yes
D
+
+ + - - QNS QNS
No
information
Not
confirmed
E + - - + ND QNS QNS No No
33
Artigo 2
Differences in Transmitted Drug Resistance and
Transmission Efficiency Between HIV-1 Subtypes in
Southern Brazil
Ricardo de Souza
1,2
, Ricardo Diaz
2*
, Kim Page-Shafer
3
, Jennifer Evans
3
, Cecilia
Sucupira
2
, Frederick M. Hecht
4
, Doug Nixon
4
, Oliver Bacon
4
, Lilian Amaral
5
,
Christopher Pilcher
4
.
1
Laboratorio de Pesquisa em HIV/AIDS, Universidade de Caxias do Sul, Caxias
do Sul, Brazil;
2
Laboratório de Retrovirologia, Universidade Federal de São
Paulo, São Paulo, Brazil,
3
Center for AIDS Prevention Studies, University of
California, San Francisco, San Francisco, California,
4
HIV/AIDS Division,
University of California, San Francisco, San Francisco, California and
4
Brazilian
AIDS Programme, Ministry of Health, Brasília, Brazil.
*Corresponding author:
Ricardo S. Diaz
Phone/fax: 55 – 11 - 55712130
Rua Pedro de Toledo,781 – 16º andar
04039-032 São Paulo – SP - Brazil
Running Title: HIV-1 Subtype and Transmitted Drug Resistance
34
Notes:
1. The study was presented in part at the XVII International AIDS Conference,
Mexico City, Mexico, August 3-8, 2008.
2. This research was funded by the following grants from the National Institutes of
Health: R03 AI64037, 2 D43 TW000017.
3. None of the authors have any commercial or other association that might pose
a conflict of interest.
4. We wish to thank the University of California, San Francisco, Center for AIDS
Prevention Studies, U.S. National Institute of Mental Health (NIMH), P30
MH062246; the International Traineeships in AIDS Prevention Studies, U.S.
NIMH, R25MH064712; and the AIDS International Training in Research Program
(AITRP), Fogarty International Center, D43TW00003.
35
Abstract
Background: A growing epidemic by clade C viruses is occurring in the south of
Brazil. We sought
to characterize the prevalence of transmitted HIV-1 among
newly diagnosed, ARV-naive HIV infections in the epicenter of the Brazilian clade
C epidemic.
Methods: We studied 114 consecutive HIV positive samples collected from
October 2006 to August 2007 at 4 anonymous HIV testing sites participating in
Projeto AMPLIAR in Rio Grande do Sul, Brazil. HIV-1 pol regions were
compared, with antiretroviral resistance defined by genotypic analysis. Recent
infections were characterized by the BED CEIA incidence assay.
Results: 15 of 114 (13%) HIV+ were classified as recently infected. 8.3% of
those with recent infection and 11,2% of long term infections showed resistance
to at least one antiretroviral class. Considering pol sequences, 63% of individuals
were infected by clade C viruses, 27% were infected by clade B, and 10% by
non-B non-C strains; proportions of recent infection were similar across subtypes.
The prevalence of primary resistance was 5.5% among clade C infected vs.
19.3% among clade B infected, and vs. 18% among non-B non-C infected
individuals. The odds of resistance were higher among HIV positives with
subtype B and non-B non-C (p=0.03). 41% of patients with resistance mutations
showed resistance to NRTI, 40% had TAMs, 60% had other NAMs, and 8.3%
showed resistance to PIs.
Conclusions: In south Brazil, where clade C infections have over-grown non-C
infections, clade C viruses appear to show a low frequency of primary ARV
resistance. These observations may be explained in part by more recent
introduction of clade C to south Brazil; by less diagnosis or ARV use in
populations affected by clade C (heterosexuals); by the relative inability of
recognizing the correlates of antiretroviral resistance in non-B strains; and/or by
other biologic factors.
36
Introduction
Human Immunodeficiency Virus Type 1 (HIV-1) genetic diversity is a hallmark of
HIV-1 infection and is characterized by three classes and nine major subtypes
distributed in geographical regions of the world.
HIV-1 subtype B has
predominated in the developed Western world and in most Latin American and
Caribbean countries, but an increasing number of non-B infections, including
subtypes F, C, D, and E [1-7], B/F recombinant [8,9] and mixed HIV-1 infections
[10] have been reported in these regions.
Subtype C predominates in southern
Africa [11] and India [12] and is increasing in frequency in China [8,9] and south
Brazil [13,14]. HIV-1 subtype C now accounts for >56% of all circulating
viruses
and is the most commonly transmitted subtype worldwide [15]. HIV-1 subtypes
also differ in its biological properties. These characteristics may influence
infectivity, transmissibility, disease progression and response to antiretroviral
treatment.
Antiretroviral resistance is a major concern in developed and developing
countries due to its potential impact on therapeutic strategies. Theoretically,
Individuals with transmitted drug resistance begin antiretroviral therapy with a
lower genetic barrier to resistance and have a higher risk of virologic failure. On
the other hand, transmitted drug resistant mutations compromise the replicative
capacity of drug resistant virus, and reduce viral transmission efficiency [16-18].
The disproportionate increase in
C viruses relative to other HIV-1 strains
suggests that subtype
C may be more easily transmitted or that it has a higher
level
of transmission "fitness" at the population level.
Brazil’s universal access to antiretroviral therapy (ART) has been remarkably
successful since its introduction in 1996 and as with many antiretroviral treatment
programs drug resistance is emerging. Despite reports of high level of secondary
resistance [19], overall transmitted resistance seems to be low in Brazil [20]
However, high levels of transmitted resistance have been reported in specific
regions of the country [21,22]. The Brazilian National AIDS Program (NAP) in
37
collaboration with the National Institute of Allergy and Infectious Diseases (NIAID)
developed the AMPLIAR program to collect prospective representative data to
monitor and to study the epidemiology and the biology of HIV in the most affected
region of the country. The co-circulation of HIV-1 subtype B, C and recombinant
forms in the south region of Brazil provides a unique opportunity to study the
dynamics of HIV infection within the same population.
In order to assess viral characteristics potentially associated to the increase of
HIV-1 subtype C in southern Brazil we compared the prevalence of transmitted
drug resistance among individuals with newly diagnosed HIV infection caused by
subtype B, C or recombinant strains in the AMPLIAR program. In addition, given
that many drug resistance mutations may compromise the replicative capacity of
the virus and might reduce transmission efficiency compared to wild-type virus
we analyzed the rate of HIV subtype B, C and mosaic infections in patients with
recent HIV-1 and compared it with the prevalence of HIV infections caused by
these clades in a group of individuals who were potential transmitters of HIV-1, all
of whom were living in the same geographical area and seeking HIV testing at
the AMPLIAR clinics.
38
Methods
Setting and Study Design
The AMPLIAR 010 protocol is a diagnostic prospective cohort study conducted in
four HIV voluntary counseling and Testing centers (VCT) in southern Brazil.
Enrollment began in November 2006, and is ongoing.
Study Subjects
We evaluated consecutive individuals presenting for HIV testing at public VCTs in
the state of Rio Grande do Sul covering approximately 45% of the state testing
population. Study population was subjects seeking HIV testing > 16 years old,
newly diagnosed with HIV infection between November 2006 and August 2007
(n=1255) and with no history of previous HIV diagnosis. Eligible individuals are
considered antiretroviral (ARV) naïve.
Testing and results disclosure were carried out according to Brazilian National
AIDS Program (NAP) guidelines. Demographic and risk behavior information
were obtained at each visit from data collection forms, administered individually
by trained counselors during the HIV voluntary counseling and testing
procedures. At each visit laboratory specimens were collected.
Testing Algorithm
HIV testing was carried out using a fourth-generation HIV enzyme-linked
immunoassay (EIA) which detects HIV p24 antigen and anti-HIV (Genscreen Plus
HIVAg-Ab; Biorad, Hercules, CA, USA) simultaneously. For specimens that were
positive on the fourth-generation assay, confirmatory anti-HIV testing using three
different anti-HIV tests were done in parallel: the Axsym HIV1/2 gO MEIA (Abbott
Laboratories, IL, USA), a third-generation EIA; the indirect
immunofluorescenceassay (IFA) from Biomanguinhos (Fiocruz, Rio de Janeiro,
Brazil); and the Determine HIV rapid test (Abbott, Abbott Park, IL, USA).
Western blot (WB) (HIV Blot 2.2, Genelabs Diagnostics, Singapore) was
performed to help resolve cases with inconclusive testing results, and was
39
interpreted according to Centers for Disease Control and Prevention (CDC)
criteria [23].
Specimens found to be negative using the fourth-generation EIA were screened
for HIV RNA (Roche Amplicor Monitor HIV RNA version 1.5; Pleasanton, CA,
USA or Versant HIV 3.0; Bayer Diagnostics, Emeryville, California, USA) in a
1:20 manual pool strategy after the technique of Busch and colleagues [24].
Specimens that were positive on the fourth-generation EIA but negative or
indeterminate on one or more follow-up anti-HIV test were submitted for
individual HIV RNA testing.
HIV laboratory staging and molecular characterization
HIV infection status was determined using all available data. Acute HIV infection
(AHI) was defined by having one or more negative antibody screening tests
results, with infection status supported by a positive HIV RNA result or
documented seroconversion. HIV antibody-positive specimens were further
characterized as recent HIV infections by BED-Capture enzyme immunoassay
[25]. Specimens were tested at the HIV Research laboratory at the Universidade
de Caxias do Sul, located in Caxias do Sul, Rio Grande do Sul, Brazil. Plasma
was stored at -70C for batched genotypic analysis at the Retrovirology
Laboratory at the Federal University of Sao Paulo, located in Sao Paulo, Brazil.
Viral RNA was extracted from plasma and complementary DNA synthesis was
carried out with random primers as described elsewhere [19]. The protease (PR)
and reverse transcriptase (RT) regions were amplified, purified and sequenced
using an automated sequencer. Identification and analysis of antiretroviral
resistance mutations were performed according to suggested consensus [26].
Statistical Analysis
Demographic, behavioral and clinical information (age, gender, schooling, marital
status, exposure category, HIV subtype, number of sex partners in the last 12
months, HIV viral load and HIV laboratory staging) were assessed using chi-
40
square and Fisher’s exact statistical test. Sequences were grouped according to
their assigned HIV-1 subtype. Existing mutations were grouped according to
antiretroviral class nucleoside/nucleotide RT, non-nucleoside RT or protease
inhibitors. The prevalence of HIV resistance mutation was estimated as the
proportion of the samples tested that presented specific mutations. The average
number of mutations and the prevalence of resistant strains in the three classes
was compared by HIV-1 subtypes. Analyses were conducted using STATA
version 10 (Stata corp, Texas).
Transmission Efficiency Analyses
The estimation of transmission efficiency for HIV subtype was represented as a
ratio originated from dividing the rate of a specific HIV clade in the group of
participants with laboratory evidence of recent HIV infection by the corresponding
rate found in the potential transmitter group. This ratio was applied to HIV
subtype groups (B, C and Mosaic). In this experimental analysis, a ratio of 1
means a relative transmission efficiency of 100%. The potential transmitter group
consisted of individuals with long-term HIV infection living in the AMPLIAR
program catchment area who had plasma HIV RNA levels >1000 copies/mL [27].
Ethical considerations
Signed informed consent was obtained for all subjects prior to enrollment into the
study. The protocol was approved by the ethical review board at each clinical
site, as well as by institutional review boards at the Universidade de Caxias do
Sul and the University of California San Francisco.
41
Results
Patient Population
Of the 1255 participants seeking HIV testing, there were a total of 149 newly
detected HIV infections. Newly identified cases represented 11.9% of the testing
population. On the basis of the BED-CEIA and discordant antibody and antigen
results we classified 15 (10%) recent HIV infections and 134 long-term infections
(Figure 1). The use of NAAT in HIV antibody negative specimens did not identify
additional cases of infection. Men compromised 54% of the testing population, of
which 27% were men who have sex with men (MSM). Prevalence of HIV was
higher for MSM (22%, OR 2.5, CI 1.4 to 4.5) than non-MSM. Among non-MSM,
having HIV was positively associated with less schooling (OR 2.0, CI 1.4 to 2.9).
Table 1 summarizes major demographic and behavioral characteristics for
subjects with and without transmitted drug resistance mutations.
Subtyping results
Of the 149 HIV infections, 8 (5.3%) specimens had undetectable HIV RNA load
and were not sequenced. One hundred and fourteen (81%) of the 141 HIV
infected participants with detectable viral load were screened for infecting HIV-1
subtype sequence analyses of partial pol sequences. Of these, 72 (63%) were
subtype C, 31 (27%) were subtype B. The 11 (10%) remaining viral isolates
corresponded to subtype F (n=5), recombinant forms F/B (n=5), and A (n=1).
Having a C vs a non-C virus was associated with being a heterosexual male vs
MSM (OR 3.5, CI 1.0 to 12.4).
Genotyping results
The prevalence of mutations at amino acid sites associated with drug resistance
among newly diagnosed drug naive individuals was present in 12 (10,5%)
subjects (Table 2). Mutations conferring drug resistance were present in 11
subjects with long-term infections and one in a recent HIV infection. We observed
a differential pattern of drug-resistant among HIV clades. Four cases (5,5%) were
42
found in subtype C and 6 cases (19,3%) in subtype B (Figure 2 and Table 2). We
further examined the frequencies of transmitted drug resistance in subtype B and
C and analyzed mutations according to antiretroviral class (ARV). Overall,
mutations conferring resistance to non-nucleoside reverse transcriptase inhibitors
(NNRTI) were the most prevalent, occurring in 7 (58%) cases. Five (41%) had
nucleoside reverse transcriptase inhibitor (NRTI) mutations. Of these, 40% had
Thymidine Analogue-associated Mutations (TAMs) and 60% had other
Nucleoside Analogue-associated Mutations (NAMs). Dual class resistance was
present in one (8,3%) case. Table 2 presents the frequency of mutations
according to ARV class and Table 3 depicts the specific mutations detected
according to subtype.
Transmission Efficiency of HIV-1 Subtypes
The proportion of recently infected subjects with subtype B and C was 19% and
11%, respectively. Overall, transmission efficiency was lower for subtype C
viruses compared to subtype B. Interestingly, when drug resistance viruses were
excluded from the analyses and only wild type virus was considered, the
transmission efficiency rate increased in 25% for subtype B and 16% for subtype
C. The effect of drug resistance on HIV subtype transmission efficiency is shown
in Figure 3A (all viruses) and 3B (wild type only). The sample size was too small
to draw conclusions from the analyses restricted to drug resistant virus (data not
shown).
The reduced transmission efficiency observed in individuals with drug resistant
virus, despite HIV subtype, could be explained by a lower average plasma HIV
RNA load than seen in individuals harboring wild type virus. To test this
hypothesis, we compared the HIV RNA for subtype B and C with and without
drug resistance mutations. Comparisons of plasma HIV RNA (median log
10
copies/mL) load in subjects with subtype B and C did not differ despite the
presence or not of drug resistance mutations. However, we observed a trend
toward lower HIV RNA levels in subjects with subtype C. The median log
10
copies/mL was 4.35 for subtype C and 4.91 for subtype B.
43
In addition, we observed that the transmission efficiency of HIV subtype C was
lower than subtype B (Figure 3A and 3B). This would not explain the expansion
of subtype C seen in our region in the last ten years. In order to understand the
observed lower transmission efficiency seen in subtype C, in the context of an
expanding subtype C epidemic in the region, we estimated the proportion of wild
type B and C viruses in the population and multiplied it by the transmission
efficiency ratio. In the proposed model (Table 4) the risk of infection by subtype
C is greater than for subtype B (8.82% vs 6.54%, p=0.17). This observation could
suggest a competitive advantage of subtype C, at a population level, when
compared to subtype B and partially explain its fast expansion in southern Brazil.
44
Discussion
Primary HIV antiretroviral resistance is an important clinical and public health
problem. The presence of resistant mutants prior to therapy contributes to a rapid
emergence of resistance when therapy is introduced [30]. To date, however, few
data are available as to how subtype diversity may affect HIV transmission and
the role of transmitted drug resistance mutations in this process.
We found a relatively low prevalence of transmitted drug resistance and a
marked differential pattern of drug resistance among HIV clades. In our study,
subtype C was 3 times less likely to harbor drug resistance mutants compared to
subtype B.
Phylogenetic analysis from Brazilian subtype C strains reveals a monophyletic
group strongly related to African and Indian viruses but with a unique signature
sequence, suggesting that subtype C entered the country as a single
introduction, or a small group of genetically related viruses in the mid 1980s [28].
The lower prevalence of drug resistance acquisition in HIV-1 subtype C combined
with the preferential selection to NNRTI mutations in this subtype may be
explained by the recent introduction of this subtype in the south region of Brazil.
Its plausible to speculate that HIV subtype C was less exposed to ARVs than
subtype B.
Acquired drug resistance in antiretroviral experienced patients has been shown to
be differentiated by HIV subtype. In a recent study, subtype C was less prone to
fix mutations associated to drug resistance over time when compared to other B
subtypes from patients experiencing the same type and length of ARV exposure
[28]. It is conceivable that differences in drug resistance acquisition reported in
this study could be, in part, related to the existence of background transmitted
mutations, as reported in our study. In addition, it has been suggested that sexual
transmission of HIV selects for highly fit drug-resistant variants that persist for
years [29].
45
The rapid spread of HIV subtype C viruses [4] worldwide has been interpreted as
indirect evidence for higher transmission efficiency of subtype C strains, although
the viral and host factors underlying subtype C spread are poorly understood.
However, it is increasingly evident that additional (nonhost)
viral factors are also
contributing to the rapid spread of HIV-1
C.
Viral studies indicate that subtype C
has distinct genetic and phenotypic properties that differentiate it from other HIV-
1 subtypes. These genetic properties may influence viral gene expression,
altering the pathogenesis of C viruses.
The transmission efficiency analyses proposed in our study are in agreement with
other studies that observed that many drug resistance mutations compromise the
replicative capacity of the virus [16]. However, specific drug resistance mutations
that particularly affect viral fitness [17,18] might impair transmission in different
ways. The observed smaller rate of transmission efficiency for subtype C is
unexpected. An important factor that might be contributive to this finding is the
viral load of the subjects studied. We observed reduced viral load in individuals
infected with subtype C when compared to subtype B, however not significant.
Our study has some limitations. First, the frequency of HIV-1 drug resistance and
acute/recent infection were too small for meaningful statistical comparisons.
Second, the utilization of NAAT for the screening of HIV negative individuals did
not identify additional cases of infection. This finding may be explained by the
small number of patients screened, by the fact that we used a fourth generation
assay for the screening procedures and alternatively by the late presentation for
testing seen in our study population. Third, we made a series of assumptions to
allow for the transmission efficiency estimation and the calculation of HIV subtype
risk in the population. An additional aspect related to subtype C in Brazil is the
high frequency of amino acid polymorphisms at positions associated to
secondary resistance mutations to protease and reverse transcriptase inhibitors.
Although experimental evidences of the impact of these substitutions in subtype
C viruses on resistance to protease inhibitors (PI) available and widely used in
Brazil is unknown [31] in our study we restricted our analyses to primary
mutations in subtype C.
46
The lower prevalence of transmitted drug resistance of HIV-1 C could be relevant
to surveillance and treatment programs in developing countries, especially in
Africa. In conclusion, In the South region of Brazil HIV-1 non B clades are the
majority (73%), with a growing prevalence of clade C infected individuals (63%).
The prevalence of primary resistance is relatively low in this region with the
absence of multi-class resistance strains. These observations may be explained
in part by a more recent introduction of clade C in the south region of Brazil; by
less diagnosis or ARV use in populations affected by clade C (heterosexuals); by
the relative inability of recognizing the correlates of antiretroviral resistance in
non-B strains; and/or by other biologic factors.
47
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Figures and tables legends
Figure 1: Testing algorithm for detecion of HIV-1 infection and transmitted
antiretroviral resistance in the AMPLIAR Program
RNA - : undetectable viral load
Ab - : HIV antibody negative
Ab + : HIV antibody positive
Figure 2:
Overall prevalence of transmitted drug resistance mutations in
newly diagnosed antiretroviral naive individuals in South Brazil (A) and
prevalence of drug resistance mutations for HIV subtype B and C according
for NRTI and NNRTI (B).
NRTI: nucleoside reverse transcriptase inhibitor
NNRTI: non nucleoside reverse transcriptase inhibitor
Figure 3: : Ratio of HIV-1 infection with both (A) wild-type (B) and drug
resistance virus among subjects with recent HIV-1 and subjects who are
potential transmitters by HIV-1 subtype.
Table 1: Patient characteristics and prevalence of risk factors for primary
resistance mutations in antiretroviral naive testing seekers in South Brazil
OR: odds ratio
P: p-value
MSM: man who have sex with man
MSW: man who have sex with woman
52
Table 2: Comparison of primary resistance mutations by HIV subtypes in
newly diagnosed antiretroviral naive individuals in South Brazil
NRTI: nucleoside reverse transcriptase inhibitor
NNRTI: non nucleoside reverse transcriptase inhibitor
PI: protease inhibitor
a. One patient was infected with a NRTI and PI resistant virus
b. Comparison NRTI and NNRTI only.
Table 3: HIV drug resistance mutations detected among newly identified
HIV cases by subtypes
a: Recent HIV infection
NRTI: nucleoside reverse transcriptase inhibitor
NNRTI: non nucleoside reverse transcriptase inhibitor
PI: protease inhibitor
Table 4 : Estimated proportion of HIV subtype B and C transmission in the
population.
Sublegend:
• *Assumes that 4.3 of Subtype B and 5% of the population of Subtype C
has 0% transmission efficiency due to drug resistance.
• **Statistical test for the comparison of proportions.
53
Figure 1.
1255HIVTesting
149HIVAb+1106HIVAb‐
1106RNA‐/Ab‐
HIVNegati ve
15RecentInfections 134Long‐term
Infections
15Sequence sAnalysed
1ResistantCase 11ResistantCases
0NotSequenced 99Se quencesAnalyse d 35 Not
Sequenced
54
Figure 2.
A
B
55
Figure 3
A. Overall
0,10
0,24
0,12
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
BCMosaics
Subtype
Percentage of Subjects
0,00
0,05
0,10
0,15
0,20
0,25
0,30
Ratio of Recently Infected
to Potential Transmitters
Recently infected Potential tramistters Ratio
B. Wild type only
0,00
0,30
0,14
0%
20%
40%
60%
80%
100%
120%
B C Mosaics
Subtype
Percentage of Subjects
0,00
0,05
0,10
0,15
0,20
0,25
0,30
0,35
Ratio of Recently Infected
to Potential Transmitters
Recently infected Potential tramistters Ratio
56
Table 1.
Mutation n (%)
Characteristic
Total
n (%)
No Yes
OR(95% CI) P
Age (years)
18 – 32 55 (48) 48 (87) 7 (13) 1.00
33 – 65 59 (52) 54 (91) 5 (9) 0.67 (0.2 – 2.1) 0.48
Gender
Male 62 (54) 56 (90) 6 (10) 1.00
Female 52 (46) 46 (88) 6 (12) 1.22 (0.3 – 3.7) 0.72
Schooling
< 8 years 55 (49) 51 (93) 4 (7) 1.00
> 8 years 58 (51) 50 (86) 8 (14) 2.65 (0.7 – 9.0) 0.11
Marital status
Married 60 (53) 52 (87) 8 (13) 1.00
Not married 54 (47) 50 (92) 4 (8) 0.57 (0.1 – 1.8) 0.35
Exposure category
Man who have sex with man (MSM) 17(16) 16 (94) 1 (6) 1.00
Man who have sex with woman (MSW) 41(39) 36 (88) 5 (12) 2.74 (0.3 – 27.7) 0.37
Heterosexual women 47(45) 41 (87) 6 (13) 2.80 (0.3 – 24.6) 0.35
Have an HIV infected partner
No 91 (80) 81 (87) 10 (13) 1.00
Yes 23 (20) 21 (91) 2 (9) 1.09 (0.2 – 4.2) 0.90
Ulcer or sores on genitals (last 12
months)
No 102 (89) 92 (90) 10 (10) 1.00
Yes 12 (11) 10 (83) 2 (17) 2.75 (0.6 - 11) 0.17
Number of sex partners in the last year
0 – 1
> 2
65 (57)
49 (43)
58 (89)
44 (90)
7 (11)
5 (10)
1.00
0.99 (0.3 – 3.1)
0.99
Unprotected sex events in the previous
3 months
Previous HIV test
No 37 (33) 34 (92) 3 (8) 1.00
Yes 76 (67) 67 (88) 9 (12) 1.25 (0.4 – 4.3) 0.72
HIV infection stage
Acute & ecent 15 (13) 14 (93) 1 (7) 1.00
Chronic 99 (87) 88 (89) 11 (11) 2.12 (0.3 – 17.5) 0.49
HIV subtype
B 31 (27) 25 (81) 6 (19) 1.00
C 72 (63) 68 (95) 4 (5) 0.47 (0.1 – 1.7) 0.25
Mosaic 11 (10) 9 (82) 2 (18) 1.95 (0.4 – 10.0) 0.42
RNA viral load (copy numbers per mL)
<1000 9 (8) 9 (100) 0 (0) --
50,000 -1000 55 (51) 48 (87) 7 (13) 1.00
>50,000 44 (41) 39 (88) 5 (12) 1.08 (0.3 – 3.4) 0.90
57
Table 2.
Antiretroviral class
n (%)
HIV subtype
No. of patients
with any mutation
n (%)
NRTI NNRTI PI
P
B (n=31) 6 (19,3) 5 (80)
a
1 (20) 1
a
0.03
b
C (n=72) 4 (5,5) 0 (0) 4 (100)
0
Mosaics (n=11) 2 (18,0) 0 (0) 2 (100) 0
58
Table 3.
HIV Subtype NRTI NNRTI PI
1 B M41L L90M
2 B M184V
3
a
B K103N
4 B V118I
5 B L210W
6 B T69NST
7 C K103N
8 C K103N
9 C V108I
10 C V108I
11 mosaic K103N
12 mosaic K103N
59
Table 4.
HIV
subtype
HIV
subtype
prevalence
%
Drug
resistance
prevalence
%
Potential
transmitters of
resistant virus *
Potential
transmittters
of wild-type
virus
Transmission
efficiency
ratio of
wild-type
virus
Risk P**
B 27 19
27% x 0.19=
5.2%
27 – 5.2 =
21.8%
0.30
21.8% x 0.3 =
6.54
C 63 5
63% x .055=
3.4%
63 – 3.4 =
59.6%
0.14
59.6% x 0.14=
8.34
.17
Mosaic 10 18
10 x 0.18=
1.8%
10 - 1.8=
8.2%
0.00
8.2% x 0=
0
60
CONCLUSÕES
1. Estratégias laboratoriais que utilizam ELISA de quarta geração
apresentaram alto desempenho analítico em nosso estudo.
2. A prevalência de infecções agudas na população que busca de
testagem e aconselhamento para o HIV na região sul foi de 1.7%.
3. Indivíduos que se apresentam para testagem na fase aguda da
infecção resultam em casos falso-negativos no esquema de
triagem que utiliza apenas ELISAS de terceira geração. A utilização
de testes confirmatórios baseados na detecção de anticorpos não
soluciona o problema.
4. Na região sul do Brasil o HIV-1 subtipo C e formas circulantes
recombinantes (FRC), juntos, são responsáveis por 78% das
infecções, com o subtipo C representando 63% das infecções.
5. A prevalência da resistência primária é relativamente baixa na
região sul como também a existência de cepas multi- resistentes.
6. A presença de mutações de resistência varia de acordo com o
subtipo. O HIV subtipo C está associado, em nosso estudo,
exclusivamente a mutações associadas a classe de anti-retrovirais
inibidores da transcriptase reversa não nucleosídeos (ITRNN).
7. Por meio da utilização de cálculos epidemiológicos como modelo
para estimar a expansão dos subtipos circulantes na região, pode-
se estimar a vantagem competitiva do HIV subtipo C a nível
populacional.
61
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