Objectives: To determine the prevalence of acute HIV infection (AHI) within the HIV-seronegative adult population presenting with reported fever in a district hospital in southern Mozambique and evaluate clinical, immunological and virological parameters of AHI.
Design: This is a prospective observational study.
Methods: Three hundred and forty-six adults presenting with reported fever at an outpatient ward at the Manhiça District Hospital in Mozambique were screened for AHI by HIV rapid serology testing, followed by HIV-RNA testing in HIV-seronegative individuals. Plasma from HIV-seronegative patients was pooled in the ratio of 1: 5 for HIV-RNA testing. Whole blood was used for Plasmodium falciparum rapid test determination at screening visit. Follow-up visits at day 7, 4 and 10 months included clinical examination, HIV serotesting and assessment of HIV-RNA, CD4 cell counts and percentage of activated CD8 T cells.
Results: HIV serotesting revealed that 37.8% (95% confidence interval 32.7–43.2) of the adults had previously undiagnosed established HIV infection. Among the HIV-seronegative patients, 3.3% (95% confidence interval 1.3–6.7) were found to have AHI as demonstrated by positive HIV-1 RNA testing. Median HIV-1 RNA levels at diagnosis of AHI were 6.21 log10 copies/ml (interquartile range 5.92–6.41) and significantly higher than median HIV-RNA load at 4 months. At day 7 after screening, patients showed a median CD4 cell count of 384 cells/μl (interquartile range 239–441) and a median percentage of activated CD8 T cells of 68.4% (interquartile range 59.6–87.8).
Conclusion: Of patients reporting with fever, 3.3% were shown to be potentially due to AHI. High prevalence of AHI in southern African populations may warrant investigation of tools and target populations for AHI screening as a novel way to address HIV prevention.
aBarcelona Centre for International Health Research (CRESIB), Hospital Clinic, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Universtitat de Barcelona, Barcelona, Spain
bManhiça Health Research Centre (CISM), Manhiça, Mozambique
cInstituto Nacional de Saude, Ministerio de Saude, Maputo, Mozambique
dCentro de Saúde de Manhiça, Manhiça, Mozambique.
Received 7 October, 2009
Revised 17 November, 2009
Accepted 20 November, 2009
Correspondence to Denise Naniche, PhD, MPH, Barcelona Centre for International Health (CRESIB), IDIBAPS/Hospital Clinic, Rossello, 132 4°, Barcelona E-08036, Spain. Tel: +34 932275706; fax: +34 932279853; e-mail: firstname.lastname@example.org
Acute HIV infection (AHI) corresponds to the initial phase of HIV infection in which virus is actively replicating but seroconversion has not yet occurred . During AHI, high levels of plasma HIV-1 RNA are accompanied by high levels of virus in genital secretions, reportedly higher for subtype C viruses present in southern Africa [2,3]. It has thus been suggested that AHI and the early months of HIV infection may contribute disproportionally to the transmission of HIV and constitute a major motor of the HIV pandemic [4–6]. AHI usually lasts for 3–4 weeks , and approximately half of the patients develop nonspecific flu-like or mononucleosis-like symptoms, including mainly fever, as well as myalgia, arthralgia, rash and sore throat. As a very nonspecific syndrome, symptomatic AHI is likely to be underreported. Furthermore, the remaining AHIs are asymptomatic and go unnoticed, making AHI very challenging to diagnose.
It has been difficult to identify AHI in Europe and other developed countries, thus it is thought to be even more challenging in Africa. Most studies [4,8–10] seeking to assess AHI have targeted sexually transmitted diseases (STDs) clinics. However, outpatient triage may be a relevant place to screen for AHI in parts of Africa with a high prevalence of malaria, where people with signs of fever are accustomed to presenting at the health post for malaria testing. Although approximately 25–35% of fevers in outpatient consultations are due to malaria , most of the remaining fevers have unknown cause, and in a population with high HIV prevalence, a proportion of fevers could be due to symptomatic AHI. The main objective of the study was to assess the prevalence of AHI in patients presenting with reported fever at an outpatient triage in southern Mozambique and to characterize the cases of AHI. Secondarily, clinical presentation and immune activation were assessed in order to shed light on characteristics of AHI subtype C infections.
Study population and visits
The participants were recruited between June and July 2008 at the Manhiça District Hospital (MDH) in Manhiça, southern Mozambique. The Centro de Investigação em Saúde de Manhiça (CISM) has been conducting continuous demographic surveillance in the district since 1996 that covered a population of 82 000 persons at the time of this study. Adults over 18 years old presenting with reported fever at the MDH outpatient ward were invited to participate if they fulfilled the following criteria: permanent residents of the demographic surveillance study area, acceptance of HIV testing and not in follow-up at the HIV day hospital.
Participants were considered for HIV-1 RNA testing only if HIV-1 serology was negative by antibody rapid test (Determine; Abbott Laboratories, Abbott Park, Illinois, USA and Unigold; Trinity Biotech Co., Wicklow, Ireland). Patients diagnosed with AHI (defined by a negative serology and positive HIV-1 RNA test) received their results 7 days after enrolment and were invited for follow-up visits at 4 and 10 months for CD4 cell count and HIV-1 RNA viral load assessment. Basic clinical and epidemiological data were recorded at the screening visit. For the AHI patients returning for 7-day visit, repeat HIV rapid testing confirmed seronegativity and a more extensive clinical questionnaire was given to describe clinical features.
Subsequent seroconversion of AHI patients was later ascertained by serotesting, and all HIV-positive individuals were referred for clinical management according to national guidelines.
Written informed consent was obtained from patients prior to participation. The study protocol was reviewed and approved by the Mozambican National Bioethics Committee and the Hospital Clinic of Barcelona Ethics Review Committee.
HIV-1 RNA levels were determined from cryopreserved plasma samples with the commercial Roche Amplicor Monitor, version 1.5 (Roche Diagnostics, Basel, Switzerland) technique for amplification and quantification of HIV-1 RNA. Lower limit of detection was 400 copies/ml.
A plasma pooling scheme was adapted from previous studies [12,13]. Briefly, plasma samples were pooled in the ratio of 1: 5 in a 200-μl aliquot. If any pool was found to be positive, individual specimens were re-tested.
Blood finger prick was used for the Plasmodium falciparum rapid test determination (ICT Diagnostics, Cape Town, South Africa) at screening according to manufacturer's instructions.
CD4 cell counting was performed after staining with labelled antibodies: CD4, CD3, CD8 and CD45 in TruCount tubes (Becton Dickinson Biosciences, San Jose, California, USA). Activated CD8 cells were identified after staining with CD3, CD8, CD38 and human leucocyte antigen (HLA)-DR antibodies. Samples were assessed by flow cytometry on a FACSCalibur (Becton Dickinson Biosciences).
Proportions for categorical variables were compared using the Pearson chi-squared test. The Wilcoxon rank-sum test was used to compare medians of continuous variables with nonnormal distribution and the Wilcoxon signed-rank test to compare paired groups. Statistical analyses were performed using STATA version 9 (STATA Corp., College Station, Texas, USA).
Characteristics of the study population
Of the 472 individuals presenting at an outpatient ward between June and July 2008 with reported fever and fulfilling recruitment criteria, 73.3% (346/472) gave informed consent to participate in the study. HIV seroprevalence based on rapid testing was found to be 37.8% [95% confidence interval (CI) 32.7–43.2] (Table 1).
Ages ranged from 18 to 86 years, with a median age of 33 years [interquartile range (IQR) 27–44 years]. Table 1 provides information about general characteristics of patients according to HIV serostatus. In the HIV serology-negative group, the median age for men was significantly younger than for women [median 30 years (IQR 24–43) versus median 38 years (IQR 26–51), respectively, P = 0.021].
The main reasons for consultation accompanying fever in both HIV-seropositive and HIV-seronegative patients were arthralgias and cough followed by diarrhoea and sore throat. Although all patients reported fever in the previous 24 h, only 9.7% of the HIV-seronegative individuals had fever on the day of attendance (Table 1).
The prevalence of P. falciparum malaria as detected by rapid testing was 16.1% (95% CI 12.5–20.4) in the overall group (n = 346). However, after stratifying by HIV serostatus, prevalence of malaria was significantly higher in HIV-seropositive patients than in HIV-seronegative individuals (21.4 and 13%, respectively, P = 0.041, χ2 test).
Prevalence of acute HIV infection
Among the 215 HIV-seronegative individuals presenting with reported fever, results for HIV-1 RNA levels were available for 211 individuals. HIV-1 RNA was detected in seven of 211 specimens giving an AHI prevalence of 3.3% (95% CI 1.3–6.7). None of the AHI cases were diagnosed with malaria. HIV-1 RNA level in AHI patients was very high at diagnosis, with a median of 6.21 log10 copies/ml (IQR 5.92–6.41 log10 copies/ml) (Table 2).
Evaluation of clinical, immunological and virological parameters of acute HIV infection
Six of the seven AHI cases identified were followed for 10 months; one was lost to follow-up after screening.
At 7-day postscreening visit, a more detailed clinical questionnaire showed that four of six of the AHI patients presented with symptoms compatible with mononucleosis-like symptoms and all six patients complained of sore throat (Table 2). Gastrointestinal symptoms were present in three of six patients, including nausea, diarrhoea or abdominal pain. Signs of sexually transmitted infection (STI) were present in four of six individuals and included vaginal discharge, genital ulcer and dysuria.
Follow-up CD4 cell count measurements at day 7 after screening showed diverse counts among individuals with a median CD4 cell count of 384 cells/μl (IQR 239–441 cells/μl). At 4 months, median CD4 cell count was 381 cells/μl (IQR 365–428 cells/μl), with an interpatient heterogeneity reduced as compared with 7 days. At 10 months after screening, CD4 cell counts increased to a median of 519 cells/μl (IQR 416–582 cells/μl). The median ratio of CD4: CD8 significantly increased from 0.38 to 0.84 between day 7 and 4 months (Table 2, P = 0.043, Wilcoxon signed-rank), primarily not only due to a significant decrease in percentage of CD8 cells but also to an increase in percentage of CD4 T cells.
The percentage of activated CD8 T cells, at day 7 after screening, showed a median of 68.4% CD8 T cells (IQR 59.6–87.8 cells) expressing CD38 and HLA-DR (Table 2).
Follow-up measures of plasma HIV-1 RNA at 4 and 10 months showed a median viral load at 4 months of 5.43 log10 copies/ml (IQR 4.72–5.83 log10 copies/ml), significantly lower than that observed at baseline (Table 2, P = 0.04, Wilcoxon signed-rank) At 10 months, median HIV-1 RNA was 5.02 log10 copies/ml (IQR 4.85–5.34 log10 copies/ml). As in the case of CD4 cell counts, HIV-1 RNA level at 10 months was not significantly different from values at 4 months.
Our findings show a high prevalence of AHI (3.3%) among patients presenting with reported fever in an outpatient ward from a rural area in southern Mozambique with high HIV burden. These symptomatic AHI patients all had extremely high viral loads, and the percentage of activated CD8+ T lymphocytes observed was similar to that reported in chronically HIV-infected African patients .
There are studies from the United States assessing prevalence of AHI in STD  clinics as well as in clinic-based populations other than STD [16–18]. AHI prevalence in patients reporting undifferentiated viral symptoms in an urgent care facility [16,17] or presenting with mononucleosis-like symptoms  was reported to range between 0.3 and 1%. In the African context, there are studies assessing prevalence of AHI in attendees of STD clinics but not from general outpatient wards. In a Malawi STD clinic, prevalence of AHI was reported to be 2.4% among HIV-seronegative men and women and 4.5% among HIV-seronegative men [9,10]. In the current study, we suggest the relevance of screening for AHI in HIV-seronegative patients with reported fever in outpatient triages in southern Africa after observing 13% of fevers potentially attributable to malaria and 3.3% to AHI. Mononucleosis-like symptoms and sore throat, although nonspecific, could be relevant syndromes to target for identification of AHI, particularly, as with a slightly more detailed questionnaire such as that performed at day 7 in our study, mononucleosis-like symptoms and sore throat were reported in four of six and six of six AHI patients, respectively.
The Manhiça district in southern Mozambique is quite representative of southern Africa, with an observed HIV seroprevalence of 29% in 2005 in pregnant women  and HIV infection predominantly of subtype C [20,21]. Our results and the few prospective studies of AHI in southern Africa [10,22,23] prompt expanded studies investigating relationships among AHI symptoms, immune activation, prognosis and transmission during AHI, particularly for HIV-1 subtype C in a population with higher baseline immune activation .
HIV-infected individuals are considered hyperinfectious from the onset of AHI up to 6 weeks thereafter. Furthermore, studies in both Uganda  and Quebec  have suggested that 50% of onward transmissions occur in the first 6 months after infection. AHI screening in the United States, with emphasis on targeting high-risk populations, has been integrated into various public health testing schemes and considered to be cost-effective in ensuring care and slowing transmission [12,15]. Plasma pooling algorithms for HIV-1 RNA testing have shown that the cost can be reduced to 2–3 US$/specimen [13,15,27]. The main limitation of HIV-1 RNA testing in resource-poor countries is the requirement for equipment, trained technicians and rapid turnaround time. However, the necessity for HIV-1 RNA monitoring is growing as the rollout of antiretroviral treatment in sub-Saharan Africa expands. Research efforts have thus increasingly been focusing on evaluating simplified methods for HIV quantification through ultrasensitive p24 or HIV-1 RNA detection from dried blood spots [9,28,29]. These tools could render AHI screening economically and technically feasible in some resource-poor settings.
Larger studies comparing AHI in various high-risk populations in sub-Saharan Africa are necessary to identify the most adapted cost-effective portals of entry into the health system. This could lead to focusing a portion of the prevention effort on AHI testing, counselling and risk modification during the earliest stage of HIV infection. In light of the relatively low impact of current HIV prevention efforts on HIV transmission in sub-Saharan Africa, research into the development of tools, interventions and the identification of target populations for AHI screening may be a novel way to address prevention.
The authors are grateful to all the patients for their participation in the study and to the continued dedication of the staff at the MDH as well as field, clinic and data management staff at the Centro de Investigaçaõ em Saúde de Manhiça, Mozambique. The authors are particularly grateful to Alface Boaventura, Apollinario Nzango, Nelito José, Lucas Nhatumbo and Elsa Banze for their contribution to logistics, patient visits, follow-up and laboratory.
Financial support was received from the Spanish Ministry of Health (grant PI071312). The Centro de Investigaçaõ em Saúde de Manhiça receives core founding from the Spanish Agency for International Cooperation and the Voluntary Counselling and Testing (VCT) clinic and day hospital from the Generalitat de Catalunya. C.S. was supported by a grant from Spanish Ministry of Health (PI070233) and D.N. was supported by a grant from the Spanish Ministry of Education and Science (Ramon y Cajal).
1. Henrard DR, Phillips JF, Muenz LR, Blattner WA, Wiesner D, Eyster ME, Goedert JJ. Natural history of HIV-1 cell-free viremia. JAMA 1995; 274:554–558.
2. Dyer JR, Kazembe P, Vernazza PL, Gilliam BL, Maida M, Zimba D, et al
. High levels of human immunodeficiency virus type 1 in blood and semen of seropositive men in sub-Saharan Africa. J Infect Dis 1998; 177:1742–1746.
3. Pilcher CD, Joaki G, Hoffman IF, Martinson FE, Mapanje C, Stewart PW, et al
. Amplified transmission of HIV-1: comparison of HIV-1 concentrations in semen and blood during acute and chronic infection. AIDS 2007; 21:1723–1730.
4. Pilcher CD, Tien HC, Eron JJ Jr, Vernazza PL, Leu SY, Stewart PW, et al
. Brief but efficient: acute HIV infection and the sexual transmission of HIV. J Infect Dis 2004; 189:1785–1792.
5. Quinn TC, Wawer MJ, Sewankambo N, Serwadda D, Li C, Wabwire-Mangen F, et al
. Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. N Engl J Med 2000; 342:921–929.
6. Cohen MS, Pilcher CD. Amplified HIV transmission and new approaches to HIV prevention. J Infect Dis 2005; 191:1391–1393.
7. Schacker T, Collier AC, Hughes J, Shea T, Corey L. Clinical and epidemiologic features of primary HIV infection. Ann Intern Med 1996; 125:257–264.
8. Bollinger RC, Brookmeyer RS, Mehendale SM, Paranjape RS, Shepherd ME, Gadkari DA, Quinn TC. Risk factors and clinical presentation of acute primary HIV infection in India. JAMA 1997; 278:2085–2089.
9. Fiscus SA, Pilcher CD, Miller WC, Powers KA, Hoffman IF, Price M, et al
. Rapid, real-time detection of acute HIV infection in patients in Africa. J Infect Dis 2007; 195:416–424.
10. Pilcher CD, Price MA, Hoffman IF, Galvin S, Martinson FE, Kazembe PN, et al
. Frequent detection of acute primary HIV infection in men in Malawi. AIDS 2004; 18:517–524.
11. WHO. World malaria report. Roll back malaria and UNICEF
. Geneva: WHO; 2005.
12. Pilcher CD, McPherson JT, Leone PA, Smurzynski M, Owen-O'Dowd J, Peace-Brewer AL, et al
. Real-time, universal screening for acute HIV infection in a routine HIV counseling and testing population. JAMA 2002; 288:216–221.
13. Quinn TC, Brookmeyer R, Kline R, Shepherd M, Paranjape R, Mehendale S, et al
. Feasibility of pooling sera for HIV-1 viral RNA to diagnose acute primary HIV-1 infection and estimate HIV incidence. AIDS 2000; 14:2751–2757.
14. Eggena MP, Barugahare B, Okello M, Mutyala S, Jones N, Ma Y, et al
. T cell activation in HIV-seropositive Ugandans: differential associations with viral load, CD4+
T cell depletion, and coinfection. J Infect Dis 2005; 191:694–701.
15. Sherlock M, Zetola NM, Klausner JD. Routine detection of acute HIV infection through RNA pooling: survey of current practice in the United States. Sex Transm Dis 2007; 34:314–316.
16. Clark SJ, Kelen GD, Henrard DR, Daar ES, Craig S, Shaw GM, Quinn TC. Unsuspected primary human immunodeficiency virus type 1 infection in seronegative emergency department patients. J Infect Dis 1994; 170:194–197.
17. Pincus JM, Crosby SS, Losina E, King ER, LaBelle C, Freedberg KA. Acute human immunodeficiency virus infection in patients presenting to an urban urgent care center. Clin Infect Dis 2003; 37:1699–1704.
18. Rosenberg ES, Caliendo AM, Walker BD. Acute HIV infection among patients tested for mononucleosis. N Engl J Med 1999; 340:969.
19. Naniche D, Lahuerta M, Bardaji A, Sigauque B, Romagosa C, Berenguera A, et al
. Mother-to-child transmission of HIV-1: association with malaria prevention, anaemia and placental malaria. HIV Med 2008; 9:757–764.
20. Bellocchi MC, Forbici F, Palombi L, Gori C, Coelho E, Svicher V, et al
. Subtype analysis and mutations to antiviral drugs in HIV-1-infected patients from Mozambique before initiation of antiretroviral therapy: results from the DREAM programme. J Med Virol 2005; 76:452–458.
21. Lahuerta M, Aparicio E, Bardaji A, Marco S, Sacarlal J, Mandomando I, et al
. Rapid spread and genetic diversification of HIV type 1 subtype C in a rural area of southern Mozambique. AIDS Res Hum Retroviruses 2008; 24:327–335.
22. Mlisana K, Auld SC, Grobler A, van Loggerenberg F, Williamson C, Iriogbe I, et al
. Anaemia in acute HIV-1 subtype C infection. PLoS One 2008; 3:e1626.
23. Novitsky V, Woldegabriel E, Kebaabetswe L, Rossenkhan R, Mlotshwa B, Bonney C, et al
. Viral load and CD4+
T-cell dynamics in primary HIV-1 subtype C infection. J Acquir Immune Defic Syndr 2009; 50:65–76.
24. Kassu A, Tsegaye A, Petros B, Wolday D, Hailu E, Tilahun T, et al
. Distribution of lymphocyte subsets in healthy human immunodeficiency virus-negative adult Ethiopians from two geographic locales. Clin Diagn Lab Immunol 2001; 8:1171–1176.
25. Wawer MJ, Gray RH, Sewankambo NK, Serwadda D, Li X, Laeyendecker O, et al
. Rates of HIV-1 transmission per coital act, by stage of HIV-1 infection, in Rakai, Uganda. J Infect Dis 2005; 191:1403–1409.
26. Brenner BG, Roger M, Routy JP, Moisi D, Ntemgwa M, Matte C, et al
. High rates of forward transmission events after acute/early HIV-1 infection. J Infect Dis 2007; 195:951–959.
27. Westreich DJ, Hudgens MG, Fiscus SA, Pilcher CD. Optimizing screening for acute human immunodeficiency virus infection with pooled nucleic acid amplification tests. J Clin Microbiol 2008; 46:1785–1792.
28. Fiscus SA, Cheng B, Crowe SM, Demeter L, Jennings C, Miller V, et al
. HIV-1 viral load assays for resource-limited settings. PLoS Med 2006; 3:e417.
29. Marconi A, Balestrieri M, Comastri G, Pulvirenti FR, Gennari W, Tagliazucchi S, et al
. Evaluation of the Abbott real-time HIV-1 quantitative assay with dried blood spot specimens. Clin Microbiol Infect 2009; 15:93–97.