For HIV prevention activities to be effective in reducing transmission by infected individuals, it is critical to intervene as close to the beginning of an individual's natural duration of infectiousness as possible. For this reason, detection of early HIV infections has been named as a priority by the WHO and UNAIDS  as well as by the US Centers for Disease Control and Prevention (CDC; Atlanta, Georgia, USA) . Diagnosing acute HIV infections – in the antibody-negative ‘window period' – can be particularly important. For public health, diagnosing acute infections maximizes the potential impact of critical prevention services [3–5] by allowing these services to be applied at the time of maximal transmission potential [6–10]. For the individual diagnosed with acute HIV, antiretroviral treatment can reduce high viral loads in blood and genital secretions , and can lead to augmented HIV-specific immune responses [11–14] and reduced viral load ‘set point’ . In developed countries, a majority of HIV infected individuals may, in fact, present for medical care at the time of acute HIV infection . Because the diagnosis is nearly always missed, however, most infected individuals remain ignorant of their HIV status until after the onset of AIDS .
Diagnosis of acute HIV infection requires a positive viral test and a negative HIV antibody test. HIV RNA can be detected in peripheral blood as early as 9 days after infection, and HIV p24 antigen can be detected a few days later [17,18]. Modern ELISA and latex-agglutination based HIV antibody tests that are used commonly for voluntary counseling and testing (VCT), remain negative until approximately 22 days after infection [18,19], although such tests are virtually 100% sensitive after this time.
Partly because the window period for detection of HIV RNA from HIV antibody-negative patients is so brief, it was previously assumed that the prevalence of antibody-negative HIV infections was extremely low in clinical testing populations. However, studies in some HIV testing populations  have detected surprising numbers of cases of acute HIV infection. Data on the prospective detection of acute HIV infection in general or specific clinical populations are scarce. Such information might be particularly important for prevention activities where the HIV epidemic is greatest. Malawi has an overall HIV antibody seroprevalence of approximately 20%, with prevalence varying markedly according to geography and age stratum . Because both inflammatory and ulcerative sexually transmitted diseases (STD) are strongly associated with HIV acquisition , we reasoned that acute HIV infection would be most readily found in Malawi among patients with acute STD. We used a highly specific  specimen pooling/HIV RNA testing approach to compare the prevalence of acute HIV infection among dermatology and STD outpatients at Lilongwe Central Hospital, Malawi.
Patients and methods
Participants and protocol
We conducted a retrospective, cross sectional study of patients screened for participation in a urethritis treatment trial  in two outpatient clinics at the Lilongwe Central Hospital in Lilongwe, Malawi between January 2000 and June 2001. All consecutive male outpatients aged 18 years or more with symptoms of acute urethritis or genital ulceration presenting for care at the STD clinic were eligible and screened for enrollment in the trial. Consecutive male patients presenting to dermatology clinic without complaints of urethritis or genital ulceration during the same time period also were eligible for participation in the trial. For all eligible subjects, HIV counseling and testing (below) was performed following informed consent. HIV RNA testing was done retrospectively in batch at completion of the study and results were provided to patients. The study protocol was approved by the UNC Committee on the Protection of the Rights of Human Subjects and the Malawi College of Medicine Health Sciences Research Committee. The study was done in compliance with the Declaration of Helsinki.
Demographic information, sexual-behavioral and medical histories were collected on all patients at study entry. Targeted physical exams, including detailed genital exam, lymph node survey and examination of the skin and oropharynx, were performed. Genital ulcer disease was defined as having active visible genital ulceration, and adenopathy was defined by enlarged, tender and/or erythematous nodes as noted on physical exam.
Urethral swabs were obtained for Gram stain and Trichomonas vaginalis culture (InPouch TV, BioMed Diagnostics, San Jose, California, USA). Urine was obtained for measurement of trichomonad DNA , Chlamydia trachomatis and Neisseria gonorhoeae ligase chain reaction (Abbott Diagnostics, Abbott Park, Illinois, USA). Syphilis serology was obtained (Rapid Plasma Reagin test; Becton Dickinson, Franklin Lakes, New Jersey, USA). Syndromic treatment for STD followed official Malawi guidelines.
Patients were screened for evidence of acute or established HIV infection using a protocol of sequential antibody and HIV RNA testing. HIV antibody screening used a latex agglutination based rapid test (100 Capillus HIV test; Cambridge Diagnostics, Cambridge, UK) with ELISA confirmation (Genetics Systems HIV-1/2 Peptide EIA; BioRad Hercules, California, USA) according to Malawi guidelines. HIV antibody-negative or -indeterminate specimens were manually pooled and screened for the presence of HIV RNA using a 50 : 10 : 1 pyramid scheme in a modification of a protocol previously described [20,25]: 200-μl aliquots from individual specimens were pooled 1 : 10; 200-μl aliquots from each of the resulting 2.0m-l ‘intermediate’ pools were then combined 1 : 5 to make master pools representing 50 specimens. Master pools were screened initially; if RNA positive, the intermediate 10-specimen pools contributing to this master pool were screened. Only RNA-positive intermediate pools were deconstructed for testing of individual specimens. EDTA-preserved specimens were screened by Roche Monitor 1.5 Ultrasensitive (Roche Diagnostics, Branchburg, New Jersey, USA) for master pools [lower limit of detection (LL), > 1000 copies/ml pooled serum with 1 : 50 dilution] and intermediate pools; then standard Roche Monitor 1.5 (LL, 200 copies/ml) individually. Specimens preserved in heparin were screened using NucliSens (NucliSens HIV-1 QT; bioMeriéux, Durham, North Carolina, USA; LL, 400 copies/ml individually or > 20 000 in master pools). In addition to these antibody-negative specimens, HIV RNA levels were determined individually on 58 consecutive HIV antibody-positive patients by NucliSens.
All specimens that were found to be HIV antibody-negative but repeatedly HIV RNA-positive individually were submitted for confirmatory antibody testing using an extremely sensitive  third generation recombinant peptide HIV ELISA (HIVAB HIV 1/2 ELISA; Abbott Diagnostics) and HIV Western blot (BioRad).
Results were reclassified for initially HIV antibody-negative but HIV RNA-positive patients following this confirmatory testing. Acute HIV infection was defined as either: (i) HIV antibody negativity at confirmatory testing, with HIV RNA repeatedly > 10 000 copies/ml ; or (ii) HIV EIA positivity with a negative or indeterminate HIV Western blot and HIV RNA repeatedly detectable at any level. For initially HIV antibody-negative specimens positive for HIV RNA, long-term (established) HIV infection was defined by both HIV EIA and HIV Western blot positivity according to US CDC standard criteria  on repeat testing of the original specimen.
Data were double entered and validated using Epi Info Software (version 6.04; CDC). Statistical analyses were performed using SAS (version 8.02, SAS Institute, Cary, North Carolina, USA) and LogXact (version 4, CYTEL Software Corporation, Cambridge, Massachusetts, USA). The sensitivity of routine HIV rapid testing/EIA confirmation for detection of all (acute and long-term) HIV infections was assessed according to the definition of acute HIV infection described above. Negative predictive value for EIA was determined for routine specimens submitted to the full antibody testing plus pooling/RNA screening protocol. HIV RNA levels were compared using the Wilcoxon rank sum test.
To consider the association of behavioral, biological and socio-demographic covariates with acute HIV infection in each of the clinic populations under study, initial bivariate analyses were conducted with calculation of prevalence odds ratios (OR) and their respective 95% confidence intervals (CI). We used multiple logistic regression to construct an exploratory, multivariate model. The model included covariates significant at P ≤ 0.2 in bivariate analyses and additional covariates deemed important for consideration a priori, regardless of their bivariate significance. The latter factors included signs and symptoms potentially related to acute retroviral syndrome (e.g., fever, headache, myalgias, inguinal lymphadenopathy) and known cofactors for HIV transmission such as genital ulcers and urethritis. Due to the limited number of outcomes, modeling was conducted in three stages reflecting symptom, clinical diagnosis, and demographic/behavioral domains. A backwards elimination procedure was used. Covariates with the largest P value were removed first. Variables were retained in the model if P ≤ 0.10, due to the small number of outcomes. To control for confounding, we also retained variables that altered any significant OR estimate by ≥ 20%. Reported multivariate prevalence OR and CI are estimated using exact methods.
Of 1361 men enrolled in the STD and dermatology clinics, 553 (40.6%) had HIV infection (as determined by a positive rapid antibody test and confirmed by EIA). HIV prevalence based on rapid antibody testing was 46.8% (434/928) in the STD clinic and 27.6% (119/432) in the dermatology clinic.
The prevalence of acute HIV infection in the overall study population was 24/1361, or 1.8%. HIV RNA was detected in 28 of 774 specimens initially reported as antibody negative, but four subjects did not meet the definition of acute HIV infection. In two specimens long-term HIV infection was recognized by repeat antibody testing, although on Western blot the p31 band was absent in one of these subjects, consistent with recent seroconversion (F.M. Hecht, unpublished data). HIV RNA was detected at levels < 10 000 copies/ml (below the threshold required in the case definition) in two other patients. HIV RNA screening could not be performed on 34 specimens because specimens were unavailable. These subjects were classified as HIV negative for the purposes of these analyses.
Of the 24 patients with acute HIV infection, 23 were from the STD clinic. In the STD clinic, these men accounted for 2.5% (23/910) of all patients, 4.5% (23/508) of HIV antibody-negative patients and 5.0% (23/457) of all HIV infections. The sole dermatology clinic patient with acute HIV (prevalence, 1/432 or 0.2%) had asymptomatic trichomoniasis.
Clinical findings suggesting an acute retroviral syndrome (such as fever, myalgias, headache, thrush or adenopathy) and not associated with STD were noted by only 11 of 24 (46%) patients. A further five patients (21%) had inguinal adenopathy in conjunction with genital ulcer disease.
The diagnosis of acute HIV was strongly associated with attendance at the STD rather than the dermatology clinic (OR, 15.2; 95% CI, 2.04–113.0). In bivariate analyses (Table 1), factors significantly associated with acute HIV in the STD clinic population included genital ulcer disease, inguinal adenopathy and non-STD related (acute retroviral) symptoms. No previous STD, an interval from last coitus > 7 days and age 24–26 years were less strongly associated with acute infection. Circumcision, sex with non-regular partners or prostitutes and condom use were not associated with having acute HIV infection.
In multivariate analysis among STD patients (Table 2), the presence of inguinal adenopathy (OR, 4.46; 95% CI, 1.78–11.15) and genital ulceration (OR, 4.48; 95% CI, 1.38–14.56) were strongly associated with acute HIV infection. The odds of acute HIV infection were nearly four times greater for persons aged 24–26 years, as compared to persons aged 18–23 years (OR, 3.67; 95% CI, 1.28–10.53). Having no previous STD history was possibly associated with acute HIV infection (OR, 3.88; 0.86–17.45).
Median (interquartile range) blood plasma viral loads for acutely HIV infected men [6.17 (5.66, 6.58) log10 copies/ml HIV RNA] were significantly higher than for 58 consecutively enrolled HIV antibody-positive men [4.42 (3.91, 4.95) log10 copies/ml; P < 0.0001] (Fig. 1). Viral load for acutely infected men with and without symptoms suggesting an acute retroviral syndrome were similar.
Considering all detectable HIV infections in the STD clinic, the sensitivity of rapid HIV antibody testing alone was 0.949 (95% CI, 0.925–0.968 for 434/457), with a negative predictive value of 0.954 (95% CI, 0.933–0.971 for 485/508). This relatively poor performance was due to the high proportion of patients with antibody-negative acute HIV infection. In the dermatology clinic, rapid HIV antibody testing alone had sensitivity of 0.992 (95% CI, 0.954–0.9998 for 119/120) and negative predictive value of 0.997 (95% CI, 0.982–0.9999 for 304/305).
Early detection of HIV infection allows better medical care for infected people and focused prevention activities. Recognizing acute HIV infection might be particularly important because the virologic ‘set point’ related to disease progression is potentially modifiable by antiretroviral therapy at this time , and acutely infected patients with high viral burden and de facto risk taking behavior may have the greatest potential for secondary transmission [6–8,10,28,29]. However, patients with antibody-negative acute HIV infection are rarely identified, even in the USA and other developed countries where extensive referral networks [30–32], screening programs and cohort studies [15,33] have been established for this specific purpose.
Acute HIV infection might be more readily detected and is of greatest importance in places with high prevalence of HIV among men and women with high risk for HIV acquisition. Accordingly, we studied patients attending the STD and dermatology clinics of Lilongwe Central Hospital in Malawi. We have previously reported HIV prevalence of 40–50% of subjects seeking care in these clinics .
In the current study, we found that 2% of all patients in these clinics had acute HIV infection. Remarkably, all but one of these patients were seeking care for symptomatic STD, and the single dermatology clinic patient identified as acutely HIV infected had asymptomatic infection with T. vaginalis. This concentration of prevalent acute HIV infections among patients with STD is consistent with both high-risk sexual behaviors of this population and the ability of STD pathogens to facilitate HIV transmission and acquisition [22,34–36]. Bivariate and multivariate analyses confirmed the association between having acute HIV infection and presenting with an STD (and, in particular, presenting with genital ulcer and/or inguinal adenopathy). These observations are consistent with the hypothesis that ulcerative diseases are important HIV transmission cofactors [22,36]. The role of specific etiologic agents in facilitating HIV transmission in Malawi is not clear; previous studies from our group suggest that chancroid (26%), syphilis (29%) and herpes simplex virus (23%) account for most genital ulceration for men with and without HIV in Lilongwe . The strong association of inguinal adenopathy with acute HIV indicates that this may be a valuable marker of infection in this particular population; however, this finding can be very non-specific and is therefore likely to be less useful as a predictor of acute infection in lower-risk populations. Acute HIV infection was also associated with age 24–26 years in this study; interestingly, this corresponds to the age of observed peak HIV incidence for Malawian men in the general population .
Many patients with acute HIV have influenza-like symptoms, and a recent cohort study involving Kenyan sex workers indicated that criteria could be developed based on acute retroviral symptoms to target subpopulations with an increased likelihood of having acute HIV infection . However, the prevalence of acute retroviral symptoms in our clinic-based study population was low (46%). This observation may be explained by patients’ presenting for STD care during the pre-symptomatic incubation period of HIV infection. Therefore, although STD themselves may be a powerful predictor of having acute HIV infection in Malawi, our results indicate that targeting of acute HIV screening based on acute retroviral symptoms in the STD clinic would be ineffective, resulting in an unacceptably large number of missed acute infections.
The prevalence of acute HIV infection in the Malawi STD clinic is particularly alarming: in that clinic, 5% of all HIV antibody-negative patients were (acutely) HIV infected. This prevalence is nonetheless consistent with previous data on acute HIV detection in clinic-based testing populations. Bollinger  found that 1.5% of antibody-negative specimens collected from an Indian STD clinic were repeatedly positive for HIV p24 antigen; Quinn and colleagues  confirmed this prevalence in the same clinic population using a more sensitive specimen pooling and HIV RNA detection scheme. Two USA studies [40,41] screened patients with undifferentiated viral symptoms—both detected acute infection in exactly 1.0% of subjects. A broad-based study encompassing all publicly funded HIV VCT sites in North Carolina  documented confirmed acute HIV infection in 0.05% of patients self-identified as at risk for HIV infection—similar to estimates obtained for less well-defined European testing populations [42,43] but 1000-fold greater than estimates obtained for low risk blood donors . While acute HIV prevalence estimates for these populations are substantially lower than for the Malawi STD clinic, it is important to note that acute infections constituted > 5% of the total number of detectable HIV infections in each of these ‘high risk’ clinical testing settings.
Acute HIV infection is generally characterized by high levels of viremia [45–47]. Even so, the observation of extremely high blood plasma HIV burden among acutely HIV infected men in this study (ranging as high as 2.4 billion copies HIV-1 RNA/ml) is consistent with the observation that viral burden in Malawi exceeds that in the West at a similar stage of disease , either as a feature of clade C infection or resulting from other host factor(s). The high viral burden associated with acute HIV infection has critical public health importance since the magnitude of viral burden is likely to predict the probability of sexual transmission of HIV. Working in Uganda, Gray and colleagues demonstrated that viral loads > 50 000 copies/ml in people with long-term infection were associated with high transmission rates , and recent studies of semen HIV dynamics indicate that viral burdens in this compartment, while somewhat lower than those in blood, change in parallel with changes measured in blood [7,11]. The transmission probabilities associated with acute disease in our study population would likely be many-fold increased over transmission probabilities in long-term HIV infection  and substantially greater than the probabilities used to justify provision of post-exposure prophylaxis in cases of needlestick injury . Furthermore, mucosal  and ulcerative STD  observed in subjects in the current study would be expected to increase the concentration of HIV in semen, further amplifying transmission risk in this setting.
The specimen pooling/HIV RNA testing algorithm used in this study has the advantages of being both highly cost efficient [20,25] and highly specific  when used in populations with low expected HIV prevalence. The disadvantages of this method include labor- and time-intensivity and potential impairment of sensitivity resulting from specimen pooling. When used to screen individual specimens, commercially available assays can reliably detect HIV RNA within 9-days of infection [18,19] but are relatively expensive and are associated with a high false-positive rate [26,30,31]. Less sensitive but more specific [30,31] ELISA-based HIV p24 antigen testing may be more readily adopted for real-time HIV screening in a developing world setting. Newer HIV detection modalities such as HIV reverse transcriptase activity assays and microchip arrays are unproven but may have a future role in detecting acute HIV infections. Janssen [2,4] and others have promoted a sensitive/less-sensitive EIA based HIV antibody testing algorithm in order to distinguish patients with likely ‘recent’ infections (< 8 months) from those with longer term infection based on antibody titer. This method does not diagnose any additional cases compared with routine testing, and therefore cannot enhance entry of patients into care and prevention. Nonetheless, it has been successfully applied for surveillance and for incidence estimation purposes in populations with predominantly clade B HIV epidemics .
Voluntary counseling and testing of people at risk for HIV infection is a central focus of worldwide HIV prevention . Samples obtained during VCT are traditionally examined for HIV antibodies. However, even with the most sensitive tests available, antibodies directed against HIV can only be detected several weeks after HIV infection, therefore missing patients with acute HIV infection [18,19]. Accordingly, the sensitivity of antibody testing to detect HIV in a population will be reduced in settings where acute HIV infections are common. In the current study sensitivity was estimated at < 95% in an STD clinic population in Malawi.
The problems with current approaches to HIV VCT remain theoretical in many areas with the greatest HIV burden. In these settings, resources for care and treatment are often limited, and both the provision and uptake of HIV testing in both high and low risk populations are still low. As HIV VCT becomes more widely implemented in this decade, however, it will probably assume a more central role in HIV prevention in the developing world. It will be critical to maximize the public health impact of such programs. In this study, under practices that reflect the current standard of care, significant numbers of patients who we believe to have the greatest short- and long-term potential for HIV transmission to susceptible partners (i.e., patients with acute HIV infection and STD) were led to believe that they were uninfected. We do not know the impact of such information on sexual behavior. However, these results suggest urgent need for reconsideration of HIV/VCT screening paradigms in settings where acute HIV infection may be common, and for further study of the role of patients with acute infection in the spread of HIV.
The authors acknowledge the clients and staff of Lilongwe Central Hospital, and the staff of the UNC Project in Lilongwe, Malawi who performed the study. S. Wise assisted with specimen pooling, and M. Kerkau and P. Joshi of the UNC Center for AIDS Research Retrovirology Core Laboratory performed the virologic testing. Confirmatory antibody testing was performed by the staff of the McLendon Clinical Laboratories at UNC-Chapel Hill.
Sponsorship: Supported in part by the UNC Center for AIDS Research (NICHD/NIAID 9-P30-AI50410-04), the UNC Fogarty Center (D43-TW01039), NIDDK (R01-49381), NIAID (AI-07001, K23-AI01781), the UNC HIV Prevention Treatment Network (U01-AI48005), and the UNC STD Cooperative Research Center (U19-AI31496).
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