With the use of HAART, it is now possible to reduce plasma HIV-1 RNA to undetectable levels and increase CD4 T-cell counts in peripheral blood in the majority of adherent patients [1,2]. T lymphocytes in peripheral blood represent only 2–5% of the total of lymphocytes in the body, whereas the majority of lymphocytes are located in lymphoid tissues. Gut-associated lymphoid tissue (GALT) represents the largest lymphoid organ. It is extremely permissive to HIV-1 infection and supportive of HIV-1 replication, providing a persistent viral reservoir [3–5]. Several studies have demonstrated that replication-competent virus persists in blood cells and lymphoid tissue after at least 2 years of HAART, despite the complete suppression of HIV-1 RNA in blood [6–8]. The presence of HIV-1 in intestinal mucosa has been demonstrated, but there are relatively few reports of the effects of HAART in these tissues [9–11].
In this study we examined the presence of HIV-1 RNA in paraffin-embedded distal duodenal mucosa biopsies from 44 HIV-positive patients by in-situ hybridization. Twenty-five out of 44 patients had been on HAART for more than 4 years. The characteristics and clinical data of the participants in the study were: 30 men, 14 women; average CD4 T-cell count 265 ± 222 cells/μl (mean ± SD); mean age 38.2 ± 7 years; mode of transmission men who have sex with men, 18 cases; heterosexual transmission, 19; intravenous drug users, six; transfusion, one. As controls, distal duodenal mucosa biopsies from 10 HIV-1-seronegative individuals were also analysed.
The duodenal mucosa biopsies were obtained for diagnostic purpose and the remaining tissue was embedded in paraffin and used for the in-situ hybridization analysis later. This protocol was reviewed and approved by the Ethics Committee of the Academia Nacional de Medicina and Juan A. Fernandez Hospital.
The HIV-RNA concentration in plasma was determined using the Amplicor HIV monitor quantitative assay (Roche Diagnostic Systems, Branchburg, New Jersey, USA). CD4 lymphocyte counts were determined by flow cytometry (Becton-Dickinson, San Jose, California, USA).
Twenty-five out of the 44 HIV-1-positive patients had HIV-1 RNA detectable in plasma (plasma viral load > 100 000 copies/ml). The remaining 19 patients had undetectable HIV-1 RNA (< 50 copies/ml).
In-situ hybridization using biotin-labeled probes and tyramide signal amplification (DakoCytomation, Gen Point; Dako Denmark A/S, Denmark) was performed to allow the qualitative detection of HIV-1 RNA in duodenal mucosa sections from patients with chronic HIV infection. Two probes were used: Gag/5′ genome 5′GGA TGT ACT CTA TCC CAT TCT GCA GCT TCC TCA 3′; Rev probe 5′TCC TGC CAT AGG AGA TGC/CAG TCG CCG CCC CTC 3′. The signal was developed by adding the chromogenic indicator dye diaminobencidine, which is oxidized by the peroxidase enzymes. The specificity of the in-situ hybridization was demonstrated by the lack of signals in the duodenal biopsies from HIV-1-seronegative patients or when the probe was omitted from the hybridization mixture. The slides were counterstained with haematoxilin. Visualization of in-situ hybridization signals was carried out using an Olympus microscope equipped with 20×, 40×, 60× and 100× objectives (Fig. 1a).
Positive HIV-1 hybridization signals were observed in the duodenal biopsies from 20 of the 44 HIV-1 patients (45.5%). Ten of the 20 HIV-1-positive duodenal biopsies corresponded to patients who had plasma viral loads below 50 copies/ml and 10 to patients who had plasma viral loads greater than 100 000 copies/ml (P = 0.4 ns, chi-square test).
Analysing the relationship of therapy to the detection of virus, HIV-1 RNA was detected in duodenal biopsies from 10 out of 19 HIV-positive patients off HAART. HIV-1 RNA was also detected in duodenal specimens collected from 10 out of 25 HIV-positive patients receiving HAART, 19 of them with confirmed viral suppression below 50 copies/ml (Fig. 1b).
Our results show that HIV RNA can be detected in patients with successful HAART, confirming that residual HIV reservoirs remain in spite of successful treatment. HAART inhibits viral replication but does not eradicate the virus from cellular reservoirs [6–8]. Studies examining peripheral blood alone provide a limited view of AIDS pathogenesis. In the present study, considering the effects of HAART on intestinal HIV-1-RNA detection, approximately half of the untreated patients with high plasma viral loads gave positive duodenal results. The results were similar in patients on successful HAART having undetectable plasma viral loads. It thus seems that the detection of HIV-1 RNA in the duodenum was not influenced by treatment. No differences in the mean percentage of HIV-1-positive biopsies were found between patients on and off HAART.
Moreover, in six patients who failed HAART no HIV-1 RNA was observed in duodenal biopsies, confirming the lack of correlation between the plasma viral load and intestinal HIV detection.
In our study the plasma viral load did not predict the presence of HIV-1 RNA in the duodenum. These data suggest that the intestinal mucosa can be a reservoir that is not influenced by levels of plasma viral load or antiretroviral therapy. Ongoing HIV replication in the intestine may be a source that repopulates the body with virus after therapy is withdrawn.
Our results confirm that it is important to study the sites of low replication in order to improve our understanding of the pathogenesis of HIV disease.
Sponsorship: This work was supported by grants from the Argentinian National Ministry of Health, Carrillo-Oñativia research fellowship.
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