In sub-Saharan Africa and parts of Asia the HIV epidemic overlaps with that of malaria. Malaria itself is causing growing morbidity and mortality, largely as a result of the spread of chloroquine-resistant Plasmodium falciparum. The interaction between these diseases is bidirectional, complex and incompletely understood. Acute malaria in HIV-infected individuals results in higher HIV viral loads . Adults with HIV infection in areas of unstable malaria transmission have higher rates of severe and fatal malaria , whereas those residing in areas with sustained transmission of malaria suffer higher rates of malaria infection . Malaria in pregnant women with HIV is more severe than among their HIV-negative counterparts , and may increase the risk of the vertical transmission of HIV [1,5–7].
Previous studies by ourselves and others have shown that a number of protease inhibitors (PI) in current use, including ritonavir, saquinavir and lopinavir, inhibit the in-vitro growth of malaria parasites at concentrations achieved in routine clinical practice [8–10]. This effect was also observed in a murine model of malaria . To define the likely clinical effect of PI-based HAART on malaria infection further, we have used a well-validated experimental approach for assessing antimalarial effect, namely the ability of serum from individuals taking the study drug to inhibit the in-vitro growth of malaria parasites [11,12].
Five groups of volunteers were studied (n = 42). Subjects in the two control groups, one (n = 16) taking no medications, and the second (n = 5) receiving chloroquine for the treatment of Plasmodium vivax infection were HIV uninfected. Subjects in the remaining three groups were receiving HAART for HIV infection. They had HIV viral loads below 5000 copies/μl and were not receiving any other drugs with known antimalarial activity. The third group were receiving non-nucleoside reverse transcriptase inhibitor (NNRTI)-based therapy (n = 6). The fourth group were receiving ritonavir-boosted saquinavir [1000 mg saquinavir twice a day with ritonavir 100 mg twice a day (400 mg twice a day in one case only), n = 6]; the fifth group were receiving co-formulated lopinavir/ritonavir (Kaletra, n = 6). To investigate for the concentration-dependent inhibition of parasite growth, two blood samples (one before dosing and one 3 h after dosing) were collected from four out of six subjects on the saquinavir/ritonavir regimen, and five out of six subjects on the lopinavir/ritonavir regimen. Serum was separated and frozen at −20°C until all samples were assayed together. The plasma concentrations of the PI were quantitated by high performance liquid chromatography as described previously .
The laboratory-adapted chloroquine-sensitive P. falciparum clone 3D7 was maintained in continuous culture as previously described . Growth inhibition of asynchronous parasites at 1% parasitaemia and 2% haematocrit was assessed using 3H-hypoxanthine incorporation . A control reference parasite culture was undertaken using pooled human sera obtained from the Brisbane Red Cross Blood Service. Each test or control serum was added to sera-free cultures constituting 40% of the total volume. After 48 h incubation, 3H-hypoxanthine incorporation was determined. All experiments were performed concurrently in triplicate, with the mean result of each triplicate used for data analysis. The mean result of the no-drug control group was used for comparison with the other groups. The non-parametric Mann–Whitney U test was used for an analysis of differences in parasite growth between the groups. The Bonferroni correction was applied to account for multiple group comparisons; thus the level of significance for between-group comparisons was set at a value of P = 0.01. The ethics committees of the Royal Brisbane and Women's and Prince Charles Hospital Health Service Districts approved the study.
Sera from the no-drug control subjects showed a variable but low-level effect on growth, compared with growth in the reference culture containing 40% pooled sera (Fig. 1). As expected, sera from chloroquine-treated subjects inhibited growth by 99% or more (P = 0.001). Sera from subjects in the lopinavir/ritonavir group inhibited growth by 59–95% (median 69%; P < 0.0001 compared with the control group), whereas that from subjects in the saquinavir/ritonavir group inhibited growth by 50–96% (median 55%; P = 0.001). Parasite growth in the sera from NNRTI-treated subjects did not significantly differ from growth in the serum of subjects taking no drugs. No correlation was observed between the PI levels and the inhibition of parasite growth.
This work demonstrates that serum taken from subjects on a HAART regimen that included ritonavir-boosted saquinavir or lopinavir shows significant antimalarial activity. Whether this effect will translate into useful clinical activity will require an observational study of patients taking this regimen in malaria-endemic areas. This methodology has previously been used as a surrogate to study the likely antimalarial activity of drugs including doxycycline, azithromycin and atovaquone, all of which have known clinical utility for the chemoprophylaxis of malaria [13,14].
A number of considerations including side-effects, costs, and the apparent moderate potency with conventional dosing make it unlikely that PI will become first-line agents in their own right for the treatment of malaria. These drugs may, however, possess activity for malaria chemoprophylaxis, and individuals taking PI-based HAART may be afforded some protection against contracting clinical malaria. This activity is in line with other drugs such as doxycycline or azithromycin, which are not useful for the treatment of malaria, but have sufficient activity to serve as useful agents for prophylaxis .
We did not observe a correlation between serum PI levels and antimalarial activity. A possible explanation is that the numbers in each group were small and the range of PI concentrations was substantial. Furthermore, the study was not designed to demonstrate a concentration–effect relationship.
The current World Health Organization-recommended strategy for the management of HIV infection in resource-limited settings with NNRTI-based combination antiretroviral therapy is likely to see many patients experiencing virological failure. PI will inevitably be required for successful antiretroviral therapy in this situation. It is therefore a priority that the clinical significance of the effect of PI therapy on malaria be determined. Such a study would best be undertaken in a clinical trial in an area co-endemic for malaria and HIV, such as sub-Saharan Africa.
The authors would like to thank the staff at the Infectious Diseases Unit of the Royal Brisbane and Women's Hospital and the AIDS Medical Unit for assistance with the recruitment of subjects, and the Brisbane Red Cross Blood Service for providing human blood and sera used for the routine culture of P. falciparum.
Sponsorship: The Australian NHMRC Program grant (290208), and Mark Nicholson, Alice Hill and the Tudor Foundation (D.L.G.); University of Queensland Women in Science Fellowship, and Ramaciotti Development Grant (T.S-A.).
Presented in part at the Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, September 2006.
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