Both parasitological diagnosis and follow-up assessments of visceral leishmaniasis were based on molecular methods; i.e. PCR on peripheral blood (PB) [4] and less frequently on bone marrow (BM). Biological diagnosis was also based on PB and BM culture selleckchem on blood agar/Novy–McNeal–Nicolle medium and direct microscopic examination of BM. Biological follow-up also included CD4 cell counts and HIV viral load measurements. Clinical follow-up of patients with visceral leishmaniasis and definitions of subclinical or clinical visceral leishmaniasis episodes have been described previously [4]. Additional quantitative real-time PCR tests were performed using a LightCycler™ instrument

with SYBRGreen (Roche, Meylan, France) for detection. All acquired fluorescence data were analysed using the LightCycler™ software. Melting curve analysis was used for characterization of the quantitative real-time PCR products. The primers, previously described in Mary et al. [7], amplified a kinetoplastic-specific sequence of 137 bp. Among the 27 Leishmania/HIV-coinfected patients followed up, 16 patients presented relapses and 11 were free of relapses. No clinical relapse occurred when CD4 cell counts were >200 cells/μL. Moreover, PCR analysis confirmed that the PB of nonrelapsing patients became PKC activation definitively PCR-negative

in the first 6 months of follow-up [4]. As regards relapsing patients, 10 of them presented a total Phosphoribosylglycinamide formyltransferase of 52 relapsing visceral leishmaniasis clinical episodes, despite adequate drug treatment of both visceral leishmaniasis and HIV-1 infections. It is noteworthy that visceral leishmaniasis relapses are responsible for serious difficulties in the monitoring of coinfected patients [3–5]. Figure 1 shows the clinical evolution of seven of these 10 patients, indicating clinically relevant and subclinical episodes or periods without any signs of visceral leishmaniasis. Anti-leishmanial treatment and HAART, CD4 cell counts, occurrences of other opportunistic infections, and Leishmania PCR and culture results are also

shown in Figure 1. The median period of follow-up was 87.5 months (ranging from 5 to 158 months). During the follow-up period, seven patients died, one was lost to follow-up and two survived. All patients experiencing visceral leishmaniasis episodes received induction treatment with amphotericin B, miltefosine or pentamidine. For all patients, during each visceral leishmaniasis clinical episode, the PCR assay used for routine diagnosis detected circulating parasites (n=153), and most CD4 counts were <200 cells/μL. Acute episodes were followed by relapse-free periods with subclinical signs or without any symptoms of visceral leishmaniasis. During these periods, the patients were not given induction treatment, but primarily received secondary prophylaxis with amphotericin B or miltefosine (Fig. 1).