Frequency and distribution of mixed Plasmodium falciparum-vivax infections in French Guiana between 2000 and 2008

Between 2000 and 2008, malaria diagnoses in the health centres and at the Hospital were performed using thin and thick blood smears, and were then confirmed by an experienced microscopist at the Department of Parasitology and Mycology at the Hospital. Given the low prevalence of mixed infections using microscopy, PCR diagnosis was implemented in order to estimate the prevalence of mixed infections.

The study included samples from patients with clinical malaria having consulted remote health centres or Cayenne Hospital between 2000 and 2008. Samples were collected for diagnostic purposes on filter paper (Whatman^®) or in EDTA vacutainers, through the Cayenne Hospital or the National Reference Centre for Malaria, respectively. When filter paper was available and when the physician on site was informed (fast rotation of professionals in these remote centers, with newcomers not aware of all ongoing protocols) the filter paper was sent to the Hospital where PCR was performed. Thus, whether a patient eventually got PCR, it was not linked to the particulars of the patient or the malaria episode leading to the consultation, but to the health care professional rotation. Thus, this was not likely to be a recruitment bias.

Among the 400 samples collected (representing 10 % of French Guiana cases), 331 came from health centres located in the eastern part of French Guiana: Cacao, Saint-Georges-de-l’Oyapock, Régina, Camopi, Trois-Sauts, and Cayenne Hospital (Fig. 1). In the latter, samples came from patients living in Cayenne or in other towns of central or eastern French Guiana.

The 69 other samples came from health centres located in the western part of French Guiana: Antecume Pata, Maripasoula, Papaïchton, Grand-Santi, and Apatou (Fig. 1). All samples were anonymized before transmission.

The DNA from the samples conserved on filter paper or at −20 °C were extracted using the DNeasy^® Blood and Tissue Kit or the Qiaamp DNA blood kit (Qiagen, Crawley, UK), as recommended by the manufacturer.

DNA was amplified using real time PCR as described by Veron et al. [20]. The amplified region corresponds to the small 18S RNA sub-unit. The P. falciparum primer sense sequence was: Pf1 5′-ATTGCTTTTGAGAGGTTTTGTTACTTT-3′, that of the antisense primer was: Pf-2 5′-GCTGTAGTATTCAAACACAATGAACTCAA-3′ and that of the probe was: Pf-probe FAM-CATAACAGACGGGTAGTCAT-MGB. The size of the amplicon was 95 pb. The sequence of the P. vivax sense primer was: Pv-1 5′-CGCTTCTAGCTTAATCCACA TAACTG-3′, that of the antisense primer was: Pv-2 5′-AATTTACTCAAAGTAA CAAGGACTTCCAAG-3′ and that of the probe was: Pv-probe VIC-CGCATTTT GCTATTATGT-MGB. The amplicon size was 142 pb. The amplification and the detection of DNA were performed in duplex, using the PCR Applied Biosystem 7300 analytic system. Real-time PCR was performed in a final volume of 25 µl, in presence of an internal positive control (IPC) (Applied Biosystem, Courtaboeuf, France). Samples were analysed in two tubes, the first one contained P. falciparum and P. vivax primers and probes and the second one the internal control of the absence of inhibitor, the IPC. Each reaction mix contained 12.5 µl of Master Mix Gene Expression (Applied Biosystem), 300 nM of each primer (P. falciparum and P. vivax), 150 nM of each probe (P. falciparum and P. vivax) and 5 µl of matrix DNA. The IPC was used as recommended by the manufacturer. PCR starts with a 10-min phase at 95 °C, followed by 50 cycles of 15 s at 95 °C and 1 min at 60 °C.

Proportions of mixed infections were compared between regions using the χ² test.

The retrospective use of anonymous patient files on the site of patient care is authorized by the French National Commission on Informatics and Liberties (CNIL). All the human blood samples and the data collected retrospectively were anonymized in a standardized case report form and in database.