Plasmodium vivax malaria in Mali: a study from three different regions

Plasmodium vivax is the most widely distributed human malaria parasite and responsible for 100–300 million clinical cases yearly, including severe disease and death. It has been estimated that 2.85 billion people are exposed to some risk of P. vivax transmission worldwide and of those circa 0.10 billion (3.5%) are in the African region where transmission is considered stable (≥0.1 case per 1,000 people per annum) [1]. Of note, it is widely accepted that P. vivax is largely absent in Central and Western Africa, mostly as a result of the absence of the Duffy blood group, a blood receptor hitherto considered indispensable for the invasiveness of P. vivax into red blood cells, as more than 90% of the population in this area are believed to be Duffy negative [2]. In recent years, however, increasing evidence of the circulation of P. vivax episodes affecting Duffy negative individuals has appeared, including one from Africa [3‐5]. Ever since, other reports have demonstrated the presence of P. vivax infections in sub-Saharan Africa [6‐9].

Mali is a landlocked nation in West Africa divided into eight regions and almost 15 million people with international borders with seven African countries. Of relevance, some reports have indicated the presence of P. vivax infections in this country [10‐12]. Here, following a survey in the northern population of Mali, where local transmission of P. vivax was also suspected, blood samples were collected from febrile patients and stained. Giemsa/Field-stained blood smears were analysed for specific P. vivax diagnosis by microscopy. Moreover, genomic DNA extracted from the same smears used in microscopy was used as templates for nested-PCR with primers for the four major Plasmodium species infecting humans. Last, amplified fragments were cloned and sequenced. Results unequivocally confirm and extend previous observations of P. vivax in Mali.

A total of 88 Giemsa/Field-stained slides were randomly selected from febrile malaria patients as part of routine National Malaria Control Programme surveillance procedures in Mali. Strikingly, out of 88 samples analysed, 25 (28.4%) contained mature stages (readily detected in less than 20 high power fields examined) suggestive of P. vivax (Figure 2A, Additional file 1, Table 1). As P. ovale and P. malariae infections are commonly found in Western and Central Africa, genomic DNA was extracted from the 25 slides for P. vivax and nested-PCR analysis was performed using probes for the four Plasmodium spp (Figure 2B). As a positive control, gDNA from a P. vivax slide from Brazil was also extracted. Fifteen samples pertaining to Goudam, Tombouctou and Gao were PCR positive for P. vivax. Among them, eleven contained mixed infections with P. falciparum, four were positive for P. malariae and none for P. ovale (Table 2).

To fully confirm these results, amplified fragments from eleven samples were cloned and sequenced. Significantly, similarity analysis with available P. vivax SSU RNA gene sequences from the SalI strain (PVX_088869, PVX_096002, PVX_097020, PVX_079693) demonstrated that sequences from Mali corresponded to P. vivax and that there were several polymorphisms (Additional file 2). Consistent with the existence of different SSU RNA genes in P. vivax[14], phylogenetic analysis tested by bootstrap showed that sequences fell into major genetic clusters of SSU RNA P. vivax genes branching-out from SSU RNA sequences of other Plasmodium species (Figure 3). Together, these results unequivocally confirmed the presence of P. vivax infections in Mali.

The presence of P. vivax malaria in sub-Saharan Africa has been largely neglected since it was demonstrated that lack of expression of the Duffy blood-group correlated with the absence of P. vivax infections [2]. Moreover, it has been suggested that P. vivax is of African origin thus driving fixation of the Duffy negative phenotype [15]. Recent evidence, however, has called upon to revise this suggestion as it strongly indicates that P. vivax may instead be of Asian origin [16, 17]. Its increasingly recognized presence in Africa may, therefore, be related to its newly acknowledged capacity of exploiting new alternative invasion pathways among populations in this continent. Regardless of the geographical origin of P. vivax and the pathway for entrance into reticulocytes, it is clear that the presence of P. vivax in Africa is probably underestimated.

A few reports, of variable quality, have described infections by P. vivax in Mali [10‐12]. An early indication of its presence came from the description of Russian doctors in the region of Gao in the 1950s (OD, unpublished). Four decades later, a case of P. vivax infection in an eight year-old girl was reported in Kidal during a survey in 1988 in Trans-Sahara [12]. The microscopy diagnosis of this patient was confirmed in two European reference laboratories at Marseille and London. More recently, Koita et al. performed two cross-sectional studies in the North-Eastern region of Mali, reporting over 10% prevalence rates of P. vivax infections as detected by Giemsa-stained smear [10]. However, and noteworthy, with the exception of one single sample, exclusion of the potentially confounding and morphologically similar P. ovale and P. malariae species were not systematically performed using molecular techniques.

Microscopy evaluation of samples examined here similarly suggested the presence of parasite blood stages resembling P. vivax. However, and as an important limitation of only using such diagnostic techniques, it seems difficult to exclude that these stages may not correspond to P. ovale species. P. ovale has been frequently reported in this particular region of Africa and even though distinct ameboid trophozoites and number of nuclei per single schizont can be used as criteria for species-specific diagnostics, this remains very challenging in most thick-blood smears. Thus, nested-PCR and DNA sequence analyses were performed to unambiguously demonstrate P. vivax infections in Mali. Amplified fragments of sizes corresponding to the SSU RNA gene of P. vivax were observed in close to 28% of infections. Moreover, sequence similarity and phylogenetic analyses unequivocally confirmed that these fragments corresponded to P. vivax sequences.

These findings are of important public health relevance. In recent years, the scientific attention of the malaria community has shifted from a focus on malaria control to specific efforts aiming at global malaria eradication [18]. However, it is important to consider that efforts needed to control P. vivax will surely exceed those necessary to control P. falciparum as transmission of this species is possible even before the appearance of clinical symptoms, and can also occur irrespective of adequate asexual parasite clearance as a result of hypnozoite-derived relapses. Indeed, the adequate treatment of P. vivax infections includes the addition of a full 14-day long course of primaquine (PQ), the only to date registered drug that can achieve the radical cure of the hepatic hypnozoites, responsible for unpredictable relapses and subsequent morbidity, and a drug that has potent gametocytocidal activity. Without the radical cure, hepatic hypnozoites will maintain those infected individuals as infectious, and thus prone to maintain transmission and develop new disease episodes. PQ is unfortunately an unsafe drug, and can lead to severe haemolysis when administered blindly to glucose-6-phosphate dehydrogenase (G6PD) deficient patients, a genetic deficiency particularly frequent in sub-Saharan Africa, limiting therefore its widespread use unless guaranteeing prior screening of this deficiency. As no rapid G6PD deficiency diagnostic tests exist to date, dismissing its risks prior to PQ administration appears unfeasible, and adequate treatment of P. vivax episodes unguaranteed, adding further complexity to the control programmes in place in many African settings. In Mali, the spread of P. vivax in the northern part of the country will complicate the possibility of malaria elimination. The current malaria control programme has no reference to vivax malaria. The findings reported here will push further to a revision of the policy document. New tools will be needed (the use of mass drug administration of primaquine for example) and access to the target population is a major public health issue. All the laboratory technicians in this endemic region of P. vivax, should be retrained for microscopic diagnostic.

An important limitation of this work is the inability to link such confirmed P. vivax infections with the Duffy and G6PD phenotypes of the individuals affected. Indeed, the presence of Duffy positive ethnic groups in central and West Africa as in the case of the Moors in Mauritania [8] or other Ethnic groups that may be present in Mali could entirely account for P. vivax transmission in the area, and would not necessarily imply that this species has found alternative mechanisms to infect Duffy-negative individuals. Prospective studies further investigating the Duffy and G6PD status and its relation to species-specific malaria risk are, therefore, urgently needed.