Genetic diversity of Plasmodium vivax isolates from Azerbaijan

Plasmodium vivax is the most widely distributed human parasite, with an estimated burden of 70–80 million cases annually [1]. In some parts of the world (Asia, South America), it is the most prevalent form of the four human malarial parasites. Although it causes a less severe disease than Plasmodium falciparum, being rarely lethal, P. vivax affects the working capacity of the population and the lack of efficient drug distribution favors the onset of drug resistant strains [2, 3]. Imported malaria is an increasing health problem in Western Europe, where about 6,500 cases are reported annually in Germany, France, Italy and the United Kingdom [4]. Although P. falciparum infections account for the majority of cases (64%), P. vivax is responsible for an additional 23% [4]. Presence in this area of residual anopheline populations susceptible to P. vivax infection represents a permanent risk for the occurrence of P. vivax indigenous malaria cases, as recently occurred in central Italy [5, 6]. Since 1970, malaria had been eradicated in central Asia, except for some residual foci in two countries belonging to the Newly Independent States (NIS), i. e. Azerbaijan and Tajikistan (WHO, Regional Office for Europe, unpublished document). At the beginning of the 1990s, the situation changed dramatically due to the re-emergence of malaria in the NIS area and especially in Tajikistan, where at present an epidemic is still in progress [7, 8]. In these countries, the existing state of the primary health care system is extremely precarious, especially in rural areas and in small villages. Malaria is a common disease, which can easily re-establish itself when a lack of control occurs.

In comparison with P. falciparum, molecular studies of the genetic diversity and dissemination of P. vivax are scanty. Recently, 33 polymorphic tandem repeats (TRs) of P. vivax and a P. vivax polymorphic microsatellite have been identified and shown to be useful in population studies [9, 10]. The merozoite surface protein 3α (MSP3-α) gene also seems to be a good candidate for studying the genetic diversity of P. vivax populations, since PCR-RFLP products indicate the presence of up to 13 alleles [11, 12]. However, the circumsporozoite protein (CSP) and merozoite surface protein 1 (MSP-1) genes still remain the most studied molecular markers in genetic epidemiological surveys carried out in P. vivax endemic areas.

In the frame of a malaria research project funded by the European Commission, a molecular study was undertaken in Azerbaijan, aimed at collecting information on the genetic make-up of P. vivax natural populations present in this endemic country. For this purpose the extent of polymorphism of CSP and MSP-1 genes were analysed in parasite isolates from five localities of central Azerbaijan by using PCR amplification and sequencing.

For CSP, the main variations already reported in the literature consist of two variant sequences, VK210 and VK247 that show a variable number of repeat units, GDRA(A/D)GQPAA and ANGAGNQPG, respectively, with some variant positions within the sequence [22, 23]. These two variants have a worldwide distribution, and locally their distribution have been also correlated with climatic gradients or with the Anophelines vector specificity [26]. Studies carried out in some Asian endemic countries, i.e. South and North Korea, China, the Philippines, the Solomon Islands and Thailand [27, 15, 28, 12] suggest that CSP has a limited value as a molecular marker for genetic variability when used alone. In the present study, all the analysed P. vivax isolates from Azerbaijan were found to belong to the type VK210 and they constitute a group of eight CSP sub-types that closely linked in the dendrogramme shown in Figure 4. Differently from what reported by other Authors [28] who showed that the CSP sequence analysis allows detecting the geographic origin of plasmodial isolates, our results did not support its use for tracking the geographic origin of Azeri isolates since we dealt with a limited number of samples studied. No genotype association with particular sampling districts was observed since, for example, the most common sub-type identified (G/4) was present in all five districts.

Our results confirmed that MSP-1 is a good polymorphic marker. In particular, the region of the gene known to be highly polymorphic and discriminative between the Belem and Sal I types was analysed [23]. A variable poly-glutamine (poly-Q) region is characteristic of the Belem type and represents the principal source of genetic diversity of this marker. Moreover, a poli-Q region is the recombination site between the two types Belem and Sal I and, as shown in the literature, interallelic recombinations between the two types are frequent [25]. The total genetic diversity observed when including the nucleotide substitutions is relatively important taking into account the low endemicity of studied area. Similar results were observed in Southeastern Iran and in Thailand [29, 12], low endemic countries for P. vivax malaria as well, where the authors detected the two types Belem and Sal I, together with several recombinant types. In particular, in the study carried out in Iran by Zakeri et al., the analysis of MSP1 genetic diversity on a total of 16 plasmodial isolates leaded to the identification of 14 genetic sub-types. It is worth noting that such a high level of diversity is probably due to the small sample size.

Our results show 17 genetic sub-types on a total of 36 isolates analysed and a quite high MSP-1 polymorphism also in Azerbaijan. As suggested in other studies [12] and also reported by Zakeri et al., it is possible to speculate that the observed genetic diversity could be also explained considering the studied area, i.e. central Azerbaijan, as transit road of the country and also of neighboring Asian country, where the circulating P. vivax populations show considerable MSP-1 genetic diversity. However, further studies aimed at collecting more information about people moving within the whole country and to closer countries are needed to verify the above hypothesis. The combined analysis of CSP and MSP1 sequence polymorphism has led to the identification of a total of 19 P. vivax sub-types, confirming that the simultaneous use of more than one genetic marker in this kind of study enhances the knowledge of genetic diversity existing in the parasite populations. The results of the current study show the circulation of multiple plasmodial clones in the studied area thus leading to the conclusion that malaria surveillance activities must be maintained in Azerbaijan in order to avoid serious disease outbreaks in the future.

The understanding of the polymorphism extent in surface antigens as CSP and MSP-1 and the resulting genetic diversity in P. vivax field populations could help in implementing malaria control activities being a crucial step for the development of a malaria vaccine.