Genotyping of pvcs
Amplification of the fragment containing the pvcs gene was attempted for blood samples obtained from patients. After two rounds of PCR, bands of 680–740 bp were amplified. This nested PCR generated three product size variants (Table 1). All the allelic variants were ordered in decreasing sizes of base pairs and coded alphabetically, starting a to c. The frequencies of type a (740 bp), type b (710 bp) and type c (680 bp) were 28%, 65% and 7%, respectively (Figure 1). The most common allele was type b (65%; 97/151). Two cases of type a were mixed with type b. There were two types of repeat region in the pvcs gene: the VK210 type of repeat unit coding for Gly-Asp-Arg-Ala-Asp/Ala-Gly-Gln-Pro-Ala , and the VK247 type coding for repeats of Ala-Asn-Gly-Ala-Gly-Asn-Gln-Pro-Gly . VK210 and VK247 were distinguished by digestion with the restriction enzymes, Alu1 and BstN1. In 151 samples 150 isolates were VK210 type and only one isolate was VK247 (Table 1).
PCR products were digested by ScrF1, which recognizes insertion on 5'CC/NGG3' restriction site in the pre repeated region and by Bbs1, which cuts the DNA on 5'GAAGACNN/NN3' site in post repeated region. Therefore, there are four possible combinations in the VK210 type (no/in, no/no, in/in and in/no) but in VK247 type two types were possible (in or no). Three variants were found in the samples (no/no, no/in, and in/in) and two of them (no/no and no/in) were predominant in the Kolkata strains (Figure 1). The frequencies of these were 43.7% for no/no and 51% for no/in respectively. Eleven alleles of pvcs (combinations of size variation and insertion presence/absence) were found in 151 isolates, and these were randomly distributed (Figure 2).
Genotyping of pvmsp1
The second fragment of pvmsp1 generated 1087–1150 bp from 151 samples.
Analysis on agarose gels revealed two different sizes, labeled a and b. Despite the lack of variation in the size of the amplification products, RFLP analysis with Alu1 and Mnl1 restriction enzymes revealed substantial diversity at the nucleotide level. The RFLP patterns of different isolates showed variation in size. Digestion with either Alu1 or Mnl1 yielded fragment sizes that were highly polymorphic. From the 151 samples 8 different Alu1 patterns and 5 different Mnl1 patterns were observed (Figure 3). When data from both analyses were combined, 35 alleles of the second fragment of pvmsp1 could be differentiated (Table 2).
The frequency distribution of the 35 alleles of the second fragment of pvmsp1 is shown in Figure 4. It was found that alleles 1 (a a1 m1) and 18 (b a1 m1), alleles 4 (a a2 m2) and 28 (b a5 m4) were particularly predominant (20 and 26, 17 and 16 respectively among 151 samples [13.2, 17.2, 11.2 and 10.6% respectively]). The remaining isolates were randomly distributed between the other variants.
Genotyping of pvmsp3-alpha
Amplification products showed a major size polymorphism with three different variants labelled a, b and c (1900-1100 bp). These were predominantly of a size (102/151 [67.5%]) (Figure 5). The frequencies of type a (1900 bp), type b (1500 bp) and type c (1100 bp) were 67.5%, 12.5% and 20% respectively. RFLP analysis with Alu1 and Hha1 restriction enzymes revealed substantial diversity at nucleotide level. Digestion with these enzymes yielded fragment sizes that were highly polymorphic. The sum of the fragment sizes did not always equal the size of the intact PCR product, indicating non-resolvable variation in size of the uncut amplification products but never more than the total size, thereby indicating a number of mixed genotypes. From the 151 samples, five different Alu1 patterns and five different Hha1 RFLP patterns were observed. When data from both analyses were combined, 37 alleles of pvmsp3-alphacould be differentiated (Table 3). The frequency distribution of the 37 alleles of pvmsp3-alpha alleles in 151 isolates is shown in Figure 6. Allele 1 (a al1 h1) was particularly dominant (22/151 [14.5%]) and the others were randomly distributed.
Analysis of three genes genotyping
The results showed marked genetic diversity in the Kolkata strains with 11 alleles for pvcs, 35 alleles of the second fragment of pvmsp1, and 37 alleles of pvmsp3-alpha. Mixed genotypes of each marker were found in pvcs 1.3%, pvmsp1 0.7% and pvmsp3-alpha 8.6%. The overall rate of mixed gene infection was 10.6% using all three genetic markers. The most common alleles for each of the three genes were seen in 31.7% of samples for pvcs (allele7-bVK210 no/in), 17% of samples for the second fragment of pvmsp1 (allele18-b a1 m1) and 14.5% for pvmsp3 (allele1-a a11 ha1) (Figure 2, 4, and 6). Therefore, when genetically homogeneous parasite lines are considered in this area, the three markers can distinguish a potential 14,245 (11 × 35 × 37) unique genotypes. In the combination of three genetic markers, The most common three marker genotype was b VK210 no/in, b a1 m1, c a1 h1 allele, which occurred at a rate of 2% (n = 3/151).