Sequence variation in the small-subunit rRNA gene of Plasmodium malariae and prevalence of isolates with the variant sequence in Sichuan, China

Abstract

By two PCR-based diagnostic methods, Plasmodium malariae infections have been rediscovered at two foci in the Sichuan province of China, a region where no cases of P. malariae have been officially reported for the last 2 decades. In addition, a variant form of P. malariae which has a deletion of 19 bp and seven substitutions of base pairs in the target sequence of the small-subunit (SSU) rRNA gene was detected with high frequency. Alignment analysis of Plasmodium sp. SSU rRNA gene sequences revealed that the 5' region of the variant sequence is identical to that of P. vivax or P. knowlesi and its 3' region is identical to that of P. malariae. The same sequence variations were also found in P. malariae isolates collected along the Thai-Myanmar border, suggesting a wide distribution of this variant form from southern China to Southeast Asia.

FIG. 1

Partial sequences of the SSU rRNA genes of malaria species. Sequences are aligned with reference to that of P. malariae. Nucleotide identity is indicated by a dot; the lack of a corresponding nucleotide is indicated by a dash. Deleted nucleotides are shown as stars. Underlined regions are targeted by the species-specific 3′ primers used in nested PCR. Sequences in boldface type are those of the primers for the first PCR (P1F/P1R), nested PCR (P2F), and MPH (MPH-1/MPH-2). Lowercase letters are the probe-targeting regions for the MPH method. The italic letter (g) in the probe region of P. ovale should be an a in the originally designed specific probe based on the previously published partial sequence (2) (DDBJ accession number D17580). Abbreviations: PfA and PfC, A type and C type of P. falciparum, respectively; PvA and PvC, A type and C type of P. vivix, respectively; Pm, P. malariae; Pk, P. knowlesi; Po, P. ovale; NewPm, isolates with a 19-bp deletion and seven substitutions compared with P. malariae.

FIG. 2

Detection of the four species of human malaria parasites by nested PCR. Results of the first PCR (A) and the second PCR (B) are shown. (A) A Sharp band(s) can be seen in positive controls and samples (lanes 2 to 6), while a faint band is seen in lane 1. No band is seen for any of the negative controls. (B) Positive controls show bands of the expected sizes, and no band is seen for the negative controls. Sample 1 is from a patient with a P. vivax infection. Samples 2 to 6 are from patients with mixed infections with P. vivax and P. malariae with a normal-size band (115 bp [sample 2]) or short band (96 bp [samples 3 to 6) of the latter species. Abbreviations: W, distilled water used as control; NC, negative control; Po, Pm, and Pf, positive controls for P. ovale, P. malariae, and P. falciparum, respectively; f, v, m, and o, products amplified by species-specific primers of P. falciparum, P. vivax, P. malariae, and P. ovale, respectively; MK, DNA size markers (pUCBM21 DNA digested by HpaII and DraI) (top to bottom, 1, 114, 900, 692, 501 and 489 [appears here as one band], 404, 320, 242, 190, 147, 124, and 110 bp).