No Genetic Bottleneck in Plasmodium falciparum Wild-Type Pfcrt Alleles Reemerging in Hainan Island, China, following High-Level Chloroquine Resistance

Abstract

Chloroquine-resistant Plasmodium falciparum was highly prevalent in Hainan, China, in the 1970s. Twenty-five years after cessation of chloroquine therapy, the prevalence of P. falciparum wild-type Pfcrt alleles has risen to 36% (95% confidence interval, 22.1 to 52.4%). The diverse origins of wild-type alleles indicate that there was no genetic bottleneck caused by high chloroquine resistance.

Chloroquine-resistant (CQR) Plasmodium falciparum became highly prevalent in Hainan Island, China, in the late 1970s, with 84% of clinical cases and 98% of samples tested for in vitro susceptibility being classified as CQR (6). In 1979, CQ was replaced by piperaquine as the first-line therapy for falciparum malaria (6). Since then, the occurrence of CQR P. falciparum has declined progressively (6), and the prevalence of the Pfcrt 76T mutation decreased from 90% in 1978 to 53 to 81% in 2001 (8). This rate of decline in prevalence of the 76T mutation is much lower than that reported for Malawi, where the prevalence of mutant Pfcrt alleles decreased from 85% in 1992 to 13% in 2000 (5). Several possible causes for the slow reversal of CQR in Hainan have been proposed, such as incomplete withdrawal of CQ and possible cross-selection by piperaquine (8). However, the genetic diversity of returning CQ-sensitive (CQS) parasites has not been examined. Any genetic bottleneck caused by the dominance of mutant Pfcrt alleles that reduces the diversity of the returning CQS parasites could impact parasite fitness and malaria epidemiology. Furthermore, the allelic type of CQR Pfcrt in Hainan has not been elucidated. In this study, we performed detailed molecular epidemiological investigations to define the allelic types of returning CQS parasites on the island as well as the allelic types of mutant Pfcrt and their origins. The results assist in understanding the mechanism and process of reversal of CQR after the cessation of CQ usage.

Fifty-one P. falciparum samples were collected from two areas in Hainan Island, China, between 2002 and 2004, including Wuzhishan City, a mountainous area central to the island (1.58% annual parasite incidence rate), and Qionghai City, an eastern hill area (0.17% annual parasite incidence rate). Ethics approval for the study was granted by the WHO Western Pacific Regional Office [(WP)WVP/ICP/MVP/1.4/001]. Parasite genomic DNA was extracted using a QIAamp DNA mini kit (Qiagen) following the manufacturer's instructions. Genotyping of msp1 and msp2 (7) indicated that 13.7% (7/51 samples) of samples had multiple-clone P. falciparum infections, with the remaining samples being clonal. Only the 44 clonal samples were analyzed further. DNA fragments of Pfcrt with reported mutations (2, 4) were amplified and sequenced. Five microsatellite (MS) markers flanking Pfcrt were also analyzed, including B5M77 (−20 kb), 2E10 (−5 kb), PE12A (+6 kb), 2H4 (+22 kb), and PE14F (+106 kb) (3, 10).

The combined msp1/msp2 genotyping revealed 31 different genotypes (Table 1). The addition of MS typing to the msp1/msp2 genotyping provided further resolution, distinguishing 41 different genotypes in the 44 samples, indicating the diverse genetic backgrounds of the parasites. Of the 44 isolates examined, 28 (64%; 95% confidence interval, 47.5 to 77.8%) had mutant Pfcrt genes (Table 1) with the same Pfcrt allelic type, i.e., the E1a type (9) commonly reported for Southeast Asia and Africa. The majority of the Pfcrt mutant isolates (27/28 isolates) had identical MS marker sizes for at least four of the five markers, and these were the same as those flanking the E1 Pfcrt allelic type (Table 1). The 28 mutant Pfcrt isolates had 21 different msp1/msp2 haplotypes. These results demonstrate that the mutant Pfcrt alleles in P. falciparum isolates collected from the two locations in Hainan share a common ancestry with Southeast Asian CQR parasites and that there has been little recombination around Pfcrt, while other antigen-encoding genes differ. CQR in Hainan was first reported in 1974, several years after CQR was reported in Southeast Asia. It is highly likely that CQR parasites were introduced to Hainan from Southeast Asia, given the close proximity of Hainan Island to Southeast Asia. CQR introduction to Hainan probably occurred in the late 1960s or early 1970s as part of the CQ selective sweep that spread across Southeast Asia and Africa (10).

TABLE 1.

Pfcrt allelic types and polymorphisms in MS markers flanking Pfcrt in P. falciparum samples collected from Hainan, China, compared to those in samples from Southeast Asia

Pfcrt allelic type (no. of samples)	Sample(s)	Corresponding msp1/msp2 haplotype(s)b	MS marker size (bp)		Amino acid in PfCrt												MS marker size (bp)
			B5M77 (−20 kb)	2E10 (−5 kb)	72	74	75	76	97	144	160	220	271	326	356	371	PE12A (+6 kb)	2H4 (+22 kb)	PE14F (+106 kb)
Samples from Hainan (44)
Wild type (16)	F12, F13, F14	1, 2, 3	147	164	C	M	N	K	H	A	L	A	Q	N	I	R	314	200	142
	6, 7	4, 5	147	186	C	M	N	K	H	A	L	A	Q	N	I	R	314	186	142
	F6, F7	6, 7	171	174	C	M	N	K	H	A	L	A	Q	N	I	R	296	206	136
	Q7	8	147	164	C	M	N	K	H	A	L	A	Q	N	I	R	314	198	142
	Q1	9	147	178	C	M	N	K	H	A	L	A	Q	N	I	R	314	218	148
	F11	10	149	188	C	M	N	K	H	A	L	A	Q	N	I	R	314	200	136
	F20	11	149	188	C	M	N	K	H	A	L	A	Q	N	I	R	314	200	142
	F18	12	151	178	C	M	N	K	H	A	L	A	Q	N	I	R	314	204	136
	Q8	13	151	178	C	M	N	K	H	A	L	A	Q	N	I	R	314	204	145
	F3	12	151	178	C	M	N	K	H	A	L	A	Q	N	I	R	314	206	136
	F16	14	171	164	C	M	N	K	H	A	L	A	Q	N	I	R	296	204	136
	F2	15	171	174	C	M	N	K	H	A	L	A	Q	N	I	R	296	192	139
E1a (28)	1, 2, 4, 5, 8, F1, Q5, Q9, Q15, Q17, Q21, Q22	16, 17, 18, 19, 1, 20, 2, 21, 22, 6, 23, 23,	149	170	C	I	E	T	H	A	L	S	E	S	T	I	314	204	145
	9, 11, F5, Q2, Q14, Q16, Q18, Q19, Q20	24, 25, 26, 27, 7, 17, 25, 18, 21	149	170	C	I	E	T	H	A	L	S	E	S	T	I	314	192	145
	3, Q11, Q12	14, 14, 28	149	170	C	I	E	T	H	A	L	S	E	S	T	I	314	204	139
	F4, F10	29, 19	149	170	C	I	E	T	H	A	L	S	E	S	T	I	314	206	145
	F15	30	149	170	C	I	E	T	H	A	L	S	E	S	T	I	314	204	142
	F9	31	149	170	C	I	E	T	H	A	L	S	E	S	T	I	296	204	142
Samples from Southeast Asiaa
E1a (3)	Dd2, AA071	NR	149	170	C	I	E	T	H	A	L	S	E	S	T	I	314	204	145
	C2B	NR	149	170	C	I	E	T	H	A	L	S	E	S	T	I	314	184	148
E1b	K1	NR	149	170	C	I	E	T	H	A	L	S	E	S	I	I	314	204	145
E1c	TM93-C1088	NR	149	170	C	I	E	T	L	A	L	S	E	S	T	I	314	204	145

^aData published previously (3), used only for comparison in the current study.

^bNR, not reported.

Sixteen isolates (36%; 95% confidence interval, 22.1 to 52.4%) were identified as having wild-type Pfcrt alleles, with no mutation in any examined fragment of Pfcrt (Table 1), and are thus susceptible to CQ. The lack of any mutation in the eight key codons identified in all mutant Pfcrt alleles suggests that the reduction in the prevalence of CQR parasites on Hainan Island is due to a resurgence of parasites with wild-type Pfcrt, not to back mutation of CQR parasites. In contrast to CQR isolates, the wild-type Pfcrt-carrying isolates showed highly diverse patterns of MS marker sizes flanking Pfcrt; 12 different MS patterns were identified in 16 wild-type Pfcrt isolates, with no apparent dominance of any pattern (Table 1). They also showed diversity in other genetic regions, with 15 different msp1/msp2 haplotypes, 5 of which were observed in Pfcrt mutant isolates. The results suggest that CQS parasites on the island have diverse origins and did not expand from one or a few clonal CQS populations. Therefore, despite the prevalence of mutant Pfcrt, nearly reaching fixation under CQ selection pressure, the number of CQS isolates did not become low enough to create a genetic bottleneck in the CQS parasite population.

The prevalence of wild-type Pfcrt alleles in our P. falciparum samples collected from central and eastern areas of Hainan Island is comparable to that previously reported by Wang et al. (8) for samples collected mostly from southern areas of the same island. Thus, it appears that wild-type CQS Pfcrt alleles are returning across the entire island. However, their frequency is still lower than that of the mutant CQR Pfcrt alleles 25 years after cessation of CQ therapy for falciparum malaria. Many factors may have contributed to the slow return of CQS parasites to Hainan Island, and our study suggests that a genetic bottleneck of CQS parasites is unlikely to be one of them. The CQR Pfcrt allelic type identified on the island matches the allele which is dominant in Africa, suggesting that the difference in CQS reemergence rates between Hainan and Malawi is not due to significant differences in the fitness of CQR parasites. There are two possible explanations for the observed difference in CQS reemergence rates. The first relates to the use of CQ for the treatment of vivax malaria. Since both Plasmodium species are present in Hainan, it is likely that some falciparum cases or mixed-species infections are diagnosed as vivax malaria and consequently treated with CQ. This would maintain some level of selection, but the full extent is difficult to ascertain. Alternatively, the use of piperaquine, a structurally similar compound to CQ, may also maintain some selection pressure, as there is evidence of weak cross-resistance between the two drugs (1). Although both factors may have contributed to the slow return of CQS parasites, it is more likely the result of continued CQ use. Importantly, the CQS parasites that are returning to the region retain diverse genetic backgrounds, which has implications for further control efforts.