INTRODUCTION
In 1979, in view of the widespread resistance of malaria strains to chloroquine treatment in Hainan, People’s Republic of China, the use of chloroquine against Plasmodium falciparum was abandoned and replaced by use of a new bisquinoline antimalarial drug, piperaquine.1 Subsequent in vitro microtest surveys suggested that the prevalence of chloroquine-resistant P. falciparum strains decreased from 98% in 1981 to 61% in 1991, and correspondingly by the four-week in vivo test the rate of chloroquine-resistant P. falciparum malaria decreased from 84% to 40% in 1991.2,3 Another study found in vitro chloroquine resistance rates had decreased from 96% in 1974–1983 to 71% in 1999–2001.4
Recent identification of the role of the P. falciparum chloroquine resistance transporter (PfCRT) mutations in chloroquine resistance has provided a molecular marker for P. falciparum strains that can fail chloroquine treatment.5–7 The PfCRT protein is altered in naturally chloroquine-resistant P. falciparum parasites by multiple mutations that always include a key charge change from a lysine-to-threonine substitution at PfCRT position 76 (K76T). The patterns of the mutations that accompany K76T depend on their geographic origin and probably represent accommodative changes that help preserve the native function of the transporter. To investigate the decrease in chloroquine-resistant P. falciparum in Hainan, we initiated a study of PfCRT 76T rates and the decreases in chloroquine resistance indicated by this marker since 1978.
MATERIALS AND METHODS
Samples.
Samples in this study included blood smears on glass slides and filter paper from subjects enrolled in the Ti-anan, Nandao, and Dongfang counties of southern Hainan province, where P. falciparum malaria is endemic. Protocols associated with sample collection were reviewed and approved by the institutional review board of Guangzhou University of Traditional Chinese Medicine (Guangzhou, People’s Republic of China). Information generated in this study was not linked to the original clinical records or in a form associable with specific individuals.
Glass slides collected in 1978, 1979, 1980, 1981, 1986, 1997, and 1998 were wrapped separately with paper and preserved in tightly sealed glass bottles. The slides from 1978, 1979, 1980, and 1981 were preserved from cases of uncomplicated and complicated P. falciparum malaria, including a case of cerebral malaria. Slides from 1986, 1997, and 1998 were from cases of uncomplicated malaria that had been used to provide samples for in vitro sensitivity tests. All samples were taken prior to the initiation of malaria treatment. Parasitemias were reconfirmed by microscopic examination of thick and thin blood smears.
Filter paper blood samples were collected in year 2001 from patients presenting with malaria symptoms and blood smears positive for P. falciparum. For this purpose, drops of peripheral blood were placed on 1.5 × 7.0-cm strips of Whatman (Brentford, United Kingdom) 3MM filter paper so that the blood covered half the length of the strip. The strips were then air-dried and kept in plastic bags until use.
Extraction of DNA.
The DNA from filter paper and glass slides was obtained by recommended protocols and reagents of QIAamp DNA mini kit (Qiagen, Valencia, CA). Supplied ATL buffer (60–90 μL) was applied to the blood smears on the slides, which were then placed on a block heater at 80°C. The buffer was taken up and reapplied by pipette several times to wash the heated smears and then collected in a 1.5-mL microcentrifuge tube. After this process was repeated 2–3 times to obtain a volume of 180 μL, the DNA was purified, and 5 μL of the resulting solution were used for polymerase chain reaction (PCR) amplification. Blank slides and blood spots from blank (non-parasitized) wells of in vitro tests were used as negative controls in the DNA extraction and PCR amplification procedures to confirm lack of contamination.
Nested PCR and mutation-specific amplifications.
Flanking primers CRTP1 5′-CCGTTAATAATAAATACACGCAG-3′ and CRTP2 5′-CGGATGTTACAAAACTATAGT-TACC-3′ (spanning 537 basepairs of the pfcrt gene sequence containing codon 76) were combined with 5 μL of DNA sample solution in standard PCR buffer mixture. After an initial two-minute denaturation step at 94°C, the DNA sequence was amplified by 35 PCR cycles at 94°C for 20 seconds, 52°C for 10 seconds, 48°C for 10 seconds, and 60°C for 1.5 minutes. Product from this PCR (1 μL) was then used in two follow-up, nested, allele-sensitive PCR amplifications to detect the codons for PfCRT 76K or 76T. These diagnostic PCR amplifications used a common inner primer (CRTP3 5′-TGACGAGCGTTATAGAG-3′) coupled with either CRTP4m 5′-GTTCTTTTAGCAAA AATTG-3′ (detects the 76T codon) or CRTP4w 5′-GTTCTTTTAGCAAAAATCT-3′ (detects the 76K codon). The PCR stages for these diagnostic amplifications were at 94°C for 2 minutes, followed by 35 cycles at 94°C for 30 seconds, 47°C for 20 seconds, and 60°C for 1 minute. Purified genomic DNA from P. falciparum clones HB3 (chloroquine sensitive) and Dd2 (chloroquine resistant) were used as positive controls, and water, extracted uninfected blood smears, and uninfected blood spots on filter paper were used as negative controls. The PCR products from the amplification reactions were evaluated by electrophoresis on 2% agarose gels.
Detection of codons for PfCRT 76K and 220A and for the P. falciparum P glycoprotein homolog 1 (Pfpgh-1) 86Y by restriction digestion of PCR products.
The wild-type codon for PfCRT 76K was detected by a primary round of PCR amplification with primers CRTP1 and CRTP2 as described above, followed by a second, nested PCR amplification in a 25-μL volume with two internal primers flanking codon 76: CRTD1 5′-TGTGCTCATGTGTTTAAAC-3′ and CRTD2 5′-CAAAACTATAGTTACC AATTTTG-3′. The PCR amplification stages were at 94°C for 2 minutes, followed by 35 cycles at 94°C for 30 seconds, 48°C for 30 seconds, and 60°C for 1 minute. Eight microliters of the product reaction mixture were treated directly with 0.5–1.0 units of the restriction enzyme Apo I for six hours at 50°C as recommended by the manufacturer (New England Biolabs, Beverly, MA). The enzyme Apo I recognizes and cuts the 76K codon, releasing a 34-basepair fragment from one end of the 145-basepair CRTD1/CRTD2 product. It does not cut the product containing the 76T codon found in chloroquine-resistant P. falciparum.
To detect the codon for PfCRT 220A, we first performed a primary round of PCR amplification using the primer pair CRT2a 5′-CCCAAGAATAAACATGCGAAAC-3′ and CRT2b 5′-ACAATTATCTGGGAGCAGTT-3′. The PCR amplification stages were at 94°C for 2 minutes, followed by 35 cycles at 94°C for 20 seconds, 52°C for 10 seconds, 48°C for 10 seconds, and 60°C for 1.5 minutes. Two microliters of the resulting product mixture were subsequently amplified under the same PCR conditions in a nested secondary reaction using the primers CRT220a 5′-TATTTATTTATTTATATATT-TTGTTTTCTTGCCATTAAGG-3′ and CRT220b 5′-ACAATTATCTCGGAGCAGTT-3′. Eight microliters of the resulting 150-basepair product were treated with 0.5 units of Bgl I for one hour at 37°C as recommended by the manufacturer (Invitrogen, Carlsbad, CA). The enzyme Bgl I cleaves 40 basepairs from the CRT220a/CRT220b product containing the wild-type 220A codon, but not from the product containing the 220S codon.
Gene segments spanning codon 86 of the P. falciparum multidrug resistance 1 (pfmdr1) gene were amplified in 25 μL of standard PCR mixture containing 5 μL of extracted DNA and primers MDR1 5′-ATGGGTAAAGAGCAGAAAGA-3′ and MDR2 5′-AACGCAAGTAATACATAAAGTCA-3′. The PCR amplification stages were at 94°C for 2 minutes, followed by 35 cycles at 94°C for 20 seconds, 52°C for 10 seconds, 48°C for 10 seconds, and 60°C for 1.5 minutes. A second, nested amplification from this segment was then performed under the same PCR conditions using 1 μL of the product solution and primers MDR3 5′-TGGTAACCTCAG-TATCAAAGAA-3′ and MDR4 5′-ATAAACCTAA-AAAGGAACTGG-3′. Presence of the mutant 86Y codon was detected by digestion of 8 μL of the second amplification product solution with 1.5 units of Afl III as recommended by the manufacturer (New England Biolabs). The products of restriction digestion were separated by electrophoresis on a 2% agarose gel and detected by staining with ethidium bromide.
Sequencing of DNA.
Products of the pfcrt gene for direct sequencing were prepared by a primary round of PCR amplification with primers F3 5′-TTGCTATATCCATGTTA-GAT-3′ and R6 5′-TCT CCATTTTATCCTAAAAG-3′, followed by a nested, second round of amplification with primers F4 5′-TTGTAACAATAGCTCTT-3′ and R5 5′-AATAA-GGATATTGCGTAATA-3′. The PCR amplification conditions were as described above for the pfmdr1 sequence. After treatment with SAP/Exo l (U.S. Biochemical Corp, Cleveland, OH), the PCR products were used directly in sequencing reactions and analyzed on an ABI 3730XL automatic sequencer as recommended (Applied Biosystems Inc., Foster City, CA).
In vitro drug susceptibility tests.
Microtest kits were provided by the Institute of Parasitic Diseases, Chinese Academy of Preventive Medical Science (Shanghai, People’s Republic of China), and susceptibility tests8 were performed as recommended by the World Health Organization. Briefly, 0.1 mL of blood was diluted with 0.9 mL of RPMI 1640 medium containing 5.94 mg/mL of HEPES, 2 mg/mL of NaHCO3, and 10% rabbit serum. After 50-μL aliquots of the resulting dilution were individually added to microplate wells precoated with serial dilutions of the antimalarial drugs, the microplates were incubated for 20–48 hours at 37 ± 1°C. Parasites were considered resistant when microscopic evaluation showed that schizont formation was not completely (100%) inhibited at a chloroquine concentration ≥ 1.6 pmol/μL.9
Statistical analysis.
Statistical analysis was performed using StatView 5.0.1 (SAS Institute Inc., Cary, NC). Statistical significance was based on an α level of 0.05.
RESULTS
Decreasing prevalence of the PfCRT 76T marker following the discontinuance of chloroquine use against P. falciparum malaria in Hainan.
Figure 1 summarizes the relative prevalence of the codons for PfCRT 76K and 76T in blood smears from 1978–1981, 1986, and 1997 and in filter papers from 2001, as determined by PCR amplification and restriction with Apo I. No or partial Apo I cutting indicating the presence of codon 76T was obtained with 101 (90%) of 112 samples from 1978–1981; results from 5 of these 101 samples suggested the mixed presence of the 76T and 76K codons. This evidence of the 76T codon decreased to 30 (70%) of 43 samples from 1986, 22 (65%) of 34 samples from 1997–1998, and 36 (53%) of 68 samples from 2001, including 3 samples with codons for both 76K and 76T). Prevalence of the 76T codon indicative of chloroquine resistance thus decreased from more than 90% to less than 60% in the 20 years following discontinuance of the use of chloroquine against P. falciparum (χ2 = 28.812, P < 0.0001).
We also performed an allele-specific PCR to confirm the Apo I restriction results from the 2001 samples. Results identified only the mutant 76T marker in the same 33 (48%) of 68 samples that showed no cutting with Apo I, whereas the mixed presence of the 76T and 76K markers was found in 4 (6%) of 68 samples. The one additional mixed infection detected by the amplification-refractory PCR but not by the Apo I restriction method can be attributed to higher sensitivity of the amplification-refractory PCR method when small subpopulations of chloroquine-resistant parasites are present in the sample (37 of 68 or 54% of the 2001 samples carrying the 76T marker).
The close association of PfCRT 76T and 220S noted in previous reports5,6,10 was also evident in these surveys. Of the DNA samples successfully amplified from the 2001 filter papers for Bgl I restriction testing, 30 of 30 that contained the 76K codon showed cutting indicative of the wild-type codon 220A. Conversely, 29 of 29 successful amplification products from samples containing the 76T codon showed no Bgl I cutting and therefore contained the 220S codon.
Sequence analysis of DNA of mutant pfcrt alleles.
Mutant pfcrt sequences spanning codon 76 were amplified from 32 selected DNA samples (selected at random from all years except 1979) and subjected to direct DNA sequencing. All were found to contain the four codons predicting the amino acid sequence 72-CVIET-76 characteristic of chloroquine-resistant parasites from southeast Asia.6,10
We also checked the pfcrt gene in six randomly selected 1986 samples for sequence differences in other regions known to harbor mutations in chloroquine-resistant parasites.5,10 In all cases, the codon data identified mutations representative of the southeast Asian origin of chloroquine resistance: M74I, N75E, K76T, A220S, Q271E, N326S, I356T, and R371I. The sequences contained none of the mutations selected in other geographic foci of chloroquine resistance (parasites from the foci in South America, The Philippines, and Papua New Guinea have such mutations as C72S, H97Q, A144T, L160Y, N326D, I356L, and R371T).5,10,11 The sequences also showed no evidence for the S163R mutation recently reported to return chloroquine-resistant parasites to the chloroquine-sensitive phenotype;12 we did not check more recent DNA samples for the S163R mutation because all 1997 and 2001 samples containing the 76T marker were chloroquine-resistant by in vitro microtests (next section and Table 1).
Association of the PfCRT 76T marker with in vitro chloroquine resistance in Hainan.
Table 1 shows the molecular marker results from 80 P. falciparum samples tested for in vitro drug response in 1997 and 2001; 22 of these samples were determined to be chloroquine-sensitive and 58 chloroquine-resistant by World Health Organization microtest criteria. Amplification products from all the chloroquine-sensitive samples carried the codon for PfCRT 76K, but not 76T. Of the 58 samples reported chloroquine-resistant by in vitro testing, 50 were found to carry the 76T codon, 5 were found to contain mixed 76T/76K codons, and 3 were discordantly found to contain the 76K codon. It was not possible to recover the frozen parasites and confirm the discordant drug test results from the three samples containing the 76K codon.
Lack of an association between Pfpgh-1 86Y polymorphism and chloroquine resistance in Hainan.
The 86Y polymorphism of Pfpgh-1 (encoded by the pfmdr1 gene) in southeast Asia has been associated with chloroquine resistance in some studies,13 but not in others.14–17 To test for possible association of this polymorphism with decreasing chloroquine resistance in Hainan, we checked pfmdr1 segments amplified from the DNA samples for Afl III restriction. Of 104 samples tested, only 2 of 20 samples from 1986 showed cutting indicative of the 86Y polymorphism (Figure 2). To ensure that the 86Y polymorphism was not being missed because of inadequate restriction conditions or other reasons, we randomly selected and sequenced 16 of the amplification products from the 1986 and 2001 DNAs. None of these samples showed evidence of the codon for Pfpgh-1 86Y.
DISCUSSION
Decreasing prevalence of the PfCRT 76T marker in Hainan is consistent with the decrease in chloroquine-resistant P. falciparum strains reported since 1981. Although the rates of resistance in Hainan are expected to vary by region, our finding of a decrease in PfCRT 76T prevalence from 90% in the late 1970s to 65% in the late 1990s and to 54% in 2001 is consistent with microtest assays showing 98% resistant strains in 1981 and 61% and 71% resistant strains in recent years.2–4 In contrast, Pfpgh-1 86Y, a polymorphism in southeast Asia that has been associated with chloroquine resistance in some studies,13 but not others,14–17 was absent from nearly all Hainan P. falciparum samples and showed no correlation with the changes in prevalence of chloroquine resistance.
The decrease in chloroquine-resistant P. falciparum in Hainan has evidently been less rapid than that reported recently from Malawi, where a switch from chloroquine was followed by a decrease in the PfCRT 76T marker from 85% in 1993 to 13% in 2000.18 The explanation for this less rapid decrease may involve several factors. First, after the 1979 discontinuation of chloroquine treatment against P. falciparum malaria, use of the drug was still recommended for P. vivax malaria in Hainan. Not all chloroquine pressure against P. falciparum would therefore have been removed since chloroquine was probably used against undifferentiated cases of malaria and perhaps some mixed P. falciparum-P. vivax infections, which occur in regions where both species are transmitted.19,20 Second, local authorities of health sectors in some areas of Hainan revived the use of chloroquine against P. falciparum for periods of time in the 1990s (Wang X, unpublished data). Third, widespread use in China of piperaquine, a bisquinoline containing structural components of chloroquine, may provide some positive selection pressure for PfCRT mutations, even though cross-resistance between these two antimalarial drugs seems to be low in P. falciparum.21,22
Data from 3 of 80 samples in our study showed discordances between their PfCRT type and their chloroquine response as determined by the World Health Organization in vitro microtest method. Independently repeated PCR experiments confirmed the PfCRT types of the three samples, but we were unable to obtain viable parasites from cryopreserved samples to retest their chloroquine responses. Discordances commonly result from inaccuracies of microtest methods on parasites from patient blood samples that have not been adapted to continuous in vitro culture.7,23 Thus, while tests can generally give good indications in epidemiologic surveys, the accuracies of individual assignments (in terms of inhibitory concentration) can be thrown off by parasite sample viability, inadequate or incorrect in vitro test conditions, inaccuracy in microscopy, and humoral factors that can carry over from the sample and act with the drug to affect parasite maturation.24 It is unlikely that the three discordances in our study can be explained by an alternative mechanism of chloroquine resistance not involving the K76T mutation in PfCRT. No such mechanism has been identified for P. falciparum parasites from any malarious region, and a single mechanism of chloroquine resistance in Hainan is consistent with the sweep of mutant parasites from a single focus in southeast Asia that eventually expanded across Asia, the Indian subcontinent, and into Africa.10
Clinical studies have consistently found the prevalence of the PfCRT 76T marker to be greater than the rate of chloroquine failure in study groups, indicating the presence of additional host and parasite factors that contribute to the clearance of chloroquine-resistant parasites.7 In 1991, studies from Hainan reported a rate of in vitro chloroquine resistance about 1.5 times higher than the rate of in vivo treatment failures.2,3 At these comparative rates, a 50–60% prevalence of PfCRT 76T in Hainan today suggests that roughly one-third to half of P. falciparum malaria cases would be expected to fail chloroquine treatment.
The recovery of chloroquine-sensitive parasite populations in Hainan after removal of drug pressure suggests a slight advantage of the native PfCRT molecule over its mutant forms and points to the possible value of drug rotation strategies in antimalarial policies. However, our view is that any reintroduction of chloroquine as monotherapy remains out of the question, while the possibility of its use in a drug combination will require much lower rates of resistance as well as a demonstrable advantage over alternatives. Combination treatments that include an artemisinin derivative with other antimalarials such as piperaquine25,26 presently provide stronger options the treatment of drug-resistant malaria in southeast Asia.
Plasmodium falciparum chloroquine resistance transporter (PfCRT) markers and reported in vitro microtest outcomes from P. falciparum samples on Hainan Island in 1997 and 2001
| Reported in vitro microtest outcome | ||||||
|---|---|---|---|---|---|---|
| Sensitive | Resistant | |||||
| 1997 | 2001 | Total (%) | 1997 | 2001 | Total (%) | |
| PfCRT 76K | 6 | 16 | 22/22 (100) | 1 | 2 | 3/58 (5.2) |
| Mixed 76K/76T | 0 | 0 | 0/22 (0) | 4 | 1 | 5/58 (8.6) |
| PfCRT 76T | 0 | 0 | 0/22 (0) | 17 | 33 | 50/58 (86.2) |
Prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76K and 76T markers in samples from malaria cases in Hainan, People’s Republic if China, 1978–2001.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 4; 10.4269/ajtmh.2005.72.410
Prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76K and 76T markers in samples from malaria cases in Hainan, People’s Republic if China, 1978–2001.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 4; 10.4269/ajtmh.2005.72.410
Prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76K and 76T markers in samples from malaria cases in Hainan, People’s Republic if China, 1978–2001.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 4; 10.4269/ajtmh.2005.72.410
Prevalence of the Plasmodium falciparum P glycopro-tein homolog 1 (Pfpgh-1) 86N and 86Y markers in samples from malaria cases in Hainan, People’s Republic of China, 1978–2001.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 4; 10.4269/ajtmh.2005.72.410
Prevalence of the Plasmodium falciparum P glycopro-tein homolog 1 (Pfpgh-1) 86N and 86Y markers in samples from malaria cases in Hainan, People’s Republic of China, 1978–2001.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 4; 10.4269/ajtmh.2005.72.410
Prevalence of the Plasmodium falciparum P glycopro-tein homolog 1 (Pfpgh-1) 86N and 86Y markers in samples from malaria cases in Hainan, People’s Republic of China, 1978–2001.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 4; 10.4269/ajtmh.2005.72.410
Authors’ addresses: Xinhua Wang, Guoqiao Li, Peiquan Chen, Xingbo Guo, Linchun Fu, and Lin Chen, Tropical Medicine Institute, Guangzhou University of Traditional Chinese Medicine, 12 Jichang Road, Guangzhou, People’s Republic of China, Telephone: 86-20-8656-6656, Fax: 86-20-8655-6726, E-mail: xinhuaw@yahoo.com. Jianbing Mu, Xinzhuan Su, and Thomas E. Wellems, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook III Building, MSC 8132, Bethesda, MD 20892-8132, Telephone: 301-496-4021, Fax: 301-402-2201, E-mails: xsu@niaid.nih.gov and tew@helix.nih.gov.
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