Chloroquine (CQ) resistant falciparum malaria makes many developing countries face one of the greatest public health challenges. (Nuwaha F, 2001, The challenge of chloroquine-resistant malaria in sub-Saharan Africa. Health Policy and Planning, 16: 1-12.)
CQ resistance was reported in Southeast Asia and South America 40 years ago, where CQ is almost abandoned. But, CQ is so cheap and safe that it is still first candidate in many countries including sub-Saharan Africa.
Traditional definition: In examination of blood-slide under continuous CQ treatment,
| Sensitive | No asexual form by day 6 and no parasite on day 7, and ring form do not reappear by day 28. |
| RI | No asexual form by day 6 and no parasite on day 7, but ring form reappear by day 28 /or/ ring forms disappear for at least 2 consecutive days, but return and are present on day 7. |
| RII | Ring forms do not disappear but are reduced to 25% or less of the pretreatment level during the first 48 hours of treatment. |
| RIII | Other (reduced to 75% or less, or remain at the same level, or increase) |
Problems of the traditional definition above:
Recent emergence and/or spread of CQ resistant falciparum malaria are reported in sub-Saharan Africa (Nuwaha F, 2001, ibid.), Melanesia (e.g. Inaba H et al., 2001, Variation of incubation time in an in vitro drug susceptibility test of Plasmodium falciparum isolates studied in the Solomon Islands. Parasitology International, 50: 9-13; where they report 22/43 by a new in vitro examination method), and even in Eastern Afghanistan (Rab MA et al., 2001, Resistance of Plasmodium falciparum malaria to CQ is widespread in eastern Afghanistan. Annals of Tropical Medicine & Parasitology, 95: 41-46; where they report 55% RI and 11% RII/RIII) In map provided by WHO, the area C almost matches with CQ resistant falciparum malaria.
The mechanism how the CQ interferes parasite's growth has recently been recognized (see Figure 1 by Warhurst). CQ changes heme metabolism in the digestive vacuole of P.f. In resistant parasites, the accumulation of CQ inside the vacuole is diminished.
The pfmdr1 gene (encoding Pgh1, a trans-membrane protein of P.f. vacuole) has been proposed as the candidate of CQ resistance in late 1980's. However, the results of follow-up studies were controversial. Recent genetic transformation experiments revealed that introducing mutated pfmdr1 gene in itself cannot give CQ resistance.
Last year, pfcrt, a gene with 13 exons, in chromosome 7 is focused as encoding another trans-membrane protein of P.f. vacuole (Fidock DA et al.: Mutations in the P. f. digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol. Cell, 6: 861-871, 2000.) One mutation, the substitution of threonine (T) for lysine (K) at position 76 (K76T=T76) was present in all resistant isolates and absent from all sensitive isolates tested in vitro. And genetic transformation experiments with plasmids expressing pfcrt T76 gave CQ resistance for 3 different CQ sensitive clones. The role of mutations in the case of failure of CQ treatment remains to be examined in clinical settings.
The present paper aimed, (1) haplotype analyses of parasites, (2) assessing the relation between CQ resistance and mutations.
514 patients, at least 2 yr, with positive blood smear of P.f. asexual forms, seeking treatment for malaria-like symptoms (fever, chills, headache, and body pain), without CQ allergy nor concurrent nonmalarial fever nor chronic malnutrition nor severe malaria (presence of coma, obtundation, seizures, prostration, respiratory distress, shock, protracted vomiting, severe parasitemia, Hct<15%, sGlu<40 mg/dl), from Mopti (population 60000) and Bandiagara (12000, famous Dogon art site), where malaria is highly endemic, attended from August 1997 to December 1997.
CQ was administered orally, 10 mg/kg/day for 2days + 5 mg/kg/day on day 3. If the patient vomited within 30 min., another full dose was given. Within 31 min to 1 hr, half dose was given.
Clinical follow-up on days 1, 2, 3, 7, 14 after treatment. Blood slide was checked on days 3, 7, 14 when malaria-like symptoms were reported or fever higher than 37.5 degree C was detected. CQ resistance was checked by traditional manner. 469 patients completed.
DNA was extracted from patients blood using dried filter papers. Nested mutation-specific PCR or nested PCR + mutation-specific endonuclease digestion was used to detect mutations in pfcrt (I74 [isoleucine for methionine], E75 [glutamic acid for N], T76, S220 [serine for alanine], E271 [for glutamine], S326 [for N], T356 [threonine for I], I371 [for arginine]) or pfmdr1 (Y86 [tyrosine for N], Y184 [for phenylalanine], C1034 [cysteine for serine], N1042[asparagine for aspartic acid], Y1246[for aspartic acid]).
Size polymorphisms of k and W region of cg2 (near pfcrt) were detected by agarose-gel electrophoresis.
Direct DNA sequencing and microsatellite analysis were also applied when needed.
Statistics: Moleculer analysis was done for randomly selected samples without any knowledge of clinical outcome. RI, RII, and RIII were pooled. Univariate analyses were done by chi-square test or Fisher's exact test. Multiple logistic regression analysis was done by STATA.
Of 469 patients, sensitive 86%, RI 11 %, RII 2%, RIII 1%. Median age of patient was 10 yr. Median parasite density was 12800/mm3
All but T356 mutations showed higher prevalence after treatment (Table 1). pfcrt T76 existed in all post-treatment infections, but pfmdr1 Y86 did not exist in 14% patients (Figure 1). Microsatellite analysis for 8 CQ resistant with pfcrt K76 (wild) and 22 sensitive with pfcrt T76 confirmed the diversity of the genetic backgrounds of P.f. (mutistrains). T356 is reported no CQ resistance by in vitro assay. In haplotype analysis, pfcrt K76 tended to be CQ sensitive with higher wild type prevalence in 220, 271, 326, 356, 371, but pfcrt T76 did not show such tendency.
T76 was the most likely to be resistant to CQ treatment (Table 2). Multiple logistic regression to control other variables also showed T76 CQ resistance (odds ratio 16.1 [95% CI ranges 5.7-45.7]).
Kaneko A et al. reported, in the 42nd annual meeting of Japanese Society of Tropical Medicine (23-24 September 2001 at Tokyo), that pfcrt T76 was not found in any sample with (8) or without (53) CQ resistance and that prevalence of pfmdr1 Y86 was significantly higher in CQ resistant (7/8) than in sensitive (19/53) in Malawi samples. This inconsistent result with Mali seems to be attributable to the fact that Malawi stopped CQ treatment 7 years ago. This result may imply, at least, existence of regional difference.