Name: Khalil, Insaf Home country: Sudan Project period: 1997-2000   Title of project Cotrimoxazole and Fansidar in the Treatment of acute uncomplicated falciparum malaria, efficacy, safety and molecular characterization of resistance    Abstract The WHO and the UNICEF have recommended cotrimoxazole, a combination of trimethoprim and sulfamethoxazole (TRM/SMX), for the treatment of suspected pneumonia, malaria and all childhood febrile illnesses in malaria endemic areas with no access to diagnostic facilities. Although this strategy might reduce mortality and morbidity in children, the intensive use of this antibiotic via cross-resistance with the structurally and functionally similar antimalarial pyrimethamine/sulfadoxine (PYR/SDX) could impose serious problems in areas where this drug is still effective. Accordingly, in vitro and in vivo sensitivities of P. falciparum isolates to the standard antibacterial dose of TRM/SMX  (2 tabs/12h x 5 days), were compared in a controlled, double blind hospital based clinical trail, with a single dose (2.5 mg/Kg) of PYR/SDX. To ensure compliance and drug absorption, plasma levels of TRM, the metabolite ACSMX, SMX & SDX were measured prior & post treatment on days 3, 7, 14 or upon recrudescence. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis were used to assess the role of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) genotypes before treatment in TRM/SMX and PYR/SDX in vivo outcome and to follow the dynamics of these genotypes post therapy.   The study was conducted in an unstable malaria area in Khartoum, the Sudan. 90 patients with acute uncomplicated falciparum malaria were recruited for the study. 47 patients were treated with TRM/SMX and 43 with PYR/SDX. The two groups were comparable in terms of demographical, parasitological, clinical and haematological data on entry to the study. Both antifols were efficient, within 48h, 91% (TRM/SMX) & 86% /PYR/SDX), became aparasitaemic, with a MPCT of 2.00 & 1.40 days respectively. With both drugs >70% of the patients became afebrile within 24h, with MFCT of 1.77 & 1.84 respectively. Cure rates were similar & high (>89% for both). Drugs were well tolerated, with good compliance. Adverse effects were only gastrointestinal & dizziness. Patients in cotrimoxazole group complained about its lengthy dosage. Treatment failure occurred in 10% of the patients. To predict the cause (s) of treatment failure, the in vivo outcome was compared with the in vitro, plasma drug levels and DHFR and DHPS genotypes. Due to the short elimination half-life of TRM/SMX, samples from patients with recurring parasites were genotyped at 3 unlinked polymorphic loci (MSP1, MSP2 and GLURP), to discriminate re-infection from recrudescence.     P. falciparum isolated from the above mentioned malaria patients, were tested in vitro against cotrimoxazole and Fansidar Ò components, TRM, PYR, SMX and SDX. DHFR and DHPS genotypes were determined to compare the isolate chemosensitivity with the point mutations in the coding regions of the 2 genes. Chemosensitivity of P. falciparum isolates to the DHFR inhibitors, PYR and TRM, decreased with the number of point mutations in the DHFR. Half-inhibitory concentrations (IC50) values between 0.01-0.128 mg/100 ml and 0.05-0.2 mg/100 ml were recorded for PYR and TRM, respectively, in isolates with the wild type at all tested codons. In vitro susceptibility of isolates with mixed infections varied between high and intermediate level of susceptibility. Isolates with double or triple mutations had IC50 values between 9-12.8 and 0.4-3.2 mg/100 ml for PYR and TRM, respectively. Despite the 100-fold increase in the IC50 of PYR between isolates with wild and mutant genotypes, the difference was only 8- fold for TRM. This suggests incomplete cross-resistance between the 2 DHFR inhibitors and that TRM remains effective even on isolates highly resistant to PYR. Half-inhibitory concentrations obtained in this study reflect the susceptibility of the tested isolates to SDX and SMX, and recommends the use of folate depleted RPMI media to increase the reproducibility of the culture method and to obtain significant differences in the IC50 values of isolate with wild and mutant DHPS alleles.   Our data showed that PYR or TRM in vitro resistance was not a determinant of the in vivo outcome to both PYR/SDX and TRM/SDX. The correlation between SDX and SMX in vitro and in vivo outcome, although based on few samples, might shed some light on the importance of these compounds in the mechanism of clinical resistance.   SDX plasma concentrations in patients treated with PYR/SDX do not suggest poor compliance or inadequate drug levels, but points to parasite resistance or increased pre-treatment parasitaemia as causes of treatment failure. DHFR and DHPS mutant genotypes (DHFR Ile-51/Asn-108/DHPS Ala-436) detected in-patients with treatment failure further documented this. The presence of Ala-436 in 3/3 resistant parasites isolated from patients with SDX concentrations, at the steady state, well above the therapeutic level (89-264 mg/ml) was found to be significantly associated with treatment failure. Although this was based on few samples, it could be considered as a preliminary evidence of the importance of DHPS Ala-436 mutation in decreasing in vivo susceptibility of P. falciparum to PYR/SDX in areas of low drug use.  Our data also suggested the insufficiency of DHFR Ile-51 and Asn-108 alone to bring about PYR/SDX in vivo failure. The same DHFR and DHPS genotypes, (Ile-51/Asn-108/Ala-436), was found in the pre-treatment samples of the resistant patients. Additional mutations were not detected upon recrudescence.   TRM, ACSMX and SMX plasma levels post therapy suggested in vivo drug insufficiency as a cause of treatment failure in 2/5 patients. In the other 3 patients, where adequate plasma drug levels were attained, treatment failure could be due to parasite resistance. MSP1, 2 and GLURP genotyping showed that all recurring parasites were a result of recrudescence rather than re-infection. This finding is important epidemiologically and clinically, as all, but one of the resistant patients was asymptomatic. In the absence of follow up silent recrudescence might not be detected or reported, yet contribute substantially to the spread of drug resistant phenotypes. The high prevalence of DHFR Ile-51 and Asn-108 alone or coupled with DHPS Ala-436 in pre-treatment samples of the in vivo sensitive parasites, reflects that this level of mutation could be overcome by cotrimoxazole. The presence of DHFR Arg-59 and DHPS Gly-437 in 2/5 resistant parasites might reflect the importance of these 2 codons in in vivo susceptibility to cotrimoxazole.  Post therapy cotrimoxazole selected for DHFR Arg-59, Asn-108, and DHPS Ser-436 and Gly-437 genotypes associated with decreased susceptibility to PYR/SDX.   Given the high prevalence of DHFR mutations in the study area and the facts that these mutations occur in a step-wise fashion and that cotrimoxazole selects for DHFR and DHPS genotypes associated with in vivo PYR/SDX resistance, TRM/SMX usage should be controlled. Due to its lengthy dosage, poor compliance may result in a low cure rate with increased tolerance of the parasite strains & suitable conditions for selection of resistant mutants.     Involved research institution(s) Centre for Medical Parasitology (CMP), University of Copenhagen   Supervisor(s) Anita M. Rønn , Department of Infectious Diseases, the National University Hospital, (Rigshospitalet) Ib C. Bygbjerg, Department of International Health, Institute of Public Health, University of Copenhagen   Correspondence