Human antibody reaction against recombinant salivary proteins of Phlebotomus orientalis in Eastern Africa.

BACKGROUND: Phlebotomus orientalis is a vector of Leishmania donovani, the causative agent of life threatening visceral leishmaniasis spread in Eastern Africa. During blood-feeding, sand fly females salivate into the skin of the host. Sand fly saliva contains a large variety of proteins, some of which elicit specific antibody responses in the bitten hosts. To evaluate the exposure to sand fly bites in human populations from disease endemic areas, we tested the antibody reactions of volunteers' sera against recombinant P. orientalis salivary antigens. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant proteins derived from sequence data on P. orientalis secreted salivary proteins, were produced using either bacterial (five proteins) or mammalian (four proteins) expression systems and tested as antigens applicable for detection of anti-P. orientalis IgG in human sera. Using these recombinant proteins, human sera from Sudan and Ethiopia, countries endemic for visceral leishmaniasis, were screened by ELISA and immunoblotting to identify the potential markers of exposure to P. orientalis bites. Two recombinant proteins; mAG5 and mYEL1, were identified as the most promising antigens showing high correlation coefficients as well as good specificity in comparison to the whole sand fly salivary gland homogenate. Combination of both proteins led to a further increase of correlation coefficients as well as both positive and negative predictive values of P. orientalis exposure. CONCLUSIONS/SIGNIFICANCE: This is the first report of screening human sera for anti-P. orientalis antibodies using recombinant salivary proteins. The recombinant salivary proteins mYEL1 and mAG5 proved to be valid antigens for screening human sera from both Sudan and Ethiopia for exposure to P. orientalis bites. The utilization of equal amounts of these two proteins significantly increased the capability to detect anti-P. orientalis antibody responses.

Plasmodium falciparum genotypes in matched peripheral, placental and umbilical cord blood in an area characterised by unstable malaria transmission in eastern Sudan.

BACKGROUND: There are few published studies on Plasmodium falciparum genotypes in peripheral, placental and umbilical cord blood in areas characterised by unstable malaria transmission. METHOD: A cross-sectional study was conducted to investigate P. falciparum genotypes in matched peripheral, placental and umbilical cord blood in eastern Sudan. Thick blood smears and P. falciparum merozoite surface protein 1 (MSP1) and 2 (MSP2) genes as polymorphic markers in polymerase chain reactions were investigated in 3 kinds of samples of 153 pregnant women at delivery. RESULTS: There was no significant difference in the prevalence of blood film-detected P. falciparum in which 5 (3.3%), 7 (4.6%) and 3 (2.0%) (P = 0.437) of the 153 samples were determined to be P. falciparum-positive by microscopy for maternal peripheral, placental and cord blood samples, respectively. Out of these 145 samples, 24 (16.6%), 39 (26.9%) and 24 (16.6%) (P = 0.039) of the peripheral, placental and cord samples, respectively, had submicroscopic parasitaemia (blood films were negative). There was no association between submicroscopic parasitaemia and age or parity. RO33 and K1 (MSP1 alleles) were detected in 21/29 (72.4%), 42/46 (85.7%), 26/27 (92.2%) and 6/29 (20.6), 16/46 (32.6) and 0(0) (P < 0.001) of the maternal, placental and cord samples, respectively. MAD20 was not detected in any of the samples. While the 3D7/IC1 allele was detected in 12 (41.3%), 30 (65.2%) and 4 (14.8%) (P < 0.001) of the peripheral, placental and cord samples, respectively, the FC (MSP2) allele was detected in only the 6 (20.6) placental samples. Multi-clonal infection was detected in 10 (34.4), 27 (58.6) and 3 (11.1) (P < 0.001) of the maternal placental and cord samples, respectively. CONCLUSION: Compared with the peripheral and cord samples, placental samples had a higher prevalence of submicroscopic parasitaemia. MSP1 alleles were predominant in the cord, while MSP2 alleles were predominant in the placental samples, which had a significant higher multiplicity of the infection.

Field-based evidence for linkage of mutations associated with chloroquine (pfcrt/pfmdr1) and sulfadoxine-pyrimethamine (pfdhfr/pfdhps) resistance and for the fitness cost of multiple mutations in P. falciparum.

Mutations in the Plasmodium falciparum pfcrt gene on chromosome 7 and possibly mutations in pfmdr1 on chromosome 5 have a role in conferring resistance against chloroquine (CQ), as do mutations of pfdhfr on chromosome 4 and pfdhps on chromosome 8 in terms of resistance against sulfadoxine/pyrimethamine (SP). The additive role of multiple mutations in the development of resistance to each drug suggests a non-random occurrence. In this study, parasite isolates were obtained from 50 patients with uncomplicated P. falciparum malaria from rural Eastern Sudan, an endemic setting with minimal overlap of infection. The parasite isolates were genotyped for detection of 12 alleles in CQ and SP resistance genes. Our main findings were: (1) the frequency of mutant alleles, pfcrt K76T, pfmdr1 N86Y, pfdhfr N51I, pfdhfr S108N, pfdhps K540E and pfdhps A581G were; 0.90, 0.86, 0.84, 0.84, 0.80 and 0.20, respectively. (2) No mutations were detected for the pfdhfr loci A16V, C59R and I164L, and for pfdhps loci S436A, A437G and A613S. (3) There was a statistically significant association between the mutations in: (i) the CQ resistance (CQR) genes, pfcrt T76 and pfmdr1 Y86 (P< or =0.001), (ii) the SP resistance (SPR) genes, pfdhfr I51, pfdhfr N108 and pfdhps E540 (P< or =0.001-0.04) and (iii) the CQ "i" and SP "ii" resistance genes (P=0.001) 4. The fitness cost of multiple mutations was revealed by a significantly reduced parasite density of isolates bearing the mutant alleles (P=0.048). However, the significantly higher gametocyte carriage rate among isolates with resistance mutations (P=0.001) is possibly an evolutionary mechanism for survival of mutant parasites.

A fixed-dose 24-hour regimen of artesunate plus sulfamethoxypyrazine-pyrimethamine for the treatment of uncomplicated Plasmodium falciparum malaria in eastern Sudan.

BACKGROUND: Artemisinin-based combination therapy is increasingly being adopted as first-line antimalarial therapy. The choice of appropriate therapy depends on efficacy, cost, side effects, and simplicity of administration. METHODS: the efficacy of fixed co-formulated (f) artesunate-sulfamethoxypyrazine-pyrimethamine (AS+SMP f) administered at time intervals of 12 hours for a 24-hour therapy was compared with the efficacy of the same drug given as a loose combination (AS+SMP l) with a dose interval of 24 hours for 3 days for the treatment of uncomplicated Plasmodium falciparum malaria in eastern Sudan. RESULTS: seventy-three patients (39 and 34 in the fixed and the loose regimen of AS+SMP respectively) completed the 28-days of follow-up. On day 3; all patients in both groups were a parasitaemic but one patient in the fixed group of AS+SMP f was still febrile. Polymerase chain reaction genotyping adjusted cure rates on day 28 were 92.3% and 97.1% (P > 0.05) for the fixed and loose combination of AS+SMP respectively. Three (4.1%) patients (one in the fixed and two patients in the loose group of AS+SMP) in the study suffered drug-related adverse effects. Gametocytaemia was not detected during follow-up in any of the patients. CONCLUSION: both regimens of AS+SMP were effective and safe for the treatment of uncomplicated P. falciparum malaria in eastern Sudan. Due to its simplicity, the fixed dose one-day treatment regimen may improve compliance and therefore may be the preferred choice.

High prevalence of drug-resistance mutations in Plasmodium falciparum and Plasmodium vivax in southern Ethiopia.

BACKGROUND: In Ethiopia, malaria is caused by both Plasmodium falciparum and Plasmodium vivax. Drug resistance of P. falciparum to sulfadoxine-pyrimethamine (SP) and chloroquine (CQ) is frequent and intense in some areas. METHODS: In 100 patients with uncomplicated malaria from Dilla, southern Ethiopia, P. falciparum dhfr and dhps mutations as well as P. vivax dhfr polymorphisms associated with resistance to SP and P. falciparum pfcrt and pfmdr1 mutations conferring CQ resistance were assessed. RESULTS: P. falciparum and P. vivax were observed in 69% and 31% of the patients, respectively. Pfdhfr triple mutations and pfdhfr/pfdhps quintuple mutations occurred in 87% and 86% of P. falciparum isolates, respectively. Pfcrt T76 was seen in all and pfmdr1 Y86 in 81% of P. falciparum. The P. vivax dhfr core mutations N117 and R58 were present in 94% and 74%, respectively. CONCLUSION: These data point to an extraordinarily high frequency of drug-resistance mutations in both P. falciparum and P. vivax in southern Ethiopia, and strongly support that both SP and CQ are inadequate drugs for this region.

Risk mapping of visceral leishmaniasis: the role of local variation in rainfall and altitude on the presence and incidence of kala-azar in eastern Sudan.

Visceral leishmaniasis (VL) is a vector-borne disease highly influenced by environmental factors. A model was developed for mapping the distribution and incidence of VL in Gedaref State, eastern Sudan, in relation to different environmental factors. Geographical information systems (GIS) were used to extract and map regression results for environmental variables of 190 villages in Gedaref State, including rainfall, vegetation status, soil type, altitude, distance from river, topography, wetness indexes, and average rainfall estimates. VL incidence in each village was calculated from hospital records. By use of logistic and linear multivariate regression analyses, models were developed to determine which environmental factors explain variability in VL presence and incidence. We found that average rainfall and the altitude were the best predictors of VL incidence. The resulting models were mapped by GIS software predicting both VL presence or absence and incidence at any locality in Gedaref State. The results are discussed in relation to VL control.