Association between interleukin-27 gene polymorphisms and Plasmodium falciparum Malaria.

Malaria is often characterized by a complicated disease course due to multifaceted intrinsic genetic factors of the host and the parasite. This study aimed to investigate the role of interleukin-27 (IL-27) gene polymorphisms in Plasmodium falciparum malaria infection in a Saudi Arabian cohort. This case-control study obtained blood samples from 250 malaria patients with P. falciparum and 200 randomly identified healthy control subjects from the Malaria Center in the Jazan area. Malaria patients were grouped into three cohorts as follow: low (<500 parasites/µl of blood), moderate (500-1000 parasites/µl of blood), and high (>1000 parasites/µl of blood) parasitemia. The results show that the IL-27 variant rs181209 was significantly associated with malaria patients (P = 0.026). Similarly, the homozygous GG genotype of rs26528 was also associated with risk of developing P. falciparum malaria (P = 0.032). The minor allele C of variant rs181206 exhibited an association with low to moderate parasitemia (P = 0.046). Furthermore, the rs181209 AA genotype was statistically significant in age group 1-5 years (P = 0.049). In conclusion, this study suggests that variant rs181209 and rs26528 could be associated with the risk of malaria infection by P. falciparum in the population studied.

Genetic Diversity and Population Genetic Analysis of Plasmodium falciparum Thrombospondin Related Anonymous Protein (TRAP) in Clinical Samples from Saudi Arabia.

The thrombospondin related anonymous protein (TRAP) is considered one of the most important pre-erythrocytic vaccine targets. Earlier population genetic studies revealed the TRAP gene to be under strong balancing natural selection. This study is the first attempt to analyze genetic diversity, natural selection, phylogeography and population structure in 199 clinical samples from Saudi Arabia using the full-length PfTRAP gene. We found the rate of nonsynonymous substitutions to be significantly higher than that of synonymous substitutions in the clinical samples, indicating a strong positive or diversifying selection for the full-length gene and the Von Willebrand factor (VWF). The nucleotide diversity was found to be π~0.00789 for the full-length gene; however, higher nucleotide diversity was observed for the VWF compared to the thrombospondin repeat region (TSP). Deduction of the amino acid sequence alignment of the PNP repeat region in the Saudi samples revealed six genotypes characterized by tripeptide repeat motifs (PNP, ANP, ENP and SNP). Haplotype network, population structure and population differentiation analyses indicated four distinct sub-populations in spite of the low geographical distance between the sampling sites. Our results suggest the likeliness of independent parasite evolution, creating opportunities for further adaptation, including host transition, and making malaria control even more challenging.

Genetic Diversity, Repeat Motifs, and Natural Selection at the C-Terminal Knob-Associated Histidine Rich Protein (KAHRP) of Plasmodium falciparum Clinical Samples from Saudi Arabia.

Background: Malaria is still a public health problem in Saudi Arabia specifically in the Jazan region. Plasmodium falciparum knob-associated histidine-rich proteins (PfKAHRPs) play an important role in cerebral malaria pathophysiology as well as pathogenesis of P. falciparum infections. The repeat region of PfKAHRP C-terminal interaction domain has been found to bind to the infected red blood cells and the vascular endothelium. Thus, this study aimed to assess the allelic variations, genetic diversity, and natural selection acting at the C-terminal PfKAHRP between parasite isolates from Saudi Arabia. Materials and Methods: The PfKHARP C-terminal interaction domain was successfully PCR-amplified and sequence data from 441 clinical isolates from Saudi Arabia were obtained. The DnaSP v5.10 software was used to determine the genetic diversity, polymorphism, haplotype, and natural selection. Haplotype network analysis was constructed by using the median-joining method in the NETWORK version 5.0.0.1 software. Results: Alignment and analysis of 441 C-terminal PfKAHRP-deduced amino acid sequences identified 5 genotypes (I-V) based on the decapeptide repeat arrangements (TKEASTSKEA, TKEASTSKGA, TKEASTTEGA, and TKEASTSKRA). Among the repeat types, Type I (49.43%, 218/441) was the most abundant in Saudi Arabia, followed by Type II (48.29%, 213/441). Overall, the nucleotide diversity in the PfKHARP C-terminal region was found to be low in Saudi Arabia (π = 0.00142); however, natural selection tests indicated positive selection (dN-dS = 1.64, P < 0.05) which was due to the variations within the repeat motifs. Genealogical relationship haplotype network of PfKAHRP from 4 different countries (i.e., Saudi Arabia, Iran, Burundi, and India) revealed 1 major shared haplotype cluster (H_1) with samples representative from all 4 countries (Saudi Arabia; n = 441, Burundi; n = 4, Iran; n = 13, and India; n = 1). Conclusion: Since this is the first study to report on genetic diversity of C-terminal PfKAHRP interaction domain and the repeat motifs from clinical samples in Saudi Arabia, it will contribute towards the rational design of antiadhesion drug therapies for P. falciparum malaria.

Associations of Single Nucleotide Polymorphisms in IL-18 Gene with Plasmodium falciparum-Associated Malaria.

INTRODUCTION: Interleukin-18 (IL-18) is a pro-inflammatory cytokine, reported to be involved in the initial immune responses against malaria. Genetic variations in the host are an important factor that influences the etiology of malaria at several disease levels. Polymorphisms within the IL-18 gene are associated with susceptibility and clinical outcome of several diseases. METHODS: We genotyped single nucleotide polymorphisms (SNPs) in IL-18 of patients infected with Plasmodium falciparum with varying extent of parasitemia and different age groups. RESULTS: SNPs rs5744292 (OR = 70.446; 95% CI = 4.318-1149.323; p<0.0001) and rs544354 (OR = 1.498; 95% CI = 1.088-2.063; p=0.013) were found to be significantly associated with parasitemia in P. falciparum-infected patients when compared with healthy control subjects. SNP rs5744292 (OR = 7.597; 95% CI=1.028-56.156; p=0.019) was associated with increased parasite density in infected patients. SNPs rs544354 (OR 0.407; 95% CI=0.204-0.812; p = 0.009) and rs360714 (OR of 0.256; 95% CI=0.119-0.554; p = 0.001) were significantly associated with parasite density in an age-dependent manner, with the risk alleles present more frequently among the younger (1-9 years) patients. Several haplotypes were found to have a significant association with parasitemia. In-vitro expression analysis using luciferase reporter assay showed that SNPs rs1946518 and rs187238 in the IL-18 gene promoter region and rs360728 and rs5744292 in the 3'-untranslated region of the IL-18 gene were associated with enhanced transcriptional activity. CONCLUSION: Our results suggest that polymorphisms within the IL-18 gene are associated with the susceptibility to P. falciparum infection and related parasitemia among groups with different parasite density and across various age groups.

Interleukin-22 Polymorphisms in Plasmodium falciparum-Infected Malaria Patients.

Background and Objectives. Malaria infection, caused by Plasmodium falciparum, is the most lethal and frequently culminates in severe clinical complications. Interleukin-22 (IL-22) has been implicated in several diseases including malaria. The objective of this study was to investigate the role of IL-22 gene polymorphisms in P. falciparum infection. Material and Methods. Ten single-nucleotide polymorphisms (SNPs), rs976748, rs1179246, rs2046068, rs1182844, rs2227508, rs2227513, rs2227478, rs2227481, rs2227491, and rs2227483, of IL-22 gene were genotyped through PCR-based assays of 250 P. falciparum infection. IL-22 gene promoter activity. RESULTS: We found that the rs2227481 TT genotype (odds ratio 0.254, confidence interval = 0.097-0.663, P. P. falciparum infection. P. P. P. P. CONCLUSION: The study suggests that IL-22 polymorphisms in rs2227481 and rs2227483 could contribute to protection against P. falciparum infection. IL-22 gene promoter activity.

Genetic diversity in the msp-1 and msp-2 alleles among Plasmodium falciparum field isolates from Jazan, Saudi Arabia.

The genetic diversity of Plasmodium falciparum infections in human is associated with the pathogenesis of malaria. It is commonly determined through amplification of the polymorphic regions of the merozoite surface proteins -1 (msp-1) and -2 (msp-2) genes. This study aimed to (1) determine the prevalence of the msp-1 and msp-2 allelic familiesand (2) identify the multiplicity of infection (MOI) in P. falciparum field isolates from the Jazan region in the Kingdom of Saudi Arabia (KSA). Blood samples from patients with microscopically confirmed malaria infections (N = 48), collected in 2010, were analysed for msp-1 and msp-2 polymorphisms.K1, MAD20 and RO33 allelic types of the msp-1 gene and 3D7 and FC27 alleles of the msp-2 gene were analysed via nested polymerase chain reaction (PCR) according to band size. The MOI was then calculated. In msp-1, 16 different alleles were identified by examining size differences in the agarose gels. These alleles-representing 5, 5 & 6 alleles-belong to K1 (120bp-420bp), RO22 (180bp-420bp) and MAD 20 (150 bp-410bp), respectively. For msp-2, a larger range of amplicon sizes was detected. A total of 13 different alleles were identified: the FC27 family had 6 alleles (380- bp1280bp), while the 3D7 family had 7 alleles (110 bp-1200bp.MOI was 1.81 for MSP-1 & 2.17 for MSP-2, with overall mean MOI of 1.99). Considerable genetic diversity was evident in the P. falciparum field isolates from the Jazan region of KSA. This diversity represents an essential step in developing effective measures to prevent malaria in KSA, as well as in assessing vaccines derived from these genes.

Molecular investigation of mixed malaria infections in southwest Saudi Arabia.

OBJECTIVE: To investigate the incidence of mixed-species (MS) malaria infection, and compare the results with microscopically confirmed cases of malaria. METHODS: During 2010, blood spots collected from 371 clinically suspected cases of malaria were microscopically examined in a cross-sectional study. The DNA was extracted from the samples, and a nested polymerase chain reaction (PCR) was performed. The results obtained by the 2 methods were compared. RESULTS: From the microscopic analysis it was determined that 369 samples (99.5%) were positive for Plasmodium falciparum (P. falciparum) and 2 were Plasmodium vivax (P. vivax) mono-infections. There were no mixed malaria infections. The PCR analysis, however, showed that in 7 cases (1.9%) the infection was caused by MS malaria comprising of P. falciparum and P. vivax, 2 of these representing the cases that were microscopically diagnosed as P. vivax mono-infections. All cases were negative for Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi. CONCLUSION: Mixed malaria infections are currently overlooked when using microscopy. The PCR assays are essential complementary techniques that should be used with microscopic examination of blood smears.

The prospect of malaria elimination in the Arabian Peninsula: a population genetic approach.

BACKGROUND: In the Arabian Peninsula malaria control is progressing steadily, backed by adequate logistic and political support. As a result, transmission has been interrupted throughout the region, with exception of limited sites in Yemen and Saudi Arabia. Here we examined Plasmodium falciparum parasites in these sites to assess if the above success has limited diversity and gene flow. METHODS: We examined 108 P. falciparum isolates in three sites in Yemen (Taiz, Dhamar and Hodeidah) and 91 isolates from Saudi Arabia (Jazan). Nine microsatellites were analyzed for allelic diversity, multi-locus haplotype and inter-population differentiation. RESULTS: Diversity at each locus (unbiased heterozygosity [H]) was relatively lower in Yemen; (Hodeidah, H=0.615, Taiz, H=0.66, Dhamar, H=0.481), compared to Saudi Arabia (Jazan, H=0.76). Microsatellites were distributed widely and private alleles, detected in a single population, were rare. Pairwise comparisons revealed that parasites population in Dhamar was relatively distanced (FST=0.19). However, Taiz (Yemen) (FST=0.065) and Hodeidah (FST=0.107) populations were closer to that in Jazan (Saudi Arabia). Nonetheless, parasites in the four sites can be considered as one population. CONCLUSION: Although malaria risk in Saudi Arabia has been cut considerably, the extent of diversity and parasite genetic structure are indicative of a large population size. Elimination strategy should target demographic factors that favor parasite dispersal and flow of imported malaria.

Development of species-specific primers for identification of Biomphalaria arabica, the intermediate host of Schistosoma mansoni in Saudi Arabia.

Schistosoma mansoni is mediated through the intermediate host Biomphalaria arabica which lives in Saudi Arabia. Molecular characterization and identification of this intermediate host are important for epidemiological studies of schistosomiasis. The present work aimed to determine the molecular variations among the populations of B. arabica found in Southern part of Saudi Arabia, and to develop species-specific primers for identification of these snails as a first step in the development of multiplex PCR for simultaneously identifying the snails and diagnosing its infections in a single step. Five populations of Saudi B. arabica snails were collected from freshwater bodies. Three populations were collected from Asser and two populations were collected from AL-Baha. Genomic DNA was extracted from snails and was amplified using five different RAPD-PCR primers. The banding patterns of amplified materials by primers P1 and P5 were identical in all populations. However, the rest primers displayed intra-specific differences among populations with variable degrees. Largest sizes of RAPD-PCR products were cloned into TA cloning vector as a preparatory step for DNA sequence analysis. After sequencing, similarity searches of obtained DNA sequences revealed that there are no similar sequences submitted to genebank data bases and its associated banks. The results obtained will be helpful in the development of simultaneous identification of B. arabica snails and diagnosis of S. mansoni infection within it in a single step by an implementation of multiplex PCR.

Detection of Toxoplasma gondii DNA by PCR in blood samples collected from pregnant Saudi women from the Aseer region, Saudi Arabia.

BACKGROUND AND OBJECTIVES: Toxoplasmosis, caused by Toxoplasma gondii, is diagnosed mainly by serological methods that are hindered by insufficient sensitivity. When it fails, it becomes necessary to rely on either direct detection of the parasite or DNA detection by polymerase chain reaction (PCR). We aimed to establish molecular tools for toxoplasmosis research in the country by using PCR targeting the B1 gene and compare it with ELISA results. DESIGN AND SETTING: Conducted at the College of Science, King Khalid University, Abha, Saudi Arabia between January 2009 and April 2010 on Saudi pregnant women attending three major hospitals in the Aseer region. PATIENTS AND METHODS: Peripheral blood samples (n=137) were collected from patients. DNA was extracted and the B1 T gondii gene was amplified by PCR. The amplicons were visualized and sequenced, and the results were analyzed. For comparison, sera were tested for anti-T gondii IgG and IgM by enzyme-linked immunosorbent assay (ELISA). RESULTS: Of the 137 samples tested, the B1 gene could be amplified in 56 cases (41%) by PCR. DNA sequencing confirmed these results. IgM-ELISA assay detected 9 (6.5%) of these cases. The results of immunoglobulin G detection were positive in 53 (38.6%) of the patients. CONCLUSION: The present study showed the need for PCR as a confirmatory assay in addition to serological assays to detect recent infection. We recommend national implementation of these molecular diagnostic tools.

Source of drug resistant Plasmodium falciparum in a potential malaria elimination site in Saudi Arabia.

A major challenge to the success of malaria control program in Saudi Arabia is the high influx of expatriates and holy visitors from malaria endemic countries. In the present study we examined whether drug resistant parasite genotypes reported in Jazan region, southwest of Saudi Arabia are imported or developed locally. We examined 178 Plasmodium falciparum isolates for alleles of dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr), associated with Sulfadoxine-Pyrimethamine (SP) resistance, and three microsatellites flanking each gene. In addition, we examined a neutral polymorphic gene (Pfg377). We compared the dhfr and dhps haplotypes in Jazan, using network analysis, to an existing similar data set of 94 P. falciparum isolates from eastern Sudan. In Jazan, double mutant dhfr allele (51I, 108N) occurred with a prevalence of 33%. The vast majority (99%) of dhps were wild-type alleles. The mean expected heterozygosity (H(e)) of microsatellites around mutant dhfr alleles (H(e)=0.312; n=60) was lower (P ≤ 0.05) than that around the wild-type allele (H(e)=0.834; n=116). Also, the mutant dhfr isolates showed high H(e) for dhps (H(e)=0.80) and the non-drug resistance locus Pfg377 (H(e)=0.63) indicative of selection for mutant dhfr only. The predominant double mutant dhfr haplotype in Jazan (73%), was prevalent among P. falciparum in east Africa. Network analysis suggests the mutant haplotype of dhfr gene was possibly introduced into Jazan from East Africa. The absence of mutations in dhps as well as triple mutant dhfr haplotype associated with SP failure support the current use of SP as a partner with artesunate as a first line therapy in Saudi Arabia. However, the close relationship between the major mutant dhfr haplotype in Sudan and Saudi Arabia, favour the hypothesis of recent migration as a source of the major resistant dhfr lineage. Thus, regular monitoring of the dhfr and dhps haplotypes is of high priority to guard possible importation of high level SP resistant lineages.

Antiviral activity of liquorice powder extract against varicella zoster virus isolated from Egyptian patients.

BACKGROUND: Varicella-zoster virus (VZV) is the etiologic agent of two diseases, varicella (chicken pox) and zoster (shingles). Varicella is a self- limited infection, while zoster is mainly a disease of adults. The present study was conducted to isolate VZV from clinically diagnosed children using cell cultures and compare the activity of liquorice powder extract, an alternative herbal antiviral agent, with acyclovir and interferon alpha 2a (IFN-α2a) against the isolated virus. METHODS: Forty-eight VZV specimens, 26 from vesicular aspirates and 22 from vesicular swabs, from children clinically diagnosed with varicella were isolated on the Vero cell line. Isolates were propagated and identified with specific antiserum using indirect immunofluorescence and immunodot blotting assays. The growth kinetics of the viral isolates was studied. The antiviral activity of liquorice powder extract, acyclovir (ACV) and IFN-α2a was evaluated against the isolated virus. RESULTS: VZV was successfully isolated in 4 of the 48 specimens, all from vesicular aspirates. The growth kinetics of the viral isolates was time dependent. The inhibitory activity of liquorice powder extract (containing 125 µg/ml glycyrrhizin) when compared to ACV (250 µg/ml) and IFN-α2a is the lowest. CONCLUSIONS: VZV isolates were successfully isolated and propagated using Vero cells. Isolates were identified using indirect immunofluorescent and immunodot blotting techniques. Growth kinetics of the isolates revealed an increase in the viral infectivity titer relative to time. Glycyrrhizin in the crude form has low antiviral activity against VZV compared with acyclovir and interferon.

Distribution of drug resistance genotypes in Plasmodium falciparum in an area of limited parasite diversity in Saudi Arabia.

Two hundred and three Plasmodium falciparum isolates from Jazan area, southwest Saudi Arabia, were typed for Pfcrt, Pfmdr1, dhps, and dhfr mutations associated with resistance to chloroquine, mefloquine, halofantrine, artemisinin, sulfadoxine-pyrimethamine, and the neutral polymorphic gene Pfg377. A large proportion (33%) of isolates harbored double mutant dhfr genotype (51I,59C,108N). However, only one isolate contained mutation dhps-437G. For Pfcrt, almost all examined isolates (163; 99%) harbored the mutant genotype (72C,73V,74I,75E,76T), whereas only 49 (31%) contained the mutant Pfmdr1 genotype (86Y,184F,1034S,1042N), 109 (66%) harbored the single mutant genotype (86N,184F,1034S,1042N), and no mutations were seen in codons 1034, 1042, and 1246. Nonetheless, three new single-nucleotide polymorphisms were detected at codons 182, 192, and 102. No differences were seen in distribution of drug resistance genes among Saudis and expatriates. There was a limited multiplicity (5%), mean number of clones (1.05), and two dominant multilocus genotypes among infected individuals in Jazan. A pattern consistent with limited cross-mating and recombination among local parasite was apparent.

Developing species-specific primers to identify Bulinus truncatus and Bulinus beccari, the intermediate hosts of Schistosoma haematobium in Saudi Arabia.

This work aimed to determine the inter- and intra-specific variations in populations of Bulinus truncatus and Bulinus beccari, the intermediate hosts of Schistosoma haematobium in Saudi Arabia, and to develop species-specific primers to identify these snails as a first step in the development of multiplex PCR for simultaneously identifying the snails and diagnosing its infections in a single step. Two populations of B. truncatus were collected from Asser and Bisha (A and B), and two B. beccari populations were collected from Mahial Asser and Merba (C and D). The snails' genomic DNA was extracted and amplified using 5 different primers. The primers displayed variable intra- and inter-specific differences across the populations. The largest RAPD-PCR fragments were cloned into a vector as a preparatory step for sequencing. Similarity searches for the sequenced cloned inserts revealed no similar sequences in the GenBank database or its associated databases. Specific primers used to target the B. truncatus and B. beccari genomes were designed using the Gene Runner program and based on the DNA sequences obtained from RAPD fragment sequence analyses. Using these primers for specific PCRs resulted in expected single-band PCR products of 536 bp for B. beccari and 478 bp for B. truncatus. These results will be helpful for simultaneously identifying B. truncatus and B. beccari snails and diagnosing S. haematobium infections within the snails using single step multiplex PCR.

Fingerprint of Biomphalaria arabica, the intermediate host of Schistosoma mansoni in Saudi Arabia, using RAPD-PCR.

In the time schistosomisis control programs are implemented in many countries, schistosomiasis continues to spread throughout the world. Among these control strategies is the vector control. Within this context, analysis of the genetic variability of the intermediate host snails is important because it allows identification of specific sequences of the genome of this mollusk related to determine their fingerprint. We investigated Biomphalaria arabica, which is found in Saudi Arabia, the intermediate host of Schistosoma mansoni infection. Genetic fingerprint was studied by RAPD-PCR using our own different random primers as well as published primers. The electrophoretic patterns resulting from amplification showed specific polymorphic markers of B. arabica. This information will be helpful in the identification of the snails and demonstrating that RAPD-PCR is an appropriate and efficient methodological approach for establishment of genetic barcode development.

Detecting mutations in PfCRT and PfMDR1 genes among Plasmodium falciparum isolates from Saudi Arabia by pyrosequencing.

The emergence of chloroquine resistance in Plasmodium falciparum is a significant public health problem where malaria is endemic. We aimed to evaluate the efficacy of pyrosequencing to assess chloroquine resistance among P. falciparum isolates from the southwestern region of Saudi Arabia by analyzing the K76T and N86Y mutations in the P. falciparum chloroquine resistance transporter (PfCRT) and P. falciparum multidrug resistance 1 (PfMDR1) genes, respectively. Blood samples (n = 121) from microscopically positive P. falciparum cases were collected. DNA was extracted, and fragments from each of the genes were amplified by PCR using new sets of primers. The amplicons were sequenced using a pyrosequencer. All of the 121 samples were amplified for assessment of the PfCRT K76T and PfMDR1 N86Y mutations. All of the samples amplified for the PfCRT 76T mutation harbored the ACA codon (121/121; 100%), indicating the presence of the 76T mutation. For the PfMDR1 N86Y mutation, 72/121 samples (59.5%) had the sequence AAT at that position, indicating the presence of the wild-type allele (86N). However, 49/121 samples (40.5%) had a TAT codon, indicating the mutant allele (Y) at position 86. This study shows that pyrosequencing could be useful as a high throughput, rapid, and sensitive assay for the detection of specific single nucleotide polymorphisms in drug-resistant P. falciparum strains. This will help health authorities in malaria-endemic regions to adopt new malaria control strategies that will be applicable for diagnostic and drug resistance assays for malaria and other life-threatening pathogens that are endemic in their respective countries.

Green tea (Camellia sinesis) ameliorates female Schistosoma mansoni-induced changes in the liver of Balb/C mice.

This study was designed to assess the effect of green tea, an aqueous extract of Camellia sinensis, on the oxidative stress, antioxidant defense system and liver pathology of Schistosoma mansoni-infected mice. Green tea at concentration of 3% (w/v) was given orally to treated mice as sole source of drinking water from the end of the 4th week to the end of 10th week post-infection; untreated mice were allowed to drink normal water. The data of the studied S. mansoni-infected mice exhibited a suppression of hepatic total antioxidant capacity, superoxide dismutase (SOD), catalase (CAT) activity and glutathione content. The liver lipid peroxidation was deleteriously elevated in S. mansoni-infected mice. The hepatic total protein content, AST and ALT activities were profoundly decreased in the S. mansoni-infected mice. Most hepatocytes were damaged and showed abnormal microscopic appearance with aggressive necrosis. Both total protein and glycogen levels have been greatly reduced as indicated by histochemical examination. The treatment of S. mansoni-infected mice with green tea succeeded to suppress oxidative stress by decreasing the lipid peroxides but failed to significantly enhance the antioxidant defense system and deteriorated changes owing to liver damage and necrosis. In consistence with biochemical data, histopathological and histochemical data indicated that treatment of S. mansoni-infected mice with green tea could ameliorate hepatocytes thus reduce cellular necrosis and partially restore both total protein and glycogen levels. Thus, the study concluded that the green tea suppresses the oxidative stress through its constituent with free radicals scavenging properties rather than through the endogenous antioxidant defense system.

Analysis of gene mutations involved in chloroquine resistance in Plasmodium falciparum parasites isolated from patients in the southwest of Saudi Arabia.

BACKGROUND AND OBJECTIVES: Chloroquine has been the drug of choice for the treatment of malaria for many decades. We aimed to examine the molecular basis of chloroquine resistance among Plasmodium falciparum isolates from the southwestern region of Saudi Arabia by analyzing the K76T and N86Y mutations in the PfCRT and PfMDR1 genes, respectively. PATIENTS AND METHODS: P falciparum-infected blood spot samples (n=121) were collected on filter papers. DNA was extracted and fragments from the above genes were amplified using nested PCR. The amplicons were digested by ApoI enzyme and sequenced. RESULTS: Of the 121 samples, 95 and 112 samples could be amplified for PfCRT K76T and PfMDR1 N86Y mutations, respectively. All of the samples amplified for the PfCRT K76T mutation were undigestible by ApoI, suggesting the presence of the K76T mutation. For the PfMDR1 N86Y mutation, 65/109 samples (59.6%) were digestible when treated with ApoI in a pattern, suggestive of the presence of the investigated wild allele (N86). However, 44/109 samples (40.4%) were digestible by ApoI, suggesting the presence of the mutated allele (Y) at position 86. DNA sequencing confirmed these results. CONCLUSION: Surprisingly, all isolates exhibited the mutated allele at codon 76 (K76T) of PfCRT. However, the mutated mutant allele at codon 86 (N86Y) of PfMDR1 was found in 40.4% of the samples studied. To our knowledge, this is the first study that has investigated the existence of the mutation in the PfMDR1 gene in the country. This study will contribute to the development of new strategies for therapeutic intervention against malaria in Saudi Arabia.

Isoenzyme electrophetic characterization of Leishmania major, the causative agent of zoonotic cutaneous Leishmaniasis in North and West Saudi Arabia.

Several expeditions were carried out to four localities (Al-Madinah Almona-warah, Tabouk region, Al-Jouf and Northern Frontiers regions) in Northern and Western Saudi Arabia for sampling zoonotic cutaneous leishmaniasis (ZCL) cases from patients and rodents. Biopsy samples were collected from 51 patients complaining of skin lesions, most of which (40 or 78.4%) proved to be ZCL. Amastigotes were detected in 33 patients (64.7%), but only 30 (58.9%) gave successful growth of promastigotes in the culture media. The positive cases were Saudis 14(35%) and non-Saudis 26 (65%). Five species of rodents were caught, Meriories libycus, Psammomys obesus, Rattus rattus, jaculus and Hystrix indica. The first species was the most dominant (90%) in which Leishmania parasites were detected. The Leishmania isolates from man and rodents were identified by isoenzyme electrophoresis and proved to be Zymodeme LON-4.

Freshwater snails in Asser region, Saudi Arabia with special refernce to the zoonotic trematode.

The present study gave information about the recent distribution of freshwater snails in Asser region, and the current status of trematode infection specially schistosomiasis within the snails. Fifteen localities were visited from Septeinber 2007 to December 2008 and the collected snails were examined for the presence of trematode infection. Seven species of snails were collected: Biomphalaria arabica, Bulinus truncatus, Bulinus beccari, Physa acuta, Lymnaea palustris, Lymnaea arabica and Melanoides tuberculata. The parasitological examination revealed none trematode immature stages.