Molecular surveillance of Plasmodium Falciparum chloroquine resistance transporter variant T76 in Jazan area, Kingdom of Saudi Arabia

The development of chloroquine as an antimalarial drug and the subsequent evolution of drug resistant Plasmodium strains had major impacts on global public health in the 20th century. In P. falciparum, the cause of the most lethal human malaria, chloroquine resistance is linked to multiple mutations in PfCRT, a protein that likely functions as a transporter in the parasite's digestive vacuole membrane. Rapid diagnostic assays for PfCRT mutations are already employed as surveillance tools for drug resistance. However, several reports have been published demonstrating cases with CO resistance. Sporadic cases have been reported as well as one large scale study demonstrated 12.4% resistance. However, all these reports were based on treatment failure [in vivo]. rather than in vitro or molecular bases. Evidence suggests a crucial role for a point mutation in the P. falciparum chloroquine resistance transporter [pfcrt] gene on chromosome 7 in conferring CQ resistance. The mutation in the K76 codon in 3 cases out of 60 [5%] using ApoI restriction enzyme was detected. Although the percentage of drug resistance was not quite disturbing, but represented the possible establishment of chloroquine-resistant P. falciparum in Saudi Arabia, or the beginning of resistant strains by labors coming from abroad. Cross-border importation of resistant strains from neighboring countries must be considered. In vivo tests must be conducted parallel with the molecular markers to estimate more precisely the actual prevalence of resistance. Validation of molecular markers is urgently required and needs strong collaborative partnerships between subregional and regional networks

Comparison of two commercial assays and microscopy with PCR for diagnosis of malaria

This study compared conventional PCR with microscopy and 2 rapid detection methods, the pLDH which detected lactic dehydrogenase enzyme produced by actively metabolizing organisms and the malaria antibody tests. The sensitivity of PCR was 1 parasite/micro l, i.e.: 50 times more sensitive than microscopy. When PCR was compared with microscopy, the sensitivity and specificity were 90% and 100% respectively. The sensitivity recorded was pLDH test in comparison to PCR [95%]. The malaria antibody test recorded the least sensitivity [68%] PCR proved as the gold standard for evaluation of applied tests and the newly introduced ones. In absence of an expert microscopist, the pLDH test could substitute for microscopy. The test proved valuable to assess clinical cure, and predict drug resistance. Its advantage over microscopy was the ability to diagnose infection with low parasitemic patients. Antibody rapid tests might be not valuable in acute cases, but still accepted as a tool in epidemiological studies and in screening patients in blood banks in malaria endemic areas

Evaluation of a real-time polymerase chain reaction assay for the diagnosis of malaria in patients from Jazan area, Saudi Arabia

A real-time PCR assay with conventional microscopy by Giemsa-stained blood films was used. PCR was completed in an hour and identified the Plasmodium species in a single reaction. Blood was collected, and DNA was extracted. A genus-specific primer set corresponding to 18S ribosomal RNA was used to amplify target sequence. Fluorescence resonance energy technology hybridization probes were designed for P. falciparum over a region containing base pair mismatches allowed Plasmodium species differentiation. Microscopically positive patients [n=60] were positive with real-time assay [100% sensitivity]. 58 were single-species infections caused by P. falciparum; mixed infections [P. falciparum and P. vivax] were shown by real-time assay. Six out of 30 negative microscopy specimens were positive by real-time PCR [80% specificity,]. The discrepant results could be due to the subjective nature of microscopy and analytical objectivity of PCR, and high analytical sensitivity of real-time assay [1 parasite/micro l] compared to microscopy [50 parasites /micro l]. Six patients were retested with ICT malaria test and 4 were positive showing that PCR results were correct. There was low correlation between parasitemia by microscopy and gene copy number for P. falciparum [r = 0.2; P =0.05 [Spearman]

Anemia, interleukin-10, tumor necrosis factor alpha, and erythropoietin levels in children with acute, complicated and uncomplicated malignant malaria in Jazan, Saudi Arabia

To gain insight into potential relationships between tumor necrosis factor alpha [TNF-alpha], interleukin 10 [IL-10], erythropoietin [EPO], and anemia in acute malaria, 90 children 3 to 11 years with acute malaria were studied. According to parasitemia and hemoglobin levels, they were divided into 3 groups: Gl [mild]: asexual low-density Plasmodium falciparum parasitemia <8000 parasites/ul and hemoglobin levels >8g/dl. G2 [high-density uncomplicated]: asexual high-density parasitemia [>8000 parasites/ul, with hemoglobin levels >8 g/dl. G3 [anemia]: with severe malaria symptoms and parasitemia with anemia [hemoglobin levels <8 g/dl]. Hospital controls included 10 children with matching age group who required inpatient management but had no malaria parasitemia. Good marrow response was in Gl and G2 showed by elevation of serum EPO and soluble transferring receptors [sTfR] and increased red cell distribution width [RDW]. In G3, bone marrow suppression was in spite of increased EPO level in response to anemia. TNF-alpha level was significantly higher G2 and G3 [P.05]. IL-10 levels in Gl were significantly higher than in hospital control group [P<0.05]. The highest level of IL-10 was in G2. The mean EL-10 to TNF- alpha ratio in G2 [4.64] was significantly higher [P<.005] than in G3 [mean ratio, 1.77]