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

Latex agglutination and indirect immunofluorescence tests in the diagnosis of Toxoplasma gondii in Saudi Arabia

Commercial latex agglutination [LA] was assessed for Toxoplasma antibody screening. The sensitivity and specificity were compared with the reference standard indirect immunofluorescence [IFA]. A total of 186 sera were collected from May 2008-October 2008 for Toxoplasma antibody by LA and IFA. Antibody to T. gondii 51/186 [27.4%] were LA-positive and 42/186 [22.6%] were IFA-positive. The sensitivity, specificity and positive predictive value of LA were 100%, 93.7% and 82.3% respectively. The nine LA-false positive sera were examined for rheumatoid factor and antinuclear antibodies, but were negative and none exhibited nonspecific polar staining as a cause for the false positivity

Polymerase chain reaction versus conventional methods in the diagnosis of vaginal trichomoniasis

A total of 200 females of whom 120 had manifestations of vaginal trichomoniasis and 80 asymptomatic ones were studied. In 54/120 symptomatic female [45%] and in 28/80 asymptomatic ones [35%], T. vaginalis was diagnosed by wet mount of bedside vaginal swab samples of 120 samples from symptomatic females, T. vaginalis was detected in 93 [77.5%] when cultured onto InPouch and 95 [79.16%] in modified thioglycolate media. Culturing 80 samples of asymptomatic females showed T. vaginalis in 35 [43.75%] onto either media. T. vaginalis genomic DNAs was amplified by PCR from 130 [65%] by using TVA5-TVA6 primer pair in 95 [79.16%] samples of 120 symptomatic females, and in 35 [43.75%] samples of 80 asymptomatic ones. Difference between groups was statistically significant. The motile trichomonads was detected by wet mount in 82/130 positive cultures giving 63.07% sensitivity and 100% positive predictive value [PPV]. Flagellates were not detected by wet mount in any negative culture, giving 100% specificity and 59.32% negative predictive value [NPV]. The wet mount diagnostic accuracy [DA] was 76%, without false-positive, but false negative was 48/130 [36.93%]. DNA was amplified from 129/130 positive culture by TVA5-TVA6 primer pair, giving 99.23% sensitivity. No amplification was detected from one positive culture. DNA was not amplified from 69/70 negative culture using TVA5-TVA6 primer pair, giving 98.57% specificity, 99.23% PPV, 98.57% NPV and 99% DA

Antiparasitic activity of cystine protease inhibitor E-64 against giardia lamblia excystation in vitro and in vivo

In this work, the therapeutic effect of E-64, a broad spectrum cysteine protease inhibitor against Giardia lamblia excystation was studied in vitro and in vivo. Purification of cysts from heavily infected human faecal samples followed by excystation and axenic cultivation of the emerging trophozoites in TYI-S-33 medium were done. In vivo, the response was evaluated experimentally through counting oocysts out-put every other day until the infection eradicated from the stools of infected E-64 treated mice compared to untreated. Also, the histopathological examination of the small intestine was compared between both of the infected groups. In the present study G. lamblia cysts incubated with E 64 in vitro completely failed in excystation in 90% while trophozoites released on 10% [partially excysted on 5% and completely excysted on 5%] compared to 90% completely excysted on other non incubated [without E-64] of cysts beside, the trophozoites didn't release on 10% [partially excysted on 5% and completely non-excysted on 5%]. In vivo, the evaluation of the therapeutic response proved that the decreasing in the oocysts out-put counting every other day until the infection eradicated from the stools of infected treated mice was very marked in comparison to untreated mice. The differences were statistically significant. The histopathological examination of the small intestine of infected non treated group proved that all the different pathological grades were found while in infected E-64 treated group, only grade I was detected. So, E-64 showed a good therapeutic effect which raises its use in the treatment of human giardiasis

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]

Protein profile and morphometry of cultured human blastocystis hominis from children with gastroenteritis and healthy ones

A total of 180 children of age group 5-12 years old in both sexes, of whom 90 were symptomatic and negative for other parasites, rota-virus or pathogenic bacteria. Another 90 children were asymptomatic, but with B. hominis in stools. Direct smear, formaline-ethyl acetate sedimentation concentration, kinyon carbol-fuchin stain, stool culture, enzyme immunoassay, culturing, morphometric study, gel electropho-resis and experimental infection of mice were done. The results showed that the central body cysts [CB], granular and multivacuolar forms isolated from symptomatic patients were larger than those from asymptomatic ones. The CB form was common compared to other forms and isolated from 104 cases. B. hominis infection was prevalent among males rather than females [60.5% versus 39.5%]. The clinical data showed that diarrhea was the most common symptom [58.9%]. The infection intensity had a direct relation with illness duration. The polyacrylamide gel electrophoresis showed that isolates from symptomatic and asymptomatic patients ranged between 24-130 kDa. All isolates showed similar banding patterns. Only minor differences was in low MW [30, 50 kDa] and in high MW [118 kDa] in samples from symptomatic patients. The histopathological examination of caecum, colon and small intestine of B. hominis mice infected from symptommatic patients showed infiltration with inflammatory cells and tissue invasion by the parasite