Antiprotozoal potential of Salvadora persica against three virulent subtypes of Blastocystis sp.

Blastocystis sp. is a group of anaerobic protozoa parasitizing the gastrointestinal tract of humans and a broad variety of animals. Evidences of Blastocystis parasites resistance development to antiprotozoal drugs urge the exploration of new therapeutics. Antiprotozoal potential of Salvadora persica, a medicinal plant traditionally used for oral hygiene, was evaluated in vitro against Blastocystis sp. human isolates. Until now, no study has described the effect of S. persica extracts on this parasitic protozoa. Blastocystis sp. positive stool samples collected from patients with gastrointestinal complaints and asymptomatic individuals diagnosed by microscopy were furthermore cultured in vitro and characterized by PCR and multiplex-PCR using sequence-tagged-site primers to determine their subtypes. Out of 21 Blastocystis sp. isolates, five were determined as ST1, 14 as ST3, and two as ST5 subtypes. Antiprotozoal activity of untreated and heat-treated S. persica roots aqueous extracts was evaluated in vitro by serial dilutions on three Blastocystis sp. subtypes; ST1, ST3, and ST5 isolated from symptomatic patients. A significant killing activity was observed with both, untreated and heat-treated aqueous extracts of S. persica at minimal concentration of 2.5 µl/ml compared to parasites' growth controls (P < 0.05). Maximal antiprotozoal effect was reached at a concentration of 20 µl/ml of S. persica aqueous extract. Means of growth inhibition effect obtained with untreated and heat-treated extracts at 40 µl/ml against the three subtypes of Blastocystis sp. were 80% (SD 2.3) and 82% (SD 1.1), respectively. No significant difference was observed in the inhibitory effect of S. persica extracts between the three Blastocystis sp. subtypes. Aqueous extract of S. persica roots contains therefore heat-stable components with significant antiprotozoal activity against Blastocystis sp. subtypes ST1, ST3, and ST5 in vitro. Further investigations are required to determine and characterize the active antiprotozoal components of S. persica roots and their evaluation in vivo.

Comparative 1D Blue-Native electrophoresis analysis of Plasmodium falciparum and human proteins associated with cytoadherence.

BACKGROUND: To understand more about changes to the molecular components that occur when host endothelium interacts with Plasmodium falciparum-infected erythrocytes, a combined technique of protein separation (1D Blue-Native electrophoresis) and mass spectrometry of infected erythrocytes with endothelial cells (EC) in a co-culture system has been used. METHODS: Native proteins were extracted from co-cultures and identified by mass spectrometry. Proteomic data from different parasite strains, either adhesion proficient (to endothelial cells) or non-adherent, were analysed in parallel to reveal protein associations linked to cytoadherence. Informatic approaches were developed to facilitate this comparison. RESULTS: Blue-Native gel separation and LC/MS/MS identification revealed major differences in samples produced from endothelial cell co-culture with adherent and non-adherent parasite strains. This approach enabled us to identify protein associations seen only with the adhesion proficient parasite strain. CONCLUSIONS: The combination of proteomic and analytical approaches has identified differences between adherent and non-adherent parasite lines in co-culture with EC, providing potential candidates for complexes or associations formed during cytoadherence involved in cell structure, signalling and apoptosis.

Subtyping of Blastocystis sp. isolated from symptomatic and asymptomatic individuals in Makkah, Saudi Arabia.

BACKGROUND: Blastocystis is a group of cosmopolitan gastrointestinal parasite of humans and a wide variety of animals. These anaerobic protozoans include more than 17 specific small-subunit ribosomal RNA subtypes, of which nine are found in humans with a variable geographical distribution. Until now, no study has described the Blastocystis subtypes present in Saudi Arabia. METHODS: In total, 1,262 faecal samples were collected from patients with gastrointestinal complaints and asymptomatic individuals visiting two major hospitals. All samples were analysed by F1/R1 diagnostic PCR, microscopy and culture methods. The subtypes of Blastocystis sp. isolates were determined by the sequenced-tagged site (STS)-based method. RESULTS: One-hundred-thirty-three positive cases were detected by F1/R1 diagnostic PCR, of which 122 were also positive by the culture method and 83 by direct microscopy. The sensitivities of direct microscopy and the culture method were 62% and 92%, respectively. Subtype (ST3) was the most prevalent (80.5%), followed by ST1 (14.5%) and ST2 (5%). ST4, ST5, ST6 and ST7 were not detected in this study. ST3 infections were significantly predominant (P < 0.05) among symptomatic patients. CONCLUSIONS: To our knowledge, this study provides the first run-through information on Blastocystis sp. epidemiology in Makkah city, revealing a rather moderate prevalence of 10.5% and the presence of three subtypes, ST1, ST2, and ST3. ST3 was the most predominant, particularly among symptomatic patients.

APPRAISAL OF PRENATAL ANTI-TOXOPLASMA GONDII (IGG+IGM)- IHA/IGM-ELISA SCREENING IN SINGLE SAMPLES VIA IGG AVIDITY TEST.

Congenital toxoplasmosis is associated with important morbidity and mortality. Since vertical transmission of Toxoplasma gondii can occur in acute cases, antenatal screening for recent infections is vital. Accurate determination of acute toxoplasmosis requires a combination of immunoassays, usually not routinely applied for screening purposes. This study evaluated the anti-T. gondii (IgG+IgM)/IgM prenatal screening procedure by IgG avidity assay. The routine prenatal screening for (IgG+IgM) anti-T. gondii by indirect hemagglutination (IHA) in serum samples was done of 2247 pregnant women who attended two hospitals between 2011 and 2013 revealed 487 (21.7%) positive samples. Examination of IHA-positive sera by IgM and IgG/IgG-avidity concurrent ELISA tests revealed 7 positive and 3 border-line IgM-ELISA titers during the initial check-up of 10 women, who were then followed up at 3-4 week-intervals. Among these, 4 (40%) showed simultaneous high avidity IgG antibodies, indicating distant infection by the parasite, and no anti-T. gondii specific IgG could be detected in follow-up sera of two cases (20%), indicating false IgM initial positive results. Only 4 (40%) women showed simultaneous IgM and low avidity IgG antibodies indicating active infections. Avoidance of an over-diagnosis of acute toxoplasmosis Anti-T. gondii (IgG+IgM)/IgM prenatal screening must be supplemented by a discriminative test like IgG avidity ELISA.

RETROSPECTIVE INVESTIGATION OF PYRIMETHAMINE-SULFADOXINE RESISTANCE INDICATORS IN FALCIPARUM-MALARIA POSITIVE BLOOD SAMPLES FROM SOUTH-WESTERN SAUDI ARABIA.

The efficacy of Sulphadoxine/Pyrimethamine (SP) in Plasmodium falciparum malaria treatment was increasingly compromised by development of parasites' resistance. Saudi Arabia shifted to new combinations including Artesunat compound during the last decade. We investigated the occurrence of mutations in P. falciparum dihydrofolate reductase (Pfdhfr) N51I, C59R and S108N and P. falciparum dihyropteroate synthetase (Pfdhps) A437G and K540D as major indicators of SP resistance in stored DNA extracts of 41 P. falciparum infected specimens collected from KSA southern endemic regions between 2012 and 2014. Analysis of alleles' polymorphisms by Nested-PCR-RFLP showed that 68%, 7%, and 24% of samples carried parasites with Pfdhfr 51I, 59R, and 108N mutant type alleles, respectively. Only one isolate's genotype shared both mutations 51I and 108N. All parasites conserved wild type alleles at codons 437 and 540 of Pfdhps gene.

IMPORTED MALARIA TO MAKKAH DISTRICT, SAUDI ARABIA: IS THERE ANY RISK OF LOCAL TRANSMISSION?

In Makkah, Saudi Arabia, there is an impending risk of imported malaria. This risk comes from the fact that millions of people, in majority from tropical and subtropical countries where malaria is endemic, visit the country to perform Hajj and Umrah every year. Moreover, millions of expatriates from endemic countries come to Makkah for work. Likewise, many Saudi citizens travel to endemic areas overseas for business and pleasure. We performed a retrospective analysis of all reported malaria cases in Makkah region, Saudi Arabia for years 2014 and 2015. In addition, sorting of mosquito populations in Makkah region was undertaken. Based on national data regarding reported malaria cases, 235 malaria cases were recorded in years 2014 and 2015. Of the reported cases 232 were non-Saudi and only 3 cases were Saudi. Those recorded Saudi cases were just returning from a travel to an endemic area. Most of the cases (79.6%) were P. falciprum and the remaining was P. vivax. Infected male represent 62% and female represent 38%. Age of the majority of reported cases (71.5%) lie between 31 and 50 years. Most of reported cases were from Chad, Pakistan, Nigeria and Sudan. Sorting of mosquito populations revealed the absence of malaria vectors in Makkah District.

COMPARISON OF A GENUS-SPECIFIC CONVENTIONAL PCR AND A SPECIES-SPECIFIC NESTED-PCR FOR MALARIA DIAGNOSIS USING FTA COLLECTED SAMPLES FROM KINGDOM OF SAUDI ARABIA.

Molecular tools are increasingly accepted as the most sensitive and reliable techniques for malaria diagnosis and epidemiological surveys. Also, collection of finger prick blood spots onto filter papers is the most simple and affordable method for samples preservation and posterior molecular analysis, especially in rural endemic regions where malaria remains a major health problem. Two malaria molecular diagnostic tests, a Plasmodium genus-specific conventional PCR and a Plasmodium species-specific Nested PCR, were evaluated using DNA templates prepared from Whatman-FTA cards' dry blood spots using both, Methanol-fixation/Heat-extraction and FTA commercial purification kit. A total of 121 blood samples were collected from six Saudi south-western endemic districts both, as thick and thin films for routine microscopic screening and onto FTA cards for molecular studies. Out of the 121 samples, 75 were P. falciparum positive by at least one technique. No other species of Plasmodium were detected. P. falciparum parasites were identified in 69/75 (92%) samples by microscopic screening in health care centers. P. genus-specific PCR was able to amplify P. falciparum DNA in 41/75 (55%) and 59/75 (79%) samples using Methanol-fixation/Heat-extraction and FTA purification kit, respectively. P. species-specific Nested PCR revealed 68/75 (91%) and 75/75 (100%) positive samples using DNA templates were isolated by Methanol-fixation/Heat- extraction and FTA purification methods, respectively. The species-specific Nested PCR applied to Whatman-FTA preserved and processed blood samples represents the best alternative to classical microscopy for malaria diagnosis, particularly in epidemiological screening.

An analysis of the binding characteristics of a panel of recently selected ICAM-1 binding Plasmodium falciparum patient isolates.

The basis of severe malaria pathogenesis in part includes sequestration of Plasmodium falciparum-infected erythrocytes (IE) from the peripheral circulation. This phenomenon is mediated by the interaction between several endothelial receptors and one of the main parasite-derived variant antigens (PfEMP1) expressed on the surface of the infected erythrocyte membrane. One of the commonly used host receptors is ICAM-1, and it has been suggested that ICAM-1 has a role in cerebral malaria pathology, although the evidence to support this is not conclusive. The current study examined the cytoadherence patterns of lab-adapted patient isolates after selecting on ICAM-1. We investigated the binding phenotypes using variant ICAM-1 proteins including ICAM-1Ref, ICAM-1Kilifi, ICAM-1S22/A, ICAM-1L42/A and ICAM-1L44/A using static assays. The study also examined ICAM-1 blocking by four anti-ICAM-1 monoclonal antibodies (mAb) under static conditions. We also characterised the binding phenotypes using Human Dermal Microvascular Endothelial Cells (HDMEC) under flow conditions. The results show that different isolates have variant-specific binding phenotypes under both static and flow conditions, extending our previous observations that this variation might be due to variable contact residues on ICAM-1 being used by different parasite PfEMP1 variants.

Vaccination with Toxoplasma lysate antigen and CpG oligodeoxynucleotides: comparison of immune responses in intranasal versus intramuscular administrations.

Toxoplasma gondii (T. gondii) is one of the most successful intracellular protozoan parasites on earth and highly prevalent in most warm-blooded vertebrates. There are no drugs that target the chronic cyst stage of this infection; therefore, development of an effective vaccine would be an important advance in disease control. Oligodeoxynucleotides (ODN) which contain immunostimulatory CG motifs (CpG ODN) can promote T-helper 1 (Th1) responses, an adjuvant activity that is desirable for vaccination against intracellular pathogen. In this study, we compare the immune responses of Toxoplasma susceptible C57BL/6 mice following intranasal and intramuscular vaccination with Toxoplasma lysate antigen (TLA) with or without CpG ODN as adjuvant. Immunized and control non-immunized mice were challenged with 85 cyst of the moderately virulent Beverley strain of T. gondii. Intranasal vaccination gave significantly a higher protection compared to other groups as indicated by prolonged survival and significantly reduced brain cyst burden (P < 0.01). Intranasal vaccination stimulated cellular immunity towards Th1 response characterized by significant INF-γ production (P < 0.01). Furthermore, fecal IgA antibody levels as an indicator of mucosal immune responses were significantly higher (P < 0.05) in intranasal vaccinated group before the challenge compared to all other groups. Intranasal vaccination was not able to upgrade the Th1 humoral arm. In contrast, intramuscular vaccination enhanced humoral immunity towards a type Th1 pattern characterized by a significant increase of specific IgG and Ig2a. Our results suggest that intranasal administration of CpG/TLA would provide a stable, pronounced, and effective vaccine against toxoplasmosis through stimulation of Th1 cellular immunity and mucosal IgA.

The role of B-cells in immunity against adult Strongyloides venezuelensis.

BACKGROUND: Strongyloides venezuelensis has been used as a tool and model for strongyloidiasis research. Elimination of S. venezuelensis adult worms from mice has been particularly associated with proliferation and activation of intestinal mast cells and eosinophils. To date, the role of B-cells in the protective mechanism against adult Strongyloides infection in experimental animals has not been reported in the literature. Therefore, the present study was carried to investigate the role of B-lymphocytes in immunity against adult S. venezuelensis infection using mice with a targeted deletion of the JH locus. METHODS: JHD knockout mice with its wild-type Balb/c mice were infected by intra-duodenal implantation of adult S. venezuelensis. Fecal egg count, intestinal worm recovery, mucosal mast cells and eosinophils were counted. RESULTS: At day 11 post infection, parasites in wild-type mice stopped egg laying, while in JHD knockout mice parasites continued to excrete eggs until the end of the observation period, day 107. The higher number of parasite eggs expelled in the feces of JHD knockout infected mice was a consequence of higher worm burdens, which established in the small intestine of these animals. On the other hand worm fecundity was comparable in both groups of mice. Both B-cell-deficient mice and wild-type mice, showed an influx of mucosal mast cells and eosinophils. The absolute numbers in JHD knockout mice were lower than those seen in wild-type mice at day 11, but not to a level of significance. JHD knockout mice could not recover from infection despite the recruitment of both types of cells. CONCLUSION: Our findings highlight a role of B cells in mucosal immunity against invasion of adult S. venezuelensis and in its expulsion. Therefore, we conclude that B-cells together with mucosal mast cells and eosinophils, contribute to immunity against adult S. venezuelensis by mechanism(s) to be investigated.

Malaria drug resistant: current situation with reference to Saudi Arabia (review).

Malaria is a chronic disease caused by parasitic protozoa of plasmodia species. Four plasmodium species are causing malaria to human (P. vivax P. ovale, P. malariae and P. falciparum). Malaria classifies as one of the most serious diseases in tropical and subtropical countries and p. falciparum represents the major cause of death by malaria species. Approximately 40% of the world population resides in areas of active malaria transmission. Treatment and prophylaxis measures are important to reduce morbidity and mortality rate of infection. In last two decades, a significant number of malaria drug resistance cases (mainly P. falciparum) were reported in endemic areas against choroquine components. Parasite showed enormous amount of antigenic variation under immune pressure leading to emergence of vaccine resistant strains. Similarly, under drug pressure it allows mutations to settle in the target genes. It is becoming more and clearer that with the continuous exposure to a drug, the parasite accumulates more and more number of mutations in these genes. Artemisinin is the only available drug that is globally effective. This review concentrates on the current situation of malaria drug resistance including epidemiological distribution, the mechanism of how the malaria resists certain drugs and the role of recent advances facilities in molecular biology to evaluate the impact on drug resistance of new drug-based strategies in Saudi Arabia.

PCR assay in malaria diagnosis using filter paper samples from Jazan region, Saudi Arabia.

PCR assay using designed primers was evaluated in detecting human malaria infection from whole blood and/or on Whatman filter-paper compared to conventional microscopy. Two DNA extraction methods were used for dried blood-spots; QIAamp mini kit and methanol-fixation/heat-extraction. A total of 118 cases were collected from 4 hospitals at Jazan district. Microscopic examination showed positivity in 66/118 samples (56%). Thin films showed parasitaemia from 1+ to 4+. In PCR assay, 79 samples (70%) were positive for the genus Plasmodium given 153 base pair PCR product. All microscopy positive samples were PCR positive but PCR detected 13 cases missed by microscopy. As for filter-paper spot samples, 68 samples (57.6%) were PCR positive when DNA was extracted by QIAamp mini kit whereas 49 (41.5%) by methanol-fixation/heat-extracion method. PCR sensitivity decreased by using DNA extracted from filter-paper compared to microscopy and whole blood PCR. But the DNA isolated from filter paper detected parasites in many microscopy negative samples.

Preliminary study of the prevalence of intestinal parasites among diarrheic inhabitants in Makkah Al-Mukarramah.

Darrheic disease is one of the greatest causes of morbidity and mortality worldwide. Intestinal parasites contribute to the disease and the well being of humans. This study was undertaken in the Holly City of Makkah Al-Mukarramah. A total of 166 diarrheic stool samples were collected. A wet smear from each specimen in normal saline and Lugol's iodine solution was examined microscopically for the trophozoites and cysts of protozoan parasites. Stools were also examined using ethyl acetate formalin concentration technique to confirm the diagnosed parasites. All stool specimens were stained by Ziehl-Neelsen stain to detect the oocysts of Cryptosporidium spp. One way ANOVA was used to analyze data. 128 persons were found to be infected, with an overall prevalence of 77.1%. 46.99% of the samples were females, and 53.01% were males. The prevalence of infection in females was 36.1%, and 40.9% in males. 16.9% of infected females were living near the Holy Masjid (down town), while 19.3% were living away from the Holy Masjid (up town). 18.7% of infected males were living down town, while 22.3% were living up town. The majority of cases fall into the young age groups (< 30 years old). There is no significant difference between the prevalence of infection down town and up town (P = 0.22), whereas the prevalence of infection between the patients over or under 30 years old was significance (P = 0.036). The rates of infection were higher in those living up town than those living down town. The results were critically discussed.

Detection of drug resistance markers for chloroquine and pyrimethamine-sulfadoxine in Jazan area, Saudi Arabia using PCR and restriction digestion.

Nested PCR and restriction analysis were used to detect mutations at codon 76 of Plasmodium falciparum chloroquine resistance transporter gene (pfcrt) and codon 59 of dihydrofolate reductase gene (dhfr) that indicate chloroquine (CQ) and pyrimethamine-sulfadoxine (PYR-SDX) resistance respectively. P. falciparum isolates from malaria-endemic area of Jazan showed CQ resistance rate (89.5%), the highest percentage of chloroquine resistance ever recorded in Saudi Arabia. One the other hand, 10.5% of isolates showed a PYR-SDX resistant allele as a first reported in the kingdom. The use of molecular markers as additional tools to map areas of chloroquine resistance was expected to contribute in the development of new strategies for therapeutic intervention towards malaria in Saudi Arabia.

Enteroparasitic occurrence in stools from residents in Southwestern region of Saudi Arabia before and during Umrah season.

OBJECTIVE: Study of the prevalence of human gastro-intestinal parasitic infections among patients living in Makkah Al-Mukkarmah city before and during Umrah season. METHODS: One hundred eighty three stool samples were collected from patients living in Makkah, between the months of March and November 2005. Eighty were collected before the Umrah season began and 103 were collected during the Umrah season. Age, sex, and address were also recorded. Samples were preserved in 10% formol saline. They were examined using the direct smear technique and the formol ether concentration method. RESULTS: The results suggest a higher prevalence of intestinal parasitic infections (70.5%) among the patients under study. Entamoeba histolytica/E. dispar and Giardia lamblia were found to be the most common intestinal parasites among patients before and during Umrah. The infection rate was higher in the under 30 age group (74.8%) and in persons living away from the Holy Masjid (77.7%). The prevalence of intestinal parasitoses during Umrah (73.8%) was higher than that before Umrah (66.3%). CONCLUSION: The present study suggests that the group of people may underline the significant increase in the prevalence of intestinal parasitic infections during Umrah season. This highly significant increase of parasitic infection rate (p=0.018) was elicited when results were compared by one-way analysis of variance (ANOVA). The present data were discussed with previous studies.

Detection of malaria in Saudi Arabia by real-time PCR.

Malaria transmission occurs in Saudi Arabia and mainly endemic in the lowlands of Asir region, the Southwester Province. Imported cases have been reported. Sensitive routine laboratory techniques for rapid and accurate malaria diagnosis are therefore desirable to facilitate the identification of individuals infected with the malarial parasites and to follow up the progress of treatment of such cases with appropriate drugs. Traditional diagnosis, based on the microscopic examination of Giemsa-stained thick and thin films remains the main standard method of diagnosis used for malaria diagnosis in Saudi Arabia. Molecular diagnostic techniques based on the detection of nucleic acids (as PCR; Real-time PCR) are now highly considered. Real time-PCR a new methodology has been recently applied to detect human malaria. In this study a total of forty four samples, using whole-blood, dried blood and thick smears were examined by PCR and Real-time PCR. Both techniques showed a higher sensitivity than the microscopy. Parasites were detected in twenty nine samples out of forty four, compared to twenty six of thirty nine were positive with thin blood film. The real-time PCR assay offers a practical and positive alternative for rapid and accurate diagnosis for malaria infection. The application of such technique will be significantly valuable especially for screening for malaria infection in endemic areas.

Molecular characterization, expression, and in vivo analysis of LmexCht1: the chitinase of the human pathogen, Leishmania mexicana.

Chitinases have been implicated to be of importance in the life cycle development and transmission of a variety of parasitic organisms. Using a molecular approach, we identified and characterized the structure of a single copy LmexCht1-chitinase gene from the primitive trypanosomatid pathogen of humans, Leishmania mexicana. The LmexCht1 encodes an approximately 50 kDa protein, with well conserved substrate binding and catalytic domains characteristic of members of the chitinase-18 protein family. Further, we showed that LmexCht1 mRNA is constitutively expressed by both the insect vector (i.e. promastigote) and mammalian (i.e. amastigote) life cycle developmental forms of this protozoan parasite. Interestingly, however, amastigotes were found to secrete/release approximately >2-4-fold higher levels of chitinase activity during their growth in vitro than promastigotes. Moreover, a homologous episomal expression system was devised and used to express an epitope-tagged LmexCht1 chimeric construct in these parasites. Expression of the LmexCht1 chimera was verified in these transfectants by reverse transcription-PCR, Western blots, and indirect immunofluorescence analyses. Further, results of coupled immunoprecipitation/enzyme activity experiments demonstrated that the LmexCht1 chimeric protein was secreted/released by these transfected L. mexicana parasites and that it possessed functional chitinase enzyme activity. Such transfectants were also evaluated for their infectivity both in human macrophages in vitro and in two different strains of mice. Results of those experiments demonstrated that the LmexCht1 transfectants survived significantly better in human macrophages and also produced significantly larger lesions in mice than control parasites. Taken together, our results indicate that the LmexCht1-chimera afforded a definitive survival advantage to the parasite within these mammalian hosts. Thus, the LmexCht1 could potentially represent a new virulence determinant in the mammalian phase of this important human pathogen.