Direct genotyping of toxoplasma gondii in blood samples from pregnant women in Jazan, Saudi Arabia

Toxoplasma gondii [T. gondii] is divided three main clonal lineages designated as type I, II, and III and atypical genotypes were also detected. The distribution of T. gondii genotypes varied from one geographic area to another. This study characterized of T. gondii isolates from pregnant women in Jazan. Genetic analysis of the GRA6-coding fragment was performed for T. gondii genotyping using PCR-RFLP method. The seropositive for Toxoplasma-specific antibodies were determined using ELISA and were 27.9% in pregnant women in Saudi Arabia. Women seropositive for Toxoplasma IgG andIgM [GI=30] and for specific IgG [GII=30] were included. Among pregnant women, 83.3% of GI [women seropositive for IgG and IgM] and 90% of GII [women seropositive for IgG] were asymptomatic and observed clinical symptoms were fever [n=4] and cachexia [n=2] and lymphadenopathy [n=1]. GRA6-nested PCR was positive in 8 blood samples [13.3%], 5 of GI and 3 of GII seropositive women. RFLP analysis showed the detection of genotype I in 8 samples with no cases coinciding to pattern of type II or type III

Limited genetic diversity among Plasmodium falciparium isolates using nested PCR in Jazan Area, Saudi Arabia

Objective: To establish molecular characterization of Plasmodium falciparum field isolates in Jazan Area of Saudi Arabia measured with highly polymorphic genetic marker, i.e. the merozoite surface protein 2 (MSP 2). Methods: Blood samples were collected from 128 clinically suspected patients attending both Jazan and Sabia hospitals, Kingdom of Saudi Arabia. Both hospitals reflected urban and rural settings respectively. Analysis of central polymorphic region of MSP 2 (3D7 and FC27 allelic families) was performed using nested PCR for malaria patients. Results: For MSP 2 allelic families of Plasmodium falciparum, 16 cases (53.3%) carried FC27 type and 14 cases (46.7%) carried 3D7 type, whereas no malaria cases harbored both allelic types. The present study showed that in urban area, 80% of FC27 fragments were 500 bp while in rural area it was 45.5% (P = 0.08). The FC27 400 bp allele was more prevalent in patients from rural than those from the urban area (P = 0.08). The most prevalent infecting 3D7 allele was the 3D7 300 bp in both areas. In the present study, there were no multiple infections. Conclusions: The limited genetic diversity which was observed in Jazan (considered as an endemic area) may be attributed to the small sample size or sustained malaria control program.

Pathological studies of different genotypes of human cryptosporidium Egyptian isolates in experimentally mice

The genotyping of cryptosporidium clinical isolates obtained from 36 gastrointestinal symptomatic patients were identified by nested PCR for amplification of 18S rRNA followed by RFLP analysis using Ssp1 and Vsp1, and then pathological changes between different cryptosporidium genotypes were evaluated in experimentally infected mice. Cryptosporidium genotypes [C. parvum, C. hominis and C. melegridies] were detected [66.7%, 27.7% and 5.6%] respectively in human isolates. Different degrees of pathological changes were found among infected mice by different Cryptosporidium genotypes. Moderate and severe degrees of pathological changes with infection score ranging from 2 to 4 were found in all infected mice with C. parvum [except one isolate], while mild degree of pathological changes with infection score of 2 was found in all mice with C. melegridies. The results showed statistically significant relation between genotype and pathological degrees. There were no differences in the average number of oocysts per smear in Group II and Group III while in Group I, there was no oocysts shedding

Identification of blastocystis hominis in patients with irritable bowel syndrome using microscopy and culture compared to PCR

There is much controversy regarding the pathogenicity of B. hominis especially in patients with irritable bowel syndrome. Detection of B. hominis in patients with irritable bowel syndrome using different diagnostic methods; microscopy, culture and PCR. The second objective is to compare between microscopy and culture using PCR as Gold Standard. Stool samples from 83 patients with irritable bowel syndrome were subjected to microscopic examination, culture on egg diphasic medium and PCR amplification using primers based on small subunit ribosomal DNA, for diagnosis of B. hominis infection. Results: Out of 83 samples, 25 [30.1%] were found positive for B. hominis by microscopy, 34 [41%] culture and 37 [44.6%] using PCR. The sensitivity of microscopy and culture versus PCR was 62.2% d 89.2%, respectively. The specificity of microscopy and culture versus PCR was 95.7% and 97.8%, respectively. The diagnostic accuracy for microscopy and culture was 83.1% and 94% respectively. Culture proved to be a more sensitive and specific tool for detection of B. hominis than microscopy

Evaluation of the nitric oxide activity against Blastocystis hominis in vitro and in vivo

The effect of exogenous nitric oxide [NO] on growth, viability and ultra-structural of B. hominis was assessed in vitro by sodium nitrite [NaNO[2]] in 0.6 mM, 0.8 mM and 1 mM concentrations. The viability of B. hominis was identified using neutral red stain. The role of NO as an endogenous oxidant was assessed by identifying its level in cecum tissue, ileum tissue, blood and stool elutes of mice infected with B. hominis symptomatic human isolates using reactive nitrogen assay compared to control. In vitro study revealed that NaNO[2] inhibited the growth and decreased viability of B. hominis with minimal lethal concentration dose 1 mM on the 4[th] day while, minimal effects were detected with 0.6 and 0.8 mM. Transmission electron microscopy study proved that apoptotic-like features were observed in growing axenic culture of B. hominis upon exposure to NaNO[2]. These changes were not only found on the vacuolar [central body] form but also they were detected on granular, multi-vacuolar and cyst forms. In vivo study proved that high levels of NO were found in infected mice compared to low changes in control group. The high levels were in cecum tissue particularly. The mean levels of NO among infected mice were 211.8 +/- 20.7 micro M in cecum, 90.4 +/- 11.6 micro M in ileum, 60.1 +/- 4.7 micro M in blood and 63.6 +/- 7.3 micro M in stool elutes while, the mean levels of NO in control mice were 70.2 +/- 3.1 in cecum, 67.8 +/- 4.7 micro M in ileum, 30.9 +/- 4.2 micro M in blood and 28.1 +/- 2.9 micro M in stool elutes. The differences were statistically highly significant. NO-donor drugs proved useful in treatment and increase the host resistance to B. hominis

Effect of exogenous adminstration of anti-oxidants on Blastocystis hominis in vivo

The effect of exogenous administration of antioxidant [Anttox] on the course of B. hominis in experimentally infected mice was studied. B. hominis isolates were obtained from 10 gastrointestinal symptomatic adult patients. Three groups of 30 infected mice [3/isolate] were used. GI was untreated infected, GII was treated by antox for 4 weeks after infection diagnosis [treatment strategy], and GIII antox treated by antox for 4 weeks before infection [prophylactic strategy]. Mild pathological changes were detected on 13.4%, 19.9% and 86.8% of mice in Gs I, II and III, respectively. Moderate pathological changes were found in 29.9%, 26.6% and 6.6% of mice in Gs I, II and III, respectively. While, the majority of severe pathological changes were in Gs I and II [56.7% and 53.5%] as compared to GIII [6.6%]. Meanwhile, 86.8% of mice in GIII had B. hominis forms >10/high power field compared to 3.3% in Gs I and II, respectively. Although 19.8% of mice in GII were positive for B. hominis by direct smear, no growth resulted in vitro and all the forms were non-viable by using neutral red stain. All the differences were statistically significant. So, antioxidant exacerbated B. hominis intensity but it decreased the pathological changes