In vivo and in vitro efficacy of Nigella sativa aqueous extract on blastocystis hominis

Metronidazole [MTZ] was the most widely accepted treatment for Blastocystis hominis [B. hominis] with high treatment failure rate, resistance and potential mutagenic and carcinogenic effects so there is urgent need to find out new, effective and safe treatment against B. hominis. The present research aimed to evaluate the therapeutic effect of the aqueous extract of Nigella sativa [NS] at different doses on B. hominis in vitro and in vivo in comparison to MTZ as a control drug. Isolates of B. hominis were obtained from patients complaining of diarrhea and abdominal pain. Isolates were cultured in egg diphasic medium [LE] for in vitro study and to adjust proper inoculating dose for in vivo study. The aqueous extract of NS at concentrations of 100 and 500?g/ml showed a potent lethal effect on B. hominis isolates in vitro. Caecal tissue of experimentally infected and treated mice with two different doses of NS [250 and 500mg/kg/d] were examined histopathologically and compared with that of mice infected and treated by two doses of MTZ [62 and 125 mg/kg/d] as control drug and Infected untreated mice as negative control group. Histopathological examination of infected untreated group showed all pathological degrees in the caecal tissue while infected treated one showed remission of pathological changes especially with higher dose [500mg/kg]. Present study proved that N. sativa had inhibitory effect on B. hominis in vitro and prevented cytopathic effect in infected mice inoculated orally with B. hominis

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.

Detection of human cryplosporidium species in surface water sources in Ismailia using polymerase chain reaction

Gryptosporidium is a water-borne parasite that has caused several outbreaks ofgastrointestinal disease worldwide. Two species of Giyptosporidium are mainly found to cause disease in man, C. hominis which shows anthroponotic transmission, and C. parvum with zoonotic transmission. The present study aimed to verify the presence of human Cryplosporidium species in surface water sources in lsmailia using Polymerase Chain Reaction [PCR]. A total of 88 water samples were collected from Ismailia canal [24 samples], and from house taps [64 samples] at different seasons of the year. After filtration and concentration, the water concentrates were examined for Cryplosporidium oocysts by modified Zichi Necisen stain. Identification of C parvum and C. hominis was performed by multiplex allele specific polymerase chain reaction [MAS-PCR] followed by high resolution melting curve [HRM] analysis. The water samples were also subjected to physiochemical and microbiological analysis. Cryplosporidium oocysts were detected in 19[79.2%] of canal water samples and in only 2 [3.1 h] tap samples. Canal water was significantly associated with higher concentration of Cryplosporidium oucysts [50-450 oocysts/L] compared to tap water [20-30 oocysts/L]. C. parvum was the most common species detected in water samples [16 of 21 positive samples, 76.2%], while C. Hominis was detected in only one sample [4.8%] Summer showed the highest percentage of positivity with Cryplosporidium oocysts, then winter followed by autumn and spring. The presence of Cryplosporidium had significant association with the turbidity levels of water samples and their basic pH. Significant associations were also found with presence of total and fecal coliforms. Presence of Cryplosporidium oocysts as significantly associated with the grade >100- 25<103 CFU for both total and fecal coliforms. Canal water was significantly associated with higher concentration of oocysts/L. compared to tap water. C. parvum was found in both sources indicating zoonotic contamination of water

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