Serological Evaluation of EgAgB16 kDa,a Recombinant Antigen from Echinococcus granulosus for Diagnosis of Human Hydatidosis

Background

Regarding that accurate diagnosis of human hydatidosis still needs more investigations, the present study was conducted to clone, express, and evaluate the gene encoding AgB subunits (EgAgB16 kDa) from Echinococcus granulosus (Iranian G1 strain) and its evaluation by ELISA test.

Methods

DNA was extracted from protoscoleces and was utilized by PCR for strain identification. Total RNA was prepared with RNeasy protect mini kit from E. granulosus (Iranian G1 strain) protoscoleces collected from naturally infected sheep with hydatid cyst. Recombinant AgB16 kDa was produced using pETDuet as vector and evaluated by ELISA method. A panel of sera including hydatid cyst-infected individuals (n=72), healthy individual (n=48), toxoplasmosis (n=4), strongyloidosis (n=4), kala-azar (n=5) and tuberculosis (n=5) were examined using this recombinant antigen.

Results

Recombinant protein was purified by affinity chromatography using His-Tag column. After purification, recombinant protein was confirmed by western blot analysis using His Tag monoclonal antibody or hydatid positive human serum. The sensitivity, specificity; positive and negative predictive values were calculated as 93.5%, 95.6%, 96% and 92.9%, in that order. The cut-off point was detected 0.3 for rAgB16.

Conclusion

While the produced recombinant AgB16 kDa showed promising results in diagnosing human hydatidosis, but more investigations should be implemented to reach an accurate gold standard.     

Evaluation of Recombinant SAG1 Protein for Detection of Toxoplasma gondii Specific Immunoglobulin M by ELISA Test

Background

Toxoplasmosis is a serious disease in immunocompromised patients and pregnant women. Differentiation of acute and chronic infection is a major challenge in serodiagnosis of the disease. Since the aim of this study was to assess the diagnostic utility of recombinant SAG1 (rec-SAG1) for the detection of Toxoplasma-specific IgM antibodies in human sera, by an enzyme-linked immunosorbent assay (ELISA).

Methods

The purified recombinant protein SAG1 was applied in house ELISA test and the ability of it in binding to specific immunoglobulin M in 30 serum samples of acute infected patients was evaluated. The results obtained by assays with the recombinant SAG1 and standard commercial assays were compared.

Results

The sensitivity and specificity of in house ELISA compared to a standard commercial ELISA (com-ELISA) were 80% and 90%, respectively.

Conclusion

It was concluded that the rec-SAG1 could be an alternative marker for detection of anti Toxoplasma-specific IgM and diagnosis of acute infection.     

Rapid Detection of Toxoplasma gondii Antigen in Experimentally Infected Mice by Dot- ELISA

Background

Toxoplasmosis is a worldwide endemic disease. In congenitally infected infants and AIDS patients, toxoplasmosis causes high rates of morbidity and mortality. In these cases antibody detection is difficult; so detection of parasite or its components could be useful tool for early detection and following treatment of the infection.

Methods

Sixty-three BALB/c mice were injected intra-peritoneal with 5×10³ tachyzoites of Toxoplasma gondii RH strain, nine mice were sacrificed daily for 7 days. Fourteen mice were injected with phosphate buffer saline as control group. Dot–ELISA was performed for detection of T.gondii antigen in mice sera and capture – ELISA was done as golden standard assay too.

Results

Toxoplasma gondii antigen was detected from day 2 in mice sera; 22% of mice sera on day 2, 33% on day 3,77% on day 4 and 100% on day 5 till their death on day 7 had shown antigenemia by dot – ELISA, no positive result was detected in control mice by dot- ELISA.

Conclusion

Dot-ELISA is a sensitive method for diagnosis of T. gondii infection in the animal model; also, this technique is more rapid and easy to perform method in comparison with capture-ELISA.     

Dientamoeba fragilis in Gaza Strip: a Neglected Protozoan Parasite

Background

The aim of this study was to detect Dientamoeba fragilis by iron haematoxylin stain, as well as its prevalence, and association between D. fragilis infection and diarrhoea among patients attending Al-Nuseirate Refugee Camp Clinic, Gaza Strip.

Methods

A cross-sectional study was conducted among 319 children and adults with age ranges from (1 to 75) years old, attending Al-Nussirat Clinic, and who were complaining from clinical symptoms, like diarrhoea and abdominal pain.

Results

28 individuals were infected with D. fragilis with a prevalence of 8.8%. The detection of 28 cases infected with D. fragilis was proved using iron haematoxylin stain, but no case was detected by direct smear or formal-ether sedimentation technique. The most frequent symptoms were abdominal pain (96.4%) and diarrhoea (71.4%) in patients with diantamoebiasis and this was statistically significant (P= 0.03). Co-infection between D. fragilis and Entamoeba histolytica/dispar was 50% and between D. fragilis and Giardia lamblia was 7.1%.

Conclusion

D. fragilis was present in the patients stool samples and was detected and proved using iron haematoxylin stain.     

Seroprevalence of Human Hydatidosis Using ELISA Method in Qom Province,Central Iran

Background

The objective of this study was to determine the prevalence of cystic echinococcosis (CE) in Qom Province, central Iran using ELISA test.

Methods

Overall, 1564 serum samples (800 males and 764 females) were collected from selected subjects by randomized cluster sampling in 2011-2012. Sera were analyzed by ELISA test using AgB. Before sampling, a questionnaire was filled out for each case. Data were analyzed using Chi-square test and multivariate logistic regression for risk factors analysis.

Results

Seropositivity was 1.6% (25 cases). Males (2.2%) showed significantly more positivity than females (0.9%) (P= 0.03). There was no significant association between CE seropositivity and age group, occupation, and region. Age group of 30-60 years encompassed the highest rate of positivity. The seropositivity of CE was 2.1% and 1.2% for urban and rural cases respectively. Binary logistic regression showed that males were 2.5 times at higher risk for infection than females.

Conclusion

Although seroprevalence of CE is relatively low in Qom Province, yet due to the importance of the disease, all preventive measures should be taken into consideration.     

Comparison of a Commercial ELISA with the Modified Agglutination Test for Detection of Toxoplasma gondii Antibodies in Sera of Naturally Infected Dogs and Cats

Background

Toxoplasma gondii can infect all warm-blooded animals. Modified agglutination test (MAT) and ELISA are widely used for the detection of T. gondii antibodies. However, there is little information on their acceptability for detecting antibodies in companion animals.

Methods

This study compared ELISA and MAT for their ability to detect T. gondii infection in naturally infected dogs and cats. Blood samples were collected from dogs and cats in different areas of Beijing, China and analyzed by ELISA and MAT. The χ² test and κ analysis were used to evaluate their efficiency and agreement.

Results

For dogs, the seroprevalence of T. gondii antibodies detected by ELISA was 34.7%, which was significantly higher than that detected by MAT (P<0.05). There was no significant difference between ELISA and MAT for detecting T. gondii antibodies in cats. Good agreements between MAT and ELISA were seen in both dogs and cats; however, inconsistent results were demonstrated by κ analysis and in MAT titer assay.

Conclusion

Serum-based ELISA may be more satisfactory for screening test of T. gondii infection in dogs, whereas both methods could be acceptable in cats.     

A Sensitive and Specific PCR Based Method for Identification of Cryptosporidium Sp. Using New Primers from 18S Ribosomal RNA

Background

The main goal of the present study was to develop a new sensitive and specific PCR based method for Identification of Cryptosporidium sp. using novel primers from 18S ribosomal RNA. Cryptosporidiosis in high-risk host groups particularly in neonates and immuno-compromised individuals may result in death. To the best of our knowledge this is the first study regarding develop a new PCR based method to diagnose the cryptosporidiosis in Iran.

Methods

A total of 850 human fecal samples from patients clinically suspected to cryptosporidiosis and 100 healthy and diarrheic cattle stool specimens were collected. The simplified formol-ether concentration method was carried out for all samples. They were then examined microscopically by modified Ziehl-Neelsen staining method. Total DNA was extracted by QIA amp DNA stool mini kit. PCR and nested-PCR was carried out by using designed primers.

Results

Twenty nine cases of cryptosporidiosis infection in human and 30 samples from cattle microscopically were positive. The described primary and nested PCR method could detect all Cryptosporidium positive samples from human and cattle. Regards to suspected negative samples in primary PCR examination, the Nested PCR could approve two more positive results. Furthermore, Nested PCR analysis was able to detect one more case which was negative in both microscopically examination and primary PCR. Specificity of the test was 100%. Sensitivity of Nested PCR in comparison to our gold standard; microscopy after Ridley concentration modified ziehl-Neelsen, was 100%.

Conclusion

Our developed PCR based method by using new primers devised from 18S ribosomal RNA revealed the ability for identification of the Cryptosporidium species such as C. parvum and C. huminis with high specificity and sensitivity.     

A Preliminary Parasitological Survey of Hepatozoon Spp. Infection in Dogs in Mashhad,Iran

Background

We attempted to determine the prevalence of Hepatozoon spp. infection in Mashhad, northeast of Iran, via blood smear parasitology.

Methods

The prevalence was investigated by examination of blood smear parasitology, using blood samples collected from 254 dogs (51 strays and 203 privately owned-dogs).

Results

Two stray dogs (2/51; 3.92%) and two privately-owned dogs (2/203; 0.98%) were infected with Hepatozoon spp. Therefore, as per blood smear parasitology, the prevalence of Hepatozoon spp. infection was 1.57% (4/254). Sixteen out of 254 dogs (6.29%) were infested with ticks; all of which were Rhipicephalus sanguineus. One of the dogs infected with Hepatozoon spp. exhibited ticks at the time of examination. Concurrent infection with Ehrlichia canis and Leishmania infantum was not detected in the four Hepatozoon spp. infected dogs.

Conclusion

This is the first epidemiological study on the prevalence of Hepatozoon spp. infection in dogs in Iran.     

Strongyloides stercoralis: The Most Prevalent Parasitic Cause of Eosinophilia in Gilan Province,Northern Iran

Background

Eosinophilia occurs in a wide variety of situations such as parasitic infections, allergic disorders, and malignancies. Most cases of eosinophilia of parasitic origin, especially those with a tissue migration life cycles consists of human infections by helminth parasites. The aim of present study was to determine the parasitic causes of eosinophilia in patients in a major endemic area of human fascioliasis in Gilan Province, northern part of Iran.

Methods

One hundred and fifty patients presenting with an elevated eosinophilia attending infectious disease clinics with or without clinical symptoms, were examined. After clinical history evaluation and physical examination, coprological examinations were performed using the formalin-ether and the Kato-Katz techniques for detection of Fasciola sp. and intestinal parasites.

Results

Forty two percent of patients were infected with S. stercoralis, nine (6%) were found to be infected with Fasciola sp. while only a single patient (0.7%) were infected by Ttrichostrongylus sp.

Conclusion

Local clinicians in Gilan may consider eosinophilia as a suggestive indication for diagnosis of human fascioliasis, especially when microscopic stool and/or serological tests are negative. Based on the results, local physicians should consider S. stercoralis as the potential causes of eosinophilia in patients with elevated eosinophilia.     

Application of the Reverse Line Blot Assay for the Molecular Detection of Theileria and Babesia sp. in Sheep and Goat Blood Samples from Pakistan

Background

The present study was designed to detect the presence of tick-borne parasites (Theileria and Babesia spp.) in 196 blood samples collected from apparently healthy sheep and goats from two provinces, Punjab and Khyber Pukhtoon Khwa, in Pakistan.

Methods

Reverse line blot (RLB) assay was applied for the parasitic detection by the amplification of hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene. A membrane with covalently linked generic and species specific oligonucleotide probes was used for the hybridization of amplified PCR products.

Results

Parasites were detected in 16% of the ruminant blood samples under study. Two Theileria species, T. lestoquardi and T. ovis, were identified in samples. 25, of the total 32, infected animals were from Khyber Pukhtoon Khwa.

Conclusion

Sheep were more prone to tick borne haemoprotozans as 81% infected samples were sheep as compared to 19% goats (P > 0.001). Risk factor analysis revealed that male (P = 0.03), animals infested by ticks (P = 0.03) and herd composed of sheep only (P = 0.001) were more infected by blood parasites.     

Introducing Alphitobius diaperinus, (Insecta: Tenebrionidae) as a New Intermediate Host of Hadjelia truncata (Nematoda)

Background

Hadjelia truncata is a nematode that causes lesions in the gizzard lining of pigeons, which may even lead to death. The aim of this study was to introduce Alphitobius diaperinus as a new intermediate host for Hadjelia truncata.

Methods

H. truncata infection was identified in a pigeon flock in Ahvaz City, Khuzestan Province, Iran by performing fecal examination and autopsy. Adult and larval stages of beetles were collected from the litter of pigeon houses, and identified morphologically. The beetle larvae were cultured in a medium, containing feces of the infected pigeons. Nematode larval stages from naturally and experimentally (culturally) infected adult beetles were fed to two groups of pigeons

Results

The collected beetles were identified as Alphitobius diaperinus. Average length and width of the adult beetles were 6.31 mm and 2.88 mm respectively. Infection rates of naturally and experimentally infected beetles with larval stages of the nematode were 66.2% and 45.1% respectively. The adult nematodes collected from gizzards of experimentally infected pigeons were identified as H. truncata. Nematode infection rates in pigeons after feeding the infective larvae collected from naturally and experimentally infected beetles were 44.7% and 32.5% respectively.

Conclusion

A. diaperinus can serve as a natural intermediate host for H. truncata.     

Urinary Schistosomiasis in Urban and Semi-Urban Communities in South-Eastern Nigeria

Background

In view of the massive rural-to-urban migration in Nigeria, investigations on transmission of urinary schistosomiasis were carried out in urban and semi-urban communities in Nike Lake area of Enugu State, Nigeria.

Methods

Urine samples of school children were tested for micro-haematuria using reagent strips followed by microscopic examination for Schistosoma haematobium eggs. Water contact sites were also identified and sampled for snails.

Results

The overall prevalence of S. haematobium eggs in school children was 4.64%. The mean intensity of infection was 1.14 ± 0.41 eggs/10ml urine. Males had insignificantly higher prevalence and intensity of S. haematobium infection than females. The youngest age group (4-7 years) had no infection. The prevalence of micro-haematuria (6.2%) was higher than that of microscopy, and this correlated positively with prevalence (r = 0.65, P < 0.01) and intensity (r = 0.50, P < 0.01) of the infection. Potential intermediate host of human shistosome collected were: Bulinus globosus, B. senegalensis and Biomphalaria pfeifferi. However, only B. globosus shed cercariae of S. haematobium, with a snail infection rate of 0.73%. Transmission was in the dry season coinciding with the drying of wells.

Conclusion

The results revealed that urinary schistosomiasis is prevalent, and that B. globosus and not B. truncatus as previously reported is the main intermediate host of urinary schistosomiasis in this part of Enugu State.     

Production and Evaluation of Toxoplasma gondii Recombinant Surface Antigen 1 (SAG1) for Serodiagnosis of Acute and Chronic Toxoplasma Infection in Human Sera

Background

The assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the Lack of a purified standardized antigen. The aim of this study was evaluation the recombinant Toxoplasma gondii SAG1 antigen for the serodiagnosis of acute and chronic toxoplasmosis.

Methods

This study describes an ELISA using recombinant SAG1 for detection of IgM and IgG antibodies against Toxoplasma gondii in human sera. Genomic DNA of T. gondii (RH Strain) was isolated and PCR reaction was performed. Recovered DNA was cloned into PTZ57R cloning vector. The recombinant plasmid was detected by restriction analysis. The SAG1 gene was subcloned in the pET- 28a expression vector. Protein production was then induced with 1 mM isopropyl-D – thiogalactopyranoside (IPTG). A total of 204 sera were tested using a commercial IgG and IgM ELISA kit (Trinity, USA) as gold standard prior to testing them with the recombinant antigen.

Results

Tested sera were divided into the following groups:(a) The 74 T. gondii IgG positive (b) 70 T.gondii IgM positive (c) 60 sera who had no serological evidence of toxoplasmosis as negative sera.To determine the specificity of the test, we used other parasitic diseases including echinococusis (N=5), malaria (N=14), leishmaniasis (N=7),fasciolasis (N=4), sterengyloidiasis (N=1). Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (Com ELISA) were 93% and 95%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 87% and 95% respectively.

Conclusion

The results acquired here show that this antigen is useful for diagnostic purposes and could be replaced by lysed, whole cell antigens for diagnosis of chronic toxoplasmosis.     

Absence of Asymptomatic Malaria Infection in Endemic Area of Bashagard District,Hormozgan Province,Iran

Background

A successful malaria elimination program calls for enough attention to parasite carriers, especially asymptomatic malaria, as well as the diagnosis and treatment of clinical cases. Asymptomatic malaria is an infection that patients do not show any symptom; thus, these patients play critical role in the concept of an elimination program. The current investigation was conducted to evaluate the presence of these cases in Bashagard District, formerly a high malaria transmission area in Hormozgan Province, Iran.

Methods

Blood samples (n = 500) were collected from symptomless individuals residing in Bashagard to evaluate Plasmodium infection by using microscopic, serological and nested-PCR techniques.

Results

Regarding the microscopic and nested-PCR analysis, no asymptomatic infection was detected among studied individuals. Totally, 1% of the studied population (5 of 500) had anti PvMSP-1₁₉-specific IgG antibody; however, only 0.2% (1 of 500) of the individuals was seropositive to recombinant PfMSP-1₁₉, using ELISA.

Conclusion

This study showed no asymptomatic malaria infection in the studied population; hence malaria elimination is feasible and can be successfully carried out in this region.     

Seroprevalence of Toxoplasma gondii in Pregnant Women and Bioassay of IgM Positive Cases in Zanjan,Northwest of Iran

Background

The aim of this study was to determine the Toxoplasma antibodies in pregnant women in Zanjan, by ELISA method.

Methods

Blood samples were taken from 500 pregnant women referred to the health centers of Zanjan City, North West Iran, IgM and IgG titers were primarily evaluated. The collected data were analyzed with SPSS 11.5 using Chi-Square test.

Results

Anti Toxoplasma IgM and IgG were positive in 1.4% and 37.2% respectively. Seropositive subjects were more frequently seen in those with >30 years old compared to younger women (<20 years old). No significant relationship was found between the seroprevalence of T. gondii infection and level of education, residence area, history of abortion and gestational age.

Conclusion

The rate of IgM positive was low; however, a large number of the studied population were IgG positive, indicative of having a latent infection due to the past exposure to Toxoplasma parasite in this region.     

Seroprevalence of Neospora caninum Infection in Dairy Cattle in Tabriz,Northwest Iran

Background

The aim of this study was to determine the seroprevalence of antibody to Neospora caninum in healthy and aborted dairy cattle in Tabriz, capital of East-Azarbaijan in northwest of Iran.

Methods

In this cross-sectional study serum samples were collected from 266 healthy and aborted Holestein-Feriesisnc cows from September 2008 to August 2009. The sera were analyzed to detect of antibody against N. caninum using the commercially ELISA kit.

Results

Seroprevalence of antibody to N. caninum was 10.5% in Tabriz dairy cattle. Also the abortion rate in all cattle sampled was 33.6% but percentage of seropositive aborted cattle was 18.4%.

Conclusion

Neosporosis could be one of the possible causes of abortion in dairy cattle in Tabriz and regarding the distribution in dogs as definitive host for the parasite, further studies in dog and cattle are recommended.     

Identification of Leishmania Species Using PCR Assay on Giemsa-Stained Slides Prepared From Cutaneous Leishmaniasis Patients

Background

Leishmaniasis is a group of diseases that are created by intracellular parasites of Leishmania. Cutaneous leishmaniasis is considered as one of the health problems in some provinces of Iran.

Methods

In this study, a total of 178 Giemsa-stained slides from confirmed cases of cutaneous leishmaniasis were examined. The slides were prepared from the patients with cutaneous leishmaniasis that referred to health centers and infected during the epidemic of cutaneous leishmaniasis in Poldokhtar city, Lorestan Province, Iran in 2006.Genomic DNA from each slide was extracted. After DNA extraction, ITS-PCR was used.

Results

Out of 178 slides, 129 (72.47%) samples had a band in the range of 485 bp and 49 (27.53%) samples 626 bp that matched L. tropica and L. major standard samples, respectively.

Conclusion

This study showed that Leishmania DNA could be efficiently extracted and amplified even from old Giemsa-stained microscopic slides that were stored more than 6 yr. In this study was shown that both L. tropica and L. major species exist in Lorestan Province.     

Development of 116 kDa Fraction for Detecting Experimental Toxoplasma gondii Infections in Mice

Background

Serological diagnosis of Toxoplasma gondii infection using crude antigens may not be more accurate. To increase the diagnostic potency of antigens, isolation of their immunogenic fractions could be useful. The current research adopted to obtain an affinity isolated fraction from RH strain using CNBr Sepharose 4B column coupled with infected mice sera helping in detection of IgM and IgG of toxoplasmosis due to RH strain and other strains.

Methods

The isolated fraction was characterized by SDS-PAGE. Moreover, the diagnostic potency of the fraction was assessed by indirect ELISA in mice experimentally infected with RH strain and two other local strains; one of sheep origin and the other of human origin.

Results

The fraction was found to be consisted of a single band of 116 kDa compared with 17 bands ranged from 116 to 16 kDa associated with crude extract. The fraction proved potent diagnostic potentials of acute and chronic mice toxoplasmosis. Where it was detected both IgM and IgG antibodies as early as two days and as late as 2 months post experimental infection with any of the three strains. The level of detected IgM and IgG by RH fraction was higher in mice infected with RH strain than with local strains except IgM due to sheep strain parasite.

Conclusions

The 116 kDa fraction of T. gondii tachyzoites can be considered as a candidate in improving of serodiagnosisof Toxoplasma infections.