Vol 11 No 1 (2016)
One Health Approach Prospect for Integrated Control and Elimination of Visceral Leishmaniasis in Ethiopia: A Narrative Review Article
Background: Leishmaniasis is an obligate intercellular protozoon that affects animals and human. It has zoonosis and/or anthroponosis transmission. Human and veterinary medicine, environmental science and wildlife conservation specialists have many commonalities in case of visceral leishmaniasis. Still the above disciplines respond against leishmaniasis in a separate way. The aim of this review is to indicate inter- and intra- sectoral collaboration for planning future control strategies.
Methods: literatures written on visceral leishmaniasis and one health approach were systematically reviewed from the year 1969 to 2014 from Pub Med, Scopus, Medline and Google scholar sources.
Result: Such a one health approach would enhance biomedical progress; improve medical and veterinary serves, entomological control and wildlife conservation for Visceral Leishmaniasis especially in endemic areas.
Conclusion: Inter- and intra – sectoral collaboration in the leishmaniasis control is limited in Ethiopia. Therefore, incorporating one health approach or integrated inter- and intra – sectoral collaboration for visceral leishmaniasis control is an effective control strategy in endemic areas.
Background: Toxoplasmosis is a common parasitic disease. There is likelihood of exposure to Toxoplasma gondii in blood donors during the periods of life. Currently, laboratory screening of blood donors for T. gondii is not routinely available. The objectives of this review were to study the effects of T. gondii on blood safety and to approach for risk reduction in blood recipients.
Methods: A literature search was performed using Cochrane library, PubMed, Scopus, Google scholar IranMedex, SID and Magiran without time limitation. All studies, which had reported the prevalence of T. gondii in Iranian blood donors in both English and Farsi languages, were evaluated and reviewed. The contents of the transfusion medicine text books related to this issue were reviewed. Searching keywords were "Blood Donors" or "Blood Transfusion" and "Toxoplasma" or "Toxoplasmosis" and Iran.
Results: In order to study the prevalence of T. gondii in Iranian blood donors, six studies have been reviewed. IgG and IgM antibodies varied between 12.3% to 52.8% and 0% to 5.47%. Some of these studies have suggested to doing the screening for all blood donors. However, based on parasitological and epidemiological evidences, there is little chance for parasite transmission by blood transfusion.
Conclusion: By considering the moderate prevalence, difficulty in the differentiation between recent and past infections, and cost-effectiveness, it is not possible and rational to perform screening of donated blood. To reduce the risk of parasite transmission, leukofilteration method is recommended.
Evaluation of a New Primer In Comparison With Microscopy for the Detection of Giardia lamblia Infection in Stool Samples
Background: Among the most important parasitic disease, causing diarrhea, Giardia lamblia is noteworthy. Nowadays detection methods for these parasites include parasitological methods such as microscopic examination. The sensitivity of these methods relies on the expertise and experience of examiners. In contrast, molecular methods such as PCR are less dependent on the expertise of the examiner. Here we developed a PCR for the detection of G. lamblia genome in stool samples in comparison with microscopy, which is the gold standard.
Methods: For the evaluation of primers, 22 positive samples and 47 negative samples were used. QIAamp DNA Stool Mini Kit (QIAGEN, Germany) was used for DNA extraction from feces. Primers for PCR were designed using Primer-BLAST which uses Primer 3 to designing specific primers (NCBI/ Primer-BLAST).
Results: Sensitivity of the PCR was done with 100% (95%CI: 84.56-100) for the detection of G. lamblia DNA isolated from patients stool samples which were positive for G. lamblia cysts and/or trophozoites using microscopy as gold standard. In comparison with microscopy, PCR had showed the specificity of 97.87% (95%CI: 88.71-99.95).Conclusion: We designed new primers for the Giardia, and PCR method for the rapid and accurate identification of Giardia parasites established. With consideration to the routine diagnosis techniques in medical parasitology and their limitations such as time consuming, laborious, less sensitivity etc. This G. lamblia PCR is a sensitive and specific application for the diagnosis of G. lamblia and provides us a reliable method in the routine intestinal parasitic infection laboratory diagnosis.
Background: The purpose of this study was to perform seroepidemiological investigation for determining the status of human fasciolosis in Pirabad Village, Lorestan Province, western Iran. Methods: Blood samples were taken from residents of the village including 801 individuals. Sera were separated and stored at -20°C until used. The samples were analyzed using ELISA.
Results: Anti-Fasciola antibodies were detected in 6 (0.7%) individuals. Difference between age, sex and drinking or swimming in the surface water with seropositivity to fasciolosis was not significant. Out of 7 shepherds, 1 (14.3%) was seropositive. Due to the small number of shepherds, comprehensive statistical inference in this regard cannot be done. Significant difference was detected between seropositivity to fasciolosis and consuming local freshwater vegetables during the last 6 months (P=0.001).Conclusion: Metacercariae carrying local freshwater plants might be the main source of contamination because consumption of these kinds of vegetables was confirmed by all participants. Awareness of local communities regarding the danger of freshwater plant consumption, through health education programs, will decrease the risk of infection.
Background: Fasciolosis, caused by the liver flukes of the genus Fasciola, is one of the most prevalent diseases of domestic livestock and human throughout the world, imposing considerable economic losses. The present study was aimed to assess the effects of different pH values on hatching rate of Fasciola miracidia.
Methods: The flukes were isolated from the infected livers of the slaughtered ruminants at the abattoir of Urmia City, Iran, crushed thoroughly and sieved for isolation of the Fasciola eggs. The eggs were washed up several times by PBS (0.01N, pH 7.2). They were incubated at different pH values of 7±0.1 (control) and 3-9.5 (treatments) at 28°C for 16 days.
Results: The maximum hatching rate was observed at pH 7 (14.93±0.65%), while no miracidia were hatched at pH 3 and/or pH 9-9.5. There were significant differences between the hatching rate of the treatments and that of the control group.
Conclusion: Water pH is proven to be a crucial factor affecting the life cycle of Fasciola and its epidemiology.
Functionally Expression of Metalloproteinase in Taenia solium Metacestode and Its Evaluation for Serodiagnosis of Cysticercosis
Background: Parasite proteases have important roles in cleavage of host proteins during the invasion of host tissues and participate in the parasite’s evasion from the host’s immune response. The aim of the present study was to estimate a metalloproteinase properties of Taenia solium metacestode (TsMP) during host-parasite interactions, and evaluate its potential as a serodiagnostic antigen for cysticercosis.
Methods: The cDNA coding for the mature catalytic domain of TsMP was cloned into pGEX-6P-1 expression vector. A recombinant glutathione S-transferase and TsMP fusion protein was induced. After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed. Immunoblot assay was processed to evaluate its potential as a serodiagnostic antigen for cysticercosis.
Results: The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates. In immunoblot assay, 87.5% of sera from patients with cysticercosis showed strong reactivity. In sera from patients with other parasitic infections and from normal controls, it showed high specificity.Conclusions: TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle. A single recombinant TsMP antigen could have a potential value for serodiagnosis of cysticercosis.
Background: Metronidazole, a 5-nitroimidazole derivative, is the main antitrichomonal agent of choice for treatment of trichomoniasis. Since 1962, some cases of treatment failure with metronidazole have been reported and recently drug resistance is now on the rise in the world. This study was aimed to determine current susceptibility of Iranian isolates of Trichomonas vaginalis to metronidazole.
Methods: This study was performed on 50 T. vaginalis isolates collected from west and central areas of Iran. After axenisation of the parasites, susceptibility testing was carried out by using serial twofold dilutions of metronidazole (400 to 0.1 µg/ml). The minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC) of the trichomonads were determined after 48 h incubation at 35.5 °C. Drug susceptibility assays of the all isolates were carried out two times in triplicate under aerobic and anaerobic conditions.
Results: Ninety-eight percent of the T. vaginalis isolates (49/50) were sensitive to metronidazole. Metronidazole resistance was defined as aerobic MIC ≥50 µg/ml, detected in one isolate. The means of aerobic MICs and MLCs and that of anaerobic MICs of the parasites were 2.91, 1.95 and 0.28 µg/ml, respectively.Conclusion: This investigation showed in vitro low-level tolerance to metronidazole in a few T. vaginalis isolates that may be leading to the development of drug resistance.
Genetic Characterization of Fasciola Isolates from West Azerbaijan Province Iran Based on ITS1 and ITS2 Sequence of Ribosomal DNA
Background: Fascioliasis, caused by Fasciola hepatica and F. gigantica, has medical and economic importance in the world. Molecular approaches comparing traditional methods using for identification and characterization of Fasciola spp. are precise and reliable. The aims of current study were molecular characterization of Fasciola spp. in West Azerbaijan Province, Iran and then comparative analysis of them using GenBank sequences.
Methods: A total number of 580 isolates were collected from different hosts in five cities of West Azerbaijan Province, in 2014 from 90 slaughtered cattle (n=50) and sheep (n=40). After morphological identification and DNA extraction, designing specific primer were used to amplification of ITS1, 5.8s and ITS2 regions, 50 samples were conducted to sequence, randomly.
Result: Using morphometric characters 99.14% and 0.86% of isolates identified as F. hepatica and F. gigantica, respectively. PCR amplification of 1081 bp fragment and sequencing result showed 100% similarity with F. hepatica in ITS1 (428 bp), 5.8s (158 bp), and ITS2 (366 bp) regions. Sequence comparison among current study sequences and GenBank data showed 98% identity with 11 nucleotide mismatches. However, in phylogenetic tree F. hepatica sequences of West Azerbaijan Province, Iran, were in a close relationship with Iranian, Asian, and African isolates.Conclusions: Only F. hepatica species is distributed among sheep and cattle in West Azerbaijan Province Iran. However, 5 and 6 bp variation in ITS1 and ITS2 regions, respectively, is not enough to separate of Fasciola spp. Therefore, more studies are essential for designing new molecular markers to correct species identification.
Identification of Leishmania Species Isolated from Human Cutaneous Leishmaniasis in Mehran, Western Iran Using Nested PCR
Background: The incidence of cutaneous leishmaniasis in the city of Mehran has risen sharply in recent years because the city borders Iraq, which has allowed entrance of different Leishmania strains. These strains have different shapes, periods of disease, and healing of lesions. The present study identified and determined cutaneous leishmaniasis species in this region.
Methods: This cross-sectional study was carried out by preparing slides from 92 patients with suspected cutaneous leishmaniasis lesions from Mehran during 2012-2013. Parasite genomic DNA was extracted and CSB2XF and CSB1XR primers were used to amplify the Leishmania minicircle kDNA regions. The parasite species were detected by specific 13Z and LIR primers by applying nested PCR technique.
Results: All banding patterns were diagnosed as L. major parasite by comparison of standard models with amplified fragments 560 bp in length from bands. The patients were 56.5% male and 43.5% female. The most frequently-infected age group was the 21-30 years group at a rate of 27.2%. About 56.3% of patients had a single lesion and a significant correlation was observed between age and number of lesions (P > 0.05).Conclusion: The nested PCR technique was shown to be an effective method with high sensitivity and specificity for identification of human Leishmania parasites. Molecular analysis revealed that parasites isolated from Mehran were identified as L. major and the disease was rural in form.
Molecular Analysis of Sarcocystis Spp. Isolated from Sheep (Ovis aries) in Babol Area, Mazandaran Province, Northern Iran
Background: To differentiate Sarcocystis macro-cyst-forming species in slaughtered sheep in Babol area, Mazandaran Province, sequence analysis of 18S rRNA gene was performed.
Methods: Overall, 150 slaughtered sheep were examined macroscopically in slaughterhouse, Babol and intra-abdominal and diaphragm muscles tissues infected with macro-cyst of Sarcocystis spp. were collected in 2013. One macro-cyst was isolated from the infected muscles of each sheep. The partial 18S rRNA gene was amplified by PCR and sequenced afterward.Results: The rate of infection with macro-cyst producing Sarcocystis spp. was 33.3% (50 / 150). The partial 18S rRNA gene of Sarcocystis species was amplified at the expected PCR product size (~1100 bp) from all 50 macroscopic cysts samples. From 30 sequences DNA samples, 20 samples (66.7%), six (20%) and four (13.3%) isolates were identified as S. gigantea, S. moulei and Sarcocystis spp., respectively. Eight and thirty-four variations in nucleotide position were seen in partial sequence of the18S rRNA gene of S. gigantea and S. moulei.Conclusion: Sheep can be considered as an alternative intermediate host for S. moulei. Furthermore, multiple alignments showed some variations in the consensus sequences of the isolates obtained in the current study compared with previously published isolates. To understand better the genetic diversity among Sarcocystis species complete sequences of the18S rRNA gene or sequence analysis of other genetic loci would be beneficial.
Background: This study was performed to induce conversion of RH strain tachyzoites of Toxoplasma gondii to bradyzoites by pH changing of the culture medium.
Methods: HeLa cell monolayers were infected at a 1:1 tachyzoite to cell ratio. Four hours after infection, the culture medium was removed and replaced with culture medium and 5% FCS, adjusted to pH 8 with NaOH. The culture was maintained at 37 °C without CO2 until the end of the experiment. Cyst-like structures were collected and stained with periodic acid schiff (PAS) staining method. The soluble antigens of cyst-like structures of RH strain, in addition to RH tachyzoite, bradyzoites of avirulent Tehran strain and uninfected HeLa cells were electrophoresed on 12.5% polyacrylamide gel. The gel was stained by coomassie brilliant blue R-250.
Results: Four days after infection of HeLa cells with tachyzoites of T. gondii, RH strain, cyst- like structures were noticed and stained with PAS. In the SDS-PAGE, protein bands of these structures had some differences with tachyzoites of RH strain, but there was quite similarity between protein bands of these structures and tissue cysts (bradyzoites) of Tehran strains. P34 and P36 (bradyzoite-specific proteins) were observed only in T. gondii bradyzoites of RH (cyst like structures) and bradyzoites of Tehran strains.
Conclusion: Alkalization of culture medium to pH 8 induced expression of bradyzoite- specific proteins and production of RH cysts in cell culture.
Microscopic and Molecular detection of Theileria (Babesia) equi infection in equids of Kurdistan Province, Iran
Background: Equine piroplasmosis (EP) is the cause of persistent tick-borne infection with no symptoms, but the most important problem of EP is due to the persistent carrier state. Carrier animals to Babesia (Theileria) equi (Laveran 1901) and B. caballi (Nuttall, 1910) infestation could be identified by extremely sensitive PCR-based method. The purpose of this study was to identify the causative agents of equine piroplasmosis based on molecular and microscopic assays in equids from Kurdistan Province, Iran.
Methods: Thirty one horse and mule blood samples were used with history of living in Kurdistan Province of Iran. The blood specimens were utilized for T. equi and B. caballi DNA identification by PCR and Giemsa stained smears for microscopic observation.
Results: The results clearly showed the presence of B. (Theileria) equi DNA in 30 of 31 blood samples (96.77%), but the microscopic examination revealed the 3 of 31 positive Babesia like organisms in the red blood cells (9.67%).Conclusion: The obtained results demonstrated the presence of hidden B. (Theileria) equi infection in horses with previous habitance in Kurdistan Province of Iran. The carrier animals became a main source of infection and can transmit the disease. Therefore, hidden infection might be considered as a health threatening and limiting factor in animals used in therapeutic antisera research and production centers.
Background: Myiasis is defined as the infestation of live human and vertebrate animals with dipterous larvae for a certain period. There are reports indicating that dogs are the most common species affected by myiasis. This study was conducted to identify myiasis-causing flies in owned and stray dogs in Mashhad (Northeastern Iran).
Methods: A total of 435 owned dogs and 800 stray dogs were examined for myiasis. Myiasis cases were cured and fly larvae were identified by microscopy using the relevant standard identification keys.
Results: Ten out of 435 owned dogs (2.29 %) and 18 out of 800 stray dogs (2.25 %) had myiasis. The causative agents of myiasis in dogs based on their frequencies were as follows: Wohlfahrtia magnifica (50%), Lucilia sericata (28.57%) and Chrysomya albiceps (21.42%).
Conclusion: W. magniﬁca was the most important myiasis-causing ﬂy among the dogs sampled here, sometimes causing very serious damages. However, when treatment was given early enough, the larvae removed and the wound disinfected, the animals usually made a full recovery.
Genetic Diversity of Dihydropteroate synthetase Gene (dhps) Of Plasmodium vivax in Hormozgan Province, Iran
Background: The present study was formulated in order to determine polymorphism of dihydropteroate synthetase gene (dhps) of Plasmodium vivax (P. vivax) in Hormozgan Province, southern Iran and mutations at codons 382, 383, 512, 553, and 585 associated with resistance of P. vivax to sulfadoxine.
Method: One-hundred eighteen isolates of P. vivax were prepared within 2007-2008 to determine dihydrofolate reductase-thymidylate synthase (dhfr-ts) gene. The isolates were determined in the study of genetic diversity of dihydropteroate synthetase gene (dhps) of P. vivax. The study was performed via PCR test and nucleotide sequencing.
Results: Of 118 blood samples infected by P. vivax, 46 and 72 samples belonged to Minab and Jask, respectively. No mutation was detected at 5 target codons. However, among these 118 samples, three isolates (2.54%) were found to have a mutation at the new codon 421.
Conclusion: Since mutation was detected in dihydrofolate reductase (Pvdhfr) gene in the same samples but no mutation was found at five main codons of Pvdhps gene, it can be concluded that P. vivax, considering their mutations in Pvdhfr, is still susceptible to sulfadoxine and therefore, to fansidar in Hormozgan Province, Southern Iran.
Background: Toxoplasmosis is considered as one of the most common infectious diseases caused by the protozoan parasite Toxoplasma gondii. Tachyzoite is the main form of Toxoplasma and continuously is maintained in cell culture or injected into the mice peritoneal cavity. This study was designed to evaluate the survival rate of RH strain of T. gondii tachyzoites in different cell free, nutrient and biological media at different temperatures.
Methods: This experimental study was performed at the Toxoplasmosis Research Center, Mazandaran University of Medical Sciences, Sari, Iran, in 2010. One ml of each solution including hypotonic saline (0.3%), normal saline (0.85%), RPMI-1640 (RPMI), RPMI with 10% fetal bovine serum (FBS), RPMI with 20% FBS, ovine hydatid cyst fluid, pasteurized milk of cow, and phosphate buffered saline (PBS) along with 4×104 T. gondii tachyzoites were added to plate wells and incubated in 4 °C, 22 °C, 37 °C, and 37 °C under 5% CO2. The survival rate and viability assessment of parasites were performed daily and the results were analyzed using Univariate tests.
Result: Tachyzoites survival rate in PBS (4 °C) and normal saline (4 °C) were considerably high, compared to other solutions in different conditions (P<0.001). The best temperature for Toxoplasma maintenance was 4 °C (P<0.001).
Conclusion: This study introduces two available and economical solutions, PBS (4 °C) and normal saline (4 °C) media, for maintenance of Toxoplasma tachyzoites as appropriate choice media for a noticeable period of time (11 days) in vitro.
Background: Schistosomiasis is one of the major communicable diseases of public health and socioeconomic importance in developing countries. This study assessed the situation of schistosomiasis among villagers of the New Halfa Agricultural Scheme, Sudan.
Methods: An epidemiological survey was carried out in three randomly selected residential sites: Village 19, Village 26 and Talat shagrat Camp, from October to December 2013. Feces and urine samples were collected from 2433 individual (1195 male and 1238 female) and examined for schistosomiasis infection. The prevalence and intensity of infection were calculated according to study sites and participants’ sex and age-group.
Results: There was no infection with Schistosoma haematobium among the examined individuals, while the overall prevalence of S. mansoni infection was 27.4% and the mean intensity among those infected was 261.1 eggs per gram (epg). A high prevalence and intensity of infection was found among the residents of Talat shagrat Camp, followed by the other two villages. The prevalence of infection among males was 41.4%, and among females was 13.9%. On the other hand, the intensity of infection among females was 293.4 epg and among males 187.6 epg. A high prevalence of infection was found in the age-groups 11-20 years and > 50 years. High intensity of infection was present in the age-groups 31-40 years and > 50 years.Conclusion: The finding of the study shows the need for an integrated control program against schistosomiasis. Mass treatment, provision of adequate clean-water supply and combating the intermediate snail host are suggested.
Malaria is a parasitic disease that is starting to be encountered in intensive care units (ICU) worldwide, owing to increasing globalisation. Severe malaria caused by Plasmodium falciparum, is characterised by cerebral malaria, acute renal failure, hypoglycaemia, severe anaemia, splenomegaly and alveolar oedema. We present the case of a 25-yr old male patient who presented to the Emergency Department of Uludag University in Bursa, Turkey in the winter of 2014 with complaints of fever for three days. His medical history revealed a 14-month stay in Tanzania. Staining of blood smears revealed characteristic gametocytes in accordance with P. falciparum infection. The day after admission, he had an epileptic seizure after which his Glasgow Coma Scale was 6, so he was intubated and transferred to the ICU. A computerized tomography scan revealed findings of cerebral oedema. Intravenous mannitol was administered for 6 days. Intravenous artemisinin was continued for 10 days. Due to refractory fevers, anti-malarial treatment was switched to quinine and doxycycline on the 14th day and on the 16th day the fevers ceased. This case emphasizes that cerebral malaria should be suspected in cases of seizures accompanying malaria, and treatment should be initiated in the ICU. Furthermore, resistance of P. falciparum to artemisinin should be in mind when a response to therapy is lacking.
Cystoisospora belli, formerly Isospora belli, as an opportunistic infection agent, is seen in immunocompromised patients like HTLV-1. We describe here cystoisosporiasis in an HTLV1 Iranian female in Mashhad, northwestern Iran in 2012 who presented with a debilitating diarrheal illness and great weight loss. C. belli was detected in her stool by modified acid-fast staining and then by molecular detection. Serologic testing was negative for HIV but she showed positivity for HTLV-1 infection. Treatment with TMP/SMX led to improvement of her diarrhea but she died after one year due to malabsorption syndrome. Adequate detection of C. belli diarrhea in immunocompromise patients of HTLV1 in endemic area can be cured by TMP/SMX.
Molecular Evaluation of a Case of Visceral Leishmaniasis Due to Leishmania tropica in southwestern Iran
We describe a case of visceral leishmaniasis (VL) due to Leishmania tropica in a 50-year-old Iranian man lived in a VL-endemic area in southwest of Iran. The patient presented with a 3-month history of fever and splenomegaly. Clinical signs and serological findings were suggestive of VL. Spleen biopsy was taken from the patient and intracellular forms of Leishmania amastigotes was seen in Giemsa stained smears. The patient was treated with pentavalent antimonial compound with complete resolution of his systemic signs and symptoms. DNA was extracted from the microscopic slides of the spleen biopsy and the nagt (N-Acetylglucosamine-1-Phosphate Transferase) gene of Leishmania was PCR-amplified. Sequence analysis of the PCR product demonstrated that the case has 99% identity with those of available sequences of L. tropica. Intra-species variation within isolate was 0-0.1%; whereas, inter-species differences of the isolate with those of L. major and L. infantum was significantly higher.
Dioctophyma renale infection is found in a wide range of mammalian species, typically in temperate areas of the world. Here, we report for the first time, the parasitism of a domestic dog by D. renale in Hamedan, Iran, a mountainous cold region, lacking significant amounts of rainfall, high humidity and temperature. A 2.5 yr old male mixed breed dog was presented with a two months history of progressive hematuria and muscle weakness. Complete blood count and serum biochemistry were performed with results indicating impaired renal function. Urinalysis, showed hematuria as well as parasitic eggs, suggestive of D. renale infection. Urinary system ultrasonography revealed a hypoecogenic tubular structure in the right kidney. The animal was treated with fenbendazole (45 mg/kg, PO, QD - five days) and ivermectin (0.02 mg/kg, SC, single dose). One week later, repeated laboratory examination confirmed presence of at least one alive worm in the affected kidney. A unilateral nephrectomy was performed; one female (60 x 5 cm) and one male (30 x 3.8 cm) live worm were taken out of the extremely thin walled right kidney. One month later, due to failure of the remained kidney and poor condition, the patient deceased. We conclude that dioctophymosis can be found in cold and or relatively dry area. Moreover, the results showed that the worm was not affected with common anthelmintic drugs.