EFECTO CLÍNICO E INTERVENCIÓN

*1- Museum of Southwestern Biology, Center for Evolutionary and Theoretical Immunology, Department of Biology, University of

New Mexico, Albuquerque, NM 87131, USA

Cercarial dermatitis is an allergic reaction caused by both mammalian and avian larval schistosomes after they penetrate the skin of a mammal or bird. Humans can contract cercarial dermatitis around the world when working or recreating in waters that have both the appropriate snail host and the bird or mammal host. While avian schistosomes are responsible for a majority of cases of dermatitis, in some areas of the world, mammalian schistosomes are most prevalent. Recent- ly, cercarial dermatitis was documented as an occupational hazard in many parts of the world, particularly where rice is grown. It was in 1928 in the United States when the causative agent of cercarial dermatitis was first described. Since that time, cercarial dermatitis has been found to occur around the world, except in Antarctica. Furthermore, the last decade has revealed a diversity of avian schistosomes and snails that have been crucial to our understanding of the current and future way in which we can understand the epidemiology of cercari- al dermatitis. The genus Trichobilharzia has achieved notoriety as the primary etiological agent for outbreaks of cercarial dermatitis around the world, especially in the temperate lati- tudes. The genus Trichobilharzia includes about 35 species that use mainly ducks as a definitive host and snails in the families Lymnaeidae and Physidae. This genus is the most speciose and widespread of the family Schistosomatidae. The focus of this presentation will be on discussing the diversity of Tricho- bilharzia, and possible mechanisms that lead to this diversity and distribution of cercarial dermatitis. It is likely that host ecology and distribution are both important in determining the global distribution and persistence of species of Trichobil- harzia and occurrence of cercarial dermatitis.

Keywords: cercarial dermatitis, Trichobilharzia, diversity, schistosomes

HEARTWORM (DIROFILARIA IMMITIS) OF DOGS – A STUDY IN CREATING RESISTANT ISOLATES

Dwight D. Bowman, MS, PhD, DACVM-Parasitology (Honorary)*

*Professor, College of Veterinary Medicine, Cornell University, [email protected]

Parasitologists are well aware that helminths become resistant to pharmacologic agents that are used in their treatment and preven- tion. In the world of veterinary parasitology, the most recent event that was a surprise to many was the development in many parts of the world of resistance of the equine roundworm, Parascaris equorum, to macrocyclic lactones. The concern has long been that similar macrolide resistance might occur with the filarioid nematodes of humans, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi, before the completion of the international eradication program – and fortunately – it has remained forestalled. Unfortunately, there have now been recovered from the field a number of isolates of

Dirofilaria immitis that are capable of infecting and developing in dogs being prescribed doses of the macrocyclic lactones that were once fully protective.This work has involved a number of different laboratories and teams in North America and Europe. The problem was first noticed in the field by US veterinary practitioners and then was seen by the FDA and pharmaceutical companies as they tried to register new products that were not successful at protecting dogs against recent field isolates of the parasites.The work has involved the collection of these isolates, verification that dogs are not pro- tected against these isolates in trials where they have been chal- lenged with isolates when on prevention, and the molecular charac- terization of markers for their identification. There have now been tests where all the current heartworm products on the market in the United States have failed to protect dogs against challenge with these resistant strains of heartworm. These isolates have been col- lected in different cities from the southeastern and central United States. New products have had trouble gaining approval in the Unit- ed States, as they are required to be tested against new field isolates, and they have failed with molecules that were once fully protective. Molecular markers have now been identified that seem capable of identifying these isolates, and there is high probability that these or similar markers will prove useful in monitoring human filarioids for potential genetic changes as related to macrocyclic lactone resistance such that alternative control methods might be strategically applied in the field if such molecular markers are identified in a given area. The prevention of canine heartworm utilized a different method of control than that utilized in human filariasis. This was a logical choice because in dogs, the adult worms, not microfilariae, are the cause of almost all pathology, while especially in onchocerciasis, the majority of disease is caused by microfilariae, not by the adult form of the parasite.In the case of D. immitis, dogs have been protected against infection with adult heartworms through the administration of the product at low doses to inhibit the development of incoming larvae, whereas, in human filariasis, the goal has been to suppress microfilaremias to prevent transmission by flies biting an infected person rather than to prevent individuals from becoming infected after the bite of an infected fly containing third-stage larvae.Now that it has been shown that filarioid nematodes, in spite of the long life cycles and vector transmission, can develop resistance, it is criti- cal that those working in human filariasis understand the potential risk posed to the world‟s control of these diseases and begin to implement increased monitoring to allow intervention if resistance appears with the human filaria in a given area.

ANTIBODY TEST OF TOXOCARIASIS AMONG HEALTHCARE EXAMINEES IN KOREA Yan J, HS Kim, JH Kim, CH Yoon, EH Hwang, H

Kang, SY An, KJ Kim, ST Hong*

*Professor, Department of Parasitology and Tropical Medicine, Institute of Endemic Diseases, Seoul National University College of

Medicine Seoul 110-799, Korea.

Korea Association of Health Promotion Seoul, Korea.

Toxocara canis is an intestinal nematode affecting dogs, which causes human infection by ingestion of embryonated eggs or larvae. Although the larvae do not develop into adult worms in the human body, they may survive for several years and migrate to various organs, presenting commonly as covert (adult) or common (child) toxocariasis, and visceral larva migrans and less frequently ocular larva migrans. Toxocariasis is inducing eosinophilia in most symptomless cases but pre- vious data are widely fluctuating. In the present study, we investigated toxocariasis ELISA subjecting healthcare exam- ines of the Korea Association of Health Promotion in Seoul and Gyeongsangnam-do. Total 610 adults (310 from Seoul, 300 from Gyeongsangnam-do, 397 men and 213 women, age 22-80 with mean 53.1±12.2 years) were subjected for multi- antigen ELISA including crude antigen of Toxocara canis lar- vae (TCLA). In total, seropositivity of toxocariasis by ELISA was 8.7% (53/610). The rates in Seoul and Gyeongsangnam- do were 6.1% (19/310) and 11.3% (34/300), respectively. Especially, according to the regional analysis, the positive rate in Gyeongsangnam-do (16.4%) was significantly higher (p<0.001) than in Seoul (6.9%) in eosinophilia group. Moreo- ver, it has been confirmed that eosinophil count from pe- ripheral blood is moderately correlated with absorbance of ELISA with TCLA. Immunoblot analysis on TCLA recog- nized antigenic proteins of 28-, 30-, 46-, 56- and 70-kDa bands in their reactivity. Taken together, the prevalence of toxocariasis is significantly high among residents with eosin- ophilia in Gyeongsangnam-do.It is recommended to screen toxocariasis for symptomless eosinophilia subjects in rural areas.

FAMILIAL TRICHOSTRONGYLIASIS OUTBREAK IN GUILAN PROVINCE, NORTHERN IRAN, MIS-

DIAGNOSED WITH ACUTE FASCIOLIASIS Keyhan Ashrafi*1_{, Ali Tahbaz}2_{, Meysam Sharifdini}3_,

Santiago Mas-Coma4

* 1- Ph.D, Department of Microbiology, School of Medicine, Gui- lan University of Medical Sciences, Rasht, Iran.

[email protected]

2- MD, Department of Infectious Diseases, Rasoul-Akram Hospital, Rasht, Iran.

3- Ph.D, Department of Microbiology, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran.

4- Ph.D, Departamento de Parasitología, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andrés Estellés s/n, 46100

Burjassot, Valencia, Spain.

The suitable climatic conditions and high amounts of rainfall, ac- companied with traditional herding, expose the livestock to parasitic diseases in Guilan province. These livestock then play a critical role in transmitting parasitic diseases to humans through infecting the environment by their fecal materials, containing ova and larvae. Guilan province has been recognised as the focus of human fascio- liasis in Iran and the whole Asia, because of the two largest out- breaks in the world, affecting more than 15000 individuals. Local physicians consider hypereosinophilia as a marker of fascioliasis, especially when accompanied with abdominal pain, fever and aller- gic manifestations. So, it is frequently confused with other parasitic diseases prevalent in the area. We are reporting an interesting case of such misdiagnosis in one family. Three sisters of the same family, aged 33, 35 and 38 years old, presented at the same time for medical care with a three-week history of abdominal and epigastric pain radiating to their back, mild to severe diarrhoea, urticaria on their hands, chest and back associated with itching, poor appetite, weak- ness and weight loss. Blood tests revealed hypereosinophilia of 20, 56 and 60%, while anti-Fasciola and anti-Strongyloides antibodies were both negative. The patients reported regular consumption of fresh vegetables, obtained from their home garden fertilized with fresh sheep manure. Three stool samples from each person were examined using formalin-ether and Kato-Katz techniques under a liver-free diet, and were negative for any ova, cyst and larvae. The patients were diagnosed as having acute fascioliasis based on clinical and epidemiological evidence and received 10 mg/kg of Egaten. One month later, they were readmitted without any sign of recov- ery. Three new stool samples were examined from each patient, under the same conditions and by the same specialist, which surpris- ingly were positive for Trichostrongylus ova. The patients were treated by combination of the usual doses of mebendazole and albendazole and their fecal materials were collected for 24 hours after therapy. Two species of Trichostrongylus, (T. colubriformis and T. vitrinus) were identified by morphological and molecular techniques. All other three family members, who were subsequently analyzed, also shed

Trichostrongylus eggs.Fascioliasis and trichostrongyliasis share many epidemiological and clinical characteristics, and are both prevalent in climates and living conditions similar to those of Guilan Province. This case highlights the need to consider trichostrongyliasis in the differential diagnosis of fascioliasis patients, particularly in the acute phase when the eggs are not present in stool, and whenever there is a history of eating fresh vegetables fertilized with fresh livestock manure, or close contact with herbivorous animals.

Keywords: trichostrongyliasis, Trichostrongylus colubriformis, T. vitrinus, familial outbreak, Iran

GENETIC VARIATION IN THE SEQUENCES OF THE INTERNAL TRANSCRIBED SPACERS (ITS) OF NUCLEAR RIBOSOMAL DNA (RDNA) AMONG AND WITHIN TOXOCARA NEMATODE OF DOGS AND CATS FROM DIFFERENT AREAS IN IRAN Fattaneh Mikaeili1_{, Hossein Mirhendi}2_{,Mehdi Mohe-}

bali3_{, Mostafa Hosseini}2_{, Zabiholah Zarei}4_{,Zahra Hei-}

dari4_{,Eshrat Beigom Kia}2

* 1-Assistant Professor, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

2-Professor, School of Medicine, Shiraz University of Medical Sci- ences, Shiraz, Iran.

3- Professor, Center for Research of Endemic Parasites of Iran (CREPI), Tehran University of Medical Sciences,

Tehran, Iran.

4-Studnt, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Toxocara canis and Toxocara cati are ascaridoid nematodes of dogs and cats. The objectives of the present study were to identify the sequence variation of the internal transcribed spacer (ITS) region within and among isolates of T. canis and

T. cati from dogs and cats in Iran. Genomic DNA was ex- tracted from 23 isolates of T. canis and 35 isolates of T. cati

collected from dogs and cats in different geographical areas of Iran. ITS region in nuclear ribosomal DNA was PCR- amplified. The PCR product of 5 isolates of T. canis and 9 isolates of T. cati were sequenced. The sequences were aligned using the BioEdit software and compared with pub- lished sequences in GenBank. Phylogenetic analysis was per- formed using Mega 5.0 software and Maximum likelihood method. The amplicons of about 1000 and 1100 bp were successfully produced for T. canis and T. cati, respectively. Pairwise comparison of the sequences did not show any dif- ferences in nucleotide sequences within T. canis isolates. Likewise, sequences of T. cati were identical and exhibiting 100% homology. However, 12% inter-species sequence dif- ference was found between the isolates of T. canis and T. cati.Regarding to no intra-species variation and a significant inter-species variation in ITS region among T. canis and T. cati

isolates, it is concluded that the region can be used for identi- fication and differentiation of Toxocara species.

Keywords: genetic variation, Toxocara canis, Toxocara cati, ITS,

EVALUATION OF NESTED-PCR AND REAL-TIME PCR METHODS FOR DETECTION OF STRONGY-

LOIDES STERCORALIS IN STOOL SAMPLES COMPARED TO CONVENTIONAL PARASITO-

LOGICAL METHODS

Meysam Sharifdini*1_{, Hossein Mirhendi}2_{, Keyhan Ash-}

rafi3_{, Mostafa Hosseini}4_{,Mehdi Mohebali}5_{, Hossein}

Khodadadi6_{,Eshrat Beigom Kia}7

* 1- Assistant Professor, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. [email protected]

2- Professor, School of Public Health, Tehran University of Medical Scienc- es, Tehran, Iran.

3- Associate Professor, Department ofMicrobiology, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran4- Professor, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. 5- Professor, School of Public Health, Tehran University of Medical Scienc-

es, Tehran, Iran.

6- Assistant Professor, School of Public Health, Tehran University of Medi- cal Sciences, Tehran, Iran.

7- Professor, School of Public Health, Tehran University of Medical Scienc- es, Tehran, Iran.

Strongyloidiasis or parasitic infection caused by intestinal nematode

Strongyloides stercoralis may lead to hyperinfection syndrome and dis- seminated infections. Without early diagnosis and proper treatment, it may even results into death of the patients. To prevent such con- sequences, incorporation of highly sensitive diagnostic methods is necessary. Therefore, the present study was performed to evaluate Nested-PCR and Real-time PCR methods for detection of S. ster- coralis in stool samples compared to conventional parasitological methods. In order to evaluate Nested-PCR and Real-time PCR methods in field condition, 466 fresh stool samples were collected from endemic areas in Iran including Guilan, Mazandaran, Khouzestan and Hormozgan Provinces and also, from patients referred to the Helminthological Laboratory of School of Public Health, Tehran University of Medical Sciences. All these samples were examined by conventional parasitological techniques (forma- lin-ether concentration and agar plate culture). From all stool sam- ples, DNA was extracted, using modified in-house (IH) method and amplified by Nested-PCR and Real-time PCR methods. Finally, results of parasitological and molecular methods were compared and validation parameters were determined. Using Nested-PCR method, from the total of 466 stool samples, 117 samples (25.1%) were de- tected as positive for S. stercoralis; among those 32 samples (7.17%) had not been detected by either formalin ether concentration or agar plate culture techniques. Considering the conventional parasitologi- cal techniques as diagnostic gold standard, sensitivity and specificity of Nested-PCR method were 100% and 91.6%, respectively. Real- time PCR method detected 88 samples (18.9%) positive for S. ster- coralis infection, among which16 samples (4.2%) had not been de- tected by either formalin ether concentration or agar plate culture techniques. In this study, 29 samples (6.22%) that were positive using Nested-PCR method could not be detected by Real-time PCR. Sensitivity and specificity of Real-time PCR method were 84.7% and 95.8%, respectively. With increasing parasitic load, CT (threshold cycle) value decreased and there was a statistically significant correla- tion between parasitic load and CT value (P<0.0001).Molecular evaluation techniques, especiallyNested-PCR with more sensetivity are more reliable for detection of Strongyloides stercoralis in stool sam- ples than conventional parasitological methods.

PRODUCTION OF TOXOCARA CATI TES120 RE- COMBINANT PROTEIN AND EVALUATION OF ITS POTENTIAL FOR SERODIAGNOSIS OF TOX-

OCARIASIS

Farzaneh Zahabiun1_{, Seyed Mahmoud Sadjjadi}2_{, Mu-}

hammad Hafiznur Yunus3_{, Anizah Rahumatullah}3_{, Mo-}

hammad Hosein Falaki Moghaddam4_{, Syazwan Saidin}3_,

Rahmah Noordin5

* 1- MSc., Institute for Research in Molecular Medicine (IN- FORMM), University Sains Malaysia, 11800 Penang, Malaysia – Parasitology and Mycology , School of Medicine, Shiraz University

of Medical Sciences, Shiraz, Iran. [email protected] 2- Ph.D, Department of Parasitology and Mycology , School of

Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. 3- MSc., Institute for Research in Molecular Medicine (IN- FORMM), Universiti Sains Malaysia, 11800 Penang, Malaysia. 4- MSc., Department of Medical Parasitology and Mycology, School

of Public Health & National Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran.

5- Ph.D, Institute for Research in Molecular Medicine (IN- FORMM), Universiti Sains Malaysia, 11800 Penang, Malaysia.

Human toxocariasis is a cosmopolitan parasitic zoonotic dis- ease caused by the infective larvae of Toxocara canis or T. cati. Diagnosis is mainly based on antibody detection by specific antigens. Although T. canis has been deemed as the causative agent of most toxocariasis cases, the role of T. cati as the etio- logic agent should not be overlooked. Thus, the present study was aimed to produce a T. cati recombinant protein (T. cati rTES120) and evaluate its serodiagnostic potential. The DNA sequence encoding TES120 of T. cati was prepared by performing RT-PCR on RNA isolated from T. cati, using primers previously used to clone T. canis TES120. After de- ducing the T. cati sequence, the E. coli codon optimized se- quence was custom-cloned into glutathione S-transferase (GST)-tagged pGEX-4T-1 expression vector (GE Healthcare, USA), then transformed into E. coli BL21 (DE3) host cell. The expressed recombinant protein was affinity- purified using GST-BindTM resin (Novagen, USA). The diagnostic potential of the T. cati rTES120 protein was de- termined using IgG4 Western blot. The DNA sequence en- coding T. cati rTES120 protein was successfully amplified and the corresponding recombinant protein prepared. The IgG4 Western blot showed the diagnostic sensitivity and specificity of T. cati rTES120 for detection of toxocariasis to be 70% and 100%, respectively.An IgG4 assay using rTES120 T. cati

showed good diagnostic potential to detect toxocariasis. A combination of rTES120 from T. cati and T. canis sources would be expected to further increase the sensitivity of the test to detect toxocariasis, for patient diagnosis or epidemio- logical studies.

Keywords: Toxocara cati, TES120 recombinant antigen (rTES120), serodiagnosis, IgG4 western blot, toxocariasis

STRONGYLOIDIASIS PRESENTING AS ULCERA- TIVE COLITIS; A CASE REPORT

Fariborz Mansour-Ghanaei1_{*, Malahat Khalili}2

*1- Professor, Gastrointestinal & Liver Diseases Research Center, Guilan University of Medical Sciences, Rasht, Iran 2-MSc., Gastrointestinal & Liver Diseases Research Center, Guilan

University of Medical Sciences, Rasht, Iran

Strongyloidiasis is a disease caused by the soil transmitted nematode, Strongyloides stercoralis, commonly distributed in tropical and subtropical areas of the world. This disease mainly affects the intestines and the symptoms sometimes mimics inflammatory bowel disease. Due to the northern