Maria Vang Johansen

Diagnostic dilemmas in helminthology: what tools to use and when?

Available data regarding the distribution, prevalence and severity of various diseases are based on the performance and operational characteristics of the diagnostic techniques applied; this is a fact that is particularly apparent in the study of helminth infections. An important lesson learnt from the efforts to rein in dracunculiasis, lymphatic filariasis and schistosomiasis is that the diagnostic approach needs to be changed as further progress is made towards control and ultimate elimination of the disease. This insight prompted the opinion piece presented here, which highlights diagnostic dilemmas in helminthology related to the stage of control achieved and sets out some research needs.

Improvement of PCR for Detection of Opisthorchis viverrini DNA in Human Stool Samples

ABSTRACT Opisthorchis viverrini is an important food-borne trematode in Southeast Asia. The infection causes significant morbidity in terms of hepatobiliary diseases and cholangiocarcinoma. The aim of this study was to improve the sensitivity of the PCR-based diagnosis of O. viverrini infection. A new fecal DNA extraction protocol for the detection of O. viverrini DNA using cetyltrimethyl-ammoniumbromide to remove PCR inhibitor was used and compared with the commercial stool kit method. The sensitivity of the new test was 79.3%, compared with the 44.8% of the previous method (P < 0.01). PCR-positive tests identified several cases judged parasite negative by the parasitological method (28.6%), indicating the new tests advantage in the diagnosis of individuals with light infections.

Prevalence and Risk Factors of Porcine Cysticercosis in Angónia District, Mozambique

Taenia solium is an important zoonosis in many developing countries. Cysticercosis poses a serious public health risk and incurs sizeable economic losses to pig production. Because data on the epidemiology of porcine cysticercosis in Mozambique are scarce, the present study was conducted to determine the prevalence and risk factors for porcine cysticercosis. A cross-sectional survey was carried out in 11 villages in Angónia district, Tete province in northwestern Mozambique. Between September and November, 2007, a total of 661 pigs were tested serologically and examined by tongue inspection. Serum samples were tested for the presence of circulating parasite antigen using a monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (Ag-ELISA). In addition, a questionnaire survey to collect information on pig production, occurrence and transmission of porcine cysticercosis, risk factors and awareness of porcine cysticercosis was conducted in the selected households from which pigs were sampled. Two hundred thirty-one samples (34.9%) were found positive by the Ag-ELISA, while by tongue inspection on the same animals cysticerci were detected in 84 pigs (12.7%). Increasing age (OR = 1.63; 95% CI = 1.13–2.37) and free-range pig husbandry system (OR = 3.81; 95% CI = 2.08–7.06) were important risk factors for porcine cysticercosis in the district. The present findings indicate that porcine cysticercosis is endemic in the region, and that increasing pig age and pig husbandry practices contribute significantly to porcine cysticercosis transmission. Further epidemiological studies on the prevalence and transmission of porcine cysticercosis in rural communities in Mozambique are needed to enable collection of more baseline data and implementation of effective control strategies within the country.

Animal Reservoir Hosts and Fish-borne Zoonotic Trematode Infections on Fish Farms, Vietnam

Cats, dogs, and pigs may be reservoir hosts.

Prevalence and Risk Factors Associated with Human Taenia Solium Infections in Mbozi District, Mbeya Region, Tanzania

Background Taenia solium cysticercosis/taeniosis is emerging as a serious public health and economic problem in many developing countries. This study was conducted to determine prevalence and risk factors of human T. solium infections in Mbeya Region, Tanzania. Methods and Findings A cross-sectional survey was conducted in 13 villages of Mbozi district in 2009. Sera of 830 people (mean 37.9±11.3 years (SD); 43% females) were tested for circulating cysticerci antigen (Ag-ELISA) and antibody (Ab-ELISA). A subset of persons found seropositive by Ag-ELISA underwent computed tomography (CT) scan of the brain for evidence of neurocysticercosis. Stool samples from 820 of the same participants were tested for taeniosis by copro-antigens (copro-Ag-ELISA) and formol-ether concentration technique. Cases of T. solium taeniosis were confirmed serologically by EITB assay (rES38). A questionnaire was used for identification of risk factors. Active cysticercosis by positive Ag-ELISA was found in 139 (16.7%) persons while anti-cysticercal antibodies were detected in 376 (45.3%) persons by Ab-ELISA. Among 55 persons positive for Ag-ELISA undergoing CT scan, 30 (54.6%) were found to have structures in the brain suggestive of neurocysticercosis. Using faecal analysis, 43 (5.2%) stool samples tested positive for taeniosis by copro-Ag-ELISA while Taenia eggs were detected in 9 (1.1%) stool samples by routine coprology. Antibodies specifically against adult T. solium were detected in 34 copro-Ag-ELISA positive participants by EITB (rES38) indicating T. solium taeniosis prevalence of 4.1%. Increasing age and hand washing by dipping in contrast to using running water, were found associated with Ag-ELISA seropositivity by logistic regression. Gender (higher risk in females) and water source were risk factors associated with Ab-ELISA seropositivity. Reported symptoms of chronic severe headaches and history of epileptic seizures were found associated with positive Ag-ELISA (p≤0.05). Conclusion The present study indicates T. solium infection in humans is highly endemic in the southern highlands of Tanzania.

Schistosoma japonicum infection in the pig as a model for human schistosomiasis japonica

Valuable information on human schistosomiasis japonica has been provided using primates and experimental rodent hosts. However, major drawbacks such as high costs and ethical concerns for the primate models and large biological deviations for the rodent models have led to the search for more appropriate models. Recent data on the pig indicate that this natural host for Schistosoma japonicum might be a realistic alternative. As only very few research groups have investigated the S. japonicum/pig model, the present review mainly deals with the experimental methods and the major host/parasite findings obtained from the authors own research group. With emphasis on a critical evaluation of the work, the results are compared to the scarce information existing on human schistosomiasis japonica. Like in humans, S. japonicum establishes mainly in the large intestinal veins, with high faecal egg counts during the acute phase of infection, which varies greatly within and between days. Concomitant resistance is another shared feature, but studies in pigs have indicated that the phenomenon is more complex than generally thought. Clinical signs as eosinophilia and diarrhoea with mucus and blood in the acute phase of infection and hepatomegaly, increased portal diameter, periportal fibrosis and ascites in chronic infections are common findings in both humans and pigs. Low protein diet aggravates the disease in pigs by increasing the establishment rates, the faecal egg excretion and the morbidity. A 100% cure rate is achieved when treating S. japonicum infected pigs with praziquantel at 40 mg/kg, and 4 weeks post treatment pigs remain resistant to reinfection. Lastly, human congenital S. japonicum infection has been confirmed in pigs but the implications of such infections for the pathogenesis of schistosomiasis japonica remain to be investigated.

PCR-based identification of individuals of Schistosoma japonicum representing different subpopulations using a genetic marker in mitochondrial DNA

A mitochondrial NADH dehydrogenase I gene fragment (NDI) was sequenced for three laboratory maintained isolates of Schistosoma japonicum. Comparison of sequences representing the isolates (originally obtained from the Anhui and Zhejiang provinces of the Peoples Republic of China, and from the Philippines) revealed inter-isolate sequence variations of 0.2-0.6% and no intra-isolate variation was found. The sequences also indicated that while the amplification products of the Zhejiang and Philippine isolates contained a recognition site for the endonuclease RsaI, there was no such site in the Anhui isolate. This was tested by digesting amplification products from a number of individual worms with RsaI. Then an infection experiment was designed to test the value of this genetic marker for studies of the population biology of S. japonicum in the final host. For this, the two Chinese isolates were used. Three groups of mice (A-C) were exposed firstly to a primary infection and then challenge-infected at weeks 4 and 7 of the experiment. In group A the first infection was done with the Anhui isolate, and the two others with the Zhejiang isolate, thereby providing a specific, detectable cohort. In groups B and C the Anhui isolate was used for the second and third infection. All mice were perfused 5 weeks after the last challenge infection, and the NDI was subsequently amplified from DNA of the perfused worms and digested with RsaI. The digestion revealed that while infection groups A and B contained mixed populations of the Anhui and Zhejiang isolates, only Zhejiang worms were present in group C. We concluded that the absence/presence of the RsaI site in the NDI provides a useful marker for the delineation of cohorts of S. japonicum.

Prevalence and risks for fishborne zoonotic trematode infections in domestic animals in a highly endemic area of North Vietnam

Fishborne zoonotic trematodes (FZT) are endemic in humans and cultured fish in Vietnam but little is known about FZT in domestic animals. A study was designed to determine FZT prevalence and species diversity, and risk factors for infection, in dogs, cats and pigs. Faecal samples from 186 dogs, 94 cats and 168 pigs belonging to 132 households in Nghia Hung district, Nam Dinh province, were examined for small trematode eggs; those were trematode eggs with length less than 50 microm. Prevalence of FZT varied significantly between cats (70.2%), dogs (56.9%) and pigs (7.7%). Forty-nine of the egg-positive animals (25 dogs, 20 cats and 4 pigs) were necropsied to obtain adult trematodes for identification. The liver fluke, Clonorchis sinensis, and 11 species of intestinal flukes including Haplorchis, Stellantchasmus, Stictodora and Centrocestus were recovered from the infected animals. The practice of feeding raw fish to the animals was a significant risk factor for infection; this risk was reduced if the animals were periodically treated with anthelmintics. Based on the high prevalence of FZT and certain risky husbandry practices, domestic animals are likely to be major contributors of FZT eggs to the environment. Therefore, education of farmers to avoid feeding raw fish and to perform regular anthelmintic treatment of dogs, cats and pigs is needed in integrated FZT control programs.

TREATMENT AND REINFECTION OF WATER BUFFALOES AND CATTLE INFECTED WITH SCHISTOSOMA JAPONICUM IN YANGTZE RIVER VALLEY, ANHUI PROVINCE, CHINA

The aim of the present study was to evaluate the efficacy of praziquantel treatment of Schistosoma japonicum infections in cattle and water buffaloes and to assess the natural rate of reinfection after treatment. The studies were conducted on 2 islands in the Yangtze River, Anhui province, China, from March 2003 to January 2004. The efficacy of praziquantel was 97% when applied orally wrapped in tree leaves at the recommended doses. The efficacy was measured using a miracidium hatching technique on fecal samples collected 20 days after treatment. The treatment did not give rise to any major side effects. Reinfection after treatment was high and occurred throughout the year in both cattle and water buffaloes. Age-related resistance was only observed in water buffaloes. It is concluded that although praziquantel is highly effective against S. japonicum in cattle and water buffaloes, a single annual treatment strategy does not effectively control transmission. New strategies for integrated control of animal schistosomiasis are needed to control schistosomiasis transmission more effectively in farm areas of China.

Taenia solium porcine cysticercosis: viability of cysticerci and persistency of antibodies and cysticercal antigens after treatment with oxfendazole.

The aim of this study was to assess the effect of treating Taenia solium infected pigs with oxfendazole (OFZ) on viability and clearance of cysticerci and the corresponding persistence of specific antibody isotypes (IgG(total), IgG1, IgG2 and IgA) and circulating cysticercal antigen (CCA). Antibody isotypes and CCA responses were measured by antibody-ELISA (Ab-ELISA) and antigen ELISA (Ag-ELISA), respectively. Correlations were made between antibodies, CCA and the total number of cysticerci enumerated at necropsy. Forty pigs with cysticercosis were randomly allocated into two groups: Treatment group (n=20) was treated with OFZ at 30 mg/kg orally while the treatment control group (n=20) was not treated. Five uninfected pigs served as negative controls. Pigs were killed at 1, 4, 8 and 26 weeks post-treatment (wkpt). Overall, the mean total cyst count in treated pigs was 2904+/-5397 (mean+/-S.D.) while in the controls it was 6235+/-6705. Mean cyst viability was 5+/-11% (mean+/-S.D.) and 97+/-4% in treated and control pigs, respectively. Results showed that OFZ killed muscular cysticerci over a period of 4 weeks but failed to kill cerebral cysticerci. Antibodies, CCA responses and clearance of dead cysts from the meat, depended on the cyst intensity of individual pigs at time of treatment since both antibody and CCA correlated with intensity of cysticerci at necropsy (r=0.441, P=0.005; r=0.654, P<0.001), respectively. IgG1 responses were the best indicator of treatment efficacy because they were predominant in both infected treated and control pigs and disappeared early after treatment. Both Ab/Ag-ELISA failed to detect cysts in the brain. Though dead cysticerci took some time (26 wkpt) to clear from the meat, treatment of porcine cysticercosis with OFZ should, in combination with other intervention measures be considered as an important, cost-effective measure in the control of taeniosis/cysticercosis.