Evidence of a species complex within the food-borne trematode Opisthorchis viverrini and possible co-evolution with their first intermediate hosts.

The food-borne trematodes, Opisthorchis viverrini, O. felineus and Clonorchis sinensis, have long been recognized as the cause of major human health problems, with an estimated 40 million infected persons. Of the three species of liver fluke, only O. viverrini is classified as a type 1 carcinogen because of its role as an initiator of chronic inflammation and the subsequent development of cholangiocarcinoma. At present, there are no techniques for the early diagnosis of cholangiocarcinoma and it is fatal for most patients. There is considerable variation in parasite prevalence and disease presentation in different geographical areas, the latter of which may be associated with genetic differences among parasites. In the present study, multilocus enzyme electrophoresis was used to provide a comprehensive genetic characterization of O. viverrini from different geographical localities in Thailand and the Peoples' Democratic Republic of Laos. Parasites from different localities were compared genetically at 32 enzyme loci. The results of the genetic analyses are sufficient to reject the null hypothesis that O. viverrini represents a single species. Therefore, O. viverrini consists of at least two genetically distinct, yet morphologically similar (i.e. cryptic) species. Moreover, there was also separation of the different populations of snails (i.e. the first intermediate hosts) into two distinct genetic groups that corresponded with the delineation of O. viverrini into two species. This suggests that there may be a history of co-evolution in this host-parasite lineage. Additionally, five distinct genetic groups of parasites were detected, each of which occurred within a different and independent river wetland system. Our findings have major implications for the implementation of effective control and surveillance programs targeted to these medically important food-borne parasites.

The influence of pregnancy on intestinal parasite infection in Thai women.

The relationship between pregnancy and both the susceptibility and pathogenicity of parasite infections is disputed. This study compares the prevalence and intensity (as measured by density of eggs in stool samples) of intestinal helminth infections in pregnant and control groups of women from Khon Kaen Province in the northeast of Thailand. Stool samples were taken at the end of the first, second and third trimesters of pregnancy as well as 2 months after parturition and compared for the two groups. There were no significant changes in the prevalence of any of the common helminth species during the course of pregnancy or between the pregnant and control groups. Nor was there any evidence that the density of helminth eggs in the stool samples differed between sample times or between the pregnant and control groups. Our study therefore supports the hypothesis that pregnancy does not influence the course of human infection with helminths.

Genetic markers for the identification and characterization of Opisthorchis viverrini, a medically important food borne trematode in Southeast Asia.

The liver fluke, Opisthorchis viverrini, is one of the major food borne trematodes in Southeast Asia, where infection causes hepatobiliary disease and subsequent development of cholangiocarcinoma. In Thailand, O. viverrini is most prevalent in the northeast where there is marked regional variation in the rate of infection in humans at provincial, district and village levels. To date, the roles of genetic variation of O. viverrini on this observed variability in infection, transmission and associated disease are not known. We have applied multilocus enzyme electrophoresis (MEE), specifically allozyme electrophoresis, to isolates of O. viverrini from Thailand and Laos to establish genetic markers to examine its systematics and population structure. Forty-six enzymes commonly found useful for genetic characterisation in parasitic helminths were screened, and of these, 33 enzymes gave sufficient staining and resolution to act as potential genetic markers. Sixteen enzymes were monomorphic and 17 enzymes were polymorphic in the pools of worms examined. Whether they are indicative of different enzyme loci, heterozygosity or unique genotypes within the pools of worms examined remains to be determined. Preliminary investigations examining five individual worms at enzyme loci where pools of worms showed multiple bands have confirmed the diagnostic value of the enzyme loci established as well as providing evidence of potential population sub structuring and heterozygosity. For the first time, we have established at least 17 enzymes that provide the basis to undertake comprehensive genetic analyses of the systematics and population structure of O. viverrini, a medically important food borne trematode in Southeast Asia.

Enzyme markers to identify and characterize Opisthorchis viverrini in Thailand and Lao PDR.

We conducted an allozyme electrophoretic study to explore potential enzyme markers to distinguish Opisthorchis viverrini in Thailand and Lao PDR. Twenty-eight enzymes encoding presumptive 32 loci were established. The enzymes glucose-6-phosphate dehydrogenase and pyruvate kinase were diagnostic between two geographically separate isolates from Thailand. Twelve enzymes, ie, aconitate hydratase, aldolase, creatine kinase, enolase, esterases, fumarate hydratase, aspartate aminotransferase, glucose-phosphate isomerase, alanine aminotransferase, isocitrate dehydrogenase, malic enzyme, and pyruvate kinase, also provided diagnostic markers for these two isolates from Thailand and one isolate from Lao PDR.

Culex quinquefasciatus in Phitsanulok as a possible vector of nocturnally periodic Wuchereria bancrofti transmission in Myanmar immigrants.

The present study was undertaken in order to study whether Culex quinquefasciatus collected in Phitsanulok Province can be an insect host for the development of Wuchereria bancrofti larvae. W. bancrofti infected blood from Myanmar workers in Mae Sot, Tak Province was fed to mosquitoes by using the artificial membrane feeding. An infection of W. bancrofti was found with the highest density of L3 in the mosquito thorax on the 14th day after feeding. The infection rate also correlated to the density of microfilaria found in the donor's blood. Our results showed that Cx. quinquefasciatus in Phitsanulok is a possible vector of nocturnally periodic W. bancrofti.

Detection of opportunistic and non-opportunistic intestinal parasites and liver flukes in HIV-positive and HIV-negative subjects.

We assessed the frequency and distribution of infection with opportunistic and non-opportunistic intestinal parasites and the liver fluke, Opisthorchis viverrini, in HIV-seropositive and HIV-seronegative subjects. Age- and sex-matched HIV-seropositive (n = 78) and HIV-seronegative patients (n = 78) from two hospitals in Khon Kaen Province, Thailand, participated in this study from November 1998 to August 2000. These subjects were divided according to the presence of diarrhea and CD4 counts. A single stool sample was obtained and analyzed by using specific techniques. Opisthorchis viverrini, was the most common parasite (19.2%) in each group. The prevalence rates of Cryptosporidium spp (11.5%) and Strongyloides stercoralis (17.9%) in the HIV-seropositive group were significantly (p < 0.05) higher than those in the HIV-seronegative group (1.0% for Cryptosporidium spp and 7.7% for S. stercoralis infections). The prevalences of these two parasites were 28% for Cryptosporidium spp and 20% for S. stercoralis in HIV-seropositives with diarrhea and CD4 counts lower than 100 cells/mm3, and were higher compared with patients without diarrhea or with high CD4 counts. These results suggest that infection with these parasites increases during HIV infection. The epidemiological distribution of Cryptosporidium and S. stercoralis may have implications for AIDS-related diseases.

Immunodiagnosis of human fascioliasis using an antigen of Fasciola gigantica adult worm with the molecular mass of 27 kDa by a dot-ELISA.

Immunodominant antigens of an approximate molecular mass of 27 kDa (FG 27) were obtained from an excretory-secretory product of adult Fasciola gigantica by a simple continuous-elution method. A dot-ELISA using the FG 27 antigen was developed for the detection of specific antibodies from patients infected with F. gigantica. Control sera were obtained from patients with other parasitic infections and healthy volunteers. The accuracy, sensitivity, specificity, and positive and negative predictive values were 98.2%, 100%, 97.4%, 76.9% and 100%, respectively. This dot-ELISA is a specific, sensitive and easy to perform method for the rapid diagnosis of fascioliasis, particularly when more complex laboratory tests are unavailable.

Rapid detection of Wuchereria bancrofti in mosquitoes by LightCycler polymerase chain reaction and melting curve analysis.

A LightCycler real-time polymerase chain reaction (PCR) assay was developed to detect Wuchereria bancrofti DNA in blood-fed mosquitoes. The assay is based on fluorescence melting curve analysis of the PCR product generated from a family of repeated DNA elements: the 182 bp SspI repeat, specific to the genus Wuchereria. According to the melting temperature, W. bancrofti infected-mosquitoes were differentiated from Brugia malayi-infected and non-infected mosquitoes as well as from genomic DNA of Dirofilaria immitis and human DNA. The method proved to be 100% sensitive in all W. bancrofti-infected mosquitoes. Melting curve analysis offers a rapid alternative for the specific detection of W. bancrofti in mosquitoes. It is very accurate and sensitive, allows a high throughput and can be performed on very small samples. The method therefore has great potential for application in epidemiological studies.

PCR diagnosis of Pneumocystis carinii on sputum and bronchoalveolar lavage samples in immuno-compromised patients.

The polymerase chain reaction (PCR) technique was compared with Wright-Giemsa (WG), Gomori methenamine silver (GMS) stains and an immunofluorescence assay (IFA) for detection of Pneumocystis carinii in immuno-compromised patients. Specimens of 21 bronchoalveolar lavages (BAL) and 139 sputum samples, were obtained from 157 patients (38 with AIDS and 119 with HIV) from four hospitals in Khon Kaen, Thailand. A true positive required at least two positives by techniques considered gold standard tests. Eleven (52.38%) BAL and 13 (9.35%) sputum specimens were positive. PCR produced the highest sensitivity and negative predictive values for the BAL (100% for each) vs. sputum samples at 84.62 and 98.41 percent, respectively. The specificity of PCR was 90% and 98.41% for BAL and sputum samples, respectively. We suggest PCR is an important tool for the epidemiological study of P. carinii in high-risk individuals.