Jiraporn Sithithaworn

Epidemiology of Strongyloides stercoralis in north-east Thailand: application of the agar plate culture technique compared with the enzyme-linked immunosorbent assay.

Cross-sectional surveys of parasitic infection were performed using the agar plate culture technique (APCT) and modified formalin-ethyl acetate concentration technique (MFECT) to assess the true prevalence of Strongyloides stercoralis relative to other parasites in north-east Thailand. The enzyme-linked immunosorbent assay (ELISA) for diagnosis of S. stercoralis infection was used to estimate the seroprevalence for comparison with coproprevalence. Faecal and serum samples were collected from study participants during October-November 2000. Within the sample population of 332 rural northeast Thais from 3 communities, S. stercoralis was the most common parasitic infection (average 28.9%, range 27.7-30.3%) as determined by APCT; by MFECT the average was 5.4% (range 1.8-8.6%). Other intestinal parasites by order of prevalence were Opisthorchis viverrini (average 14.2%, range 8.6-19.4%), hookworm (average 12.3%, range 4-20.2%), Echinostoma sp. (7.5%), Giardia intestinalis (0.9%), Trichuris trichiura (0.6%), and Taenia sp., Hymenolepis nana and Entamoeba coli (all 0.3%). In an analysis of a subset of the sample population for which serum samples were available (n = 120), coproprevalence by APCT was 33.3% (range 27-53.8%) and seroprevalence was 47.5% (range 29.7-57.9%) by modified unit-based ELISA and 34.2% (range 21.6-42.1%) by conventional optical density (OD)-based ELISA. Taking APCT as the reference method for diagnosis of strongyloidiasis, the sensitivity and specificity of the OD-based ELISA were 65% and 81.3%, respectively, and of the unit-based ELISA were 77.5% and 71.3%, respectively. Our results indicate that S. stercoralis is the predominant parasite in rural north-east Thailand, and that APCT and ELISA should be used as complementary diagnostic methods for community-based parasite surveys, at least among those in high-risk groups.

Exceptionally High Prevalence of Infection of Bithynia siamensis goniomphalos with Opisthorchis viverrini Cercariae in Different Wetlands in Thailand and Lao PDR

The carcinogenic liver fluke, Opisthorchis viverrini, requires Bithynia snail intermediate hosts in its life cycle. However, the prevalence of O. viverrini in snail intermediate hosts is typically low (< 1%). Here, we examined B. siamensis goniomphalos from 48 localities in Thailand and The Lao Peoples Democratic Republic (Lao PDR) and reported high-prevalence levels of O. viverrini. The highest-prevalence levels per locality were 6.93% (mean = 3.04%) in Thailand and 8.37% (mean = 2.01%) in Lao PDR; 4 of 13 localities examined showed prevalence higher than any prevalence previously recorded. The number of cercariae infecting snails and their prevalence were positively correlated with the size of the snails. High prevalence occurred in the Songkram River wetland (Thailand) and the Nam Ngum River wetland (Lao PDR). Our results show that transmission of O. viverrini from humans as well as animal reservoir hosts to snail intermediate hosts is ongoing and potentially increasing in endemic areas across Thailand and Lao PDR.

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.

Microsatellite loci in the carcinogenic liver fluke, Opisthorchis viverrini and their application as population genetic markers

Opisthorchis viverrini is a carcinogenic foodborne trematode endemic in Southeast Asia especially in Thailand and the Lao Peoples Democratic Republic. Opisthorchiasis causes hepatobiliary diseases and cholangiocarcinoma (bile duct cancer). Currently there is substantial evidence on genetic variation of O. viverrini but the information on population genetic structure is lacking. Because microsatellite DNA of this parasite is not available, we for the first time isolated and utilized microsatellite DNA as genetic markers to examine genetic diversity and the population structure of O. viverrini. Partial genomic DNA libraries were constructed by conventional and enrichment methods which yielded microsatellite-containing clones of 0.18-0.25% and 16.84%, respectively. Within 41 microsatellite loci isolated 36.59% were perfect, 60.98% were interrupted and 2.44% were compound microsatellites. The CA repetitions were the most frequent, followed by GT and CAT. Primers specific to the flanking regions of 12 microsatellite loci were developed to genotype 150 O. viverrini individuals from geographical localities in Thailand and Lao PDR. Allele numbers per locus ranged from 2 to 15, with the mean expected heterozygosity of 0.03-0.66. Analyses of O. viverrini from 5 localities revealed a high level of genetic diversity and had significant deviation from Hardy-Weinberg equilibrium. Significant heterozygote deficiency as well as heterozygote excess was detected across all localities indicating the possibility of selfing (inbreeding) as a predominant reproductive mode. Significant genetic differentiation (F(ST)) was also detected between worms from different localities with varying levels of genetic heterogeneity. We discuss our results in terms of what these novel microsatellite markers reveal about the epidemiology and transmission dynamics of this medically important parasite, both in terms of the current study and their potential for future comprehensive population genetic studies O. viverrini sensu lato in Southeast Asia.

Diagnostic values of parasite-specific antibody detections in saliva and urine in comparison with serum in opisthorchiasis.

Infection by the liver fluke (Opisthorchis viverrini) causes hepatobiliary disease and bile duct cancer (cholangiocarcinoma, CCA) in endemic areas in Southeast Asia. Measurements of humoral immune response particularly parasite-specific antibodies are useful not only for serodiagnosis but they have been implicated as risk factors of CCA. In this study, we used indirect Enzyme Immunosorbent Assay (ELISA) to measure O. viverrini-specific immunoglobulins in serum, urine and saliva and assessed efficacies in diagnosis of opisthorchiasis and evaluated the relationship of antibodies among clinical specimens in a sample population in endemic areas in Khon Kaen, Thailand. By employing the Receiver Operation Characteristics (ROC) analysis, diagnostic efficacy based upon the area under the curve (AUC) revealed that serum, salivary IgG and IgA performed better than urine for diagnosis of opisthorchiasis. Seropositive cases were found in both parasite egg-negative as well as O. viverrini egg-positive groups. The levels of serum IgG correlated with intensity of O. viverrini infection (P<0.05). Diagnostic sensitivities based on serum and salivary IgG, IgA also positively associated with the intensity of infection. Correlations between serum antibodies and those in saliva were found to be greater in egg-negative than egg-positive individuals for O. viverrini. Our findings indicated a complex interrelation between antibody responses in different clinical specimens triggered by liver fluke infection. More comprehensive examinations are needed to determine the potential utility of salivary antibody detection which, in combination with the conventional fecal examination method, may better assist in the identification of individuals with opisthorchiasis. Furthermore, it may provide a better indicator of the risk of disease, particularly CCA.

Microproteinuria during Opisthorchis viverrini infection: a biomarker for advanced renal and hepatobiliary pathologies from chronic opisthorchiasis.

Approximately 680 million people are at risk of infection with Opisthorchis viverrini (OV) and Clonorchis sinensis, with an estimated 10 million infected with OV in Southeast Asia alone. While opisthorchiasis is associated with hepatobiliary pathologies, such as advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA), animal models of OV infection show that immune-complex glomerulonephritis is an important renal pathology that develops simultaneously with hepatobiliary pathologies. A cardinal sign of immune-complex glomerulonephritis is the urinary excretion of immunoglobulin G (IgG) (microproteinuria). In community-based studies in OV endemic areas along the Chi River in northeastern Thailand, we observed that over half of the participants had urine IgG against a crude OV antigen extract (OV antigen). We also observed that elevated levels of urine IgG to OV antigen were not associated with the intensity of OV infection, but were likely the result of immune-complex glomerulonephritis as seen in animal models of OV infection. Moreover, we observed that urine IgG to OV antigen was excreted at concentrations 21 times higher in individuals with APF and 158 times higher in individuals with CCA than controls. We also observed that elevated urine IgG to OV antigen could identify APF+ and CCA+ individuals from non-cases. Finally, individuals with urine IgG to OV antigen had a greater risk of APF as determined by Odds Ratios (OR = 6.69; 95%CI: 2.87, 15.58) and a greater risk of CCA (OR = 71.13; 95%CI: 15.13, 334.0) than individuals with no detectable level of urine IgG to OV antigen. Herein, we show for the first time the extensive burden of renal pathology in OV endemic areas and that a urine biomarker could serve to estimate risk for both renal and hepatobiliary pathologies during OV infection, i.e., serve as a “syndromic biomarker” of the advanced pathologies from opisthorchiasis.

Genetic relationships within the Opisthorchis viverrini species complex with specific analysis of O. viverrini from Savannakhet, Lao PDR by multilocus enzyme electrophoresis.

Recent systematic studies of Opisthorchis viverrini based on multilocus enzyme electrophoresis (MEE) have shown that there are at least five genetic groups and possibly two cryptic species occurring in Thailand and Lao PDR each associated with a specific wetland system. A study based on mitochondrial DNA (mtDNA) gene analyses of an O. viverrini population from Savannakhet (SV, Lao PDR) clustered with several isolates from Thailand and Lao PDR although they originated from different river wetland systems. We used MEE to re-examine whether O. viverrini from SV was similar genetically to isolates from Thailand and Lao PDR. The allelic profiles of O. viverrini from SV and five different wetlands representing defined genetic groups of O. viverrini were recorded at 24 enzyme loci as opposed to only two loci of mtDNA. Contrary to previous studies, O. viverrini from SV was found to have fixed genetic differences at six to eight of the 24 loci examined (24.50–35.42%). Allelic data indicated that O. viverrini from SV differed from isolates in the Nam Ngum River wetland in Lao PDR (29.33% fixed genetic differences) and clustered with O. viverrini from Nakhon Phanom and Sakon Nakhon within the Songkram River wetland in Thailand but had fixed genetic differences from these at 24.5% of loci examined. Our data confirm the association between genetic groups of O. viverrini and specific wetland systems, and raise important questions regarding the significance of the genetic differences and relationships of O. viverrini from these wetlands.

Comparative Assessment of the Gelatin Particle Agglutination Test and an Enzyme-Linked Immunosorbent Assay for Diagnosis of Strongyloidiasis

ABSTRACT The performances of the gelatin particle agglutination test (GPAT) and enzyme-linked immunosorbent assay (ELISA) for the diagnosis of strongyloidiasis with reference to the results of the agar plate culture technique (APCT) were evaluated with samples from 459 individuals from communities in northeast Thailand where strongyloidiasis is endemic. The prevalence of strongyloidiasis in five sample groups determined by GPAT varied between 29.3 and 61.5% (mean, 38.8%). ELISA and APCT, employed concurrently, gave lower prevalence rates of 27.5% (range, 21.6 to 42.1%) and 22.7% (range, 12.7 to 53.8%), respectively. By using APCT as the standard method, the sensitivity of GPAT was generally higher than that of ELISA (81 versus 73%). The specificity of GPAT was slightly lower than that of ELISA (74 versus 86%). The resulting GPAT titers exhibited positive linear relationships with the ELISA values (optical density at 490 nm) (P < 0.05), which suggests that the GPAT titer also reflects the levels of specific antibody comparable to those reflected by the ELISA values. Based on the relative ease and simplicity of use of the technique as well as the acceptable rates of sensitivity and specificity of the test, GPAT is more practical for screening for strongyloidiasis than the conventional ELISA.

Levels of 8-OxodG Predict Hepatobiliary Pathology in Opisthorchis viverrini Endemic Settings in Thailand

Opisthorchis viverrini is distinct among helminth infections as it drives a chronic inflammatory response in the intrahepatic bile duct that progresses from advanced periductal fibrosis (APF) to cholangiocarcinoma (CCA). Extensive research shows that oxidative stress (OS) plays a critical role in the transition from chronic O. viverrini infection to CCA. OS also results in the excision of a modified DNA lesion (8-oxodG) into urine, the levels of which can be detected by immunoassay. Herein, we measured concentrations of urine 8-oxodG by immunoassay from the following four groups in the Khon Kaen Cancer Cohort study: (1) O. viverrini negative individuals, (2) O. viverrini positive individuals with no APF as determined by abdominal ultrasound, (3) O. viverrini positive individuals with APF as determined by abdominal ultrasound, and (4) O. viverrini induced cases of CCA. A logistic regression model was used to evaluate the utility of creatinine-adjusted urinary 8-oxodG among these groups, along with demographic, behavioral, and immunological risk factors. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive accuracy of urinary 8-oxodG for APF and CCA. Elevated concentrations of 8-oxodG in urine positively associated with APF and CCA in a strongly dose-dependent manner. Urinary 8-oxodG concentrations also accurately predicted whether an individual presented with APF or CCA compared to O. viverrini infected individuals without these pathologies. In conclusion, urinary 8-oxodG is a robust ‘candidate’ biomarker of the progression of APF and CCA from chronic opisthorchiasis, which is indicative of the critical role that OS plays in both of these advanced hepatobiliary pathologies. The findings also confirm our previous observations that severe liver pathology occurs early and asymptomatically in residents of O. viverrini endemic regions, where individuals are infected for years (often decades) with this food-borne pathogen. These findings also contribute to an expanding literature on 8-oxodG in an easily accessible bodily fluid (e.g., urine) as a biomarker in the multistage process of inflammation, fibrogenesis, and infection-induced cancer.

Concomitant and protective immunity in mice exposed to repeated infections with Echinostoma malayanum

Concomitant immunity and its consequence against infection play roles in regulating worm burdens in helminthiasis. Under natural conditions, this immunity is generated by exposure to repeated low dose or trickle infection. In this study, concomitant immunity was induced in mice exposed repeatedly to infection with Echinostoma malayanum and its protective effect on a challenge infection evaluated. A profile of worm burden from exposure to 10 metacercariae/mouse/week rose rapidly during the first 2 weeks reaching a plateau from week 3 to 8 post infection. Based on a cumulative dose of infection, worm recoveries were around 75% in the first 2 weeks, dropped to 50% at week 3 and 19% at week 8. After week 2, adult worm burden was constant and no juvenile worms were found after week 3 of the experiment. To examine the effect of resistance against reinfection, mice in the experimental group were primarily infected with 10 metacercariae/week for 5 weeks, treated with praziquantel and were challenged with 75 metacercariae/animal. The number of worms recovered from the experimental groups was significantly lower than that from naïve control groups beginning from 24 h to 28 days post challenge. The worms in the experimental group showed growth retardation and the proportion of adult worms was lower than that in the control animals especially during the first 3 weeks of the experiment. Parasite fecundity was also suppressed compared with that in the control group. The selective effects of protective immunity on establishment, growth, and fecundity of challenged worms affected the population dynamics of E. malayanum which is a similar phenomenon to concomitant immunity in schistosomiasis.