Wanchai Maleewong

Development of a PCR-based method for the detection of Opisthorchis viverrini in experimentally infected hamsters

Opisthorchis viverrini infection is an endemic disease that causes a serious public health problem in southeast Asia, especially in northeast Thailand. We have developed a PCR method using a pair of primers named OV-6F/OV-6R for detecting O. viverrini eggs in stool samples and compared it with Stolls egg-count method. The primers were designed based on the pOV-A6 specific DNA probe sequence which gave a 330 base pair product. The PCR method can detect a single egg in artificially inoculated faeces or as little as 2 x 10(-17) ng of O. viverrini genomic DNA. The method gave 100% sensitivity in all hamster groups except in animals exposed to the lowest intensity of infection (1 metacercaria/hamster). In the first month of infection, the PCR method was more sensitive than using the egg-count method in all infected groups especially in the light infections. The PCR method was also successfully used in monitoring a therapeutic study. Since the PCR method showed no cross-reaction with Heterophyid flukes, it can be useful for specific identification of O. viverrini eggs in stool samples without the risk of false positives. It also has great potential for application in clinical epidemiological studies.

Detection of Opisthorchis viverrini in Human Stool Specimens by PCR

Opisthorchiasis is a liver fluke infection, found mainly in Southeast Asia (8) but increasingly in developed countries due to an influx of Asian immigrants (7). The diagnostic methods are based on the demonstration of eggs in stools, although there are still difficulties in distinguishing eggs from heterophyid and lecithodendriid parasites (2). We have successfully developed a PCR-based method for detection of Opisthorchis viverrini in experimentally infected hamsters (6). The aim of the present study was to determine the value of PCR for detection of O. viverrini eggs in human feces. Stool specimens from 85 parasite-infected individuals, as confirmed by the formalin-ether technique, were collected from Srinagarind Hospital, Faculty of Medicine, Khon Kaen University. The specimens were composed of 40 O. viverrini parasites, 10 minute intestinal flukes, 10 hookworms, 10 echinostomes, 7 Taenia spp. parasites, 3 Strongyloides stercolaris parasites, 2 Entamoeba coli parasites, and 3 specimens from individuals with mixed infections. To evaluate the sensitivity of the method, normal stool specimens were spiked with either 1, 10, 25, 50, 100, 200, 500, and 1,000 O. viverrini eggs or 0.02, 0.2, 2, 20, and 200 pg and 2 ng of genomic DNA, respectively. The sensitivity of the detection for stool specimens was compared with that for Stolls egg count technique (5). To extract the DNA from stool specimens, 100 mg of feces in 1 ml of sterile distilled water was extracted with 200 μl of ether and centrifuged at 6,500 × g for 2 min. The pellet was mixed with 500 μl of 0.5 N NaOH and left for 1 h at room temperature. After being autoclaved, the supernatant was collected and invertedly mixed with 250 μl of 3 M sodium acetate and 500 μl of absolute ethanol before being cleaned up with the QIAamp DNA Mini kit (Qiagen, Hilden, Germany), and 10 μl was used in the PCR (6). The sensitivity test in the spiking experiment demonstrated that the limit of our PCR detection is 200 eggs or 200 pg of genomic DNA. The expected 330-bp product and its doublet of 660 bp were in 32 of the 40 fecal specimens (80.0%) from patients with O. viverrini (Table ​(Table1).1). Compared with that of Stolls egg count, the sensitivity of the PCR was found to be 100, 68.2, and 50% in the fecal specimens containing >1,000, 200 to 1,000, and <200 eggs per g of feces, respectively. Our method gave one false positive (97.8% specificity) in the specimen containing eggs of echinostomes (Table ​(Table1).1). The positive and negative predictive values are 96.9 and 84.6%, respectively. TABLE 1. Results of PCR and Stolls egg count compared to formalin-ether technique Our PCR method is sensitive and highly specific, with no amplification occurring when samples contained small intestinal flukes or other parasitic eggs. It was more sensitive than the previous method using a DNA probe as a diagnostic tool (3). The detection limit of 200 eggs can be assumed to be produced by one adult fluke, since an adult worm can release 50 to 200 eggs per g of feces (1, 4). The sensitivity value of PCR is somewhat lower than those of the formalin-ether and Stoll egg count techniques. This may be caused by the use of low amounts of stool sample (0.1 g for PCR and 2 and 1 g for formalin-ether and Stolls egg count, respectively). However, mild infection (200 to 1,000 eggs per g of feces) could be easily missed by Stolls dilution technique (4), whereas our technique gave a 68.2% positive rate. The false-positive result occurring for a specimen containing eggs of echinostomes may not have been an actual positive result, since the specimen was the only one positive among the 10 stool samples containing echinostomes. Thus, the results in this study suggest that our PCR method is a suitable tool for detection of O. viverrini infection, especially in a large number of samples at one time. With some improvement in sample preparation, it will be very useful for sensitive and specific diagnosis and epidemiological studies and offers a potential means for detection of the parasite in the intermediate hosts.

The Opisthorchis viverrini genome provides insights into life in the bile duct

Opisthorchiasis is a neglected, tropical disease caused by the carcinogenic Asian liver fluke, Opisthorchis viverrini. This hepatobiliary disease is linked to malignant cancer (cholangiocarcinoma, CCA) and affects millions of people in Asia. No vaccine is available, and only one drug (praziquantel) is used against the parasite. Little is known about O. viverrini biology and the diseases that it causes. Here we characterize the draft genome (634.5 Mb) and transcriptomes of O. viverrini, elucidate how this fluke survives in the hostile environment within the bile duct and show that metabolic pathways in the parasite are highly adapted to a lipid-rich diet from bile and/or cholangiocytes. We also provide additional evidence that O. viverrini and other flukes secrete proteins that directly modulate host cell proliferation. Our molecular resources now underpin profound explorations of opisthorchiasis/CCA and the design of new interventions.

Comparison of the Quantitative Formalin Ethyl Acetate Concentration Technique and Agar Plate Culture for Diagnosis of Human Strongyloidiasis

ABSTRACT The quantitative formalin ethyl acetate concentration technique (QFEC) was compared to agar plate culture (APC) for the detection of Strongyloides stercoralis larvae. QFEC could substitute for APC only when the parasite load was higher than 50 larvae per g of stool. This study serves as a good reminder to those conducting stool exams about the sensitivity and specificity limitations of both techniques.

Detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes by a PCR-based method.

A PCR procedure for the detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes was developed. This procedure was based on primers designed from a pOV-A6 specific probe sequence giving a 330 base-pair product. The detection was accomplished in host tissue homogenates to which a single cercaria or metacercaria was introduced. PCR can detect as little as a single cercaria artificially inoculated in a snail or a single metacercaria artificially inoculated in a fish sample. The method gave a 100% positivity rate for all infected snails or fishes. The method did not yield a 330 base-pair amplified product with other digenean fluke DNAs such as Haplorchis taichui, Centrocestus spp., Echinostoma malayanum, Fasciola gigantica, animal schistosomes, Paragonimus heterotremus or Haplorchoides spp. The assay has great potential for application in epidemiological surveys of both snail and fish intermediate hosts as well as for investigation of foodborne parasites in freshwater fishes.

Immunoblot Diagnostic Test for Neurognathostomiasis

Neurognathostomiasis is a rare but severe form of human gnathostomiasis. Diagnosis of neurognathostomiasis is made presumably by using clinical manifestations. Serologic tests for neurognathostomiasis are not widely available and limited. We studied 12 patients with diagnoses of neurognathostomiasis at Srinagarind Hospital, Khon Kaen University, Thailand. There were three types of neurognathostomiasis (five patients with intracerebral hemorrhage, one patient with subarachnoid hemorrhage, and six patients with myelitis). All patients were tested for antibodies against Gnathostoma spinigerum by an immunoblotting technique. The sensitivity and specificity of the 21-kD and 24-kD diagnostic bands were 83.3% and 100%, and 91.7% and 100%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for the 21-kD and 24-kD diagnostic bands were all 100%. Both diagnostic bands are a helpful diagnostic tool for neuro gnathostomiasis and show good diagnostic properties.

Cerebrospinal fluid cytokine responses in human eosinophilic meningitis associated with angiostrongyliasis

The levels of interleukin 5 (IL5), IL10, and IL13 in the cerebrospinal fluid (CSF) were markedly higher in 30 patients with eosinophilic meningitis associated with angiostrongyliasis (EOMA) than in the controls (P<0.001). IL2, IL4, interferon gamma (IFNgamma), and tumor necrosis factor alpha (TNFalpha) levels were not significantly different (P>0.05). IL5, IL10, and TNFalpha levels correlated with eosinophil levels (P=0.023, P=0.018, and P=0.005, respectively) while IL2, IL4, IL13, and IFNgamma did not (P>0.05). Our data suggest that local T-helper-2 (TH2) cytokine responses are predominant in the CSF of patients with EOMA. Data on T lymphocyte-parasite interactions are important for the design of effective vaccines and immunotherapies. The measurement of T-helper-1 (TH1)/TH2 cytokines in the CSF may also have some potential for the diagnosis of parasite associated meningitis.

Factors Affecting Recovery of Strongyloides stercoralis Larvae: an Approach to a Newly Modified Formalin-Ether Concentration Technique for Diagnosis of Strongyloidiasis

ABSTRACT To improve the diagnosis efficiency of human strongyloidiasis by using formalin-ether concentration technique (FECT), the effects of various factors on the recovery rates of Strongyloides stercoralis larvae were comparatively evaluated. Fresh stool and a short time exposure of larvae to formalin yielded significantly higher numbers of larvae than preserved stool and 10-min exposure. Likewise, straining through wire mesh yielded a significantly higher number of larvae recovered than straining through gauze did. In addition, centrifugation for 5 min for separation of larvae from debris yielded a significantly greater number of larvae recovered than centrifugation for 2 min did. The efficacies of the five versions of FECT with different factors-FECT 1, FECT 2, FECT 3, FECT 4, and FECT 5-were then compared. It was found that FECT 5 was 1.8, 2.0, 1.9, and 1.4 times more effective than FECT 1, FECT 2, FECT 3, and FECT 4, respectively. FECT 5 is a modified FECT method, whose modifications included using fresh stool without a preservative substance; a short-time rather than 10-min formalin exposure; and the use of wire mesh instead of gauze.

Detection of Paragonimus heterotremus Eggs in Experimentally Infected Cats by a Polymerase Chain Reaction–Based Method

A polymerase chain reaction (PCR) procedure for the detection of Paragonimus heterotremus eggs in stool samples was developed and compared with Stolls egg count method. The primers were designed on the basis of a previously constructed pPH-13–specific DNA probe, which produced an approximate 0.5-kb amplified product. This PCR method could detect as few as 5 eggs in 0.6 g of artificially inoculated feces of a healthy control cat or as little as 1 × 10−4 ng of P. heterotremus genomic DNA. The assay had 100% sensitivity in all infected cats. The method did not yield an approximate 0.5-kb product with DNA from other parasites such as Gnathostoma spinigerum, Trichinella spiralis, Fasciola gigantica, Echinostoma malayanum, Opisthorchis viverrini, Dirofilaria immitis, and Taenia saginata; exceptions were Paragonimus siamensis and Paragonimus westermani. In addition, no genomic DNA from Escherichia coli, Burkholderia pseudomallei, Acinetobacter anitratus, Mycobacterium tuberculosis, Staphylococcus aureus, β-Streptococcus grA, and Proteus mirabilis or from the vertebrate and invertebrate hosts of P. heterotremus was amplified in the PCR assay. This assay has great potential for application in clinical epidemiological studies.

Evaluation of Immunoglobulin G Subclass Antibodies against Recombinant Fasciola gigantica Cathepsin L1 in an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Human Fasciolosis

ABSTRACT A cystatin capture enzyme-linked immunosorbent assay (ELISA) using recombinant Fasciola gigantica cathepsin L1 antigen was developed to detect specific immunoglobulin G (IgG) subclass antibodies (IgG1, IgG2, IgG3, and IgG4) and was evaluated for its diagnostic potential for human fasciolosis. In an analysis of the sera of 13 patients infected with F. gigantica, 209 patients with other parasitic infections, 32 cholangiocarcinoma patients, and 42 healthy controls, the IgG4-ELISA gave the highest diagnostic values. The sensitivity, specificity, accuracy, and positive and negative predictive values of this method based on the detection of IgG4 antibody were 100%, 99.3%, 99.3%, 86.7%, and 100%, respectively. The results revealed that restricting the ELISA to the detection of specific IgG4 antibody enhanced the specificity and accuracy for the serodiagnosis of human fasciolosis.