An Alternative Method for Extracting Plasmodium DNA from EDTA Whole Blood for Malaria Diagnosis.

Molecular techniques have been introduced for malaria diagnosis because they offer greater sensitivity and specificity than microscopic examinations. Therefore, DNA isolation methods have been developed for easy preparation and cost effectiveness. The present study described a simple protocol for Plasmodium DNA isolation from EDTA-whole blood. This study demonstrated that after heating infected blood samples with Tris-EDTA buffer and proteinase K solution, without isolation and purification steps, the supernatant can be used as a DNA template for amplification by PCR. The sensitivity of the extracted DNA of Plasmodium falciparum and Plasmodium vivax was separately analyzed by both PCR and semi-nested PCR (Sn-PCR). The results revealed that for PCR the limit of detection was 40 parasites/µl for P. falciparum and 35.2 parasites/µl for P. vivax, whereas for Sn-PCR the limit of detection was 1.6 parasites/µl for P. falciparum and 1.4 parasites/µl for P. vivax. This new method was then verified by DNA extraction of whole blood from 11 asymptomatic Myanmar migrant workers and analyzed by Sn-PCR. The results revealed that DNA can be extracted from all samples, and there were 2 positive samples for Plasmodium (P. falciparum and P. vivax). Therefore, the protocol can be an alternative method for DNA extraction in laboratories with limited resources and a lack of trained technicians for malaria diagnosis. In addition, this protocol can be applied for subclinical cases, and this will be helpful for epidemiology and control.

Promoter hypermethylation of early B cell factor 1 (EBF1) is associated with cholangiocarcinoma progression.

DNA hypermethylation in a promoter region causes gene silencing via epigenetic changes. We have previously reported that early B cell factor 1 (EBF1) was down-regulated in cholangiocarcinoma (CCA) tissues and related to tumor progression. Thus, we hypothesized that the DNA hypermethylation of EBF1 promoter would suppress EBF1 expression in CCA and induce its progression. In this study, the DNA methylation status of EBF1 and mRNA expression levels were analyzed in CCA and normal bile duct (NBD) tissues using a publicly available database of genome-wide association data. The results showed that the DNA methylation of EBF1 promoter region was significantly increased in CCA tissues compared with those of NBD. The degree of methylation was negatively correlated with EBF1 mRNA expression levels. Using methylation-specific PCR technique, the DNA methylation rates of EBF1 promoter region were investigated in CCA tissues (n=72). CCA patients with high methylation rates of EBF1 promoter region in the tumor tissues (54/72) had a poor prognosis. Higher methylation rates of EBF1 promoter region have shown in all CCA cell lines than that of an immortal cholangiocyte cell line (MMNK1). Upon treatment with the DNA methyltransferase inhibitor 5-Aza-dC, increased EBF1 expression levels and reduced DNA methylation rates were observed in CCA cells. Moreover, restoration of EBF1 expression in CCA cells led to inhibition of cell growth, migration and invasion. In addition, RNA sequencing analysis suggested that EBF1 is involved in suppression of numerous pathways in cancer. Taken together, DNA hypermethylation in the EBF1 promoter region suppresses EBF1 expression and induces CCA progression with aggressive clinical outcomes.

Establishment of Highly Transplantable Cholangiocarcinoma Cell Lines from a Patient-Derived Xenograft Mouse Model.

Cholangiocarcinoma (CCA) is a deadly malignant tumor of the liver. It is a significant health problem in Thailand. The critical obstacles of CCA diagnosis and treatment are the high heterogeneity of disease and considerable resistance to treatment. Recent multi-omics studies revealed the promising targets for CCA treatment; however, limited models for drug discovery are available. This study aimed to develop a patient-derived xenograft (PDX) model as well as PDX-derived cell lines of CCA for future drug screening. From a total of 16 CCA frozen tissues, 75% (eight intrahepatic and four extrahepatic subtypes) were successfully grown and subpassaged in Balb/c Rag-2-/-/Jak3-/- mice. A shorter duration of PDX growth was observed during F0 to F2 transplantation; concomitantly, increased Oct-3/4 and Sox2 were evidenced in 50% and 33%, respectively, of serial PDXs. Only four cell lines were established. The cell lines exhibited either bile duct (KKK-D049 and KKK-D068) or combined hepatobiliary origin (KKK-D131 and KKK-D138). These cell lines acquired high transplantation efficiency in both subcutaneous (100%) and intrasplenic (88%) transplantation models. The subcutaneously transplanted xenograft retained the histological architecture as in the patient tissues. Our models of CCA PDX and PDX-derived cell lines would be a useful platform for CCA precision medicine.

Opisthorchis viverrini: Gene expression profiling of carcinogenic adult liver fluke worms using 5' SAGE.

Opisthorchis viverrini is the only liver fluke that has been proved to be associated with cholangiocarcinoma (CCA). However, the mechanisms by which O. viverrini participates in the carcinogenesis of CCA are still unclear. To understand the biology and host-parasite interaction related to O. viverrini infection, gene expression profiling of this parasite is required. Here, we constructed the first 5' serial analysis of gene expression (5' SAGE) library of the adult O. viverrini and matched the data with the public data of O. viverrini, Clonorchis sinensis and other related Platyhelminthes and Nematodes. We obtained 12,401 unique tag sequences, of which 6515 (53%) could be matched with the 3419 transcript sequences. The two most abundant tag sequences were vitelline B precursor protein and myoglobin. Often several transcription start sites (TSS) were observed for one transcript. This finding may reflect the dynamic nature of transcriptional initiation events of O. viverrini genes in vivo.

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 Stoll's 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 x 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, beta-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.

Novel mutations in cholangiocarcinoma with low frequencies revealed by whole mitochondrial genome sequencing.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations have been shown to be associated with cancer. This study explored whether mtDNA mutations enhance cholangiocarcinoma (CCA) development in individuals. MATERIALS AND METHODS: The whole mitochondrial genome sequences of 25 CCA patient tissues were determined and compared to those of white blood cells from the corresponding individuals and 12 healthy controls. The mitochondrial genome was amplified using primers from Mitoseq and compared with the Cambridge Reference Sequence. RESULTS: A total of 161 mutations were identified in CCA tissues and the corresponding white blood cells, indicating germline origins. Sixty-five (40%) were new. Nine mutations, representing those most frequently observed in CCA were tested on the larger cohort of 60 CCA patients and 55 controls. Similar occurrence frequencies were observed in both groups. CONCLUSIONS: While the correspondence between the cancer and mitochondrial genome mutation was low, it is of interest to explore the functions of the missense mutations in a larger cohort, given the possibility of targeting mitochondria for cancer markers and therapy in the future.

Comparative proteomic analysis of juvenile and adult liver fluke, Opisthorchis viverrini.

We used comparative two-dimensional gel electrophoresis to highlight proteins that are differentially expressed in the maturation stage of the parasite Opisthorchis viverrini (OV). The proteins differentially expressed in the juvenile/adult forms of the parasite are thought to be important for survival and pathogenesis. We used a nonlinear gradient pH ranged 3-10 strips for isoelectric focusing to resolve soluble proteins from four different maturation periods of OV from 1 week juvenile to 4 week adult. Approximately 210-240 protein spots were resolved by 2-DE in two ranges of pI (4.5-5.8 and 6.0-8.0). At least 35 protein spots were differentially expressed in 4 week adult compared to 1 week juvenile fluke. These proteins may involve in sex organ development and egg production. Comparative analysis of the OV proteome of different aged parasites during maturation may help to better understand parasite biology, pathogenesis/carcinogenesis related to this parasite and lead to the identification of new targets of vaccines and drugs.

A novel predictive equation for potential diagnosis of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is the second most common-primary liver cancer. The difficulties in diagnosis limit successful treatment of CCA. At present, histological investigation is the standard diagnosis for CCA. However, there are some poor-defined tumor tissues which cannot be definitively diagnosed by general histopathology. As molecular signatures can define molecular phenotypes related to diagnosis, prognosis, or treatment outcome, and CCA is the second most common cancer found after hepatocellularcarcinoma (HCC), the aim of this study was to develop a predictive model which differentiates CCA from HCC and normal liver tissues. An in-house PCR array containing 176 putative CCA marker genes was tested with the training set tissues of 20 CCA and 10 HCC cases. The molecular signature of CCA revealed the prominent expression of genes involved in cell adhesion and cell movement, whereas HCC showed elevated expression of genes related to cell proliferation/differentiation and metabolisms. A total of 69 genes differentially expressed in CCA and HCC were optimized statistically to formulate a diagnostic equation which distinguished CCA cases from HCC cases. Finally, a four-gene diagnostic equation (CLDN4, HOXB7, TMSB4 and TTR) was formulated and then successfully validated using real-time PCR in an independent testing set of 68 CCA samples and 77 non-CCA controls. Discrimination analysis showed that a combination of these genes could be used as a diagnostic marker for CCA with better diagnostic parameters with high sensitivity and specificity than using a single gene marker or the usual serum markers (CA19-9 and CEA). This new combination marker may help physicians to identify CCA in liver tissues when the histopathology is uncertain.

Genome size estimation of liver fluke Opisthorchis viverrini by real-time polymerase chain reaction based method.

The human liver fluke, Opisthorchis viverrini, has been categorized as a class one carcinogenic organism according to its strong association with cholangiocarcinoma, bile duct cancer which has high incidence in the northeast of Thailand. The lack of genome database of this parasite limited the studies aimed to understand the basic molecular biology of this carcinogenic liver fluke. The determination of the genome size is an initial step prior to the full genome sequencing. In this study, we applied an absolute quantitative real-time polymerase chain reaction for this aspect. Our results indicated the genome size of O. viverrini is 75.95 Mb or C value 0.083. The information of O. viverrini genome size is useful for estimation of sequence coverage and the cost of the parasite's whole genome sequencing using next-generation sequencing technologies.

Molecular identification of a case of Paragonimus pseudoheterotremus infection in Thailand.

Paragonimiasis is an important food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. In Southeast Asia, Paragonimus heterotremus is the only proven causative pathogen. Recently, a new Paragonimus species, P. pseudoheterotremus, was found in Thailand. This species is genetically similar to P. heterotremus and is considered as a sister species. However, infectivity or pathogenicity of P. pseudoheterotremus to humans remains unclear. We report the first confirmed human pulmonary paragonimiasis case caused by P. pseudoheterotremus infection. After polymerase chain reaction/sequencing of the DNA extracted from Paragonimus eggs in the sputum of the patient, partial internal transcribed spacer 2 and cytochrome c oxidase subunit 1 sequences were approximately identical (98-100%) with those of P. pseudoheterotremus. For P. heterotremus, the partial internal transcribed spacer 2 sequence was approximately identical (99-100%), but the partial mitochondrial cytochrome c oxidase subunit 1 sequence showed a similarity of 90-95%.