• 제목/요약/키워드: PCR diagnosis

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First Record of Paramphistomes Fischoederius cobboldi and Paramphistomum epiclitum Detected in Bovine Rumen from a Local Market of Savannakhet Province, Lao PDR

  • Sanguankiat, Surapol;Sato, Marcello Otake;Sato, Megumi;Maipanich, Wanna;Yoonuan, Tippayarat;Pongvongsa, Tiengkham;Boupha, Boungnong;Chigusa, Yuichi;Moji, Kazuhiko;Waikagul, Jitra
    • Parasites, Hosts and Diseases
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    • v.54 no.4
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    • pp.543-547
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    • 2016
  • In the present study, we report on the occurrence of paramphistomes, Fischoederius cobboldi and Paramphistomum epiclitum, in Lao PDR with the basis of molecular data. Parasite materials were collected from bovines bred in Ban Lahanam area, Savannakhet Province, Lao PDR at Lahanam public market. Morphological observations indicated 2 different species of paramphistomes. The mitochondrial gene cox1 of the specimens was successfully amplified by PCR and DNA sequencing was carried out for diagnosis of 11 specimens. Pairwise alignment of cox1 sequences were performed and confirmed F. cobboldi and P. epiclitum infecting bovines in Laos. Although there were many limiting points, as the small number of worm samples, and the restricted access of the animal host materials, we confirmed for the first time that 2 species of paramphistomes, F. cobboldi and P. epiclitum, are distributed in Lao PDR. More studies are needed to confirm the paramphistome species present in Savannakhet and its hosts to clear the natural history of these parasites of ruminants in the region and measure the impact of this parasite infection in the life and health of the local people.

An Autochthonous Case of Canine Visceral Leishmaniasis in Korea

  • Bhang, Dong Ha;Choi, Ul Soo;Kim, Hyun Jeong;Cho, Kyoung-Oh;Shin, Sung-Shik;Youn, Hee-Jeong;Hwang, Cheol-Yong;Youn, Hwa-Young
    • Parasites, Hosts and Diseases
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    • v.51 no.5
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    • pp.545-549
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    • 2013
  • A 12-year-old spayed female mixed-bred dog presented with nasal bleeding of 2 days duration and a skin nodule in the left flank. No abnormalities were found in coagulation profiles and blood pressure. Cytological evaluation of the nodule revealed numerous characteristic round organisms having a nucleus and a bar within macrophages and in the background, consistent with leishmaniasis. In vitro culture was unsuccessful but PCR of the nodular aspirate identified the organisms as Leishmania infantum, and the final diagnosis was canine leishmaniasis. No history of travel to endemic countries was noted. Because the dog had received a blood transfusion 2 years before the illness, serological screening tests were performed in all donor dogs of the commercial blood bank using the commercial Leishmania ELISA test kit, and there were no positive results. Additional 113 dogs with hyperglobulinemia from Seoul were also screened with the same kits but no positive results were obtained. To the best of the author's knowledge this is the first autochthonous case of canine leishmaniasis in Korea.

Mutational Analysis of Korean Patients with Phenylketonuria

  • Koo, Soo Kyung;Lee, Kwang-Soo;Jung, Sung-Chul;Lee, Jong-Eun;Lee, Dong Hwan
    • Journal of The Korean Society of Inherited Metabolic disease
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    • v.4 no.1
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    • pp.5-12
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    • 2004
  • Purpose Phenylketonuria is an inborn error of metabolism, which is inherited as an autosomal recessive trait. PKU is resulting from deficiency of phenylalanine hydroxylase. PAH gene spans about 90 kb on chromosome 12q and comprises 13 exons. In order to define the genetic basis of PKU and the frequencies and distribution of PAH mutations in the Korean population, we analyzed PAH gene in independent 80 patients with PKU. Methods All 13 exons including exon-intron boundaries and 2 kb of 5' upstream region of the PAH gene were analyzed by PCR-direct sequencing methods. Results PAH gene analysis revealed 39 different mutations including 10 novel mutations. The novel mutations consisted of 9 missense mutations (P69S, G103S, N207D, T278S, P281A, L293M, G332V, S391I and A447P) and a novel splice site variant (IVS10-3C>G). R243Q, IVS4-1G>A, and E6-96A>G were the most relevant mutations and they accounted in the whole for 38% of the mutant alleles identified in this study. We also observed that. $BH_4$ responsibility was. associated with genotype of R241C, R53H and R408Q. Conc1ustion Our present study with 80 participants extends the previous results to more comprehensive understanding of PAH allele distribution and frequency in Koreans. Although Korean mutation profile of PAH is similar to those of the nearest oriental populations (Japanese, Chinese, and Taiwanese), several different characteristic features are revealed. The characterization of the genotype-phenotype relationship was also performed. Our data would be very useful information for diagnosis, genetic counseling and planning of dietary and therapeutic strategies in Korean PAH patients.

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JAK-2 V617F Mutational Analysis in Primary Idiopathic Myelofibrosis: Experience from Southern Pakistan

  • Sultan, Sadia;Irfan, Syed Mohammed
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.17
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    • pp.7889-7892
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    • 2015
  • Background: Primary idiopathic myelofibrosis (PMF) is a clonal Ph-chromosome negative myeloproliferative neoplasm characterized by dysregulated kinase signaling and release of abnormal cytokines. In the recent past, following JAK2 V617F mutation invention, important revolution has been made in the molecular diagnostic biology of this disease. The rational of this study was to determine the mutational status of JAK2 V617F in Pakistan patients with PMF. Materials and Methods: In this cross sectional study, 20 patients with PMF were enrolled from January 2011 to December 2014. Diagnosis was based on WHO criteria for PMF. All patients were screened for G-T point mutation (V617F) in the JAK2 gene on chromosome 9 by allele specific PCR. Results: The mean age was $57.9{\pm}16.5years$. The male to female ratio was 3:1. The frequency of JAK2 V617F positivity in our PMF patients was found to be 55%. Positive correlations of JAK2 V617F mutation were established with high TLC count, raised LDH and marked splenomegaly (P<0.05). No correlation of JAK2 V617F could be established with age and gender (P>0.05). Conclusions: The JAK2 V617F mutation frequency in our PMF patients was similar to those reported previously. In our hands JAK2 V617F mutated patients expressed an aggressive disease phenotype. Screening for the mutation in all suspected PMF cases could be beneficial in differentiating patients with reactive and clonal marrow fibrosis.

Smad4 Expression in Hepatocellular Carcinoma Differs by Hepatitis Status

  • Yao, Lei;Li, Fu-Jun;Tang, Zhi-Qiang;Gao, Shuang;Wu, Qe-Quan
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.4
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    • pp.1297-1303
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    • 2012
  • Aims: Primary hepatocellular carcinoma (HCC) is a common malignancy often related to hepatitis viral infection. Smad4 is known to mediate the TGF-${\beta}$ pathway to suppress tumorigenesis. However, the function of Smad4 in HCC is still controversial. In this study we compared levels of Smad4 in HCC tissues with or without hepatitis virus infection and adjacent normal-appearing liver. Methods: Samples from HCC patients were analyzed for Smad4 protein and mRNA expression by immunohistochemistry (IHC), RT-PCR and Western blotting. Results: We found that tumor tissues expressed less Smad4 mRNA and protein than the adjacent tissues. Most HCC tumor tissues were negative for Smad4 in IHC staining, while the majority of adjacent tissues were positively stained. Interestingly, protein levels were higher in HCC tissues with viral hepatitis than those without virus infection. Suppression of expression appeared closely related to HCC, so that Smad4 appears to function as a tumor suppressor gene (TSG). Conclusion: Patients with hepatitis viral infection, at higher risk for HCC, exhibited increased Smad4 protein expression suggesting hepatitis virus may modulate Smad4 expression, which is functionally distinct from its putative role as a TSG. Smad4 expression may thus be an applicable marker for diagnosis and/or a target to develop therapeutic agents for HCC.

Differential microRNA Expression by Solexa Sequencing in the Sera of Ovarian Cancer Patients

  • Ji, Ting;Zheng, Zhi-Guo;Wang, Feng-Mei;Xu, Li-Jian;Li, Lu-Feng;Cheng, Qi-Hui;Guo, Jiang-Feng;Ding, Xian-Feng
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.4
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    • pp.1739-1743
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    • 2014
  • MicroRNAs are a class of small noncoding RNA which play important regulatory roles in a variety of cancers. MiRNA-specific expression profiles have been reported for several pathological conditions. In this study, we combined large scale parallel Solexa sequencing to identify 11 up-regulated miRNAs and 19 down-regulated miRNAs with computational techniques in the sera of ovarian cancer patients while using healthy serum as the control. Among the above, four miRNAs (miR-22, miR-93, miR-106b, miR-451) were validated by quantitative RT-PCR and found to be significantly aberrantly expressed in the serum of ovarian cancer patients (P<0.05). There were no significant differences between samples from cancer stage I/II and III/IV. However, the levels of miR-106b (p=0.003) and miR-451 (p=0.007) were significantly different in those patients under and over 51 yearsof age. MiR-451 and miR-93 were also specific when analyzed with reference to different levels of CA125. This study shows that Solexa sequencing provides a promising method for cancer-related miRNA profiling, and selectively expressed miRNAs could be used as potential serum-based biomarkers for ovarian cancer diagnosis.

Frequency of FLT3 (ITD, D835) Gene Mutations in Acute Myelogenous Leukemia: a Report from Northeastern Iran

  • Allahyari, Abolghasem;Sadeghi, Masoud;Ayatollahi, Hossein;Yazdi, Hamed Najjaran;Tavakol, Mohammad
    • Asian Pacific Journal of Cancer Prevention
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    • v.17 no.9
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    • pp.4319-4322
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    • 2016
  • Background: FLT3 is mutated in about 1/3 of acute myelogenous leukemia (AML) patients. The aim of the present study was to report the prevalence of FLT3 mutations and comparison with prognostic factors in AML patients in the Northeastern of Iran. Materials and Methods: This cross-sectional study concerned 100 AML cases diagnosed based on bone marrow aspiration and peripheral blood. DNA for every AML patient was extracted and underwent PCR with FLT3-ITD primers. Results: The mean age at diagnosis was 28.5 years (range, 1-66 years), 52 patients (52%) being male. Out of 100 AML patients, 21 (21%) had FLT3 mutation, (17 with FLT3-ITD, 81%, and 4 with FLT3-D825, 19%). Of the 21, 14 (66.7%) had heterozygous mutation. There was no significant difference between age, sex and organomegaly between patients with FLT3 mutation versus FLT3 wild-type. Conclusions: Our frequency of FLT3 is in line with earlier fidnings of approximately 20 to 30% and also the prevalence of FLT3-ITD is more than FLT3-D35 mutation. There was no significant difference between prognostic factors (age and sex) in the patients with FLT3 mutation versus FLT3 wild-type. The prevalence of FLT3 heterozygous mutations is more that homozygous mutations in AML patients.

Expression and Antigenicity of Replicase Protein from Snow Mountain-Like Caliciviruses, Korean Isolates (한국형 사람 Calicivirus Replicase 단백의 발현 및 항원성 평가)

  • Chang, Mi-Yoon;Yang, Jai-Myung;Kim, Kyung-Hee
    • The Journal of Korean Society of Virology
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    • v.27 no.2
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    • pp.151-160
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    • 1997
  • In view of the potential of replicase protein as a diagnostic reagent for human caliciviruses (HuCVs), we have cloned and over-expressed this gene from the Snow Mountain-like Korean strains in Escherichia coli as a fusion protein with glutathione S-transferase (GST), and described the preliminary antigenic characterization of the recombinant products. Each 470bp fragment corresponding to highly conserved region of RNA-dependent RNA polymerase was generated by RT-PCR from stools of two diarrheal children, cloned in pMOSBlue T-vector, and subcloned between the EcoRI and SalI restriction sites of pGEX-4T-3, a GST gene fusion vector, yielding $pGCV_{pol}$. This construct expressed a Snow Mountain-like HuCV replicase under the control of the IPTG-inducible tac promoter. An extract prepared by sonication of the E. coli cell inclusion bodies bearing $pGCV_{pol}$ products was purified and analyzed by SDS-PAGE. After Coomassie blue staining, it was shown that the recombinant replicase migrated on the gels with an approximate molecular mass of 46.5 kDa, that was subsequently cleaved into a 26 kDa GST fragment and a 20.5 kDa replicase protein upon digestion with thrombin protease. The replicase was recognized on immunoblotting with the sera from symptomatic children with the HuCV-associated diarrhea but not by asymptomatic sera from adults. The results presented the first biological activity of individually expressed HuCV replicase subunit and provided important reagents for diagnosis of HuCV infection.

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Seropositivity and Serointensity of Toxoplasma gondii Antibodies and DNA among Patients with Schizophrenia

  • Omar, Ainsah;Bakar, Osman Che;Adam, Nor Fatini;Osman, Hakim;Osman, Arina;Suleiman, Ahmad Hatim;Manaf, Mohd Rizal Abdul;Selamat, Mohd Ikhsan
    • Parasites, Hosts and Diseases
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    • v.53 no.1
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    • pp.29-34
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    • 2015
  • The aim of this cross sectional case control study was to examine the serofrequency and serointensity of Toxoplasma gondii (Tg) IgG, IgM, and DNA among patients with schizophrenia. A total of 101 patients with schizophrenia and 55 healthy controls from Sungai Buloh Hospital, Selangor, Malaysia and University Malaya Medical Center (UMMC) were included in this study. The diagnosis of schizophrenia was made based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). The presence of Tg infection was examined using both indirect (ELISA) and direct (quantitative real-time PCR) detection methods by measuring Tg IgG and IgM and DNA, respectively. The serofrequency of Tg IgG antibodies (51.5%, 52/101) and DNA (32.67%, 33/101) among patients with schizophrenia was significantly higher than IgG (18.2%, 10/55) and DNA (3.64%, 2/55) of the controls (IgG, P=0.000, OD=4.8, CI=2.2-10.5; DNA, P=0.000, OD=12.9, CI=2.17-10.51). However, the Tg IgM antibody between patients with schizophrenia and controls was not significant (P>0.005). There was no significant difference (P>0.005) in both serointensity of Tg IgG and DNA between patients with schizophrenia and controls. These findings have further demonstrated the strong association between the active Tg infection and schizophrenia.

Biological characterization of Brucella spp. isolated from cattle in Gyeongbuk, Korea (국내 경북지역 소에서 분리된 브루셀라 분리주의 생물학적 특성)

  • Kim, Jeong-Hwa;Lim, Jeong Ju;Kim, Dong Hyeok;Lee, Jin Ju;Kim, Dae Geun;Jun, Moo-Hyung;Kim, Sang Hun;Chang, Hong Hee;Lee, Hu Jang;Min, Won-Gi;Kim, Suk
    • Korean Journal of Veterinary Research
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    • v.50 no.2
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    • pp.117-124
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    • 2010
  • Members of the genus Brucella are facultative intracellular bacteria and cause brucellosis, a chronic disease in humans and abortion in animals. In this study, we tested sera for brucellosis of 15 Hanwoo farms in the western part of Gyeong-buk province, resulting 5 farms were brucellosis positive in 2008. We collected blood from 277 heads in the brucellosis positive 5 farms, and performed serological diagnosis, brucella positive cattle which had shown higher than 200 antibody titer in tube agglutination test were slaughtered, supramammary lymph nodes were collected, and Brucella spp. wild type isolation and identification were performed. From these results, 15 of Brucella spp. wild type strains were isolated and all strains were identified as B. abortus biotype 1 by biological and molecular analysis. In the antimicrobial susceptibility test, all 15 strains had a similar susceptibility and resistance pattern. This study may be useful for bacteriological and epidemiological understanding of cattle brucellosis in Korea.