• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.27 no.2
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• pp.95-103
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• 2024

Purpose: Diarrhea is one of the leading causes of mortality in children living in developing countries. The etiology of acute diarrhea in each healthcare center varies depending on place, time, and population. This study aimed to identify pathogen patterns in human immunodeficiency virus (HIV)-infected and non-HIV children suffering from acute diarrhea, using multiplex real time reverse transcriptase polymerase chain reaction (RT-PCR), in an Indonesian tertiary hospital. Methods: This cross-sectional study was conducted at Dr. Cipto Mangunkusumo National Hospital from March 2019 to April 2020. Results: The study showed that multiplex RT-PCR results were positive in 58.9% of the specimens, with more positive results in HIV-infected children than in non-HIV-infected children (70% vs. 54.7%). Altogether 72 enteropathogens were detected from all specimens. Enteropathogens in non-HIV children with acute diarrhea consisted of bacteria (70.6%) and viruses (29.4%) with a predominance of enteroaggregative Escherichia coli (25.4%), followed by Campylobacter spp. (11.8%), enteropathogenic E. coli (9.8%), Norovirus GII (7.8%), and Clostridium difficile (7.8%). Enteropathogens in HIV-infected children consisted of viruses (57.1%), bacteria (28.6%), and parasites (14.3%) comprising Norovirus GII (24%), Cryptosporidium spp. (14.3%), Campylobacter spp. (14.3%), Norovirus GI (14.3%), and Astrovirus (14.3%). Cryptosporidium spp. was the only parasite found in this study and was found only in HIV-infected children. In non-HIV children with acute diarrhea, most pathogens were invasive bacteria, while in HIV-infected children, more viral and parasite infections occurred, primarily caused by opportunistic pathogens. Conclusion: The pattern of enteropathogens can help clinicians determine further examinations and appropriate empirical antimicrobial therapy for the patient.

• Tuberculosis and Respiratory Diseases
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• v.39 no.6
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• pp.517-525
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• 1992

Background: Since its development by Saiki et al, polymerase chain reaction (PCR) has been very useful in various fields of molecular biology. PCR can be used for the detection of a very small amount of microbial agent, and is especially useful in those patients who are difficult to diagnose microbiologically or serologically. Mycobacterium tuberculosis is a very slowly growing organism and AFB staining frequently shows false negative results, and therefore PCR would be a very rapid, easy, and sensitive diagnostic method for the diagnosis of Mycobacterium tuberculosis. Method: To compare PCR with conventional methods in diagnosing Mycobacterium tuberculosis in sputum, we used sputa of patients who visited or were admitted to Seoul National University Hospital. The amplification targets were 383 base pair DNA, a part of 2520 base pair DNA encoding 65 kD Mycobacterium tuberculosis specific protein (the primers are TB-1, -2), and 123 base pair DNA, a part of IS6110 fragment, which multiple copies are known to exsist PCR one genome (the primers are Sal I-1, -2). We also requested AFB staing and culture to the lab of Seoul National University Hospital with the same sample and compared the results. Results: 1) Using TB-1, -2 primers, PCR was positive in 73.1% (19/26) of culture positive sputa, in 12.5% (1/8) of culture negative. but clinically diagnosed tuberculous sputa, and was negative in all sputa of patients who were clinically diagnosed as non-tuberculous etiology. 2) Using Sal I-I, -2 primers, PCR was positive in 94.1% (32/34) of culture positive sputa, in 23.1% (6/26) of culture negative, but clinically diagnosed tuberculous sputa, and was negative in 87.5% (14/16) of sputa from patients who were clinically diagnosed as non-tuberculous etiology. Conclusion: PCR could be a very rapid, sensitive and specific method for the diagnosis of Mycobacterium tuberculosis in sputa, and further studies should be followed for the development of easier method.

• The Journal of Korean Society of Virology
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• v.29 no.1
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• pp.39-44
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• 1999

Warts, or verrucae, are benign epithelial proliferations of the skin and mucosa caused by infection with human papillomaviruses (HPV). It is now recognized that there are many different HPV types. Especially type3 is most frequently observed in flat wart. Other types, such as type2, 10, 14, 27, 28, 29, 38, and 41 are rarely encounted in flat wart. We describe here a simple and economic method for detection and identification of epidermodysplasia verruciformis-associated HPV. The method is based on polymerase chain reaction (PCR) amplification and restriction analysis. The method has been developed with cloned HPV DNA and DNA from clinical samples. Clinical samples are from either frozen tissue or paraffin-embedded tissue. Genomic fragments were obtained from two different HPV types (3 and 10). The amplification fragments were identified by a form of miniature fingerprinting, with a set of restriction enzymes that gave a unique digestion pattern for each HPV type. We have tested 74 clinical samples. Only type3 among these clinical samples is detected, and one sample is involved in neither type3 nor type10.

• Journal of Dairy Science and Biotechnology
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• v.41 no.3
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• pp.103-112
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• 2023

This study aimed to assess the effectiveness of 10 different pre-enrichment methods using Real-Time polymerase chain reaction (PCR) in support of the FDA method. When the initial Cronobacter spp. (Enterobacter sakazakii) inoculation was 7.2 CFU/g, the Ct values were observed in the following order: 21.37 (Enterobacteriaceae enrichment [EE] broth), 21.95 (brain heart infusion [BHI]), 22.72 (tryptic soy broth [TSB]), 23.02 (violet red bile lactose [VRBL]), 22.31 (TSB-0.1% sodium pyruvate [SP]), 23.43 (distilled water [DW]), 24.34 (phosphate buffered saline [PBS]), 24.95 (nutrient broth [NB]), 25.82 (TSB-0.6% yeast extract [YE]), and 28.27 (violet red bile glucose [VRBG]). For an inoculation of 1.82% CFU/g of Cronobacter spp. (E. sakazakii), the Ct values were recorded in this sequence: 20.34 (EE broth), 22.16 (TSB-0.6% YE), 22.37 (BHI), 22.71 (VRBL), 22.88 (TSB), 23.01 (DW), 23.19 (NB), 23.79 (TSB-0.1% SP), 24.66 (VRBG), and 24.70 (PBS). Finally, when the inoculum of Cronobacter spp. (E. sakazakii) was 0.182 CFU/g, the Ct values followed this order: 21.93 (VRBL), 23.07 (TSB-0.6% YE), 23.31 (DW), 23.47 (PBS), 23.70 (BHI), 24.14 (TSB-0.1% SP), 25.14 (TSB), 29.00 (VRBG), 31.55 (EE broth), and were undetected in the case of NB. Consequently, these results indicate that there were no significant differences among the 10 different pre-enrichment broths. Future studies should focus on exploring pre-enrichment broths that can improve the limit of detection at very low Cronobacter spp. (E. sakazakii) concentrations and enhance the selective recovery of Cronobacter spp. (E. sakazakii) under acid, antibiotic, cold, and heat damage conditions.

• Clinical and Experimental Pediatrics
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• v.51 no.11
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• pp.1191-1197
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• 2008

Purpose : Previously, Epstein-Barr virus (EBV) infection was diagnosed by serological examination; currently, many EBV antigen detection methods have been developed and applied clinically for diagnosing EBV infection. To delineate the clinical characteristics of EBV infection, clinical and laboratory findings were evaluated for patients who tested positive in EBV polymerase chain reaction (PCR). Methods : EBV PCR was conducted in 352 patients admitted to the pediatric ward from January 2004 to December 2006, with more than 2 clinical signs such as fever (${\geq}37.5^{\circ}C$), exudative throat infection, lymphadenopathy, hepatitis of unknown etiology, and splenomegaly. The EBV viral gene was detected by PCR in 115 patients (32%), and the clinical characteristics of these patients were evaluated. Laboratory findings such as leukocytosis, thrombocytopenia, atypical lymphocyte, and alteration in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in peripheral blood were examined. The EBV-specific immunoglobulin M antibody (EBV-IgM Ab) was also tested. Results : Most of the children were younger than 8 years (89%), and the male to female ratio was 1.3:1. Exudative throat infection and fever (${\geq}37.5^{\circ}C$) were observed in all patients. Cervical lymph node enlargement was seen in 36 patients (31 %); leukocytosis ($WBC{\geq}10,000/mm^3$), in 54 patients (47%); and atypical lymphocyte (${\geq}20%$), in 28 patients (24%). EBV-IgM Ab was positive in 33 patients (29%). The younger patients had higher ALT levels and higher incidence of positive EBV-IgM Ab than the older patients. Conclusion : The cumulative number of patients diagnosed to have EBV infection by PCR increased markedly for those under 8 years. ALT was higher and EBV-IgM Ab was detected more in younger patients with EBV infection.

• Microbiology and Biotechnology Letters
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• v.48 no.2
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• pp.230-235
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• 2020

The global evolution of antibiotic resistance has threatened the efficacy of available treatment options with ravaging impacts observed in developing countries. As a result, investigations into the prevalence of antibiotic resistance and the role of plasmids are crucial. In this study, we investigated the presence and distribution of bla_TEM and gyrA genes, plasmid profiles, and the antimicrobial susceptibility pattern of Salmonella strains isolated from raw meat and stool sources across Niger State, Nigeria. Ninety-eight samples, comprising 72 raw meat and 26 stool samples, were screened for Salmonella spp. The antimicrobial susceptibility of Salmonella isolates to 10 commonly used antimicrobial agents was determined using the KirbyBauer disc diffusion method. Isolates were further analyzed for plasmids, in addition to PCR amplification of beta-lactamase (bla_TEM) and gyrA genes. A total of 31 Salmonella spp. were isolated, with 22 from raw meat (70.97%) and 9 from stool (29.03%). Salmonella spp. with multiple resistance patterns to ceftazidime, cefuroxime, ceftriaxone, erythromycin, ampicillin, cloxacillin, and gentamicin were detected. Ofloxacin and ciprofloxacin were found to be the most effective among the antibiotics tested, with 67.7% and 93.5% susceptible isolates, respectively. Nine (29.03%) isolates harbored plasmids with molecular sizes ranging between 6557 bp and 23137 bp. PCR amplification of gyrA was detected in 1 (3.23%) of the 31 isolates while 28 isolates (90.32%) were positive for bla_TEM. This study shows the incidence of antibiotic resistance in Salmonella isolates and the possible role of plasmids; it also highlights the prevalence of ampicillin resistance in this local population.

• The Journal of Korean Society of Virology
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• v.30 no.1
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• pp.29-37
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• 2000

The aim of this study was to investigate viral etiology in dilated cardiomyopathy (DCM) by polymerase chain reaction (PCR) or nested reverse transcription PCR (RT-PCR), and characterize the enteroviral RNA presented in the clinical specimens. Twenty-eight paraffin-embedded heart tissue samples were assayed to detect cytomegalovirus, herpes simplex virus type 1, type 2, parvovirus, adenovirus, and enterovirus (EV) with each specific primer. Of these 28 patients (mean age: 27, M: 24, F: 4), 26 were histologically diagnosed as DCM and 2 as myocardial infarction (MI). Nested RT-PCR detected enteroviral RNA in 7 (26.9%) of 26 patients with DCM, and none of patients with MI. And none of DNA viruses tested were detected from the samples. Amplified products were also genotyped by single-strand conformation polymorphism (SSCP). Three subtypes can be differentiated from 7 clinical specimens. Furthermore, direct sequence analysis was performed to determine whether genetic variation of EV is present in the explanted heart tissues from patients with DCM. Although most of the sequences among the wild isolates have the greatest similarity to those of coxsackievirus B3, there are specific regions of variable sequences (no 490 - no 510). The data suggest that enterovirus may be a major viral pathogen for the DCM in Korea and nucleotide sequence data indicate that coxsackievirus B3 may be a leading etiologic agent of DCM.

• Parasites, Hosts and Diseases
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• v.34 no.2
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• pp.135-142
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• 1996

Antigenic domain of jai or surface protein (p30) of Toxoplosmc Sondii was analyzed after polymerase chain reaction (PCR) of its gene fragments. Hydrophilic or hydrophobic moiety of amino acid sequences were expressed as glutathione S-transferase (G57) fusion proteins. Fragments of p30 gene were as follows: 737, total p30 open reading frame (ORF) ; S28, total ORF excluding N-terminal signal sequence and C-terminal hydrophobic sequence; Al9, N-terminal 2/3 parts of A28; A19, N-terminal 2/3 of S28; P9, C-terminal 2/3 part of S28; Z9. middle 1/3 of S28; and 29, C-terminal 1/3 of S28. respectively. Primer of each fragment was synthesized to include clamp sequence of EcoR I restriction site. PCR amplified DNA was inserted info GST (26 kDa) expression vector, PGEX-47-1 to transform into Escheri,hia coei (.JM105 strain). G57 fusion proteins were expressed with IPTG induction as 63. 54, 45, 45, 35, 36. and 35 kDa proteins measured by SDS-PAGE. Each fusion protein was confirmed with G57 detection kit. Western blot analysis with the serum of a toxoplasmosis patient revealed antigenicity in proteins expressed by T37. S28, and Al9 but not those by Pl8. X9, Y10, and Z9. Antigenicity of p30 seems to be located either in N-terminal 115 part in the presence of middle 1/3 part or in the oligopeptides between margins of the first and second 1/3 parts.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.8
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• pp.3471-3476
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• 2014

Background: Aberrant expression of the microRNA-29 family is associated with tumorigenesis and cancer progression. As transport carriers, tumor-derived exosomes are released into the extracellular space and regulate multiple functions of target cells. Thus, we assessed the possibility that exosomes could transport microRNA-29c as a carrier and correlations between microRNA-29c and apoptosis of bladder cancer cells. Materials and Methods: A total of 28 cancer and adjacent tissues were examined by immunohistochemistry to detect BCL-2 and MCL-1 expression. Disease was Ta-T1 in 12 patients, T2-T4 in 16, grade 1 in 8, 2 in 8 and 3 in 12. The expression of microRNA-29c in cancer tissues was detected by quantitative reverse transcriptase PCR (QRT-PCR). An adenovirus containing microRNA-29c was used to infect the BIU-87 human bladder cancer cell line. MicroRNA-29c in exosomes was measured by QRT-PCR. After BIU-87 cells were induced by exosomes-derived microRNA-29c, QRT-PCR was used to detect the level of microRNA-29c. Apoptosis was examined by flow cytometry and BCL-2 and MCL-1 mRNA expressions were assessed by reverse transcription-polymerase chain reaction. Western blotting was used to determine the protein expression of BCL-2 and MCL-1. Results: The expressions of BCL-2 and MCL-1 protein were remarkably increased in bladder carcinoma (p<0.05), but was found mainly in the basal and suprabasal layers in adjacent tissues. The expression of microRNA-29c in cancer tissues was negatively correlated with the BCL-2 and MCL-1. The expression level of microRNA-29c in exosomes and BIU-87 cells from the experiment group was higher than that in control groups (p<0.05). Exosome-derived microRNA-29c induced apoptosis (p<0.01). Although only BCL-2 was reduced at the mRNA level, both BCL-2 and MCL-1 were reduced at the protein level. Conclusions: Human bladder cancer cells infected by microRNA-29c adenovirus can transport microRNA-29c via exosomes. Moreover, exosome-derived microRNA29c induces apoptosis in bladder cancer cells by down-regulating BCL-2 and MCL-1.

• The Korean Journal of Nuclear Medicine Technology
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• v.23 no.1
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• pp.35-39
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• 2019

Purpose Hepatitis B virus (hepatitis B virus, HBV) infection is a worldwide major public health problem and it is known as a major cause of chronic hepatitis, liver cirrhosis and liver cancer. And serologic tests of hepatitis B virus is essential for diagnosing and treating these diseases. In addition, with the development of molecular diagnostics, the detection of HBV DNA in serum diagnoses HBV infection and is recognized as an important indicator for the antiviral agent treatment response assessment. We performed HBeAg assay using Immunoradiometric assay (IRMA) and Chemiluminescent Microparticle Immunoassay (CMIA) in hepatitis B patients treated with antiviral agents. The detection rate of HBV DNA in serum was measured and compared by RT-PCR (Real Time - Polymerase Chain Reaction) method Materials and Methods HBeAg serum examination and HBV DNA quantification test were conducted on 270 hepatitis B patients undergoing anti-virus treatment after diagnosis of hepatitis B virus infection. Two serologic tests (IRMA, CMIA) with different detection principles were applied for the HBeAg serum test. Serum HBV DNA was quantitatively measured by real-time polymerase chain reaction (RT-PCR) using the Abbott m2000 System. Results The detection rate of HBeAg was 24.1% (65/270) for IRMA and 82.2% (222/270) for CMIA. Detection rate of serum HBV DNA by real-time RT-PCR is 29.3% (79/270). The measured amount of serum HBV DNA concentration is $4.8{\times}10^7{\pm}1.9{\times}10^8IU/mL$($mean{\pm}SD$). The minimum value is 16IU/mL, the maximum value is $1.0{\times}10^9IU/mL$, and the reference value for quantitative detection limit is 15IU/mL. The detection rates and concentrations of HBV DNA by group according to the results of HBeAg serological (IRMA, CMIA)tests were as follows. 1) Group I (IRMA negative, CMIA positive, N = 169), HBV DNA detection rate of 17.7% (30/169), $6.8{\times}10^5{\pm}1.9{\times}10^6IU/mL$ 2) Group II (IRMA positive, CMIA positive, N = 53), HBV DNA detection rate 62.3% (33/53), $1.1{\times}10^8{\pm}2.8{\times}10^8IU/mL$ 3) Group III (IRMA negative, CMIA negative, N = 36), HBV DNA detection rate 36.1% (13/36), $3.0{\times}10^5{\pm}1.1{\times}10^6IU/mL$ 4) Group IV(IRMA positive, CMIA negative, N = 12), HBV DNA detection rate 25% (3/12), $1.3{\times}10^3{\pm}1.1{\times}10^3IU/mL$ Conclusion HBeAg detection rate according to the serological test showed a large difference. This difference is considered for a number of reasons such as characteristics of the Ab used for assay kit and epitope, HBV of genotype. Detection rate and the concentration of the group-specific HBV DNA classified serologic results confirmed the high detection rate and the concentration in Group II (IRMA-positive, CMIA positive, N = 53).