Background: Facing urgent requirements of the rapid diagnosis of tuberculosis nowadays, our laboratory have improved the multiple PCR for 3 target genes spontaneously (IS 1081, IS 6110, 23S rDNA) and developed into the multiplex PCR kit to improve the effects of diagnosis of tuberculosis. Objective: To evaluate the multiplex PCR kit for the 3 target genes (IS 1081, IS 6110, 23S rDNA) for the diagnosis of tuberculosis on standard panel and clinical specimens. Subjects and method: DNA extracted from M. tuberculosis strains and clinical specimens were amplified by multiplex PCR kit, comparison with singleplex PCR for each target gene. Results: On standard panel, sensitivity and specificity reached 100% and detected limitation was 100 fg DNA template (about 3 M. tuberculosis cells). Multiplex PCR kit detected 195/307 (63.52%) suspected clinical specimens compared with singleplex PCR which detected 106/307 (34.53%) for IS 1081, 182/307 (59.82%) for IS 6110 and 136/307 (44.30%) for 23S rDNA. Conclusions: PCR amplification with one target gene was insufficient for diagnosis of the tuberculosis strains in Viet Nam. Multiplex PCR kit for 3 target genes simultaneously (IS 1081, IS 6110, 23S rDNA) could detect all tuberculosis strains.
Tuberculosis ; PCR

Introduction: Carpanenamase-producing Enterobacteriaceae (CPE) has emerged as a threat to hospitalized patients. Phenotypic test such as Modified hodge test was less sensitive and specific especially to detect blaNDM-1 which is the most predominant genotype in this region. Nucleic acid amplification technology offers improved specificity and sensitivity. Failed amplification due to the presence of inhibitors is a limitation. In this study, we tried to use previous method described by Villumseen et al with some modification using another DNA extraction kit. Methods: Ten mls of sterile whole blood taken from nearly expired blood bag from blood bank was spiked with 200 µl of 0.5mcFarland bacterial suspension from thirty-six confirmed isolates of blaNDM-1 carbapenamase-producing Klebsiella pneumoniae in an aerobic Bactec Plus and incubated until the growth was detected. The blood specimen was subjected to DNA extraction method using Macherey-Nachel, Nucleospin® Blood QuickPure followed with multiplex PCR. Results: Out of the 36 isolates, 12 isolates revealed blaNDM-1 , 9 isolates revealed blaNDM-1 and blaOXA-48, 7 isolates revealed blaNDM-1, blaVIM and blaKPC genotypes that were amplified at cycle threshold of less than 30. Another 8 isolates could not pick up any genotypes possibly due to pipetting error as all the internal control were amplified. Eight true negative gram negative isolates underwent same procedure and none amplified at a cycle threshold less than 30. Conclusion: This modified method was proved to give a high yield of CPE genotypes with the cycle threshold was set at less than or equal to 30 and able to overcome the presence of PCR inhibitors.
PCR inhibitors

Introduction: Carpanenamase-producing Enterobacteriaceae (CPE) has emerged as a threat to hospitalized patients. Phenotypic test such as Modified hodge test was less sensitive and specific especially to detect blaNDM-1 which is the most predominant genotype in this region. Nucleic acid amplification technology offers improved specificity and sensitivity. Failed amplification due to the presence of inhibitors is a limitation. In this study, we tried to use previous method described by Villumseen et al with some modification using another DNA extraction kit. Methods: Ten mls of sterile whole blood taken from nearly expired blood bag from blood bank was spiked with 200 µl of 0.5mcFarland bacterial suspension from thirty-six confirmed isolates of blaNDM-1 carbapenamase-producing Klebsiella pneumoniae in an aerobic Bactec Plus and incubated until the growth was detected. The blood specimen was subjected to DNA extraction method using Macherey-Nachel, Nucleospin® Blood QuickPure followed with multiplex PCR. Results: Out of the 36 isolates, 12 isolates revealed blaNDM-1 , 9 isolates revealed blaNDM-1 and blaOXA-48, 7 isolates revealed blaNDM-1, blaVIM and blaKPC genotypes that were amplified at cycle threshold of less than 30. Another 8 isolates could not pick up any genotypes possibly due to pipetting error as all the internal control were amplified. Eight true negative gram negative isolates underwent same procedure and none amplified at a cycle threshold less than 30. Conclusion: This modified method was proved to give a high yield of CPE genotypes with the cycle threshold was set at less than or equal to 30 and able to overcome the presence of PCR inhibitors
PCR inhibitors

Background: To distinguish the different types of pathogenic E. coli with other non-pathogenic E.coli in the intestine is extremely important in diagnosis. Up to date there are at least six types of E. coli that causes diarrhea. Objectives: We have designed a multiplex PCR assay for the direct detection of 6 categories of diarrheagenic Escherichia coli. Subjects and method: This techniques proved to be specific and rapid for detecting virulence genes from Shiga toxin-producing (stx and eae), enteropathoogenic (eae), enterotoxigenic (elt, est), ennteroinvasive (ipaH), enteroaggregative (aggR), and diffuse adherent (daaE) Esscherichia coli. The technique was applied to 295 clinical stool specimens. Results: The highest prevalence is EAggEC with 51 positive samples.(17.29%), 48 EIEC (16.27%), 17 EPEC (5.76%), 8 ETEC (LT) (2.71%), 5 ETEC (ST) (1.69%), 1 DAEC (0.34%), no STEC positive and 19 mix infections (6.44%). Conclusion: Multiplex PCR assay is a quick and highly accuurate technique. It is not only specific but can also amplify 7 virulence genes of diarrrheagenic E.coli at the same time. This method would offer an effective alternative to traditional culture methods for the identification and differentiation of human diarrhaegenic Escherichia coli.
direct PCR ; E.coli

Background: Fungal keratitis is a serious ocular infection that can cause corneal scarring and blindness. Currently, diagnosis of fungal pathogens remains a difficult problem. Objectives: To investigate the application of semi-nested PCR targeted ITS genes for detection of fungal agents causing keratitis. Material and method: Ten identified fungal strains, 4 bacterial strains, 20 scraping samples from patients with suspected fungal keratitis and 2 scraping samples from patients with suspected bacterial keratitis were tested using semi-nested PCR. Results: Semi-nested PCR showed positive results for the samples of identified fungal strains and for the 20 scraping samples from patients with suspected fungal keratitis. Neither samples of bacterial strains nor scraping samples from suspected bacterial keratitis patients gave positive PCR results. Conclusion: Semi-nested PCR is a robust tool for specific and rapid detection of fungal agents causing keratitis.
Fungal keratitis ; semi-nested PCR

Background: It is important to diagnosis and properly treat patients with diarrhea is having a highly sensitive and specific technique to rapidly identify the caused bacteria, especially Diarrheagenic Escherichia Coli (DEC) isolates. Objectives: To develop and complete the bacterial DNA extraction procedure and optimize the DNA concentration for multiplex PCR for DEC. Materials and method: 7 reference strains of DEC and 10 fecal samples taken randomly were tested using DNA extraction and PCR techniques. Results. A bacterial DNA extraction procedure has been developed and optimized. This is a simple process and does not require expensive equipment. The test result is available after 90 \u2013 100 minutes. The minimum DNA content required for PCR to give positive results is 100ng per reaction. Conclusion: The development and completion of DNA extraction procedure plays an important role in early detection of DEC in fecal samples and serves as a base for further research on diarrheagenic bacteria.
DNA extraction ; PCR ; Escherichia coli

Background: Deletion and duplication mutations of dystrophin gene make up from 70 to 75% of patients with Duchenne Muscular Dystrophy (DMD). Two thirds of children with DMD inherited from the heterozygous mothers the mutated gene which is located on one of the sex chromosomes. Objective: To detect the asymptomatic carriers of dystrophin gene mutation using molecular techniques. Subject and methods: 3 DMD patients and their 9 relatives. Using techniques: DNA extraction and quantitative Polymerase Chain Reaction (PCR). Results: Successfully detected 4 heterozygous individuals from 9 female members of three different families that have already confirmed DMD patients. Conclusion: This method could lead to a new way of prenatal diagnosis of DMD as well as other genetic disorders that are caused by deletion or duplication mutation.
Duchenne muscular dystrophy ; carrier ; quantitative PCR

Background: The multiplex polymerase chain reaction can rapidly differentiate taenia spp adults and cysticercose cysts.\r\n', u'Objective: The study aimed to identify taenia spp adults and cysticercose cysts in human by using multiplex PCR\r\n', u'Subjects and methods: The multiplex PCR was applied by using the 4 forward and one reverse primer to amplify the target gene cytochrome c oxydase subunit I (COXI) of the Taenia spp in human. T\r\n', u'Results:4 molecular sizes of PCR products were appeared: 269 bp,720 bp, 827 bp, 984 bp. Seventy six samples including 65 flat worms and 11 cysticercose cysts which collected from the patients who are living in 19 different provinces and cities of Northern part of Viet Nam and treated in the clinic of NIMPE were examined. Of 65 flat worms analyzed 35 samples were T.asiatica (58.46%), 27 were T.saginata (41.54%). All 11 cysticercose cysts were T.solium (100%). \r\n', u'Conclusion: The result also indicated that some time 3 Taenia species were found at the same area. A remarkable difference of infection rate was found between men and women, also adults and children. \r\n', u'\r\n', u'
Taenia spp adults ; cysticercose cysts ; multiplex PCR

INTRODUCTION: The role of the laboratory during the COVID-19 pandemic is not limited to just diagnosis of the disease, but also in clinical decision-making, by providing information on relevant laboratory biomarkers. Clinicians also use Ct value to guide patient management. There are limited studies available locally regarding the significance of Ct value and pertinent laboratory biomarkers in COVID-19 patients. This study aimed to assess the aforementioned laboratory data, along with the clinicopathologic characteristics of affected patients, and determined if this information may be useful for robust clinical decision-makin METHODOLOGY: In this retrospective analytic study, we identified 325 out of 1,049 adult Filipino inpatients diagnosed with COVID-19 and analyzed their Ct values and pertinent laboratory biomarkers such as neutrophil and lymphocyte count, platelet count, LDH, ferritin, procalcitonin, CRP, AST/SGOT, ALT/SGPT, PT/ INR, and D-dimer, and correlated them with the severity of the disease. RESULTS: Two hundred twenty (67.7%) patients had non-severe disease, while 105 (32.3%) had severe disease. Lower Ct values of ORF1ab (median = 26.4) and N (median = 24.8) genes were seen in the severe group compared to the non-severe group and were found to be significant (p<0.001). Laboratory markers (neutrophil, platelet counts, LDH, ferritin, procalcitonin, CRP, AST, PT/INR, and D-dimer) were associated with severe COVID-19. On the other hand, ALT was not associated with severe disease. CONCLUSION The laboratory biomarkers together with Ct value and overall clinical picture may provide valuable information to physicians for more robust clinical decision-making.
COVID-19 ; laboratory biomarkers ; SARS-CoV-2 ; RT-PCR