Partly 5` Untranslated Region (5` UTR)-Based Phylogenetic Analysis of Three Hepatitis C Virus Isolates From Jakarta, Indonesia: a Preliminary Study

Currently, we reported results of a nested PCR assay specific 5` UTR region of HCV genome that showed three different patterns of DNA fragments (single expected specific DNA band, single DNA band higher in size than an expected band, and multiple DNA bands). Three isolates (Isolate A, B, and C), representing all the three DNA bands, were analyzed by using phylogenetic trees. The results showed that the Isolate A, B, and C were classified into HCV genotypes 2, 1, and 3, respectively. The Isolate A and B were very closely related to viral isolates from Madagascar and Brazil, respectively and were not closely related to other Indonesia isolates. In contrast with the Isolate A and B, the Isolate C was very closely related to another Indonesia isolate. Among all there isolates, the Isolate C was very closely related to an Indonesia isolate detected from a cirrhosis patient, indicating that the Isolate C might be more virulence than the Isolate B and C. However, a complete genome-based comprehensive genetic characterizationfor all the three isolates needs to be conducted in future research to confirm all findings in this study

Untranslated region-5' and viral protein 1-based genetic stability analysis of bulk polio in Indonesia 2010-2019

Cases of vaccine-associated paralytic poliomyelitis (VAPP) continued increasingly from 2010-2019 in the world. Oral polio vaccine (OPV) is the live attenuated virus-based vaccine that could genetically revert to neurovirulent during the vaccine production process or when the virus replicates in the human body. The poliovirus neurovirulence is determined by the UTR-5' region and VP1 coding region. UTR-5' played a role in protein translation and VP1 was responsible for the immunogenicity of the virus. Some reported mutations in UTR-5' and VP1 could affect the neurovirulence of poliovirus. In this study, we analyzed the genetic stability of the UTR-5' and VP1 in the bulk of OPV types -1 and -3 produced in 2010 - 2019. The results of the analysis of UTR-5' sequences in Sabin strain types-1 and -3 and VP1 sequences on Sabin virus type 1 did not show any mutations. Meanwhile, the VP1 sequences in Sabin strain type 3 showed nucleotide mutation C2493U that caused the substitution amino acid Thr6Ile amino acid in all samples of the type 3 bulk polio test. Based on the results of in silico analysis, this mutation in VP1 did not contribute significantly to the neurovirulence of the virus

Optimizing Real-Time PCR methods for detection of ssaN gene Salmonella enterica subsp.enterica in the blood specimen

Background Typhoid fever caused by Salmonella typhi is a common acute infection of the reticuloendothelial system, intestinal lymphoid tissue, and gall bladder. Detection of Salmonella spp. is still based on cultures and serological methods.Widal test is one of the serological tests that is still widely used, especially in developing countries including Indonesia.Widal tests have low sensitivity and specificity. They often produce false positive or false negative results.ObjectiveThe aim of this study were i) real time PCR optimization to develop a Salmonella enterica detection system. ii) molecular detection of new target gene (ssaN gene) from blood specimens in typhoid fever patients.Methods An experimental laboratory study was performed from March to October 2016. Extraction of Salmonella typhi DNA is used as templates for the optimization of real time PCR reaction.The blood sample was from patients suspected with typhoid fever obtained from the Menteng Sub-district Health Center according to the inclusion criteria.ResultsSpecificity test of real time PCR showed that the primers and probes used are not cross-react against other microorganisms. Sensitivity test obtained minimal detection is at least 10 cfu/ml of blood specimen. In blood clinical specimens, real time PCR could detect 19 (38%) positive samples of 50 blood specimen from suspected typhoid fever patients. Eleven samples with negative Widal serology gives positive results in real time PCR.ConclusionReal time PCR used in this study can increase the level of rate of positive testing by 22% of the total specimens.Keywords : Salmonella enterica subsp.enterica, typhoid fever, ssaN gene, real time PC

Detection of Human Group a and C Rotaviruses in Pediatric Patients with Acute Gastroenteritis by Real TIME RT-PCR Assay: a Preliminary Study

Rotavirus causes 25–55% of all hospital admissions for diarrhea and approximately 611.000 deaths every year in developing countries. Clinically, it is not possible to recognize the diarrhea caused by rotavirus and other infections. To know a causative agent of rotavirus gastroenteritis, availability of an accurate diagnosis assay is necessary. Therefore, we developed real time RT-PCR assay (rRT-PCR) assay for confirmation of infections of Group A or C rotaviruses simultaneously. A total of 54 stool samples obtained from pediatric patients (< 5 years old) was used in this study. All samples were tested for Group A rotavirus by Serological rapid test. Result of serological rapid test was compared with rRT-PCR assay to obtain the test accuracies of both assays. Result of this study showed that rates of positive testing for Group A rotavirus by serological rapid test and the rRT-PCR assay were 22.22% and 18.50%, respectively. Forty-two serology-negative specimens for Group A rotavirus were also PCR negative (100% specificity). Two serology-positive specimens for Group A rotavirus was rRT-PCR negative (confirmed by electrophoresis gel); therefore, rRT-PCR assay represents the decrease of 3.70% in the number of specimens that are positive for Group A rotavirus. For Group C rotavirus, all tested samples were no rRT-PCR positive and the results need to be confirmed in the future

Comparison of RT-PCR-Dot Blot Hybridization Based on Radioisotope 32P with Conventional RT-PCR and Commericial ELISA Assays for Blood Screening of HIV-1

There are many commercial ELISA and rapid test kits that have been used for blood screening; however, the kits can give false positive and negative results. Therefore, RT-PCR (Reverse Transcription Polymerase Chain Reaction) - Dot Blot Hybridization based on radioisotope 32P (RDBR) method was developed in this research, to compare the method with the conventional RT-PCR and commercial ELISA Enzyme-Linked Immunosorbent Assay) kit. This method is efficient for screening of large blood specimens and surveillance study. Eighty seven samples were used and serum of the samples were tested by ELISA to detect HIV-1. The HIV-1 RNA genome was extracted from plasma samples and tested using the RT-PCR and RDBR methods. Of 87 samples that were tested, the rates of positive testing of the RT-PCR, the RDBR, and the ELISA were 71.26%, 74.71%, and 80.46%, respectively. The RDBR (a combination of RT-PCR and dot blot hybridization) was more sensitive than conventional RT-PCR by showing 3.45% in increase number of positive specimens. The results showed that of 9 samples (10.34%) were negative RDBR and positive ELISA, while 4 samples (4.60%) werenegative ELISA and positive RDBR. The two methods showed slightly difference in the results but further validation is still needed. However, RDBR has highpotential as an alternative method for screening of blood in large quantities when compared to method of conventional RT-PCR and ELISA

The effect of dadih in BALB/c mice on pro-inflammatory and anti-inflammatory cytokine productions

The normal microflora formed as commensal bacteria have roles in maintaining homeostasis in the intestine tract. The reduction in the amount and on the diversity of the commensal bacteria lead to gastrointestinal dysbiosis which increase number of pathogens, induce inflammatory and can drive to colorectal cancer. Probiotics can be used to prevent, regulate, and modulate immune response by triggering the development of pathogen-specific memory. Currently, many foreign probiotic products are available in the market that cause the domestic products are less well known. Dadih is an original probiotic’s products originally from West Sumatra, Indonesia. It is made from fermented buffalo milk containing lactic acid bacteria (LAB). The objective of the study was to investigate the effect of dadih pro-inflammatory and anti-inflammatory cytokine production. The study was conducted using male BALB/c mice aged 6-8 weeks with body weight (BW) 20-30 g. Mice were given dadih at doses of 112 mg/20g BW for eight weeks. The results indicated that LAB bacteria in dadih are coccus, Gram-positive bacteria with 3x107 colony-forming units (CFU) and dominated by Lactococcus lactis subsp. lactis. In addition, the increase of both the anti-inflammatory cytokines (IL-10) and pro-inflammatory cytokines (TNF-α and IL-1β) was observed. In conclusion, the dadih can be used to maintain the immune system of mice

Detection of Opportunistic Fungus Pneumocystis jirovecii Major Surface Glycoprotein (MSG) gene in HIV-AIDS Patients with Pneumoniae in Jakarta

Pneumocystis jirovecii is known to cause opportunistic infections in the lower respiratory tract in individuals with low immune systems, especially patient with HIV infection. The prevalence of P. jirovecii pneumonia (PjP) in various countries show varying numbers. In Indonesia, HIV cases continue to rise. However, the data in Indonesia concerning the case of PjP is very limited. Until now the prevalence of PjP in Indonesia is only based on clinical symptoms of the patient. Currently, diagnosis of PjP relies on microscopic examination. The disadvantage of this examination is not easy to do and has a high negative predictive value. Thus, this study was conducted to develop a molecular test to diagnose PjP infection in HIV-AIDS suspected pneumonia. Molecular diagnostic test aimed for Major Surface Glycoprotein (MSG) gene of P. jirovecii detection was done through real-time PCR against 100 sputum samples. Demographic data show that the prevalence of PjP infection in HIV-AIDS suspected pneumonia patients in Jakarta is 20.0%, male 75% within 31-40 y.o (35%), dominant (80%) from patients with CD4+ T-lymphocytes of 200-349 cells/µL. Molecular real-time PCR methods were shown to give five times sensitivity higher than Giemsa stain.   Keywords: P. jirovecii, HIV, real-time PC

Gene S Characterization of Hantavirus Species Seoul Virus Isolated From Rattus Norvegicuson an Indonesian Island

Background: Hantavirus lives and reproduces in the body of rodents. Rattus norvegicuswas one found in the Kepulauan Seribu islands of Indonesia. Hantavirus species Seoul virus (SEOV) is a negative single chain RNA viruses included in the family Bunyaviridae. It has a few specific genes, especially genes S that can be developed for a diagnostic test. The aim of this study was to ascertain the character of gene S of hantavirus species Seoul virus. Methods: Gene sequencing of S Seoul virus from lung tissue of rodents was conducted. DNA fragment sequencing used primer pairs of SEOS-28F and SEOS -360R, VNS-1501F and VNS-CSR. The results of sequencing were analyze by seqscapeprogram to obtain a sequence of nucleotides, and analyzed by Mega5 programs. Phylogenetic analysis was done for homology nucleotides and amino acids which were compared to other hanta virus species from the gene bank. Results: The comparison analysis showed, the highest homology from strain KS74 was 88.4% and the lowest from strain KS90 was 87.2%. The highest homology of amino acids sequence compared with Seoul virus came from strain KS74 was 91.3% and the lowest came from strain KS90 was 89.5%. Conclusion: Gene S of viruses was found in Kepulauan Seribu in Indonesia and it was comparable to that found in Singapore and Korea. (Health Science Indones 2014;1:1- 6)