In vitro transcription of HIV-1 RNA for standard RNA.

Background: The quantitative assays are important tests in the management of patients with HIV-1/AIDS. The important step in developing the assay is the availability of the standard HIV-1 RNA. For this purpose, we optimized in vitro HIV-1 RNA transcription to produce the standard HIV-1 RNA. Methods: The HIV-1 DNA was amplified from pNL43 by PCR using a primer pair that was specific for conserved region of HIV-1 Gag gene. The PCR product was further cloned into pBluescript II KS. The recombinant plasmid was restricted with EcoRI enzyme. Then, the linearized plasmid was used as template for RNA transcription. RT-PCR and PCR were performed simultaneously for confirmation of synthesized RNA fragment. Results: A 115 bp DNA of HIV-1 Gag gene has been cloned into pBluescript II SK with the exact true orientation. The reaction of the RNA transcription was also successfully performed. The RNA transcripts have been confirmed and showed the accuracy of the transcripts. Conclusion: We successfuly constructed the recombinant plasmid containing a conserved region of HIV-1 Gag gene, and the HIV-1 RNA has been transcribed in vitro as well.

A second generation of RT-PCR assay for detection of human immunodeficiency virus type 1 (HIV-1) infection.

Aim A spesific and rapid diagnosis such as RT-PCR asay is the most needed to minimize transmission of HIV-1 infection. Therefore, in this study we developed the RT-PCR assay that was spesifi c against the gag gene of HIV-1. Methods The developed RT-PCR assay was evaluated against 46 specimens that were obtained from voluntary counseling and testing for HIV (VCT) in Rumah Sakit Umum Pemerintah (RSUP) Sanglah, Bali. To get the sensitivity and specifi city of RT-PCR assay, the results of assays were compared with the results of commercially serologic tests that were commonly used in Indonesia. Results The RT-PCR assay could detect 21 of 26 serologic test-positive specimens and showed 19 negative results of 20 serologic test-negative specimens. There was one specimen that was positive in RT-PCR but negative in serologic assay, which might depict a true yield at particular condition when the serologic assay was unable to detect. Five serologic positive-test specimens were negative by RT-PCR that was possibly caused by low detection level of the assay. Conclusion The RT-PCR assay is potential to be used for the detection of HIV-1 infection with a sensitivity and specificity of 80.8% and 95.0% respectively.

Antibody anti-H5N1 detection in poultry farmers and workers in poultry collection facilities in Indonesia, 2007.

Aim Between July 2005 and May 2008, Indonesia reported 133 H5N1 confi rmed human cases with a case fatality proportion of 81%. Fifty-four percent of cases had a history of direct contact with poultry (chickens). Therefore, it is important to defi ne the detection of antibody of H5N1 among people who have intensive contact with poultry have been exposed to H5N1 viruses. Methods We collected sera from healthy poultry-collecting-facility (PCF) workers in Jakarta and healthy poultryfarmers in Sukabumi which have close contact with poultry. Anti-H5N1 antibodies were tested with modifi ed Haemagglutination Inhibition (HI) assay using A/Ck/Banten/05-1116/05(H5N1) antigen and with Neutralization (NT) assay using A/H5N1/Indo/05/IBCDC-RG virus. Results Among the 216 PCF worker sera and the 495 poultry-farmer sera that we collected, we found that all poultryfarmers were seronegative and one percent of poultry-collecting-facilities workers were seropositive by both HI and 1% by NT assays. Conclusions This study detected asymptomatic H5N1 virus infection among poultry workers in PCFs with intensive contact with various types of different poultry who had different titers of antibody, but no antibodies were detected among poultry farmers.

Development of multiplex-PCR assay for rapid detection of Candida spp.

Aim Candida spp. infection commonly occur in immunocompromised patients. Biochemical assay for identifi cation of Candida spp. is time-consuming and shows many undetermined results. Specifi c detection for antibody, antigen and metabolites of Candida spp. had low sensitivity and specifi city. In this study, we developed a rapid diagnostic method, Multiplex-PCR, to identify Candida spp. Methods Five Candida spp. isolates were cultured, identifi ed with germ tube and API® 20 C AUX (BioMerieux® SA) kit. Furthermore, DNA was purifi ed by QIAamp DNA mini (Qiagen®) kit for Multiplex-PCR assay. Results DNA detection limit by Multiplex-PCR assays for C. albicans, C. tropicalis, C. parapsilosis, C. krusei and C. glabrata were 4 pg, 0,98 pg, 0,98 pg, 0,5 pg and 16 pg respectively. This assay was also more sensitive than culture in that Multiplex-PCR could detect 2.6-2.9 x 100 CFU/ml, whereas culture 2.6-2.9 x 102 CFU/ml. Conclusion Multiplex-PCR is much more sensitive than culture and thus, can be recommended as a sensitive and specifi c assay for identifi cation of Candida spp.