1. NAME; BEDAN W KANGETHE BSC. BIOCHEMISTRY, NPHLS NHRL

3. During the past decade there has been a dramatic reduction in the transmission of Human Immunodeficiency Virus Type 1 Types tests carried out are;  Viral antibody, antigen,  Nucleic acids  Viral culture Body fluids used to test are; Saliva, CSF, blood and organs The sources of risk of HIV-1 and HCV transmission are;  Marker-negative “window period”  Blood donors infected with immunovariant viral strains  persistent antibody-negative (immunosilent) carriers,  Laboratory test procedure errors.

4.  A total of 800 plasma samples were obtained from 4000 blood donors in NBTC in Nairobi Kenya  Samples were collected as whole blood in EDTA tubes  Samples size was achieved by taking every 5th sample in random sampling to reduce bias rates

5.  All the ELISA negative samples for HIV, HCV,RPR and HCV qualified for the study  All samples were retested for ELISA for confirmation  The sample tracking forms were filled each affixed with blood donors’ barcode number and study number  The rejected samples were removed and sample rejection form filed with reason clearly stated  The plasma in the cryovials were stored at -20 degrees centigrade awaiting extraction  The samples that took longer time were transferred to -80 degrees

6.  RNA extractions was done using Cobas Amplicor version 1.5 protocol  600ul of lysing was used to lyse the plasma cells to release the virions  The tubes were labeled with study number and the controls as; NC, LPC, and HPC, 50 ul of the controls and 200ul of normal plasma were added to their respective tubes and 200ul of the plasma samples were added to tubes labeled with the study number

7.  The mixtures were vortexed for 5min and incubated at RT for 10 min  800 mls of isopropanol was added to clean the RNAs  The tubes were vortexed for 5min and centrifuge in a micro centrifuge at 14000 RPM for 15 min  The isopropanol was aspirated using a sterile fine tip pipettes.  1000mls of 70% ethanol was added to the pellets to solidify them and the tubes span at 14000 RPM for 5min.  The ethanol was completely aspirated making sure no amount is left.  400mls of HIV diluent was added and the pellet was dissolved.  The extracted RNA was headed to the amplification or stored at -20 for 7 days but if longer at -80 degrees.

8. The amplification was done by RT PCR. The order was created starting with NC, LPC, HPC and the samples according to the A-ring maps,  The A-rings were loaded in the thermocyclers.  The A- rings contained 50ul of the extract and 50ul of master mix which carries the primers and the nucleotides.  The cycling took about 2 hrs and detection took 3 hrs. The quantified copies of the viral copies are reflected in the software in copies/ml or not detectable if negative.

9.  All the 800 sample were subjected to the 2nd ELISA and initial PCR run.  And the results tabulated.  798 sample showed both Elisa and PCR negative  2 samples tested positive

11.  The RNA extraction procedure described here is tolerable and easy to perform.  The detection limit of the Amplicor RT PCR on plasma was 400 to 700,000 copies per ml hence giving a good range of detection.  The number of indeterminate samples probably due to the presence of PCR inhibitors was significantly reduced.

12.  The sensitivities of the Amplicor RNA RT PCR test and of the 4th generation were 100%, respectively.  The RT-PCR primers were able to amplify fragments from the pol, env, and gag regions, respectively.  Previous results have shown that most of the patients in Kenya are infected with HIV-1 subtype A. Therefore, one can speculate that both the RT PCR and 4th ELISA could be used for HIV-1 detection in regions where non-B subtypes are predominant.

13.  In conclusion, the RNA extraction method from plasma, is easy to perform, and permits reliable diagnosis of HIV-1 infection when combined with RT PCR.  Moreover, no difference of sensitivity was observed in the assays in blood donors.  The most ideal time to perform HIV-1 diagnosis would be shortly after sample collection for better quantification.

14.  The choice of one test instead of the other depends on the setting in which it is to be used.  Further evaluation of this has been taken by some partners on DBS and the results correlated well with this study  The entire procedure is now being performed in NHRL and few regional laboratories in management of HIV infection.  Viral load is ran to assess the viremia levels on patients who are not responding to treatment.

15.  Although the risk of transmitting infection to transfusion recipients in Kenya has been drastically reduced the window period menace is still a hindrance.  The current estimated risks for transmission of HCV and HIV by a single antibody negative component are 1/150,000 and 1/676,000 respectively (see Goodnough et al).

16.  Because viremia precedes seroconversion by several days to weeks, tests that detect viral nucleic acids are more sensitive than current screening tests.  While nucleic acid amplification test technologies (NAT) are available, they have not been fully applied in MOH Kenya.  The recent development of commercially- available assays using NAT has made it possible for blood centers to consider applying these tests to blood donor screening.

17.  In this study even though showed there was no much difference between the PCR and Elisa in screening of donors, there is high demand of PCR to create airtight safety to our blood recipients.  In Kenya this study has noticed that very few experts are trained on the NAT technology.

18.  Thanks to my NHRL manger, Mr. Mamo Umuro for allowing use of the laboratory for the study  To director NBTC, Dr. Margaret Oduor for giving me permission to use the samples from her institution  To NASCOP blood safety officer, Mr. Mwalo for timely availing the tests kits for this study  To Kelvin Alumasi who was research assistant