A pilot program of a dry blood spot HIV-1 RNA method for ...

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  • Good afternoon. I’m very pleased to talk to you today about our pilot program using a dry blood spot HIV RNA method for early infant diagnosis and viral load monitoring in rural Tanzania.
  • During the next 20 minutes I will talk about the challenges for early infant diagnosis and viral load monitoring in sub-Saharan Africa that led us to design the program Then I’ll discuss some potential solutions to these challenges. Next I’ll talk about the HIV RNA capacity in northern Tanzania. Then I’ll talk about the design of the program that I was the study coordinator for. I’ll look at some of the results and how the performance of DBS holds up against liquid plasma. I will review the overall program performance and then focus on how clinicians and patients felt about the program. Then I’ll talk about challenges we encountered while completing the program. Then some of the next steps after we have finished this pilot program and finally I’ll acknowledged the large group of collaborators who worked on this program.
  • So what are some of the challenges with early infant diagnosis of HIV infection? As we know, antibody-based diagnostic methods are useful for adults and older children, but are not reliable for infants <18 months due to the presence of maternal antibody. Early infant diagnosis can be central to ensuring that lifesaving antiretroviral therapy can be initiated and other management decisions can be taken. Nucleic acid amplification tests provide a means for early infant diagnosis of HIV but traditionally have relied on liquid plasma samples. However, liquid plasma samples pose challenges for remote healthcare services because they must either be tested locally using expensive and complex equipment or frozen at -80 C and transported on dry ice to a central laboratory.
  • Now let’s look at challenges for viral load monitoring in sub-Saharan Africa. As we know, there has been a rapid expansion of HIV treatment and care programs in sub-Saharan Africa. Most patients receiving ART through these programs are monitored using clinical and immunologic assessments. However, we know that virologic failure usually occurs sometime before treatment failure can be detected on clinical or immunologic grounds. Thus, viral load monitoring would be useful to assess response to ART and the maintenance of virologic suppression. However, viral load monitoring faces similar technologic and logistic concerns to those just described for early infant diagnosis.
  • So what are some potential solutions to the problems of providing early infant diagnosis and viral load monitoring services to rural and remote parts of sub-Saharan Africa? One possibility would be the development of alternative nucleic acid tests that are simple and cheaper and could feasibly be deployed to the district hospital level. A number of groups are working on such technologic advances. Another potential solution is an alternative sample type to liquid plasma that could be easily prepared, stored, and transported to a central laboratory that maintains equipment and expertise for nucleic amplification testing. Dry blood spots or DBS represent a potential alternative sample type that may be better suited for use in rural and remote areas. As we know, DBS DNA PCR for early infant diagnosis is well validated and widely used. DBS RNA PCR for early infant diagnosis has been less well studied, but the possibility of a single DBS RNA PCR platform for early infant diagnosis and potentially for viral load monitoring that works under field conditions is appealing. This is the focus of our work in northern Tanzania.
  • Let me briefly describe current HIV RNA capabilities in northern Tanzania. Under our management, the Kilimanjaro Christian Medical Centre or KCMC Biotechnology Laboratory in Moshi has established the only HIV RNA service in northern Tanzania. In February 2006 we acquired an Abbott m2000rt instrument which we operated with manual sample preparation. In February 2008, the service was upgraded to the Abbott m2000system by adding automated sample preparation. The instrument was used initially for patient care early infant diagnosis and viral load monitoring and more recently for research studies. Since establishing the HIV RNA service, we have received a large number of requests to expand the reach of the service beyond the greater Moshi area.
  • These pictures shows our staff operating the Abbott m2000system; automated sample preparation on the left and amplification and detection on the right. At present we perform between 500 and 1,000 HIV RNA tests per month. In order to expand the reach of this service, we designed a pilot program of a dry blood spot HIV RNA method to assess the feasibility of expanding access of our HIV RNA service to rural and remote healthcare facilities in northern Tanzania and to evaluate the performance of a DBS program under field conditions.
  • We partnered with two rural and remote hospitals in northeastern Tanzania shown on this map. Magunga Hospital is located in Korogwe District 300 kilometers from our laboratory in Moshi and and Teule Hospital is located in Muheza District 350 kilometers from Moshi. Both hospitals are located on a highway between the larger towns of Moshi and Tanga. The hospitals are small and would not otherwise have access to HIV RNA services despite operating large HIV treatment and care programs.
  • The pilot program design in two parts: In Part A, we enrolled HIV-1 exposed infants and infants clinically suspected to have HIV aged <18 months of age who needed a nucleic amplification tests to diagnose HIV infection. And in Part B we enrolled HIV-infected patients over 18 months of age who needed a nucleic amplification tests to manage their HIV infection. .
  • The Methods were as follows. Team members at sites filled out brief standardized questionnaire that collected information about sociodemographic and clinical characteristics of participants. EDTA blood was collected and was used to prepare dry blood spots that were sent by Tanzania Expedited Mail Service or EMS to our laboratory at KCMC. In addition, plasma was separated, frozen at -80 ºC , and shipped weekly to KCMC on dry ice. The DBS were tested at our laboratory for HIV-1 RNA using the m2000system. Early infant diagnosis DBS also were sent to South Africa to Contract Lab Services for HIV DNA PCR. HIV RNA levels were assessed at baseline and at approximately 10 weeks to assess stability under local storage conditions. The liquid plasma sample was tested for HIV-RNA using the m2000 system in Moshi. Plasma HIV RNA results were returned to sites by EMS for patient care.
  • This slide visually shows the flow of samples and results between the sites and the laboratories. As you can see the samples originate at our rural sites with the patients. The frozen plasma is sent weekly to the biotech lab in Moshi. The DBS are sent through the Tanzanian mail service weekly. Then the samples are tested for HIV RNA in Moshi. After the DBS from the Part A infant diagnosis samples were tested they were sent to South Africa for DNA PCR at CLS and the results are sent back to Moshi.
  • In terms of results, please see poster P_28 for technical performance of dry blood spot versus liquid plasma HIV RNA. But to summarize the poster, under field conditions in Tanzania, dry blood spot HIV RNA performed well for early infant diagnosis against both liquid plasma HIV RNA and against DBS DNA PCR. There was strong agreement between DBS and plasma HIV RNA above 4 logs HIV RNA and reasonable agreement down to 400 copies/mL. Furthermore, dry blood spots produce stable HIV RNA levels over 80 days of storage under local conditions. Now for the rest this presentation I would like to focus on the overall performance of the dry blood spot program.
  • First I will talk about the Performance of the Tanzania Expedited Mail Service. The median transit time between sites and the KCMC Biotechnology Laboratory for dry blood spot samples was 1.5 days with a range of 1 to 2 business days. In terms specimens or results lost by EMS, 28 packages with DBS were sent from the rural sites and all were received at the KCMC Biotechnology Laboratory. 27 packages containing results, case report form and consent form clarifications, and supplies were sent from the Laboratory to the rural sites and all packages were received at the sites.
  • Now we will talk about dry blood spot training at the KCMC Biotechnology Laboratory required to begin testing this sample type. As mentioned, our laboratory staff had more than 2 years experience using the Abbott m2000 instrument for HIV-1 RNA measurement prior to the implementation of dry blood spot testing. Laboratory staff needed less than one day of training from an Abbott Field Application and Service Specialist to learn about testing dry blood spots. Then they were proficient using the machine. How about the training of sites for sample collection, preparation, and shipping?
  • Training for staff from sites was done over two days at the KCMC Biotechnology Laboratory. One clinician and one laboratory worker was trained from each site. Trainees returned to sites to train other program workers using study materials. Training and trouble-shooting support was available from the Moshi-based Study Coordinator, me.
  • In order to give you a sense for total turn about times achieved under our program conditions, let me break down the steps from sample collection to result receipt at the site The interval from sample collection to shipping from the sites ranged from 0 to 7 days. Transit time to the laboratory was 1 to 2 days. The interval from sample receipt to testing at the KCMC Biotechnology laboratory was 1 to 51 days. If the ‘Christmas Period’ period is excluded, the interval from sample receipt to testing is 1-21 days. And from testing to shipping results back to the sites was 0 to 7 days. Again the transit time back to the sites was 1-2 days. So the total turnaround time was 4 to 69 days or 4-39 if Christmas period is excluded As you can see, most delay can be attributed to issues in the KCMC Biotechnology Laboratory.
  • Let’s look at reasons for delays in testing samples at the KCMC Biotechnology Laboratory. First, there were a number of plasma run failures; details are shown here. In addition, national grid problems in December 2008 conspired with a laboratory generator malfunction to result several weeks without testing. And we interrupted study activities during the ‘Christmas’ period due to major reductions in clinic activities at sites.
  • During our program we had a number of rejected and lost samples 35 samples were lost in run failures, either plasma or DBS 3 were lost because of successive machine errors. There were 6 samples that were hemolyzed and 7 sample were of insufficient volume when they arrived from the sites. 4 samples were rejected because they were collected outside the study period. 7 patients enrolled in Part A were greater than or equal to 18 months at the time of enrollment. And as mentioned, no samples were lost in transport.
  • Now I’d like to share some impressions of the program from some physicians and their feedback from patients. Here are some quotes obtained after the study ended: ‘The doctors found the program helpful for capacity building for staff.’ ‘Participants thought DBS were helpful in deciding management (of exposed infants and infected adults).’ ‘We advice if results are promising to hurry in the next phase of (program) so that we can prolong life of infected infants and save lives of non-infected as mothers can stop breast feeding and be very carefully in raising their babies to avoid other ways of transmission.’
  • … and some impressions of patients… To quote a physician from Magunga Hospital, the ‘majority of patients were highly impressed by the study especially mothers of infants as they were very ambitious in knowing the results of their babies and that was clearly seen when we gave results.’
  • We asked doctors at each sites if they would use a HIV RNA service if it were available via DBS and if not what barriers there would be to using it. Malabeja Anangisye from Magunga in Korogwe ‘I think in the future these diagnostic tools viral load/infant diagnosis via DBS very helpful, but usage it depends on guidelines from the Ministry of Health.’
  • In response to the same question, Sally Edmonds from Teule Hospital in Muheza. Yes, yes, yes, please. Having early infant diagnosis is great and the mothers really like it. The main barrier is in the difficulties of getting the results back effectively which would have to be improved loads.
  • Some of our challenges included issues in the KCMC Biotechnology Laboratory such as failed runs, power outages, and other problems with testing. These meant that a number of samples were lost in testing and led to a backlog of samples. In turn, this led to extended times to get results back to physicians at sites. Since physicians had asked patients to return in a month for their results, this led to frustration.
  • In terms of next steps, we hope that the results of our program can be used by policy makers to inform decisions about the use of dry blood spots to provide HIV-1 RNA services to people living in rural and remote areas.
  • I would like to acknowledge the members of the dry blood spot study team listed on this slide. Pause. Thank you for your attention.
  • I would be happy to take questions.
  • A pilot program of a dry blood spot HIV-1 RNA method for ...

    1. 1. A pilot program of a dry blood spot HIV-1 RNA method for early infant diagnosis and viral load monitoring in rural Tanzania Sarah M. Lofgren, BA Research Fellow KCMC-Duke University Collaboration Moshi TANZANIA
    2. 2. Overview <ul><li>Challenges for early infant diagnosis and viral load monitoring in sub-Saharan Africa </li></ul><ul><li>Potential solutions </li></ul><ul><li>HIV RNA capacity in northern Tanzania </li></ul><ul><li>Pilot program of a dry blood spot method </li></ul><ul><li>Performance against liquid plasma </li></ul><ul><li>Program performance </li></ul><ul><li>Impressions of clinicians and patients </li></ul><ul><li>Challenges </li></ul><ul><li>Next steps </li></ul>
    3. 3. Challenges for early infant diagnosis <ul><li>Antibody-based methods </li></ul><ul><ul><li>Useful for adults </li></ul></ul><ul><ul><li>Not reliable for infants <18 months </li></ul></ul><ul><li>Early infant diagnosis </li></ul><ul><ul><li>Initiation of ART and other management </li></ul></ul><ul><ul><li>Nucleic acid amplification tests (NAT) </li></ul></ul><ul><li>Challenges of NAT for rural areas </li></ul><ul><ul><li>Test locally using expensive, complex equipment </li></ul></ul><ul><ul><li>Freeze and transport on dry ice to central laboratory </li></ul></ul>
    4. 4. Challenges for viral load monitoring <ul><li>Expansion of HIV treatment and care programs </li></ul><ul><ul><li>Monitoring by clinical and immunology assessments </li></ul></ul><ul><ul><li>Virologic failure occurs earlier </li></ul></ul><ul><ul><li>Viral load monitoring useful </li></ul></ul><ul><li>Technologic and logistic concerns </li></ul><ul><ul><li>Similar to early infant diagnosis </li></ul></ul>
    5. 5. Solutions to challenges <ul><li>Alternative NAT assays </li></ul><ul><ul><li>Simpler, cheaper systems that could be deployed to district hospital level </li></ul></ul><ul><ul><li>Groups working on technologic advances </li></ul></ul><ul><li>Alternative sample type </li></ul><ul><ul><li>Easy to prepare, store, transport </li></ul></ul><ul><ul><li>Tested in a central laboratory </li></ul></ul><ul><li>Dry blood spots (DBS) </li></ul><ul><ul><li>DBS DNA PCR for early infant diagnosis well validated and widely used </li></ul></ul><ul><ul><li>DBS RNA PCR for early infant diagnosis less studied </li></ul></ul><ul><ul><li>Single DBS RNA PCR platform for early infant diagnosis and viral load monitoring </li></ul></ul>
    6. 6. HIV RNA capabilities in northern Tanzania <ul><li>KCMC Biotechnology Laboratory, Moshi </li></ul><ul><ul><li>February 2006: Abbott m2000rt with manual sample preparation </li></ul></ul><ul><ul><li>February 2008: Abbott m2000system with automated sample preparation </li></ul></ul><ul><ul><li>Patient care and research </li></ul></ul><ul><ul><li>Expand the reach of the service </li></ul></ul>
    7. 7. HIV RNA capabilities at Moshi Tanzania Automated sample preparation and amplification/detection using m 2000 system
    8. 8. Sites Moshi Magunga Hospital Korogwe Teule Hospital Muheza
    9. 9. Pilot program design <ul><li>A two-part program </li></ul><ul><ul><li>Part A: HIV-1 exposed or suspected infants <18 months of age </li></ul></ul><ul><ul><li>Part B: HIV-infected patients ≥ 18 months </li></ul></ul>
    10. 10. Methods <ul><li>Brief standardized questionnaire </li></ul><ul><li>EDTA blood collected </li></ul><ul><ul><li>Prepare DBS, sent by EMS to KCMC </li></ul></ul><ul><ul><li>Plasma separated, frozen, shipped weekly to KCMC </li></ul></ul><ul><li>Dry blood spot </li></ul><ul><ul><li>HIV-1 RNA by Abbott m2000system </li></ul></ul><ul><ul><li>HIV DNA PCR in South Africa (infant diagnosis) </li></ul></ul><ul><ul><li>HIV RNA levels assessed at baseline and 10 weeks </li></ul></ul><ul><li>Liquid plasma sample </li></ul><ul><ul><li>HIV-1 RNA by Abbott m2000system </li></ul></ul><ul><ul><li>Results returned to collection site for patient care </li></ul></ul>
    11. 11. Sample and result flow Part A samples to CLS in South Africa for DNA DBS testing KCMC Biotechnology Laboratory Molecular Section Teule Hospital Muheza Results Results Frozen liquid plasma Dry blood spots Dry blood spots Magunga Hospital Korogwe
    12. 12. Results <ul><li>See poster P_28 for technical performance of DBS vs. liquid plasma HIV RNA </li></ul><ul><ul><li>DBS HIV RNA performed well for early infant diagnosis against both liquid plasma HIV RNA and DBS DNA PCR </li></ul></ul><ul><ul><li>Strong agreement between DBS and plasma HIV RNA for above 4 logs and reasonable agreement down to 400 cp/mL </li></ul></ul><ul><ul><li>DBS HIV RNA levels were stable over 80 days of storage </li></ul></ul>
    13. 13. Performance of the Tanzania Expedited Mail Service (EMS) <ul><li>Transit times </li></ul><ul><ul><li>Median 1.5 days (range 1-2 business days) </li></ul></ul><ul><li>Specimens or results lost </li></ul><ul><ul><li>28 packages with DBS were sent from the rural sites and all were received at the KCMC Biotechnology Laboratory </li></ul></ul><ul><ul><li>27 packages with results, case report form and consent clarifications, and supplies were sent to rural sites and all were received </li></ul></ul>
    14. 14. Dry blood spot training at the KCMC Biotechnology Laboratory <ul><li>Laboratory staff had >2 years experience using m2000 instrument for HIV-1 RNA measurement on liquid plasma samples </li></ul><ul><li>Staff needed <1 day of training from an Abbott Field Application and Service Specialist for DBS testing </li></ul>
    15. 15. Training for sites <ul><li>2 day training at KCMC Biotechnology Laboratory </li></ul><ul><ul><li>1 clinician and 1 lab worker from each site </li></ul></ul><ul><li>Trainees returned to sites to </li></ul><ul><ul><li>Train other program workers </li></ul></ul><ul><ul><li>Training and trouble-shooting support was available from the Moshi-based Study Coordinator </li></ul></ul>
    16. 16. Total turnaround times <ul><li>Turnaround times from collection to result at site </li></ul><ul><ul><li>Collection to shipping: 0-7 days </li></ul></ul><ul><ul><li>Transit site to laboratory: 1-2 days </li></ul></ul><ul><ul><li>Sample receipt to testing: 1-51 days </li></ul></ul><ul><ul><ul><li>Excluding Christmas 1-21 days </li></ul></ul></ul><ul><ul><li>Testing to shipping: 1-7 days </li></ul></ul><ul><ul><li>Transit laboratory to site: 1-2 days </li></ul></ul><ul><li>Total turnaround time: 4-69 days </li></ul><ul><ul><li>Excluding Christmas Period 4-39 days </li></ul></ul><ul><li>Most delays results from testing issues at the KCMC Biotechnology Laboratory </li></ul>
    17. 17. Reasons for delays in the KCMC Biotechnology Laboratory <ul><li>Run failures of liquid plasma </li></ul><ul><ul><li>At liquid handler- 2 run failures </li></ul></ul><ul><ul><li>At amplification and detection- 1 run failures </li></ul></ul><ul><li>National grid power problems in December 2008 combined with generator malfunction </li></ul><ul><li>Interruption of study activities during ‘Christmas’ period </li></ul>
    18. 18. Rejections and lost samples <ul><li>35 samples lost in run failures </li></ul><ul><li>3 lost because of successive machine errors </li></ul><ul><li>6 samples were hemolyzed </li></ul><ul><li>7 were of insufficient volume </li></ul><ul><li>4 rejected because collected outside study period </li></ul><ul><li>7 patients were ≥18 months at time of enrollment </li></ul><ul><li>No samples were lost in transport </li></ul>
    19. 19. Impressions of physicians <ul><li>‘ The program has helped in capacity building for staff’ </li></ul><ul><li>‘ Participants thought DBS were helpful in deciding management (of exposed infants and infected adults)’ </li></ul><ul><li>‘ We advice if results are promising to hurry in next phase of (program) so that we can prolong life of infected infants and save lives of non-infected as mothers can stop breastfeeding and be very carefully in raising their babies to avoid other ways of transmission’ </li></ul>
    20. 20. Impressions of patients <ul><li>‘ Majority of patients were highly impressed by the study especially mothers of infants (part A) as they were very ambitious in knowing results of their babies and that was seen clearly when we gave results’ </li></ul>
    21. 21. <ul><li>‘ I think in future these diagnostic tools viral load/infant diagnosis via DBS very helpful, but usage it depends on guidelines from Ministry of Health.’ </li></ul><ul><ul><li>Malabeja Anangisye </li></ul></ul><ul><ul><li>physician at Magunga Hospital </li></ul></ul>Would you use this service if it was available via DBS? If not what barriers would there be to using it?
    22. 22. Would you use this service if it was available via DBS? If not what barriers would there be to using it? <ul><li>‘ YES, YES, YES, PLEASE. Having early diagnosis is great and the mothers really like it. The main barrier is in the difficulties of getting the results back effectively which would have to be improved loads.’ </li></ul><ul><ul><li>Sally Edmonds </li></ul></ul><ul><ul><li>physician at Teule Hospital </li></ul></ul>
    23. 23. Challenges <ul><li>Issues in the KCMC Biotechnology Laboratory </li></ul><ul><li>Time to get results back to sites </li></ul>
    24. 24. Next steps <ul><li>Results can be used by policy makers to inform decisions about use of DBS to provide HIV-1 RNA services to rural and remote areas </li></ul>
    25. 25. Acknowledgements Moshi John Crump Caroline Chevallier Anne Morrissey David Sifuna Emma Msuya Werner Schimana John Shao Korogwe Malabeja Ananagisye Omary Abdul Samwel Gesase Gerald Kulwa Alvan Butichi Sr. Upendo Mvungi Muheza Sally Edmonds Ben Amos Hawa Chubwa Naftali Vadwiga Monica Mhilu Abbott Daan Potgieter Sven Thamm Rob Dintruff CLS Wendy Stevens
    26. 26. Any Questions?
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