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Infant Diagnosis and Patient Monitoring

Infant Diagnosis and Patient Monitoring. Question.

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Infant Diagnosis and Patient Monitoring

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  1. Infant Diagnosis and Patient Monitoring

  2. Question • Your 21 year old HIV-infected patient with a CD4+ cell count of 285 cells/mm3 who you placed on a HAART regimen in the third trimester has an uneventful spontaneous vaginal delivery. You want to know if the infant has been infected with HIV. You order: • A) DNA PCR • B) RNA PCR • C) HIV Antibody test • D) p24 antigen test • When do you order the test? • A) Immediately • B) 6 weeks • C) 3 months • D) 12 months • E) 18 months

  3. Question • You have chosen to continue the patient on her HAART regimen. What are your options for monitoring the success of her ARV therapy?

  4. Nucleic acid testing in remote areas: a field study John A. Crump, MB, ChB, DTM&H

  5. Overview • Challenges for early infant diagnosis and viral load monitoring in remote and resource-constrained settings • Potential solutions • Pilot program of a dry blood spot method • Performance against liquid plasma • Program performance • Challenges • Conclusions • Next steps

  6. Challenges for early infant diagnosis • Antibody-based methods • Useful for adults • Not reliable for infants <18 months • Early infant diagnosis • Initiation of ART and other management • Nucleic acid amplification tests (NAT) • Challenges of NAT for rural areas • Test locally using expensive, complex equipment • Send to a reference laboratory for testing within 6 hours • Freeze and transport on dry ice to central laboratory

  7. Challenges for viral load monitoring • Expansion of HIV treatment and care programs • Monitoring by clinical and immunologic assessments • Virologic failure occurs earlier • Viral load monitoring useful • Technologic and logistic concerns • Similar to early infant diagnosis

  8. Solutions to challenges • Alternative NAT assays • Simpler, cheaper systems that could be deployed to district hospital level • Groups working on technologic advances • Alternative sample type • Easy to prepare, store, transport • Tested in a central laboratory • Dry blood spots (DBS) • DBS DNA PCR for early infant diagnosis well validated and widely used • DBS RNA PCR for early infant diagnosis less studied • Single DBS RNA PCR platform for early infant diagnosis and viral load monitoring • Simultaneous early infant diagnosis and baseline HIV RNA level

  9. HIV RNA capabilities in northern Tanzania • KCMC Biotechnology Laboratory, Moshi • February 2006: Abbott m2000rt with manual sample preparation • February 2008: Abbott m2000system with automated sample preparation • Patient care and research • Expand the reach of the service

  10. HIV RNA capabilities at Moshi, Tanzania Automated sample preparation and amplification/detection using m2000 system

  11. Sites Moshi Magunga Hospital Korogwe Teule Hospital Muheza

  12. Dry blood spot HIV RNA pilot program design • A two-part program • Part A: HIV-1 exposed or suspected infants <18 months of age • Part B: HIV-infected patients ≥18 months

  13. Methods • Brief standardized questionnaire • EDTA blood collected • Prepare DBS, sent by mail to KCMC • Plasma separated, frozen, shipped weekly to KCMC • Dry blood spot • HIV-1 RNA by Abbott m2000system • HIV DNA PCR in South Africa for early infant diagnosis • HIV RNA levels assessed at baseline and 10 weeks • Liquid plasma sample • HIV-1 RNA by Abbott m2000system • Results returned to collection site for patient care

  14. Magunga Hospital Korogwe Frozen liquid plasma Teule Hospital Muheza Dry blood spots Dry blood spots KCMC Biotechnology Laboratory Molecular Section Results Results Sample and result flow Part A samples to CLS in South Africa for DNA DBS testing

  15. Dry blood spot study flow diagram Initial Enrollment: 375 Patients Part A: 223 Patients Part B: 152 Patients Hemolyzed: 6 QNS: 7 Usable samples: 152 Usable samples: 210 Collected outside study period: 4 patients Age ≥18 mo: 7 patients Age <18 mo: 203 patients 148 patients Lost in run failure: 10 samples Lost in sample errors: 1 sample Lost in run failure: 25 samples Lost in sample errors: 2 samples 176 samples remaining 137 samples remaining

  16. Rejections and lost samples • 35 samples lost in run failures • 3 lost because of successive machine errors • 6 samples were hemolyzed • 7 were of insufficient volume • 4 rejected because collected outside study period • 12 patients were ≥18 months at time of enrollment • No samples were lost in transport

  17. Demographic and clinical characteristics of participants • Part A participants 176 • Median (range) age 6 months (1 day to 17 months) • 82 (46%) female • 101 (58%) enrolled because HIV-exposed, 5 (3%) because of clinical suspicion for HIV, and 70 (40%) both • Part B participants 137 • Median (range) age 34 years (21 months to 77 years) • 108 (79%) female • CD4 count median (range) 253 (6-2,586) cells/mm3 • 73 (53%) reported receiving ART

  18. Plasma versus DBS log HIV-1 RNA concentration, Tanzania, 2008-9 R value = 0.9709 DBS HIV-1 RNA concentration log copies/mL Plasma 2,084 copies/mL DBS not detected Plasma HIV-1 RNA concentration log copies/mL

  19. Results • See poster MoPeB010 • DBS HIV RNA performed well for early infant diagnosis against both liquid plasma HIV RNA and DBS DNA PCR • DBS HIV RNA levels were stable over 80 days of storage

  20. Performance of the Tanzania mail service • Transit times • Median 1.5 days (range 1-2 business days) • Specimens or results lost • 28 packages with DBS were sent from the rural sites and all were received at the KCMC Biotechnology Laboratory • 27 packages with results, case report form and consent clarifications, and supplies were sent to rural sites and all were received

  21. Dry blood spot training at the central laboratory • Laboratory staff had >2 years experience using Abbott m2000 instrument for HIV-1 RNA measurement on liquid plasma samples • Staff needed <1 day of training from an Abbott Field Application and Service Specialist for DBS testing • Proficiency in DBS testing was attained after completing two successful runs

  22. Training for sites • 2 day training at the central laboratory • 1 clinician and 1 lab worker from each site • Trainees returned to sites to • Train other program workers • Training and trouble-shooting support was available from the Moshi-based coordinator

  23. Total turnaround times • Turnaround times from collection to result at site • Collection to shipping: 0-7 days • Transit site to laboratory: 1-2 days • Sample receipt to testing: 1-51 days • Excluding vacation period 1-21 days • Testing to shipping: 0-7 days • Transit laboratory to site: 1-2 days • Total turnaround time: 4-69 days • Excluding vacation period 4-39 days • Most delays results from testing issues at the KCMC Biotechnology Laboratory

  24. Reasons for delays in the KCMC Biotechnology Laboratory • Run failures • 2 DBS runs failed • 3 DBS runs had failed controls • 1 DBS runs had >50% samples with errors • National grid power problems in December 2008 combined with generator malfunction • Interruption of study activities during a December vacation period

  25. Challenges • Issues in the central laboratory • Time to get results back to sites

  26. Conclusions • Dry blood spot HIV RNA performs well against plasma HIV RNA and dry blood spot HIV DNA for early infant diagnosis of HIV when collected and transported under field conditions in Tanzania • Dry blood spot HIV RNA performs well against plasma HIV RNA for measurement of HIV-1 RNA levels down to 5,000 copies/mL and quite well to 400 copies/mLwhen collected and transported under field conditions in Tanzania • A dry blood spot HIV RNA service is feasible using a sound central laboratory, appropriately trained staff, and the mail service available in Tanzania • Turnaround times were adequate but could be improved by addressing issues at the central laboratory

  27. Next steps • 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

  28. Acknowledgements Moshi Sarah M. Lofgren Caroline C. Chevallier Anne B. Morrissey David J. Sifuna Emma Msuya Werner Schimana John A. Bartlett John F. Shao Korogwe Malabeja Ananagisye Omary Abdul Samwel Gesase Gerald Kulwa Alvan Butichi Upendo Mvungi Muheza Sally Edmonds Ben Amos Hawa Chubwa Naftali Vadwiga Abbott Daan Potgieter Sven Thamm Robert Dintruff CLS Wendy S. Stevens

  29. Question • Your 21 year old HIV-infected patient with a CD4+ cell count of 285 cells/mm3 who you placed on a HAART regimen in the third trimester has an uneventful spontaneous vaginal delivery. You want to know if the infant has been infected with HIV. You order: • A) DNA PCR • B) RNA PCR • C) HIV Antibody test • D) p24 antigen test • When do you order the test? • A) Immediately • B) 6 weeks • C) 3 months • D) 12 months • E) 18 months

  30. Question • You have chosen to continue the patient on her HAART regimen. What are your options for monitoring the success of her ARV therapy?

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