Dried Blood Spot for Early Infant Diagnostic Using DNA PCR: Rwanda Experience

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Dried Blood Spot for Early Infant Diagnostic
Using DNA PCR Rwanda Experience
International Center for AIDS Care and Treatment
Programs Annual Meeting, March 2006 Dar Es
Salaam, Tanzania

• Luis M. Felipe Gonzalez MD PhD
• Amilcar Tanuri MD PhD
• Rugimbanya Pierre, Bsc
• Tuesday, March 7, 2006

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What Is a Dried Blood Spot (DBS)?

• Whole blood dried on filter paper
• Usually obtained from pricking skin, not from
  phlebotomy
• Requires only a small amount of blood
• Easy to store
• Easy to transport

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Required Supplies for DBS

• Blood collection card (filter paper)
• Lancet
• Alcohol
• Gauze or cotton wool
• Sealable plastic bags
• Humidity cards
• Desiccant packs

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How to Collect DBS

• Use Universal Safety Precautions for handling
  blood
• Clearly label card with appropriate
  identification number
• Do not touch circles

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Position baby with foot down
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Clean the area, dry 30 seconds
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Press lancet into foot, prick skin
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Allow large drop to collect
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Fill entire circle with drop
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Clean area, leave with no bandage
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Foot hold squeeze for heel stick
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Done! Lets do another one!
Do what??
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Dry Completely Before Packaging
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Insert Into Sealable Plastic Bag
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Add Desiccant Packets
Minimum 10 packets per bag
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Add Humidity Cards and Seal Bag
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How to Package DBS for Shipping

• Insert into envelope
• Include lab requisitions
• Include specimen delivery checklist
• Label outside clearly
• Send to reference laboratory

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Early Infant Diagnosis Why is it Important?

• For the health of infants
• High mortality among HIV infected infants
• Rapid progressor infected in utero or
  perinatally
• First significant illness likely to end in death
• Infants with HIV do not grow well, and may suffer
  brain damage
• Early treatment improves outcomes treat before
  they are sick

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Diagnosing HIV in Infants

• ELISA tests
• Inexpensive
• Accurate only after 18 months
• Require phlebotomy
• Rapid tests
• Inexpensive
• Accurate only after 18 months
• PCR (DNA or RNA)
• More expensive
• Accurate even at birth
• In the past, required phlebotomy
• Now can be done on blood from a heel prick

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Early Infant Diagnosis Technical Background

• Polymerase Chain Reaction (PCR) testing
• Looks for HIV virus (DNA or RNA), not antibody
• Not a new test
• Used in US for more than 15 years
• Used in Rwanda since late 2003, only in 3 health
  facility required phlebotomy
• Quite accurate even in newborns
• Almost 100 accurate after 6 weeks old

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Background Work in Rwanda Pilot

• We developed a highly efficient method for HIV
  proviral DNA extraction from DBS samples using
  the extraction procedure from Roche Amplicor v
  1.5
• We also proved that DNA is very stable in DBS.
• We also proved in field evaluation conducted in
  Rwanda between Feb-August 05 that the DNA PCR can
  be used with success in patients samples.

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1st phase DBS Evaluation
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2nd phase DBS Evaluation

• We planned a 2nd phase evaluation for DBS where
  the results obtained from the samples extracted
  from DBS will be compared with the gold standard
  tests material (whole blood) in infants between
  0-18 months old. We will calculate the
  performance parameters such as sensitivity,
  specificity, NVP, and PPV.
• We have engaged 3 clinics in Kigali which
  normally send samples for DNA PCR to the National
  Reference Lab.
• The whole blood sample will be tested with
  Amplicor v 1.5 (Gold Standard) and the results
  returned to the patients. The sample left over
  utilized to generate 5 DBS spots. The DBS will be
  preserved for at least 7 days at room temperature
  before testing.

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Results

• Analyzing the results from 345 samples we could
  find 54 positive and 291 negative samples. The
  table below shows all parameters of this
  evaluation. There is good sensitivity (98.02),
  specificity (100), NVP, and PPV of DBS results
  when compared with gold standard (GS) test.
• 1 false negative sample (406) which will be
  further analyzed for clarification.

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Results 2nd phase DBS Evaluation
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Serology
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Conclusion

• The DBS prove to be a reliable way to collect and
  transport infant samples. The preliminary
  analysis of test results from the 2nd phase shown
  similar performance when the results are compared
  with DNA extracted from whole blood (GS). Taking
  all in account , the DBS is a nice tool to expand
  the access for PCR test in exposed children in
  Rwanda.
• One lab and one tech working full time can test
  11000 samples per year.

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Monitoring the Implementationof DBS in 5 HC in
Kigali

• 5 sites were chosen in Kigali to evaluate the
  implementation of DBS
• October to December
• Analyzing and implementing all logistics aspects
  involved (critical part), training, monitoring,
  etc.
• 500 DBS DNA PCR tests from HIV exposed infants
  during this time

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DNA PCR Infant Diagnosis NRL Rwanda
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Were is DBS IMD in Rwanda now?

• 13 Sites sending DBS
• 12 Sites sending whole blood
• 1000 DBS process so far 110 positives
• 3700 whole blood samples process 530 positives
• In total 230 infant patients on ART treatment 30
  from DBS diagnostics
• Technical group created to calculate cost and
  need.
• National algorithm created for IMD first test (
  6 weeks) is DBS, after 9 month, first is RT and
  PCR to confirm positive RT.

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The Whole Team
ICAP New York ( Columbia Family) Dra. Wafaa el
Sadr ( Big Boss ICAP-CU) Dra. Elaine Abrams
(pediatric guru) Dra. Miriam Rabkin Dr. David
Hoos Dra. Jessica Justman Mr. Peter Twyman Mr
Michael Reich ( Money Man) CDC Rwnada Mr. Jeff
Hanson CDC Atlanta Dra. Emilia Rivadeneira (
Molly)

• NRL Rwanda
• Mr. Pierre Rugimbanya ( Rwanda Big Boss)
• Dr. Jhon Rusine
• Tech. Nathan Makonbe ( master in PCR)
• Tech Emmanuel Rumbenya ( master in PCR)
• TRAC Rwanda
• Dra. Anita Asimwe
• Dra. Elevani
• ICAP Rwanda
• Dr. Ruben Sahabo (ICAP Rwanda Big Boss)
• Dra. Cristiane Adje( Laboratory Management)

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• THANK YOU
• Murakoze
• Asanti Sana
• GRACIAS
• 
  P.D. Hakuna Matata