Evaluation of different RNA extraction methods and storage conditions of dried plasma or blood spots for human immunodeficiency virus type 1 RNA quantification and PCR amplification for drug resistance testing.

Author(s): Monleau M, Montavon C, Laurent C, Segondy M, Montes B, Delaporte E, Boillot F, Peeters M

Publication: J Clin Microbiol, 2009, Vol. 47, Page 1107-18

PubMed ID: 19193835 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to evaluate different extraction kits for isolation of HIV from fresh plasma and DPS. Both spiked plasma and patient specimens were used for extraction with the QIAamp viral RNA mini kit (QIAamp), Abbot, Nuclisens, and High Pure viral nucleic acid kit (HighPure).

   Summary of Findings:

   Using fresh plasma specimens, variability between extraction methods was similar to interassay variability. Abbot and Nucliesens kits performed well for all DPS viral loads tested, while HighPure and QIAamp performed poorly with low viral load specimens. All kits had some undetected virus in DPS, but this was particularly a problem for the QIAamp and HighPure kits. PCR amplification of the PR and RT regions of the polymerase gene worked well for the Nuclisens and Abbot kits, but had a high level of failure with the QIAamp and HighPure kits. Results using DPS from patients were similar to those from spiked plasma. In conclusion while no difference between extraction kits were identified when fresh plasma was used, QIAamp and HighPure extraction kits did not perform as well as Abbot and Nuclisens kits when using DPS.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • AIDS/HIV-related

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Prognostic factor	3.3 log(10) viral copies/mL 4.3 log(10) viral copies/mL 5.3 log(10) viral copies/mL
   Analyte Extraction and Purification	Analyte isolation method	QIAamp viral RNA mini kit (Qiagen) Abbot sample preparation system (Abbot) Nuclisens manual extraction kit (BioMerieux) HighPure viral nucleic acid kit (Roche)
   Biospecimen Preservation	Dehydration duration/condition	Dried plasma spot Fresh plasma

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2. Study Purpose

   The purpose of this study was to compare extraction of HIV RNA from DPS and DBS using the Abbot and Nuclisens kits.

   Summary of Findings:

   Use of the Nuclisens kit yielded no false negative results from DBS, while use of the Abbot kit led to only one false negative for a low viral load specimen. A specimen that had been previously found to be negative by both methods using DPS was found to be positive using DBS, a finding the authors attribute to proviral load. Also possibly attributable to proviral load, when the results were broken down by viral load it was found that if the viral load was less than 3.7 log(10) copies/mL both kits caused an overestimation in DBS. When the viral load was more than 3.7 log(10) copies/ml, the Nuclisens kit caused a slight underestimation of viral load. Overall, use of the Abbot kit led to slight underestimations of viral load (p=0.032) in DBS while the Nuclisens kit did not.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative

   Diagnoses:

   • AIDS/HIV-related

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Whole blood
   Biospecimen Preservation	Dehydration duration/condition	Dried spot
   Preaquisition	Prognostic factor	3.3 log(10) viral copies/mL 4.3 log(10) viral copies/mL 5.3 log(10) viral copies/mL
   Analyte Extraction and Purification	Analyte isolation method	Abbot sample preparation system (Abbot) Nuclisens manual extraction kit (BioMerieux)

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3. Study Purpose

   The purpose of this study was to determine the effects of storage of DPS at 20 or 37 degrees C for up to 3 months on HIV RNA load. The specimens used in this study were spiked with HIV virus and extracted using the Abbot and Nuclisens kits.

   Summary of Findings:

   Storage of DPS specimens for 8 weeks at 20 degrees C did not significantly alter viral load as compared to fresh plasma specimens using either kit. After 12 weeks of storage at 20 degrees C, 0.71 log(10) copies/mL and 0.36 log(10) copies/mL decreases were found using the Abbot and Nuclisens kits, respectively. Importantly PCR amplification in specimens with low viral loads was found to be altered after only 1 week of storage at 20 degrees C and decreased in higher viral loads after storage for 4 weeks. This was particularly true for long amplicons. Storage at 37 degrees C resulted in decreased measurement of viral load after 1 month and by 8 weeks the lowest viral loads were no longer detectable. Further, PCR amplification declined rapidly after storage for 1 week at 37 degrees C.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Other Preservative

   Diagnoses:

   • AIDS/HIV-related

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Abbot sample preparation system (Abbot) Nuclisens manual extraction kit (BioMerieux)
   Biospecimen Preservation	Dehydration duration/condition	Dried plasma spots Fresh plasma
   Preaquisition	Prognostic factor	3.3 log(10) viral copies/mL 4.3 log(10) viral copies/mL 5.3 log(10) viral copies/mL
   Storage	Storage temperature	20 degrees C 37 degrees C
   Storage	Storage duration	0 weeks 1 week 2 weeks 4 weeks 8 weeks 12 weeks

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