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01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

Increased sensitivity for detecting malaria parasites in human umbilical cord blood using scaled-up DNA preparation

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Spencer D Polley, Colin J Sutherland, Fiona Regan, Maha Hassan, Peter L Chiodini
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-62) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

Study design by CJS, SDP, PLC, MH and FR; maternal malaria risk data collected by MH; DNA extraction and amplification was performed by SDP; Manuscript was written by SDP, CJS, FR and PLC. All authors read and approved the final manuscript.

Abstract

Background

All mothers donating umbilical cord blood units to the NHS cord blood bank undergo an assessment for the likelihood of prior exposure to malaria infection. Those deemed at risk due to a history of travel to, or residence in, malaria endemic regions are screened serologically to detect anti-malaria antibodies. A positive result excludes the use of the cord blood for transplant therapy unless a risk assessment can ensure that malaria transmission is extremely unlikely. This paper details the screening of cord blood units from malaria serology positive mothers to detect malaria parasite DNA using a highly sensitive nested PCR.

Methods

Uninfected blood from a healthy volunteer was spiked with known quantities of malaria parasites and 5 millilitre and 200 microlitre aliquots were subjected to DNA extraction using QIAamp DNA maxi and DNA mini kits respectively. Nested PCR, to detect malarial SSU rRNA sequences, was performed on the purified DNA samples to determine the limit of detection for this assay with both extraction methodologies. Following assay validation, 54 cord blood units donated by mothers who were positive for anti-malaria antibodies were screened by this approach.

Results

When DNA was purified from 5 millilitres of blood it was possible to routinely detect as few as 50 malaria parasites per millilitre using nested PCR. This equates to a significant increase in the sensitivity of the current gold standard nucleic acid amplification technique used to detect malaria parasites (routinely performed from > 200 microlitre volumes of blood). None of the 54 donated cord blood units from serology positive mothers tested positive for malaria parasites using this scaled up DNA preparation method.

Conclusion

Serological testing for malaria parasites may be overly conservative, leading to unnecessary rejection of cord blood donations that lack malaria parasites and which are, therefore, safe for use in stem cell therapy.
Zusatzmaterial
Authors’ original file for figure 1
12936_2010_2040_MOESM1_ESM.pdf
Literatur
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