Establishment of the 1st World Health Organization International Standard for Plasmodium falciparum DNA for nucleic acid amplification technique (NAT)-based assays.

BACKGROUND: In order to harmonize results for the detection and quantification of Plasmodium falciparum DNA by nucleic acid amplification technique (NAT)-based assays, a World Health Organization (WHO) collaborative study was performed, evaluating a series of candidate standard preparations. METHODS: Fourteen laboratories from 10 different countries participated in the collaborative study. Four candidate preparations based upon blood samples parasitaemic for P. falciparum were evaluated in the study. Sample AA was lyophilized, whilst samples BB, CC and DD were liquid/frozen preparations. The candidate standards were tested by each laboratory at a range of dilutions in four independent assays, using both qualitative and quantitative NAT-based assays. The results were collated and analysed statistically. RESULTS: Twenty sets of data were returned from the participating laboratories and used to determine the mean P. falciparum DNA content for each sample. The mean log10 "equivalents"/ml were 8.51 for sample AA, 8.45 for sample BB, 8.35 for sample CC, and 5.51 for sample DD. The freeze-dried preparation AA, was examined by accelerated thermal degradation studies and found to be highly stable. CONCLUSION: On the basis of the collaborative study, the freeze-dried material, AA (NIBSC code No. 04/176) was established as the 1st WHO International Standard for P. falciparum DNA NAT-based assays and has been assigned a potency of 10(9) International Units (IU) per ml. Each vial contains 5 x 10(8) IU, equivalent to 0.5 ml of material after reconstitution.

Elimination of false-negative hepatitis C virus RNA results by removal of inhibitors in cadaver-organ donor blood specimens.

Detection of viral nucleic acids in blood samples from cadavers is often difficult because of inhibition of the reverse transcriptase (RT) or polymerase chain reaction (PCR) steps by substances present in the samples. A robust method for the extraction and detection of hepatitis C virus (HCV) RNA from cadaver blood samples by polymerase chain reaction RT-PCR has been developed on the basis of the Qiagen QIAamp DNA mini kit extraction system (Basel, Switzerland). Twenty of 36 samples tested were positive for HCV RNA. Six of the 16 HCV-antibody- and RNA-negative samples contained inhibitors that were successfully removed by pretreatment of samples with the Qiagen AX matrix before extraction.