NEW YORK (GenomeWeb) — An Italian research group has developed a direct PCR-based approach for more simple and accessible pharmacogenomic testing to determine whether HIV patients carry the HLA-B*5701 marker associated with risk of serious side effects to the antiretroviral drug abacavir.
A paper published online this week in The Pharmacogenomics Journal describes the group's method, which relies on Thermo Fisher Scientific's Phusion Human Specimen Direct PCR kit to combine DNA extraction and amplification in a single step performed directly on the biological sample without the need for extraction.
According to the authors, the approach also shaved time and cost off of traditional HLA-B*5701 testing by coupling an initial simpler testing step focused on ruling out the large proportion of patients who are HLA-B*57-negative, and then a second nested PCR protocol to identify whether an HLA-B*57-positive sample carries the *5701 allele sequence.
Of the known pharmacogenomic associations and markers, HLA-B*5701 is one of the best characterized. Medical groups have adopted guidelines strongly recommending or even requiring HLA testing prior to prescribing abacavir (marketed by ViiV healthcare as Ziagen), and the US Food and Drug Administration-approved drug label for abacavir recommends pre-therapy screening for HLA-B*5701 and the use of alternative therapy in those positive for the allele. However, the lack of availability and relatively high cost of standard HLA typing has been a barrier to the widespread implementation of this PGx approach, especially in less industrialized countries, according to the study authors.
Emiliano Giardina, a member of the department of biomedicine and prevention at the University of Rome, and the study's senior author, told PCR Insider in an email that while other groups have also tried to make HLA typing easier and more accessible, his lab thought direct PCR was a particularly promising platform.
Direct PCR, he wrote, "allows diffusion [of the team's assay] even in the laboratories which do not have the required technologies for HLA-B*5701 genotyping by traditional methods." In the study, the team estimated the approach should cost less than a Euro per test.
In the publication, Giardina and his colleagues — including a scientist from ViiV Healthcare — described their adaptation of direct PCR to the task of detecting HLA-B*5701 and shared results of a validation of the approach.
Because frequency of HLA-B*5701 can vary widely, the group decided to use direct PCR and gel electrophoresis as a first-line screen to detect positivity/negativity of HLA-B*57. Then, in HLA-B*57-positive cases, they used nested PCR to detect the presence of the HLA-B*5701 allele.
The group found that the direct PCR step was able to accurately discriminate HLA-B*57-negative subjects — approximately 90 percent of the individuals tested — from the remaining seven percent of HLA-B*57-positive individuals. Nested PCR for the presence of HLA-B*5701 then distinguished three subjects as HLA-B*5701-positive and a fourth as negative. The researchers sequenced these samples, and determined that their results were correct in all four cases.
The researchers then moved on to a larger validation using 150 blinded buccal swab samples from 97 healthy patients and 53 HIV-positive subjects, selected to favor the HLA-B*5701-genotype. The group processed the swabs in parallel, both by direct/nested PCR and using the gold standard direct sequencing methodology.
The concordance between the two methods was 100 percent, the team reported.
The authors argued that because their method is performed directly on a biological sample without preliminary DNA extraction and sequencing, it is both faster and simpler than current standard testing, which generally involves separate extraction and sequencing steps. Moreover, the group wrote, dividing testing into two phases allows the discrimination of a large proportion of HLA-B*57-negative patients in half the time, precluding further analysis for this group and thus significantly reducing necessary time and testing costs.
By publishing a description of the method, and successful validation using blinded samples, Giardina said the team hopes it can inform other laboratories that can then adopt the technique.
"Taken altogether, these results provide evidence of the direct PCR as an inexpensive and efficient approach just requiring a PCR thermocycler and an electrophoretic chamber to perform the entire genotyping analysis," the authors concluded.