This study describes, to our knowledge, the first large-scale, multilaboratory, international comparison of HIVDR genotyping assay performance using DBS. Results provide confidence that DBS-based genotyping is similarly reproducible and reliable, although less sensitive, compared with plasma-based genotyping [10
]. Reduced sensitivity is most likely attributable to reduced template copy number input for DBS, compared with plasma.
One of the goals of the DBS validation panel was to generate experimental data on which to base a recommended DBS-based genotyping protocol that could be used by all laboratories performing testing for WHO HIVDR surveys. Key variables of each assay were compared and analyzed in view of the sensitivity and reproducibility results presented here. Variables included were use of nested PCR, number (1 or 2) of PCR amplicons, amplicon length, RNA extraction chemistry or kit type, automated or manual RNA extraction, RNA extraction input (plasma volume equivalent, based on blood volume per DBS and assuming hematocrit of 50%), RT reaction input (fraction of RNA extracted), PCR input (fraction of RT reaction products), second-round PCR input (if nested, fraction of first-round PCR), RT and PCR enzyme, magnesium ion and deoxyribonucleotide triphosphate concentration in RT and PCR, number of PCR cycles, PCR product detection method, type of PCR machine (make and model), and RT and PCR primer length and sequence. However, no single variable could be clearly associated with better assay performance, most likely because of the multifactorial influence of >1 variable. Nonetheless, on the basis of the common use of several variables by all laboratories with good performance, some themes can be identified. These include use of a nested PCR amplification strategy, silica-based nucleic acid purification, a preference for manual vs automated extraction procedure, use of a 1-tube RT-PCR strategy (ie, all of the RT reaction is used for first-round PCR), and 35–40 cycles of PCR at each round. The specific procedures used by the 2 laboratories with the best performance using the validation panel that also gave good results in the proficiency panel will be made available through the WHO laboratory network for laboratories that are developing the capacity to use DBSs for genotyping; these procedures will complement the consensus protocol already available [12
Assessment of assay performance based on the proficiency panel results was largely influenced by sensitivity (ability to amplify PR-RT for sequencing), because sequence accuracy per se was very similar to previous results obtained using plasma specimens and was heavily influenced by the degree of heterogeneity of the virus specimen used [10
]. Thus, efforts to optimize assay success when using DBSs as the specimen type in laboratories already proficient at performing genotyping using plasma should be focused on this aspect of the procedure. For laboratories with no experience handling DBSs that would like to develop this capacity, WHO recommends starting with one of the protocols shown to work well through the studies described here, which are available on request.
An important caveat to conclusions based on testing of DBSs prepared by diluting cell-free virus in blood from HIV-negative donors is that sensitivity limits may be underestimated because of the lack of proviral DNA in the cellular compartment [13
]. However, because the RNA component is less stable during extended storage times under suboptimal temperature or humidity conditions [4
], results using DBS panels that are prepared, stored, and shipped under ideal conditions may overestimate sensitivity that may be expected during implementation of HIVDR surveys in many rural settings when DBSs are prepared from HIV-infected patient anticoagulated blood or directly without venipuncture. In the present study, the possible contribution of proviral DNA to the sequence results in clinical whole blood specimens was not studied.
In the present study, international shipment at ambient temperature did not affect amplification success rates in a consistently detrimental manner. The differences observed when comparing amplification rates for specimens shipped on dry ice vs at ambient temperature were small and inconsistent, and they sometimes favored ambient shipment. However, the interpretation of the amplification success data presented here is tempered by the small number of specimens tested with VL in a range that would be expected to be on the verge of being associated with amplification failure when handled under extreme conditions and by differences among the shipping conditions (distance, time, and actual temperature) to each laboratory. Larger field studies designed to rigorously test the effect of international shipment of clinical specimens at ambient temperature are under way.
Assay validation and proficiency testing are necessary but not sufficient for WHO accreditation of a laboratory to perform genotyping using DBSs. To obtain WHO accreditation for DBS testing, a laboratory must fulfill the following criteria: existing accreditation by WHO for the performance of HIVDR genotyping of plasma specimens at least 6 months of experience in DBS-based genotyping; at least 100 DBS specimens successfully amplified and sequenced, with a success rate of at least 80% when VL is >5000 copies/mL and DBSs have been properly handled (ie, according to WHO recommendations for preparation, storage, and shipping); successful testing of a WHO-recognized proficiency panel consisting of DBS specimens; successful validation of a DBS-based in-house assay for genotyping using the standardized criteria described elsewhere [12
]; and submission to WHO of a report summarizing the aforementioned elements for review.
WHO surveys of acquired HIVDR require genotyping to be performed on specimens with VL (in plasma) of ≥1000 copies/mL; it is a goal of the laboratory network to implement methods that will enable amplification of DBS specimens with VLs in this range. The laboratories with the best performance described here provide optimism that this is achievable.