We describe proof-of concept for a novel method for point-of-care (POC) detection of HIV-1 viremia. We show that RT pH-LAMP is feasible in low buffer conditions, performs well across a wide range of clinical isolates and, through electronic detection of pH change, is able to distinguish positive from negative samples when run on a CMOS chip. The system as evaluated incorporates many of the requirements for an assay that can be used in field conditions with minimal laboratory infrastructure, including the lack of a need for refrigeration. The USB mounted device can potentially operate with any suitably configured hand-held computer device and has no other requirement for a mains power supply. In addition, the system needs no complex equipment requiring skilled calibration or maintenance and utilises a self-contained, disposable detection system which is potentially very cheap to manufacture by standard silicon-chip production methods. The underlying approach has been applied to Ion Torrent DNA sequencing technology11 and underpins a commercial DNA testing service12. However, this is the first report of pH mediated on-chip detection of RNA.

The development of HIV RT pH-LAMP was chosen over other amplification methods for three reasons; the isothermal nature of the reaction simplifies the technical challenges of thermal cycling on the chip (though such cycling is possible) and, more importantly, the use of LAMP generates higher concentrations of amplification products13 which, through greater changes in pH, improves the sensitivity and speed of the assay. Thirdly, LAMP is less perturbed by non-specific inhibitors than PCR14, allowing the potential for less stringent RNA preparation in a final integrated device, unlike PCR. LAMP has previously been used to detect HIV-1 RNA using alternative read-out platforms, such as turbidimetry and fluorimetry15. One advantage of the approach described here is the potential to multiplex different assays on one CMOS chip. After evaluating targets (and combinations of targets) in the HIV-1 gag and pol genes, our method uses primer sequences targeted to the integrase gene in common with published LAMP methods and laboratory-based commercial PCR assays. In addition, we were able to meet the challenges or viral diversity and improve sensitivity across a wide range of clinical isolates through the use of redundant bases and inosine.

The data generated show that the RT pH-LAMP reaction, as designed, is capable of detecting low copy numbers (down to 10 RNA copies/reaction) of RNA transcripts cloned from the HIV-1 integrase gene. The time to generate a positive reaction was 30 minutes using fluorescent detection, extending to a maximum of 50 minutes when the reaction was performed on the pH-sensitive silicon chip with low copy number. The cases of false negative results at this detection limit were not biased to any particular HIV-1 clade (Supplementary Table 3a,b) and sequencing studies (not published) could not determine any LAMP primer mismatches that could explain the failure to amplify.

While laboratory based HIV-1 viral load detection systems in well-resourced clinical settings are able to detect an HIV-1 viral load of 50 copies/ml or lower, such sensitivity is not required for the monitoring of ART in resource-poor settings. Modelling demonstrates that thresholds of 1000 copies/ml or even 5000 copies/ml may be acceptable16 and more cost-effective and WHO recommendations suggest that detection of viral loads >1000 copies/ml have sufficient sensitivity17. The detection rate of 95% for the LAMP reaction at >1000 copies/reaction using fluorescent detection is encouraging, but before the method can be considered suitable for field use, it will need to be integrated with an efficient RNA extraction method capable of achieving high yields of nucleic acid from peripheral blood. The optimisation data here shows that high sensitivity can be achieved if sufficient target RNA is in the assay. The sensitivity of the assay on the chip was slightly lower than that seen in vitro (88.8% and 95% respectively) (Table 1) which is most likely a consequence of the low reaction volume (12 × 0.6 μl/reaction) compared to the fluorescent output (1 × 25 μl) and this may account for some reduction in the rate of detection. As well as maximising the quantity of viral nucleic acid that can be delivered to the chip, further optimisation of the size of the reaction chambers is planned which could potentially improve sensitivity further. A second generation chip that can hold more than 7.2 μl/reaction is being tested for deployment and it is anticipated that it would match the performance of the fluorescence based RT-LAMP for HIV-1.

Whilst further development is required, including an evaluation of specificity across a wide range of clinical isolates before field evaluations, our HIV specific pH-LAMP assay coupled with novel CMOS chip technology shows great potential as a route to a point of care diagnostic suitable for use in clinical settings without access to a laboratory infrastructure, as is often the case in the developing world. The technology has the potential to be scalable for the detection of multiple pathogens simultaneously and this will be part of future programme of work.