An integrated isothermal nucleic acid amplification test to detect HPV16 and HPV18 DNA in resource-limited settings

Abstract

High-risk human papillomavirus (HPV) DNA testing is widely acknowledged as the most sensitive cervical cancer screening method but has limited availability in resource-limited settings, where the burden of cervical cancer is highest. Recently, HPV DNA tests have been developed for use in resource-limited settings, but they remain too costly for widespread use and require instruments that are often limited to centralized laboratories. To help meet the global need for low-cost cervical cancer screening, we developed a prototype, sample-to-answer, point-of-care test for HPV16 and HPV18 DNA. Our test relies on isothermal DNA amplification and lateral flow detection, two technologies that reduce the need for complex instrumentation. We integrated all test components into a low-cost, manufacturable platform, and performance of the integrated test was evaluated with synthetic samples, provider-collected clinical samples in a high-resource setting in the United States, and self-collected clinical samples in a low-resource setting in Mozambique. We demonstrated a clinically relevant limit of detection of 1000 HPV16 or HPV18 DNA copies per test. The test requires six user steps, yields results in 45 min, and can be performed using a benchtop instrument and minicentrifuge by minimally trained personnel. The projected per-test cost is <$5, and the projected instrumentation cost is <$1000. These results show the feasibility of a sample-to-answer, point-of-care HPV DNA test. With the inclusion of other HPV types, this test has the potential to fill a critical gap for decentralized and globally accessible cervical cancer screening.

Advanced technologies towards improved HPV diagnostics

“…Besides PCR, lateral flow assays for HPV detection are increasingly combined also with IATs, mostly RPA, which was described in chapter 3 95–98 . For instance, a study by Ma et al utilized RPA to detect 25 HPV types from as low as 100 fg of genomic DNA per reaction, using a panel of 450 cervical clinical samples.…”

“…Another significant drawback is that most of these technologies were not yet fully tested in low‐resource settings outside well‐equipped laboratories or hospitals. Only few relevant studies, mostly utilizing LFA combined with RPA 98 or paperfludic device with hybrid capture technology, 161 showed performance in low‐resource settings in developing countries. Authors of the former study even estimated a cost of a single test to be around $5, a very affordable price due to their extraction‐free and PCR‐free approach 98 .…”

“…Good concordance between the detection methods and routine cervical screening was achieved, reaching 94.7% agreement for lateral flow dipstick and 97.8% agreement for the reversed dot blot 97 . A very recent study by Kundrod et al utilized an inventive low‐cost NATflow platform (commercially available from Axxin Pty Ltd.) for RPA‐based detection of HPV16 and HPV18 98 . The platform did not require DNA extraction step, but instead involved a streamlined sample preparation technique that could be directly added to the amplification reaction by using achromopeptidase enzyme (ACP) for enzymatic lysis of the cells, reducing overall assay time to only 45 min.…”

“…Only few relevant studies, mostly utilizing LFA combined with RPA 98 or paperfludic device with hybrid capture technology, 161 showed performance in low‐resource settings in developing countries. Authors of the former study even estimated a cost of a single test to be around $5, a very affordable price due to their extraction‐free and PCR‐free approach 98 . Other authors did not estimate projected costs of their assays, but it can be expected that by implementing CRISPR‐Cas system, microfluidic devices or nanomaterials, overall price per test would increase.…”

“…Besides PCR, lateral flow assays for HPV detection are increasingly combined also with IATs, mostly RPA, which was described in chapter 3. [95][96][97][98] For instance, a study by Ma et When compared with qPCR, the sensitivity of detecting HPV16 was 100% for samples containing a minimum of 1000 copies per reaction and 93% for those with at least 500 copies per reaction. The specificity of the test for HPV16 was 100%, the positive predictive value was 86% and the negative predictive value was 56%.…”