What does it take to accurately find all true infections while missing none?

Successful control of an infectious disease requires accurate identification of all infections, whether symptomatic or not. Achieving this within a reasonable budget in a timely manner is of great importance for most infectious diseases.

One typical example is HIV. Since many HIV-positive people are unaware that they are infected with the virus, the virus can be passed on long before the patient is diagnosed. Of particular concern is the spread of the disease through blood donation, making it necessary to screen ALL donated blood in blood banks. For HIV screening, nucleic acid testing (NAT) is currently the most effective approach as it reduces the risk of false negatives during the window period. As NAT is relatively expensive, blood is usually screened using a pooling strategy, which results in a dramatic increase in throughput and decrease in cost. But the dilution of virus in pooled samples decreases the effective sensitivity of the test, lengthening the window period by 4 days.

Malaria is another example. Having achieved remarkable success, malaria control programs seem to be heading toward elimination. However, malaria is spread through a mosquito vector, and asymptomatic or “reservoir” infections may account for 20%-50% of all transmissions. Malaria screening of walk-in patients typically employs microscopy or rapid diagnostic tests (RDT). Reservoir infections cannot be identified using passive testing, and eradication can never be attained if programs focus on testing walk-in patients. Therefore, active screening with NAT must be adopted in malaria eradication efforts. Current NATs, however, require specialized skills and are usually time-consuming. In Yunnan, China, more than 350,000 samples were screened to find hundreds of infections in 2014. Finishing this task with current NATs is impractical.

Such diagnostic dilemmas are faced for most infectious diseases: NAT gives better results, but is usually low-throughput and requires higher skills and greater expense. High-throughput molecular diagnostic assays with simplified workflow and improved cost-effectiveness are desperately needed.

In our recent study, we designed a novel RNA quantification technology, CLIP-PCR, which significantly increased the throughput for clinical molecular diagnostics with simplified procedures. The operation involves only mixing, dispensing and decanting - steps that are readily amenable to automation. And thanks to the selective capture during the test, irrelevant nucleic acids can be washed off before the signal production procedure, making possible the adoption of a pooling strategy without compromising assay performance. These benefits together make CLIP-PCR an ideal approach for sensitive, convenient and affordable large-scale molecular tests for infectious diseases.

In our abstract for the 2015 AACC annual meeting and our recent article in Clinical Chemistry, we described an application of CLIP-PCR to large-scale screening of malaria in elimination settings. CLIP-PCR identified 14 infections, including 4 reservoirs, from 3358 samples with <500 tests (in ten 96-well plates), costing <US$0.60 for each sample and demonstrating an improved approach for malaria control. We believe, after identifying the proper target for a disease, such as HIV-I GAG or the 3-X-tail element for HCV, CLIP-PCR is amenable for high-throughput, sensitive and convenient diagnosis of most, if not all, diseases. This is all achievable in an ELISA-like workflow!