Emerging Point-of-Care Diagnostics for Infectious Diseases

Advances in nucleic acid-based detection of pathogens supports rapid sample-to-answer diagnostics for infectious disease

Sensitive, specific, and rapid diagnostic tests are critical to reducing morbidity and mortality of infectious disease patients. Viral, bacterial, and other types of infections can develop from a vast number of pathogenic and opportunistic organisms, indicating a need for versatile diagnostics that can be adapted to a wide variety of infectious etiologies. 

"Sensitive and specific POC diagnostics for infectious diseases not only facilitate effective treatment, but also minimize the development of antibiotic-resistant pathogens."

Nucleic acid amplification tests (NAATs) have become a mainstay of diagnostics for infectious diseases. Through amplification and detection of target organism nucleic acids (NAs), NAATs provide highly sensitive detection of pathogens in many different patient samples. Earlier generations of NAATs depend on labor intensive protocols and bulky equipment that require centralized labs, making it difficult to obtain rapid results and often impede immediate clinical responses. 

However, emerging NAAT technology uses novel protocols with integrated sample preparation, faster amplification and detection methods, and innovative microfluidic designs to support point-of-care testing (POCT) for an array of infectious diseases including SARS-CoV-2 and globally prevalent sexually transmitted infections (STIs). 

These advances support rapid sample-to-answer diagnostics and personalized clinical responses and can therefore significantly improve patient outcomes.

Adapting NAATs for current challenges

NAATs are typically classified into two major types of NA amplification: PCR and isothermal amplification (iNAATs). PCR traditionally requires cumbersome laboratory equipment to cycle samples through different temperatures for denaturation and amplification, which inherently limits its use in POC settings. 

To circumvent such limitations, iNAATs were developed using enzymatic-mediated denaturation, avoiding the need for repeated thermocycling.1 Since the advent of iNAATs, various platforms that are potentially adaptable for POCT have emerged. Loop-mediated amplification (LAMP), for example, uses a series of inner and outer primers in combination with DNA polymerase to amplify target DNA.2 Though LAMP is not new technology, it has proved to be a valuable platform for the development of new NAATs. 

Of current relevance to the ongoing COVID-19 pandemic, LAMP was used to develop a highly sensitive and specific SARS-CoV-2 assay, enabling easy-to-read bedside results in under one hour.3 Mammoth Biosciences combined revolutionary CRISPR technology with a LAMP-based SARS-CoV-2 assay to improve detection following NA amplification of the novel coronavirus. 

The CRISPR-Cas12-based DETECTRTM assay provides a sample-to-result time of approximately 45 minutes compared to four hours for established qRT-PCR methods and is currently under review for FDA approval pending clinical validation.4 Critically, POC NAAT-based diagnostics facilitate immediate initiation of treatment protocols for COVID-19 to prevent severe disease and mortality. 

Evolving landscape for STI diagnostics

Sensitive and specific POC diagnostics for infectious diseases not only facilitate effective treatment, but also minimize the development of antibiotic-resistant pathogens. Reported cases of globally prevalent STIs such as chlamydia and gonorrhea have demonstrated significant annual increases in recent years, indicating a need for improved screening methods and more personalized treatment protocols.5 As a result, STI diagnostics have become a substantial source of innovation for POCT technology. 

The application of microfluidics, which allows sensitive fluid regulation within a single portable component, to existing technology such as PCR amplification, has dramatically expanded POC testing for STIs and other infectious diseases. 

"Through amplification and detection of target organism nucleic acids (NAs), NAATs provide highly sensitive detection of pathogens in many different patient samples."

These platforms not only alleviate the need for laboratory equipment but can also integrate NA extraction, minimizing sample preparation protocols.6 For example, the FDA-approved binx health io platform uses a microfluidics chip for ultrafast PCR combined with a proprietary electrochemical detection technology, enabling highly sensitive and specific POC screening for gonorrhea and chlamydia in approximately 30 minutes.5

Several promising new POC NAATs using similar ultrarapid microfluidics platforms remain in development or in clinical trials, demonstrating the high demand for rapid sample-to-answer diagnostics. The Visby Medical Sexual Health Test uses PCR for detection of chlamydia, gonorrhea, and trichomonas in under 30 minutes, and the MobiNAAT performs PCR using droplet microfluidics in a stand-alone cartridge including all necessary reagents for NA extraction and amplification.5 Remarkably, MobiNAAT can screen for gonorrhea and determine antibiotic susceptibility in 15 minutes, facilitating personalized treatment protocols that may reduce the prevalence of antibiotic-resistant infections.5 

The CDC’s recently released 2021 STI Treatment Guidelines suggests the use of POC NAATS for chlamydia and gonorrhea detection,7 and future guidelines will likely include more rapid sample-to-answer tests that screen for a variety of STIs as they gain FDA approval.

Getting ultrarapid sample-to-answer tests to the clinic

Despite the promise of new POC NAATs on the horizon, decentralization of diagnostics from laboratories to clinics comes with challenges. POC tests need to achieve FDA Clinical Laboratory Improvement Amendments (CLIA) waiver status to be performed by non-laboratory personnel. As such, access to CLIA-certified laboratories for clinical validation may be a significant impediment to the approval of some POC NAATs. 

Nonetheless, advancing portable technology and innovative approaches to NA amplification and detection point to an exciting new generation of POC diagnostics. These tests have the potential to bring ultrarapid sample-to-answer tests directly to the clinic, support more personalized diagnostics and treatment, and improve outcomes for patients with infectious diseases.

References:

1. Lee SH, Park SM, Kim BN, Kwon OS, Rho WY, Jun BH. Emerging ultrafast nucleic acid amplification technologies for next-generation molecular diagnostics. Biosens Bioelectron. 2019;141:111448. 

2. Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):E63. 

3. Yan C, Cui J, Huang L, et al. Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay. Clin Microbiol Infect. 2020;26(6):773-779.

4.  Broughton JP, Deng X, Yu G, et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020;38(7):870-874. 

5.  Gaydos CA, Manabe YC, Melendez JH. A narrative review of where we are with point-of-care sexually transmitted infection testing in the United States. Sex Transm Dis. i 2021;48(8S):S71-S77. 

6.  Sachdeva S, Davis RW, Saha AK. Microfluidic point-of-care testing: commercial landscape and future directions. Front Bioeng Biotechnol. 2021;8:602659. 

7.  Workowski KA, Bachmann LH, Chan PA, et al. Sexually Transmitted Infections Treatment Guidelines, 2021. MMWR Recomm Rep. 2021;70(4):1-187.