How qPCR Changed Infectious Disease Detection

Making qPCR molecular diagnostics accessible at point-of-care changed how we detect and treat major infectious diseases.
Zihua Wang, MBA., PhD, Diane Stewart, PhD

 

Infectious diseases have always been a part of human life and their impacts are a major driver of our history. SARS-CoV-2 may be the most notable, recent example, both in its human impact and the way it accelerated clinical diagnostics technology.

Other infectious diseases such as the bubonic plague, smallpox, Spanish flu, polio, HIV/AIDS, and malaria have affected humans throughout history. In fact, smallpox killed 400,000 individuals in Europe each year during the 18th century. By the 20th century, smallpox caused 500 million deaths. New diagnostics and treatments finally eradicated it in the late 1970s.

The World Health Organization (WHO) estimates that infectious diseases cause approximately 25% of the 60 million deaths each year worldwide. This results in a tremendous economic burden throughout the world. The CDC estimates infectious diseases cost the US health care system over $120 billion in a single year (2014).

Early Disease Detection Methods

Early infectious disease diagnoses were based on imaging techniques using microscopy. Optical microscopy, combined with cell culture, was used to examine morphology, isolate the disease-causing bacterial or viral agent, and assess possible drug resistance.

Prior to electron microscopy, only larger microorganisms greater than 150 nm could be imaged. E. coli were identified by growing a bacterial sample on a gelatin-like media, harvesting the bacteria, staining the culture, and preparing a slide for viewing with an optical microscope (Figure 1A). Smaller microorganisms, such as viruses, were visualized only after electron microscopy was developed (Figure 1B).

Figure 1. Optical and electron microscopy images. A) Optical microscopy–optical image of stained E. coli. B) Electron microscope image of coronavirus. The field of view (FOV) is approximately 200 nm.

Imaging and culture tests take several days to produce results and often have low sensitivity. In addition, the infectious agent may be difficult to culture or isolate.

Antibody-Based Pathogen Detection

Antibody-based tests using immunofluorescence were introduced 50 years ago to detect the presence or absence of protein structures produced in the body after an infection occurs. This technology is used to diagnose active pulmonary Mycobacterium tuberculosis infection by detecting the MPT64 protein.

However, early detection is an issue with antibody testing because it may take several weeks for a detectable amount of antibody to appear in the blood, especially with slow-growing organisms. Clinical labs used these techniques prior to nucleic acid diagnostics or qPCR molecular testing invented in the 1980s.

Comparing qPCR and Other Detection Methods

Real-time polymerase chain reaction (qPCR) detects the DNA or RNA sequences of the infectious microorganisms at a very early stage of infection, thus offering greater sensitivity and time to actionable result. In addition, qPCR has become more affordable and accessible because of recent advances in miniaturization, microfluidics, and nanotechnology. Table 1 shows the evolution of diagnostic methods and the relative time to pathogen identification.

Diagnostic method evolution
Table 1. Diagnostic method evolution, time for pathogen identification, and strengths and limitations.

 

qPCR is widely used in routine infectious disease diagnostics such as blood banking, viral screening, HIV viral load monitoring, and oncological detection of key mutations in small-cell carcinoma. It is favored due to the quick turnaround time for results. Tests are quickly designed and validated using available pathogen genetic information.

This can be compared with a slow turnaround time for immunological tests, which rely on available antibodies against the pathogens. Antibodies can take up to several weeks to build a sufficient concentration in patient’s blood (seroconversion) for reliable detection. qPCR is known for being reliable in the clinical lab and has measurable impacts on economic and patient outcomes.

How qPCR Changed Infectious Disease Detection

As qPCR matured and made point-of-care diagnostics accessible in the clinic, it showed immediate impacts in how major infectious diseases are detected and treated. Syndromic testing became practically possible, as well, with multiplex assay panels that rapidly identify specific pathogens or antimicrobial resistance markers.

Hepatitis C Virus (HCV)

According to the CDC, an estimated 50,000 acute human hepatitis C (HCV) infections contributed to 15,000 deaths in United States in 2018. The anti-HCV seroconversion will occur 8 to 11 weeks after exposure to the virus in most cases and even longer for individuals with immunosuppressed systems. Therefore, immunological agent-based assays cannot detect the HCV infection in the initial 8 to 11 weeks.

However, qPCR can aid in early diagnosis since HCV RNA is detectable within 1 to 2 weeks post-HCV exposure. This allows the patient to be treated at an initial stage of infection and helps to limit the spread of HCV and reduces the damage to liver caused by liver cancer development, cirrhosis, and liver failure.

If physicians detect HCV early, they can use antiviral drugs like Harvoni, which provides greater than 95% cure rate. Early diagnosis using qPCR can be a valuable tool for physicians to manage treatment options.

Human Immunodeficiency Syndrome (HIV)

Early detection using qPCR can also help physicians manage viral infection with acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV). The HIV/AIDS pandemic caused 35 million deaths by the end of 2015.

According to the CDC, over 37,000 positive HIV cases were diagnosed in the United States in 2018. Although some people infected with HIV can recover without treatment, more than half will become chronically impaired with no cure.

Early diagnosis and treatment reduce the risk of developing AIDS-related symptoms and death. Like anti-HCV seroconversion, anti-HIV seroconversion occurs around 23 to 90 days post HIV exposure. HIV antigens can be detected around 18 to 45 days.

Nucleic acid-based tests can detect HIV RNA in 10 to 33 days post exposure (Figure 2), providing opportunities for early diagnosis and treatment.

Figure 2. Window of detection of HIV markers early in HIV infection and window period of different generations of immunoassays compared to nucleic acid amplification testing.

SARS-CoV-2

In 2015, the WHO assembled a team of scientists and public health experts to prioritize pathogens with the greatest risk of epidemic or pandemic potential. The pathogenic coronavirus family, which includes SARS-CoV, MERS-CoV, and SARS-CoV-2 variants, was included in the pathogens that required urgent action to contain or prevent epidemics.

In December 2019, a cluster of patients with pneumonia in Wuhan, China was reported. The causal virus’s genetic sequence became public two weeks later.

By the end of January 2020, the CDC and FDA developed the emergency use authorization (EUA) strategy for the coronavirus disease 2019 (COVID-19). On the 4th of February 2020, the CDC obtained an EUA for its reverse transcription qPCR-based COVID-19 tests. Because of the sensitivity and minimal false-positive results, a molecular test is a better choice than a rapid antigen test.   The qPCR tests for detection of SARS-CoV-2 and pandemic surveillance illustrate how quickly molecular diagnostic tests can be developed.

Tracking New COVID Variants

Like many other RNA viruses, SARS-CoV-2 can mutate quickly. Many variants such as 20I/501Y.V1, 20H/501Y.V2, 20J/501Y.V3, and CAL.20C have evolved from the original virus. These variants rely on expensive and labor intense next-generation sequencing technology for continual surveillance.

Because of the developing situation, a research use only testing platform offering multiplex capability, such as the MiQLab, could provide an excellent tool for checking the SARS variants. MiQLab is a sample-to-answer qPCR instrument with automated sample preparation and real-time PCR monitoring along with 27-plex capability from a single viral RNA (or DNA) preparation.

The system has the potential to detect over 20 variants of a virus in a single sample. MiQLab provides a less expensive tool for variant monitoring by using assays that can detect known sequenced mutations. In addition to being easy to use, the open-access capability allows the laboratories to validate and design their own tests in response to the variants in their area.

Multiplex qPCR

Multiplex panels are a mainstay in developing countries where the high cost of testing and treatment require fast and sensitive techniques for pathogen detection. In addition, qPCR has the potential to diagnose complex antimicrobial resistance (AMR) in both human and veterinary medicine.

Multiplex assay panels for multiple syndromic diagnostics show clean economic and healthcare benefits. A study in Japan that examined hospitalized pediatric infections showed a measurable reduction in therapeutics and length of stay when a multiplex qPCR test for respiratory infection was prescribed.

Antimicrobial Resistance (AMR)

Antimicrobial resistance is a global economic and public health concern because the spread of AMR traits outpaces new antibiotic development. The CDC’s Antibiotic Resistance Threats Report states that “more than 2.8 million antibiotic-resistant infections occur in the U.S. each year, and more than 35,000 people die as a result”.

Culture and sensitivity testing takes several days from sampling to results and has poor accuracy due to culturing conditions. Thus, qPCR assays are important for providing resistance profiling information.

The most widely known case of resistance is methicillin resistance Staphylococcus aureus (MRSA), which is mediated by the mecA gene. Direct, early, and sensitive detection of the mecA gene by qPCR is desirable for identifying MRSA to avoid the use of the wrong antibiotic or to prescribe ineffective antibiotics.

Resistance testing by qPCR has been used with measurable success in diagnosing strains of TB and allowing for early treatment decisions with the correct antibiotic.

Conclusion

As the global population increases, infectious diseases are becoming more common and their impacts more widely felt. Advances in rapid molecular testing with qPCR have enabled faster and more effective diagnostics and early use of correct therapeutics.

Related blog post: Read how Modernizing POC Diagnostics could help readiness for future infectious disease outbreaks

 

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