From Cell Cultures to Multiplex PCR

Infectious diseases are among the most challenging and urgent issues facing healthcare delivery worldwide. With diseases like Ebola, influenza and Middle East Respiratory Syndrome (MERS) making headlines every day, the spotlight is increasingly bright. But despite these headliners, diseases that have slipped out of the public eye (like malaria or glandular fever), as well as the traditional burdens on clinicians (for instance, infections in patients immunocompromised by transplant or chemotherapy) remain just as important.

As treatments develop, there is a growing need for fast and efficient techniques to identify pathogens. From the early days of detecting disease agents through cultures, to more modern polymerase chain reaction (PCR) methods, early and efficient diagnosis continues to be crucial.

Recent developments in infectious disease diagnostics mean we can identify rare conditions in a fraction of the time it once took, and with much greater accuracy. Nonetheless, there are still bridges to be crossed to ensure the best possible results not only for clinicians, but for patients.

The traditional approach

Traditionally, cultures have been used to detect pathogens in clinical samples. Many bacteria, fungi, parasites and viruses can be grown in a lab under the correct conditions, and the precise nature of the culture can identify the characteristics of the microbe itself. For example, successfully growing Staphylococcus aureus in a culture that contains beta-lactam antibiotics indicates methicillin-resistant S. aureus (MRSA).

Techniques used to identify microbes include solid and liquid cultures, as well as cell cultures, and these are commonly used with samples isolated from urine, stool, the genital tract, the throat or the skin. Though culture is traditionally the benchmark for identifying organisms, it has one major drawback – time. Results may not be available for days or weeks, and not all pathogens can be cultured. In a situation where effective treatment depends on rapid and accurate diagnosis, this kind of delay is simply not an option. Therefore, it’s clear that there is a real need for alternative methods.

PCR-based systems

More and more, we’re seeing molecular diagnostics used in clinical settings. These techniques are revolutionizing infectious disease diagnosis; heralded as a diagnostic tool for the new millennium, some researchers believe molecular diagnostics could render traditional hospital laboratories obsolete (1).

Quantitative real-time PCR (qPCR)-based systems detect the agents of disease directly from clinical samples, without the need to wait for a culture to grow. This is particularly valuable in the rapid detection of fastidious microorganisms, along with those that can’t be grown in the laboratory at all. Additionally, sequence analysis of amplified microbial DNA allows for better identification and characterization of pathogens.

One of the earliest recognized applications of PCR in clinical practice was for the detection of Mycobacterium tuberculosis. Before its advent, there were week- to month-long delays associated with standard testing for M. tuberculosis infection – despite the crucial public health impact of early recognition, isolation, and treatment of infected patients. Although PCR assays resulted in significantly decreased time to diagnosis, the only FDA-approved use was when employed alongside a conventional smear and culture. It was suggested, though, that more widespread use of these assays might significantly improve both cost efficacy and clinical outcomes.

Today, qPCR is the most well-developed molecular technique, more widely used than ligase chain reaction and delivering more sensitivity than signal amplification techniques (2). It has a wide range of fulfilled and potential clinical applications, including the evaluation of emerging infections, specific and broad-spectrum pathogen detection, and antimicrobial resistance profiling. But as molecular methods continue to make headway in disease diagnosis, the technology must keep up with growth. We need further development to improve automation, optimize detection sensitivity and specificity, and expand the capacity to detect multiple targets simultaneously – an advancement known as “multiplexing.”

Multiplex PCR

As the number of microbial agents detectable by qPCR increases, the ideal end goal is the simultaneous detection of multiple agents that cause similar or identical symptoms. The term “multiplex” refers to the fact that numerous pathogens are detected in a single assay. This technique is useful for synchronized identification of viruses, bacteria, fungi and parasites, and it can save significant time, effort and money – making it a practical choice for busy laboratories.

Multiplex PCR involves amplifying unique regions of DNA in either individual pairs or combinations of many primers. All of these amplifications take place under a single set of reaction conditions. In a real-time PCR thermocycler that allows the analysis of each frequency specific to the individual amplicon, it is possible to identify specific pathogens.

Using multiplex PCR, clinicians are able perform complete screenings for pathogens known to cause specific clinical syndromes. One syndromic approach in current use screens for the underlying causes of most infections encountered in clinical labs – respiratory infections; gastroenteritis; meningitis; sexually transmitted infections; fever, rash and childhood infections; eye infections; infections of the immunosuppressed; hepatitis; and tropical fever. The benefit of such a broad-spectrum approach is that clinicians don’t have to request testing for a specific pathogen, but can identify a single cause, or rule out multiple options, in a single test. This level of detail can go a long way toward influencing treatment decisions.

Since adopting multiplex PCR approaches, clinical laboratories have found themselves better equipped to provide fast, reliable analysis. Within our own laboratory, Laboratoires Réunis in Luxembourg, qPCR multiplex technology has helped us to ensure that 85 to 90 percent of our reports are completed on the same day. Every day, we perform three runs with plates that include viruses, bacteria, parasites, yeasts and dermatophytes. But despite the extent of our analyses, we are still able to keep turnaround at an all-time low.

Working in the diagnostic sector, I’ve seen a remarkable difference in the way clinical tests are carried out over recent years. It’s good news, though – these latest developments have made a marked increase in the number of patients and clinicians served. Since adopting qPCR multiplex kits, we’ve increased the number of bacterial gastroenteritis multiplex tests we complete from 2,600 in 2010 to approximately 6,000 in 2014 – all without any increase in the hands-on time required from our staff. Furthermore, our turnaround times are dramatically reduced and urgent samples are still guaranteed to be processed within four to five hours of their arrival (see infographic). These positive effects on our laboratory’s day-to-day operation have made us able to provide clinicians with the option of starting any necessary treatment with first choice antibiotics.

Looking forward

However, even with these advancements, continuous development is still underway. Although each multiplex qPCR assay must be individually optimized for its specific reagents, sample-based techniques, such as lyophilization, can enhance stability and dramatically increase ease of use, making a real difference to molecular diagnostic methods in a laboratory setting. Whatever approach further refinements to multiplex qPCR take, we can certainly expect to see exciting developments in the near future – propelling the field of infectious disease diagnostics even farther into the 21st century.

Udo Margraff is a pharmacist and clinical pathologist at Laboratoires Réunis, Junglinster, Luxembourg.