Multiplex PCR Panel Tests for Infections, March 2018

There has been extensive growth and development of molecular tests for the diagnosis of infections. What has driven the development of these tests is that many of the disease states e.g gastroenteritis, influenza like illness, pneumonia, meningitis and encephalitis etc that patients present to their doctors may be caused by numerous different micro-organisms. Identifying the specific organism causing the infection often has therapeutic implications. If it is a viral infection, the illness is usually self limiting and the patient can be reassured that he/she is expected to recover in days without the need for the use of antibiotics. In contrast, a bacterial infection may progress and intervening with the appropriate antibiotic early will shorten the course of illness. Until recently, to accurately diagnose the etiology of pneumonia, several sputum samples (or other respiratory samples) will be sent for bacterial culture, viral culture or viral molecular testing. These would be sent to different laboratories. The turnaround time to obtain results of these may be 2-3 days for bacterial culture, 10-14 days for viral culture and 1-3 days for molecular tests. The availability of multiplex syndromic panel tests has begun to replace much of what been previously done at multiple diagnostic microbiology laboratories. With one properly obtained respiratory sample (sputum, nasopharyngeal aspirate) a multiplex PCR test can be ordered for anything from 4 to almost 30 tests looking for different bacteria, viruses or fungi and parasites (in the appropriate setting). The sample will be subjected to nucleic acid amplification test (NAT), usually by polymerase chain reaction (PCR) within a closed system. With improved technology, the turnaround time has been reduced to several hours or within 24 hours of receipt of the sample. With further miniaturization and further technological advances in the future, sample testing using a multiplex PCR platform will become available in the clinic setting (point of care tests) rather than having to send the sample to the laboratory which may be “off-site”.

As of 2017, multiplex PCR tests are available in several laboratories in Singapore for infections in the respiratory, gastrointestinal tracts and central nervous system (for meningitis and encephalitis). These tests are increasingly used in everyday clinical practice. The “multiplex panel” tests that we use frequently are as follows:

Respiratory Virus Panel:this is usually done on a sputum or pharyngeal sample and covers almost 20 different viruses that commonly cause infection of the respiratory tract.

Pneumonia Multiplex PCR: this is usually done on sputum or bronchoalveolar lavage sample and helps to identify Bordetella pertussis, Mycoplasma pneumoniae, Chlamydophilia pneumoniae and Legionella.

Gastrointestinal PCR:this is done on a stool sample and covers common micro-organisms causing infection in the gastrointestinal tract including bacteria, viruses and parasites.

Meningitis/Encephalitis multiplex PCR: This is done on a cerebrospinal fluid sample and used to diagnose central nervous system (CNS) infections such as meningitis and encephalitis. In Singapore, some laboratories use their own in-house tetraplex PCR and others use commercial multiplex PCR kits that cover numerous bacteria, viruses and fungi.

In the future, comprehensive multiplex PCR tests are expected to become available in Singapore for the diagnosis of sexually transmitted infections, bone and joint infections and fever in the returned traveller.
It is important to provide some cautionary remarks about the use and roll out of these tests. Firstly, they are expensive but have the advantage of using one sample to target multiple organisms which should potentially partially replace the costs of multiple samples that are sent to different laboratories. In the case of bacterial infections, another disadvantage is that these multiplex PCR tests do not provide susceptibility results (ie the tests that determine which is the appropriate antibiotic to choose). Some of these panel tests do provide information about genetic markers of antibiotic resistance in the sample and can provide some guidance in antibiotic selection. A recently introduced “Hospital Pneumonia Panel” will detect 21 bacteria, 1 fungus and 19 drug resistance markers and the charge for this single test will be over SGD1000. Thirdly, the tests are highly sensitive in the detection of viruses, bacteria and sometimes fungi. With increased sensitivity of such tests, concerns have been raised about the interpretation and false positive tests. Individuals may be colonized by certain bacteria and may also shed some viruses asymptomatically without the presence of any serious illness. Another possible explanation of false positive tests includes “contamination” of the collected sample.

Multiplex PCR tests will become more commonly used for the diagnosis of infections. For infections such as pneumonia, the multiplex PCR test has improved our understanding of the epidemiology of community acquired pneumonia. Multiplex PCR will improve our diagnostic accuracy and in the case of febrile illnesses due to viruses, it will allow the doctor to withhold the use of antibiotics in many instances.