Molecular Diagnostics in Pediatric Infectious Disease: A Practical Guide to PCR Testing

Molecular Diagnostics in Pediatric Infectious Disease: A Practical Guide to PCR Testing

More than a decade has passed since the use of polymerase chain reaction (PCR) for diagnosis of neonatal herpes simplex virus (HSV) disease was reported and has become standard of care.1Before PCR use, brain biopsy with histologic examination and viral culture of brain tissue were the definitive methods required to diagnose HSV central nervous system disease.

PCR is a molecular technique used to amplify nucleic acids of a potential pathogen in a sample to determine the presence or absence of a pathogen. Unlike basic microbiologic culture methods, PCR does not require the pathogen to be viable or to grow.

The past 10  to 15 years have yielded an explosion of discoveries in the field of molecular diagnostics that have translated into faster and—in most cases—more sensitive testing options for pediatric patients who are being evaluated for infectious disease. This brief review summarizes some of the more clinically relevant advances in PCR testing that you may want to consider employing for some of the patients you see every day.

Although Bordetella pertussis infection remains a clinical diagnosis, laboratory confirmation may be sought when symptoms are not definitive. The detection of B pertussis DNA by PCR is increasingly used for laboratory confirmation. Culture of B pertussis requires specific media (which may not be readily available) and also requires samples to be transported promptly to the laboratory after proper collection which may lead to false negative results. PCR is more sensitive than bacteriologic culture and has a high specificity. It is recommended, however, that both tests be obtained when PCR is ordered because possible sample contamination can lead to false positive results.2  Nasopharyngeal samples for PCR and culture collected within the first 7 to 10 days from the onset of symptoms may yield a better sensitivity than samples collected after that time. However, PCR test results may be available within 24 to 48 hours in some labs, whereas the culture may take up to 10 days to grow.3  The PCR test also may detect bacterial DNA for 3 weeks whereas the culture is unlikely to be positive after 2 weeks of cough.4

The rapidity of B pertussis PCR test results may allow you to quickly implement appropriate prophylaxis for contacts close to the source case. The rapid results also have implications for hospital isolation and cohorting because B pertussis requires Droplet Isolation Precautions (mask) for hospitalized patients until standard treatment—a 5-day course of azithromycin2—is complete.

You may want to link to a regional medical center laboratory that performs PCR testing for B pertussis. When used in appropriately obtained samples, this testing can help identify a local outbreak of pertussis in real time.

PCR testing for B pertussis has some disadvantages. Given the larger quantity of bacterial DNA obtained in the sample, the optimal specimen for Pertussis PCR testing is a nasopharyngeal  aspirate (nasal wash) of the posterior nasopharynx with a saline. A nasal swab sample of the posterior oropharynx is also acceptable.4 These samples have variable amounts of bacterial DNA and are not standardized with equal amounts of bacteria, which may impact the PCR results. Pretreatment with antibiotics may yield false negative PCR results4 and sample contamination may yield a false positive reading.3 In addition, PCR requires trained personnel to perform the testing.3

Enteroviral Infection
In the United States, the broad group of viruses that make up the Enteroviruses causes a wide array of illness, including meningitis and encephalitis, typically in summer to late fall.5

PCR testing can also be helpful in the diagnosis of Enteroviral infections, specifically Enterovirus (viral) meningitis. In the setting of meningitis, Enteroviral PCR appears to be more sensitive and have a more rapid turnaround time than viral culture.6,7 Patients with viral meningitis found to have Enterovirus in their CSF by PCR have had reduced hospital costs including shorter hospitalizations, reduced evaluations and interventions, and less antibiotic use.6 The latter benefit is particularly noteworthy as the threat of multi-drug resistant bacterial organisms moves to the fore and the number of safe and effective antibiotics approved for children dwindles.

Previously, Enterovirus PCR analysis of CSF was only available as a send-out test performed at reference laboratories. Now, real-time PCR for Enterovirus detection in CSF samples has been brought to the local hospital and medical center laboratory settings; at some sites, results may be available in less than 24 hours.6 Experts in the field note that there is a risk that sample contamination can yield false positive results, but rates of false positives are not reported.5

Clostridium difficile colitis
C difficile colitis can now be detected by PCR testing. Patients with abdominal complaints and diarrhea typically have a recent history of systemic antibiotic use that may lead to C difficile overgrowth in the gut. Additionally, a strain of C difficile referred to as the NAP-1 strain has been associated with greater severity of disease, most likely the result of the strain’s greater toxin production.8

Earlier C difficile toxin assays lacked sensitivity and frequently required repeat samples to confirm the diagnosis. Newer PCR techniques detect for the presence of the bacterial gene required for toxin production-- and not the actual toxin. The test yields result in less than 24 hours. Sensitivity is reported at greater than 95% in some assays.9
C difficile PCR assay results from stool may be falsely positive in infants under 12 months of age. Therefore, testing should only be performed in infants when other possible causes of diarrhea have been excluded. Additionally, patients under 12 months could be colonized with the organism and still have diarrhea from other causes.

More accurate and faster diagnostic results can lead to prompt discontinuation of unnecessary systemic antibiotics that may be exacerbating C difficile symptoms. These accurate and faster test results may also prompt consideration of specific therapies directed against C difficile.8

PCR Diagnostic Tests for Upper Respiratory Infection
Several tests are now available for prompt diagnosis of upper respiratory infection. Most pediatricians are familiar with nasal wash or nasal swab samples previously used for rapid identification of RSV and influenza viruses by antigen detection methods. Now, nasopharyngeal samples—including nasal washes—may be obtained and tested by multiplex nucleic acid testing (multiplex PCR), which allows for simultaneous detection and identification of multiple respiratory pathogens in 1 to 10 hours.10 

The cost of performing multiplex PCR tests on all patients during the respiratory illness season may be prohibitive. However, certain subgroups (eg, the immunocompromised) may benefit from the prompt diagnosis and initiation of therapy (when appropriate) that this testing makes possible. Additionally, when a viral etiology is rapidly identified, antibiotic therapy may be discontinued, and proper hospital isolation and cohorting steps can be taken, 2 measures that may save money over time.11

Samples are not standardized with multiplex PCR testing, however, and multiple pathogens may be identified on one sample, raising concern about true co-infection versus false positive results.10   

The Way Forward
Matrix Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS), now being tested, holds promise for rapid identification of a variety of pathogens in clinical samples in the future. This testing modality uses unique protein profiles (spectra) of bacteria and fungus to rapidly identify organisms in clinical specimens from known cataloged databases of these profiles.

Routine microbiology laboratory testing requires 2 days or more for organism growth and isolation on culture and complete antibiotic susceptibility testing. MALDI-TOF-MS allows for the detection of bacterial and fungal macromolecules in complex mixtures without purification. Results suggest that it is possible to get blood and urine culture results to the treating clinician in real-time with identification down to the species level—making preliminary results available in hours—not days.

Work is now being done to evaluate whether the MALDI-TOF-MS can detect antibiotic resistance by analyzing specific spectra from different strains of known resistant and susceptible pathogens, such as Staphylococcus aureus isolates. Although this method is not yet widely available, it will likely change the basic management of bacteremia, urinary tract infection, and possibly many other infections, when it becomes clinically available.12

The Bottom Line
Molecular diagnostics have led to the more rapid and accurate diagnosis of infectious diseases in children and are changing the way pediatric medicine is practiced—from the clinic to the ICU. The future appears full of potential, including the further clinical development and use of the MALDI-TOF-MS, which—at least theoretically—could make the “48-hour rule-out sepsis” admission much shorter. These modalities aid in diagnosis and treatment of infections for the betterment of the children and their families. However, as these diagnostics become more readily available, test results must continue to be interpreted within the context of the patient’s signs, symptoms, and epidemiologic risk factors because false positive results do occur.

Table. Pros and Cons of PCR Testing
Infection Pros Cons
Bordetella pertussis Rapid results allow for quick implementation of prophylaxis for contacts close to source case; may detect pathogen presence 3 weeks from cough onset (only 2 weeks with culture) Non-standardized samples; false positives secondary to sample contamination; trained personnel required for testing
Enterovirus Increased sensitivity; rapid turn-around times; decreased antimicrobial use; shorter hospital stays; reduced hospital costs Sample contamination may yield false positive results
Clostridium difficile Results available in <24 h; sensitivity >95% in some assays C. difficile PCR assay results from stool may be falsely positive in infants younger than 12 months; must be run on liquid stools
Upper respiratory infection
(multiplex PCR)
Rapid turn-around time if “in-house”; may allow for prompt discontinuation of antibiotics (viral etiology); may help prompt isolation of hospitalized patients Expensive; non-standardized samples; more than one pathogen may be reported as identified (co-infection vs. false-positive results)



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