Traditional Culture vs Molecular PCR
by Lee Schmidt, PhD
Overview
Culture and sensitivity testing is a clinical diagnostic test that has existed for decades with very little advancements or improvements in how the test is performed. Most commonly, culture testing involves taking a clinical sample and allowing it to grow for several days and identifying/analyzing any pathogens that outgrow on artificial media. This process can take 48-96 hours for bacterial cultures and up to 30 days for fungal cultures. Some pathogens are also extremely difficult or even impossible to grow in a lab setting. Sensitivity testing cannot be performed until an adequate amount of bacterial or fungal colonies have grown, separated for unique identification, and grown again in a pure culture. The ability to directly identify pathogens in less than 2 days by DNA analysis has a distinct advantage in both time and accuracy. Here we will discuss a technical comparison of culture methodology via PCR testing, as well as provide a summary of medical utility of these tests from the physicians who intend to use this data to determine treatment for their patients.
Technical Comparison
Below are technical comparisons using published clinical study data explaining the advantages of PCR testing for nail fungus and wound pathogens compared to older methodology, specifically culture and sensitivity methods.
Nail Fungus
Superficial mycoses are becoming a major public health concern and have become one of the most common infections globally, noted back as far as 2006 in Warnock et al.1 and echoed in Gupta et al.2 in 2021. Challenges arise here where superficial fungal infections can lead to invasive infections due to an increase in at-risk populations driven by several co-morbidities including diabetes, cardiovascular issues, and increased population age. Systemic antifungal agents are the most effective treatment for onychomycosis, but unfortunately, they are also associated with serious side effects including hepatotoxicity, drug-drug interactions (DDIs), and renal toxicity.
Successful treatment options for onychomycosis requires a long treatment regimen (typically months) in duration along with active infection management. As it turns out, up to 50% of onychomycosis patients experience a relapse during treatment3. Several factors are known to contribute to this medical issue, but one of the more prominent is incorrect diagnosis at baseline by missing co-infections with non-dermatophyte molds (NDM) or missing the diagnosis of a mixed dermatophyte and NDM infection. It is commonly accepted in the clinical realm that identification of fungi down to the species is important when selecting appropriate anti-fungal therapy, especially infections comprised of NDMs and dermatophytes.
PCR has been used to improve the ability to detect the causative fungi in nail specimens from patients with suspected onychomycosis. Results of a commercial multiplex PCR for the detection of dermatophytes, especially Trichophyton rubrum (the main dermatophyte implicated), as compared to conventional methods were already reported in a published study4. A total of 418 nail scrapings obtained from dermatological outpatients were handled in the Laboratory of Microbiology between May 2010 and May 2013. Among them, multiplex PCR detected 126 (30.1 %) dermatophyte-positive samples, whereas culture revealed 44 (10.5 %). Direct microscopy revealed 63 (15.1 %) positive specimens. T. rubrum was identified in 116 out of 126 (92 %) positive PCR samples and 40 out of 44 (91 %) dermatophyte-positive cultures. Implementation of PCR increased species-specific detection of dermatophytes by 21.1 %, leading to a threefold increase as compared to culture alone. Multiplex PCR offers a time-saving diagnostic tool for tinea unguium and augments laboratory assistance to clinical evaluation for proper treatment.
Another study, Lin et al.,5 provides improved diagnosis of skin and nail dermatophyte infections compared to microscopy and culture: a laboratory study and review of the literature. This study details that dermatophytes are the most common cause of superficial mycosis, estimated to affect 20% to 25% of the general population. They assessed the performance of a novel real-time polymerase chain reaction (RT-PCR) multiplex assay for diagnosis of dermatophytosis. To evaluate sensitivity and specificity, known fungi commonly found on skin, as well as 105 samples with culture confirmed dermatophytosis were tested using Dermatophyte and Fungi assay (AusDiagnostics, Sydney, Australia), a novel multiplex assay for diagnosis of dermatophytosis in skin and nail. This was followed by prospective evaluation of 195 clinical samples for dermatophytosis by both conventional methods and RT-PCR. RT-PCR showed almost two-fold higher sensitivity and high specificity in the diagnosis of skin and nail dermatophytosis compared to traditional microscopy and culture. In addition, RT-PCR demonstrated markedly reduced turnaround time from 4 to 6 weeks to 4 to 6 hours and ability for high throughput.
Every day, more and more studies are published demonstrating the advances in patient care, especially for fungal infections, that revolve around the shift to molecular technology. In summary, PCR has several advantages when used for detection of nail fungus pathogens in lieu of culture methods. As demonstrated in the above clinical studies, PCR is faster, more sensitive, more accurate, and provides the physician with actionable information to improve patient care.
Wounds
Infections can significantly delay the healing process in chronic wounds, placing an enormous economic burden on health care resources. Identification of infection biomarkers and imaging modalities to observe and quantify them has seen progress over the years. While traditional diagnostic approaches provide valuable information, they are time-consuming and depend on clinicians’ experiences. There is a need for noninvasive wound infection diagnostics that are highly specific, rapid, and accurate, and do not require extensive training.
Culture and sensitivity have many major shortcomings in healthcare diagnosis. Failures in bacteria isolation (due to swarms or non-growth on artificial media) are common and lead to loss or non-detection of microorganisms nonrecoverable in culture media. Furthermore, the presence of non-dividing or static bacteria cannot be ruled out as they are still capable of promoting a host immunological response. On culture, it is known that antibiotic therapy might induce a block of bacterial division and the impossibility of recovering cells in culture media. In these cases, a molecular method targeting DNA is advantageous and leads to a much higher likelihood of fully revealing the causative agents in a wound infection. In a 2014 study6, 230 clinical samples with a culture-negative report obtained from 182 patients were examined with a protocol of PCR targeting the bacterial 16S rRNA gene to evaluate the usefulness of molecular methods in differencing culture-negative infections from other pathologies. Amplicons were obtained in 14% of the samples, although this percentage increased (27%) in a subgroup of patients with presumptive diagnosis of infection and ongoing antibiotic therapy. By multiplex PCR, it was shown that detected DNA belonged mostly to Enterobacteriaceae and enterococcal species. Multiple culture-negative, PCR-positive samples and isolation of the same bacterial species in culture in additional samples from the same patient support the clinical significance of the data obtained and highlight the complementary role and usefulness of applying molecular methods in diagnostic microbiology.
In short, molecular PCR testing for wounds is fast, sensitive, and provides actionable information to the physician for same day prescription of the correct antibiotic, greatly enhancing patient care.
