Molecular diagnostics are more sensitive and specific than traditional culture-based testing and there are many successful examples where nucleic acid amplification testing has replaced culture.
Current, commercially available molecular testing has the capability to be performed in traditional microbiology laboratories, but laboratories would have to decide what makes sense for their facility.
A thoughtful approach to the implementation of molecular testing is necessary when deciding to transition from culture to molecular testing.
Despite significant advances in molecular testing platforms, in vitro cultivation of microbial pathogens on artificial media remains a hallmark of infectious disease diagnostics. Culture-based approaches allow for the recovery, identification, and antimicrobial susceptibility testing of a specific pathogen. However, there are many limitations to the use of culture. The growth of an organism can take days to weeks; definitive testing for identification can be performed only on pure culture or isolated colony, which may introduce additional delays; some organisms cannot be cultivated in vitro (eg, some viruses, parasites) or have specialized growth requirements that cannot be met by most routine clinical microbiology laboratories (eg, Mycoplasma species). Ultimately, culture is labor intensive and is associated with long turnaround times. Molecular testing has exquisite sensitivity and specificity and is associated with faster turnaround times. Limitations of molecular assays include high cost, the need for specific primer sequences based on an a priori knowledge of the pathogen target sequence, and laboratory personnel with specific expertise in molecular techniques. Nonetheless, there are many areas where laboratories have transitioned away from culture-based pathogen detection to molecular methods. This article discusses the considerations for adoption of molecular assays as a replacement (in whole or in part) for culture, and to highlight examples where this approach has been successfully implemented.
The movement away from culture-based testing to molecular testing for infectious diseases largely began in academic medical centers and specialized reference laboratories, which implemented laboratory-developed molecular testing. Recently, there has been a shift in adoption of commercially available, sample-to-answer testing platforms for more routine pathogens. The nature of these tests allows use by smaller community hospitals and other laboratories located outside of large systems and academic centers. The decision to migrate culture-based diagnostic testing to molecular testing must include an assessment of several factors, including test volume, analytical performance, workflow and throughput, availability of trained personnel, and cost. Regulatory and accreditation factors must also be taken into consideration.
Early adoption of molecular testing as a replacement for culture occurred in the diagnostic virology laboratory. The ability to read cytopathic effect in cell culture and identify specific viruses based on the cytopathic effect observed in various cell lines is an expert skill that has waned in laboratories over time owing to staff attrition and a shortage of new medical laboratory scientists who are interested in microbiology. This situation has caused a shift in virology testing to shorter turnaround time and shell vial cultures, which require their own level of expertise to read immunofluorescent stains. The loss of skilled personnel and the inability to propagate some viruses in culture in vitro led to the replacement of culture-based virology testing with molecular testing. Viruses such as human immunodeficiency virus, hepatitis A, hepatitis B, and hepatitis C were among the first molecular tests used both for the diagnosis of infection and monitoring of viral load over time to determine the effectiveness of antiviral therapy. Testing for other clinically important viruses soon shifted to molecular testing. Respiratory virus testing, for example, has largely transitioned to multiplex molecular panels that allow for a diagnosis of influenza virus A, influenza virus B, and respiratory syncytial virus (as well as other respiratory viruses for which there are no specific antiviral therapies, such as human metapneumovirus) from a single specimen in a single test. The diagnosis of infectious diarrhea has also shifted from culture, specialized microscopy, and rapid antigen testing methods to multiplex molecular gastrointestinal panels. Etiologic causes of infectious gastroenteritis include bacteria, viruses, and parasites, and clinical presentation typically does not allow for a specific diagnosis because diarrhea is the predominant symptom. Therefore, a battery of tests must be ordered, yet often the causative agent remains unidentified. Gastrointestinal panels provide the laboratory with a single test for a single specimen and yield results for a breadth of infectious agents. Performance of these culture-independent diagnostic tests is reported to be excellent
, with a sensitivity of greater than 95% and a specificity of greater than 97%. Despite the improved turnaround time and improvement in the use of infection control practices , culture-independent diagnostic tests have created a quandary with regard to the public health
. The replacement of culture with culture-independent diagnostic tests has resulted in laboratories no longer routinely recovering isolates of Salmonella and other pathogens of public health importance, which must be submitted to public health laboratories for outbreak tracking and other epidemiologic purposes. Some states continue to require submission of an isolate to the public health laboratory, whereas others have allowed submission of the primary specimen that tested positive by a culture-independent diagnostic test. As laboratories work through this diagnostic transitional period, it is critical that the lines of communication between diagnostic laboratories and public health laboratories remain open.
One of the earliest transitions to molecular diagnostics for bacteria was for the sexually transmitted pathogens Neisseria gonorrhoeae and Chlamydia trachomatis . Molecular probe assays and polymerase chain reaction provide exquisite sensitivity and specificity and provide faster time to result compared with culture. Most laboratories now use molecular methods for the screening of patients with suspected C trachomatis – N gonorrhoeae infection. Several molecular assays cleared by the US Food and Drug Administration (FDA) are commercially available and demonstrate excellent performance for urogenital specimens from both males and females ( Table 1 ). Infections in men who have sex with men and other high-risk patients can be diagnosed by testing oropharyngeal, ocular, and/or rectal specimens
. These alternative specimen types have not received FDA clearance; therefore, testing would require validation by individual laboratories. The drawback to molecular testing for these pathogens is that there is no isolate available for antimicrobial susceptibility testing. However, current standard therapeutic regimens recommended by the Centers for Disease Control and Prevention include ceftriaxone, 250 mg administered intramuscularly, and azithromycin, 1 g orally, for uncomplicated infection . The gonococcal isolate surveillance project tracks resistance patterns in the United States by partnering with sexually transmitted disease clinics and publishing resistance patterns for various regions. Clinicians in these locales may rely on gonococcal isolate surveillance project data to provide appropriate empiric therapy of suspected gonorrhea infections. In cases of suspected failure of empiric treatment, the Centers for Disease Control and Prevention recommend culture of appropriate specimens, with antimicrobial susceptibility testing of recovered gonococcal isolates ; however, with the transition to molecular testing, many laboratories no longer have the expertise or reagents to perform this testing on site and would have to send specimens to a reference laboratory.