Nucleic acid hybridization technologies utilize complementary properties of the DNA double-helix structures to anneal together DNA fragments from different sources. These techniques are utilized in polymerase chain reaction (PCR) and fluorescent resonance energy transfer (FRET) techniques to identify microorganisms (Khan, 2014). 

A discussion of every infectious agent that might be detected with a probe technique is beyond the scope of this policy. Many probes have been combined into panels of tests. For the purposes of this policy, only individual probes are reviewed.

For guidance on nucleic acid identification of Candida in vaginitis, please refer to CAM 269-Diagnosis of Vaginitis Including Multi-Target PCR Testing.

Regulatory Status 
As of May 11, 2020, a list of current U.S. Food and Drug Administration (FDA, 2020) approved or cleared nucleic acid-based microbial tests is available at: https://www.fda.gov/medical-devices/vitro-diagnostics/nucleic-acid-based-tests..

Policy 

Application of coverage criteria is dependent upon an individual’s benefit coverage at the time of the request. 

1. The coverage status of nucleic acid identification using direct probe, amplified probe, or quantification for the microorganism’s procedure codes is summarized in Table 1 below. "MCC" in the table below indicates that the test is considered MEDICALLY NECESSARY; while “DNMCC” tests indicates that the test is considered NOT MEDICALLY NECESSARY.

2. Simultaneous ordering of any combination of direct probe, amplified probe, and/or quantification for the same organism in a single encounter is considered NOT MEDICALLY NECESSARY.

Policy Guidelines
A discussion of every infectious agent that might be detected with a probe technique is beyond the scope of this policy. Many probes have been combined into panels of tests. For the purposes of this policy, other than the respiratory virus panel, only individual probes are reviewed.

Table of Terminology

Rationale
Nucleic acid hybridization technologies, including polymerase chain reaction (PCR), ligase- or helicase-dependent amplification, and transcription-mediated amplification, are beneficial tools for pathogen detection in blood culture and other clinical specimens due to high specificity and sensitivity (Khan, 2014). The use of nucleic acid-based methods to detect bacterial pathogens in a clinical laboratory setting offers “increased sensitivity and specificity over traditional microbiological techniques” due to its specificity, sensitivity, reduction in time, and high-throughput capability; however, “contamination potential, lack of standardization or validation for some assays, complex interpretation of results, and increased cost are possible limitations of these tests” (Mothershed & Whitney, 2006).

World Health Organization (WHO)
For detection of mpox, the WHO recommends “detection of viral DNA by polymerase chain reaction (PCR)” as the preferred laboratory test and recommends that any individual with a suspected case should be offered testing. They note that the best specimens for diagnosis are taken directly from the rash. Antigen and antibody detection may not be able to distinguish between orthopoxviruses (WHO, 2022).

2018 Infectious Diseases Society of America (IDSA)
Specific guidelines for testing of many organisms listed within the policy coverage criteria is found in the updated 2018 Infectious Diseases Society of America (IDSA) guidelines and recommendations titled, “A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiology” (Miller et al., 2018). “This document is organized by body system, although many organisms are capable of causing disease in > 1 body system. There may be a redundant mention of some organisms because of their propensity to infect multiple sites. One of the unique features of this document is its ability to assist clinicians who have specific suspicions regarding possible etiologic agents causing a specific type of disease. When the term “clinician” is used throughout the document, it also includes other licensed, advanced practice providers. Another unique feature is that in most chapters, there are targeted recommendations and precautions regarding selecting and collecting specimens for analysis for a disease process. It is very easy to access critical information about a specific body site just by consulting the table of contents. Within each chapter, there is a table describing the specimen needs regarding a variety of etiologic agents that one may suspect as causing the illness. The test methods in the tables are listed in priority order according to the recommendations of the authors and reviewers” (Miller et al., 2018).

Centers of Disease Control and Prevention (CDC) 
Candida Auris (C. auris)
The CDC writes that “Molecular methods based on sequencing the D1-D2 region of the 28s rDNA or the Internal Transcribed Region (ITS) of rDNA can identify C. auris.” The CDC further notes that various PCR methods have been developed for identifying C. auris (CDC, 2024d).

Chlamydia Pneumoniae (C. pneumoniae)
The CDC writes that RT-PCR is the “preferred” method of detecting an acute C. pneumoniae infection (CDC, 2024e).

Clostridioides difficile (C. diff)
The CDC states that there are four laboratory tests that can be used to diagnose Clostridioides difficile infection (CDI). “FDA-approved PCR assays are same-day tests that are highly sensitive and specific for the presence of a toxin-producing C. diff organism.” The CDC does note that “molecular assays can be positive for C. diff in asymptomatic individuals and those who do not have an infection” and “when using multi-pathogen (multiplex) molecular methods, read the results with caution as the pre-test probability of C. diff infection might be less” (CDC, 2024b).

Cytomegalovirus (CMV)
The CDC states that “The enzyme-linked immunosorbent assay is the most common serologic test for measuring antibody to CMV.” The CDC also notes that “congenital CMV infection cannot be diagnosed with antibody testing (IgG and IgM)” and recommends “the standard laboratory test for diagnosing congenital CMV infection is a PCR on saliva, with a confirmatory test on urine.” (CDC, 2024f). 

Mpox Virus
The CDC defines a suspect case of Mpox as a “new characteristic rash or meets one of the epidemiologic criteria and has a high clinical suspicion for mpox.” A probable case is defined as “no suspicion of other recent Orthopoxvirus exposure (e.g., Vaccinia virus in ACAM2000 vaccination) AND demonstration of the presence of Orthopoxvirus DNA by polymerase chain reaction of a clinical specimen OR Orthopoxvirus using immunohistochemical or electron microscopy testing methods OR Demonstration of detectable levels of anti-orthopoxvirus IgM antibody during the period of 4 to 56 days after rash onset.” A confirmed case of Mpox is defined as “demonstration of the presence of Mpox virus DNA by polymerase chain reaction testing or Next-Generation sequencing of a clinical specimen OR isolation of Mpox virus in culture from a clinical specimen” (CDC, 2024k).

The CDC states that “Mpox is diagnosed using real time PCR tests” and further notes “clinicians should collect two swabs from each lesion (generally from 2-3 lesions) in case additional testing, such as clade-specific testing, is needed for these patients” (CDC, 2024l).

MRSA
The CDC remarks that “Providers can test some patients to see if they carry MRSA in their nose or on their skin. This test involves rubbing a cotton-tipped swab in the patient's nostrils or on the skin. The only way to know if MRSA is the cause of an infection is to test for the bacteria in a laboratory.” The CDC further states “There are many methods laboratorians can use to test for MRSA” and lists that “Phenotypic methods recommended for the detection of MRSA include: cefoxitin broth microdilution, oxacillin broth microdilution, and cefoxitin disk diffusion testing.” The CDC includes additional methods including “Nucleic acid amplification tests, such as the polymerase chain reaction (PCR), to detect the mecA gene, which mediates oxacillin resistance in staphylococci” but notes “mecA PCR tests will not detect novel resistance mechanisms or uncommon phenotypes (e.g., mecC or borderline-resistant oxacillin resistance)” (CDC, 2024h).

Non-Polio Enterovirus
The CDC remarks that their laboratories “routinely” perform qualitative testing for enteroviruses, parechoviruses, and uncommon picornaviruses and states that “CDC and some health departments test with molecular sequencing methods, or a real-time reverse transcription polymerase chain reaction (rRT-PCR) lab test” (CDC, 2024j).

Respiratory Syncytial Virus (RSV)
The CDC writes that “PCR tests can be used to diagnose anyone for RSV. Antigen tests are only effective when testing infants and young children” (CDC, 2024c).

Miscellaneous
The CDC does not mention the need to quantify [through PCR] Bartonella, Legionella pneumophila, or Mycoplasma pneumoniae. However, PCR can be performed for both Bartonella, Legionella pneumophila, and Mycoplasma pneumoniae specimen (CDC, 2024a, 2024g, 2024i). “Nucleic Acid Amplification Tests (NAATs) are the preferred method of diagnostic testing for M. pneumoniae infections” (CDC, 2024i). No guidance was found on Hepatitis G.

Committee on Infectious Diseases, American Academy of Pediatrics, 31st Edition (2018-2021, Red Book)
The Committee on Infectious Diseases released joint guidelines with the American Academy of Pediatrics. In it, they note that “the presumptive diagnosis of mucocutaneous candidiasis or thrush usually can be made clinically.” They also state that FISH probes may rapidly detect Candida species from positive blood culture samples, although PCR assays have also been developed for this purpose (AAP Committee on Infectious Diseases, 2018).

European Centre for Disease Prevention and Control (ECDC)
On May 23, 2022, the ECDC released a rapid risk assessment of the Mpoxmulti-country outbreak. They recommend that patients with probable cases should be tested with a “Mpox virus specific PCR or an orthopoxvirus specific PCR assay which is then confirmed through sequencing” (ECDC, 2022b).

On June 2, 2022, ECDC released interim advice on risk communication and community engagement during the 2022 Mpox outbreak in Europe. This is a joint report with the WHO regional office for Europe. They recommend speaking to your doctor about getting tested for Mpox if you develop a rash with a fever or feeling of discomfort or illness (ECDC, 2022a). 

United Kingdom Heath Security Agency (UKHSA)
The UKHSA states that “Mpox is diagnosed by PCR test for the Mpox virus (MPXV) on a viral swab taken from one or more vesicles or ulcers.” Specifically, it is recommended that healthcare workers “Take a viral swab in viral culture medium or viral transport medium (for example Virocult^®) from an open sore or from the surface of a vesicle. If other wounds are present, ensure that the sample is definitely taken from a vesicle, an ulcer or a crusted vesicle. Rub the swab over the lesion and place the swab in the collection tube. If there are pharyngeal lesions, a throat swab should also be taken” (UKHSA, 2024). UKHSA also suggests that “A viral throat swab can be taken for high-risk contacts of a confirmed or highly probable case who have developed systemic symptoms but do not have a rash or lesions that can be sampled. Please note that even if the throat swab is negative, the individual must continue with monitoring and isolation as instructed by their local health protection team, and should be reassessed and sampled if further symptoms develop.” Lastly, “If follow-up testing is required from a confirmed or highly probable case, either because of clinical deterioration or to inform discharge from isolation to an inpatient setting, additional samples should be taken and should include the following:

• a lesion swab and throat swab in viral transport medium
• a blood sample in an EDTA tube
• a urine sample in a universal sterile container” (UKHSA, 2024).

The UKHSA states that “Following the identification of a cluster of sexually transmitted HCID Clade I mpox in 2023, there is an increased risk of mpox HCID infection circulating unrecognized on the background of Clade II infections.” They therefore recommend “All diagnostic samples from all individuals testing positive for mpox should now be subject to clade confirmation. Positive mpox samples should be sent to RIPL for clade specific testing if clade differentiation is not available through local mpox testing services” (UKHSA, 2024).

The UKHSA states that mpox DNA viruses can be detected in semen up to 11 days after acute infection, and recommends that: “Following the initial 12 weeks and up to 6 months after recovery from infection, UKHSA recommends performing MPXV PCR on semen samples (and where necessary, oropharyngeal and/or rectal swabs) if the patient:

• is undergoing fertility treatment or planning pregnancy
• is undergoing planned semen storage (for example prior to chemotherapy)
• has an immunocompromised sexual partner (including a pregnant partner)
• is concerned about transmission to sexual partner or partners for any other reason and requests a test from their clinician” (UKHSA, 2024).

HHV-6 Foundation
The human herpesvirus 6 (HHV-6) foundation also states that “a negative finding in the plasma does not rule out a localized active infection in an organ (e.g. uterus, brain, thyroid, liver). Persistent HHV-6 infections have been found in the liver, brain, lungs, heart tissue and uterus, with no trace of HHV-6 DNA in the plasma. Quantitative testing on blood and tissues is preferred because it can differentiate between the very low levels occasionally found in healthy controls and high levels found in diseased tissues” (HHV-6 Foundation, 2024).

The HHV-6 foundation states that qualitative PCR DNA tests on whole blood are “useless for differentiating active from latent infection” but notes that the test may be useful for differentiating between herpes virus-6A and herpes virus-6B. The HHV-6 foundation states that quantitative PCR DNA tests on whole blood can differentiate active from latent infection “If the viral load is >200 copies per ml or 20 copies per microgram of DNA then this is an active infection.”

References:

1. AAP Committee on Infectious Diseases. (2018). Red Book® 2018. https://publications.aap.org/aapbooks/book/546/Red-Book-2018-Report-of-the-Committee-on
2. CDC. (2024a, January 10). Clinical Guidance for Bartonella henselae. https://www.cdc.gov/bartonella/hcp/bartonella-henselae/
3. CDC. (2024b). Clinical Testing and Diagnosis for CDI. https://www.cdc.gov/c-diff/hcp/diagnosis-testing/
4. CDC. (2024c, October 28). Diagnostic Testing for RSV. https://www.cdc.gov/rsv/hcp/clinical-overview/diagnostic-testing.html
5. CDC. (2024d, May 29). Identification of Candida auris. https://www.cdc.gov/candida-auris/hcp/laboratories/identification-of-c-auris.html
6. CDC. (2024e, November 15). Laboratory Testing for Chlamydia pneumoniae. https://www.cdc.gov/cpneumoniae/php/laboratories
7. CDC. (2024f). Laboratory Testing for CMV and Congenital CMV. https://www.cdc.gov/cytomegalovirus/php/laboratories/index.html
8. CDC. (2024g, March 25). Laboratory Testing for Legionella. https://www.cdc.gov/legionella/php/laboratories
9. CDC. (2024h). Laboratory Testing for Methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA). https://www.cdc.gov/mrsa/php/laboratories/index.html
10. CDC. (2024i, June 5). Laboratory Testing for Mycoplasma pneumoniae. https://www.cdc.gov/mycoplasma/php/laboratories
11. CDC. (2024j, November 14). Laboratory Testing for Non-Polio Enterovirus. https://www.cdc.gov/non-polio-enterovirus/php/laboratories/index.html
12. CDC. (2024k, July 22). Mpox Case Definitions. https://www.cdc.gov/poxvirus/monkeypox/clinicians/case-definition.html
13. CDC. (2024l). Mpox Clinical Testing. https://www.cdc.gov/poxvirus/mpox/clinicians/clinical-testing.html
14. ECDC. (2022a). Interim advice on Risk Communication and Community Engagement during the monkeypox outbreak in Europe, 2022. https://www.ecdc.europa.eu/sites/default/files/documents/Joint-ECDC-WHO-interim-advice-on-RCCE-for-Monkeypox-2-June-2022.pdf
15. ECDC. (2022b). Risk assessment: Monkeypox multi-country outbreak. https://www.ecdc.europa.eu/en/publications-data/risk-assessment-monkeypox-multi-country-outbreak
16. FDA. (2022, April 19). Nucleic Acid Based Tests. https://www.fda.gov/medical-devices/vitro-diagnostics/nucleic-acid-based-tests
17. HHV-6 Foundation. (2024). Overview on Testing for HHV-6 infection. https://hhv-6foundation.org/patients/hhv-6-testing-for-patients
18. Khan, A. (2014). Rapid Advances in Nucleic Acid Technologies for Detection and Diagnostics of Pathogens. J Microbiol Exp, 1(2). https://doi.org/10.15406/jmen.2014.01.00009
19. Miller, J. M., Binnicker, M. J., Campbell, S., Carroll, K. C., Chapin, K. C., Gilligan, P. H., Gonzalez, M. D., Jerris, R. C., Kehl, S. C., Patel, R., Pritt, B. S., Richter, S. S., Robinson-Dunn, B., Schwartzman, J. D., Snyder, J. W., Telford, I. I. I. S., Theel, E. S., Thomson, J. R. B., Weinstein, M. P., & Yao, J. D. (2018). A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiology. Clinical Infectious Diseases, ciy381-ciy381. https://doi.org/10.1093/cid/ciy381
20. Mothershed, E. A., & Whitney, A. M. (2006). Nucleic acid-based methods for the detection of bacterial pathogens: present and future considerations for the clinical laboratory. Clin Chim Acta, 363(1-2), 206-220. https://doi.org/10.1016/j.cccn.2005.05.050
21. UKHSA. (2024, February 15). Monkeypox: diagnostic testing. https://www.gov.uk/guidance/monkeypox-diagnostic-testing
22. WHO. (2022). Monkeypox. https://www.who.int/health-topics/monkeypox