Medical Policy

Policy Num:      11.001.007
Policy Name:    Identification of Microorganisms Using Nucleic Acid Probes
Policy ID:          [11.001.007]  [Ac / B / M+ / P-]  [2.04.10]

Last Review:       July 16, 2024
Next Review:      July 20, 2025
 

Related Policies:
11.001.038. - Multitarget Polymerase Chain Reaction Testing for Diagnosis of Bacterial Vaginosis
05.001.039. - Intravenous Antibiotic Therapy and Associated Diagnostic Testing for Lyme Disease

Identification of Microorganisms Using Nucleic Acid Probes 

summary

Description

Nucleic acid probes are available for the identification of a wide variety of microorganisms. Nucleic acid probes can also be used to quantitate the number of microorganisms present. This technology offers advantages over standard techniques when rapid identification is clinically important, microbial identification using standard culture is difficult or impossible, and/or treatment decisions are based on quantitative results.

Summary of Evidence

For individuals who have signs and/or symptoms of meningitis and/or encephalitis who receive a nucleic acid-based central nervous system pathogen panel, the evidence includes a systematic review and a pivotal prospective study. Relevant outcomes include test accuracy and validity, other test performance measures, medication use, symptoms, and change in disease status. Access to a rapid method that can simultaneously test for multiple pathogens may lead to the faster initiation of more effective treatment and conservation of cerebrospinal fluid. The available central nervous system panel is highly specific for the included organisms, but the sensitivity for each pathogen is not well-characterized. More than 15% of positives in the largest clinical validity study were false-positives. A negative panel result does not exclude infection due to pathogens not included in the panel. The evidence is insufficient to determine that the technology results in an improvement in the net health outcome.

For individuals who have signs and/or symptoms of gastroenteritis who receive a nucleic acid-based gastrointestinal pathogen panel, the evidence includes prospective and retrospective evaluations of the tests’ sensitivity and specificity and prospective studies on utility. Relevant outcomes include test accuracy and validity, other test performance measures, medication use, symptoms, and change in disease status. The evidence suggests that pathogen panels are likely to identify both bacterial and viral pathogens with high sensitivity, compared with standard methods. Access to a rapid method for etiologic diagnosis of infections may lead to more effective early treatment and infection control measures. However, in most instances, when a specific pathogen is suspected, individual tests could be ordered. There may be a subset of patients with an unusual presentation who would warrant testing for a panel of pathogens at once, but that subset has not been well defined. The evidence is insufficient to determine that the technology results in an improvement in the net health outcome.

For individuals who have signs and/or symptoms of respiratory infection who receive a nucleic acid-based respiratory pathogen panel, the evidence includes a systematic review and 2 randomized controlled trials (RCTs). Relevant outcomes include test accuracy and validity, other test performance measures, medication use, symptoms, and change in disease status. The systematic review reported that all 3 reviewed multiplex polymerase chain reaction systems were highly accurate. One RCT and 1 quasi-RCT evaluated utility of a respiratory panel and found benefits in time-to-treat and length of hospital stay. In addition, 1 subanalysis found fewer antibiotics being prescribed for patients diagnosed with the panel. The panel did not significantly affect duration of antibiotic use, readmission, or mortality rates. The evidence is sufficient to determine that the technology results in an improvement in the net health outcome.

Additional Information

Not applicable.

OBJECTIVE

The objective of this evidence review is to determine whether testing for microorganisms using nucleic acid probes improves the net health outcome in individuals with suspected infections.

POLICY statements

The use of nucleic acid testing using a direct or amplified probe technique (without quantification of viral load) may be considered medically necessary for the following microorganisms (see Policy Guidelines):

• Bartonella henselae or quintana

• Bordetella pertussis

• Candida species

• Chlamydia pneumoniae

• Chlamydia trachomatis

• Clostridium difficile

• Enterococcus, vancomycin-resistant (eg, enterococcus vanA, vanB)

• Enterovirus

• Herpes simplex virus

• Human papillomavirus

• Influenza virus

• Legionella pneumophila

• Mumps

• Mycobacterium species

• Mycobacterium tuberculosis

• Mycobacterium avium-intracellulare

• Mycoplasma genitalium (MG)

• Mycoplasma pneumoniae

• Neisseria gonorrhoeae

• Rubeola (measles)

• Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

• Staphylococcus aureus

• Staphylococcus aureus, methicillin-resistant

• Streptococcus, group A

• Streptococcus, group B

• Trichomonas vaginalis

• Zika virus.

The use of nucleic acid testing using a direct or amplified probe technique (with or without quantification of viral load) may be considered medically necessary for the following microorganisms:

• Cytomegalovirus

• Hepatitis B virus

• Hepatitis C virus

• HIV 1

• HIV-2

• Human herpesvirus 6.

The use of nucleic acid testing with quantification of viral load is considered investigational for microorganisms that are not included in the list of microorganisms for which probes with or without quantification are considered medically necessary.

The use of nucleic acid testing using a direct or amplified probe technique is considered investigational for the following microorganisms:

• Gardnerella vaginalis

• Hepatitis G.

The use of the following nucleic acid testing panel (without quantification of viral load) may be considered medically necessary:

• Respiratory virus panel.

The use of the following nucleic acid testing panels (with or without quantification of viral load for viral panel elements) is considered investigational:

• Central nervous system pathogen panel

• Gastrointestinal pathogen panel.

POLICY GUIDELINES

For the purposes of this policy, other than the respiratory pathogen panel, gastrointestinal pathogen panel, and central nervous system panel, nucleic acid testing for individual organisms is informed by published guidelines and is not subject to evidence review (see Supplemental Information). Many probes have been combined into panels of tests. Multi-target tests are commercially available and some are FDA cleared (e.g., Alinity mSTI). The FDA maintains a list of 'Cleared or Approved Nucleic Acid Based Tests’ at https://www.fda.gov/medical-devices/in-vitro-diagnostics/nucleic-acid-based-tests. New tests may become available between policy updates.

The use of molecular diagnostics for the diagnosis and management of Borrelia burgdorferi infection (Lyme disease) is addressed in evidence review 5.01.08.

Vaccine-preventable disease surveillance for outbreaks and diagnosis of isolated cases: the Centers for Disease Control and Prevention (CDC) Pertussis and Diphtheria Laboratory has developed its own polymerase chain reaction (PCR) and serological assays to diagnose pertussis, mumps, and rubeola (measles) and has recommendations for their appropriate use.

For bacterial vaginosis, this evidence review addresses the use of single organism direct or amplified nucleic acid probes with or without quantification. Evidence review 2.04.127 (Multitarget Polymerase Chain Reaction Testing for Diagnosis of Bacterial Vaginosis) addresses the use of multitarget PCR tests for bacterial vaginosis.

For Candida species, culture for yeast remains the criterion standard for identifying and differentiating these organisms. Although sensitivity and specificity are higher for nucleic acid amplification tests (NAATs) than for standard testing methods, the CDC and other association guidelines do not recommend NAATs as first-line testing for Candida species. The CDC (2015) classifies uncomplicated vulvovaginal candidiasis as being sporadic or infrequent; or mild to moderate; or, in nonimmunocompromised individuals, as likely to be caused by C. albicans. A presumptive diagnosis can be made in the clinical care setting. However, for complicated infections, the CDC states that NAATs may be necessary to test for multiple Candida subspecies. Complicated vulvovaginal candidiasis is classified as being recurrent or severe; or, in individuals with uncontrolled diabetes, debilitation, or immunosuppression, as less likely to be caused by a C. albicans species.

Antibiotic sensitivity of streptococcus A culture is generally not performed for throat cultures. However, if an antibiotic sensitivity is considered, then the most efficient method of diagnosis would be a combined culture and antibiotic sensitivity.

In the evaluation of group B streptococcus, the primary advantage of a DNA probe technique compared with traditional culture techniques is the rapidity of results. This advantage suggests that the most appropriate use of the DNA probe technique is in the setting of impending labor, for which prompt results could permit the initiation of intrapartum antibiotic therapy.

Use of NAAT for SARS-CoV-2 is for confirming coronavirus disease 2019 (COVID-19) diagnoses. This medical policy does not address antibody testing (serological IgG assays).

It should be noted that the technique for quantification includes both amplification and direct probes; therefore, simultaneous coding for both quantification with either amplification or direct probes is not warranted.

Many probes have been combined into panels of tests. For the purposes of this policy, other than the respiratory pathogen panel, gastrointestinal pathogen panel, and central nervous system panel, only individual probes are reviewed.

Coding

Please see the Codes table for details.

BENEFIT APPLICATION

BlueCard/National Account Issues

Plans may want to identify specific sites of service (ie, hospital, reference laboratory). Plans may also review benefits for test panels by evaluating individual components of the panel.

State or federal mandates (eg, Federal Employee Program) may dictate that certain U.S. Food and Drug Administration (FDA) approved devices, drugs, or biologics may not be considered investigational, and thus these devices may be assessed only by their medical necessity.

Benefits are determined by the group contract, member benefit booklet, and/or individual subscriber certificate in effect at the time services were rendered. Benefit products or negotiated coverages may have all or some of the services discussed in this medical policy excluded from their coverage.

BACKGROUND

Nucleic Acid Probes

A nucleic acid probe is used to detect and identify species or subspecies of organisms by identifying nucleic acid sequences in a sample. Nucleic acid probes detect genetic materials, such as RNA or DNA, unlike other tests, which use antigens or antibodies to diagnose organisms.

The availability of nucleic acid probes has permitted the rapid direct identification of microorganism DNA or RNA. Amplification techniques result in exponential increases in copy numbers of a targeted strand of microorganism-specific DNA. The most used amplification technique is polymerase chain reaction (PCR) or reverse transcriptase PCR. In addition to PCR, other nucleic acid amplification techniques have been developed, such as transcription-mediated amplification, loop-mediated isothermal DNA amplification, strand displacement amplification, nucleic acid sequence-based amplification, and branched-chain DNA signal amplification. After amplification, target DNA can be readily detected using a variety of techniques. The amplified product can also be quantified to assess how many microorganisms are present. Quantification of the number of nucleic acids permits serial assessments of response to treatment; the most common clinical application of quantification is the serial measurement of HIV RNA (called viral load).

The direct probe technique, amplified probe technique, and probe with quantification methods vary based on the degree to which the nucleic acid is amplified and the method for measurement of the signal. The direct probe technique refers to detection methods in which nucleic acids are detected without an initial amplification step. The amplified probe technique refers to detection methods in which either target, probe, or signal amplification is used to improve the sensitivity of the assay over direct probe techniques, without quantification of nucleic acid amounts.

• Target amplification methods include PCR (including PCR using specific probes, nested or multiplex PCR), nucleic acid-based sequence amplification, transcription-mediated amplification, and strand displacement amplification. Nucleic acid-based sequence amplification and transcription-mediated amplification involve amplification of an RNA (rather than a DNA) target.

• Probe amplification methods include ligase chain reaction.

• Signal amplification methods include branched DNA (bDNA) probes and hybrid capture methods using an anti-DNA/RNA hybrid antibody.

The probe with quantification techniques refers to quantitative PCR or real-time PCR methods that use a reporter at each stage of the PCR to generate absolute or relative amounts of a known nucleic acid sequence in the original sample. These methods may use DNA-specific dyes (ethidium bromide or SYBR green), hybridization probes (cleavage-based [TaqMan] or displaceable), or primer incorporated probes.

Direct assays will generally have lower sensitivity than amplified probes. In practice, most commercially available probes are amplified, with a few exceptions. For this evidence review, indications for direct and/or amplified probes without quantification are considered together, while indications for a probe with quantification are considered separately.

Classically, identification of microorganisms relies either on the culture of body fluids or tissues or identification of antigens, using a variety of techniques including direct fluorescent antibody technique and qualitative or quantitative immunoassays. These techniques are problematic when the microorganism exists in very small numbers or is technically difficult to culture. Indirect identification of microorganisms by immunoassays for specific antibodies reactive with the microorganism is limited by difficulties in distinguishing between past exposure and current infection.

Potential reasons for a nucleic acid probe to be associated with improved clinical outcomes compared with standard detection techniques include the following (note: in all cases, for there to be clinical utility, making a diagnosis should be associated with changes in clinical management, which could include initiation of effective treatment, discontinuation of other therapies, or avoidance of invasive testing):

• Significantly improved speed and/or efficiency in making a diagnosis.

• Improved likelihood of obtaining any diagnosis in cases where standard culture is difficult. Potential reasons for difficulty in obtaining standard culture include low numbers of the organisms (eg, HIV), fastidious or lengthy culture requirements (eg, Mycobacteria, Chlamydia, Neisseria species), or difficulty in collecting an appropriate sample (eg, herpes simplex encephalitis).

• There is no way to definitively make a diagnosis without nucleic acid testing.

• The use of nucleic acid probe testing provides qualitatively different information than that available from standard cultures, such as information regarding disease prognosis or response to treatment. These include cases where quantification of viral load provides prognostic information or is used to measure response to therapy.

The risks of nucleic acid testing include false-positive and false-negative results, inaccurate identification of pathogens by the device, inaccurate interpretation of test results, or incorrect operation of the instrument.

• False-positive results can lead to unnecessary treatment, with its associated toxicities and side effects, including allergic reaction. In addition, true diagnosis and treatment could be delayed or missed altogether.

• False-negative results could delay diagnosis and initiation of proper treatment.

• It is possible that these risks can be mitigated by the use of a panel of selected pathogens indicated by the clinical differential diagnosis while definitive culture results are pending.

Regulatory Status

The U.S. Food and Drug Administration maintains a list of nucleic acid amplification tests (NAATs) that have been cleared by the Center for Devices and Radiological Health. These NAATs have been cleared for many of the microorganisms discussed in this review and may be reviewed on this site.

Table 1 summarizes the NAATs cleared for central nervous system panels when diagnosing meningitis and/or encephalitis, for panels when diagnosing gastroenteritis, and for respiratory panels.

Table 1. FDA Cleared Nucleic Acid Amplification Tests for Central Nervous System, Gastrointestinal, and Respiratory Panels

;CNS: central nervous system; DEN: de novo; FDA: Food and Drug Administration.

Clinical laboratories may develop and validate tests in-house and market them as a laboratory service; laboratory-developed tests must meet the general regulatory standards of the Clinical Laboratory Improvement Amendments (CLIA). Laboratories that offer laboratory-developed tests must be licensed by the CLIA for high-complexity testing.

RATIONALE

This evidence review was created in July 1998 and has been updated regularly with searches of the PubMed database. The most recent literature update was performed through May 2, 2024.

Evidence reviews assess whether a medical test is clinically useful. A useful test provides information to make a clinical management decision that improves the net health outcome. That is, the balance of benefits and harms is better when the test is used to manage the condition than when another test or no test is used to manage the condition.

The first step in assessing a medical test is to formulate the clinical context and purpose of the test. The test must be technically reliable, clinically valid, and clinically useful for that purpose. Evidence reviews assess the evidence on whether a test is clinically valid and clinically useful. Technical reliability is outside the scope of these reviews, and credible information on technical reliability is available from other sources.

The evidence review section of this policy update focuses on pathogen panels. The supplemental information section contains supporting information for the medical necessity of the use of the organism-specific nucleic acid amplification tests (NAATs) which have guideline support. Guidelines from the Centers for Disease Control and Prevention, National Institute of Health, Infectious Diseases Society of America, or America Academy of Pediatrics were used to evaluate appropriate indications for the following individual microorganisms: Bartonella henselae or quintana, Candida species, Chlamydia pneumoniae, Chlamydia trachomatis, Clostridium difficile, cytomegalovirus, enterovirus, hepatitis B virus, hepatitis C virus, herpes simplex virus, human herpesvirus 6, human papillomavirus, HIV-1, influenza virus, Legionella pneumophila, Mycobacteria species, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Staphylococcus aureus, Streptococcus group A and group B, vancomycin-resistant Enterococcus, and Zika virus.

Promotion of greater diversity and inclusion in clinical research of historically marginalized groups (e.g., People of Color [African-American, Asian, Black, Latino and Native American]; LGBTQIA (Lesbian, Gay, Bisexual, Transgender, Queer, Intersex, Asexual); Women; and People with Disabilities [Physical and Invisible]) allows policy populations to be more reflective of and findings more applicable to our diverse members. While we also strive to use inclusive language related to these groups in our policies, use of gender-specific nouns (e.g., women, men, sisters, etc.) will continue when reflective of language used in publications describing study populations.

Population Reference No. 1

Central Nervous System Bacterial and Viral Panel

Clinical Context and Test Purpose

The purpose of nucleic acid-based central nervous system (CNS) pathogen panels is to provide a diagnostic option that is an alternative to or an improvement on existing tests for patients with signs and/or symptoms of meningitis and/or encephalitis.

The following PICO was used to select literature to inform this review.

Populations

The relevant population of interest is individuals with signs and/or symptoms of meningitis and/or encephalitis.

Interventions

The test being considered is nucleic acid-based CNS pathogen panel.

Patients with signs and/or symptoms of meningitis and/or encephalitis are managed by infectious disease specialists and emergency medicine professionals in an emergency or inpatient clinical setting. Testing with a CNS pathogen panel leads to reduced time to diagnosis compared with standard laboratory techniques (approximately 1 to 8 hours).^1,

The FilmArray Meningitis/Encephalitis (ME) Panel (BioFire Diagnostics, Salt Lake City, UT) is a nucleic acid-based test that simultaneously detects multiple bacterial, viral, and yeast nucleic acids from cerebrospinal fluid (CSF) specimens obtained via lumbar puncture from patients with signs and/or symptoms of meningitis and/or encephalitis. The test has been cleared for marketing through the FDA 510(k) process. The test identifies 14 common organisms responsible for community-acquired meningitis or encephalitis:

Bacteria: Escherichia coli K1; Haemophilus influenzae; Listeria monocytogenes; Neisseria meningitidis; Streptococcus agalactiae; Streptococcus pneumoniae;
Viruses: Cytomegalovirus; enterovirus; herpes simplex virus 1; herpes simplex virus 2; human herpesvirus 6; human parechovirus; varicella-zoster virus;
Yeast: Cryptococcus neoformans/gattii.

Run-time is approximately 1 hour per specimen.

Comparators

Comparators of interest include culture or serologic tests and CNS pathogen-specific testing (nucleic acid-based testing for individual pathogens).

The standard approach to the diagnosis of meningitis and encephalitis is culture and pathogen-specific polymerase chain reaction (PCR) testing of CSF based on clinical characteristics. These techniques have a slow turnaround time, which can delay administration of effective therapies and lead to unnecessary empirical administration of broad-spectrum antimicrobials.

Outcomes

The general outcomes of interest are test accuracy, test validity, other test performance measures, medication use, symptoms, and change in disease status.

True-positive and true-negative results lead to faster diagnosis and correct treatment, or no unnecessary treatment, as well as fewer repeated tests.

False-positive and false-negative results, inaccurate identification of a pathogen by the testing device, failure to correctly interpret test results, or failure to correctly operate the instrument may lead to misdiagnosis resulting in inappropriate treatment while postponing treatment for the true condition. Such a situation could lead to incorrect, unnecessary, or no treatment, necessity for additional testing, and delay of correct diagnosis and treatment.

Though not completely standardized, follow-up for suspected meningitis and/or encephalitis would typically occur in the days to weeks after a diagnosis decision and initiation of treatment.

Study Selection Criteria

Methodologically credible studies were selected using the following principles:

• The study population represents the population of interest. Eligibility and selection are described.

• The test is compared with a credible reference standard.

• If the test is intended to replace or be an adjunct to an existing test , it should also be compared with that test.

• Studies should report sensitivity, specificity, and predictive values. Studies that completely report true- and false-positive results are ideal. Studies reporting other measures (eg, receiver operating characteristic [ROC], area under the receiver operating characteristic curve [AUROC], c-statistic, likelihood ratios) may be included but are less informative.

• • Reported on a validation cohort that was independent of the development cohort.

• Studies should also report reclassification of diagnostic or risk category.

Clinically Valid

A test must detect the presence or absence of a condition, the risk of developing a condition in the future, or treatment response (beneficial or adverse).

Review of Evidence

The systematic review and meta-analysis by Tansarli and Chapin (2019) examined the diagnostic accuracy of the BioFire FilmArray ME panel.^2, Thirteen prospective and retrospective studies conducted from 2016 through 2019 were reviewed (N=3764 patients); 8 were included in the meta-analysis (n=3059 patients). Included in the meta-analysis is the study by Leber et al [2016]^3,, which is discussed below. Risk of bias among the studies was mixed but tended toward low risk, with the index test aspect being most questionable. No applicability concerns were found in any studies. To be eligible, studies had to provide sensitivity and specificity data compared with a reference standard. Patients in the studies had infections caused by a variety of components found on the panel (bacterial, viral, Cryptococcus neoformans/gatti). Table 2 summarizes the sensitivity, specificity, and other measurements of accuracy. The highest proportions of false-positive results were for Streptococcus pneumoniae (17.5%) and Streptococcus agalactiae (15.4%). The highest proportion of false negatives was seen for herpes simplex virus types 1 and 2, enterovirus, and C neoformans/gatti. The rate of false-positive results with the ME panel suggests this method should be used with caution, and additional diagnostic methods should be used to confirm panel results.

Table 2. Accuracy of BioFire FilmArray Meningitis/Encephalitis Panel

Source: Tansarli and Chapin (2019)2,CI: confidence interval; NPV: negative predictive value; NR: not reported; PPV: positive predictive value.a Adjudication is further investigation of results, which could include further testing, clinician input, or chart review. In this study, it was performed for discordant results between index and reference tests.

The study by Leber et al (2016) was a FDA pivotal study, as well as the largest and 1 of the only prospective studies available.^3, A total of 1560 samples were tested, which were taken from children and adults with available CSF, but not limited to those with high pretest probability for an infectious cause for meningitis or encephalitis. (Table 3 ) Even the most prevalent organisms were present only a small number of times in the samples. The specificities ranged from 98% to 100% and, given the high number of true negatives, the specificities were estimated with tight precision. However, given the small number of true positives, the sensitivities to detect any given organism could not be estimated with precision. A total of 141 pathogens were detected in 136 samples with the FilmArray and 104 pathogens were detected using comparator methods; 43 FilmArray results were false-positive compared with the comparator method and 6 were false-negative. For 21 of the 43 false-positives, repeat testing of the FilmArray, comparator, or additional molecular testing supported the FilmArray results. The remaining 22 false-positives (16% of all positives) were unresolved. Codetections were observed in 3.7% (5/136) of positive specimens. All 5 included a bacterial and viral positive result, and all 5 specimens were found to have a false-positive result demonstrated by comparator testing. (Table 4 ) The investigators suggested that the discrepancies could have been due to specimen contamination or another problem with the assay configuration or testing process.

Smaller studies^4,5, were consistent with Leber (2016) in estimating the specificities for all included pathogens to be greater than 98%. However, there were also a very low number of true-positives for most pathogens in these studies and thus the estimates of sensitivities were imprecise. Relevance, study design, and trial conduct limitations are shown in Tables 5 and 6.

Cuesta et al (2024) prospectively evaluated the performance of a multiplex PCR assay (QIAstat-Dx ME panel) compared to conventional diagnostic methods and the Biofire FilmArray ME Panel for diagnosing meningoencephalitis in 50 CSF samples.^6, Conventional methods identified a pathogen in 29 CSF samples (58%), with 41% bacterial and 59% viral etiologies. The QIAstat-Dx ME panel demonstrated a sensitivity of 96.5% (95% CI, 79.8% to 99.8%) and specificity of 95.2% (95% CI, 75.2% to 99.7%), with high positive predictive values (PPV) and negative predictive values (NPV) (96.4% and 95.2%) and complete agreement (91.8%) with conventional methods based on Cohen's kappa. In contrast, the FilmArray ME panel had a lower sensitivity (85.1%; 95% CI, 55.9% to 90.2%), specificity (57.1%; 95 %CI, 29.6% to 70.3%), positive and negative predictive values and only moderate agreement (43.5%) with conventional methods. The FilmArray ME panel reported 7 single-pathogen and 5 polymicrobial false positive results, most commonly for herpes simplex virus (HSV)-1, while the QIAstat-Dx ME panel had only one false positive (VZV) and one false negative (HSV-1) result. Limitations include the enrichment of positive samples in the QIAstat-Dx ME analysis and the inability to evaluate all panel targets due to a lack of some positive CSF samples.

López et al (2024) retrospectively reviewed the performance of the Biofire FilmArray ME panel compared to conventional diagnostic methods in 313 patients with suspected ME seen at a single-center from 2018 to 2022.^7, FilmArray was positive in 84 cases (26.8%) (HSV-1 [10.9%], varicella zoster virus (VZV) [5.1%], Enterovirus [2.6%], and S. pneumoniae [1.9%]). In the 136 cases where both FilmArray and routine methods were performed, there was a 25.7% lack of agreement. In the overall tested population, the sensitivity was estimated to be 81% (95% CI, 70.6% to 89%) with a specificity of 89% (95% CI, 85.4% to 93.4%). The authors reported a high NPV (93.4%; 95 % CI, 89.9% to 95.7%) and modest PPV (73%; 95 %CI, 64.6% to 80.1%). While FilmArray had a low false negative rate of 6.6%, it reported a high false positive rate of 28.6%, mainly due to HSV-1. The authors observed that the PPV dropped to 36.9% in cases without pleocytosis and 70.2% in those lacking high CSF protein levels; other test characteristics were less impacted by individual CSF characteristics. Limitations include the retrospective single-center design and that conventional testing could not be performed on all samples due to insufficient volume.

Table 3. Characteristics of Clinical Validity Studies of Central Nervous System Panel

CNS: central nervous system; CSF: cerebrospinal fluid; LDT: laboratory-developed test; NR: not reported; PCR: polymerase chain reaction.

Table 4. Results of Clinical Validity Studies of Central Nervous System Panel

CI: confidence interval; CNS: central nervous system; NA: not available; NR: not reported.a Positives and negatives retrospectively selected from a convenience sample with different selection criteria; prevalence is unknown.b Confidence intervals not provided in publication; estimated based on available information.

Tables 5 and 6 display notable limitations identified in each study.

Table 5. Study Relevance Limitations of Studies of Central Nervous System Panels

CNS: central nervous system.The evidence limitations stated in this table are those notable in the current review; this is not a comprehensive gaps assessment.a Population key: 1. Intended use population unclear; 2. Clinical context is unclear; 3. Study population is unclear; 4. Study population not representative of intended use.b Intervention key: 1. Classification thresholds not defined; 2. Version used unclear; 3. Not intervention of interest.c Comparator key: 1. Classification thresholds not defined; 2. Not compared to credible reference standard; 3. Not compared to other tests in use for same purpose.d Outcomes key: 1. Study does not directly assess a key health outcome; 2. Evidence chain or decision model not explicated; 3. Key clinical validity outcomes not reported (sensitivity, specificity and predictive values) ; 4. Reclassification of diagnostic or risk categories not reported; 5. Adverse events of the test not described (excluding minor discomforts and inconvenience of venipuncture or noninvasive tests).e Follow-Up key: 1. Follow-up duration not sufficient with respect to natural history of disease (true positives, true negatives, false positives, false negatives cannot be determined).

Table 6. Study Design and Conduct Limitations

The evidence limitations stated in this table are those notable in the current review; this is not a comprehensive gaps assessment.a Selection key: 1. Selection not described; 2. Selection not random or consecutive (ie, convenience).b Blinding key: 1. Not blinded to results of reference or other comparator tests.c Test Delivery key: 1. Timing of delivery of index or reference test not described; 2. Timing of index and comparator tests not same; 3. Procedure for interpreting tests not described; 4. Expertise of evaluators not described.d Selective Reporting key: 1. Not registered; 2. Evidence of selective reporting; 3. Evidence of selective publication.e Data Completeness key: 1. Inadequate description of indeterminate and missing samples; 2. High number of samples excluded; 3. High loss to follow-up or missing data.f Statistical key: 1. Confidence intervals and/or p values not reported; 2. Comparison to other tests not reported.

Clinically Useful

A test is clinically useful if the use of the results informs management decisions that improve the net health outcome of care. The net health outcome can be improved if patients receive correct therapy, more effective therapy, or avoid unnecessary therapy, or testing.

Review of Evidence

Direct Evidence

Direct evidence of clinical utility is provided by studies that have compared health outcomes for patients managed with and without the test. Because these are intervention studies, the preferred evidence would be from randomized controlled trials (RCTs).

No RCTs were available that evaluated clinical utility.

Chain of Evidence

Indirect evidence on clinical utility rests on clinical validity. If the evidence is insufficient to demonstrate test performance, no inferences can be made about clinical utility.

Section Summary: Central Nervous System Bacterial and Viral Panel

The FilmArray ME Panel provides fast diagnoses compared with standard culture and pathogen-specific PCR, and because it combines multiple individual nucleic acid tests, clinicians can test for several potential pathogens simultaneously. The test uses only a small amount of CSF, leaving remaining fluid for additional testing if needed. The test is highly specific for the included organisms. However, due to the low prevalence of these pathogens overall, the sensitivity for each pathogen is not well-characterized. More than 15% of positives in the largest study were reported to be false-positives, which could cause harm if used to make clinical decisions. Also, a negative panel result does not exclude infection due to pathogens not included in the panel.

For individuals who have signs and/or symptoms of meningitis and/or encephalitis who receive a nucleic acid-based central nervous system pathogen panel, the evidence includes a systematic review and a pivotal prospective study. Relevant outcomes include test accuracy and validity, other test performance measures, medication use, symptoms, and change in disease status. Access to a rapid method that can simultaneously test for multiple pathogens may lead to the faster initiation of more effective treatment and conservation of cerebrospinal fluid. The available central nervous system panel is highly specific for the included organisms, but the sensitivity for each pathogen is not well-characterized. More than 15% of positives in the largest clinical validity study were false-positives. A negative panel result does not exclude infection due to pathogens not included in the panel. The evidence is insufficient to determine the effects of the technology on health outcomes.

Population Reference No. 2

Gastrointestinal Pathogen Panel

Clinical Context and Test Purpose

The purpose of nucleic acid-based gastrointestinal (GI) pathogen panels is to provide a diagnostic option that is an alternative to or an improvement on existing tests in patients with signs and/or symptoms of gastroenteritis.

The following PICO was used to select literature to inform this review.

Populations

The relevant population of interest is individuals with signs and/or symptoms of gastroenteritis.

The most common 2 types of GI pathogens are either bacterial or viral, including but not limited to the following^8,9,10,:

• Bacterial (common to U.S. and may be foodborne): Bacillus cereus, Campylobacter, Clostridioides (Clostridium) difficile, Clostridium botulinum, Clostridium perfringens, Cronobacter sakazakii, Esherichia coli, Listeria monocytogenes, Salmonella spp., Shigella spp., Staphylococcus aureus, Yersinia enterocolitica.

• Viral: norovirus, rotavirus, adenovirus, astrovirus, sapovirus.

Norovirus is the most common cause of foodborne illness in the U.S.^11,

Interventions

The intervention being considered is testing with a nucleic acid-based GI pathogen panel.

These panels are capable of qualitatively detecting the DNA or RNA of multiple pathogens, including but not limited to Campylobacter, Clostridioides (Clostridium) difficile, Plesiomonas shigelloides, Salmonella spp., Yersinia spp., enteroaggregative Escherichia coli, enteropathogenic E coli, enterotoxigenic E coli (ETEC), Shiga toxin-producing E coli (STEC), E coli O157, Shigella/enteroinvasive E coli, adenovirus F 40/41, astrovirus, norovirus, rotavirus, and sapovirus.

For community-acquired diarrheal illness, extensive GI panels for parasites and viruses may be unnecessary because these illnesses are usually self-limited and, as viruses, are treated with supportive care and hydration.^12, In situations in which the GI condition is likely foodborne based on patient history, GI pathogen panels may be limited to the most common pathogens typically found with foodborne illness. For patients who are immune competent, such a panel could include Salmonella, Campylobacter, Shigella, Cryptosporidium (parasite), STEC, and E coli O157. More pathogen targets may be included if testing for C difficile or testing patients who are critically ill or immunocompromised.^12,

Time to a result of testing with a pathogen panel is reduced compared with standard laboratory techniques (<6 hours).^13,

Comparators

Comparators of interest include culture or serologic tests and GI pathogen-specific testing (nucleic acid-based testing for individual pathogens).

Outcomes

The general outcomes of interest are test accuracy, test validity, other test performance measures, medication use, symptoms, and change in disease status.

True-positive and true-negative results lead to faster diagnosis and correct treatment, or no unnecessary treatment, as well as fewer repeated tests.

False-positive and false-negative results, inaccurate identification of a pathogen by the testing device, failure to correctly interpret test results, or failure to correctly operate the instrument may lead to misdiagnosis resulting in inappropriate treatment while postponing treatment for the true condition. Such a situation could lead to incorrect, unnecessary, or no treatment, subsequent testing, and delay of correct diagnosis and treatment. ^14,

Though not completely standardized, follow-up for suspected gastroenteritis or GI conditions would typically occur in the weeks to months after a diagnosis decision and initiation of treatment.

Study Selection Criteria

Methodologically credible studies were selected using the following principles:

• The study population represents the population of interest. Eligibility and selection are described.

• The test is compared with a credible reference standard.

• If the test is intended to replace or be an adjunct to an existing test , it should also be compared with that test.

• Studies should report sensitivity, specificity, and predictive values. Studies that completely report true- and false-positive results are ideal. Studies reporting other measures (eg, ROC, AUROC, c-statistic, likelihood ratios) may be included but are less informative.

• • Reported on a validation cohort that was independent of the development cohort.

• Studies should also report reclassification of diagnostic or risk category.

Clinically Valid

A test must detect the presence or absence of a condition, the risk of developing a condition in the future, or treatment response (beneficial or adverse).

Review of Evidence

Infectious gastroenteritis may be caused by a broad spectrum of pathogens resulting in the primary symptom of diarrhea. Panels for GI pathogens use multiplex amplified probe techniques and multiplex reverse transcription for the simultaneous detection of many GI pathogens such as C difficile, E coli, Salmonella, Shigella, norovirus, rotavirus, and Giardia. The performance study of the first FDA-cleared GI panel (xTAG Pathogen Panel [GPP], Luminex Molecular Diagnostics, Inc, Toronto, Ontario, CA), showed high sensitivity and specificity and overall strong positive percent agreement for the organisms on the panel (Table 7). ^15,

Table 7. Prospective Performance Data by Organism

Source: FDA Decision Summary..15,

 CI: Confidence interval; ETEC: enterotoxigenic Escherichia coli; STEC: Shiga toxin–producing E coli.

Several studies of GI pathogen panels have demonstrated overall high sensitivities and specificities and indicated the panels might be useful for detecting causative agents for GI infections, including both foodborne and infectious pathogens. Claas et al (2013) assessed the performance characteristics of the xTAG Pathogen Panel (GPP; Luminex, Toronto, ON, Canada) compared with traditional diagnostic methods (i.e., culture, microscopy, enzyme immunoassay/direct fluorescent antibody, real-time PCR , or sequencing) using 901 stool samples from multiple sites.^16, The sensitivity of GPP against real-time PCR was >90% for nearly all pathogens tested by real-time PCR; the 1 exception was adenovirus at 20%, but sensitivity could be higher because real-time PCR did not distinguish between adenovirus species. Kahre et al (2014) found similar results when they compared the FilmArray GI panel (BioFire Diagnostics, Salt Lake City, UT, USA) with the xT AG GPP.^17, Both panels detected more pathogens than routine testing. Of 230 prospectively collected samples, routine testing identified 1 or more GI pathogens in 19 (8.3%) samples; FilmArray detected 76 (33.0%), and xT AG detected 69 (30.3%). Two of the most commonly detected pathogens in both assays were C difficile (12.6% to 13.9% prevalence) and norovirus (5.7% to 13.9% prevalence). Both panels showed >90% sensitivity for the majority of targets.

Using the xTAG GPP, Beckmann et al (2014) evaluated 296 patients who were either children with gastroenteritis (n=120) or patients who had been to the tropics and had suspected parasite infestation (adults, n=151; children, n=25).^13, Compared with conventional diagnostics, the GPP showed 100% sensitivity for rotavirus, adenovirus, norovirus, C difficile, Salmonella species, Cryptosporidium, and Giardia lamblia. Specificity was >90% for all but norovirus (42%) and G lamblia (56%); both also had lower PPV at 46% and 33%, respectively. Salmonella species also had low PPV at 43%; all others had 100% PPV. Negative predictive value was 100% for all pathogens.

Buchan et al (2013) evaluated a multiplex real-time PCR assay (ProGastro SSCS, Gen-Probe Prodesse, San Diego, CA) limited to Campylobacter spp., Salmonella spp., and Shigella spp. against culture; and they tested for STEC against broth enrichment followed by enzyme immunoassay.^18, A total of 1244 specimens from 4 U.S. clinical laboratories were tested. Bidirectional sequencing was used to resolve discrepancies between ProGastro and culture or enzyme immunoassay. The overall prevalence of pathogens detected by culture was 5.6%, whereas the ProGastro assay and bidirectional sequencing showed an overall prevalence of 8.3%. The ProGastro SSCS assay showed a sensitivity of 100% and a specificity of 99.4% to 100% for all pathogens. This is compared with a sensitivity of 52.9% to 76.9% and a specificity of 99.9% to 100% for culture compared with ProGastro SSCS assay.

Al-Talib et al (2014) assessed the diagnostic accuracy of a pentaplex PCR assay with specific primers to detect hemorrhagic bacteria from stool samples.^19, The primers, which were mixed in a single reaction tube, were designed to detect Salmonella spp., Shigella spp., enterohemorrhagic E. coli, and Campylobacter spp., all of which are a particular danger to children in developing countries. The investigators used 223 stool specimens from healthy children and spiked them with hemorrhagic bacteria. All primers designed had 100% sensitivity, specificity, PPV, and NPV.

Jiang et al (2014) developed a reverse transcription and multiplex real-time PCR assay to identify 5 viruses in a single reaction.^20, The viruses included norovirus genogroups I and II; sapovirus genogroups I, II, IV, and V; human rotavirus A; adenovirus serotypes 40 and 41; and human astrovirus. Compared with monoplex real-time PCR, multiplex real-time PCR assay had sensitivity ranging from 75% to 100%; specificity ranged from 99% to 100%.

The health technology assessment and systematic review by Freeman et al (2017) evaluated multiplex texts to identify GI pathogens in people suspected of having infectious gastroenteritis.^21, Tests in the assessment were xTAG® GPP and FilmArray GI Panel. Eligible studies included patients with acute diarrhea, compared multiplex GI pathogen panel tests with standard microbiology tests, and assessed patient, management, and/or test accuracy outcomes. Of the 23 identified studies, none provided an adequate reference standard for comparing the accuracy of GI panels with standard tests, so sensitivity and specificity analyses were not performed. Positive and negative test agreement were analyzed for individual pathogens for the separate panel products and are not detailed in this review. The meta-analysis of 10 studies found high heterogeneity in participants, country of origin, conventional methods used, and pathogens considered. Using conventional methods as the determinant of clinically important disease, the meta-analysis results suggested GI panel testing is reliable and could supplant current microbiological methods. An increase in false positives would result, along with the potential for overdiagnosis and incorrect treatment. However, if GI panel testing is identifying important pathology being missed with conventional methods, the result could be more appropriate treatments. The clinical importance of these findings is unclear, and an assessment of GI panel testing effect on patient management and outcomes, compared with conventional testing, is needed.

Kosai et al (2021) evaluated the Verigene Pathogens Nucleic Acid Test (Luminex Corporation), testing 268 clinical stool samples for bacteria and toxins and 167 samples for viruses.^22, Of these samples, 256 and 160 samples, respectively, (95.5% and 95.8%) had fully concordant results between the Verigene EP test and the reference methods (which were culture for bacteria and toxins and xTAG GPP for viral detection). Overall sensitivity and specificity were 97.0% and 99.3%, respectively. Sensitivity for individual pathogens ranged from 87.5% to 100%, and specificity ranged from 98.7% to 100%. A total of 13 false-positive and 6 false-negative results were reported.

Ahmed et al (2024) evaluated the performance of the BioFire FilmArray GI Panel for diagnosing infectious diarrhea caused by parasitic and bacterial infections in intensive care unit patients in Egypt.^23, The study included 50 stool samples subjected to conventional identification (microscopic examination, stool culture, and bacterial identification) and molecular diagnosis by the FilmArray Panel. For parasitic infections, the sensitivity and specificity of the panel compared to microscopy were 83.3% and 100% for Cryptosporidium oocysts and 100% and 92.5% for Giardia lamblia cysts, respectively. For bacterial infections, the BioFire FilmArray GI Panel demonstrated 100% sensitivity and specificity for both E. coli and Salmonella compared to stool culture. The overall agreement between the BioFire FilmArray GI Panel and conventional methods was 98% for Cryptosporidium, 94% for G. lamblia, and 100% for both E. coli and Salmonella.

Clinically Useful

A test is clinically useful if the use of the results informs management decisions that improve the net health outcome of care. The net health outcome can be improved if patients receive correct therapy, more effective therapy, or avoid unnecessary therapy or testing.

Direct Evidence

Direct evidence of clinical utility is provided by studies that have compared health outcomes for patients managed with and without the test. Because these are intervention studies, the preferred evidence would be from RCTs.

Meltzer et al (2022) conducted a single-center RCT investigating antibiotic use in patients with moderate to severe suspected infectious diarrhea presenting to the emergency department.^24, Patients were randomized to receive multiplex PCR testing with the BioFire FilmArray GI panel (n=38) or standard care (usual testing or no testing; n=36). In the PCR arm, subjects received antibiotics in 87% of bacterial or protozoal diarrheal infections (13/15) compared to 46% (6/13) in the control arm (p=.042). No significant differences were found between groups in follow-up symptoms as assessed on days 2, 7, and 30, or emergency department length of stay. The study was terminated early due to the COVID-19 pandemic and thus was underpowered. Additional limitations include potential antibiotic prescribing at subsequent healthcare visits that was not captured and lack of a standardized reference test for the control arm.

Chain of Evidence

Indirect evidence on clinical utility rests on clinical validity.

A 9-month, prospective, multi-center study by Cybulski et al (2018) assessed the effect of the BioFire FilmArray GI PCR panel on clinical diagnosis and decision-making. It also compared the diagnostic accuracy for patients with positive results obtained exclusively using the GI panel with results obtained using conventional stool culture^25, (Table 8). Testing on 1887 consecutive fecal samples was performed in parallel using the GI panel and stool culture. The GI panel detected pathogens in significantly more samples than culture; median time from collection to results and collection to initiation of treatment was also significantly less. The use of a GI panel also led to a significant trend toward targeted rather than empirical therapy (r²=0.65; p=.009 by linear regression). Results of the GI panels resulted in discontinuation of antimicrobials in 8 of 9 STEC, with just 1 example of GI panel results affecting clinical decision-making (other results summarized in Table 9). Limitations of the study include the limit to 2 hospitals within a single healthcare system and certain subgroups that were too small for analysis. In addition, it was unclear how the historic controls were used since the current samples were tested with both a GI panel and culture.

The prospective study by Beal et al (2017) also aimed to assess the clinical impact of the BioFire FilmArray GI panel^26, (Table 8). Stool samples from 241 patients (180 adults and 61 children) were tested with the GI panel and compared with 594 control patients from the previous year who were tested via culture. The most common pathogens detected by the GI panel were enteropathogenic E. coli (n=21), norovirus (n=21), rotavirus (n=15), sapovirus (n=9), and Salmonella (n=9). The GI panel patients had significantly fewer subsequent infectious stool tests compared with the control group. The GI panel patients also had 0.18 imaging studies per patient compared with 0.39 (p=.0002) in the control group. The GI panel group spent fewer days on antibiotic(s) per patient: 1.73 versus 2.12 in the control group. In addition, average length of time from stool culture collection to discharge was 3.4 days for the GI panel group and 3.9 days for the controls (p=.04) (other results summarized in Table 9). The GI panel improved patient care in several ways: (1) it identified a range of pathogens that might not have been detected by culture, (2) it reduced the need for other diagnostic tests, (3) it resulted in less unnecessary use of antibiotics, and (4) it led to shorter length of hospital stay. Some limitations of the study include not confirming the results in which the GI panel did not agree with standard testing and using a historical cohort as a control group.

Table 8. Summary of Key Observational Comparative Study Characteristics

ED: emergency department; GI: gastrointestinal; U.S.: United States

Table 9. Summary of Key Observational Comparative Study Results

CI: confidence interval; GI: gastrointestinal; NA: not applicable; NR: not reported.

Section Summary: Gastrointestinal Pathogen Panel

Most GI panels combining multiple individual nucleic acid tests provide faster results compared to standard stool culture. Sensitivity and specificity are generally high, but the yield of testing may be affected by the panel composition. Results of comparisons of conventional methods for ova and parasites to nucleic acid tests are limited. Prospective observational studies were available to evaluate the clinical utility of a GI panel, which was shown in faster turnaround times leading to quicker treatment and a trend away from empirical treatment toward targeted therapy. However, both studies were limited by lack of adjudication of discordant results or the use of only a historical control. A small RCT found a higher rate of appropriate antibiotic prescribing in patients managed with a GI panel, but was terminated early and thus underpowered. Access to a rapid method for etiologic diagnosis of GI infections may lead to more effective early treatment and infection control measures. However, in most instances, when there is suspicion for a specific pathogen, individual tests could be ordered or a limited pathogen panel could be used. There may be a subset of patients with an unusual presentation who would warrant testing for a larger panel of pathogens at once, but that subset has not been well defined.

For individuals who have signs and/or symptoms of gastroenteritis who receive a nucleic acid-based gastrointestinal pathogen panel, the evidence includes prospective and retrospective evaluations of the tests’ sensitivity and specificity and prospective studies on utility. Relevant outcomes include test accuracy and validity, other test performance measures, medication use, symptoms, and change in disease status. The evidence suggests that pathogen panels are likely to identify both bacterial and viral pathogens with high sensitivity, compared with standard methods. Access to a rapid method for etiologic diagnosis of infections may lead to more effective early treatment and infection control measures. However, in most instances, when a specific pathogen is suspected, individual tests could be ordered. There may be a subset of patients with an unusual presentation who would warrant testing for a panel of pathogens at once, but that subset has not been well defined. The evidence is insufficient to determine that the technology results in an improvement in the net health outcome.

Population Reference No. 3

Respiratory Pathogen Panel

Clinical Context and Test Purpose

The purpose of the nucleic acid-based respiratory pathogen panel is to provide a diagnostic option that is an alternative to or an improvement on existing tests in patients with signs and/or symptoms of respiratory infection.

The following PICO was used to select literature to inform this review.

Populations

The relevant population of interest is individuals with signs and/or symptoms of respiratory infections.

The available evidence also notes that respiratory pathogen panels are particularly effective for high-risk individuals.

High-risk individuals can include:

• Immunocompromised adult or pediatric patients, such as

  • Hematopoietic stem cell or solid organ transplant recipients

  • Individuals receiving high-dose chemotherapy and/or steroids

• Adults who appear acutely ill with respiratory conditions—particularly in certain settings such as influenza outbreaks

• Critically ill adult individuals—particularly intensive care unit (ICU) patients

Interventions

The test being considered is the nucleic acid-based respiratory pathogen panel.

The respiratory pathogen panel is used to diagnosis respiratory infection due to bacteria or viruses and to help guide management of the infection. This panel is performed primarily when a patient is seriously ill, hospitalized, and/or at an increased risk for severe infection with complications or multiple infections. Not everyone with symptoms is tested (eg, fever, aches, sore throat, and cough). Samples are collected by nasopharyngeal swab in universal transport medium or respiratory wash (ie, nasal wash, nasal aspirate, or bronchoalveolar lavage wash). Examples of these pathogens include adenovirus, coronavirus (HKU1, NL63, 229E, OC43), human metapneumovirus, human rhinovirus/enterovirus, influenza A (H1, H1-2009, H3), influenza B, parainfluenza (1, 2, 3, 4), respiratory syncytial virus, Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae.

Patients are tested in an outpatient setting.

Comparators

Comparators of interest include culture or serologic tests and respiratory pathogen-specific testing (nucleic acid-based testing for individual pathogens).

Outcomes

The general outcomes of interest are test accuracy, test validity, other test performance measures, medication use, symptoms, and change in disease status.

True-positive and true-negative results lead to faster diagnosis and correct treatment, or no unnecessary treatment, as well as fewer repeated tests.

False-positive and false-negative results, inaccurate identification of a pathogen by the testing device, failure to correctly interpret test results, or failure to correctly operate the instrument may lead to misdiagnosis resulting in inappropriate treatment while postponing treatment for the true condition. Such a situation could lead to incorrect, unnecessary, or no treatment, subsequent testing, and delay of correct diagnosis and treatment.

Follow-up typically occurs in the days and weeks after diagnosis decision and initiation of treatment.

Study Selection Criteria

Methodologically credible studies were selected using the following principles:

• The study population represents the population of interest. Eligibility and selection are described.

• The test is compared with a credible reference standard.

• If the test is intended to replace or be an adjunct to an existing test , it should also be compared with that test.

• Studies should report sensitivity, specificity, and predictive values. Studies that completely report true- and false-positive results are ideal. Studies reporting other measures (eg, ROC, AUROC, c-statistic, likelihood ratios) may be included but are less informative.

  • Reported on a validation cohort that was independent of the development cohort.

• Studies should also report reclassification of diagnostic or risk category.

• The RCT conducted by Darie et al (2022) was excluded as the Unyvero Hospitalized Pneumonia panel is not currently commercially available in the United States.^24,

Clinically Valid

A test must detect the presence or absence of a condition, the risk of developing a condition in the future, or treatment response (beneficial or adverse).

Review of Evidence

Clark et al (2023) conducted a systematic review and meta-analysis of the impact of multiplex PCR testing among individuals with a suspected acute respiratory tract infection in the hospital setting.^28, Twenty-seven studies representing 17,321 patients were identified for analysis. Multiplex testing was associated with a reduction in both time to results (-24.22 h; 95% CI, -28.70 to -19.74 h) and hospital length of stay (-0.82 days; 95% CI, -1.52 to -0.11). Antivirals were more likely to be prescribed among influenza positive individuals (risk ratio [RR], 1.25; 95% CI, 1.06 to 1.48) as was use of an appropriate infection control facility (RR, 1.55; 95% CI, 1.16 to 2.07).

Huang et al (2018) published a systematic review and meta-analysis of a multiplex PCR system for the rapid diagnosis of respiratory virus infections.^29, Authors summarized diagnostic accuracy evidence on the detection of viral respiratory infections for BioFire FilmArray RP (Film Array), Nanosphere Verigene RV+ test, and Hologic Gen-Probe Prodesse assays. The study reviewed 20 studies with 5510 patient samples. Multiplex PCRs were found to have high diagnostic accuracy with AUROC >0.98 for all reviewed viruses except adenovirus (AUROC 0.89). All 3 reviewed multiplex PCR systems were shown to be highly accurate.

Clinically Useful

A test is clinically useful if the use of the results informs management decisions that improve the net health outcome of care. The net health outcome can be improved if patients receive correct therapy, more effective therapy, or avoid unnecessary therapy or testing.

Review of Evidence

Direct Evidence

Direct evidence of clinical utility is provided by studies that have compared health outcomes for patients managed with and without the test. Because these are intervention studies, the preferred evidence would be from RCTs.

Several studies of various respiratory viral panels have demonstrated that the multiplex assay detected clinically important viral infections in a single genomic test and thus, may be useful for detecting causative agents for respiratory tract disorders. ^30,31,32,

Randomized Controlled Trials

Cartuliares et al (2023) conducted a prospective, multicenter, randomized controlled trial to evaluate the impact of point-of-care multiplex PCR on antibiotic prescribing for patients admitted with suspected community-acquired pneumonia in Denmark.^33, Lower respiratory tract samples were collected from 294 patients randomized to either the PCR group (Biofire FilmArray Pneumonia Panel plus added to standard care) or the standard care only group. The primary outcome, prescription of no or narrow-spectrum antibiotics at 4 hours, did not differ significantly between the PCR (62.8%) and standard of care (59.6%) groups (OR, 1.13; 95% CI, 0.96 to 1.34; p=.134). However, the PCR group had significantly more targeted antibiotic prescriptions at 4 hours (OR, 5.68; 95% CI, 2.49 to 12.94; p<.001) and 48 hours (OR, 4.20; 95% CI, 1.87 to 9.40; p<.001), and more adequate prescriptions at 48 hours (OR, 2.11; 95% CI, 1.23 to 3.61; p=.006) and day 5 (OR, 1.40; 95% CI 1.18 to 1.66; p<.001). There were no significant differences in ICU admissions, 30-day readmissions, length of stay, 30-day mortality, or in-hospital mortality.

Andrews et al (2017) published a quasi-randomized study assessing the impact of multiplex PCR on length of stay and turnaround time compared with routine, laboratory-based testing in the treatment of patients aged ≥16 years presenting with influenza-like illness or upper or lower respiratory tract infection (Table 10).^34, Patients were selected at inpatient and outpatient clinics in 3 areas of a hospital. FilmArray RP PCR systems were used. Of eligible patients (N=606), 545 (89.9%) were divided into a control arm (n=211) and an intervention arm (n=334). While PCR testing was not associated with a reduction in length of stay, turnaround time was reduced (see Table 11 for detailed results). Limitations of the study included design and patient allocation (patients were allocated to the intervention arm on even days). Additionally, the patients considered in the study were not noted to be high-risk individuals as defined above, only those with pertinent symptoms.

The parallel-group, open-label RCT by Brendish et al (2017) evaluated the routine use of molecular point-of-care testing (POCT) for respiratory viruses in adults presenting to a hospital with acute respiratory illness (Table 10).^35, In a large U.K. hospital, over 2 winter seasons, investigators enrolled adults within 24 hours of presenting to the emergency department or acute medical unit with acute respiratory illness or fever >37.5°C, or both. A total of 720 patients were randomized (1:1) to either molecular POCT for respiratory viruses (FilmArray Respiratory Panel; n=362) or routine care (n=358), which included diagnosis based on clinical judgment and testing by laboratory PCR at the clinical team’s discretion. All patients in the POCT group were tested for respiratory viruses; 158 (45%) of 354 patients in the control group were tested. Because patients presenting with symptoms are often put on antibiotics before tests can be run, the results of the POCTs were unable to influence the outcome in many patients; therefore, a subgroup analysis was necessary for those who were only given antibiotics after test results were available. The results of the analysis showed antibiotics were prescribed for 61 (51%) of 120 patients in the POCT group and for 107 (64%) of 167 in the control group (difference, -13.2%; 95% confidence interval [CI], -24.8% to -1.7%; p=.0289). Mean test turnaround time for POCT was 2.3 hours (standard deviation [SD], 1.4) versus 37.1 hours (SD, 21.5) in the control group. The percentage of patients prescribed a neuraminidase inhibitor who tested positive for influenza was significantly higher for the POCT group than the control group (82% vs. 47%), and it was significantly lower for the percentage who tested negative for influenza (18% vs. 53%). In addition, the time to first dose was 8.8 hours (SD, 15.3) for POCT and 21.0 hours (SD, 28.7) for the control group (see Table 11 for more results). Blinding of the clinical teams to which group a patient had been randomized to was not possible because the purpose of the study was to inform the clinical team of POCT results. In addition, the limit of the study to the winter months means the findings cannot be extrapolated to the rest of the year.

Table 10. Summary of Key RCT Characteristics

 ED: emergency department; PCR: polymerase chain reaction; POCT: point of care testing (using FilmArray Respiratory Panel); RCT: randomized controlled trial; RTI: respiratory tract infection.a Quasi-randomized study.b The dates do not make sense because they overlap, likely due to an error in the article. Another place in the article says the “winter seasons in 2014-15 and 2015-16.”

Table 11. Summary of Key RCT Results

CI: confidence interval; IQR: interquartile range; NR: not reported; OR: odds ratio; RCT: randomized controlled trial; SD: standard deviation.a 30 days post-enrollment.b Within 30 days of study participation.c Mean risk difference.d Unadjusted odds ratio.

Tables 12 and 13 display notable limitations identified in each study.

Table 12. Study Design and Conduct Limitations

The study limitations stated in this table are those notable in the current review; this is not a comprehensive gaps assessment.a Selection key: 1. Selection not described; 2. Selection not random or consecutive (ie, convenience).b Blinding key: 1. Not blinded to results of reference or other comparator tests.c Test Delivery key: 1. Timing of delivery of index or reference test not described; 2. Timing of index and comparator tests not same; 3. Procedure for interpreting tests not described; 4. Expertise of evaluators not described.d Selective Reporting key: 1. Not registered; 2. Evidence of selective reporting; 3. Evidence of selective publication.e Data Completeness key: 1. Inadequate description of indeterminate and missing samples; 2. High number of samples excluded; 3. High loss to follow-up or missing data.f Statistical key: 1. Confidence intervals and/or p values not reported; 2. Comparison with other tests not reported.

Table 13. Study Relevance Limitations

The study limitations stated in this table are those notable in the current review; this is not a comprehensive gaps assessment.a Population key: 1. Intended use population unclear; 2. Clinical context is unclear; 3. Study population is unclear; 4. Study population not representative of intended use.b Intervention key: 1. Classification thresholds not defined; 2. Version used unclear; 3. Not intervention of interest.c Comparator key: 1. Classification thresholds not defined; 2. Not compared to credible reference standard; 3. Not compared to other tests in use for same purpose.d Outcomes key: 1. Study does not directly assess a key health outcome; 2. Evidence chain or decision model not explicated; 3. Key clinical validity outcomes not reported (sensitivity, specificity and predictive values); 4. Reclassification of diagnostic or risk categories not reported; 5. Adverse events of the test not described (excluding minor discomforts and inconvenience of venipuncture or noninvasive tests).e Follow-Up key: 1. Follow-up duration not sufficient with respect to natural history of disease (true-positives, true-negatives, false-positives, false-negatives cannot be determined).

Chain of Evidence

Indirect evidence on clinical utility rests on clinical validity. If the evidence is insufficient to demonstrate test performance, no inferences can be made about clinical utility.

Section Summary: Respiratory Pathogen Panels

The evidence for the clinical validity or clinical utility of respiratory pathogen panels in diagnosing respiratory infections includes systematic reviews, 2 RCTs, and a quasi-randomized study. The systematic review reported that all 3 reviewed multiplex PCR systems were highly accurate. The clinical utility demonstrated by the RCTs showed benefits in test results turnaround time, time to receive treatment, targeted antibiotic prescriptions, and length of hospital stay. Significant differences were not seen in readmission, or mortality.

For individuals who have signs and/or symptoms of respiratory infection who receive a nucleic acid-based respiratory pathogen panel, the evidence includes a systematic review and 2 randomized controlled trials (RCTs). Relevant outcomes include test accuracy and validity, other test performance measures, medication use, symptoms, and change in disease status. The systematic review reported that all 3 reviewed multiplex polymerase chain reaction systems were highly accurate. One RCT and 1 quasi-RCT evaluated utility of a respiratory panel and found benefits in time-to-treat and length of hospital stay. In addition, 1 subanalysis found fewer antibiotics being prescribed for patients diagnosed with the panel. The panel did not significantly affect duration of antibiotic use, readmission, or mortality rates. The evidence is sufficient to determine that the technology results in an improvement in the net health outcome

SUPPLEMENTAL INFORMATION

The purpose of the following information is to provide reference material. Inclusion does not imply endorsement or alignment with the evidence review conclusions.

Practice Guidelines and Position Statements

Guidelines or position statements will be considered for inclusion in ‘Supplemental Information’ if they were issued by, or jointly by, a US professional society, an international society with US representation, or National Institute for Health and Care Excellence (NICE). Priority will be given to guidelines that are informed by a systematic review, include strength of evidence ratings, and include a description of management of conflict of interest.

Numerous guidelines have been identified concerning the use of nucleic acid amplification tests (NAATs) for the diagnosis of the pathogens discussed in this review. Table 14 provides an index of NAAT recommendation by virus/ infection.

Table 14. Index of NAAT Recommendations by Virus/Infection

AAP: American Academy of Pediatrics; ACG: American College of Gastroenterology; ASM: American Society for Microbiology; AST: American Society of Transplantation; CDC: Centers for Disease Control and Prevention; CNS: central nervous system; GI: gastrointestinal; HIV: human immunodeficiency virus; IDSA: Infectious Disease Society of America; NA: not applicable (none found); NAAT: nucleic acid amplification test; NIH: National Institutes of Health; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2.a Guidelines Not Recommending includes not only guidelines that recommend against NAATs but also those that were neutral on the use of NAATs.b CDC recommends culture for first-line identification of Candida species; it recommends NAAT for complicated infections and for second-line diagnosis.c Indicates guidelines in which the issuing body specifically recommends that U.S. Food and Drug Administration (FDA)-cleared NAATs be used.

Centers for Disease Control and Prevention

The Centers for Disease Control and Prevention (CDC) have published multiple recommendations and statements regarding the use of NAATs to diagnose the viruses and infections discussed in this evidence review since 2009.

1.1 The CDC published guidance for laboratory testing for cytomegalovirus (CMV); the guideline stated that the standard laboratory test for congenital CMV is polymerase chain reaction (PCR) on saliva, with confirmation via urine test to avoid false-positive results from ingesting breast milk from CMV seropositive mothers. Serologic tests were recommended for persons >12 months of age.^36,

1.2 The CDC published diagnostic methods for mycoplasma pneumoniae.^37, They cited NAAT as a method of diagnosis, along with culture or serology.

1.3 The CDC published updated guidelines on Zika virus testing.^38, Routine testing for Zika virus in asymptomatic pregnant patients is not recommended, but NAAT testing may still be considered for asymptomatic pregnant women with recent travel to an area with risk of Zika outside the U.S. and its territories. Symptomatic pregnant patients should receive NAAT testing if they have recently traveled to areas with a risk of Zika virus or if they have had sex with someone who lives in or recently traveled to areas with risk of Zika virus. If a pregnant woman (with risk of Zika virus exposure) has a fetus with prenatal ultrasound findings consistent with congenital Zika virus infection, Zika virus NAAT and IgM testing should be performed on maternal serum and NAAT on maternal urine. If amniocentesis is being performed as part of clinical care, Zika virus NAAT testing of amniocentesis specimens should also be performed.

1.4 In 2017, the CDC updated its guidelines on norovirus gastroenteritis outbreak management and disease prevention.^39,40, Real-time reverse transcription-PCR assays, specifically, TaqMan-based real-time assays, which can contain multiple probes, is considered the effective laboratory diagnostic protocol for testing suspected cases of viral gastroenteritis.

1.5 In 2015, the CDC made recommendations for the use in NAATs in diagnosing numerous sexually transmitted infections. ^41, These recommendations were most recently updated in 2021, with the publication of new guidelines and the following recommendations:^42,

1.5.1 For Candida species:

• • • "The majority of PCR tests for yeast are not FDA [U.S. Food and Drug Administration] cleared, and providers who use these tests should be familiar with the performance characteristics of the specific test used."

1.5.2 For Gonococcal Infections:

• • • "Culture, NAAT, and POC [point of care] NAAT, such as GeneXpert (Cepheid), are available for detecting genitourinary infection with N. gonorrhoeae."

    • "NAATs and POC NAATs allow for the widest variety of FDA-cleared specimen types, including endocervical and vaginal swabs and urine for women, urethral swabs and urine for men, and rectal swabs and pharyngeal swabs for men and women. However, product inserts for each NAAT manufacturer should be consulted carefully because collection methods and specimen types vary."

1.5.3 For Chlamydial Infection:

• • • "NAATs are the most sensitive tests for these specimens and are the recommended test for detecting C. trachomatis infection. NAATs that are FDA cleared for use with vaginal swab specimens can be collected by a clinician or patient in a clinical setting. Patient collected vaginal swab specimens are equivalent in sensitivity and specificity to those collected by a clinician using NAATs, and this screening strategy is highly acceptable among women. Optimal urogenital specimen types for chlamydia screening by using NAAT include firstcatch urine (for men) and vaginal swabs (for women). Recent studies have demonstrated that among men, NAAT performance on self-collected meatal swabs is comparable to patient-collected urine or provider-collected urethral swabs."

1.5.4 For Gardnerella vaginalis:

• • • "Multiple BV [bacterial vaginosis] NAATs are available for BV diagnosis among symptomatic women. These tests are based on detection of specific bacterial nucleic acids and have high sensitivity and specificity for BV (i.e., G. vaginalis, A. vaginae, BVAB2, or Megasphaera type 1) and certain lactobacilli (i.e., Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri)...Five quantitative multiplex PCR assays are available...Two of these assays are FDA cleared (BD Max Vaginal Panel and Aptima BV), and the other three are laboratory-developed tests."

1.5.5 For hepatitis C infection (HCV):

• • • In addition, “testing for HCV infection should include use of an FDA-cleared test for antibody to HCV…followed by NAAT to detect HCV RNA for those with a positive antibody result. Persons with HIV infection with low CD4+ T-cell count might require further testing by NAAT because of the potential for a false-negative antibody assay.”

1.5.6 For diseases characterized by genital, anal, or perianal ulcers (eg., herpes simplex virus [HSV], syphilis):

• • • "Specific evaluation of genital, anal, or perianal ulcers includes syphilis serology tests and darkfield examination from lesion exudate or tissue, or NAAT if available; NAAT or culture for genital herpes type 1 or 2; and serologic testing for type-specific HSV antibody. In settings where chancroid is prevalent, a NAAT or culture for Haemophilus ducreyi should be performed;" and

    • "PCR is also the test of choice for diagnosing HSV infections affecting the central nervous system (CNS) and systemic infections (e.g., meningitis, encephalitis, and neonatal herpes). HSV PCR of the blood should not be performed to diagnose genital herpes infection, except in cases in which concern exists for disseminated infection (e.g., hepatitis)."

1.5.7 For Human immunodeficiency virus 1 (HIV-1):

• • • The use of NAAT is not mentioned; serologic tests are recommended for detecting antibodies against HIV-1 and by virologic tests that detect HIV antigens or RNA.

1.5.8 For human papillomavirus (HPV):

• • • There are several FDA-cleared HPV tests that detect viral nucleic acid or messenger RNA; however, there are currently no algorithms for HPV 16/18/45 testing in the clinical guidelines;

    • Testing for nononcogenic HPV (types 6 and 11) is not recommended; and

    • “HPV assays should be FDA-cleared and used only for the appropriate indications” and should not be performed if the patient is “deciding whether to vaccinate against HPV;” when “providing care to persons with genital warts or their partners;” when “testing persons aged <25 years as part of routine cervical cancer screening;” or when “testing oral or anal specimens.”

1.5.9 For Trichomonas vaginalis:

• • • NAAT is recommended for detecting T vaginalis in women due to its high sensitivity and specificity. Multiple assays are FDA-cleared to detect T vaginalis from vaginal, endocervical, or urine specimens for women.

    • Although there is not a currently FDA-cleared assay test available for use in men, assays "...should be internally validated in accordance with CLIA [Clinical Laboratory Improvement Amendments] regulations before use with urine or urethral swabs from men."

1.6 In 2014, the CDC published recommendations regarding the laboratory-based detection of C. trachomatis and N. gonorrhoeae infections.^43, It stated:

• • NAATs are superior other available diagnostic tests in “overall sensitivity, specificity, and ease of specimen transport;”

  • The use of “NAAT to detect chlamydia and gonorrhea except in cases of child sexual assault involving boys and rectal and oropharyngeal infections in prepubescent girls” is supported by evidence; and

  • Only NAATs that have been cleared by the FDA for detection of C. trachomatis and N. gonorrhoeae should be used “as screening or diagnostic tests because they have been evaluated in patients with and without symptoms.”

1.7 In 2009, the CDC published updated guidelines for the use of NAATs in diagnosing Mycobacterium tuberculosis bacteria.^44, The CDC recommended that “NAA testing be performed on at least one respiratory specimen from each patient with signs and symptoms of pulmonary TB [tuberculosis] for whom a diagnosis of TB is being considered but has not yet been established, and for whom the test result would alter case management or TB control activities.” Although it noted that “culture remains the gold standard for laboratory confirmation of TB and is required for isolating bacteria for drug-susceptibility testing and genotyping,” the guideline stated that “NAA testing should become standard practice for patients suspected to have TB, and all clinicians and public health TB programs should have access to NAA testing for TB to shorten the time needed to diagnose TB from 1 to 2 weeks to 1 to 2 days.”

1.8 In 2021, the CDC published Sexually Transmitted Infections Treatment Guidelines. The CDC recommendations note that: "M. genitalium is an extremely slow-growing organism. Culture can take up to 6 months, and technical laboratory capacity is limited to research settings. NAAT for M. genitalium is FDA cleared for use with urine and urethral, penile meatal, endocervical, and vaginal swab samples. Molecular tests for macrolide (i.e., azithromycin) or quinolone (i.e., moxifloxacin) resistance markers are not commercially available in the United States. However, molecular assays that incorporate detection of mutations associated with macrolide resistance are under evaluation. Men with recurrent NGU should be tested for M. genitalium using an FDA-cleared NAAT. If resistance testing is available, it should be performed and the results used to guide therapy. Women with recurrent cervicitis should be tested for M. genitalium, and testing should be considered among women with PID. Testing should be accompanied with resistance testing, if available. Screening of asymptomatic M. genitalium infection among women and men or extragenital testing for M. genitalium is not recommended. In clinical practice, if testing is unavailable, M. genitalium should be suspected in cases of persistent or recurrent urethritis or cervicitis and considered for PID."^45,

National Institutes of Health et al

2.1 The National Institute of Health (NIH), CDC, and HIV Medicine Association of the Infectious Diseases Society of America (IDSA) published guidelines for the prevention and treatment of opportunistic infections in adults and adolescents with HIV.^46, The most recent update took place in 2024. In these guidelines, NAATs are discussed in the following situations:

2.1.1 Bartonella species

• • • For patients with suspected bacillary angiomatosis, serologic tests are the standard of care and the most accessible test for diagnosing Bartonella infection. There are PCR methods that have been developed for identification and speciation of Bartonella and are becoming increasingly available through private laboratories, as well as the CDC and may aid in diagnosis of Bartonella in freshly biopsied tissue samples or whole blood.

2.1.2 Clostridioides (Clostridium) difficile

• • • Detection of either the C. difficile toxin B gene, using NAAT, or the C. difficile toxin B protein, using an enzyme immunoassay, is required for diagnosis. PCR assays have high sensitivity and can detect asymptomatic carriers.

2.1.3 Cytomegalovirus

• • • For patients with suspected CMV disease, diagnosis is based on clinical symptoms and the presence of CMV in cerebral spinal fluid (CSF) or brain tissue. “In rare cases, the diagnosis may be unclear, and PCR of aqueous or vitreous humor specimens for CMV and other pathogens—especially herpes simplex virus, varicella zoster virus, and Toxoplasma gondii—can be useful for establishing the diagnosis."

2.1.4 Hepatitis B

• • • The CDC, the United States Preventive Services Task Force, and the American Association for the Study of Liver Disease (AASLD) recommend that patients with HIV infection should be tested for hepatitis B; however, NAATs are not recommended for initial testing in patients with HIV.

2.1.5 Hepatitis C

• • • Patients with HIV are recommended to undergo routine hepatitis C screening, initially “performed using the most sensitive immunoassays licensed for detection of antibody to HCV in blood.” The use of NAATs are not mentioned for initial testing in patients with HIV.

2.1.6 Herpes Simplex Virus

• • • “HSV DNA PCR and viral culture are preferred methods for diagnosis of mucocutaneous lesions potentially caused by HSV.”

2.1.7 Mycobacterium tuberculosis infection and disease

• • • “ NAA tests provide rapid diagnosis of TB, and some assays also provide rapid detection of drug resistance.”

    • "NAA assays, if positive, are highly predictive of TB disease when performed on Acid-Fast Bacillus (AFB) smear-positive specimens. However, because nontuberculous mycobacterial infections (NTM) may occur in people with HIV with advanced immunodeficiency, negative NAA results in the setting of smear-positive specimens may indicate NTM infection and can be used to direct therapy and make decisions about the need for respiratory isolation."

    • "NAA tests are more sensitive than AFB smear, being positive in 50% to 80% of smear negative, culture-positive specimens and up to 90% when three NAA tests are performed. Therefore, it is recommended that for all patients with suspected pulmonary TB, a NAA test be performed on at least one specimen. NAA tests also can be used on extrapulmonary specimens with the caveat that the sensitivity is often lower than with sputum specimens."

Infectious Disease Society of America et al

Since 2008, the IDSA has partnered with various societies to publish 9 recommendations regarding the use of NAATs to diagnose the viruses and infections discussed in this evidence review.

3.1 In 2024, the IDSA and the American Society for Microbiology published a guide on the diagnosis of infectious diseases.^47, In this guideline, NAATs were recommended diagnostic procedures for enterovirus, hepatitis C, hepatitis B, cytomegalovirus, bacterial vaginosis, herpes simplex virus, human herpesvirus 6, HIV, influenza virus, and Zika virus. In addition to providing guidance on diagnosing these diseases, the guidelines also provided recommendations on testing for other conditions by testing for common etiologic agents. Table 15 describes selected conditions for which IDSA recommends NAATs for diagnosing etiologic agents.

Table 15. IDSA Recommended Conditions for Use of NAATs in Identifying Etiologic Agents of Other Conditions*

* The IDSA provided recommendations for many situations in which NAATs are recommended for diagnosing certain etiologic agents commonly seen, with the listed conditions noted under the Recommended Conditions for Use of NAATs in Diagnosis Column.HIV: human immunodeficiency virus; IDSA: Infectious Disease Society of America; MRSA: methicillin-resistant Staphylococcus aureus; NAAT: nucleic acid amplification test: NTM: nontuberculous mycobacteria.a Recommended as first choice if available. b Where applicable and laboratory-validated. c The guidelines caution that NAAT is not 100% sensitive in individuals with established HIV infection due to viral suppression; therefore, if NAAT is used, subsequent serologic testing is recommended.

Use of NAATs for diagnosing Candida species, Gardnerella vaginalis, Streptococcus group B, and vancomycin-resistant Enterococcus as etiologic agents was not recommended.

3.2 In 2017, the IDSA published clinical practice guidelines for the management of healthcare-associated ventriculitis and meningitis.^48, When making diagnostic recommendations, the IDSA notes cultures as the standard of care in diagnosing healthcare-associated ventriculitis and meningitis, but that “nucleic acid amplification tests, such as PCR, on CSF may both increase the ability to identify a pathogen and decrease the time to making a specific diagnosis (weak, low).” (Strength of recommendation and quality of evidence established using the GRADE [Grading of Recommendations Assessment, Development and Evaluation] methodology).

3.3 In 2008, the IDSA published clinical practice guidelines for the management of encephalitis.^49, The following recommendations were made:

• • “Biopsy of specific tissues for culture, antigen detection, nucleic acid amplification tests (such as PCR), and histopathologic examination should be performed in an attempt to establish an etiologic diagnosis of encephalitis (A-III).” (Strength of recommendation level “A indicates good evidence to support recommendation for use.” Quality of evidence level III indicates “evidence from opinions of respected authorities based on clinical experience, descriptive studies, or reports of expert committees.”^50,)

  • “Nucleic acid amplification tests (such as PCR) of body fluids outside of the CNS may be helpful in establishing the etiology in some patients with encephalitis (B-III).” (Strength of recommendation level B indicates “moderate evidence to support recommendation.” Quality of evidence level III indicates “evidence from opinions of respected authorities based on clinical experience, descriptive studies, or reports of expert committees.”^50,)

  • “Nucleic acid amplification tests (such as PCR) should be performed on CSF specimens to identify certain etiologic agents in patients with encephalitis (A-III). Although a positive test result is helpful in diagnosing infection caused by a specific pathogen, a negative result cannot be used as definitive evidence against the diagnosis.”

  • The use of NAATs was recommended for diagnosing CMV, herpes simplex virus 1 and 2, human herpesvirus 6, Bartonella henselae, Mycoplasma pneumoniae, and Mycobacterium tuberculosis.

3.4 In 2018, the IDSA and the Society for Healthcare Epidemiology of America (SHEA) published weak recommendations with low quality evidence for the use of NAATs to diagnose Clostridioides (Clostridium) difficile.^51,

• • “The best-performing method (i.e., in use positive and negative predictive value) for detecting patients at increased risk for clinically significant C. difficile [CDI] infection” is use of a “stool toxin test as part of a multistep algorithm…rather than NAAT along for all specimens received in the clinical laboratory when there are no preagreed institutional criteria for patient stool submission.”

  • “The most sensitive method of diagnosis of CDI in stool specimens from patients likely to have CDI based on clinical symptoms” is use of “a NAAT alone or a multistep algorithm for testing…rather than a toxin test alone when there are preagreed institutional criteria for patient stool submission.”

3.5 In 2017, the IDSA published clinical practice guidelines for the diagnosis and management of infectious diarrhea.^52, The following recommendations were made:

• • In situations where enteric fever or bacteremia is suspected, “culture-independent, including panel-based multiplex molecular diagnostics from stool and blood specimens, and when indicated, culture-dependent diagnostic testing should be performed” (GRADE: strong, moderate).

  • In testing for Clostridioides (Clostridium) difficile in patients >2 years of age, “a single diarrheal stool specimen is recommended for detection of toxin or toxigenic C. difficile strain (e.g., nucleic acid amplification testing)” (GRADE: strong, low).

  • NAATs are not recommended for diagnosing CMV.

  • It was also noted that “clinical consideration should be included in the interpretation of results of multiple-pathogen nucleic acid amplification tests because these assays detect DNA and not necessarily viable organisms” (GRADE: strong, low).

3.6 In 2016, the IDSA published updated clinical practice guidelines for managing candidiasis.^53, The guideline noted many limitations of PCR testing. No formal recommendation was made, but the guidelines did state that “the role of PCR in testing samples other than blood is not established.”

3.7 In 2020, the IDSA established a panel composed of 8 members including frontline clinicians, infectious diseases specialists and clinical microbiologists who were members of the IDSA, American Society for Microbiology, SHEA , and the Pediatric Infectious Diseases Society (PIDS). Panel members represented the disciplines of adult and pediatric infectious diseases, medical microbiology, as well as nephrology and gastroenterology. The panel created a coronavirus disease 2019 (COVID-19) diagnosis guideline using the GRADE approach for evidence assessment; and, given the need for rapid response to an urgent public health crisis, the methodological approach was modified according to the GIN/McMaster checklist for development of rapid recommendations. The panel published recommendations for COVID-19 diagnosis in an online format, as when substantive new information becomes available the recommendations will require frequent updating.^54, The current recommendations (published December 23, 2020) support Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid testing for the following groups:

• • all symptomatic individuals suspected of having COVID-19;

  • asymptomatic individuals with known or suspected contact with a COVID-19 case;

  • asymptomatic individuals with no known contact with COVID-19 who are being hospitalized in areas with a high prevalence of COVID-19 in the community;

  • asymptomatic individuals who are immunocompromised and being admitted to the hospital, regardless of COVID-19 exposure;

  • asymptomatic individuals prior to hematopoietic stem cell transplant or solid organ transplantation, regardless of COVID-19 exposure;

  • asymptomatic individuals without known exposure to COVID-19 undergoing major time-sensitive surgeries;

  • asymptomatic individuals without a known exposure to COVID-19 who are undergoing a time-sensitive aerosol generating procedure (e.g., bronchoscopy) when personal protective equipment (PPE) is limited, and testing is available;

  • asymptomatic individuals without known exposure when the results will impact isolation/quarantine/ PPE usage decisions, dictate eligibility for surgery, or inform administration of immunosuppressive therapy.

The IDSA panel further recommends the following:

• • collecting nasopharyngeal swab, mid-turbinate swab, anterior nasal swab, saliva or a combined anterior nasal/oropharyngeal swab rather than oropharyngeal swabs alone for SARS-CoV-2 RNA testing in symptomatic individuals with upper respiratory tract infection or influenza like illness suspected of having COVID-19 (conditional recommendation, very low certainty of evidence).

  • nasal and mid-turbinate swab specimens may be collected for SARS-CoV-2 RNA testing by either patients or healthcare providers, in symptomatic individuals with upper respiratory tract infection or influenza like illness suspected of having COVID-19 (conditional recommendation, low certainty of evidence).

  • a strategy of initially obtaining an upper respiratory tract sample (e g , nasopharyngeal swab) rather than a lower respiratory sample for SARS-CoV-2 RNA testing in hospitalized patients with suspected COVID-19 lower respiratory tract infection. If the initial upper respiratory sample result is negative, and the suspicion for disease remains high, the IDSA panel suggests collecting a lower respiratory tract sample (e.g., sputum, bronchoalveolar lavage fluid, tracheal aspirate) rather than collecting another upper respiratory sample (conditional recommendations, very low certainty of evidence).

  • performing a single viral RNA test and not repeating testing in symptomatic individuals with a low clinical suspicion of COVID-19 (conditional recommendation, low certainty of evidence).

  • repeating viral RNA testing when the initial test is negative (versus performing a single test) in symptomatic individuals with an intermediate or high clinical suspicion of COVID-19 (conditional recommendation, low certainty of evidence).

  • using either rapid reverse-transcriptase (RT)-PCR or standard laboratory-based NAATs over rapid isothermal NAATs in symptomatic individuals suspected of having COVID-19 (conditional recommendation, low certainty of evidence).

American Society of Transplantation

4.1 In 2019, the American Society of Transplantation Infectious Diseases Community of Practice published guidelines which addressed vancomycin-resistant enterococci (VRE) infections in solid organ transplant patients.^55, The guidelines noted the cost-effectiveness and accuracy of “emerging molecular diagnostics for VRE colonization, including multiplexed PCR performed after culture on selective media,” compared with culture alone.

American Academy of Pediatrics

5.1 The thirty-second edition of the American Academy of Pediatrics (AAP) Red Book (2021) describes the diagnostic and treatment options for many infectious diseases in the pediatric population.^56, Their recommendations for appropriate diagnostic tests for the viruses and infections discussed in this policy are detailed in Table 16.

Table 16. Red Book Diagnostic Test Recommendations for the Pediatric Population

BCYE: buffered charcoal yeast extract; CNS: central nervous system; CSF: cerebrospinal fluid; DNA: deoxyribonucleic acid; EIA: enzyme immunoassay; FDA: Food and Drug Administration; GDH: glutamate dehydrogenase; HIV: human immunodeficiency virus; HPV: human papillomavirus; HSE: herpes simplex encephalitis; IFA: indirect fluorescent antibody; MERS-CoV: Middle East respiratory syndrome coronavirus; MSRA: methicillin-resistant Staphylococcus aureus; NAAT: nucleic acid amplification test; NTM: nontuberculous mycobacteria; PCR: polymerase chain reaction; PNA FISH: peptide nucleic acid fluorescent in situ hybridization; RNA: ribonucleic acid; RT: reverse transcriptase; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

5.2 In 2019, the AAP published guidelines on managing infants at risk for group B streptococcus (GBS).^57, It recommends antenatal vaginal-rectal culture performed by using a broth enrichment “followed by GBS identification by using traditional microbiologic methods or by NAAT-based methods.” However, point-of-care NAAT-based screening should not be the primary method of determining maternal colonization status due to reported variable sensitivity as compared with traditional culture, as well as “because most NAAT-based testing cannot be used to determine the antibiotic susceptibility of colonizing GBS isolates among women with a penicillin allergy.”

American College of Gastroenterology

6.1 In 2016, the American College of Gastroenterology published clinical guidelines on the diagnosis, treatment, and prevention of acute diarrheal infections in adults.^58, It recommended that, given that “traditional methods of diagnosis (bacterial culture, microscopy with and without special stains and immunofluorescence, and antigen testing) fail to reveal the etiology of the majority of cases of acute diarrheal infection,… the use of FDA-approved culture-independent methods of diagnosis can be recommended at least as an adjunct to traditional methods. (Strong recommendation, low level of evidence).” These are described in the rationale as multiplex molecular testing.

American Society for Microbiology

7.1 In 2020, the American Society for Microbiology updated the 2010 guidelines on detecting and identifying GBS that were originally published by the CDC, with plans to continue updating regularly.^59, The most recent update took place July 2021. The guidelines state that "intrapartum NAAT without enrichment has an unacceptably high false negative rate...As such we do not recommend the use of intrapartum NAAT without enrichment to rule out the need for prophylaxis." All GBS screening specimens should be incubated in selective enrichment broth prior to agar media plating or NAAT. "Nucleic acid amplification-based identification of GBS from enrichment broth is acceptable" for GBS screening, "but not sufficient for all patients" due to high false-negative rates.

U.S. Preventive Services Task Force Recommendations

Not applicable.

Medicare National Coverage

There is no national coverage determination. In the absence of a national coverage determination, coverage decisions are left to the discretion of local Medicare carriers.

Ongoing and Unpublished Clinical Trials

Some currently ongoing trials that might influence this review are listed in Table 17.

Table 17. Summary of Key Trials

ISRCTN: International Standard Randomised Controlled Trial Number; NCT: national clinical trial.aDenotes industry-sponsored or cosponsored trial

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Codes

Applicable Modifiers

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Policy History