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Designing a Full Infectious Disease Panel

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In vitro diagnostics (IVD) are valuable tests that are used to detect and identify many pathogens that can cause infections within the human body. The diagnosis and selection of appropriate treatment depends on the correct identification of the bacteria, virus, or parasite, including any antibiotic resistance contained by these organisms. Laboratories can either use on market IVDs or develop their own laboratory developed tests (LDT) to identify these pathogens that are commonly detected in infectious disease in patients. Laboratories that want to offer their own LDT must carefully design infectious diseases panels for molecular diagnostics to meet clinical testing standards or the laboratories need to choose on market IVDs that include the markers they need to meet their clinical testing needs. Multiplex polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) are laboratory techniques used in infectious disease testing because of their high sensitivity and specificity compared to culture, microscopy, and immunoassays.1,2 Multiplex PCR panels can detect numerous pathogens simultaneously from a patient’s sample instead of each individually. This molecular technology can be used to detect pathogens in many parts of the body, such as a respiratory pathogen panel (RPP), wound pathogens, gastrointestinal (GI) pathogens, sexually transmitted infections (STI), and urinary tract infections (UTI). Common pathogens identified in infectious disease areas are illustrated below (not an exhaustive list):

Respiratory Pathogens3,4 Wound Pathogens5,6 Gastrointestinal Pathogens7,8 Sexually Transmitted Pathogens9,10 Urinary Tract Pathogens11
Rhinovirus Staphylococcus aureus Salmonella enterica Chlamydia trachomatis Escherichia coli
Influenza virus Pseudomonas aeruginosa Campylobacter species Herpes simplex virus 1(HSV-1) or Herpes simplex virus 2 (HSV- 2) Klebsiella pneumoniae
Adenovirus Proteus mirabilis Yersinia enterocolitica Neisseria gonorrhoeae Proteus miribilis
Enterovirus Escherichia coli Escherichia coli Human Papillomavirus (HPV) Enterobacter faecalis
Respiratory syncytial virus (RSV) Corynebacterium spp. Shigella species Mycoplasma genitalium Entercoccus spp.
SARS-CoV-2 Coagulase-negative staphylococci   Treponema pallidum Staphylococcus saprophyticus
S. pyogenes (Group A streptococcus) Enterococcus   Trichomonas vaginalis

Accurate and rapid clinical diagnostic tests help providers quickly and accurately diagnose what is causing many common infections and help providers select the appropriate antimicrobial treatments to help fight off the pathogens and ensure a faster patient recovery.

When designing or selecting an infectious disease panel, a laboratory must evaluate the pathogens they will include in their testing. Laboratories can perform this step by reading through the current and earlier scientific literature to understand the common pathogens in the infectious disease they are examining and to identify the bacteria, fungi, or viruses that are relevant to the area of infection. It is also essential to stay up to date with other laboratory tests for that area of infectious disease to see what others are testing for. Most testing leverages peer reviewed literature as well as historical detection rates to check that tests are capturing the clinically relevant pathogens essential to patient testing. Questions that should be answered by the laboratory when designing or selecting an infectious disease panel include the following:

  • Is the test capturing the most commonly identified pathogens for that area of infectious disease?
  • What are some other pathogens that others are testing? Are they relevant to the area of focus?
  • Are there emerging pathogens that you would like to detect?
  • Which pathogens have known resistance to antibiotics? Do you want to detect these in the test and identify the antibiotic resistance (ABR) genes?
  • Is there a clinical need for the detection of the specific organisms at present?

Once the scientific staff completes a draft of the testing menu, the laboratory must validate the molecular test. A validation study is essential to determine the sensitivity, selectivity, precision, and accuracy in test development. A validation team develops a detailed validation plan that should include the following:

  • Description of the disease, scientific basis of the test, and explanation of the assay, including regions to be amplified;
  • Assay principle including a description of the assay list of primers/ probes (oligonucleotides) to be used;
  • The clinical purpose of the assay describing the clinical relevance of the assay, why the assay is needed, variants in the population, including the percentage of variants in ethnic populations;
  • Performance characteristics include accuracy, precision, linearity, analytical sensitivity, analytic specific/selectivity, reportable range of test results, and reference intervals;
  • Specimen information includes sample type, procurement, unacceptable specimen criteria, handling of specimens, and sample stability;
  • Reagents and equipment to be used in the test;
  • Measurements of quality control of the test, such as positive, negative, and internal controls, and evaluation of the performance of the test. This section will include the calculations of the accuracy, precision, standard curve, analytical sensitivity/specificity/ selectivity, reportable range and interpretations, and reference intervals.12,13
  • Clinical validation understanding limitations of the test with real world clinical trial data should follow analytical validation.

Once the validation studies are complete, the Laboratory Director approves the results, and the test is ready for final review before the laboratory can offer the test to the public.

Laboratories should also evaluate how commercial and federal payers will assess clinical need for a validated LDT or IVD test. Each payer will have requirements that will need to be met by providers using the test for their patients. Test developers must review testing codes such as Current Procedural Terminology (CPT) codes and International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10) codes that determine patient testing coverage and reimbursement per test. A review of commercial payer coverage policies and National Coverage Determination (NCD) and Local Coverage Determination (LCD) of the appropriate Medicare Administrative Contractor (MAC) will help a laboratory understand medical policy related to the test. Upon review of coverage policies, there also may be the absence of policies or denial of coverage surrounding testing specific pathogens on the test. In that case, the laboratory may need to generate evidence to support its testing for the payors to review. This step may take time to produce this new evidence supporting the pathogen’s testing. The laboratory will need to work with the payor to submit the supporting information to enable possible approval and coverage of their infectious disease test.

Once an infectious disease test developed with molecular technology is offered to physicians by the laboratory, the scientific staff must continuously review the new test for its continued relevance in the clinical setting, test performance, clinical trial information, and insurance payment of the

testing panel. It is necessary to check the test results after the launch to make sure the test identifies the pathogens as seen in its validation. The laboratory will need to review the current scientific literature for any updates to the pathogens on the panel and any new or missing pathogens that the staff should add to the existing testing menu. Feedback from physicians using the infectious disease testing panel and clinical trial data are both constructive in understanding its usefulness in patient treatment plans. The laboratory should monitor reimbursement of the test from payors and communicate with them as needed to receive payment for the test. It also may be helpful to consider studies that demonstrate the increased specificity and sensitivity of the test compared to the other testing methods for infectious diseases. The laboratory and staff should continuously monitor successful molecular infectious disease panels for their usefulness and update the testing menu as needed.

The selection and/or design of a molecular infectious disease panel is an exciting process that can result in a clinically relevant diagnostic that providers in the field of infectious disease utilize. Building a testing panel is a thoughtful process from researching the testing menu, validation studies of the test, reimbursement of the test by payors, and the surveillance of how the test is performing and any updates necessary once the testing launch occurs. Laboratories that use these guidelines to develop an infectious disease will produce a testing product that will meet the needs of physicians in the field of infectious disease and support patients that require accurate and rapid testing.

Watch Our On-Demand Webinar: How to Choose Clinically Relevant Target for Your Infectious Disease Panels.

About the Author

Dr. Margo Lee is a dedicated Senior Medical Science Liaison with over a decade of experience supporting molecular clinical diagnostics in the fields of infectious disease and genetic testing. She has worked for many years both in Medical Affairs and as a laboratory scientist. Dr. Lee is driven by the need to share her clinical and laboratory expertise with the medical community and to help improve patient outcomes. Dr. Lee has worked at the National Institutes of Health, both at the National Cancer Institute and Human Genome Research Institute.

References:

  1. Nolte, Frederick & Woolworth Hirschhorn, Julie & Hill, Charles. (2017). Polymerase Chain Reaction and other Nucleic Acid Amplification Technology.
  2. Shen, Chang-Hui. “Amplification of Nucleic Acids.” Diagnostic Molecular Biology (2019): n. pag.
  3. Thomas M, Bomar PA. Upper Respiratory Tract Infection. [Updated 2022 Jun 27]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK532961/
  4. Viral and Bacterial Interactions in the Upper Respiratory Tract. Bosch AATM, Biesbroek G, Trzcinski K, Sanders EAM, Bogaert D (2013) Viral and Bacterial Interactions in the Upper Respiratory Tract. PLOS Pathogens 9(1): e1003057. https://doi.org/10.1371/journal.ppat.1003057
  5. Bessa, L.J., Fazii, P., Di Giulio, M. and Cellini, L. (2015), Bacterial isolates from infected wounds and their antibiotic susceptibility pattern: some remarks about wound infection. Int Wound J, 12: 47-52. https://doi.org/10.1111/iwj.12049
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  7. Morten Helms, Jacob Simonsen, Kåre Mølbak, Foodborne Bacterial Infection and Hospitalization: A Registry-Based Study, Clinical Infectious Diseases, Volume 42, Issue 4, 15 February 2006, Pages 498–506, https://doi.org/10.1086/499813
  8. Porter CK, Choi D, Cash B, Pimentel M, Murray J, May L, Riddle MS. Pathogen-specific risk of chronic gastrointestinal disorders following bacterial causes of foodborne illness. BMC Gastroenterol. 2013 Mar 8;13:46. doi: 10.1186/1471-230X-13-46. PMID: 23510245; PMCID: PMC3599665.
  9. Garcia MR, Leslie SW, Wray AA. Sexually Transmitted Infections. [Updated 2022 Nov 28]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK560808/
  10. Low N, Broutet N, Turner R (2017) A Collection on the prevention, diagnosis, and treatment of sexually transmitted infections: Call for research papers. PLoS Med 14(6): e1002333. https://doi.org/10.1371/journal.pmed.1002333
  11. Bono MJ, Leslie SW, Reygaert WC. Urinary Tract Infection. 2022 Nov 28. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan–. PMID: 29261874.
  12. Food and Drug Administration. Draft Guidance “Framework for Regulatory Oversight of Laboratory Developed Tests. October 3, 2014. http://www.fda.gov/downloads/ MedicalDevices/DeviceRegulationandGuidance/
  13. Centers for Medicare and Medicaid Services. Background document on CLIA oversight of LDTs https://www.cms.gov/Regulations-and-Guidance/ Legislation/CLIA/Downloads/ LDT-and-CLIA_FAQs.