Credit CDC PHIL
#16,989
Although RT-PCR tests for pathogens (for which well-matched primers are available) are normally extremely accurate - and can approach 99% - no lab test is 100% perfect. False negatives, and less frequently, false positives can occur.
As we've previously seen (see EID Journal: Sensitivity and Specificity Of MERS-CoV Antibody Testing and at the start of the COVID pandemic Problematic Lab Testing For The Novel Coronavirus), PCR testing is very good, but it is not infallible.
How and when in the course of an illness patient samples are taken, the time between taking a sample and testing it, the accuracy of the primers used, and even cross contamination can all negatively impact the test's accuracy.
Two weeks ago the CDC issued a Laboratory Advisory on avoiding false positives when testing for Monkeypox.
08/23/2022: Lab Advisory: Monkeypox Virus Testing Considerations to Prevent False Positive Test Results
Audience: Clinical Laboratory Professionals
Level: Laboratory Advisory
To prevent false positive test results, CDC recommends that laboratory professionals perform repeat testing to verify positive diagnostic results for Orthopoxvirus or Monkeypox virus DNA in specimens with high Cycle Threshold (Ct) values from persons who do not meet identified epidemiologic risk criteria for monkeypox.
Review laboratory test results (including raw data, PCR curves, etc.) carefully before you report results. Because molecular tests (e.g., real-time PCR tests) are very sensitive, cross-contamination is possible. If you obtain a high Ct value (generally ~34 or higher), CDC recommends to immediately re-extract and re-test to ensure there was no cross-contamination. CDC suggests this approach based on high Ct value alone, even in the absence of epidemiologic information. When possible, re-extract and re-test before you report results. Obtain approval from your CLIA director before you implement this recommendation in your laboratory.
Refer to the Health Alert Network (HAN) Health Update for more information on testing for monkeypox.
On Friday the CDC's MMWR published a review of 3 recent Monkeypox `false positive' results that likely inspired the above advisory, all obtained from low risk individuals with atypical rashes, and whose RT-PCR tests required a high Ct value (cycle threshold) before returning a positive result.
Due to its length I've only posted some excerpts, so follow the link to read it in its entirety.
Early Release / September 2, 2022 / 71
Faisal S. Minhaj, PharmD1,2; Julia K. Petras, MSPH1,2; Jennifer A. Brown, DVM3; Anil T. Mangla, PhD4; Kelly Russo, MD5; Christina Willut4; Michelle Lee, MPH4; Jason Beverley, MS4; Rachel Harold, MD4; Lauren Milroy, MPH3; Brian Pope3; Emily Gould, MD6; Cole Beeler, MD6; Jack Schneider, MD6; Heba H. Mostafa, MD, PhD7; Shana Godfred-Cato, DO1; Eleanor S. Click, MD1; Brian F. Borah, MD1,2; Romeo R. Galang, MD1; Shama Cash-Goldwasser, MD1,2; Joshua M. Wong, MD1; David W. McCormick, MD1; Patricia A. Yu, PharmD1; Victoria Shelus, PhD1,2; Ann Carpenter, DVM1,2; Sabrina Schatzman, PhD1,8; David Lowe, PhD1; Michael B. Townsend, PhD1; Whitni Davidson, MPH1; Nhien T. Wynn, MS1; Panayampalli S. Satheshkumar, PhD1; Siobhán M. O’Connor, MD1; Kevin O’Laughlin, MD1; Agam K. Rao, MD1; Andrea M. McCollum, PhD1; María E. Negrón, DVM, PhD1; Christina L. Hutson, PhD1; Johanna S. Salzer, DVM, PhD1; CDC Monkeypox Emergency Response Team (View author affiliations)View suggested citation
Summary
What is already known about this topic?
Testing for Monkeypox virus, using Food and Drug Administration 510(k)–cleared non-variola Orthopoxvirus real-time polymerase chain reaction (PCR) test and laboratory developed real-time PCR tests, is critical for diagnosis of suspected cases.
What is added by this report?
Three persons with atypical rashes, uncharacteristic illnesses, and absence of risk factors or an epidemiologic link to a known monkeypox case received false-positive real-time PCR test results; late cycle threshold values were all ≥34.
What are the implications for public health practice?
When testing specimens from patients with atypical signs and symptoms or without epidemiologic links or risk factors or where these are unknown, laboratories should reextract and retest specimens with real-time PCR Ct values that are high (≥34) to avoid unnecessary medical treatment and expenditure of public health resources.
Since May 2022, approximately 20,000 cases of monkeypox have been identified in the United States, part of a global outbreak occurring in approximately 90 countries and currently affecting primarily gay, bisexual, and other men who have sex with men (MSM) (1). Monkeypox virus (MPXV) spreads from person to person through close, prolonged contact; a small number of cases have occurred in populations who are not MSM (e.g., women and children), and testing is recommended for persons who meet the suspected case definition* (1). CDC previously developed five real-time polymerase chain reaction (PCR) assays for detection of orthopoxviruses from lesion specimens (2,3). CDC was granted 510(k) clearance for the nonvariola-orthopoxvirus (NVO)–specific PCR assay by the Food and Drug Administration. This assay was implemented within the Laboratory Response Network (LRN) in the early 2000s and became critical for early detection of MPXV and implementation of public health action in previous travel-associated cases as well as during the current outbreak (4–7). PCR assays (NVO and other Orthopoxvirus laboratory developed tests [LDT]) represent the primary tool for monkeypox diagnosis.
These tests are highly sensitive, and cross-contamination from other MPXV specimens being processed, tested, or both alongside negative specimens can occasionally lead to false-positive results.
This report describes three patients who had atypical rashes and no epidemiologic link to a monkeypox case or known risk factors; these persons received diagnoses of monkeypox based on late cycle threshold (Ct) values ≥34, which were false-positive test results.
The initial diagnoses were followed by administration of antiviral treatment (i.e., tecovirimat) and JYNNEOS vaccine postexposure prophylaxis (PEP) to patients’ close contacts. After receiving subsequent testing, none of the three patients was confirmed to have monkeypox. Knowledge gained from these and other cases resulted in changes to CDC guidance. When testing for monkeypox in specimens from patients without an epidemiologic link or risk factors or who do not meet clinical criteria (or where these are unknown), laboratory scientists should reextract and retest specimens with late Ct values (based on this report, Ct ≥34 is recommended) (8). CDC can be consulted for complex cases including those that appear atypical or questionable cases and can perform additional viral species- and clade-specific PCR testing and antiorthopoxvirus serologic testing.
The three patients described in this report were not MSM, and all had an atypical rash (i.e., without the characteristic progression over 2–4 weeks from pustular to deep-seated, umbilicated lesions). The patients initially received positive Orthopoxvirus real-time PCR test results, with high Ct values (≥34); the positive PCR results were followed by implementation of clinical and public health recommendations for monkeypox, including antiviral treatment and PEP.† This activity was reviewed by CDC and was conducted consistent with applicable federal law and CDC policy.§
(SNIP)
Discussion
Evaluation of these three patients for monkeypox highlights the need for caution in interpreting single laboratory test findings in patients with a low pretest probability of infection; this includes lack of an epidemiologic link, non-MSM populations (e.g., women and children, who currently account for <2% of confirmed monkeypox cases), and signs, symptoms, or rash progression inconsistent with monkeypox. This approach is similar to the caution recommended in evaluating other laboratory tests when pretest probability is low (e.g., D-dimer results for a deep vein thrombosis or serology for Lyme disease)†† (10). Multiple clinical features in each of these three patients were inconsistent with monkeypox, including an atypical rash that was inconsistent with the characteristic progression of monkeypox lesions, as well as the absence of an epidemiologic link to a known case of monkeypox. The Ct values of all initial positive test results were high (≥34) indicating a low level of viral DNA. Cautious interpretation of test results is warranted when the pretest probability of monkeypox is low. As monkeypox testing has expanded, CDC recommends that laboratory professionals verify positive diagnostic results (8) for Orthopoxvirus or MPXV DNA in specimens with high Ct values, especially from persons who do not meet epidemiologic risk criteria for monkeypox or for whom lesions do not progress as expected. Molecular tests (e.g., real-time PCR tests) are highly specific and sensitive; however, when epidemiologic criteria are absent or unknown and the Ct value is high (generally ≥34), CDC recommends reextraction and retesting of the specimen.
Monkeypox currently occurs predominantly among MSM, although infection can occur in any person after close physical contact with persons with monkeypox or items that have been in contact with lesions, such as clothing or bedding. Because the positive predictive value in populations with low disease incidence is lower than that in populations with a higher disease incidence, laboratory results in persons with low pretest probability of infection should be carefully examined and reviewed, and other plausible diagnoses (e.g., hand, foot, and mouth disease; varicella; molluscum contagiosum) should be considered. The clinical course of illness should be reviewed, including documenting the lesions with photographs. CDC can be consulted for atypical or questionable cases and can perform additional viral-specific and clade-specific PCR testing and antiorthopoxvirus serology.
CDC Monkeypox Emergency Response Team
Ramona Bhatia, CDC; Anne Kimball, CDC; Brett Petersen, CDC; Yon Yu, CDC; Kevin Chatham-Stephens, CDC; Kia Padgett, CDC; Maureen J. Miller, CDC; Isaac Zulu, CDC; William C. Carson, CDC; Sapna Bamrah Morris, CDC; Caroline Schrodt, CDC; Amy Beeson, CDC; David Kuhar, CDC; Zeshan Chisty, CDC.
