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Copyright 1999 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.1999American Medical AssociationThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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From November 1997 through March 1998, the number of positive tests for Cryptosporidium increased in several locations in the United States. Several laboratories (e.g., the New York state laboratory and the Medical Science Laboratories in Wisconsin) retested original stool specimens and could not confirm the original positive test result. Following reports to the manufacturer by the Massachusetts, New York, and Wisconsin state health departments about possibly inaccurate test results, Alexon-Trend* (Ramsey, Minnesota) notified its laboratory customers in a March 25, 1998, letter that three lots of its enzyme-linked immunosorbent assay (ELISA) 24 well (catalog number 540-24) ProSpecT® Cryptosporidium Microplate Assay (lot numbers 970717, 975011, and 980401) and seven lots of its ELISA 96 well (catalog number 540-96) ProSpecT® Cryptosporidium Microplate Assay (lot numbers 970696, 970775, 970883, 975006, 980402, 980808, and 980809) were subject to a "non-specific reaction between some stool specimens and the microplate assay" (i.e., a false-positive test result) (K. Hood, Alexon-Trend, personal communication, March 25, 1998). Alexon-Trend directed laboratories to discontinue using kits with implicated lot numbers. This report summarizes an analysis of reports of false-positive tests and describes identification of apparent clusters in three states.
On April 2, 1998, CDC requested state epidemiologists and state laboratory directors to report suspected cases and clusters of false-positive tests. Six states (California, Idaho, Maine, Massachusetts, New York, and Wisconsin) reported apparent clusters and/or an increase in the overall number of positive test results. A working group of state and local public health laboratorians and epidemiologists from these six states participated in a conference call on May 18, 1998, to review their experiences. The findings from five states were reviewed; an apparent false-positive cluster in Idaho was omitted because it involved an ELISA kit not referenced in the manufacturer's letter.
The working group established three case definitions. A confirmed false-positive (CFP) case was one in which a stool specimen that originally tested positive by an implicated lot of the Alexon-Trend kit before March 25, 1998, was available for retesting, subsequently tested negative by an alternate ELISA kit, and if additional testing was performed (e.g., acid-fast and/or fluorescent antibody staining), tested negative by the additional method(s). A possible false-positive (PFP) case was one in which a stool specimen that originally tested positive by an implicated lot of the Alexon-Trend kit before March 25, 1998, was not available for retesting by an alternate ELISA kit but tested negative by an additional method(s) (e.g., acid-fast and/or fluorescent antibody staining). An indeterminate case was one in which a stool specimen tested positive by an implicated lot of the Alexon-Trend kit before March 25, 1998, but for which no original stool specimen was available for retesting, and the original stool specimen was not tested by any other method. Participating laboratories were given a letter designation (e.g., New York has reports from five laboratories, which are designated NY-A, NY-B, NY-C, NY-D, and NY-E).
A total of 62 CFP, eight PFP, and 155 indeterminate cases, including four clusters, were reported in the five states. Five laboratories provided information regarding their rate of positivity (i.e., the number of positive tests for Cryptosporidium expressed as a percentage of the total number of tests for Cryptosporidium) for January 1997-April 1998. For each laboratory, CFP, PFP, and indeterminate cases occurred at the same time as the highest rates of positivity. Information was not available regarding how false-positive test results may have affected patients (e.g., additional diagnostic testing or experimental therapy). Maine, Massachusetts, and Wisconsin provided details regarding their investigations to determine the cause of their suspected disease cluster.
During November-December 1997, laboratory MA-A reported four stool specimens positive by ProSpecT® Cryptosporidium Microplate Assay from residents of one town in Massachusetts. The local health department found no link between cases, and testing of the town's water supply was negative for Cryptosporidium. During January-March 1998, 27 additional positive test results were reported from this laboratory, compared with one to two positive tests per month during the same 3-month period in 1997. No stool specimens were available for retesting. The physicians who ordered the stool tests were notified that positive test results should be considered indeterminate.
During November-December 1997, laboratory WI-A noted that 10 stool specimens that were positive by ProSpecT® Cryptosporidium Microplate Assay were all negative when retested by direct fluorescent antibody (DFA); four also were negative when retested by repeat ELISA. This increase could not be explained by an increase in effluent turbidity at the water treatment plant or by an increase in morbidity measured by other surveillance systems in place in Milwaukee County since the 1993 Cryptosporidium outbreak.1,2 WI-A had noted a gradual increase in the rate of positive ELISAs for Cryptosporidium from a background of ≤2% in the fall of 1997 to 5% in March 1998, with peaks of ≥25% positive on March 6 and 19. The other 11 laboratories involved in statewide Cryptosporidium surveillance, all of which use DFA routinely, reported no increases in absolute number of tests or increases in the rate of positive tests. The physicians who ordered the stool tests were notified of CFP or indeterminate results.
From late January to early February 1998, 41 of 50 elderly male residents on one ward and one of 50 residents on a second ward at a 100-bed extended-care facility experienced gastrointestinal illness. The first cases of illness began approximately 10 days after a severe ice storm caused a power failure lasting several days at the facility and in surrounding communities. Stool samples were negative for bacterial pathogens. Additional persons with diarrhea were reported in mid-February; two of four initial stool specimens from these persons tested positive by ProSpecT® Cryptosporidium Microplate Assay. Stool specimens from 35 of 79 facility patients in both wards and from one outpatient tested positive for Cryptosporidium by this method. A public health investigation and water testing were performed at the facility. Because clinical and epidemiologic characteristics of this outbreak were inconsistent with cryptosporidiosis, the 36 antigen-positive specimens were re-evaluated at ME-A, a reference laboratory, and all were negative by ELISA. Water tests were negative for coliform bacteria.
JR Miller, MD, B Mojica, MD, City Epidemiologist, New York City Dept of Health. J Nadle, MPH, California Emerging Infections Program; DJ Vugia, MD, SH Waterman, MD, State Epidemiologist, California Dept of Health Svcs. B Mamer, PhD, C Hahn, MD, State Epidemiologist, Idaho Dept of Health and Welfare. KM Doing, PhD, Affiliated Laboratories, Inc, Bangor; JL Hamm, N Buker, Togus Veterans Administration Hospital, Togus; GA Beckett, MPH, KF Gensheimer, MD, State Epidemiologist, Maine Dept of Human Svcs. P Kludt, MPH, A DeMaria, MD, State Epidemiologist, Massachusetts Dept of Public Health. J Ennis, MS, J Keithly, PhD, S Kondracki, D Ackman, MD, P Smith, MD, State Epidemiologist, New York State Dept of Health. D Warshauer, PhD, Medical Science Laboratories, Milwaukee; M Proctor, PhD, J Davis, MD, State Epidemiologist, Wisconsin Dept of Health and Social Svcs. Div of Parasitic Diseases, National Center for Infectious Diseases, CDC.
ELISA and other immunoassays offer advantages over diagnostic tests based on microscopic methods, especially for laboratories that perform large numbers of tests. ELISA can be used to test multiple stool specimens simultaneously, and ELISA does not require the same high level of technical skill needed to identify parasites based on the morphologic and staining characteristics observed during microscopic examination. However, when a laboratory depends solely on ELISA for detection of Cryptosporidium, false-positive test results may go unrecognized for long periods of time because of problems associated with the kit reagents or technician error.
Retaining stool specimens, or preparing a permanent microscopic slide whenever an ELISA result is positive has implications for cost, staffing, and storage. In laboratories that rely solely on antigen tests of stool specimens for parasites and that do not routinely retain stool specimens or make permanent slides, management should consider monitoring the rate of positive test results and, when this rate noticeably increases above a certain level (e.g., two or more times the laboratory's mean positivity rate for an organism), implement confirmatory testing by microscopic methods and/or begin archiving stool specimens. Alternatively, all stool specimens could be split before testing so that an aliquot of a specimen positive by ELISA could be sent to a reference diagnostic laboratory for confirmation. This method is analogous to using ELISA as a screening test for human immunodeficiency virus, with Western blot testing used to confirm specimens positive by ELISA.3 Another advantage of retaining stool specimens is its availability for polymerase chain reaction-based genotyping, as might be warranted in an outbreak. In New York, laboratories using ELISA must either prepare a permanent microscopic slide or retain a portion of the original stool specimen, and laboratories are required to hold slides or stool specimens for 1 year. As a result of the investigation described in this report, New York state has reminded laboratories of this existing requirement and has used this incident in a statewide educational workshop for laboratorians.
In many communities, a cluster of laboratory-reported cases of cryptosporidiosis elicits a multidisciplinary investigation to find the cause. Every community should develop a plan for responding quickly and efficiently to increases in the number of reported cases of cryptosporidiosis.4 Essential components of an effective response plan include confirming the diagnosis, comparing current disease data with baseline data, and developing a strategy for critically and systematically determining whether there is a community outbreak. Having access to good laboratory records and stored specimens facilitates confirmation of the diagnosis and reduces the likelihood that limited health department resources will be redirected to an unnecessary community-wide epidemiologic investigation on the basis of false-positive laboratory results.
When evidence suggests a commercial laboratory diagnostic kit is yielding inaccurate test results, this information should be forwarded to the kit manufacturer and the appropriate local and state health department. These departments will inform the state certifying authority for laboratory practice, the Food and Drug Administration, and CDC.
False-Positive Laboratory Tests for Cryptosporidium Involving an Enzyme-Linked Immunosorbent Assay—United States, November 1997-March 1998. JAMA. 1999;281(5):411–412. doi:10.1001/jama.281.5.411-JWR0203-2-1