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Methods

Setting. Amphia Hospital is a 1,370‐bed teaching hospital in Breda, The Netherlands. The Laboratory for Microbiology and Infection Control uses the AxSYM analyzer to test, on average, approximately 100 samples per week for HBsAg.² In the second week of September 2002 (week 36 of 2002), 12 (75%) of 16 samples in one batch tested positive for HBsAg. All 12 HBsAg‐positive samples tested negative for anti‐HBc. Because no patients had any signs or symptoms compatible with HBV infection (all samples were from pregnant women during routine screening), a pseudo‐outbreak of HBV infection due to cross‐contamination was suspected. We decided to perform an epidemiologic investigation to elucidate the source of the cross‐contamination.

Epidemiologic investigation. A descriptive study based on laboratory results and patient clinical data was conducted to determine the times, locations, and patients associated with the pseudo‐outbreak. Case patients were defined as follows: patients who were HBsAg positive and anti‐HBc negative, irrespective of the result of the confirmatory test for HBsAg (case definition 1); patients who were HBsAg positive and anti‐HBc negative, with a negative result of the confirmatory test for HBsAg (case definition 2); and patients who were HBsAg positive and anti‐HBc negative, with a positive result of the confirmatory test for HBsAg (case definition 3).

Laboratory investigation. For each sample that tested positive for HBsAg and negative for anti‐HBc, a confirmatory test for HBsAg was performed using a different analyzer (Organon Teknika). If the result of the confirmatory test for HBsAg was negative, the initial result was considered to be a false‐positive finding. If the result of the confirmatory test for HBsAg was positive, the initial result was considered to be a true‐positive finding.

The possibility of cross‐contamination was studied by obtaining a serum sample from a patient who was HBsAg and anti‐HBc positive. A semiautomatic cap remover was used to remove the cap of this sample and subsequently used to remove the cap of 4 tubes containing a sterile salt solution. This experiment was repeated 3 times, and all samples were tested in the AxSYM for HBsAg and anti‐HBc.

Results

Epidemiologic investigation. On the basis of case definition 1, the pseudo‐outbreak started in week 35 (Figure 1), just after major maintenance of the AxSYM analyzer had been performed, implicating it as the most likely source of HBV. However, according to case definition 2, there was no pseudo‐outbreak. This finding probably represents the rate of false‐positive results of HBsAg tests in our laboratory, which are based on the aspecific reactivity of this test. On the basis of case definition 3, the pseudo‐outbreak started in week 31. In week 30, a semiautomatic cap remover for blood collection tubes (Electa Lab) was introduced in the laboratory to improve employee safety.

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Figure 1.  Epidemiologic curve of a pseudo‐outbreak of hepatitis B virus infection, according to 3 different case definitions (see Methods for a description of the case definitions).

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Laboratory investigation. The samples obtained during week 36 that had false‐positive results of tests for HBsAg all tested positive for HBsAg upon confirmatory testing, suggesting that case definition 3 correctly described HBV infection during the pseudo‐outbreak. Cross‐contamination with HBsAg, which resulted in false‐positive test results, was demonstrated repeatedly in cross‐contamination experiments. Cross‐contamination with anti‐HBc did not result in false‐positive test results. A semiautomatic cap remover similar to the device that underwent analysis is shown in Figure 2.

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Figure 2.  Semiautomatic cap remover for blood collection tubes (Electa Lab). The cap remover is used as follows: the device is placed on a blood collection tube, a needle enters the aluminium film that covers the open end of the tube, a circular blade enters and cuts the film, and the film is removed from the tube. Four blood collection tubes can be opened in this fashion before the films need to be removed from the device.

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Discussion

An unusual high rate of HBsAg positivity in a low‐risk population was found in our laboratory. Epidemiological methods were used to elucidate the source of the problem. A good case definition proved to be of vital importance for identifying the correct cause of the outbreak. From the epidemiological data, together with the cross‐contamination experiments, it was concluded that the pseudo‐outbreak was most likely caused by the introduction and use of the cap remover. After the investigations, the cap remover was abandoned, and the pseudo‐outbreak ended.

A proportion of all infections diagnosed in the laboratory are believed to be falsely identified as such and can be part of a pseudo‐outbreak.^3,4 Pseudo‐outbreaks are an ongoing problem for infection control personnel, infectious disease physicians, and hospital epidemiologists. The implications of false‐positive test results, whether caused by the limitations of the test itself or by errors in procedure, can be substantial. For example, Laurel et al.⁵ described a pseudo‐outbreak of Aspergillus niger infection that followed building construction in the clinical microbiology laboratory. Patients were prescribed toxic antifungal medication (amphotericin B). Additionally, these inaccurate diagnoses generated 6 consultations: 3 to the infectious diseases service and 1 each to the renal, allergy, and pulmonary services. Multiple laboratory studies were required either to clarify the diagnosis or to monitor adverse effects of therapy (eg, renal function tests when amphotericin B was prescribed).

The cap remover that caused the cross‐contamination problem is used throughout Europe. Surprisingly, this adverse affect has not, to our knowledge, been reported in literature. According to the package insert of the cap remover, the manufacturer is aware of this phenomenon but considers it to be of no importance: “laboratory tests proved that the cap remover does not contaminate samples (or in minimum and no considerable values).”

Similar to other reports of pseudo‐outbreaks in which clusters of false‐positive test results have been identified, the false‐positive HBsAg results we report did not correlate with the clinical conditions of the patients, and no incorrect diagnoses were made.⁶ All results of initial tests were reported as equivocal by the medical microbiologist. It was advised that patients should be retested before a diagnosis was made. This underscores the importance of having a professional on staff who reports laboratory results with the awareness of the laboratory procedures that have been performed and the knowledge of the consequences of these results for the patients involved.