Original Article
Detection of Circulating Candida Enolase by Immunoassay in Patients with Cancer and Invasive Candidiasis
N Engl J Med 1991; 324:1026-1031April 11, 1991
- Abstract
- Abstract
Background.
Invasive candidiasis is a major nosocomial infection that is difficult to diagnose. Few biochemically defined markers of invasive candidiasis are known. Initial findings suggested that the presence of candida enolase in the blood may be a novel marker for invasive candidiasis.
Methods.
We tested 170 patients at high risk for invasive candidiasis for candida enolase antigenemia. All the patients had cancer and neutropenia. We detected antigen using a double-sandwich liposomal immunoassay for candida enolase in serially collected serum samples. Invasive candidiasis was proved by finding candida species in deep nonmucosal tissue, blood cultures, or both. Antigen testing was performed with the investigator blinded to tissue or culture diagnosis.
Results.
Among 24 patients with proved invasive candidiasis, 149 serum samples were tested for enolase antigenemia; 80 were positive and 69 negative (sensitivity per sample, 54 percent). Multiple sampling improved the detection of antigenemia, which was found in 11 of 13 proved cases of deep tissue infection (85 percent) and in 7 of 11 proved cases of fungemia (64 percent). Specificity was 96 percent as measured against control groups including patients with mucosal colonization, bacteremia, and other deep mycoses. Antigenemia was detected in the absence of fungemia in 5 cases of deep tissue candidiasis, but was not detected in 6 cases of fungemia alone.
Conclusions.
Candida enolase antigenemia is a novel marker for invasive candidiasis. It may be a useful indicator of deep infection in patients with cancer and neutropenia and may complement the diagnostic usefulness of blood cultures. (N Engl J Med 1991; 324:1026–31.)
Media in This Article
Table 1
Enolase Antigenemia in Patients with Cancer and Invasive Candidiasis and in Those without the Infection, According to Number of Patients.*Table 2
Enolase Antigenemia in Patients with Cancer and Invasive Candidiasis and in Those without the Infection, According to Number of Serum Samples.*Article Activity
- Article
INVASIVE candidiasis is difficult to diagnose and is the cause of substantial morbidity and mortality in patients with cancer.1 2 3 4 5 The current approach to the diagnosis of invasive candidiasis consists of clinical suspicion and the use of blood cultures, diagnostic imaging techniques (e.g., CT scanning and magnetic resonance imaging), and tissue biopsy. These approaches, however, often lack sensitivity, may produce positive results only in more advanced stages of infection, and may be unduly invasive in immunocompromised patients.
Newer diagnostic approaches for invasive candidiasis include the detection of candida cell-wall antigens, such as mannans,6 , 7 and candida metabolites, such as D-arabinitol.8 , 9 Little is known, however, about the diagnostic usefulness of detecting candida cytoplasmic antigens.10 11 12 13 14
Strockbine et al. identified an immunodominant (eliciting high concentrations of antibody) cytoplasmic 48-kd antigen of candida for which antibody production correlated with deep candidiasis in patients who did not have cancer.11 This 48-kd antigen was subsequently found to be candida enolase.15 , 16 Studies in experimental disseminated candidiasis in mice and rabbits demonstrated that antigenemia due to the 48-kd antigen detected by enzyme-linked immunosorbent assay was expressed in the absence of fungemia, correlated with deep tissue infection, distinguished superficial involvement from deep involvement, and decreased in response to antifungal therapy.17
In order to investigate the expression of this candida cytoplasmic antigen in the serum of patients with cancer who are at high risk for deeply invasive candidiasis and to investigate its potential for diagnosis of such infections, we conducted a prospective clinical trial among patients from four medical oncology centers over a two-year period.
Methods
Study Population
Patients were considered to be at high risk for invasive candidiasis if they had cancer and had received cytotoxic chemotherapy and broad-spectrum antibiotics, had granulocytopenia, and had central venous catheters. Such patients from the following centers were enrolled in this study: Clinical Center, National Cancer Institute, Bethesda, Maryland; Duke University Medical Center, Durham, North Carolina; Harper Hospital, Wayne State University, Detroit; and Johns Hopkins Hospital, Baltimore. Patients with candidiasis were entered as having invasive candidiasis (proved by tissue or blood culture) or colonization (as defined below). Other patients with cancer were enrolled in one of the following control groups: those with bacteremia, those with noncandida fungal infections, and those with no microbiologic evidence of infection (as defined below). The patients were followed prospectively by investigators at each of the participating institutions, who paid particular attention to the methods of establishing a diagnosis of candidiasis, timing and reporting of blood cultures, initiation of antifungal therapy, sites of candida infection, species of candida, and patient outcome.
For the purposes of this study, invasive candidiasis was defined as infection due to candida species in blood or deep nonmucosal tissue. Invasive candidiasis was classified further as either deep candidiasis (tissue-proved invasive candidiasis) or fungemia (invasive candidiasis proved by blood culture only). In this study, tissue-proved deep candidiasis excluded mucosal candidiasis by definition. Candida infection of submucosal sites was classified separately. Colonization was defined as the presence of candida isolated from mucosal surfaces (oropharynx, rectum, or vagina) with no evidence of deep visceral infection in patients with cancer and neutropenia. Five patients with neutropenia who had urine cultures positive for candida were specifically excluded from this study because upper urinary tract infection could not be reliably ruled out or proved.
In order to determine the specificity of enolase antigenemia in patients without evidence of candidiasis, three groups of patients with neutropenia were evaluated as controls: those with bacteremia, those with noncandida fungal infections, and those with no microbiologic evidence of infection. Patients with neutropenia were defined as having bacteremia if they had fever, bacteria isolated from blood cultures, and no evidence of candidiasis. Patients were defined as having noncandida fungal infections if they had fever and microbiologically or histopathologically proved deep fungal infection due to fungi other than candida species (e.g., pulmonary aspergillosis). Those defined as having no microbiologic evidence of infection were patients with cancer, neutropenia, and fever who were considered to have no microbiologically or clinically proved infection. To evaluate the specificity of enolase antigenemia in control subjects without infection or cancer, serum from normal blood-bank donors was collected and analyzed.
Microbiologic Monitoring
Cultures of blood, sterile body fluids, and mucosal surfaces were obtained as clinically indicated in order to establish a microbiologic diagnosis in febrile patients with cancer. All specimens were cultured for fungi. Blood was cultured by the Bactec method (Becton Dickinson Diagnostic Instrument Systems, Baltimore), by lysis centrifugation (Isolator system, Dupont, Wilmington, Del.), or both. Specimens from other anatomical sites were plated onto Sabouraud's glucose agar. Yeast species were identified in each center with use of germ tube, cornmeal agar, the API-20C system (Analytab, White Plains, N.Y.). or all three.
Collection and Handling of Specimens
Serum samples were collected prospectively from all control and infected patients. Once a patient in this study proved on histopathologic examination of tissue or by blood culture (or both) to be infected, serial serum samples were obtained prospectively and the course of infection was followed closely by the investigators. Data and serum samples from all patients in the study were collected by day-to-day monitoring.
Serum samples were collected, numbered, and frozen at –70°C on site by the clinical coordinator. Coded specimens were submitted in batches to laboratory personnel, who analyzed the samples in a blinded fashion. A patient's identification was not revealed until all that patient's specimens had been processed. The data were not known by the clinician following the patient and were not used in the diagnosis or management of patients during the study. At each site all laboratory personnel underwent training with a blinded panel of samples with known concentrations of antigen ranging from negative to highly positive in order to ensure proficiency and consistency in reading test results.
Assay Principles
The antigen-detection system used in this study was a liposomal immunoassay for the direct qualitative detection of candida enolase antigen, a 48-kd protein, in serum (Becton Dickinson Microbiology Systems, Baltimore). Murine IgA monoclonal antibody specific for the antigen was immobilized on a porous nitrocellulose membrane. Antigen in serum was bound to the immobilized antibody as the specimen passed through the membrane. Polyclonal rabbit IgG antibodies directed against the antigen were bound to the trapped antigen to enhance the number of reactive sites. Liposome detector particles containing a red dye and coated with goat IgG antirabbit antibodies were then bound, resulting in the formation of a colored triangular image on the membrane in a positive reaction. Results were considered positive if a completely formed triangle was created in this reaction. The intensity of the color reaction was scored on a scale of 1 to 4. Negative results were defined as the absence of a visible triangle. The sensitivity of the assay in detecting antigen in serum seeded with known quantities of purified antigen was 1 to 2 nmol (0.5 to 1.0 μg) per liter.
Statistical Analysis
Differences in proportions in categories were measured with Fisher's exact test or chi-square analysis. Differences in means were determined by Student's t-test. All P values are two-tailed, and a P value <0.05 was considered significant. Confidence intervals were determined with the methods of Brown and Hollander.18 The sensitivity of the assay was defined as TP/(TP + FN), the specificity as TN/(FP + TN), the positive predictive value as TP/(TP + FP), and the negative predictive value as TN/(TN + FN), where TP is true positive results, FP false positive results, TN true negative results, and FN false negative results.
Results
Study Patients
One hundred seventy patients with cancer were studied. Twenty-four patients were found to have invasive candidiasis; 13 of these patients had deep candidiasis (tissue-proved invasive candidiasis), and 11 had fungemia (invasive candidiasis proved by blood culture). Fifty patients with a diagnosis of candida colonization and 96 patients with no evidence of candidiasis made up the negative control groups.
Patients with Cancer and Invasive Candidiasis
The sensitivity and specificity of enolase antigenemia and the data used for their calculation (see Statistical Analysis) are shown in Tables 1Table 1
Enolase Antigenemia in Patients with Cancer and Invasive Candidiasis and in Those without the Infection, According to Number of Patients.* and 2Table 2Enolase Antigenemia in Patients with Cancer and Invasive Candidiasis and in Those without the Infection, According to Number of Serum Samples.*. In patients with tissue-proved invasive candidiasis (Table 1), the sensitivity was 85 percent, the positive predictive value 65 percent, and the negative predictive value 99 percent. In patients with candidiasis proved by blood culture alone, the sensitivity of enolase antigenemia was 64 percent, the positive predictive value 54 percent, and the negative predictive value 97 percent. Among all the patients with invasive candidiasis proved by deep tissue biopsy or blood culture, the sensitivity was 75 percent, the positive predictive value 75 percent, and the negative predictive value 96 percent. Specificity was 96 percent.When the expression of enolase antigenemia was analyzed according to individual serum sample, sensitivity was reduced to 52 percent among patients with tissue-proved invasive candidiasis (Table 2). In patients with candidiasis proved by blood culture alone, the sensitivity of enolase antigenemia in a single serum sample was 63 percent. Among all the patients with invasive candidiasis proved by biopsy or blood culture, the sensitivity of an individual serum sample remained relatively low (54 percent). By comparison, the specificity for each sample was 98 percent.
Table 3Table 3
Candida Enolase Antigenemia in Patients with Cancer and Tissue-Proved Invasive Candidiasis. summarizes the results in patients with cancer and deep candidiasis proved on biopsy or at autopsy. Eleven of the 13 patients were antigen-positive, and 2 were antigen-negative. One hundred twenty-two serum samples were tested for antigen, and 63 were positive. Ten of the 11 patients with enolase antigenemia had proved hepatic candidiasis, whereas the 2 antigen-negative patients did not. Patient 8 had involvement of the skin, spleen, lung, kidney, and gastrointestinal tract proved at autopsy. Patient 15 had a candida septic arthritis of the shoulder and was discharged receiving antifungal therapy. Among the 11 patients with enolase antigenemia, blood cultures were negative in 5.Table 4Table 4
Candida Enolase Antigenemia in Patients with Cancer and Invasive Candidiasis Proved by Blood Culture. summarizes the results in patients with cancer and candidiasis proved by blood culture. Seven of the 11 patients in this group were antigen-positive, and 4 were antigen-negative. Twenty-seven serum samples were collected, and 17 were antigen-positive. All four antigen-negative patients survived and were discharged from the hospital.Patients with Cancer and Candida Colonization
We studied 50 patients with cancer who had candida colonization along oral or rectal mucosal surfaces (or both) but who were not considered to have deep candidiasis. Among the species of yeast isolated were C. albicans, C. tropicalis, C. parapsilosis, and C. krusei. Among the 50 patients studied, 3 (6 percent) were antigen-positive. Among 236 serum samples from these patients, 5 (2 percent) were positive. One patient with neutropenia and acute leukemia had positive results on three consecutive serum samples; this patient was colonized repeatedly in stool with C. albicans and C. krusei and received empirical amphotericin B therapy for recurrent fever, suggesting possible invasive candidiasis.
Patients and Controls without Candidiasis
In order to determine the specificity of the assay further, we studied two other populations: normal blood-bank donors and patients with cancer and no evidence of candidiasis. Among the patients with cancer and no evidence of candidiasis, three groups were studied: 53 patients with no evidence of bacterial or fungal infection, 32 with bacteremia, and 11 with noncandida deep mycoses due to aspergillus species, Cryptococcus neoformans, and Saccharomyces delbrueckii Lindner. None of these noncandida mycoses involved the liver.
In this group with cancer and no evidence of candidiasis, enolase antigenemia was detected in 3 of 96 patients (3 percent) and in 5 of 299 serum samples (2 percent). Among the 96 patients with cancer without candidiasis, none of the 53 with no evidence of infection had enolase antigenemia. In addition, among 140 single samples from normal blood-bank donors, 3 (2 percent) were weakly positive for antigen (data not shown). Of the 11 patients with a noncandida deep mycosis, 1 with deep infection due to S. delbrueckii had enolase antigenemia (9 percent). Also, 2 of 32 patients with bacteremia (6 percent) had enolase antigenemia. The other patients with pulmonary aspergillosis or disseminated cryptococcosis had no detectable antigen.
Relation between Blood Cultures and Antigenemia
In this trial, blood cultures and enolase antigenemia appeared to be complementary in cases of invasive candidiasis (Table 5Table 5
Relation between Blood Cultures and Enolase Antigenemia in the Detection of Invasive Candidiasis in Patients with Cancer.). Among the 24 patients with invasive candidiasis, blood cultures alone were positive in 6, enolase antigenemia alone was present in 5, and both blood cultures and the antigen assay were positive in the remaining 13. Among the patients with tissue-proved invasive candidiasis, 2 had positive blood cultures alone, 5 had enolase antigenemia alone, and 6 had both positive blood cultures and enolase antigenemia.In five patients (Patients 1, 3, 7, 13, and 16) the presence of invasive candidiasis was established only by tissue diagnosis. All these patients had a number of negative blood cultures, radiographic findings consistent with hepatic candidiasis, and biopsy-proved infection. Enolase antigenemia was present during multiple sampling in all but one of the patients (Patient 3), in whom the only sample drawn was positive.
Yield of Blood Cultures and Serum Sampling
Among the 13 cases of tissue-proved deep candidiasis, blood cultures were positive in 18 of 135 samples (13 percent). Multiple serum samples were also obtained for the detection of candida enolase antigen in these cases; antigenemia was detected in 63 of 122 serum samples (52 percent; P≤0.0001; difference in percentages, 39 percent; 95 percent confidence interval, 28 to 49). Among the cases of candidiasis proved by blood culture, the cultures were positive in 36 of 46 samples (78 percent) and enolase antigenemia was detected in 17 of 27 samples (63 percent; P = 0.25; difference in percentages, 15 percent; 95 percent confidence interval, –6.5 to 37).
Discussion
This study demonstrated that enolase antigenemia was often present in patients with cancer and deep candidiasis, particularly in those with proved tissue invasion. Enolase antigenemia was seldom present in patients colonized with candida or in those who were at high risk but who had no evidence of candidiasis. Multiple serum samples were necessary to maximize detection. Antigenemia was present in the absence of fungemia in some patients with tissue-proved deep candidiasis, but it was absent in the presence of fungemia in others. The detection of antigen therefore complemented rather than replaced blood cultures in the determination of deep candidiasis.
The purpose of this study was to characterize the expression of candida enolase antigenemia in several carefully defined populations of patients with cancer. We studied high-risk groups in which the pattern or stage of candidiasis could be well defined radiographically, microbiologically, and histologically. The patients with antigen-positive invasive candidiasis were identified according to strict criteria, including histopathologic examination, blood cultures, or both. The preponderance of tissue-proved cases involving hepatic infection may be due to the fact that the liver is a commonly recognized site of invasive candidiasis in patients with cancer. Other groups at high risk, including patients with burns and those undergoing solid-organ transplantation and other surgical procedures complicated by infection, are currently being studied in other centers.
The only gold standard for the detection of deeply invasive candidiasis is demonstration of the organism in tissue obtained through biopsy or postmortem examination. These criteria are problematic when one attempts to diagnose invasive candidiasis when only fungemia exists. Although the combination of neutropenia and fungemia may increase the probability that the patient has deeply invasive candidiasis, tissue invasion remains unproved in such cases. This problem of classification becomes more apparent in patients with neutropenia and candiduria in whom mucosal colonization in the bladder cannot be distinguished from deep kidney invasion. Such groups of patients require more investigation.
Evaluation of the control population demonstrated no antigen in the serum of most patients with bacterial infections or noncandida mycoses, including aspergillosis and cryptococcosis, supporting the specificity of this antigen for candida species. Antibodies against the enolase of candida species are known to cross-react with the enolase of saccharomyces species, however.15 It is therefore possible that patients with saccharomyces infections19 express this antigenic activity. The only patient with enolase antigenemia associated with other mycoses had fungemia and pulmonary infection due to S. delbrueckii. Unpublished observations suggest that the enolase of all species of candida is recognized, with the possible exception of that of Torulopsis (C.) glabrata. There were weakly positive reactions in samples from 3 of the 140 normal blood-bank donors, possibly because of preexisting antibodies to the component reagents of the assay.
Ninety-four percent of the patients with superficial candidiasis were antigen-negative. Among the three patients with positive assays and superficial candidiasis, two had a single weakly positive reaction, possibly due to cross-reacting antibody. The third received empirical amphotericin B for possible clinically occult invasive candidiasis.
Antigenemia was present in several patients with tissue-proved candidiasis without fungemia. The finding of enolase antigenemia in the absence of fungemia during the course of deep visceral candidiasis is consistent with the results of earlier in vivo studies showing that mice infected intraperitoneally or intravenously with C. albicans had deep visceral infection and enolase antigenemia but minimal or no fungemia.17 These studies found a good correlation between the level of circulating antigen and the extent of deep tissue infection (as measured by colonyforming units per gram of tissue), suggesting that enolase antigenemia may be a reflection of deep visceral candidiasis. The mechanisms of release and regulation and the plasma kinetics of candida enolase are not known. If candida enolase is released in vivo during deep tissue infection, then enolase antigenemia may be an important marker of deep visceral infection in the absence of fungemia. In view of the sensitivity of 85 percent and the specificity of 96 percent for enolase antigenemia in tissue-proved deep visceral infection, a febrile patient with cancer who does not have fungemia but who has hepatic and splenic lesions consistent with hepatosplenic candidiasis on CT scanning might be spared a biopsy to confirm the diagnosis.
In contrast, 4 of the 11 patients with cancer and fungemia were antigen-negative. Moreover, data from ongoing studies in surgical patients without neutropenia indicate that "transient fungemia," as determined by the blinded investigator, was not associated with enolase antigenemia. These data would further indicate that enolase antigenemia may be more reflective of the tissue load of organisms in deep visceral candidiasis than of fungemia itself, and that detection of enolase antigenemia would complement rather than replace blood culture.
Several factors may have contributed to false negative determinations of invasive candidiasis in this study: low tissue concentrations of candida, whether because of antifungal therapy or recovery from granulocytopenia; antibody-mediated clearance of antigen; and infrequent sampling. Antibody-mediated clearance of candida enolase may be an important factor in false negative results of the assay. Patients who are not as profoundly immunocompromised as those in this study may be able to mount a sufficient level of neutralizing antibody. Strockbine et al.11 found that candida enolase antigen was the immunodominant cytoplasmic antigen of candida in relatively immunocompetent patients with invasive candidiasis, in whom high titers of antibody were detected by Western blot analysis. Indeed, as patients with granulocytopenia recover from the effects of cytotoxic chemotherapy, levels of circulating antigen may decline as a result of rising antibody titers as well as the clearance of candida from tissue by neutrophils.
The sensitivity of the current assay for the detection of candida enolase may be improved by several means. First, enolase antigen may be detectable in the urine of patients with invasive candidiasis. For example, granulocytopenic rabbits with disseminated candidiasis were found to have antigenuria due to candida enolase,17 and a 47-kd cytoplasmic antigen consistent with candida enolase was recently detected in the urine of patients with fungemia on immunoblotting, although this requires further investigation.20 The detection of antigenuria is noninvasive, may be advantageous in the presence of neutralizing antibody, and may complement the detection of enolase antigenemia. Second, the detection of rising titers of neutralizing antibody to candida enolase also may complement the usefulness of antigen detection, especially in less immunocompromised patients.
Blood cultures and the antigen-detection method appear to be complementary. As Table 5 demonstrates, approximately one half the cases were recognized by both methods, and the remaining half were recognized by one system or the other alone. The sensitivity of positive blood cultures in this study was relatively high as compared with that in earlier studies of fungemia and candidiasis, possibly because of the improved methods of detection in blood culture.21 22 23 When lysis centrifugation was compared with conventional broth systems, for example, fungemia was detected earlier and more frequently.21 , 22 When lysis centrifugation was used in combination with the Bactec radiometric method, more episodes of fungemia were detected than by either method alone.23
Although this study was not specifically designed to address whether a positive blood culture or enolase antigenemia was the first indicator of deep candidiasis, several cases demonstrate that enolase antigenemia was present before fungemia was detected. Patients 4, 5, 20, and 21, for example, had simultaneous sets of blood cultures and serum samples at the time of initial evaluation. Antigenemia was detected in Patient 20 one day before the detection of fungemia, and in Patients 4, 5, and 21 antigenemia was detected two days before fungemia. Antigenemia was detected in another patient (Patient 14) seven days before the detection of fungemia. In Patient 9, after five sets of negative blood cultures and tests for antigen, enolase antigenemia was detected eight days before fungemia. Thus, six patients were found to have antigenemia one to eight days before positive blood cultures.
When determining whether blood cultures or tissue examination revealed candida species before enolase antigenemia appeared, we identified one patient (Patient 16) in whom a biopsy demonstrated hepatic candidiasis at least six days before antigenemia was detected. Moreover, six patients had fungemia or tissue-proved invasive candidiasis with no detectable enolase antigenemia.
The timing of serum collection was particularly important. A single negative serum sample did not exclude a diagnosis of deep candidiasis. Had the study involved only a single serum sample drawn weekly, many patients who proved on almost daily sampling to have antigenemia would have been deemed antigen-negative. Collecting serum samples almost daily also coincides with the clinical realities in febrile patients with neutropenia, who are evaluated at least that often for sources of infection. The prospective nature of this study also ensured more reliable handling of the serum samples. Since repeated cycles of freezing and thawing are known to denature the enolase protein and diminish detectable antigenic activity, processing should involve at most a single freeze-thaw cycle. Given the apparent complementarity between the detection of fungemia and enolase antigenemia, sensitivity in the detection of candidiasis may be improved by drawing serum samples for antigen assay with each set of blood cultures. Specificity may perhaps be improved and cost contained by limiting the testing for enolase antigenemia to patients considered to be at high risk for invasive candidiasis.
We are indebted to Diana Schlosky, B.S., R.N., Janet Cress, B.S., R.N., and Dorie Marshall, M.S., R.N., for data collection; to David Cates, B.S., for assistance with data management; to John Soule, B.S., Vanessa Thomas, B.S., and Mark Miller, B.A., for laboratory technical assistance; to Seth Steinberg, Ph.D., for assistance in statistical analysis; to Lawrence R. Crane, M.D., and Marc Rubin, M.D., for helpful suggestions; and to John E. Bennett, M.D., for constructive review of the manuscript.
Source Information
From the Infectious Diseases Section, Pediatric Branch, National Cancer Institute, Bethesda, Md. (T.J.W., R.S., E.N., J. Lecciones, J. Lee, P.A.P.); Duke University Medical Center, Durham, N.C. (J.W.H.); Harper Hospital, Wayne State University Medical Center, Detroit (J.D.S., V.S., P.C.); the Johns Hopkins Medical Institutions, Baltimore (W.G.M.); Becton Dickinson Advanced Diagnostics, Baltimore (S.M.M.); the Department of Microbiology and Immunology, Temple University, Philadelphia (H.R.B.); the University of Iowa Hospitals and Clinics, Iowa City (M.A.P.); and the Department of Laboratory Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Md. (C.S.). Address reprint requests to Dr. Walsh at the Infectious Diseases Section, Pediatric Branch, National Cancer Institute, Bldg. 10, Rm. 13N–240, Bethesda, MD 20892.
Dr. Maret is employed by Becton Dickinson Microbiology Systems, and Dr. Buckley also has financial interests in the company.
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