DIAGN MICROBIOLINFECTDIS 1991;14:17-20
17
VIROLOGY
Comparison of the Kallested Pathfinder EIA, Cytocentrifuged Direct Fluorescent Antibody, and Cell Culture for the Detection of Chlamydia trachomatis William LeBar, Howard Schubiner, Claudia Jemal, Barry Herschman, Kay Criswell, Nancy Curtin, and Marcia Sherbeck
A total of 203 duplicate endocervical samples collected from patients at an adolescent health care center were tested for the presence of Chlamydia trachomatis by cell culture, Pathfinder enzyme immunoassay (EIA) (Kallestad) and cytocentri-
fuged direct fluorescent antibody (DFA). Compared to cell culture, the Pathfinder assay demonstrated a sensitivity and specificity of 85.2% and 100%, whereas the DFA procedure demonstrated to be 92.6% sensitive and 99.4% specific.
Enzyme immunoassays (EIA) and direct fluorescent antibody (DFA) staining for the detection of Chlamydia trachomatis have become widely accepted for routine use, in part, because they may overcome some of the technical difficulties associated with cell culture (Mahony et al., 1989; Mumatz et al., 1989; Thomas et al., 1989). These methods also may offer the advantage of speed, labor savings, and the ability to detect live organisms as well as soluble antigens. Their performance, however, may be affected by many of the same factors considered to be the shortcomings of cell culture procedures (Mahony et al., 1989). These factors include specimen collection, the type of collection device, transport and storage,
the presence of inhibitory substances, and detection methods of a particular antigen (Hipp et al., 1987; Mahony et al., 1989). In addition, the cutoff value chosen to separate negative from positive tests may have an effect on the sensitivity and specificity of both EIA and DFA when compared to cell culture (Freke et al., 1987; Pastorek et al., 1988). In this study, we compare the results of the Kallestad Pathfinder EIA Detection Kit (EIA) to cell culture and DFA staining of concentrated culture transport material.
MATERIALS A N D METHODS Patient Population
From the Departments of Pathology (W.L.B., C.J., B.H., K.C., N.C., N.C., M.S.), Providence Hospital, Southfield, Michigan; and the Department of Internal Medicine and Pediatrics (H.S.), Wayne State University, Detroit, Michigan. Address reprint requests to: Dr. W. LeBar, Department of Pathology, Providence Hospital, 16001West Nine Mile, Southfield, MI 48037. Received March 30, 1990; revised and accepted July 2, 1990 © 1991Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50
The study population consisted of adolescent and young adult females w h o presented to a primary care health center associated with Wayne State University's Department of Pediatrics and the Detroit Health Department between May and July 1989. A total of 203 consecutive patients between the ages of 12 and 25, w h o had pelvic exams, were included into the study.
18
Specimen Collection The exocervix was cleansed with a cotton swab to remove excess mucus and exudate. Cultures were obtained by insertion and rotation of a Dacron-tipped swab into the endocervical canal. All specimens were placed into 2-sucrose phosphate (2-SP) transport medium. Specimens tested by the Pathfinder were collected in the same manner with transport kits supplied by the manufacturer. For the first 100 patients, the culture swab was collected first followed by the Pathfinder. The order was reversed during the remainder of the study.
Cell Culture Specimens were transported to the laboratory where they were processed or were stored at 2°-4°C for no longer than 24 hr. The sample (200 ~1) was added to each of two 1-dram shell vials containing McCoy cells. The vials were centrifuged (3000 g) for 1 hr at 30°C, and the supernatant fluid was aspirated. Chlamydia culture medium containing cycloheximide (1 p,g/ml) was added and the cultures were incubated at 35°C under 5% CO2. After 48 hr of incubation, one vial was fixed with methanol and stained with a fluorescein-labeled monoclonal antibody conjugate (Kallestad Diagnostics, Chaska, MN), and the second vial was disrupted by scraping and vortexing and then passaged onto fresh McCoy cells. A positive cell culture was any specimen containing one or more fluorescent-stained inclusions in the primary or second passage culture.
W. LeBar et al.
conjugated anti-rabbit immunoglobulin for an additional 1 hr. The tubes were then washed and incubated with tetramethylbenzidine substrate solution for 15 min. After addition of a stopping solution, the tubes were evaluated spectrophotometrically at 450 nm. A positive result was greater than the absorbance of the negative control plus 0.100. No gray zone was used.
Cytocentrifuged DFA DFA slides were prepared by spinning 200 ~I of wellmixed culture transport fluid at 1100 rpm for 10 min in a Cytospin II centrifuge (Shandon, Pittsburgh, PA). After centrifugation, 0.5 ml of methanol was added to the slides and allowed to evaporate. Slides were stained with fluorescein-labeled monoclonal major outer membrane protein antibody (Kallested) for 15 min and then examined with an epifluorescence microscope. Slides were scored as positive if 5 or more elementary bodies were visualized. All smears were read prior to the availability of culture and EIA results.
Statistics Specimens were considered true positives if C. trachomatis was isolated in culture or, if the culture was negative, both DFA and EIA were positive. Sensitivities, specificities, and predictive values positive and negative (PVP, PVN) were determined by the method of Galen and Gambino (1975).
Pathfinder EIA
RESULTS
The Pathfinder Chlamydia EIA detection system is an enzyme immunoassay that utilizes a monoclonal antibody capture and polyclonal antibody detector system, which will detect all serovars of C. trachomatis as well as C. psittaci. The assay was performed according to the manufacturer's instructions. All samples were prepared for testing by addition of 1.0 ml of a NaOH-detergent solution to each tube containing a specimen swab, followed by incubation at room temperature for 10 min. Next the samples were vortexed, and then liquid was expressed from the swab by pressing and rotating it against the side of the tube; the swab was discarded. The sample was then neutralized with a buffered HC1 solution and vortexed again. Positive and negative controls were treated in a similar manner. The treated specimen or control (200 ~l) was then added to tubes coated with monoclonal antibody to chlamydia and incubated for 1 hr. Next the tubes were reacted with a polyclonal rabbit antibody to chlamydia for 1 hr, followed by reaction with horseradish-peroxidase-
A total of 203 female patients from an adolescent health care center had endocervical samples tested for the presence of C. trachomatis by culture, EIA, and cytocentrifuged DFA. The prevalence of infection in this patient population was 13.3% (27/203). Cell culture detected 26 of 27 of the positive specimens. One sample was positive only on blind passage. Compared to consensus results, the sensitivity, specificity, predictive values positive and negative for cell culture were 96.2%, 100%, 100%, and 99.4%, respectively. The Pathfinder EIA detected 23 of 27 positive specimens and there were no false-positive results. This EIA assay demonstrated 85.2% sensitivity, 100% specificity, 100% PPV, and 97.7% PVN. Cytocentrifuged DFA detected 25 of 27 positive specimens. The sensitivity, specificity, PVP and PVN compared to the consensus results were 92.5%, 99.4%, 96.1%, and 98.8%, respectively. These results are summarized in Table 1. In total, there were seven samples that gave dis-
Kallested Pathfinder Evaluation
TABLE 1
19
Comparison of Culture, DFA and EIA for Detection of Chlamydia trachomatis
Cell culture~ Pathfinder EIA Cytocentrifuged DFA
Sensitivity
Specificity
PVP
PVN
96.2% (26/27) 85.2% (23/27) 92.5% (25/27)
100% (176/176) 100% (176/176) 99.4% (175/176)
100% (26/26) 100% (23/23) 96.1% (25/26)
99.4% (176/177) 97.7% (176/180) 98.8% (175/177)
qncludes specimen negative by culture, but positive by both EIA and DFA.
crepant results. Data on these samples are summarized in Table 2. One sample (176) was negative by both EIA and DFA, and the culture on this sample was positive only after passage. When considering the remaining three false-negative EIAs (nos. 15, 42, 65), two had counts of five elementary bodies per slide, and the other, thirty per slide. Each of these three cultures were positive on primary inoculation. One sample (no. 64) contained four elementary bodies, but was negative by both culture and EIA. An additional sample (no. 128) was negative by DFA, but positive by both culture and EIA. There was also a sample negative by culture, but positive by both EIA and DFA (no. 85).
DISCUSSION This study compared the Pathfinder EIA and cytocentrifuged DFA to cell culture for the detection of C. trachomatis in endocervical specimens. When comparing chlamydia detection methods, it is assumed that the sensitivity of cell culture is
17
VIROLOGY
Comparison of the Kallested Pathfinder EIA, Cytocentrifuged Direct Fluorescent Antibody, and Cell Culture for the Detection of Chlamydia trachomatis William LeBar, Howard Schubiner, Claudia Jemal, Barry Herschman, Kay Criswell, Nancy Curtin, and Marcia Sherbeck
A total of 203 duplicate endocervical samples collected from patients at an adolescent health care center were tested for the presence of Chlamydia trachomatis by cell culture, Pathfinder enzyme immunoassay (EIA) (Kallestad) and cytocentri-
fuged direct fluorescent antibody (DFA). Compared to cell culture, the Pathfinder assay demonstrated a sensitivity and specificity of 85.2% and 100%, whereas the DFA procedure demonstrated to be 92.6% sensitive and 99.4% specific.
Enzyme immunoassays (EIA) and direct fluorescent antibody (DFA) staining for the detection of Chlamydia trachomatis have become widely accepted for routine use, in part, because they may overcome some of the technical difficulties associated with cell culture (Mahony et al., 1989; Mumatz et al., 1989; Thomas et al., 1989). These methods also may offer the advantage of speed, labor savings, and the ability to detect live organisms as well as soluble antigens. Their performance, however, may be affected by many of the same factors considered to be the shortcomings of cell culture procedures (Mahony et al., 1989). These factors include specimen collection, the type of collection device, transport and storage,
the presence of inhibitory substances, and detection methods of a particular antigen (Hipp et al., 1987; Mahony et al., 1989). In addition, the cutoff value chosen to separate negative from positive tests may have an effect on the sensitivity and specificity of both EIA and DFA when compared to cell culture (Freke et al., 1987; Pastorek et al., 1988). In this study, we compare the results of the Kallestad Pathfinder EIA Detection Kit (EIA) to cell culture and DFA staining of concentrated culture transport material.
MATERIALS A N D METHODS Patient Population
From the Departments of Pathology (W.L.B., C.J., B.H., K.C., N.C., N.C., M.S.), Providence Hospital, Southfield, Michigan; and the Department of Internal Medicine and Pediatrics (H.S.), Wayne State University, Detroit, Michigan. Address reprint requests to: Dr. W. LeBar, Department of Pathology, Providence Hospital, 16001West Nine Mile, Southfield, MI 48037. Received March 30, 1990; revised and accepted July 2, 1990 © 1991Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50
The study population consisted of adolescent and young adult females w h o presented to a primary care health center associated with Wayne State University's Department of Pediatrics and the Detroit Health Department between May and July 1989. A total of 203 consecutive patients between the ages of 12 and 25, w h o had pelvic exams, were included into the study.
18
Specimen Collection The exocervix was cleansed with a cotton swab to remove excess mucus and exudate. Cultures were obtained by insertion and rotation of a Dacron-tipped swab into the endocervical canal. All specimens were placed into 2-sucrose phosphate (2-SP) transport medium. Specimens tested by the Pathfinder were collected in the same manner with transport kits supplied by the manufacturer. For the first 100 patients, the culture swab was collected first followed by the Pathfinder. The order was reversed during the remainder of the study.
Cell Culture Specimens were transported to the laboratory where they were processed or were stored at 2°-4°C for no longer than 24 hr. The sample (200 ~1) was added to each of two 1-dram shell vials containing McCoy cells. The vials were centrifuged (3000 g) for 1 hr at 30°C, and the supernatant fluid was aspirated. Chlamydia culture medium containing cycloheximide (1 p,g/ml) was added and the cultures were incubated at 35°C under 5% CO2. After 48 hr of incubation, one vial was fixed with methanol and stained with a fluorescein-labeled monoclonal antibody conjugate (Kallestad Diagnostics, Chaska, MN), and the second vial was disrupted by scraping and vortexing and then passaged onto fresh McCoy cells. A positive cell culture was any specimen containing one or more fluorescent-stained inclusions in the primary or second passage culture.
W. LeBar et al.
conjugated anti-rabbit immunoglobulin for an additional 1 hr. The tubes were then washed and incubated with tetramethylbenzidine substrate solution for 15 min. After addition of a stopping solution, the tubes were evaluated spectrophotometrically at 450 nm. A positive result was greater than the absorbance of the negative control plus 0.100. No gray zone was used.
Cytocentrifuged DFA DFA slides were prepared by spinning 200 ~I of wellmixed culture transport fluid at 1100 rpm for 10 min in a Cytospin II centrifuge (Shandon, Pittsburgh, PA). After centrifugation, 0.5 ml of methanol was added to the slides and allowed to evaporate. Slides were stained with fluorescein-labeled monoclonal major outer membrane protein antibody (Kallested) for 15 min and then examined with an epifluorescence microscope. Slides were scored as positive if 5 or more elementary bodies were visualized. All smears were read prior to the availability of culture and EIA results.
Statistics Specimens were considered true positives if C. trachomatis was isolated in culture or, if the culture was negative, both DFA and EIA were positive. Sensitivities, specificities, and predictive values positive and negative (PVP, PVN) were determined by the method of Galen and Gambino (1975).
Pathfinder EIA
RESULTS
The Pathfinder Chlamydia EIA detection system is an enzyme immunoassay that utilizes a monoclonal antibody capture and polyclonal antibody detector system, which will detect all serovars of C. trachomatis as well as C. psittaci. The assay was performed according to the manufacturer's instructions. All samples were prepared for testing by addition of 1.0 ml of a NaOH-detergent solution to each tube containing a specimen swab, followed by incubation at room temperature for 10 min. Next the samples were vortexed, and then liquid was expressed from the swab by pressing and rotating it against the side of the tube; the swab was discarded. The sample was then neutralized with a buffered HC1 solution and vortexed again. Positive and negative controls were treated in a similar manner. The treated specimen or control (200 ~l) was then added to tubes coated with monoclonal antibody to chlamydia and incubated for 1 hr. Next the tubes were reacted with a polyclonal rabbit antibody to chlamydia for 1 hr, followed by reaction with horseradish-peroxidase-
A total of 203 female patients from an adolescent health care center had endocervical samples tested for the presence of C. trachomatis by culture, EIA, and cytocentrifuged DFA. The prevalence of infection in this patient population was 13.3% (27/203). Cell culture detected 26 of 27 of the positive specimens. One sample was positive only on blind passage. Compared to consensus results, the sensitivity, specificity, predictive values positive and negative for cell culture were 96.2%, 100%, 100%, and 99.4%, respectively. The Pathfinder EIA detected 23 of 27 positive specimens and there were no false-positive results. This EIA assay demonstrated 85.2% sensitivity, 100% specificity, 100% PPV, and 97.7% PVN. Cytocentrifuged DFA detected 25 of 27 positive specimens. The sensitivity, specificity, PVP and PVN compared to the consensus results were 92.5%, 99.4%, 96.1%, and 98.8%, respectively. These results are summarized in Table 1. In total, there were seven samples that gave dis-
Kallested Pathfinder Evaluation
TABLE 1
19
Comparison of Culture, DFA and EIA for Detection of Chlamydia trachomatis
Cell culture~ Pathfinder EIA Cytocentrifuged DFA
Sensitivity
Specificity
PVP
PVN
96.2% (26/27) 85.2% (23/27) 92.5% (25/27)
100% (176/176) 100% (176/176) 99.4% (175/176)
100% (26/26) 100% (23/23) 96.1% (25/26)
99.4% (176/177) 97.7% (176/180) 98.8% (175/177)
qncludes specimen negative by culture, but positive by both EIA and DFA.
crepant results. Data on these samples are summarized in Table 2. One sample (176) was negative by both EIA and DFA, and the culture on this sample was positive only after passage. When considering the remaining three false-negative EIAs (nos. 15, 42, 65), two had counts of five elementary bodies per slide, and the other, thirty per slide. Each of these three cultures were positive on primary inoculation. One sample (no. 64) contained four elementary bodies, but was negative by both culture and EIA. An additional sample (no. 128) was negative by DFA, but positive by both culture and EIA. There was also a sample negative by culture, but positive by both EIA and DFA (no. 85).
DISCUSSION This study compared the Pathfinder EIA and cytocentrifuged DFA to cell culture for the detection of C. trachomatis in endocervical specimens. When comparing chlamydia detection methods, it is assumed that the sensitivity of cell culture is
