Vol. 30, No. 11
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1992, p. 2938-2940
0095-1137/92/112938-03$02.00/0
Comparison of Two Culture Approaches, Blind Passage and Dual Observation, for Detecting Chlamydia trachomatis in Various Prevalence Populations SCOTT J. ZIMMERMAN, 1'2 ESTELLE MOSES,' NILOPHER SOFAT,"12 WILLIAM R. BARTHOLOMEW,12t AND DANIEL AMSTERDAM' 2* Division of Clinical Microbiology and Immunology, Erie County Medical Center,' and Department of Microbiology, State University of New York at Buffalo, 2 Buffalo, New York 14215 Received 10 February 1992/Accepted 6 August 1992
Chlamydia trachomatis diagnosis in our laboratory consisted of dual inoculation of shell vials and detection of inclusions by using fluorescein-consjugated monoclonal antiserum; the second culture vial was conventionally used for blind passage when the first vial was negative. We compared the increase in positivity using blind passage with that of a strategy utilizing observation of two stained monolayers (dual observation) without blind passage, in an effort to reduce the reporting time and labor associated with the conventional approach. A total of 4,329 specimens were obtained from an obstetrics and gynecology (OB-GYN) clinic (2,563 specimens) and the sexually transmitted disease clinic (1,766 specimens). These specimens were used to compare the two strategies. Blind passage of 1,269 initially culture-negative specimens from the OB-GYN clinic resulted in an additional 6 positive chlamydial diagnoses. In comparison, a similar number of specimens (1,294) from the OB-GYN clinic collected subsequently to the first group were tested by dual observation. There were five additional positive findings. A similar evaluation of specimens from the sexually transmitted disease clinic was performed. Blind passage of 313 initially culture-negative specimens yielded 3 additional positive diagnoses, whereas dual observation of 1,435 similar specimens resulted in 9 positive diagnoses. On the basis of analysis of 4,332 specimens, sensitivity of dual observation is comparable to that of blind passage; labor, cost, and reporting time of dual observation are reduced in comparison to those of blind passage. additional burden of performing blind passage increases the assay time and cost of materials, as well as delays the report. We chose to assess two strategies, blind passage and a modified procedure, dual observation, described in Materials and Methods, in our chlamydia culture methods. Different prevalence populations were used to determine the impact on sensitivity, cost, and reporting time.
According to the Centers for Disease Control, genital infections by Chlamydia trachomatis are the most frequent cause of bacterial sexually transmissible disease in the United States, with an estimated 4 million cases occurring annually (2, 3). Such infections can lead to serious sequelae when left untreated; hence, accurate laboratory diagnosis is essential to initiate antichlamydia chemotherapy and to decrease the morbidity associated with these infections. Cell culture isolation of C. trachomatis is the most sensitive and specific test for the diagnosis of chlamydia infection and can be used for all anatomical and specimen types (1). Unlike noncultural tests, it is not limited to specimens for which the test is approved. In cases of sexual assault, culture is the recommended test modality (3). Although many noncultural assays are available, cell culture, because of its sensitivity and specificity, is considered the "gold standard" for chlamydia diagnosis. Several cell lines have been applied for the successful isolation of chlamydiae; however, McCoy cells are most commonly used (5). Cells are grown as a monolayer either on a coverslip in shell vials or on the bottom of 96-well microdilution plates. Studies indicate that the shell vial technique offers greater sensitivity than does the microdilution plate procedure, especially for specimens with low inclusion counts (i.e., normal or asymptomatic populations) (5). To further enhance the sensitivity of culture, second or blind passage has been used to detect chlamydiae in specimens initially inoculated and considered negative. Culture is expensive and labor intensive, and the
*
MATERIALS AND METHODS
Specimens. During a 1-year period, 4,329 urogenital specimens were obtained for C. trachomatis diagnosis from an obstetrics and gynecology (OB-GYN) clinic (2,563 specimens) and the local sexually transmitted disease (STD) clinic (1,766 specimens). The results of culture of these specimens were used to determine prevalence for each patient population. Specimens obtained from the OB-GYN and STD clinics were used to compare two chlamydia culture strategies: blind passage and dual observation. For each population, two separate time spans were used to determine the impact of dual observation and blind passage. Chlamydia specimen transport and storage. Specimens were transported on ice within 24 h of collection in tubes containing sucrose phosphate medium, pH 7.0 to 7.2 (Ortho Diagnostics, Raritan, N.J.) and glass beads. Upon arrival in the laboratory, specimens were stored at 2 to 6°C for up to 24 h or at -70°C when stored for longer than 24 h but less than 72 h before processing. Conventional C. trachomatis culture (blind passage). Specimens were inoculated in duplicate within 72 h of collection. Antimicrobial agents (gentamicin [100 ,g/ml], vancomycin [100 ,ug/ml], and amphotericin B [5 ,ug/ml]) were added to the specimen tubes. Tubes were then vortexed and sonicated
Corresponding author.
t Present address: VA Medical Center, Kansas City, MO 64128;
and University of Kansas Medical Center, Kansas City, KS 66103.
2938
VOL. 30, 1992
TWO CULTURE METHODS FOR DETECTING C. TRACHOMATIS
prior to the addition of 0.25-ml aliquots to shell vials containing McCoy cell monolayers grown on coverslips. Inoculated cultures were centrifuged at 2,800 x g for 1 h and incubated for an additional hour at 35°C. Following aspiration of the supernatant, 1.0 ml of maintenance medium containing cycloheximide and 10% fetal calf serum was added to each shell vial. After 48 to 72 h of incubation at 35°C, cultures were fixed and stained with fluorescent monoclonal antibody directed against the lipopolysaccharide portion of the cell (Kallestad, Austin, Tex.) and examined at a magnification of x400 (x40 objective) for the presence of inclusion bodies with an epifluorescence microscope. A positive culture was defined as cells containing apple-green inclusions and was reported. Negative cultures were blind passaged by scraping cells from the second tube and inoculating a new shell vial; the culture method was then repeated. Results were reported on the basis of the finding for the passaged shell vial. Modified C. trachomatis culture (dual observation). Specimens were tested in duplicate in a manner identical to the conventional (blind passage) culture method. Instead of passing cells from the second negative vial, the vial was fixed and stained with fluorescent monoclonal antibody (as above). Specimens that were first-vial-negative were not reported until the second vial was observed. Material costs for culture of chlamydiae by using McCoy cells in shell vials. The materials (costs) per specimen used in this study, excluding labor costs, were swab ($0.08), transport tube ($2.00), two McCoy cell shell vials ($2.20), immunofluorescent stain for two slides ($1.75), and growth media for two vials ($0.16). On the basis of duplicate inoculations, the cost for materials totaled $6.19 per specimen. Statistical analysis. A statistical analysis of the populations was applied by using 95% confidence intervals calculated to determine whether the sample size was adequate for accurate interpretation of the results (12). Additionally, evaluations of the two culture modalities were made by using Student's t test. RESULTS
During the study period, we tested 4,329 specimens for C. trachomatis, using cell culture, and detected 539 positive results. The overall prevalence of infection in these specimens was 12.5%. The annual prevalence of C. trachomatis infection for each site during the study period was 17.7% for the STD clinic and 6.5% for the OB-GYN clinic. Table 1 gives the number of positive specimens identified by blind passage and by dual observation in the STD clinic and OB-GYN populations. Of 313 specimens from the STD clinic processed by using conventional culture (including blind passage), 3 (3.3% [3/901) additional positive results for C. trachomatis were detected as a result of the second passage. In comparison, 1,453 specimens from the same clinic were tested by using dual observation, yielding an additional 9 (3.3% [9/2701) positive specimens. There was no statistical difference between the numbers of additional positive specimens detected by blind passage and dual observation in the STD populations. Similarly, 1,269 specimens submitted from the OB-GYN clinic yielded an additional 6 (6.7% [6/90]) positive results when blind passage was utilized, whereas 1,294 specimens tested from the same clinic produced an additional 5 (5.6% [5/89]) specimens positive for C. trachomatis by using dual observation. As for the STD population, no significant difference (P = 0.756) in
2939
TABLE 1. Numbers of positive specimens identified by blind passage (BP) or dual observation (DO) for two prevalence populations No. tested
No. positive
Prevalence"
STD clinic
313 1,453
87 261
27.8 18.0
1,269
84
1,294 Cumulativeg BP 1,582
OB-GYN clinic
DO
2,747
No. (%) of additional positive byb: BP DO
%
Source
(3.3%)d
3 (3.3%)C NT
6.6
NT
6 (6.7%)e
84
6.5
5 (5.6%)
NT
171
10.8
NT
9 (5.0%)
345
12.6
14 (3.9%)
NT
NT 9
a Calculated by using the additive result of DO or BP. b NT, not tested. c Confidence limits, 0.4 to 7.0%. d Confidence limits, 1.2 to 5.4%. ' Confidence limits, 1.5 to 11.7%; P = 0.756 between groups denoted by footnotes d and e. f Confidence limits, 1.2 to 10.3%; P = 0.756 between groups denoted by footnotes d and e. g Cumulative results for STD plus OB-GYN specimens tested by blind passage and dual observation; P = 0.551.
results between blind passage and dual observation was detected. We analyzed the material costs and time savings associated with dual observation and blind passage (Table 2). On the basis of material costs described above, an overall prevalence of C. trachomatis infection of 10.0% at this institution, and an annual specimen load of 8,473, we calculated annual cost savings to range from $2,159.82 to $2,641.86 for high-prevalence populations and from $4,856.67 to $5,174.91 for low-prevalence populations. Likewise, annual "hands-on" time savings ranged from 83.1 to 101.6 h for high-prevalence populations and 187.0 to 199.2 h for low-prevalence populations. Overall, for a specimen load of 8,473, the annual cumulative material cost savings for all specimens would range from $8,925.93 to $9,913.41 and the annual cumulative hands-on time savings for all specimens would range from 343.5 to 381.4 h. The populations studied were statistically evaluated and determined to be an adequate size for accurate data interpretation, on the basis of 95% confidence limits. Additionally, there was no statistical difference (P = 0.551) between TABLE 2. Annual material cost ($) and hands-on savings (h) of dual observation versus blind passage based upon Erie County Medical Center chlamydia data (8,473 specimens) and a
10.0% prevalence
Parameter
Positive culture'
Material costs ($)
0.00-987.48
Hands-on savings (h)
0.00-37.90
a b
Savings Negative culture
Cumulativeb
8,925.93 8,925.93-9,913.41 343.50
No savings would be realized for first-vial-positive Total savings for positive and negative results.
343.50-381.40
specimens.
2940
ZIMMERMAN ET AL.
the numbers of additional positive specimens detected by using blind passage and by dual observation. DISCUSSION The presumptive clinical diagnosis of genital infection due to C. trachomatis can be difficult and requires the support of laboratory evidence, especially for women and asymptomatic patients. Accurate diagnosis and treatment are crucial in these groups to reduce the risk of complications associated with infection. Cell culture is the gold standard for chlamydia diagnosis but has had limited acceptance because of technical difficulties, time requirements, and cost. While the effort to improve chlamydia detection has focused on alternative methods to culture, certain clinical, epidemiologic, and legal scenarios mandate the use of culture (3, 4, 11). Culture is recognized as prima facie evidence for legal cases of rape and sexual abuse; most nonculture techniques have not been approved for use with specimens collected from nonurogenital sites. Recent epidemiologic evidence has indicated an increase in the prevalence of sexually transmitted agents in the United States, including C trachomatis (2, 11), which has increased the occurrence of pelvic inflammatory disease (a sequelae of chlamydia infection) (11) and validated the association of C. trachomatis infections and maternal deaths due to complications from ectopic pregnancy (4). We believe that culture is the best choice for the identification of chlamydia infections and chose to investigate modifications to improve our culture technique by reducing labor, time, and cost. Reports in the literature regarding many of the premises of culture techniques are conflicting. The McCoy cell is the most commonly used cell line for chlamydial isolation procedures (5). Schachter (8) and Stamm et al. (10) have reported that the fluorescent-antibody technique of staining inclusions is the most sensitive. However, some investigators (6, 7) have demonstrated that blind or multiple passaging increases the recovery of organisms from known-positive specimens. Schachter and Martin (9) have indicated a failure of multiple passages to increase chlamydial recovery. It has been our experience that blind passage increases the yield of chlamydial recovery (Table 1). We chose to evaluate two culture techniques, blind passage and dual observation, both of which are capable of increasing the yield of identifiable organisms over that of routine culture. Our results indicate that the two techniques are comparable in the additional number of positive specimens that each yields. However, it is impossible to determine with the protocol used whether the increased yield of positive specimens from blind passage or dual observation is due to random distribution of infectious particles or to increased sensitivity of one format over the other. Further analysis reveals that dual observation can reduce both material cost and time associated with culture. During this investigation, four study periods were used to evaluate the effect of blind passage and dual observation on the detectability of C. trachomatis. The prevalence of infec-
J. CLIN. MICROBIOL.
tion in the two STD populations varied form 18.0 to 27.8%, whereas the two OB-GYN populations demonstrated a difference in prevalence of 4. Additionally, the individuals tested within each group were independent of one another. This study demonstrates that blind passage and dual observation cumulatively increased the detection of C. trachomatis-positive specimens by 5.0 and 3.9%, respectively, compared with the use of a single shell vial culture, and that, comparatively, dual observation did not diminish the capability to detect C. trachomatis compared with blind passage (P = 0.551). Dual observation also has the capacity to decrease hands-on time, reporting time, and the cost per specimen for both positive and negative results in various prevalence populations. The results of this study indicate that the sensitivity of dual observation is comparable to that of blind passage and that this culture approach should be considered because of the potential savings in material costs, labor, and reporting time. REFERENCES 1. Centers for Disease Control. 1991. False-positive results with the use of chlamydia tests in the evaluation of suspected sexual
abuse-Ohio, 1990. Morbid. Mortal. Weekly Rep. 39:932-935. 2. Centers for Disease Control. 1985. Chlamydia trachomatis infections: policy guidelines for prevention and control. Morbid. Mortal. Weekly Rep. 34(Suppl. 35):S53-S74. 3. Centers for Disease Control. 1989. Sexual assault and STD. Morbid. Mortal. Weekly Rep. 38:40-43. 4. Centers for Disease Control. 1990. Ectopic pregnancy-United States, 1987. Morbid. Mortal. Weekly Rep. 39:401-404. 5. Clyde, W. A., Jr., G. E. Kenny, and J. Schachter. 1984. Cumitech 19: laboratory diagnosis of chlamydial and mycoplasmal infections. Coordinating ed., W. L. Drew. American Society for Microbiology, Washington, D.C. 6. Jones, R. B., B. P. Katz, B. Van Der Pol, V. A. Caine, B. E. Batteiger, and W. J. Newhall V. 1986. Effect of blind passage and multiple samplings on recovery of Chlamydia trachomatis from urogenital specimens. J. Clin. Microbiol. 24:1029-1033. 7. Jones, R. B., B. Van Der Pol, and B. P. Katz. 1989. Effect of differences in specimen processing and passage technique on recovery of Chlamydia trachomatis. J. Clin. Microbiol. 27:894898. 8. Schachter, J. 1979. Diagnosis of chlamydial infection. Curr. Ther. Res. 26(Suppl. 6S):675-686. 9. Schachter, J., and D. H. Martin. 1987. Failure of multiple passages to increase chlamydial recovery. J. Clin. Microbiol. 25:1851-1853. 10. Stamm, W. E., M. Tam, M. Koester, and L. Cles. 1983.
Detection of Chlamydia trachomatis inclusions in McCoy cell
cultures with fluorescein-conjugated monoclonal antibodies. J. Clin. Microbiol. 17:666-668. 11. Thompson, S. E., and A. E. Washington. 1983. Epidemiology of sexually transmitted Chlamydia trachomatis infections. Epidemiol. Rev. 5:96-123. 12. Washington, J. A. 1990. Confidence intervals: an important component of data presentation. Clin. Microbiol. Newsl. 12: 109-110.
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1992, p. 2938-2940
0095-1137/92/112938-03$02.00/0
Comparison of Two Culture Approaches, Blind Passage and Dual Observation, for Detecting Chlamydia trachomatis in Various Prevalence Populations SCOTT J. ZIMMERMAN, 1'2 ESTELLE MOSES,' NILOPHER SOFAT,"12 WILLIAM R. BARTHOLOMEW,12t AND DANIEL AMSTERDAM' 2* Division of Clinical Microbiology and Immunology, Erie County Medical Center,' and Department of Microbiology, State University of New York at Buffalo, 2 Buffalo, New York 14215 Received 10 February 1992/Accepted 6 August 1992
Chlamydia trachomatis diagnosis in our laboratory consisted of dual inoculation of shell vials and detection of inclusions by using fluorescein-consjugated monoclonal antiserum; the second culture vial was conventionally used for blind passage when the first vial was negative. We compared the increase in positivity using blind passage with that of a strategy utilizing observation of two stained monolayers (dual observation) without blind passage, in an effort to reduce the reporting time and labor associated with the conventional approach. A total of 4,329 specimens were obtained from an obstetrics and gynecology (OB-GYN) clinic (2,563 specimens) and the sexually transmitted disease clinic (1,766 specimens). These specimens were used to compare the two strategies. Blind passage of 1,269 initially culture-negative specimens from the OB-GYN clinic resulted in an additional 6 positive chlamydial diagnoses. In comparison, a similar number of specimens (1,294) from the OB-GYN clinic collected subsequently to the first group were tested by dual observation. There were five additional positive findings. A similar evaluation of specimens from the sexually transmitted disease clinic was performed. Blind passage of 313 initially culture-negative specimens yielded 3 additional positive diagnoses, whereas dual observation of 1,435 similar specimens resulted in 9 positive diagnoses. On the basis of analysis of 4,332 specimens, sensitivity of dual observation is comparable to that of blind passage; labor, cost, and reporting time of dual observation are reduced in comparison to those of blind passage. additional burden of performing blind passage increases the assay time and cost of materials, as well as delays the report. We chose to assess two strategies, blind passage and a modified procedure, dual observation, described in Materials and Methods, in our chlamydia culture methods. Different prevalence populations were used to determine the impact on sensitivity, cost, and reporting time.
According to the Centers for Disease Control, genital infections by Chlamydia trachomatis are the most frequent cause of bacterial sexually transmissible disease in the United States, with an estimated 4 million cases occurring annually (2, 3). Such infections can lead to serious sequelae when left untreated; hence, accurate laboratory diagnosis is essential to initiate antichlamydia chemotherapy and to decrease the morbidity associated with these infections. Cell culture isolation of C. trachomatis is the most sensitive and specific test for the diagnosis of chlamydia infection and can be used for all anatomical and specimen types (1). Unlike noncultural tests, it is not limited to specimens for which the test is approved. In cases of sexual assault, culture is the recommended test modality (3). Although many noncultural assays are available, cell culture, because of its sensitivity and specificity, is considered the "gold standard" for chlamydia diagnosis. Several cell lines have been applied for the successful isolation of chlamydiae; however, McCoy cells are most commonly used (5). Cells are grown as a monolayer either on a coverslip in shell vials or on the bottom of 96-well microdilution plates. Studies indicate that the shell vial technique offers greater sensitivity than does the microdilution plate procedure, especially for specimens with low inclusion counts (i.e., normal or asymptomatic populations) (5). To further enhance the sensitivity of culture, second or blind passage has been used to detect chlamydiae in specimens initially inoculated and considered negative. Culture is expensive and labor intensive, and the
*
MATERIALS AND METHODS
Specimens. During a 1-year period, 4,329 urogenital specimens were obtained for C. trachomatis diagnosis from an obstetrics and gynecology (OB-GYN) clinic (2,563 specimens) and the local sexually transmitted disease (STD) clinic (1,766 specimens). The results of culture of these specimens were used to determine prevalence for each patient population. Specimens obtained from the OB-GYN and STD clinics were used to compare two chlamydia culture strategies: blind passage and dual observation. For each population, two separate time spans were used to determine the impact of dual observation and blind passage. Chlamydia specimen transport and storage. Specimens were transported on ice within 24 h of collection in tubes containing sucrose phosphate medium, pH 7.0 to 7.2 (Ortho Diagnostics, Raritan, N.J.) and glass beads. Upon arrival in the laboratory, specimens were stored at 2 to 6°C for up to 24 h or at -70°C when stored for longer than 24 h but less than 72 h before processing. Conventional C. trachomatis culture (blind passage). Specimens were inoculated in duplicate within 72 h of collection. Antimicrobial agents (gentamicin [100 ,g/ml], vancomycin [100 ,ug/ml], and amphotericin B [5 ,ug/ml]) were added to the specimen tubes. Tubes were then vortexed and sonicated
Corresponding author.
t Present address: VA Medical Center, Kansas City, MO 64128;
and University of Kansas Medical Center, Kansas City, KS 66103.
2938
VOL. 30, 1992
TWO CULTURE METHODS FOR DETECTING C. TRACHOMATIS
prior to the addition of 0.25-ml aliquots to shell vials containing McCoy cell monolayers grown on coverslips. Inoculated cultures were centrifuged at 2,800 x g for 1 h and incubated for an additional hour at 35°C. Following aspiration of the supernatant, 1.0 ml of maintenance medium containing cycloheximide and 10% fetal calf serum was added to each shell vial. After 48 to 72 h of incubation at 35°C, cultures were fixed and stained with fluorescent monoclonal antibody directed against the lipopolysaccharide portion of the cell (Kallestad, Austin, Tex.) and examined at a magnification of x400 (x40 objective) for the presence of inclusion bodies with an epifluorescence microscope. A positive culture was defined as cells containing apple-green inclusions and was reported. Negative cultures were blind passaged by scraping cells from the second tube and inoculating a new shell vial; the culture method was then repeated. Results were reported on the basis of the finding for the passaged shell vial. Modified C. trachomatis culture (dual observation). Specimens were tested in duplicate in a manner identical to the conventional (blind passage) culture method. Instead of passing cells from the second negative vial, the vial was fixed and stained with fluorescent monoclonal antibody (as above). Specimens that were first-vial-negative were not reported until the second vial was observed. Material costs for culture of chlamydiae by using McCoy cells in shell vials. The materials (costs) per specimen used in this study, excluding labor costs, were swab ($0.08), transport tube ($2.00), two McCoy cell shell vials ($2.20), immunofluorescent stain for two slides ($1.75), and growth media for two vials ($0.16). On the basis of duplicate inoculations, the cost for materials totaled $6.19 per specimen. Statistical analysis. A statistical analysis of the populations was applied by using 95% confidence intervals calculated to determine whether the sample size was adequate for accurate interpretation of the results (12). Additionally, evaluations of the two culture modalities were made by using Student's t test. RESULTS
During the study period, we tested 4,329 specimens for C. trachomatis, using cell culture, and detected 539 positive results. The overall prevalence of infection in these specimens was 12.5%. The annual prevalence of C. trachomatis infection for each site during the study period was 17.7% for the STD clinic and 6.5% for the OB-GYN clinic. Table 1 gives the number of positive specimens identified by blind passage and by dual observation in the STD clinic and OB-GYN populations. Of 313 specimens from the STD clinic processed by using conventional culture (including blind passage), 3 (3.3% [3/901) additional positive results for C. trachomatis were detected as a result of the second passage. In comparison, 1,453 specimens from the same clinic were tested by using dual observation, yielding an additional 9 (3.3% [9/2701) positive specimens. There was no statistical difference between the numbers of additional positive specimens detected by blind passage and dual observation in the STD populations. Similarly, 1,269 specimens submitted from the OB-GYN clinic yielded an additional 6 (6.7% [6/90]) positive results when blind passage was utilized, whereas 1,294 specimens tested from the same clinic produced an additional 5 (5.6% [5/89]) specimens positive for C. trachomatis by using dual observation. As for the STD population, no significant difference (P = 0.756) in
2939
TABLE 1. Numbers of positive specimens identified by blind passage (BP) or dual observation (DO) for two prevalence populations No. tested
No. positive
Prevalence"
STD clinic
313 1,453
87 261
27.8 18.0
1,269
84
1,294 Cumulativeg BP 1,582
OB-GYN clinic
DO
2,747
No. (%) of additional positive byb: BP DO
%
Source
(3.3%)d
3 (3.3%)C NT
6.6
NT
6 (6.7%)e
84
6.5
5 (5.6%)
NT
171
10.8
NT
9 (5.0%)
345
12.6
14 (3.9%)
NT
NT 9
a Calculated by using the additive result of DO or BP. b NT, not tested. c Confidence limits, 0.4 to 7.0%. d Confidence limits, 1.2 to 5.4%. ' Confidence limits, 1.5 to 11.7%; P = 0.756 between groups denoted by footnotes d and e. f Confidence limits, 1.2 to 10.3%; P = 0.756 between groups denoted by footnotes d and e. g Cumulative results for STD plus OB-GYN specimens tested by blind passage and dual observation; P = 0.551.
results between blind passage and dual observation was detected. We analyzed the material costs and time savings associated with dual observation and blind passage (Table 2). On the basis of material costs described above, an overall prevalence of C. trachomatis infection of 10.0% at this institution, and an annual specimen load of 8,473, we calculated annual cost savings to range from $2,159.82 to $2,641.86 for high-prevalence populations and from $4,856.67 to $5,174.91 for low-prevalence populations. Likewise, annual "hands-on" time savings ranged from 83.1 to 101.6 h for high-prevalence populations and 187.0 to 199.2 h for low-prevalence populations. Overall, for a specimen load of 8,473, the annual cumulative material cost savings for all specimens would range from $8,925.93 to $9,913.41 and the annual cumulative hands-on time savings for all specimens would range from 343.5 to 381.4 h. The populations studied were statistically evaluated and determined to be an adequate size for accurate data interpretation, on the basis of 95% confidence limits. Additionally, there was no statistical difference (P = 0.551) between TABLE 2. Annual material cost ($) and hands-on savings (h) of dual observation versus blind passage based upon Erie County Medical Center chlamydia data (8,473 specimens) and a
10.0% prevalence
Parameter
Positive culture'
Material costs ($)
0.00-987.48
Hands-on savings (h)
0.00-37.90
a b
Savings Negative culture
Cumulativeb
8,925.93 8,925.93-9,913.41 343.50
No savings would be realized for first-vial-positive Total savings for positive and negative results.
343.50-381.40
specimens.
2940
ZIMMERMAN ET AL.
the numbers of additional positive specimens detected by using blind passage and by dual observation. DISCUSSION The presumptive clinical diagnosis of genital infection due to C. trachomatis can be difficult and requires the support of laboratory evidence, especially for women and asymptomatic patients. Accurate diagnosis and treatment are crucial in these groups to reduce the risk of complications associated with infection. Cell culture is the gold standard for chlamydia diagnosis but has had limited acceptance because of technical difficulties, time requirements, and cost. While the effort to improve chlamydia detection has focused on alternative methods to culture, certain clinical, epidemiologic, and legal scenarios mandate the use of culture (3, 4, 11). Culture is recognized as prima facie evidence for legal cases of rape and sexual abuse; most nonculture techniques have not been approved for use with specimens collected from nonurogenital sites. Recent epidemiologic evidence has indicated an increase in the prevalence of sexually transmitted agents in the United States, including C trachomatis (2, 11), which has increased the occurrence of pelvic inflammatory disease (a sequelae of chlamydia infection) (11) and validated the association of C. trachomatis infections and maternal deaths due to complications from ectopic pregnancy (4). We believe that culture is the best choice for the identification of chlamydia infections and chose to investigate modifications to improve our culture technique by reducing labor, time, and cost. Reports in the literature regarding many of the premises of culture techniques are conflicting. The McCoy cell is the most commonly used cell line for chlamydial isolation procedures (5). Schachter (8) and Stamm et al. (10) have reported that the fluorescent-antibody technique of staining inclusions is the most sensitive. However, some investigators (6, 7) have demonstrated that blind or multiple passaging increases the recovery of organisms from known-positive specimens. Schachter and Martin (9) have indicated a failure of multiple passages to increase chlamydial recovery. It has been our experience that blind passage increases the yield of chlamydial recovery (Table 1). We chose to evaluate two culture techniques, blind passage and dual observation, both of which are capable of increasing the yield of identifiable organisms over that of routine culture. Our results indicate that the two techniques are comparable in the additional number of positive specimens that each yields. However, it is impossible to determine with the protocol used whether the increased yield of positive specimens from blind passage or dual observation is due to random distribution of infectious particles or to increased sensitivity of one format over the other. Further analysis reveals that dual observation can reduce both material cost and time associated with culture. During this investigation, four study periods were used to evaluate the effect of blind passage and dual observation on the detectability of C. trachomatis. The prevalence of infec-
J. CLIN. MICROBIOL.
tion in the two STD populations varied form 18.0 to 27.8%, whereas the two OB-GYN populations demonstrated a difference in prevalence of 4. Additionally, the individuals tested within each group were independent of one another. This study demonstrates that blind passage and dual observation cumulatively increased the detection of C. trachomatis-positive specimens by 5.0 and 3.9%, respectively, compared with the use of a single shell vial culture, and that, comparatively, dual observation did not diminish the capability to detect C. trachomatis compared with blind passage (P = 0.551). Dual observation also has the capacity to decrease hands-on time, reporting time, and the cost per specimen for both positive and negative results in various prevalence populations. The results of this study indicate that the sensitivity of dual observation is comparable to that of blind passage and that this culture approach should be considered because of the potential savings in material costs, labor, and reporting time. REFERENCES 1. Centers for Disease Control. 1991. False-positive results with the use of chlamydia tests in the evaluation of suspected sexual
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