Evaluation of a New Selective Enrichment Broth for
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微生物临床杂志 2005年第2期
Departments of Pathology and Laboratory Medicine, Memorial Hospital of Rhode Island, Pawtucket
Brown Medical School
Women and Infants Hospital of Rhode Island, Providence, Rhode Island
ABSTRACT
Studies at two Brown Medical School-affiliated hospitals were undertaken to evaluate a new selective broth medium (GBS broth) and to compare it to the LIM broth currently used to culture for group B streptococci. Beta-hemolytic group B streptococci produce a carotenoid pigment that turns GBS broth an orange color. From a total of 580 pregnant women, duplicate vaginal-rectal swabs were collected at 35 to 37 weeks of gestation and cultured for group B streptococci, using either LIM broth (a selective broth containing antibiotics) or GBS broth for enrichment. Specimens were either transported to the laboratory or immediately placed in the respective enrichment broths and delivered to the laboratory. GBS broth medium had sensitivity, specificity, and positive and negative predictive values of 87.8, 100, 100, and 95.1% when planted in the laboratory and 90.3, 100, 100 and 97.6%, respectively, when inoculated at bedside. Use of GBS broth would satisfy Centers for Disease Control and Prevention requirements and would provide faster, more-sensitive, and cost-effective detection of group B streptococci in pregnant women.
TEXT
Group B streptococci (GBS) cause serious, life-threatening infections in the newborn. Mortality of GBS sepsis in neonates may exceed 50%, and preterm infants are especially at risk. Intrapartum prophylaxis for pregnant women colonized with GBS has been recommended for several years, since clinical trials showed that the administration of antibiotics to women in labor had a drastic effect in reducing early-onset invasive GBS infection in the neonate (1). Centers for Disease Control and Prevention guidelines issued in 2002 (2, 3) recommend that all pregnant women be screened for vaginal-rectal colonization with GBS at 35 to 37 weeks of gestation. These new guidelines for predelivery testing of pregnant women may have a significant impact on clinical microbiology laboratories, which may expect to see an increase in the volume of test requests for GBS screening for pregnant women. The recommended method for detecting carriage of GBS in pregnant women is to use a selective enrichment broth medium with subsequent subculture on a sheep blood agar plate (BAP) (3). Some laboratories inoculate a BAP before placing the swab in the enrichment broth. The enrichment broth is subcultured only if the primary BAP is negative. A more rapid, less labor-intensive, and more cost-effective method than the traditional culture is needed to screen patients for GBS colonization. Granada medium has long been used in Europe to detect GBS in pregnant women. Strains of beta-hemolytic GBS produce a carotenoid pigment, visible as an orange color, in this medium, usually within 24 h. Variable results on the sensitivity of this medium have been reported, with some investigators considering it to be unacceptably low (4, 5). A new, improved enrichment broth, modified from the original Granada medium, called GBS broth, is now also available (7). This medium has the advantage of reducing both incubation and technical time. Our studies were undertaken to evaluate these media.
(This material was presented in part at the 104th General Meeting of the American Society for Microbiology, New Orleans, La., 23 to 27 May 2004.)
From October 2002 to May 2004, duplicate swabs were used to collect specimens from the lower vagina and rectum of 250 pregnant women at 35 to 37 weeks of gestation who were seen in the Family Medicine Department of Memorial Hospital of Rhode Island (study 1). Swabs were sent to the laboratory. One swab was used to inoculate a BAP (Becton Dickinson Co., Cockeysville, Md.) and then placed in a tube of LIM enrichment broth (Becton Dickinson Co.), which is a selective broth containing antibiotics. The second swab was used to inoculate a BAP and placed in a tube of GBS broth (Northeast Laboratory Services, Waterville, Maine).
During the time period of July 2002 to Oct 2002, duplicate vaginal-rectal swabs from 330 pregnant women at Women and Infants Hospital of Rhode Island (study 2) were collected as described above but placed directly in medium (LIM or GBS broth) at the bedside. Tubes were transported to the laboratory. All media were incubated in CO2 at 35°C for 24 h. Primary plates were examined for the presence of GBS colonies. The GBS broths in both institutions were examined for the presence of any orange pigment. Each GBS and LIM broth was then subcultured to a BAP. Plates were inspected for the presence of colonies suggestive of GBS after 24 h of incubation at 35°C. Streptoccocus agalactiae (GBS) was identified by the CAMP test or by latex agglutination with streptococcal group B antisera.
Study 1. Of the 250 pregnant women studied, 30% (75 of 250) were found to be colonized by GBS. LIM broth and GBS broth were negative for 175 patients. All colonized women were identified by growth of GBS on the primary BAP or on subculture from LIM or GBS broth to a BAP. GBS was detected in 66 of the GBS broths; orange pigment was visible in 53 (80%) of these tubes. The remaining 13 tubes showed no pigmentation, but GBS was grown upon subculture on a blood agar plate. Nine GBS broths from the GBS-positive cultures were negative for GBS. Since all pigmented GBS broths grew GBS upon subculture, no false-positive GBS broths were detected. Sensitivity, specificity, positive predictive value, and negative predictive value for GBS broth were 87.8, 100, 100, and 95.1%, respectively (Table 1).
Study 2. Of the 330 pregnant women studied, 25.2% (83 of 330) were found to be colonized by GBS. LIM broth and GBS broth gave negative results for 247 patients. All colonized women were identified by subculture from LIM or GBS broth to a BAP. GBS was detected in 77 of the GBS broths; orange pigment was visible in 55 (71.4%) of these tubes. The remaining 22 tubes showed no pigmentation, but GBS was grown upon subculture on a blood agar plate. Six GBS broths from the GBS-positive cultures were negative for GBS. As in study 1, no false-positive GBS broths were detected. Sensitivity, specificity, positive predictive value, and negative predictive value for GBS broth were 90.3, 100, 100, and 97.6%, respectively (Table 2). No false-positive GBS broths were detected in our studies. In study 1, the nine false-negative GBS broth cultures were found in lightly colonized women. In these cases, no primary BAP was positive for GBS, which was isolated only from subcultures of LIM broth. GBS broth is more apt to be pigmented and shows the greatest sensitivity when incubated for a full 24 h.
Since GBS broth may be used as a transport medium, it can be inoculated at the bedside. Direct inoculation of specimens immediately into transport medium has been reported to significantly increase the yield of positive GBS culture results (6) and would be especially useful for lightly colonized women. Our studies support this idea, with a slightly higher sensitivity and negative predictive value (90.3 and 97.6% versus 87.8 and 95.1%) when bedside testing was employed. LIM broth, which had 21 false negatives, does not appear to be as useful in serving as a transport medium. Direct bedside inoculation would save labor on the part of the laboratory staff. Another possible reason for increased sensitivity in study 2 might be the fact that in study 1, each swab was rubbed onto an agar plate before being placed in the broth. As suggested in earlier reports (7), for nonhemolytic isolates of GBS, methods that do not rely on hemolysis or pigment production must be used for detection. Subculture of a nonpigmented GBS broth on a BAP would accomplish this task.
In conclusion, GBS broth provides a means for transport, enrichment, and visual recognition of the presence of GBS. Since positive results (orange pigmentation) may be seen with overnight incubation and our study showed a specificity of 100%, there is no need to subculture positive broth cultures, decreasing turnaround time by 24 h and eliminating further workup. Considering the time saved in labor and media, use of this medium could result in considerable cost savings and decrease overall turnaround time.
ACKNOWLEDGMENTS
We thank the microbiology laboratory staff at Memorial Hospital of Rhode Island and Women and Infants Hospital of Rhode Island for their cooperation and assistance in this study.
REFERENCES
American Academy of Pediatrics Committee on Infectious Diseases/Committee on Fetus and Newborn. 1997. Revised guidelines for prevention of early-onset group B streptococcal (GBS) infection by chemoprophylaxis. Pediatrics 99:489-496.
Baron, E. J. 2003. Laboratory support for prevention of perinatal group B streptococcal disease: commentary on the new guidelines on screening for group B streptococci during pregnancy. Clin. Microbiol. Newslett. 25:65-68.
Centers for Disease Control and Prevention. 2002. Prevention of perinatal group B streptococcal disease. Morb. Mortal. Wkly. Rep. 51:1-22.
Gil, E. G., M. C. Rodriguez, R. Bartolome, B. Berjano, L. Cabero, and A. Andreu. 1999. Evaluation of the Granada agar plate for detection of vaginal and rectal group B streptococci in pregnant women. J. Clin. Microbiol. 37:2648-2651.
Overman, S. B., D. D. Eley, B. E. Jacobs, and J. A. Ribes. 2002. Evaluation of methods to increase the sensitivity and timeliness of detection of Streptococcus agalactiae in pregnant women. J. Clin. Microbiol. 40:4329-4331.
Silver, H. M., and J. Struminsky. 1996. A comparison of the yield of positive antenatal group B streptococcus cultures with direct inoculation in selective growth medium versus primary inoculation in transport medium followed by delayed inoculation in selective growth medium. Am. J. Obstet. Gynecol. 175:155-157.
Votava, M., M. Tejkalova, M. Drabkova, V. Unzweitig, and I. Bravny. 2001. Use of GBS media for rapid detection of group B streptococci in vaginal and rectal swabs from women in labor. Eur. J. Microbiol. Infect. Dis. 20:120-122.(Judith S. Heelan, Judith )
Brown Medical School
Women and Infants Hospital of Rhode Island, Providence, Rhode Island
ABSTRACT
Studies at two Brown Medical School-affiliated hospitals were undertaken to evaluate a new selective broth medium (GBS broth) and to compare it to the LIM broth currently used to culture for group B streptococci. Beta-hemolytic group B streptococci produce a carotenoid pigment that turns GBS broth an orange color. From a total of 580 pregnant women, duplicate vaginal-rectal swabs were collected at 35 to 37 weeks of gestation and cultured for group B streptococci, using either LIM broth (a selective broth containing antibiotics) or GBS broth for enrichment. Specimens were either transported to the laboratory or immediately placed in the respective enrichment broths and delivered to the laboratory. GBS broth medium had sensitivity, specificity, and positive and negative predictive values of 87.8, 100, 100, and 95.1% when planted in the laboratory and 90.3, 100, 100 and 97.6%, respectively, when inoculated at bedside. Use of GBS broth would satisfy Centers for Disease Control and Prevention requirements and would provide faster, more-sensitive, and cost-effective detection of group B streptococci in pregnant women.
TEXT
Group B streptococci (GBS) cause serious, life-threatening infections in the newborn. Mortality of GBS sepsis in neonates may exceed 50%, and preterm infants are especially at risk. Intrapartum prophylaxis for pregnant women colonized with GBS has been recommended for several years, since clinical trials showed that the administration of antibiotics to women in labor had a drastic effect in reducing early-onset invasive GBS infection in the neonate (1). Centers for Disease Control and Prevention guidelines issued in 2002 (2, 3) recommend that all pregnant women be screened for vaginal-rectal colonization with GBS at 35 to 37 weeks of gestation. These new guidelines for predelivery testing of pregnant women may have a significant impact on clinical microbiology laboratories, which may expect to see an increase in the volume of test requests for GBS screening for pregnant women. The recommended method for detecting carriage of GBS in pregnant women is to use a selective enrichment broth medium with subsequent subculture on a sheep blood agar plate (BAP) (3). Some laboratories inoculate a BAP before placing the swab in the enrichment broth. The enrichment broth is subcultured only if the primary BAP is negative. A more rapid, less labor-intensive, and more cost-effective method than the traditional culture is needed to screen patients for GBS colonization. Granada medium has long been used in Europe to detect GBS in pregnant women. Strains of beta-hemolytic GBS produce a carotenoid pigment, visible as an orange color, in this medium, usually within 24 h. Variable results on the sensitivity of this medium have been reported, with some investigators considering it to be unacceptably low (4, 5). A new, improved enrichment broth, modified from the original Granada medium, called GBS broth, is now also available (7). This medium has the advantage of reducing both incubation and technical time. Our studies were undertaken to evaluate these media.
(This material was presented in part at the 104th General Meeting of the American Society for Microbiology, New Orleans, La., 23 to 27 May 2004.)
From October 2002 to May 2004, duplicate swabs were used to collect specimens from the lower vagina and rectum of 250 pregnant women at 35 to 37 weeks of gestation who were seen in the Family Medicine Department of Memorial Hospital of Rhode Island (study 1). Swabs were sent to the laboratory. One swab was used to inoculate a BAP (Becton Dickinson Co., Cockeysville, Md.) and then placed in a tube of LIM enrichment broth (Becton Dickinson Co.), which is a selective broth containing antibiotics. The second swab was used to inoculate a BAP and placed in a tube of GBS broth (Northeast Laboratory Services, Waterville, Maine).
During the time period of July 2002 to Oct 2002, duplicate vaginal-rectal swabs from 330 pregnant women at Women and Infants Hospital of Rhode Island (study 2) were collected as described above but placed directly in medium (LIM or GBS broth) at the bedside. Tubes were transported to the laboratory. All media were incubated in CO2 at 35°C for 24 h. Primary plates were examined for the presence of GBS colonies. The GBS broths in both institutions were examined for the presence of any orange pigment. Each GBS and LIM broth was then subcultured to a BAP. Plates were inspected for the presence of colonies suggestive of GBS after 24 h of incubation at 35°C. Streptoccocus agalactiae (GBS) was identified by the CAMP test or by latex agglutination with streptococcal group B antisera.
Study 1. Of the 250 pregnant women studied, 30% (75 of 250) were found to be colonized by GBS. LIM broth and GBS broth were negative for 175 patients. All colonized women were identified by growth of GBS on the primary BAP or on subculture from LIM or GBS broth to a BAP. GBS was detected in 66 of the GBS broths; orange pigment was visible in 53 (80%) of these tubes. The remaining 13 tubes showed no pigmentation, but GBS was grown upon subculture on a blood agar plate. Nine GBS broths from the GBS-positive cultures were negative for GBS. Since all pigmented GBS broths grew GBS upon subculture, no false-positive GBS broths were detected. Sensitivity, specificity, positive predictive value, and negative predictive value for GBS broth were 87.8, 100, 100, and 95.1%, respectively (Table 1).
Study 2. Of the 330 pregnant women studied, 25.2% (83 of 330) were found to be colonized by GBS. LIM broth and GBS broth gave negative results for 247 patients. All colonized women were identified by subculture from LIM or GBS broth to a BAP. GBS was detected in 77 of the GBS broths; orange pigment was visible in 55 (71.4%) of these tubes. The remaining 22 tubes showed no pigmentation, but GBS was grown upon subculture on a blood agar plate. Six GBS broths from the GBS-positive cultures were negative for GBS. As in study 1, no false-positive GBS broths were detected. Sensitivity, specificity, positive predictive value, and negative predictive value for GBS broth were 90.3, 100, 100, and 97.6%, respectively (Table 2). No false-positive GBS broths were detected in our studies. In study 1, the nine false-negative GBS broth cultures were found in lightly colonized women. In these cases, no primary BAP was positive for GBS, which was isolated only from subcultures of LIM broth. GBS broth is more apt to be pigmented and shows the greatest sensitivity when incubated for a full 24 h.
Since GBS broth may be used as a transport medium, it can be inoculated at the bedside. Direct inoculation of specimens immediately into transport medium has been reported to significantly increase the yield of positive GBS culture results (6) and would be especially useful for lightly colonized women. Our studies support this idea, with a slightly higher sensitivity and negative predictive value (90.3 and 97.6% versus 87.8 and 95.1%) when bedside testing was employed. LIM broth, which had 21 false negatives, does not appear to be as useful in serving as a transport medium. Direct bedside inoculation would save labor on the part of the laboratory staff. Another possible reason for increased sensitivity in study 2 might be the fact that in study 1, each swab was rubbed onto an agar plate before being placed in the broth. As suggested in earlier reports (7), for nonhemolytic isolates of GBS, methods that do not rely on hemolysis or pigment production must be used for detection. Subculture of a nonpigmented GBS broth on a BAP would accomplish this task.
In conclusion, GBS broth provides a means for transport, enrichment, and visual recognition of the presence of GBS. Since positive results (orange pigmentation) may be seen with overnight incubation and our study showed a specificity of 100%, there is no need to subculture positive broth cultures, decreasing turnaround time by 24 h and eliminating further workup. Considering the time saved in labor and media, use of this medium could result in considerable cost savings and decrease overall turnaround time.
ACKNOWLEDGMENTS
We thank the microbiology laboratory staff at Memorial Hospital of Rhode Island and Women and Infants Hospital of Rhode Island for their cooperation and assistance in this study.
REFERENCES
American Academy of Pediatrics Committee on Infectious Diseases/Committee on Fetus and Newborn. 1997. Revised guidelines for prevention of early-onset group B streptococcal (GBS) infection by chemoprophylaxis. Pediatrics 99:489-496.
Baron, E. J. 2003. Laboratory support for prevention of perinatal group B streptococcal disease: commentary on the new guidelines on screening for group B streptococci during pregnancy. Clin. Microbiol. Newslett. 25:65-68.
Centers for Disease Control and Prevention. 2002. Prevention of perinatal group B streptococcal disease. Morb. Mortal. Wkly. Rep. 51:1-22.
Gil, E. G., M. C. Rodriguez, R. Bartolome, B. Berjano, L. Cabero, and A. Andreu. 1999. Evaluation of the Granada agar plate for detection of vaginal and rectal group B streptococci in pregnant women. J. Clin. Microbiol. 37:2648-2651.
Overman, S. B., D. D. Eley, B. E. Jacobs, and J. A. Ribes. 2002. Evaluation of methods to increase the sensitivity and timeliness of detection of Streptococcus agalactiae in pregnant women. J. Clin. Microbiol. 40:4329-4331.
Silver, H. M., and J. Struminsky. 1996. A comparison of the yield of positive antenatal group B streptococcus cultures with direct inoculation in selective growth medium versus primary inoculation in transport medium followed by delayed inoculation in selective growth medium. Am. J. Obstet. Gynecol. 175:155-157.
Votava, M., M. Tejkalova, M. Drabkova, V. Unzweitig, and I. Bravny. 2001. Use of GBS media for rapid detection of group B streptococci in vaginal and rectal swabs from women in labor. Eur. J. Microbiol. Infect. Dis. 20:120-122.(Judith S. Heelan, Judith )