- Email: [email protected]
Evaluation of aerobic and anaerobic methods for recovery of streptococci from throat cultures
CLINICAL A N D LABORATORY OBSERVATIONS
Articles in this section should require no more than three Journal pages, the text 1000 words or less. A combined total of two illustrations or tables and up to 10 references will be accepted. An abstract is not necessary.
Evaluation of aerobic and anaerobic methods for recovery of streptococci from throat cultures Martin F. Randolph, M.D., John J. Redys, M.S., and John B. Cope, B.S. Farmington, Conn.
IN CLINICAL PRACTICE, the diagnosis of group A B-hemolytic streptococcal pharyngitis rests on a compatible clinical syndrome and the recovery of GABHS by throat culture. Because of the variability of the clinical signs, the culture remains the mainstay of diagnosis, which for many clinicians and laboratories does not go beyond the use of standard blood agar plates incubated aerobically at 35 ~ C. Several years ago we reported the successful use of a streptococcal culture kit developed specifically for office or clinical use. (Strep Kit, Detekta-Kit Inc., Rocky Hill, Conn.). Features of the kit were selective inhibitors in the blood agar and a clear plastic surface overlay strip to serve as an oxygen barrier to the inoculum? More recently, reports z+ have described improved recovery of G A B H S when blood agar plates were incubated under anaerobic conditions (Gas Pak 100 Anaerobic System, BBL No. 60626, Becton, Dickinson & Co., Cockeysville, Md.). Because the streptococcal culture kit witti its oxygen-barrier strip appeared to be a much simpler way to achieve reduced oxygen tension, a comparison of these two methods with each other and with the standard blood agar aerobic method was undertakem METHODS The siudy was begun'on October 1, 1982, and ended on June 1, 1983. Specimens were cultured only from patients From the Department o f Pediatrics, University o f Connecticut ttealth Center, and the Connecticut State Health Laboratories. Reprint requests: Martin F. Randolph, M.D.. 70 Deer Hill Ave., Danbury, CT 06810.
in whom G A B t l S pharyngitis was strongly Suspected. Instead of standard Petri dishes, I u • 3" clear polystyrene kits were used for all media in the interest of surface area uniformity. Earlier studies ~ showed that these kits were as satisfactory as Petri dishes for streptococcal screening, and required much less incubator and refrigerator space. All media were prepared with a glucose-free blood agar base to which 4.8% to 5.2% sheep blood agar was added, based on the hematocrit of each lot of sheep blood. A comparison set included three cultures in each patient. All cultures were taken and interpreted by one of us (M.F.R.); two of the cultures were standard blood agar, and the third was the Strep Kit with selective inhibitors (0.3 pg gentamicin and 10 pg nalidixie acid per milliliter) GABHS
Group A B-hemolytic streptococci
]
in standard blood agar medium. A single throat swab, directly from the patient, was used to inoculate all three cultures. The first culture was incubated aerobically (office incubator), the second culture anaerobically in a Gas Pak Jar, and the third culture (Strep Kit) With the oxygen barrier strip on its surface. The strip w~is peeled back, the surface was inoculated, and the strip was replaced. The Strep Kit was incubated aerobically (office incubator). To exclude the potential for bias related to the order of inoculation, the order was methodically varied for each successive patient; one third of each comparison set was inoculated first, second, and third. Hence, it would be expected that any bias would be equally distributed between comparisons. Group A differentiation disks were The Journal o f P E D 1 A T R 1 C S Vol. 104. No. 6. June 1984
897
898
Clinical and laboratory observations
The Journal of Pediatrics June 1984
Table. Recovery of group A B-hemolytic streptococci from throat cultures: Comparison of three methods
I
Standard blood agar
Gas Pak Jar
300
300
300
178 59.5
207 69
217 72
75 103
85 122
81 136
Total cultures Positive cultures n % Positivity of culture I+ to 2+ 3+ to 4+
Strep Kit
applied to all cultures. Cultures were incubated in one incubator (35 ~ C) and examined in 18 to 24 hours. Positive cultures were quantified using the method of Breese et al. 6 B-Hemolytic streptococci were preliminarily identified as group A or not, on the basis of bacitracin susceptibility; a 0.04 unit bacitracin disk was placed on a heavily inoculated area and observed for any inhibition after 18 hours to 24 hours', incubation. Final identification was done by fluorescent antibody test. Culture sets were submitted to the Connecticut State Department of Laboratories for review, subculture, and confirmation of group A by fluorescent ant!body method. Results were analyzed by the chi-square test of dissimilar parts. 7 Parental consent was obtained for each child admitted to the study. RESULTS Three hundred throat cultures were surveyed for B-hemolysis using the three different incubation techniques (Table). Of the 300 throat cultures, 239 (80%) were found to contain B-hemolytic streptococci by at least one of the three incubation techniques; 218 (73%) were found to be group A by the fluorescent antibody test. GABHS yield was as follows: Strep Kit 217 (72%), Gas Pak Jar 207, and standard blood agar aerobic method 178 (59.5%). Of the 218 GABHS isolated, the Strep Kit detected 99%, the Gas Pak Jar 95%, and the standard aerobic method 80%. Differences between the Strep Kit and the standard blood agar methods, and between standard methods and the Gas Pak Jar were statistically significant: x 2 11.264 and 6.098, respectively. The difference between the Gas Pak Jar and the Strep Kit was not significant: X2 0.8370 (Xz >3.841 significant at P = 0.05). Ten cultures falsely negative for GABHS by the Gas Pak method were strongly positive by Strep Kit. A heavy, confluent overgrowth of unidentified organisms on the Gas Pak medium was noted in each case, masking or interfering with the growth of streptococci. It is probable that overgrowth could have been prevented and false negative cultures avoided if appropriate selective inhibitors had been incorporated in the Gas Pak medium.
A total of 39 cultures positive by Strep Kit or Gas Pak Jar were undetected by the standard aerobic method. Of these, 33 were quantified as 3+ to 4+, and only six as 1+ to 2+. Of the 21 non-group A streptococci isolated, all were positive by Strep Kit, 17 by Gas Pak Jar, and only seven by standard aerobic method. The increased sensitivity of the Strep Kit for the growth of non-group A streptococci did not pose a problem; differentiation was accomplished on the primary culture by use of group A differentiation disks. No cultures positive by the standard aerobic method were negative by Strep Kit or Gas Pak Jar methods. DISCUSSION These results support the view that either anaerobic incubation or reduced oxygen tension as provided by the oxygen barrier strip, used in combination with selective inhibitors in the media, significantly enhances recovery of streptococci from throat cultures. These results are not unexpected. Anaerobic conditions enhance hemolysis by B-hemolytic streptococci and contribute to the suppression of the growth of aerobic commensal flora. 24. 7 Inhibitors in the media selectively suppress the growth o f susceptible organisms in mixed cultures that would otherwise obscure the growth of B-hemolytic streptococci and that may themselves produce B-hemolysis (e.g., staphylococci, Neisseria spp, and Escherichia coli). 8-'~ These two factors, singly or in combination, optimize the growth of /3-hemolytic streptococci and reduce the number of false negative cultures. 2-4'7-j~ In practice we find the Strep Kit less costly (Gas Pak Jar $165, Gas Pak envelopes $1 each, Strep Kit $0.85) and less cumbersome for office use than the Gas Pak Jar. The Jar needs to be charged and sealed with each batch of throat cultures; incubation is usually delayed until sufficient cultures have been accumulated to fill the jar. The Strep Kit is better suited to office use. It is inoculated directly with the throat swab, incubated (aerobically) in an ordinary office incubator, and read the next morning (12 to 18 hours). The selective nature of the medium and the reduced oxygen levels afforded by the barrier strip allow for easy interpretation even by nontechnical office staff.' GABHS are cultured from the throats of at least 35% of children with acute pharyngitis in our daily practice. The larger recovery rate achieved in this study undoubtedly relates to a highly selected study population surveyed during the months of October through April, when streptococcal infections are common. Whether the additional 12.5% of cultures identified as positive by the Strep Kit and missed by the standard blood agar aerobic method represent true streptococcal infection or the carrier state was not addressed. Selective use of the throat culture
Volume 104 Number 6
remains the mainstay of diagnosis and our only insurance in practice against overdiagnosis of streptococcal pharyngitis. We thank Michael A. Gerber, M.D., for critical review of the manuscript. REFERENCES 1. Randolph MF, Redys J J, Cope J, Morris KE: Streptococcal pharyngitis: Evaluation of a new diagnostic kit for clinic and office use. Am J Dis Child 130:171, 1976. 2. Dykstra MA, McLaughlin, Bartlett RC: A comparison of media and techniques for detection of group A streptococci in throat swab specimens. J Clin Microbiol 9:236, 1976. 3. Beerman CA, Goldblatt SA: Screening for group A streptococcus by means of anaerobic primary plate technique. J PEDIATR10:70, 1982.
Clinical attd laboratory observations
899
4. Lauer BA, Relier BL, Mirrett S: Effect of atmosphere and duration of incubation on the primary isolation of group A from throat cultures. J Clin Microbiol 17:388, 1983. 5. Redys J J: Role of throat cultures: Laboratory diagnosis of streptococcal disease. Minn Med Special Issue, August 1975. 6. Breese BB, Disney FA, Talpey WB, Green JL: Beta-hemolytic streptococcal infection. Am J Dis Child 119:18, 1970. 7. Colton T: Colton's statistics in medicine. Boston, 1974, Little, Brown, p 177-179. 8. McGonagle LA: Evaluation of a screening procedure for the isolation of beta-hemolytic streptococci, tieallh Lab Sci 11:61, 1974. 9. Kurzynski TA, Meise C, Daggs R, ttelstad A: A new selective surface plating medium for recovery of group A streptococci. J Clin Microbiol 9:189, 1979. 10. Gutekunst RR: Selective medium designed to nurture 5 pyogenes. Clin Lab Forum June 1973.
Benign bacteremia caused by Sahnonella typhi and paratyphi in children younger than 2 years Catterine Ferreccio, M.D., Myron M. Levine, M.D., D.T.P.H., Alejandro Manterola, M.D., German Rodriguez, M.D., Isabel Rivara, M.D., Ingeborg Prenzel, M.D., Robert E. Black, M.D., M.P.H., Thomas Mancuso, M.D., and Dorothy Bulas, M.D. Baltimore, Md., and Santiago, Chile
TYPHOID FEVER tIAS REMAINED ENDEMIC in Santiago, Chile, for decades; since 1977 the incidence has exceeded 150 cases per 100,000 population. Typhoid fever occurs mainly in persons 5 to 25 years of age (Table I), and is generally manifested as a classic clinical syndrome including fever, abdominal discomfort and distention, headache, malaise, constipation, and hepatosplenomegaly. Few cases of typhoid fever are reported in children younger than 2 years. Thus it was necessary to determine whether the very low reported incidence of typhoid fever in
From the Centerfor Vaccine Development, University of Mcu'yland School of Medicine; Roberto de Rio Children's Hospital; and Servicio de Salud Area Norte, Ministry of Health, Santiago. Supported b)" grants fronl the World Health Organization attd the Pan Anleriean Health Org'anization and by research contract DAMD-81-C-III5 fronl the U.S. Arnly Medical Research and Development Command. Reprint requests: Myron M. Levine, M.D.. Center for Vaccine Development. University of Maryland School of Medicine, 29 S. Greene St.. Balthnore, hiD 21201.
young children represents a lack of consumption of the vehicles that transmit Salmonella typhi to older children or whether infection occurs but the infant host manifests an atypical response that is not readily recognized clinically. To help resolve this question, we systematically performed blood cultures in children younger than 2 years with fever who were seen at two health centers in Santiago during the 3 peak months of the typhoid fever season. METHODS Rectal temperatures were recorded in all children younger than 2 years who were seen at Pincoya and Consultorio Dos health centers in the northern administrative area (Area Norte) of Santiago from January through March 1983. In all children with a temperature >--38 ~ C, 2 ml blood was drawn for culture and inoculated into a flask containing 35 ml brain-heart infusion with 0.01% sodium polyanethol sulfonate. The reason for the blood culture was explained to the parents, and verbal informed consent was obtained, according to local custom. The study was discontinued in the last week of March, by which time blood from 197 consecutive children had been cultured. Cultures were
Articles in this section should require no more than three Journal pages, the text 1000 words or less. A combined total of two illustrations or tables and up to 10 references will be accepted. An abstract is not necessary.
Evaluation of aerobic and anaerobic methods for recovery of streptococci from throat cultures Martin F. Randolph, M.D., John J. Redys, M.S., and John B. Cope, B.S. Farmington, Conn.
IN CLINICAL PRACTICE, the diagnosis of group A B-hemolytic streptococcal pharyngitis rests on a compatible clinical syndrome and the recovery of GABHS by throat culture. Because of the variability of the clinical signs, the culture remains the mainstay of diagnosis, which for many clinicians and laboratories does not go beyond the use of standard blood agar plates incubated aerobically at 35 ~ C. Several years ago we reported the successful use of a streptococcal culture kit developed specifically for office or clinical use. (Strep Kit, Detekta-Kit Inc., Rocky Hill, Conn.). Features of the kit were selective inhibitors in the blood agar and a clear plastic surface overlay strip to serve as an oxygen barrier to the inoculum? More recently, reports z+ have described improved recovery of G A B H S when blood agar plates were incubated under anaerobic conditions (Gas Pak 100 Anaerobic System, BBL No. 60626, Becton, Dickinson & Co., Cockeysville, Md.). Because the streptococcal culture kit witti its oxygen-barrier strip appeared to be a much simpler way to achieve reduced oxygen tension, a comparison of these two methods with each other and with the standard blood agar aerobic method was undertakem METHODS The siudy was begun'on October 1, 1982, and ended on June 1, 1983. Specimens were cultured only from patients From the Department o f Pediatrics, University o f Connecticut ttealth Center, and the Connecticut State Health Laboratories. Reprint requests: Martin F. Randolph, M.D.. 70 Deer Hill Ave., Danbury, CT 06810.
in whom G A B t l S pharyngitis was strongly Suspected. Instead of standard Petri dishes, I u • 3" clear polystyrene kits were used for all media in the interest of surface area uniformity. Earlier studies ~ showed that these kits were as satisfactory as Petri dishes for streptococcal screening, and required much less incubator and refrigerator space. All media were prepared with a glucose-free blood agar base to which 4.8% to 5.2% sheep blood agar was added, based on the hematocrit of each lot of sheep blood. A comparison set included three cultures in each patient. All cultures were taken and interpreted by one of us (M.F.R.); two of the cultures were standard blood agar, and the third was the Strep Kit with selective inhibitors (0.3 pg gentamicin and 10 pg nalidixie acid per milliliter) GABHS
Group A B-hemolytic streptococci
]
in standard blood agar medium. A single throat swab, directly from the patient, was used to inoculate all three cultures. The first culture was incubated aerobically (office incubator), the second culture anaerobically in a Gas Pak Jar, and the third culture (Strep Kit) With the oxygen barrier strip on its surface. The strip w~is peeled back, the surface was inoculated, and the strip was replaced. The Strep Kit was incubated aerobically (office incubator). To exclude the potential for bias related to the order of inoculation, the order was methodically varied for each successive patient; one third of each comparison set was inoculated first, second, and third. Hence, it would be expected that any bias would be equally distributed between comparisons. Group A differentiation disks were The Journal o f P E D 1 A T R 1 C S Vol. 104. No. 6. June 1984
897
898
Clinical and laboratory observations
The Journal of Pediatrics June 1984
Table. Recovery of group A B-hemolytic streptococci from throat cultures: Comparison of three methods
I
Standard blood agar
Gas Pak Jar
300
300
300
178 59.5
207 69
217 72
75 103
85 122
81 136
Total cultures Positive cultures n % Positivity of culture I+ to 2+ 3+ to 4+
Strep Kit
applied to all cultures. Cultures were incubated in one incubator (35 ~ C) and examined in 18 to 24 hours. Positive cultures were quantified using the method of Breese et al. 6 B-Hemolytic streptococci were preliminarily identified as group A or not, on the basis of bacitracin susceptibility; a 0.04 unit bacitracin disk was placed on a heavily inoculated area and observed for any inhibition after 18 hours to 24 hours', incubation. Final identification was done by fluorescent antibody test. Culture sets were submitted to the Connecticut State Department of Laboratories for review, subculture, and confirmation of group A by fluorescent ant!body method. Results were analyzed by the chi-square test of dissimilar parts. 7 Parental consent was obtained for each child admitted to the study. RESULTS Three hundred throat cultures were surveyed for B-hemolysis using the three different incubation techniques (Table). Of the 300 throat cultures, 239 (80%) were found to contain B-hemolytic streptococci by at least one of the three incubation techniques; 218 (73%) were found to be group A by the fluorescent antibody test. GABHS yield was as follows: Strep Kit 217 (72%), Gas Pak Jar 207, and standard blood agar aerobic method 178 (59.5%). Of the 218 GABHS isolated, the Strep Kit detected 99%, the Gas Pak Jar 95%, and the standard aerobic method 80%. Differences between the Strep Kit and the standard blood agar methods, and between standard methods and the Gas Pak Jar were statistically significant: x 2 11.264 and 6.098, respectively. The difference between the Gas Pak Jar and the Strep Kit was not significant: X2 0.8370 (Xz >3.841 significant at P = 0.05). Ten cultures falsely negative for GABHS by the Gas Pak method were strongly positive by Strep Kit. A heavy, confluent overgrowth of unidentified organisms on the Gas Pak medium was noted in each case, masking or interfering with the growth of streptococci. It is probable that overgrowth could have been prevented and false negative cultures avoided if appropriate selective inhibitors had been incorporated in the Gas Pak medium.
A total of 39 cultures positive by Strep Kit or Gas Pak Jar were undetected by the standard aerobic method. Of these, 33 were quantified as 3+ to 4+, and only six as 1+ to 2+. Of the 21 non-group A streptococci isolated, all were positive by Strep Kit, 17 by Gas Pak Jar, and only seven by standard aerobic method. The increased sensitivity of the Strep Kit for the growth of non-group A streptococci did not pose a problem; differentiation was accomplished on the primary culture by use of group A differentiation disks. No cultures positive by the standard aerobic method were negative by Strep Kit or Gas Pak Jar methods. DISCUSSION These results support the view that either anaerobic incubation or reduced oxygen tension as provided by the oxygen barrier strip, used in combination with selective inhibitors in the media, significantly enhances recovery of streptococci from throat cultures. These results are not unexpected. Anaerobic conditions enhance hemolysis by B-hemolytic streptococci and contribute to the suppression of the growth of aerobic commensal flora. 24. 7 Inhibitors in the media selectively suppress the growth o f susceptible organisms in mixed cultures that would otherwise obscure the growth of B-hemolytic streptococci and that may themselves produce B-hemolysis (e.g., staphylococci, Neisseria spp, and Escherichia coli). 8-'~ These two factors, singly or in combination, optimize the growth of /3-hemolytic streptococci and reduce the number of false negative cultures. 2-4'7-j~ In practice we find the Strep Kit less costly (Gas Pak Jar $165, Gas Pak envelopes $1 each, Strep Kit $0.85) and less cumbersome for office use than the Gas Pak Jar. The Jar needs to be charged and sealed with each batch of throat cultures; incubation is usually delayed until sufficient cultures have been accumulated to fill the jar. The Strep Kit is better suited to office use. It is inoculated directly with the throat swab, incubated (aerobically) in an ordinary office incubator, and read the next morning (12 to 18 hours). The selective nature of the medium and the reduced oxygen levels afforded by the barrier strip allow for easy interpretation even by nontechnical office staff.' GABHS are cultured from the throats of at least 35% of children with acute pharyngitis in our daily practice. The larger recovery rate achieved in this study undoubtedly relates to a highly selected study population surveyed during the months of October through April, when streptococcal infections are common. Whether the additional 12.5% of cultures identified as positive by the Strep Kit and missed by the standard blood agar aerobic method represent true streptococcal infection or the carrier state was not addressed. Selective use of the throat culture
Volume 104 Number 6
remains the mainstay of diagnosis and our only insurance in practice against overdiagnosis of streptococcal pharyngitis. We thank Michael A. Gerber, M.D., for critical review of the manuscript. REFERENCES 1. Randolph MF, Redys J J, Cope J, Morris KE: Streptococcal pharyngitis: Evaluation of a new diagnostic kit for clinic and office use. Am J Dis Child 130:171, 1976. 2. Dykstra MA, McLaughlin, Bartlett RC: A comparison of media and techniques for detection of group A streptococci in throat swab specimens. J Clin Microbiol 9:236, 1976. 3. Beerman CA, Goldblatt SA: Screening for group A streptococcus by means of anaerobic primary plate technique. J PEDIATR10:70, 1982.
Clinical attd laboratory observations
899
4. Lauer BA, Relier BL, Mirrett S: Effect of atmosphere and duration of incubation on the primary isolation of group A from throat cultures. J Clin Microbiol 17:388, 1983. 5. Redys J J: Role of throat cultures: Laboratory diagnosis of streptococcal disease. Minn Med Special Issue, August 1975. 6. Breese BB, Disney FA, Talpey WB, Green JL: Beta-hemolytic streptococcal infection. Am J Dis Child 119:18, 1970. 7. Colton T: Colton's statistics in medicine. Boston, 1974, Little, Brown, p 177-179. 8. McGonagle LA: Evaluation of a screening procedure for the isolation of beta-hemolytic streptococci, tieallh Lab Sci 11:61, 1974. 9. Kurzynski TA, Meise C, Daggs R, ttelstad A: A new selective surface plating medium for recovery of group A streptococci. J Clin Microbiol 9:189, 1979. 10. Gutekunst RR: Selective medium designed to nurture 5 pyogenes. Clin Lab Forum June 1973.
Benign bacteremia caused by Sahnonella typhi and paratyphi in children younger than 2 years Catterine Ferreccio, M.D., Myron M. Levine, M.D., D.T.P.H., Alejandro Manterola, M.D., German Rodriguez, M.D., Isabel Rivara, M.D., Ingeborg Prenzel, M.D., Robert E. Black, M.D., M.P.H., Thomas Mancuso, M.D., and Dorothy Bulas, M.D. Baltimore, Md., and Santiago, Chile
TYPHOID FEVER tIAS REMAINED ENDEMIC in Santiago, Chile, for decades; since 1977 the incidence has exceeded 150 cases per 100,000 population. Typhoid fever occurs mainly in persons 5 to 25 years of age (Table I), and is generally manifested as a classic clinical syndrome including fever, abdominal discomfort and distention, headache, malaise, constipation, and hepatosplenomegaly. Few cases of typhoid fever are reported in children younger than 2 years. Thus it was necessary to determine whether the very low reported incidence of typhoid fever in
From the Centerfor Vaccine Development, University of Mcu'yland School of Medicine; Roberto de Rio Children's Hospital; and Servicio de Salud Area Norte, Ministry of Health, Santiago. Supported b)" grants fronl the World Health Organization attd the Pan Anleriean Health Org'anization and by research contract DAMD-81-C-III5 fronl the U.S. Arnly Medical Research and Development Command. Reprint requests: Myron M. Levine, M.D.. Center for Vaccine Development. University of Maryland School of Medicine, 29 S. Greene St.. Balthnore, hiD 21201.
young children represents a lack of consumption of the vehicles that transmit Salmonella typhi to older children or whether infection occurs but the infant host manifests an atypical response that is not readily recognized clinically. To help resolve this question, we systematically performed blood cultures in children younger than 2 years with fever who were seen at two health centers in Santiago during the 3 peak months of the typhoid fever season. METHODS Rectal temperatures were recorded in all children younger than 2 years who were seen at Pincoya and Consultorio Dos health centers in the northern administrative area (Area Norte) of Santiago from January through March 1983. In all children with a temperature >--38 ~ C, 2 ml blood was drawn for culture and inoculated into a flask containing 35 ml brain-heart infusion with 0.01% sodium polyanethol sulfonate. The reason for the blood culture was explained to the parents, and verbal informed consent was obtained, according to local custom. The study was discontinued in the last week of March, by which time blood from 197 consecutive children had been cultured. Cultures were