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Journal of Clinical Microbiology, March 2001, p. 1021-1024, Vol. 39, No. 3
Respiratory Diseases Branch, Division of Bacterial and
Mycotic Diseases,1 and Epidemic
Intelligence Service, Epidemiology Program
Office,2 Centers for Disease Control and
Prevention, and Department of Pediatrics, Egleston
Children's Hospital, Emory University,6
Atlanta, Georgia; Soroka University Medical
Center3 and Faculty of Health
Sciences,4 Ben Gurion University, Beer
Sheva, Israel; and Beijing Children's Hospital, Beijing,
People's Republic of China5
Received 10 October 2000/Returned for modification 20 November
2000/Accepted 22 December 2000
Field studies of Streptococcus pneumoniae
(pneumococci) nasopharyngeal (NP) colonization are hampered by the need
to directly plate specimens in order to ensure isolate viability. A
medium containing skim milk, tryptone, glucose, and glycerin (STGG) has been used to transport and store NP material, but its ability to
preserve pneumococci has not been evaluated. Our objective was to
qualitatively and semiquantitatively evaluate the ability of STGG to
preserve pneumococci in NP secretions. Entwined duplicate calcium
alginate NP swab samples were obtained from children. One swab was
plated directly onto a gentamicin blood agar plate; the other was
placed in STGG. Growth from the directly plated specimen was compared
with growth from an STGG aliquot immediately cultured or stored at
Streptococcus
pneumoniae (pneumococci) is the most important cause of bacterial
otitis media, pneumonia, bacteremia, and meningitis among children
worldwide (12, 15, 17). Pneumococci are also important
because the rate of nonsusceptibility to various classes of
antimicrobial agents, such as penicillins and cephalosporins, is rising
throughout the United States and worldwide (4, 18, 19).
Prevention efforts have been hampered by the lack of a vaccine which is
immunogenic for important serotypes in children younger than 2 years of
age. A seven-valence pneumococcal conjugate vaccine (Prevnar; Wyeth
Lederle Vaccines) which is immunogenic and efficacious in this age
group recently has been licensed in the United States for use among
children through 9 years of age and is recommended routinely for those
under 2 years of age (1, 3, 16). The effect of this and
other conjugate pneumococcal vaccines on nasopharyngeal (NP)
colonization is a subject of intense investigation.
It is well known that pneumococci are spread from person to person via
the respiratory route. NP colonization studies have shown that people
acquire pneumococci at a young age, carry these organisms for various
periods of time, may carry more than one serotype at a time, and
transmit these organisms to others with whom they are in close contact
(2, 5, 9-11, 14). Many studies of the dynamics and
ecology of pneumococcal NP carriage, particularly in the setting of new
conjugate pneumococcal vaccines, will be performed in settings where
microbiologic facilities are not readily available.
An optimal medium has not been validated for the transport,
preservation, and recovery of pneumococci from NP material. One medium,
STGG (skim milk-tryptone-glucose-glycerin), has been used in some
epidemiologic field studies of pneumococcal carriage (8, 13). However, culturing of NP material stored in STGG has not been compared with direct plating (DP) of NP material on selective blood agar, considered the standard method for isolating pneumococci.
In this study, we aimed to determine (i) what proportions of NP
specimens yield pneumococci by direct inoculation onto culture plates
compared with storage in STGG, (ii) what proportion of pneumococci
collected on an NP swab is recovered by direct plate inoculation, (iii)
whether qualitative and semiquantitative recoveries of pneumococci from
NP secretions suspended in STGG are at least equivalent to those from
direct plate inoculation, and (iv) the least stringent and optimum
conditions for storage of NP secretions containing pneumococci in STGG.
(This research has been presented in abstract form [M. A. Bronsdon, K. L. O'Brien, P. Yagupsky, J. Janco, R. Dagan,
J. Elliott, C. G. Whitney, Y.-H. Yang, L. G. Robinson, R. Facklam, G. Carlone, and B. Schwartz, Abstr.
38th Intersci. Conf. Antimicrob. Agents Chemother., Abstr. D-5,
p. 128, 1998].)
Subjects.
A convenience sample of 186 children younger than
5 years of age and who were attending clinics or emergency rooms for
any complaint was obtained in China (n = 36), Israel
(n = 132), and the United States (n = 18).
STGG transport medium.
We followed the previously published
recipe for the preparation of STGG medium (7), modified
from Gherna (6). We mixed and dissolved 2.0 g of skim
milk powder (Difco, Detroit, Mich.), 3.0 g of Oxoid tryptone soy
broth, 0.5 g of glucose, and 10 ml of glycerol in 100 ml of
distilled water. The solution was dispensed in 1.0-ml amounts into
screw-cap 1.5-ml vials (Sarstedt Microtube, Newton, N.C.). These were
autoclaved at 15 lb/in2 and 121°C for 10 min.
After the vials were cooled, the caps were screwed on tightly and the
vials were stored at Sample collection and processing.
We collected NP specimens
on duplicate pediatric calcium alginate swabs (Fisherbrand, catalog
number 14-959-78; Fisher Scientific, Pittsburg, Pa.) which were twined
together in a sterile manner and passed into the nasopharynx of the
subject. The combined swabs were rotated 180°, removed from the nose,
and untwined in a sterile fashion.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.3.1021-1024.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Evaluation of a Medium (STGG) for Transport and Optimal Recovery
of Streptococcus pneumoniae from Nasopharyngeal
Secretions Collected during Field Studies
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C for 9 weeks, 20°C for 9 weeks, or 4°C for 5 days. Of 186 specimens, 96 (52%) were positive for pneumococci from the direct
plating; 94 (98%) of these were positive from the fresh STGG specimen.
Pneumococci were recovered from all 38 positive specimens frozen at
70°C, all 18 positive specimens frozen at 20°C, and 18 of 20 positive specimens stored at 4°C. Recovery of pneumococci after
storage of NP material in STGG medium at 70°C is at least as good
as that from direct plating. Storage at 20°C is also acceptable.
Storage at 4°C for 5 days is not ideal.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
20°C or refrigerated at 4°C until used. All
vials were used within 6 months of preparation. The medium was tested
for sterility by plating the entire volume of one vial from each lot
onto Trypticase soy agar with 5% sheep blood (BBL Microbiology
Systems, Cockeysville, Md.) and incubating the plate at 37°C
for 48 h. If the growth of any organism was observed, the lot was
discarded. STGG vials were vortexed for approximately 20 s
immediately before being inoculated with an NP swab specimen to draw
into suspension the precipitate which develops during storage.
20°C for 9 weeks, or 70°C for 9 weeks). After storage,
the vial was brought to room temperature and vortexed for approximately
30 s. A 100-µl aliquot was inoculated, streaked, and incubated
as described above. This sample was termed the stored STGG transport
(STGG-Stored) sample.
Pneumococcal identification.
Alpha-hemolytic colonies
phenotypically suspicious for pneumococci were confirmed as such using
optochin disk susceptibility and bile solubility assays. Growth was
scored on a semiquantitative scale: scant growth, <25 colonies in
quadrant 1; 1+ growth, 
25 colonies in quadrant 1 and <25 in quadrant
2; 2+ growth, 25 colonies in quadrant 2 and <25 in quadrant 3; 3+
growth, 25 colonies in quadrant 3 and <25 in quadrant 4; and 4+
growth, 25 colonies in quadrant 4.
Ethics and informed consent. This study was approved by the institutional review boards of the Centers for Disease Control and Prevention, Emory University, Soroka University Medical Center, and Beijing Children's Hospital. Written informed consent was obtained from parents, as required by the local institutional review board.
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RESULTS |
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Materials and Methods
Results
Discussion
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Recovery of pneumococci by DP.
Fifty-four NP specimens were
tested for the qualitative and semiquantitative recoveries of
pneumococci from both the DP and the DP-Residual samples. Of these, 38 (70%) were positive for pneumococci from the DP samples. The
qualitative isolation of pneumococci was fully concordant between the
DP and the DP-Residual samples. Among the 38 specimens positive for
pneumococci, 18 (47%) produced growth from the DP-Residual samples
equal or greater than that from the DP samples. Of the 17 specimens which had growth of 
2+ from the DP samples, 13 (76%) had equivalent or greater growth from the DP-Residual samples.
Recovery of pneumococci from STGG versus DP. Pneumococci were recovered from 96 (52%) of 186 DP samples and from 94 (51%) of STGG-Fresh samples. Six specimens were discordant for the recovery of pneumococci between the DP and the STGG-Fresh samples; in two, pneumococci grew only from the STGG-Fresh samples, and in four, they grew only from the DP samples. Growth was categorized as 1+ in four discordant cases and as 2+ in the remaining two discordant cases.
Seventy-eight pneumococcal specimens were used for the semiquantitative growth comparison between DP and STGG-Fresh samples. Thirty-six (44%) of the STGG-Fresh samples had growth greater than or equal to the growth from the DP samples. Among 26 specimens with growth categorized as2+ from the DP samples, 15 (58%) had equivalent or greater growth from the STGG-Fresh samples.
Recovery of pneumococci from STGG-Stored samples.
Of the
132 specimens used to evaluate storage conditions, 76 (58%) were
positive for pneumococci from the DP samples; of these, 72 (95%) were
also positive from the STGG-Fresh samples. Compared with the results
obtained with DP and STGG-Fresh samples, we found no substantial
differences in the qualitative recoveries of pneumococci under any of
the storage conditions tested (Table
1). Semiquantitative recovery of
pneumococci stored at 70°C for 9 weeks and at 20°C for 9 weeks
was at least as good as that from the DP samples (Table 2). There was some reduction of
pneumococcal growth in the samples stored at 4°C for 5 days.
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DISCUSSION |
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Materials and Methods
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Until now, studies have not been conducted to evaluate the qualitative and quantitative abilities of STGG to sustain pneumococci in NP specimens. To our knowledge, no other transport media have been evaluated in this way either. Previously, STGG has been used for populations with extremely high and dense colonization rates (8, 13). Those studies focused on the gross recovery of pneumococci rather than on low-level colonization or small proportions of multiple serotypes, which are now issues of considerable interest. In many settings, the laboratory facilities necessary for the processing of such specimens are not located on site. The performance of such studies, even when laboratory facilities are readily available, could be enhanced if NP specimens could be tested in batch form. We found that STGG performed well compared to DP, the standard technique for NP carriage studies.
We have shown that substantial NP material remains on a swab after it
is used to inoculate a culture plate. We recovered pneumococci from the
residual swab material of specimens found positive for pneumococci by
DP. The enhanced recovery of pneumococci from the DP-Residual samples
compared with the DP samples was especially noted for specimens which
had 2+ growth in the DP samples.
We also compared the recoveries of pneumococci from the DP samples and from a duplicate swab suspended in STGG. Use of the transport medium resulted in enhanced recovery of pneumococci compared with recovery by DP, especially at low colony counts. We identified a few samples that were negative for pneumococci from the DP samples but positive for pneumococci from the STGG samples and vice versa. The discrepancies between the STGG and the DP samples all occurred at low colony counts and may have represented sampling inconsistency or incomplete mixing of samples. There were no qualitative discrepancies between recoveries of pneumococci from the DP and STGG samples at higher colony counts.
On a qualitative basis, all storage conditions studied were acceptable
for the recovery of pneumococci compared with DP. When evaluated on a
semiquantitative basis, the specimens maintained at 4°C did not
preserve pneumococci as well as those maintained at 20°C or
70°C but were nevertheless acceptable when compared with DP.
Although the limited follow-up time did not distinguish between storage
at 20°C and that at 70°C, the experience of others has shown
that maintaining specimens at 70°C results in long-term survival
of pneumococci (E. Stubbs, M. McKinnon, T. M. Shelby James,
H. Smith Vaughan, and A. J. Leach, 2nd Int. Symp. Pneumococci
Pneumococcal Dis., abstr. P104, 2000). Transporting a specimen in STGG
on wet ice (simulated by 4°C) followed by storage at 20°C in the
short term (up to 9 weeks) and by maintenance of the specimen at
70°C for the long term is likely acceptable. Optimal conditions
consist of freezing the specimen as quickly as possible at 70°C.
The benefits of the STGG storage medium include (i) enhanced recovery
of pneumococci compared with DP, especially at low organism concentrations; (ii) ability to conduct multiple assays on a single NP
specimen; (iii) long-term storage of the original NP specimen at
70°C without loss of CFU; (iv) manipulation of the inoculum size to
adjust the density of colonies on a plate; (v) transport of NP
specimens from the site of collection to a distant laboratory; and (vi)
assay of NP specimens in batch form.
In conclusion, we have shown equivalent recoveries of pneumococci after suspension in STGG compared with DP of the swab, even at low organism numbers (<50 CFU). Pneumococci from NP secretions are preserved in the short term and in the long term by suspension in STGG when transported and stored at low temperatures. STGG is inexpensive and remains stable for at least 6 months after sterilization. We recommend STGG for transport and storage of field-collected NP specimens in epidemiologic studies.
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FOOTNOTES |
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* Corresponding author. Present address: Center for American Indian and Alaskan Native Health, Johns Hopkins School of Hygiene and Public Health, 621 N. Washington St., Baltimore, MD 21205. Phone: (410) 614-3806. Fax: (410) 955-2010. E-mail: klobrien{at}jhsph.edu.
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Discussion
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Journal of Clinical Microbiology, March 2001, p. 1021-1024, Vol. 39, No. 3
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.3.1021-1024.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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