Heelan et al. (5) recently presented data on the sensitivity of a liquid Granada medium (9) (GBS broth) for detection of Streptococcus agalactiae. We have tested this medium, and it contains, as Granada medium, starch, proteose peptone 3, methotrexate, a Good's buffer, serum, glucose, and selective agents. So, we agree with the authors that detection of orangered colonies in GBS broth is 100% specific for the presence of beta-hemolytic group B streptococci (GBS). Nevertheless, some statements in this paper need clarification.The authors claim that sometimes Granada agar lacks sensitivity for GBS detection, but no comparison between GBS broth and Granada agar is reported. In the two references cited to support this claim (3, 7), it is acknowledged that the batches of Granada agar used were probably spoiled (10). Moreover, the flurry of references (1,2,4,6,8,9,14,15) that support a very good sensitivity of Granada agar to detect GBS are not mentioned.Regarding sensitivity of GBS broth, the figures reported are somewhat confusing. One-hundred fifty-eight GBS strains were recovered from 580 swabs, but only in 108 cases were red colonies detected, so GBS broth sensitivity is 68.3% instead of the 87.8% or 90.3% reported. GBS pigment was not observed in 35 tubes that later (after subculture) proved to be positive (25% of all positives), and this figure makes the usefulness of this medium for direct GBS detection doubtful. When (study 1) GBS broth was used as a detection medium, 9 of 75 GBS strains (12%) were completely lost (positive in subculture from LIM broth but negative in subculture from GBS broth). When (study 2) GBS broth was used as a detection and transport medium, 6 of 83 GBS (7%) were also completely lost. This failure to detect (even after subculture) some GBS-positive vaginorectal samples can be an important drawback of GBS broth. Another fact is the dramatic failure of LIM broth to recover GBS (21 of 83 positive samples were lost in study 2) found in Heelan's work. This finding needs further evaluation, as enrichment in LIM broth is an accepted procedure for GBS detection in the Centers for Disease Control and Prevention guidelines (13).Poor performance in detecting GBS could be explained by the fact that when liquid Granada media are kept refrigerated the starch precipitates (retrogradation) (16), producing an opaque white mass, where GBS colonies are sometimes barely noticeable. However, if not kept refrigerated the medium deteriorates in a few days (12). We have observed that when media containing folate antagonists (as methotretaxate) are used as transport media and swabs are kept in these media for several hours before starting incubation, the number of GBS CFU nosedives. This renders the use of such media as transport media unsafe. For these reasons, in our view, the use of liquid selective Granada media as transport media for detection of GBS colonization in pregnant women cannot be recommended.
Recovery of group B streptococci (GBS) was assessed in 1,204 vaginorectal swabs stored in Amies transport medium at 4 or 21°C for 1 to 4 days either by direct inoculation onto Granada agar (GA) or by culture in blood agar (BA) and GA after a selective broth enrichment (SBE) step. Following storage at 4°C, GBS detection in GA was not affected after 72 h by either direct inoculation or SBE; however, GBS were not detected after SBE in the BA subculture in some samples after 48 h of storage and in GA after 96 h. After storage at 21°C, loss of GBS-positive results was significant after 48 h by direct inoculation in GA and after 96 h by SBE and BA subculture; some GBS-positive samples were not detected after 24 h of storage followed by SBE and BA subculture or after 48 h of storage followed by SBE and GA subculture. Storage of swabs in transport medium, even at 4°C, produced after 24 h an underestimation of the intensity of GBS colonization in most specimens. These data indicate that viability of GBS is not fully preserved by storage of vaginorectal swabs in Amies transport medium, mainly if they are not stored under refrigeration.The recommendations of the Centers for Disease Control and Prevention (10) for prevention of infections with neonatal group B streptococci (GBS) include cultures from anogenital swabs on all pregnant women, collected at 35 to 37 weeks of gestation. The recommendations state that swabs could be placed into a nonnutritive transport medium (such as Amies or Stuart without charcoal) and that transport media will maintain GBS viability for up to 4 days at room temperature or under refrigeration. Nevertheless, the accepted practice (3) of placing vaginal and rectal swabs in a transport medium and transporting them (even after several days) to the clinical laboratory has been questioned, because it has been reported that the yield of GBS-positive specimens increases when swabs are inoculated directly into selective media, thereby avoiding transport media (11). Moreover, some reports suggest a rapidly declining number of GBS for GBS-positive swabs that are stored in transport media (8).This study examines the effect of time and temperature on GBS viability and GBS recovery rates for vaginorectal swabs stored in Amies transport medium either when the swabs are inoculated directly in Granada agar (GA) plates (9) or a selective broth enrichment (SBE) step (1, 3) is used before inoculating GA and blood agar (BA) plates.Swab collection and processing. Two vaginorectal swabs were collected from each of 1,204 pregnant women (at 35 to 37 weeks of gestation); after collection, the swabs were placed in Amies transport medium (Biomedics, Madrid, Spain) and sent to the laboratory. All specimens were processed within 4 h of collection.In the first phase of the study, we studied 300 samples, using one vaginal swab from each woman. Each swab was placed in a tube containing 0.8 ml of 0.85% NaCl and swirled vigorously. Nine additional swabs were immersed in this tube and then placed in tubes of Amies transport mediu...
The use of 1% unmodified rice starch and 1% horse serum instead of 2% soluble starch and 5% serum in Granada medium is described. These components result in a medium of increased stability, preventing spoilage after a few days of storage at room temperature.The detection of orange-red colonies in Granada medium (GM) (5) is an easy way of identifying hemolytic Streptococcus agalactiae (a group B streptococcus [GBS]) in clinical samples (1,3,9,11,15,17,20). Proteose Peptone 3 (BD/Difco, Franklin Lakes, N.J.) (12,18), soluble starch (6), and horse serum (10, 13) are necessary components of culture media aimed to detect GBS by pigment production. In addition to these components, GM contains a folate pathway inhibitor (methotrexate) (4, 5), a Good's buffer (MOPS [morpholinepropanesulfonic acid]) (5, 8), and glucose (5) to trigger GBS pigment production.However, an important drawback of GM is its poor stability if not stored refrigerated. The use of improperly stored GM can cause failure in the detection of GBS (7,14,20). We hypothesized that hydrolysis of soluble starch caused by the amylase in the serum (16) may be an important factor in the spoilage of nonrefrigerated GM (M. De La Rosa-Fraile, Letter, J. Clin. Microbiol. 41:4007, 2003). Here we report on the use of unmodified starch, instead of soluble starch, and the reduction or removal of the serum in order to increase the stability of nonrefrigerated GM.An overnight culture of GBS strain ATCC 12386 in brain heart broth diluted 1/100 in 0.85% NaCl was used for initial testing. The GBS pigment was graded 0 (no pigment), 1ϩ (yellow), 2ϩ (pale orange), 3ϩ (orange-red), or 4ϩ (deep red). Colony size and pigment score were assessed after 18 h of anaerobic incubation at 36°C.Several formulations of GM were prepared with 1 or 2% soluble starch (catalog no. 1252; Merck HGaA, Darmstadt, Germany) and the following unmodified starches (from SigmaAldrich Corp., St. Louis, Mo.): cornstarch (catalog no. S 4126), rice starch (catalog no. S 7260), and wheat starch (catalog no. S 5127). Each medium was prepared with 5, 2, or 1% horse serum or without horse serum. Plates were inoculated as they were prepared or after storage for 6 days at 4, 22, or 30°C.When GM plates were inoculated with GBS strain ATCC 12386, either as they were prepared or after 6 days of storage in the refrigerator, the pigment production scores were 4ϩ for all media, but GBS colonies were smaller in the media prepared without serum (1-versus 2-mm diameters). Nevertheless, after 6 days at either 22 or 30°C, the media performed differently (Fig. 1). Media prepared with unmodified starches and serum supported good GBS pigment production (scores of 3ϩ and 4ϩ). Media prepared with either 1 or 2% soluble starch and serum deteriorated, and pigment production diminished (scores of 1ϩ and 2ϩ). All the media prepared without serum supported good GBS pigment production (scores of 3ϩ and 4ϩ), but the colonies were smaller (about 1 mm in diameter). Owing to these results, and because the rice starch was easier to dissolv...
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