Rapid presumptive identification of streptococci directly from blood cultures by serologic tests and the L-pyrrolidonyl-beta-naphthylamide reaction.

The value of the Strepslide kit for the rapid presumptive identification of streptococci directly from blood cultures without prior determination of hemolysis patterns was assessed and compared with that of the Streptex and Phadebact streptococcus kits. Studies involved 94 simulated and 60 clinical isolates of 83 streptococci. The Streptex and Strepslide kits had excellent sensitivity and specificity for group A, B, F, and G organisms, and the Phadebact kit had excellent sensitivity and specificity for groups B and G. Group C reactions usually occurred with all of the streptococcus kits with pneumococci and occasionally with alpha-hemolytic streptococci. Although these kits were unacceptable for group C and D organisms, enterococci which were common clinical isolates could be directly identified in blood cultures by a supplementary rapid L-pyrrolidonyl-beta-naphthylamide biochemical test. Direct application of the Phadebact pneumococcus kit to blood cultures was also assessed with 29 isolates of 20 organisms. The specificity was good, but the sensitivity was only 65.5%.     

Rapid method for identification of group B streptococci in neonatal blood cultures.

A rapid technique used for the identification of Streptococcus agalactiae, Lancefield group B, from the blood cultures of two neonatal infants is reported. The method utilized the Phadebact Streptococcus Test System (Pharmacia Diagnostics, Piscataway, N.J.) and the supernatant from 13- and 14-h blood cultures. Additional studies with simulated neonatal blood cultures revealed that this method was reproducible. Additional studies also revealed that some non-specific agglutination did occur, which could not be eliminated with dithiothreitol, but was visibly reduced by treatment with soluble staphylococcal protein A.     

Evaluation of techniques for isolation of group A streptococci from throat cultures.

In the first study, selective sulfamethoxazole-trimethoprim blood agar (SXT-BA) and conventional blood agar (BA) plates incubated under CO2 and anaerobically were compared for their ability to recover group A streptococci from throat cultures. Recovery rates were: SXT-BA (anaerobic), 100%; SXT-BA (CO2), 98%; BA (anaerobic), 89.2%; and BA (CO2), 76.5%. Primary plate bacitracin test results could be read on significantly more of the SXT-BA plates. Readability rates were: SXT-BA (anaerobic), 97%; SXT-BA (CO2), 96%; BA (anaerobic), 70.6%; and BA (CO2), 32.4%. A second study compared with the SXT-BA method versus a BA-double-disk (BA-DD) method which utilizes conventional media with addition of a bacitracin differentiation and a sulfamethoxazole-trimethoprim susceptibility disk placed adjacent to one another in the heavy area of inoculation. Isolation rates were: SXT-BA, 100% and BA-DD, 88%. Readability rates for direct bacitracin tests were: SXT-BA, 92% and BA-DD, 76.2%. In our hands, the SXT-BA method was superior for yielding highest isolation rates and for yielding highest readability rates of direct bacitracin test results.     

Evaluation of anaerobic incubation for recovery of group A streptococci from throat cultures.

No statistical differences were found in the recovery of group A streptococci from throat culture specimens after overnight incubation of blood agar plates in 5% CO2 compared with anaerobiosis. Anaerobic incubation required many more subcultures and resulted in considerably greater technical time and expense.     

Serological identification of group A streptococci from throat scrapings before culture.

The use of a microtechnique (modified nitrous acid extraction) to test samples from 150 school children and from patients with acute follicular tonsillitis has indicated that group A streptococci in the throat can be identified from tonsillar scrapings in 30 min. The results are comparable to the grouping results obtained by standard throat culture techniques and the Lancefield procedure for grouping. No cross-reaction with other bacteria or cellular material occurs. Study has also shown that the nitrous acid extraction yields three- to fourfold more polysaccharides than the Lancefield hot-HCl of Fuller formamide techniques. The use of the microtechnique leads to another 20-fold concentration of the antigen. Immune salting-out effect could be obtained with 1.00 M sodium acetate. Such molarity is too low to cause nonspecific slating out. It leads to a strong ampliciation of the precipitin reaction.     

Rapid species identification of group C streptococci isolated from horses.

Two commercial systems, the API 20S (Analytab Products, Plainview, N.Y.) and the Rapid Strep (API System S.A., Montalieu-Vercieu, France), were evaluated for ease of use and accuracy in the rapid identification of group C streptococci isolated from horses. A total of 85 Streptococcus isolates were tested, including S. equi (67 isolates), S. zooepidemicus (13 isolates), and S. equisimilis (5 isolates). All S. equi and S. zooepidemicus isolates were correctly identified within 24 h by the Rapid Strep system. Specific grouping sera was necessary to distinguish between S. equisimilis and group G or L strains. The API 20S system did not provide species identification of any of these isolates. An identification of randomly selected isolates to species level was performed by conventional methods and confirmed the identification derived through the Rapid Strep system. Our results indicate that the Rapid Strep system is a valuable aid for species identification of equine isolates of group C streptococci.     

Liposome immunoassay for rapid identification of group A streptococci directly from throat swabs.

The Q Test Strep (Becton Dickinson and Co., Franklin Lakes, N.J.) is a new solid-phase liposome immunoassay for the rapid diagnosis of group A beta-hemolytic streptococcal pharyngitis. Compared with blood agar plate cultures, the Q Test Strep had a sensitivity of 91%, a specificity of 83%, a positive predictive value of 88%, and a negative predictive value of 87%. Liposome technology can be used to facilitate the rapid diagnosis of group A beta-hemolytic streptococcal pharyngitis.     

Rapid identification of group B streptococci by counterimmunoelectrophoresis.

Beta-hemolytic streptococcal isolates have been examined by counterimmunoelectrophoresis (CIE) with group B antiserum to determine whether this techinque is of value in the rapid identification of group B strains. Ninety stock cultures and 100 clinical isolates of beta-hemolytic streptococci including representatives of groups A, D, C, G, and B were inoculated into Todd-Hewitt broth; after incubation at 37 C for 1, 2, 3, and 4 h, aliquots of the whole broth cultures were removed and tested by CIE. Antigen was not regularly detected in the 1-, 2-, and 3-h samples, but after 4 h all 126 group B streptococcal strains identified by the capillary precipitin reaction gave CIE precipitin bands with group B antiserum. None of the 58 non-group B strains gave precipitin reactions with this antiserum. Cerebrospinal fluid from an infant with group B streptococcal meningitis and peritoneal fluid from a patient with group B streptococcal peritonitis had free group B antigen detected by the CIE technique. CIE of broth cultures and direct body fluids appears to be a rapid and sensitive method for the identification of group B streptococcal strains.     

Rapid coagglutination test for the direct detection of group A streptococci from throat swabs

A one-minute antigen detection test was compared with a conventional culture method for detecting group A -hemolytic streptococci. The test detects coagglutination between protein A and streptococcal antigen extracted directly from throat swabs. Of the 307 specimens tested, 66 (21.5%) were positive for group A streptococci by culture and 16 specimens (5.2%) were positive for other -hemolytic streptococci. The direct test agreed with the culture in 274 of 307 specimens (accuracy 89.3 %). The sensitivity of the test was 86.4 % (57/66), the specificity 90 % (217/241), the positive predictive value 70.4 % and the negative predictive value 96 %. If only throat cultures with more than 100 colonies of group A streptococci per plate, were considered, the sensitivity of the direct test rose to 96 %. If only a strong agglutination was considered positive, the specificity of the direct test rose to 98 %. Further studies are needed to determine whether this test could be used alone or in addition to culture.     

Identification of the site on IgG Fc for interaction with streptococci of groups A, C and G.

The interaction between living groups A, C and G streptococci and IgG Fc was studied using human IgG, IgG Fc and IgG Fc-intermediate (Fci) fragments, chemically modified human IgG and fragment D of staphylococcal protein A (SPA). Diethylpyrocarbonate modification of His or N-acetylimidazole modification of Tyr of human IgG resulted in the loss of its capacity to inhibit the binding of radiolabelled human IgG Fc to the group A streptococci types M1 and M55, and to the group C strain SC-1, indicating that the amino acids His and Tyr are involved in the binding. Lys seems not to participate in the binding of IgG to these bacteria, however, since reductive methylation of Lys did not reduce its inhibitory capacity. Fragment D of SPA also inhibited the binding of radiolabelled human IgG Fc to strains M1, M55 and SC-1. We have previously shown that these bacteria do not bind to IgG fragments consisting of only the C gamma 2 or C gamma 3 domains. On the basis of these results, and the known relative positions in space of the His and Tyr residues on IgG Fc, it is speculated whether streptococci with IgG Fc receptors, like SPA and rheumatoid factors, interact with IgG in the interface between the C gamma 2 and C gamma 3 domains and involve His 435 and one or more of Tyr 436, His 433 and His 310. The similarities in binding sites on IgG for RFs and these bacterial Fc binding proteins suggest structural similarities between them that may be relevant to the production of rheumatoid factors in rheumatoid arthritis.     

Selective and enhanced recovery of group A and B streptococci from throat cultures with sheep blood agar containing sulfamethoxazole and trimethoprim.

Sheep blood agar containing 23.75 microgram of sulfamethoxazole and 1.25 microgram of trimethoprim (SXT-BA) per ml was compared with conventional sheep blood agar (SBA) for isolating group A and B streptococci from throat cultures. This selective medium allowed much better recovery of group A and B streptococci and suppressed the growth of the normal flora, including "viridans" streptococci. In an initial study of 700 throat cultures, SXT-BA recovered 42% more group A and 49% more group B streptococci than did SBA. When SXT-BA was introduced into the routine microbiology laboratory and used by a number of medical technologists. SXT-BA recovered 28% more group A and 37% more group B streptococci than did SBA. In addition, the selective medium inhibited 83% of the non-group A and B streptococci that were recovered by SBA.     

Effect of atmosphere and duration of incubation on primary isolation of group A streptococci from throat cultures.

The optimal incubation conditions for isolation of group A streptococci from throat cultures are controversial. Therefore, we compared the effects of aerobic and anaerobic incubations after 24 and 48 h on the recovery of group A streptococci. Throat swabs submitted to the clinical laboratory were inoculated onto duplicate 5% sheep blood agar plates, incubated aerobically or anaerobically (GasPak jar) at 35 degrees C, and examined semiquantitatively after 24 and 48 h. Group A streptococci were identified by the fluorescent-antibody technique. Of 1,040 specimens, 506 (48.6%) grew beta-hemolytic streptococci, including 200 (19.2%) group A streptococci. Group A streptococci were recovered significantly more often with anaerobic incubation than with aerobic incubation after 24 h (182 versus 138; P less than 0.001) and after 48 h (193 versus 174; P less than 0.05). Non-group A beta-hemolytic streptococci also were recovered significantly more often with anaerobic incubation after 24 and 48 h (P less than 0.001). Colony counts were not affected by the incubation atmosphere. We conclude that incubation of throat cultures in an anaerobic atmosphere is superior to incubation in air for detection of group A streptococci. The greater sensitivity of anaerobic incubation, however, may not justify the extra laboratory effort and cost required to differentiate group A streptococci from the non-group A streptococci detected as a result of anaerobic incubation. Throat cultures should be examined after 24 and 48 h, especially if plates are incubated aerobically.     

Rapid biochemical tests for characterization of the Mycoplasmatales.

Methods are described for the rapid detection of beta-D-glucosidase and phosphatase in mycoplasma cultures using fluorogenic 4-methylumbelliferone substrates. These methods were applied to a selection of mycoplasma cultures and were compared with the conventionally used tests for these enzymes. Results were similar by both methods, but the fluorogenic tests could be read after 1 h, whereas the conventional tests took several days.     

Rapid presumptive identification of gram-negative rods directly from blood cultures by simple enzymatic tests.

Gram-negative rods were presumptively identified directly from blood cultures within 15 min as Escherichia coli, a member of the Klebsiella-Enterobacter group, or oxidase positive. Samples of artificially seeded blood cultures (193 cultures) and patient blood cultures (78 cultures) were filtered into a Dynadepth test card with the Bac-T-Screen instrument (Vitek, Inc., Hazelwood, Mo.). Triton X-100 was then filtered into the test card to lyse the blood cells but not the entrapped bacteria, and either methylumbelliferone-labeled substrates or oxidase reagent was applied to the filter surface. The oxidase test was read within 30 s, and the methylumbelliferone and indole tests were read after a 10-min incubation at room temperature. Positive beta-galactosidase, beta-glucuronidase, and indole test results predicted the identification of E. coli with a 96 to 100% sensitivity and a 99 to 100% specificity. Positive beta-xylosidase and beta-galactosidase test results and negative oxidase and beta-glucuronidase test results were 85 to 93% sensitive and 100% specific for a Klebsiella-Enterobacter organism. A positive oxidase test result and negative beta-glucuronidase, beta-xylosidase, and indole test results were highly predictive of Pseudomonas aeruginosa (sensitivity, 100%; specificity, 99%). The procedures described are rapid and simple and provide a direct presumptive identification of the gram-negative rods most commonly found in blood cultures.     

Rapid commercial test for direct detection of group A streptococci in throat swabs

The Hybritech Strep A ICON was used for direct testing of 1016 throat specimens for group A betahemolytic streptococci. Both the test and culture were negative in 829 specimens (81.6 %); both were positive in 164 cases (16.1 %); the test was positive and culture negative in 9 cases (0.9 %); and the test negative and culture positive in 14 cases (1.4 %).     

Distribution of B streptococcal type antigens among streptococci of serological groups B, G and L

Serotyping of streptococci of serological group B isolated from humans revealed mainly the type patterns Ia/c and III/R. The protein antigen c components were c alpha beta, c alpha and less frequently, c beta. Extracts of two group B streptococcal cultures reacted with specific antiserum against type candidate 7271. Bovine group B streptococci mainly expressed the type antigens IV/X and NT/X. Protein antigen c occurred as c alpha beta, c alpha and less frequently as c beta component. Among the non group B streptococcal cultures used in this study, only those of serological groups G and L reacted with specific antisera against protein antigens R and X.