CLINICAL MICROBIOLOGY AND INFECTIOUS ...

CLINICAL MICROBIOLOGY

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INFECTIOUS

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PATRICIA MUNOZ, MD, MARIA DOLORES DiAZ, MD, EMILIA CERCENADO, PHARMD, MARTA RODRiGUEZ-CREIXEMS, MD, JUAN BERENGUER, MD, AND EMILIO BOUZA, MD, PHD

91.8%). Neither the culture medium used nor the production of H2S affected the accuracy of the test. The results from this study showed that the MUCAP test is a rapid, simple, and sensitive method for the presumptive identification of Salmonella species. (Key words: Salmonella; MUCAP test; Stool cultures; Rapid diagnosis) Am J Clin Pathol 1993;100:404-406.

and Brilliant Green agar (Becton Dickinson Laboratories, United Kingdom), which were incubated overnight. In addition, Aeromonas- and Campylobacter-selective media were used. Samples also were inoculated into a selenite enrichment broth from which a subculture was made on SS agar after 18 hours of incubation. All colonies suspected of being Salmonella (lactose-negative or H2S-producing colonies detected on MacConkey, SS, or Brilliant Green agar) were picked off for a Triple Sugar Iron agar test and subculture. They also were identified with the MicroScan AutoSCAN-4 (Baxter Laboratories, Sacramento, CA) and confirmed by serologic tests with commercial antisera (Difco Laboratories, Detroit, MI). 10 " Approximately 25% of these Salmonella colonies were sent to a referral laboratory for additional serologic characterization. If no lactose-negative or H2S-positive colonies were present, plates were discarded. The remaining colonies were checked afterward with the MUCAP test. For the fluorescence test, plates were examined first in semidarkness under a Wood's lamp (wavelength, 365 nm), provided by the manufacturer, to identify naturally fluorescent strains. Suspect colonies then were flooded aseptically with one drop of MUCAP test reagent and incubated for three to five minutes in semidarkness; then they were examined MATERIALS AND METHODS under the Wood's lamp. The development of a blue fluorescence over or around the colony was considered a positive reDuring a three-month period, all clinical stool specimens sult. received in our laboratory for bacteriologic culture were inocuThe sensitivity, specificity, and predictive value of the posilated onto MacConkey agar, Salmonella-Shigella agar (SS), tive and negative test results were calculated by standard formulae.12 The identification steps were repeated so that the biochemical characteristics of the colonies before and after From the Servicio de Microbiologic! Clinica, Hospital Generaladdition "Gre- of MUCAP reagent could be compared. The previgorio Maraflon," Madrid, Spain. ously flooded colonies were subcultured at 24 and 48 hours to investigate the possible influence over the strain viability. Presented in part at the 30th ICAAC, Atlanta, Georgia, September 1990. Received June 18, 1992; revised manuscript accepted for publication RESULTS February 8, 1993. Address reprint requests to Dr. Mufioz: Servicio de Microbiologia During the study period, 976 colonies from 555 clinical stool Clinica, Hospital General "Gregorio Maraflon," Dr. Esquerdo 46, specimens from 480 patients were examined, and 176 colonies 28007 Madrid, Spain. Salmonella infections are still a major public health and economic problem in many countries. The standard laboratory procedure for identifying presumptive Salmonella colonies requires at least 48 hours. It is especially tedious and time-consuming when cultures are heavily contaminated, such as those from fecal specimens.1 Various rapid diagnostic methods have been used to simplify and speed up the procedure for stool cultures, but most methods require a large capital outlay for equipment.2"5 The MUCAP test (Biolife Italiana, Milan, Italy) is a rapid fluorogenic screening method for Salmonella. It provides the substrate, an eight-carbon ester conjugated with 4-methyl-umbelliferone, in a sterile organic solvent. In the presence of a Salmonella-specific enzyme (C8-esterase), the substrate is cleaved, and umbelliferone, which is strongly fluorescent, is released.6 The technique has been used to identify various microbial species rapidly,7,8 but there appears to be little information regarding its application to the identification of Salmonella.9 Therefore, we evaluated the potential usefulness of the MUCAP test in the routine screening for Salmonella in clinical stool cultures.

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The authors compared a three-minutefluorescencemethod (MUCAP test; Biolife Italiana, Milan, Italy) with standard microbiologic procedures for the presumptive identification of Salmonella from stool cultures on three solid culture media. From 976 suspect colonies, 176 proved to be Salmonella by the traditional methods. They all were detected by the MUCAP test (sensitivity, 100%). There were 65 false-positive results, corresponding mostly to Pseudomonas (specificity,

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Salmonella on tool Cultures TABLE 1. MUCAP TEST RESULTS OBTAINED ON DIFFERENT SELECTIVE MEDIA

DISCUSSION Other rapid techniques for the presumptive identification of Salmonella have been described, but they are not as fast and sensitive as this one.13,14 In our experience, the MUCAP test was equally effective on the three culture media used, although SS agar provided the best recovery of Salmonella. Some authors found that the specificity of the test was significantly higher on MacConkey agar than on SS agar,9 but another study, performed with stock cultures of Salmonella, obtained similar results on Hektoen, XLD, Levine modified, and SS media.15 According to these data, the test could prove useful in any laboratory, regardless of the culture medium used. Although we did not find any naturally fluorescent strains, they probably should not be tested with the MUCAP test. There was concern regarding the efficacy of the test in a labora-

TABLE 2. STRAINS RESPONSIBLE FOR FALSE-POSITIVE RESULTS

Microorganisms

MacConkey Agar SS Agar BG Agar Total (n = 25) (n = 30) (n = 10) (n = 65)

Pseudomonas aeruginosa 16 Citrobacler freundii 2 Proteus mirabilis 1 Escherichia coli 3 Klebsiella pneumoniae 1 Kluyvera spp. 1 Enterobacter cloacae 1

25 2 2 1 — — —

SS = Salmonella-Shigella agar; BG = Brilliant Green agar. No. 4


tory with a low prevalence of Salmonella isolates. For instance, the expected false-positive rate in a laboratory with only a 2% prevalence of salmonellosis would be 8% instead of the 6.6% we No. of Results by MUCAP Test on: obtained with our 20% incidence of salmonellosis; however, because most false-positive findings result from Pseudomonas MacConkey Agar SS Agar BG Agar species, this apparently high false-positive rate could be reto 1.6% with an oxidase test on MUCAP-positive (n = 317) (n = 532) (n = 127) duced easily colonies.16 Bacteria (+) (-) (+) (-) (+) (-) In our opinion, clinical laboratories should consider including the MUCAP test in their stool culture procedure as a preSalmonella spp. 41 0 103 0 32 0 sumptive identification tool for the rapid screening of SalmoOther organisms 25 251 30 399 10 85 nella on solid culture media. It can enhance the rate of recognition of Salmonella colonies in the presence of mixed SS = Saltnonella-Shigella agar; BG = Brilliant Green agar. non-lactose-fermenting bacteria on the agar surface. It also facilitates the detection of rare colonies with uncommon biochemical characteristics (lactose fermenting or non-H2S producing) without increasing the laboratory work load. Because (18%) from 103 patients were identified as Salmonella species. of its high sensitivity, it could completely take the place of The serogroups were as follows: serogroup D (Salmonella enroutine bacteriologic procedures for the screening of Salmoteritidis, 100 cases), serogroup B (Salmonella typhimurium and Salmonella bredeney, 53 cases), serogroup C (Salmonella vir- nella from stool culture plates from possible carriers or infected chow, 21 cases), and nongroupable Salmonella (2 cases). In our patients during an outbreak and for the detection of Salmonella-contaminated food. The test also could be very useful in study, no patient had more than one serogroup. monitoring infected patients during or after therapy. All the Salmonella colonies were detected accurately with In our experience, as well as that of others, a negative MUthe MUCAP test, so there were no false-negative results (sensiCAP test result ruled out the presence of Salmonella.1617 tivity, 100%; negative predictive value, 100%); however, there Aguirre and associates9 reported four false-negative results, but were 65 false-positive results (6.6%), 49 corresponding to Pseuthree occurred in single colonies mixed with other enteric flora domonas species. The test showed a specificity of 91.8% and a and the results of the MUCAP test became positive after the positive predictive value of 74%. If the easily detectable oxicolonies were subcultured on a new SS agar plate to obtain a dase-positive colonies (Pseudomonas) were excluded, the test pure culture. This excellent sensitivity (100%) makes its use would have only a 1.6% false-positive rate. No spontaneous particularly suitable for clinical, epidemiologic, and industrial fluorescent strains were encountered. screening procedures. On this basis, the MUCAP test can be It is interesting that we found no significant difference in the remarkably useful for the routine screening of Salmonella in efficacy of the test on the three culture media used, although SS especially predisposed people, such as human immunodefiagar was the most useful in recovering Salmonella species from ciency-positive or renal transplant patients, in whom Salmostool specimens (Table 1). The false-positive results from the nella also can be recovered frequently from blood and urine three media are listed in Table 2. The specificity and predictive cultures. value of the positive test results for the three culture media were The reporting of either a negative or positive presumptive 93% and 77% for SS agar, 90% and 62% for MacConkey agar, result only 18 hours after the stool sample has been collected and 89% and 76% for Brilliant Green agar, respectively. provides the clinician with useful information regarding the The MUCAP test correctly identified three lactose-fermentcause of the diarrhea, with a minimum delay. Naturally, for ing (all H2S positive) and one non-H2S-producing Salmonella clinical purposes, other enteric pathogens, mostly Shigella, strain. The biochemical characteristics of the colonies were not must be investigated further and results reconfirmed or altered by the previous application of the MUCAP test reagent corrected the following day. In Spain, the estimated cost is nor viability because flooded colonies grew adequately on the approximately $ 1.00 per test. Laboratories with a lower incisubcultures performed at 24 and 48 hours. These data suggest dence of salmonellosis should determine whether it is worththat there seems to be no need for a previous subculture. while to incorporate the test in routine procedures or reserve it for special circumstances. Additional studies are warranted.

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CLINICAL MICROBIOLOGY AND INFECTIOUS DISEASE Original Article Kelly MT, Brenner DJ, Farmer JJ III. Enterobacteriaceae. In: Lennette EH, Ballows A, Hausler WJ, Shadomy HJ, eds. Manual of Clinical Microbiology. Washington, DC: American Society for Microbiology, 1990, pp 263-277. Rhoden DL, Smith PB, Baker CN, Schable B, Stocker SA. AutoSCAN-4 system for identification of gram-negative bacilli. J Clin Microbiol 1985;22:915-918. Sackett DL, Haymes RB, Tugwell L. Epidemiology: A Basic Science for Clinical Medicine. Boston: Little Brown and Company, 1985. Nordlander E, Phuphaisan S, Bodhidatta L, Arthur J, Echeverria P. Microscopic examination of stools and a latex slide agglutination test for the rapid identification of bacterial enteric infections in Khmer children. Diagn Microbiol Infect Dis 1990;13:273-276. Metzler J, Nachamkim 1. Evaluation of a latex agglutination test for the detection of Salmonella and Shigella spp. by using broth enrichment. J Clin Microbiol 1988;26:2501-2504. Pontello M, Russolo S, Carozzi F, Bottiroli U. Evaluation of a new, rapid method (MUCAP test) for the presumptive identification of Salmonella on primary isolation media. In: Fifth International Symposium on Rapid Methods and Automation in Microbiology and Immunology; November 4-6, 1987; Florence, Italy. Ruiz J, Varela MC, Sempere MA, et al. Presumptive identification of Salmonella enterica using two rapid tests. Eur J Clin Microbiol Infect Dis 1991; 10:649-651. Ollson M, Sik A, Wollin R. Identification of salmonellae with the 4-methylumbelliferyl caprilate fluorescence test. J Clin Microbiol 1991;29:2631-2632.

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10. REFERENCES 1. Sack BR, Tilton RC, Weissfeld AS. Laboratory Diagnosis of Bacterial Diarrhea. Cumitech 12. Washington, DC: American Society for Microbiology, 1980. 11. 2. Greene L, Appelbaum PC, Kellogg JA. Evaluation of a two hour method for screening pathogens from stool specimens. J Clin Microbiol 1984;20:285-287. 12. 3. Teti G, Burdash NM, Zamboni C, et al. Evaluation of a rapid method to exclude the presence of certain enteric pathogens in stool specimens. J Clin Microbiol 1984;20:1049-1052. 13. 4. Villasante PA, Aguilar A, Merino FJ, et al. Rapid automated method for screening of enteric pathogens from stool specimens. J Clin Microbiol 1987;25:584-585. 5. Yong D, Thompson TS, Prytula A. Rapid microbiochemical method for presumptive identification of gastroenteritis asso14. ciated members of the family Enterobacteriaceae. J Clin Microbiol 1985;21:914-918. 6. Maddocks JL, Greenam MJ. Technical method: A rapid method 15. for identifying bacterial enzymes. J Clin Pathol 1975;28:686687. 7. Godsey JH, Matteo RM, Shen D, Tolman G, Gohlke JR. Rapid identification of Enterobacteriaceae with microbial enzyme activity profiles. J Clin Microbiol 1981; 13:483-490. 8. Littel KJ, Hartman PA. Fluorogenic selective and differential me16. dium for isolation of fecal streptococci. Appl Environ Microbiol 1983;45:622-627. 9. Aguirre PM, Cacho JA, Folgueira L, et al. Rapid fluorescence 17. method for screening Salmonella spp. from enteric differential agars. J Clin Microbiol 1990;28:148-149.