Apr 15, 1985 - Stuart's medium and Middlebrook TB (12A) medium ... were detected in human blood in 2 to 5 days, a notably shorter time period than that.
Vol. 23, No. 2
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1986, p. 401-403 0095-1137/86/020401-03$02.00/0 Copyright © 1986, American Society for Microbiology
Radiometric Method for the Rapid Detection of Leptospira Organisms N. MANCA, R. VERARDI, D. COLOMBRITA, G. RAVIZZOLA, E. SAVOLDI, AND A. TURANO* Instituto of Microbiology, University of Brescia, 25100 Brescia, Italy Received 15 April 1985/Accepted 15 October 1985
A rapid and sensitive radiometric method for detection of Leptospira interrogans serovar pomona and Leptospira interrogans serovar copenhageni is described. Stuart's medium and Middlebrook TB (12A) medium supplemented with bovine serum albumin, catalase, and casein hydrolysate and labeled with "4C-fatty acids were used. The radioactivity was measured in a BACTEC 460 (Johnston Laboratories). With this system, Leptospira organisms were detected in human blood in 2 to 5 days, a notably shorter time period than that required for the majority of detection techniques.
BACTEC model 460 (Johnston Laboratories) and expressed as the growth index (GI). The GI is defined as the quantity of radioactive 14CO2 produced which reflects the rate and amount of growth occurring in the bottle. Initial inoculum. Cultures of leptospira were inoculated into Stuart's medium and incubated at 30°C. After 6 days, twofold serial dilutions were made in appropriate medium to quantitate the organisms. Twenty microscopic fields were counted and the results were averaged (11). A 10-1 dilution was uncountable, whereas a 10-2 dilution usually contained seven to eight leptospires per microscopic field and a 10-3 dilution had less than one organism per field. One milliliter of each of these three dilutions was added to each of three different media: 10 ml of Stuart's medium with 8 ,uCi of [t4Cjasparagine, 10 ml of Stuart's medium with 4 ,uCi of [14C]asparagine, and 8 ml of Stuart's medium supplemented with 2 ml of Middlebrook TB (12A) medium containing 2 ,uCi of "4C-labeled fatty acids. In one experiment the GI was compared with the number of leptospires per microscopic field (Table 1). At the completion of the experiment, 0.1 ml of inoculum from each bottle was plated on Columbia agar plates with 5% sheep blood to detect contamination. Growth of L. interrogans serovar pomona in Stuart's medium supplemented with fatty acids, as determined by the GI, was maximum compared with Stuart's medium supplemented with [14C]asparagine (Fig. 1). A GI of 142 after 24 h of incubation and one of 991 after 5 days were seen in the 10-1 dilution. A GI of 20 after 24 h and a GI of 327 after 7 days were observed in the 10-2 dilution. After 6 days, the 10- dilution showed a GI of only 23 (data not shown). Growth could not be maintained after 7 days because death of leptospires occurred, possibly due to the metabolic
Human leptospirosis is characterized by the dissemination of the leptospires through both organs and body fluids. Indeed, leptospires can be isolated from blood, urine, and tissues by using semisynthetic media or inoculation of susceptible animals or both (4-6, 10). To detect the microorganism in blood or urine during leptospiremia or in a growth medium after inoculation with appropriate clinical specimens, it is necessary, after incubation, to visualize the organisms by dark-field microscopy (11). The sensitivity of microscopic observation is low, and it is also time-consuming, labor intensive, and potentially hazardous to laboratory personnel. To reduce the time of incubation (usually 6 to 14 days, although in some cases, leptospires may be seen after day 3) (1), labor, and the risk of infection, we have developed a rapid and sensitive radiometric assay for Leptospira interrogans serovar pomona and Leptospira interrogans serovar copenhageni, those leptospires most frequently seen as pathogens in Italy. Media. L. interrogans serovar pomona and L. interrogans serovar copenhageni were grown on Stuart's medium (10) (Difco Laboratories, Detroit, Mich.) and Middlebrook TB (12A) (Johnston Laboratories; Becton-Dickinson). The Middlebrook TB (12A) medium is an enriched Middlebrook 7H9 broth base which has been supplemented with bovine serum (fraction V), catalase, casein hydrolysate, and 14Clabeled fatty acid substrates not specified by the suppliers. L-[14C]asparagine (CFB95; Amersham International, Prodotti Gianni, Italy) was added to 10 ml of Stuart's medium to a final total concentration of 4 or 8 ,uCi. (Here and throughout the text we refer to the total concentration.) In other experiments, 2 ml of Middlebrook TB (12A) medium containing 2 ,uCi of "4C-labeled fatty acids, the primary carbon and energy source for leptospires (7), was added to 8 ml of Stuart's complete medium. Strains. L. interrogans serovar pomona and L. interrogans serovar copenhageni were selected from the stock culture collection of the Institute of Microbiology of the University of Brescia, Brescia, Italy, or were isolated from clinical specimens during the study. The isolates were checked for purity in Stuart's agar medium (11). Determination of growth. Leptospires were observed and counted with a Leitz Orthoplan microscope equipped with a dark-field condenser, a planar 40/0.65 170/0.17 objective, and periplan GW1OxM oculars. 14C activity was measured with a *
TABLE 1. Correlation between number of leptospires per microscopic field and GIa No. of leptospires (±SD)b Radiometric method (GI) 5 50 100 30 2 50 20 1 20
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L. interrogans serovar pomona was seeded in Stuart's medium supplemented with Middlebrook TB (12A) medium. b Values represent the average of five counts. c The reading was performed on day 5 of incubation. a
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NOTES
VOL. 23, 1986
products of fatty acid metabolism (9). With Stuart's medium supplemented with 4 ,uCi of ['4C]asparagine, however, growth could be maintained for longer than 7 days. In this medium, GIs of 33 and 98 were seen after 3 and 9 days (10-1 dilution). These results may be due to the insufficient concentration of labeled [14C]asparagine. When the concentration of ['4C]asparagine was raised to 8 ,Ci, a GI of 24 was observed after 24 h of incubation and one of 109 was seen after 6 days (10-' dilution). GIs of 23 after 4 days, 32 after 9 days, 29 after 9 days, and 34 after 14 days were seen for the 10-2 and 10-3 dilutions, respectively. The growth response of L. interrogans serovar copenhageni, using the same media as with L. interrogans serovar pomona, was virtually identical to that of the latter organism. Leptospirosis may be a life-threatening disease, and its rapid detection is relevant to both clinical diagnosis and prognosis. It has been noted that the number of leptospires in blood is very low (11). To simulate in vivo conditions, we have performed the experiments with different leptospiral inocula ranging from high to very low. Growth was detected more rapidly and easily than with traditional culture methods by using various media supplemented with 14C-labeled compounds, which can be used by leptospires as both a carbon and an energy source. If one considers the difficulty in growing Leptospira spp. and then the expertise required to detect growth microscopically, it appears that the radiometric method is advantageous. Comparable results have been observed for L. interrogans serovar pomona and L. interrogans serovar copenhageni. However, Stuart's medium, supplemented with Middlebrook medium with 14C-labeled fatty acids, appears to be optimal for growth. Stuart's medium supplemented with 8 ,uCi of [14C]asparagine may also be suitable, but 4 ,uCi of asparagine is insufficient. In four cases of human leptospirosis, using Stuart's medium supplemented with Middlebrook medium, we detected the leptospires in blood within 2 to 5 days. GIs ranged from 20 to 401. The death of leptospires after 7 days in Stuart's medium supplemented with labeled fatty acids does not affect the GI and is inconsequential once the growth has been detected and the diagnosis has been made. When urine or blood samples were seeded in Stuart's
403
medium, growth was not detected by dark-field examination before day 7 to 14 of incubation, on the average. In five additional cases of human leptospirosis, growth was detected from urine cultures in two cases after 2 days and from blood cultures in three cases, one each after 5, 6, and 14 days. As a result, we now routinely inoculate Stuart's medium supplemented with Middlebrook TB medium (12A). LITERATURE CITED 1. Alexander, A. D. 1980. Spirochetes, p. 376-382. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and J. P. Truant (ed.), Manual of clinical microbiology, 3rd ed. American Society for Microbiology, Washington, D.C. 2. Buddemeyer, E. U., G. M. Wells, R. Hutchinson, and G. S. Johnson. 1978. Radiometric estimation of the replication time of bacteria in culture: an objective and precise approach to quantitative microbiology. J. Nuclear Med. 19:619-625. 3. Deland, F. H., and H. N. Wagner. 1969. Early detection of growth with carbon 14-labelled glucose. Radiology 92:154. 4. Ellinghausen, H. C., Jr., and W. C. Cullogh. 1967. Nutrition of Leptospira pomona and growth of 13 other serotypes using fractionated oleic albumin complex (DAC) and medium of bovine albumin and polysorbate 80. Am. J. Vet. Res. 26:45-51. 5. Fischer, G. W., D. K. Powers, and M. M. Galton. 1958. Studies of techniques for the isolation of leptospires. I. The suitability of 1-2 day old baby chicks as compared to hamsters for the isolation of various leptospiral serotypes. J. Infect. Dis.
103:150-156. 6. Fletcher, W. M. 1928. Recent work on leptospirosis, tsutsugamushi disease and tropical typhus in the Federal Malay States. Trans. R. Soc. Trop. Med. Hyg. 21:265-287. 7. Johnson, R. C., V. C. Harris, and J. K. Walby. 1969. Characterization of leptospires according to the fatty acids requirements. J. Gen. Microbiol. 55:399-407. 8. Middlebrook, G., Z. Reggiardo, and W. H. Tigert. 1977. Automated radiometric detection of Mycobacterium tuberculosis. Am. Rev. Respir. Dis. 115:66-69. 9. Smibert, R. M. 1973. The Spirochaetales, p. 161-190. In A. I. Laskin and H. A. Lechevalier (ed.), Handbook of microbiology, vol. 1. CRC Press, Inc., Cleveland. 10. Stuart, R. D. 1946. The preparation and use of a simple culture
medium for Leptospirae. J. Pathol. Bacteriol. 58:343-349. 11. Sulzer, C. R., and W. L. Jones. 1980. Leptospirosis: methods in laboratory diagnosis. DHEW pub. (CDC) 16-8275 (rev.) Centers for Disease Control, Atlanta.
