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been seen in university students (9). We believe that 2 g per day of tetracycline or erythromycin for 14 days is often an effective therapy. T h e relatively long time (nearly four months) n e e d e d for spread of the infection through the four families is similar to the slow spread observed in military trainee o u t b r e a k s (10). T h e prolonged case-to-case intervals suggest that Chlamydia pneumoniae has a long incubation period.

Eur. J. Clin. Microbiol. Infect. Dis.

10. Kleemola M, Saikku P, Visakorpi R, Wang SP, GraysIon JT: Epidemics of pneumonia caused by TWAR, a new Chlamydiaorganism, in military trainees in Finland. Journal of Infectious Diseases 1988,157: 230-236.

Detection of Chlamydia trachomatis by the Polymerase Chain Reaction in Young Patients with Acute Epididymitis

References 1. Grays!on JT, Campbell LA, Kuo CC, Mordhorst CH, Saikku P, Thorn D, Wang SP: A new respiratory tract pathogen: Chlamydia pneumoniae, strain TWAR. Journal of Infectious Diseases 1990, 161: 618-625. 2. Wang SP, Grayston JT: Population prevalence antibody to Chlamydia pneumoniae, strain TWAR. In: Bowie WR, Caldwell HD, Jones RP, M~trdh PE, Ridgway GL, Schachter J, Stamm WE, Ward ME (ed): Chlamydial infections. Cambridge University Press, Cambridge, 1990, p. 402--405. 3. Aldous MB, Wang SP, Foy HM, Grayston J-T: Chlamydia pneumoniae, strain TWAR, infection in Seattle children and their families, 1965-1979. In: Bowie WR, Caldwell HD, Jones RP, M~trdh PE, Ridgway GL, Schachter J, Stamm WE, Ward ME (ed): Chlamydial infections. Cambridge University Press, Cambridge, 1990, p. 437-440. 4. Gvayston JT, Mordhors! CH, Bruu AL, Vene S, Wang SP: Countrywide epidemics of Chlamydia pneumoniae, strain TWAR, in Scandinavia, 1981-1983. Journal of Infectious Diseases 1989, 159: 1111-1114. 5. MordhorsI CH, Wang SP, Myhra W, Grayston JT: Chlamydia pneumoniae, strain TWAR, infections in Denmark 1975-1987. In: Bowie WR, Caldwell HD. Jones RE Mhrdh PE, Ridgway GL, Sehaehter J, Stamm WE, Ward ME (ed): Chlamydial infections. Cambridge University Press, Cambridge, 1990, p. 418421. 6. Myhra W, Mordhorst CH, Wang SP, Grays!on JT: Clinical features of Chlamydia pneumoniae, strain TWAR, infection in Denmark 1975-1987. In: Bowie WR, Caldwell HD, Jones RP, Mhrdh PE, Ridgway GL, Schachter J, Stamm WE, Ward ME (ed): Chlamydial infections. Cambridge University Press, Cambridge, 1990, p. 422-425. 7. Volkert M, Christensen PM: Two ornithosis complement-fixing antigens from infected yolk sacs. I. The phosphatide antigen, the virus antigen and methods for their preparation. Acta Pathologica et Microbiologica Scandinavica i956, 37: 211-218. 8. Wang SP, Grayston JT: Immunologic relationship between genital TRIC, lymphogranuloma venereum, and related organisms in a new microtiter indirect immunofluorescence test. American Journal Ophthalmology 1970, 70: 367-374. 9. Grayslon JT, Kuo CC, Wang SP, Altman J: A new Chtamydia psittaci strain, TWAR, isolated in acute respiratory tract infections. New England Journal of Medicine 1986, 315: 161-168.

A. Eley 1., K.M. Oxley 1, R.C. S p e n c e r 1, G.R. K i n g h o r n 2, E.T. B e n - A h m e i d a 1, C.W. P o t t e r 1

Specimens from 11 patients presenting with acute epididymitis were tested for the presence of Chlamydia trachomatis by an e n z y m e i m m u n o assay ( E I A ) , growth in M c C o y cells and the polym e r a s e chain reaction (PCR), and for other microorganisms by standard laboratory techniques. Chlamydia trachoma!is urethral infection was detected in four patients by tissue culture, in three patients by E I A and in nine patients by P C R , These findings confirm the usually low detection rate of Chlamydia trachomatis by conventional tissue culture and E I A . D e t e c t i o n by P C R indicated both the diagnostic value o f this technique and the i m p o r t a n c e o f this organism in epididymitis.

Patients with acute epididymitis can be divided into two age groups: in older persons (> 45 years) the disorder is usually associated with urinary

tract infections and the presence of urinary tract pathogens such as m e m b e r s of the Enterobacteriaceae (1, 2), whilst in younger persons (< 45

years) infection is commonly associated with sexually transmitted disease (3, 4). Chlamydia trachomatis has now b e e n established as the commonest cause of epididymitis in young men, the detection rate often being in the range of 30-50 % (5-7). However, in a high proportion of these cases detection has been based solely on raised 1Department of Experimental and Clinical Microbiology, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, UK. 2Genitourinary Medicine, Royal Hallamshire Hospital, Sheffield, UK.

Vol. 11, 1992

621

of C h l a m y d i a t r a c h o m a t i s (12). These primers generate a 517bp amplified product.

antibody titres. This still leaves a majority of patients in which no infective cause can be found. One reason for this may be the relative insensitivity of conventional laboratory methods used for the detection of chlamydia infection. In this StUdy we assess the potential of PCR to detect C h l a m y d i a t r a c h o m a t i s in patients presenting With acute epididymitis.

The technique of PCR was performed on 5 pl of processed specimen DNA in a total volume of 50 ~1 of reaction mixture containing 10 mM Tris HC1 of pH 8.3, 50 mM KC1, 2.5 mM MgC1, 0.01% gelatin, 200 M of dNTP and 1 unit of Ampli Taq DNA polymerase (Perkin-Elmer Cetus, USA) plus 100 pmol of each primer (reconstituted according to the manufacturer's instructions). This mixture was subjected to UV light irradiation for 10 min in an Amplirad (Genetic Research Instrumentation, UK) to inactivate possible contaminated DNA before the addition of template DNA. Acknowledged procedures were performed to reduce sample-to-sample contamination in a safety hood including the use of positive displacement pipettes. Solutions were overlayed with three drops of mineral oil (Sigma, UK) to prevent evaporation, and subjected to 40 cycles of amplification using a Hybaid intelligent heating block (Hybaid, UK); each cycle consisted of 1 min at 94 °C, 2 min at 42 °C and 3 min at 74 °C. Water and the 7.5 kb purified C h l a m y d i a t r a c h o m a t i s plasmid designated PGLV 440 (12) were used as negative and positive controls in every PCR run, respectively. Furthermore, material from an uninoculated swab in transport medium was confirmed as a target-negative PCR control. The PCR product was electrophoresed in 2 % agarose gel stained with ethidium bromide and visualised using a UV-transilluminator. After electrophoresis the amplified products were transferred by Southern blotting and hybridization performed using the method of Claas et al. (13) with the DNA probe 3' labelled

a n d M e t h o d s . Urine samples and urethral swabs were collected from 11 patients presenting at the Department of Genito-Urinary Medicine, Royal Hallamshire Hospital, Sheffield, UK. Only five patients produced expressed prostatic secretions. For detection of C h l a m y d i a t r a c h o m a t i s urethral swabs were placed in transport medium (8) and cultured in McCoy cells without passage and on shell vials in Eagle medium using the technique of Ripa and Mardh (9). A second urethral swab was tested by an EIA (Chlamydiazyme, Abbott Diagnostics, USA). Materials

Samples of expressed prostatic secretion from four patients were also tested in tissue culture. PCR was performed on DNA extracted from the Urethral swabs collected in transport medium, and four prostatic secretion samples which were Subsequently stored at -70 °C. DNA was extracted from a 200 pl sample of urethral swab material in transport medium or 200 /al of expressed prostatic secretion using the method of Ossewaarde et al. (10) and stored at -20 "C for SUbsequent testing. Two oligonucleotide sequences, each of 18 nucleotides (British Biotechnology, UK) were used as primers for the PCR; these Were G G A C A A A T C G T A T C T C G G and GAAACCAACTCTACGCTG as described and used by Claas et al. (11) and taken from the published nucleotide sequence of the endogenous plasmid

Table 1: Resultsof tests to detect microorganismsin specimensfrom 11patientspresentingwith acute epididymitis. Patient no.

Age

Duration of Testsfor C. trachomatis in urethral swabs symptoms (days) PCR Culture EIA

Tests for other organisms Urine

Urethral swabs

----__._.

1 2 3 4

5 6 7 8 9 10 I1

22 31 25 31 20 36 28 20 30 38 25 , ,,,,,,,,,,

7

.

1 4 10 2

+ + + +

.

+ . . +

.

2

+

+

14

+

7

+

1 14 2

+ + .

. . .

.

G. vaghlalis

-

-

+

. . -

+

E. coli

-

+

+

-

-

-

-

-

N. gonorrhoeae

. .

. .

. . .

. . .

. . .

-

622

by the E C L chemiluminescence kit (Amersham, UK). Signal generation and detection was performed following the manufacturer's instructions. Conventional bacteriological examinations were also carried out on urine samples, urethral swabs and prostatic secretion samples to detect other genitourinary pathogens.

Results and Discussion. After culture in McCoy cells Chlamydia trachomatis was recovered from four patients, three of whom were also positive in the E I A (Table 1). Only one sample of prostatic secretion was suitable for testing by tissue culture and was negative for Chlamydia trachomatis; three samples were toxic for McCoy cells and the remaining specimen was not tested. Previous unpublished studies p e r f o r m e d in t h i s laboratory have shown that prostatic secretion and semen samples are commonly toxic for McCoy cells and are generally unsuitable for testing by tissue culture methods. However, it may be advantageous to centrifuge samples and to inoculate the cell monolayers with a resuspended pellet, although we have not tried this method ourselves. Confirmation of P C R results with D N A hybridization indicated that in 9 of the 11 patients with epididymitis, Chlamydia trachomatis was detected (Table 1); this was the only method positive for Chlamydia trachomatis in five patients. Two prostatic secretion samples were positive for Chlamydia trachomatis by P C R confirming the urethral swab PCR findings; however both samples were toxic on tissue culture, and one was negative in the E I A and in culture of the urethral swab. In three cases Chlamydia trachomatis was identified together with Neisseria gonorrhoeae, Escherichia coli or Staphylococcus aureus: the latter was found in culture of a prostatic secretion sample. However, the use of more sensitive techniques such as PCR may shed further light on the clinical relevance of these other organisms in epididymitis. We suggest that the presence of gonococci is relevant to the aetiology of the epididymitis, as reported by others (1), but that the presence of the other organisms is more likely to indicate secondary infection or contamination.

Eur. J. Clin. Microbiol. Infect. Dis.

conventional techniques or their inappropriate use in the detection of Chlamydia trachomatis. However, in an earlier study (3), surprisingly, Chlamydia trachomatis was detected on tissue culture in 8 of 11 urethral swabs from patients with epididymitis. In this instance it is unclear why the isolation rate for tissue culture was so high as the only known difference in technique was their use of 5-iodo-2-deoxyuridine-treated cells instead of cytochalasin B as we did. Our PCR results confirm that such a high detection rate for Chlamydia trachomatis is possible, although why other workers do not seem able to attain such high isolation rates is presumably due to a number of factors such as poor techniques for sampiing, and storage after sampling, use of incorrect swabs, and poor culture methods. Although Chlamydia trachomatis was only detected in the urethra and not at the site of infection these P C R results indicate that the organism may be important in the causation of epididymitis, especially in young adults. However, in order to verify these interesting findings a large study should be undertaken comparing several detection methods including the PCR.

References 1. Oriel JD, Ridgway GL: Genital infection in man.

British Medical Bulletin 1983, 39: 133-137. 2. De Jong Z, Pontonnier F, Plante P, Gautier JR, loualalen A, Archambaud M, Chabanon G: The fre-

quency of Chlamydia trachomatis in acute epididymitts. British Journal of Urology 1988, 62: 76--78. 3. Berger RE, Alexander ER, Monda GD, Ansell J, McCormick G, Holmes KK: Chlamydia trachomatis as

a cause of acute "idiopathic" epididymitis. New England Journal of Medicine 1978, 298: 301-304. 4. Schiebei JH, Anderson JT, Brandenhoff P, Geerdsen JP, Bay.Nielsen A, Sclmttz BA, Walter S: Chlamydia

trachomatis and acute epididymitis. Scandinavian Journal of Urology and Nephrology 1983, 17: 47-51. 5. Pearson RC, Baumber CD, McGhie D, Thamber V:

The relevance of Chlamydia trachomatis in acute epididymitis in young men. British Journal of Urology 1988, 62. 72-75.

6. Doble A, Taylor-Robinson D, Thomas B J, Jalil N, Harris JRW, Wilherow RON: Acute epididymitis: a

Gardnerella vaginalis was identified in one patient in the absence of evidence of infection by any other organism, and in one patient no organisms were detected. Bacteroides spp., Ureaplasma urealyticum or Mycoplasma hominis were not isolated.

7. Robinson A J, Grant JBF, Spencer RC, Potter CW, Kinghorn GR: Acute cpididymitis: why patient and

The results of the present study suggest that the failure to identify an aetiological agent in epididymitis is probably due to the insensitivity of

certain infections of man: comparison of yolk sac and cell cultures for detection and isolation. Journal of Infectious Diseases 1969, 120: 451-462.

microbiological and ultrasonographie study. British Journal of Urology 1989, 63: 90-94. consort must be investigated. British Journal of Urology 1990, 66: 642--645. 8. Gordon FB, Harper TA, Quan AL, Treharne JD, Dwyer RSC, Garland JA: Detection of Chlamydia in

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623

9. Ripa KT, Mardh PA: New simplifiedculture technique for Chlamydia trachomatis. In: Hobson D, Holmes KK (ed): Non-gonococcalurethritis and related infections. American Society for MicrobiologyWashington,DC, 1977, p. 323-327. 10. Ossewaarde JM, Rieffe M, Buisman NJF, Van Loon AM: Diagnosis of Chlamydia trachomatis conjunctivitis by PCR and detection of secretory IgA. In: Bowie WR, Caldwell HD, Jones RP, Mardh PA, RidgwayGL, SchachterJ, Stamm WE, WardME (ed): Chlamydia infections. Cambridge University Press, Cambridge, 1990, p. 495-498. 11. Claas HCJ, Mekhers WJG, de Bruyn Ill, de Graaf M, van Dijk WC, Lindeman J, Quint WGV-"Detection of Chlamydia trachomatis in clinical specimens by the polymerase chain reaction. European Journal of Clinical Microbiology& Infectious Diseases 1990, 9: 864868. 12. Halt C, Ward ME, Clarke IN: Analysesof the entire nucleotide sequence of the cryptic plasmid of Chlamydia trachomatis serovar LI. Evidence of involvement in DNA replication. Nucleic Acids Research 1988, 16: 4053-4067. 13. Ciaas HCJ, Wagenvoort JHT, Niesters HGM, Tio "IT, van Rijsoort-Vos JH, Quint WGV: Diagnostic value of the polymerasechain reaction for Chlamydia detection as determined in a follow-up study. Journal of Clinical Microbiology 1991. 29: 42-45.

,,

imm

Septicemia and Hepatic Abscess Caused by Pediococcus acidilactici J.M. Sire*, RY. D o n n i o , R. Mesnard, R Pou~dras, J.L. Avril

A case of postoperative Pediococcus acidilactici septicemia with parallel isolalion of the organism from hepatic specimens is presented. Laboratory nlethods to identify this vancomycin-resistant gram-positive cocci are described. Very few cases of documented infections due to this bacterium have been reported in the literature.

Pediococci are lactic bacteria found on fermenting vegetables and in silage, dairy products and beer (1). Pediococcus acidilactici and PediococCUs pentosaceus have been isolated from human Specimens of saliva (2), stool (3, 4), blood (5) and abdominal abscess material (6), but only two reports have associated pediococci with human Laboratoire de Bact6riologie-Virologie,Centre Hospitalier et Universitaire Pontehaillou,35033 Rennes, France.

disease (7, 8), including one case of septicemia. We report the simultaneous isolation of Pediococcus acidilactici from multiple blood cultures and from bile and hepatic abscess material following abdominal surgery.

Case Report. A 51-year-oId man with a history of cutaneous Recklinghausen's disease was admitted to a community hospital in Brittany with abdominal pain, nausea, melena and a temperature of 39 °C for 24 h. Ultrasonography and computed tomography scans performed at this time showed a heterogenous mass in the left colon area. The patient was transferred to our university hospital to undergo laparotomy which revealed a large gastric tumor and smaller jejunaI tumors adherent to the colon. A partial gastrectomy with jejunocolostomy and cotocolostomy was performed. Histologic examinations identified the tumorous lesions as schwannomas, related to Recklinghausen's neurofibromatosis. Culture of a subphrenic abscess sample on day I4 after surgery yielded Escherichia coli and Pseudomonas aeruginosa. This abscess was drained and the patient received ceftazidime (1 g t.i.d.), gentamicin (60 mg t.i.d.) and metronidazole (0.5 g t.i.d.) for 23 days. On day 53, Escherichia coli was isolated from three blood cultures. This septicemia was treated with ceftazidime (1 g t.i.d.) and amikacin (400 mg b.i.d.). Furthermore, because Staphylococcus epidermidis was isolated from a central venous catheter, the patient received vancomycin (750 mg b.i.d.) from day 53 to day 64. He was operated on once again on day 79 because of a newly detected retrohepatic abscess. On this occasion, the surgical procedure consisted of evacuation of the abscess, cholecystectomy (the gallbladder was necrotic), and ablation of necrotic tissue from the tail of the pancreas. Vesicular bile and abscess fluid samples were obtained at this time. Direct examination of both specimens showed numerous gram-positive cocci with a few gram-negative and gram-positive bacilli. Culture yielded Enterococcus faecalis, Pseudomonas aeruginosa, Lactobacillus casei subsp, rhamnosus and Pediococcus acidilactici. The patient was transferred to an intensive care unit and six blood cultures were obtained within 48 h (six aerobic and six anaerobic culture flasks; bioM6rieux, France). Pediococcus acidilactici was isolated from all bottles. In addition to this bacterium, one aerobic bottle yielded Pseudomonas aeruginosa and two anaerobic bottles yielded Enterococcus faecatis. The patient

Detection of Chlamydia trachomatis by the polymerase chain reaction in young patients with acute epididymitis.

Specimens from 11 patients presenting with acute epididymitis were tested for the presence of Chlamydia trachomatis by an enzyme immunoassay (EIA), gr...
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