Vol. 11, 1992

References 1. Swarlz MN: Myositis. In: Mandell GL, Douglas RG, Bennet JE (ed): Principles and practice of infectious diseases. Churchill Livingstone, New York, 1990, p. 812--818. 2. Gaut P, WongPK, Meyer RD: Pyomyositisin a patient with the acquired immunodeficiencysyndrome. Archives of Internal Medicine 1988, 148: 1608-1610. 3. Swarts RL, Martlnez LA, Robson HG: Gonococcal pyomyositis. Journal of the American Medical Association 1981, 246: 246. 4. Brennessei DJ, Robbins N, Hindman S" Pyomyositis caused by Yersinia enterocolitica; Journal of Clinical Microbiology 1984, 20: 293-294. 5. Schwab R, Panwalker AP: Klebsiella pyomyositis. American Journal of Medicine 1986, 81: 1116-1117. 6. Jordan JM, Hsu VW, Allen NB: Pyomyositis and polyarticular septic arthritis from Haemophilus influenzae in a non-immunoeompromisedadult. Journal of Rheumatology 1987, 14: 1190-1192. 7. Kmtzke RA, Golenbock DT: Pyomyositisand hepatic abscess in associationwithAeromonas hydrophila sepsis. American Journal of Medicine 1987, 83: 347-349. 8. Sarubbi FA, Gafford GD, Bishop DR: Gram-negative bacterial pyomyositis:uniqueease and review.Reviews of Infectious Diseases 1989, 11: 789-792. 9. Pascual J, Liafio F, Rivera M, Carriilo R, Ortufio J: Necrotizing myositissecondaryto Serratia marcescens in a renal allograft recipient. Nephron 1990, 55: 329331. 10. Fincher RM, Jackson MW, Fisher AQ" Citrobacter freundii" a newly reported cause of pyomyositis. American Journal of Medical Sciences 1990, 299: 331333. 11. SchiffRG, Silver L: Tropical pyomyositis.Demonstration of extent and distribution of disease by gallium scintigraphy. ClinicalNuclear Medicine 1990, 15: 542544. 12. Sharif HS, Clark DC, Aabed MY, Aideyan OA, Haddad ME, Mattsson TA: MR imagingof thoracic and abdominal wall infections:comparisonwith other imagingprocedures. AmericanJournal of Roentgenology 1990, 154: 989-995. 13. Back SA, O'Neill T, Fishbein G, Gwinup G: A case of group B streptococcalpyomyositis.Reviews of Infectious Diseases ]990, 12: 784-787. 14. Beck W, Grose C: Pyvmyositis presenting as acute fit~lommfilpare. Pediatric InfectiousDiseases Journal 1984, 3: 445--448.

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in order to rule out factors such as p o o r compliance in taking medication or continued sexual contact during the study period. Alternatively, development of antimicrobial resistance in the microorganism involved may account for treatment failure. In this study we investigated cases of treatment failure occurring in an earlier therapy study of patients with non-gonococcal urethritis who were treated with eiprofloxaein or doxycycline (1). Randomly selected Chlamydia trachomatis strains isolated before therapy and all available pairs of strains isolated before and after therapy with either drug were serotyped, and the in vitro activity of these two drugs and erythromycin against the strains was determined. Five Chlamydia trachomatis strains isolated only before therapy with ciprofloxaein or doxycycline were serotyped, and the MICs and MBCs of ciprofloxaein, erythromycin and doxycycline for these strains determined. Likewise, serotype, MIC and MBC were determined for four pairs of strains isolated before and after therapy with ciprofloxacin (1 g daily in a single oral dose for seven days) and one strain isolated before and after therapy with doxycycline (200 mg on day 1, followed by 100 mg daily for six days). The MIC of each drug was determined using the method described by Segreti et al. (2). DEAEdextran treated McCoy cells were inoculated with 103-104 inclusion-forming units of each chlamydial isolate per well, centrifuged for 60 rain, and incubated at 37 *C in a 5 % CO2 atmosphere for two days. The inoculum was then replaced with culture media (0.1 ml per well) containing serial twofold dilutions of each antimicrobial agent and 0.1 lag/ml cycloheximide. The drugs were diluted from 16 to 0.015 lag/ml. After 48 h of incubation two wells per dilution were fixed and stained with a fluorescine-conjugated monoclonal antibody to ChIamydia trachomatis (3). The MIC was defined as the lowest concentration at which 90 % inhibition of inclusion body formation was observed. The remaining wells were incubated for another 48 h in culture media without antibiotics. The MCB was defined as the lowest concentration of antimicrobial agent yielding no inclusions after passage (2).

Antimicrobiai Susceptibility and Serotyping of Chlamydia trachomatisStrains Isolated before and after Treatment with Ciprofloxacin and Doxycyline

Staphylococcus areus ATCC 25923 and 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 with known MIC values for the test drugs were used as control strains.

In evaluating new therapies for treatment of sexually transmitted diseases it is necessary to examine carefully patients in whom treatment fails

The procedure for serotyping was as follows. The culture medium containing suspended Chtamydia trachomatis and host cell debris was centri-

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

Table 1: Serotypes of and MICs of doxycydine, ciprofloxaein and erythromycin for five Chlamydia trachomatis strains iso-

lated only before antibiotic treatment or before and after treatment. Agent

MIC (/ag/ml) Serotype Before After

Ciprofloxaein

Doxycycline

D I E H F K I F D D E E D J G

Ciprofloxacin Before After

K I F D

2 2 2 2 2 2 1 2 2

*

2 2 2 2 2 1

Doxycyeline Before After

2 1 2 2

0.25 0.12 0.25 0.12 0.06 0.25 0.12 0.25 0.25

*

0.12 O.O6 0.12 0.12 0.12 0.12

Erythromyein Before After

0.25 0.12 0.25 0.25

0.5 0.25 0.5 0.25 0.25 0.5 0.25 0.12 0.12

0.5 0.25 0.12 0,12

*

0.25 0.25 0.25 0.25 0.25 0.5

*

*Strain not viable for testing.

fuged at 11,000 x g for 15 min. The resulting pellet was suspended in phosphate buffered saline of pH 7.6 containing 0.02 % formalin. Suspensions of prototype antigens or clinical isolates to be typed were applied to nitrocellulose membranes and dried. The dot enzyme immunoassay for serotyping was performed as described previously (4). The MICs of doxycycline before treatment were 0.06-0.25/ag/ml (Table 1). Unfortunately the only post-treatment strain in the doxycycline group died in storage and could not be tested further. The MICs of ciprofloxacin before and after therapy were 1-2 lag/ml (Table 1). No elevation of MICs was observed in the strains isolated after treatment. The MICs of erythromycin for all strains were 0.12-0.5/ag/ml (Table 1). All of the MIC and MBC values determined in this study were identical or within one dilution step. All strains isolated before and after therapy were of the same serotype (Table 1). The MICs of doxycycline, ciprofloxacin and erythromycin were within the ranges previously reported (5). The fact that the pre-treatment and post-treatment strains were the same does not rule out a new infection, but makes it less probable, as condom use was advised during the treatment and follow-up period. Thus we conclude that these cases should be regarded as genuine instances of treatment failure of ciprofloxacin without development of resistance.

Recently, Jones et al. (6) isolated Chlamydia trachomatis strains resistant to tetracycline from five patients. These isolates were also resistant to doxycycline and erythromycin. The clinical significance of this resistance is not known. To our knowledge there are no reports of resistance to ciprofloxacin in Chlamydia trachomatis. Although successful treatment of Chlamydia trachomatis infections with ciprofloxacin depends not only on the MIC/MBC values but also on other pharmacokinetic parameters, clinical studies have demonstrated that ciprofloxacin in increasing dosages did not display increased efficacy (1, 7, 8). Thus, the findings in this investigation support our previous conclusion that Chlarnydia trachomatis infections in these patients were not cured with ciprofloxacin therapy (1). A.H. van der Willigen 1. T. van Rijsoord-Vos 2 J.H.T. Wagenvoort 2 W.E. S t a m m 3 R.J. Suchland 3 E. Stolz 1 Department of Dermato-Venereology, and 2Department of Clinical Microbiology, University Hospital RotterdamDijkzigt, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. 3Chlamydia Laboratory, University of Washington, Seattle, Washington 98195, USA.

Vol. 11, 1992

References 1. van der Willigen AH, Polak-Vogelzang AA, Habbema L, Wagenvoort JHT: Clinical efficacy of ciprofloxacin

versus doxycyclinein the treatment of non-gonococcal urethritis in males. European Journal Clinical Microbiology and Infectious Diseases 1985, 5: 658--661. 2. Segreti J, Kessler HA, Kapell KS, Trenholme GM:

In vitro activity of A-56268 (TE-031) and four other antimicrobial agents against Chlamydia trachomatis. Antimicrobiai Agents and Chemotherapy 1987, 31: I00-101. 3. Slamm WE, Tam M, Koester CM, Cles L: Detection of Chlamydiatrachomatisinclusions in McCoycell cultures with fluorescein-conjugated monodonal antibodies. Journal of Clinical Microbiology1983,17: 666668. 4. Barnes RC, Wang SP, Kua CC, Slamm WE: Rapid

immunotyping of Chlarnydia trachomatis with monoclonal antibodies in a solid-phase enzyme-immunoassay. Journal of Clinical Microbiology 1985, 22: 609--613. 5. M~rflh PA, Paavonen J, Puolakkainen M: Chlamydia. Plenum Publishing, New York, 1989, p. 103-114.

6. Jones RB, van der Pol B, Martin DH, Shepard MK:

Partial characterization of Chlamydia trachomatis isolates resistant to multiple antibiotics. Journal of Infectious Diseases 1990, 162: 1309-1315. 7. Arya OP, Hobson D, Hart CA, Bartzokas C, Pratt BC: Evaluation of ciprofloxacin 500 mg twice daily

for one week in treating uncomplicated gonococcal, chlamydial and non-specific urethritis in men. Genitourinary Medicine 1986, 62: 170--174. 8. Jeskanen L, Karppinen L, Ingervo L, Reitamo $, Happonen HP, Lassus A: Ciprofloxacin versus doxycycline

in the treatment of uncomplicated urogenital Chlwnydia trachornatis infections. A double-blind comparative study. Scandinavian Journal of Infectious Diseases 1989, 60: 62--65.

E v a l u a t i o n o f an E n z y m e l m m u n o a s s a y for D e t e c t i o n o f Clostridium difficile T o x i n A

The laboratory diagnosis of enteric disease due to Clostridiurn difficile is usually established by fecal culture and/or tissue culture assay (1). We report the results of an evaluation of a new enzyme immunoassay (EIA) for the rapid detection of Clostridium difficile toxin A. Fecal specimens were spread on cycloserinecefoxitin-fructose egg yolk agar (Oxoid, UK) plates which were incubated anaerobically at 37 °C for 48 h. Isolates with the characteristic colonial morphology and odor were identified as Clostridiurn difficile with the R a p i D A N A II system (Innovative Diagnostic Systems, USA) and

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the Serobact C. difficile latex agglutination test kit (Wellmark Diagnostics, Canada). Fecal filtrates and broth cultures of Clostridium difficile isolates were assayed f o r cytotoxin using the Toxi-titer microtiter plate system (2) with human foreskin fibroblast cells (Bartels Immunodiagnostic Supplies, USA) according to the manufacturer's instructions. The plates were examined daily for 48 hours; in positive specimens showing the characteristic cytopathic effect results were confirmed by neutralization with antitoxin.

Clostridium diffictTe toxin A was detected with the Premier C. difficile toxin A E I A (Meridian Diagnostics, USA), according to the manufacturer's instructions except that seven washes were used to remove unbound conjugate. Using only five washes, as recommended by the manufacturer, there was an unacceptably high rate ( 1 8 % ) of indeterminate results. Results were analyzed using a spectrophotometer (Whittaker M.A. Bioproducts, USA) at A45o (reading < 0.100, negative; > 0.100 to 0.149, indeterminate; > 0.150, positive). Clostridium difficile was isolated from 170 specimens from 94 patients and cytotoxin was detected in 92 specimens (65 patients). Toxigenic Clostridium difficile was recovered from 100 specimens (73 patients). Only 18 patients had symptoms associated with antibiotic-associated diarrhea. Seven (1.4 % ) fecal specimens negative in the culture and cytotoxin assay gave indeterminate results in the EIA to detect toxin A and were excluded from further analysis. The E I A results compared to those obtained in the cytotoxin assay are shown in Table 1. The toxin A E I A was very sensitive (90 %) and specific (98 %). The EIA was also positive in five of eight specimens negative in the cytotoxin assay but from which toxigenic Clostridium difficile was recovered. Although both toxin A and toxin B are probably involved in the pathogenesis of Clostridium difficile-associated diseases, toxin A is presumed to be responsible for most of the symptoms of pseudomembranous colitis as only toxin A induces gastrointestinal tissue damage and a fluid response in animal models (3-6). An E I A for detection of toxin A was first described by Lyerly et al. (7) and was subsequently found to have 100 % specificity but only 6 1 % sensitivity in patients thought to have Clostridium difficile-related enteric disease according to clinical criteria (8). Further refinements in Clostridium difficile toxin A antibody preparations have led to the development of several commercial EIAs for

Antimicrobial susceptibility and serotyping of Chlamydia trachomatis strains isolated before and after treatment with ciprofloxacin and doxycycline.

Vol. 11, 1992 References 1. Swarlz MN: Myositis. In: Mandell GL, Douglas RG, Bennet JE (ed): Principles and practice of infectious diseases. Churchil...
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