Y.G. Tsai, C.C. Wang, D.M.

Chu, et al

CASE REPORTS

PRIMARY STAPHYLOCOCCAL INFECTION AND TOXIC SHOCK SYNDROME DIAGNOSED BY POLYMERASE CHAIN REACTION
Yi-Giien Tsai, Chih-Chien Wang, Der-Ming Chu, Ming-Chih Tsai, and Mong-Ling Chu

Abstract: Primary staphylococcal pneumonia complicated with toxic shock syndrome (TSS) is relatively uncommon in children. Staphylococcus aureus exotoxins are thought to function as superantigens, and seem to promote disease manifestations. The identification of staphylococcal toxin genes by polymerase chain reaction (PCR) offers a specific and rapid diagnostic method for TSS. We describe a 7-year-old child with TSS resulting from staphylococcal pneumonia. S. aureus enterotoxins A and B were detected in the sputum of this patient by PCR.

(J Formos Med Assoc 2000;99:9424) Key words:

toxic shock syndrome Staphylococcus aureus polymerase chain reaction

Toxic shock syndrome (TSS) is an acute, life-threatening, multiple-system disease characterized by fever, rash, and shock with subsequent desquamation [1]. Diagnosis of TSS depends on the clinical features satisfying the case definition. Recent advances in techniques for identifying staphylococcal toxins have greatly helped in understanding the causes of TSS [2]. We describe an unusual case of TSS in a child with primary staphylococcal pneumonia. Staphylococcal enterotoxins A (SEA) and B (SEB) were detected using a polymerase chain reaction (PCR) toxin assay. To the best of our knowledge, detection of staphylococcal toxins by PCR in TSS has not been previously reported in Taiwan.

Case Report
A 7-year-old boy was in good health until 7 days prior to admission, when he developed a sore throat and cough. One day before admission, he suffered from vomiting, diarrhea, fever, and erythematous rash over the entire body surface. During the next 12 hours, he was increasingly dyspneic and lethargic. Vital signs upon arrival at the

hospital were as follows: body temperature, 41C; blood pressure, 84/42 mm Hg; respiratory rate, 58/min; and heart rate, 178 beats/min. He appeared ill and irritable when aroused. His conjunctiva were intensely injected; the oropharyngeal and buccal mucosal membranes also showed hyperemia. Facial flushing with oral fissure and scarlet-like rash with petechiae over the entire body were noted. Rales were audible bilaterally in the chest. Laboratory findings included a hemoglobin level of 9 1.95 mmol/L and a white blood count of 11.7 x 10 /L, with 86% segmented neutrophils, 12% monocytes, and 9 1.5% lymphocytes. The platelet count was 72 x 10 /L within the first 24 hours of hospitalization and remained low until the ninth day. Electrolytes were within normal limits. Serum biochemical analysis showed the following: blood urea nitrogen, 6.1 mmol/L; creatinine, 66.3 mmol/L; creatine phosphokinase, 765 U/L; aspartate aminotransferase, 56 U/L; and alanine aminotransferase, 11 U/L. The prothrombin time was 13.1 seconds (control, 12.1 sec) and the partial prothrombin time was 47.1 seconds (control, 28.6 sec). Urine analysis revealed proteinuria (2+) and hematuria (46 red blood cells/high power field) under microscopic examination. The chest roentgenogram

Department of Pediatrics, Tri-Service General Hospital, National Defense Medical Center, Taipei. Received: 10 December 1999. Revised: 3 January 2000. Accepted: 7 March 2000. Reprint requests and correspondence to: Dr. Chih-Chien Wang, Department of Pediatrics, Tri-Service General Hospital, 40, Sec. 3, Ting-Chow Road, Taipei, Taiwan.

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revealed patch consolidation in the right middle lobe and peribronchial wall thickness. The patient subsequently developed generalized tonic-clonic seizure with hypotension (76/26 mm Hg). He was referred to our pediatric intensive care unit and underwent endotracheal intubation with ventilation support. He was resuscitated with fluids and received empiric antibiotic therapy with oxacillin (150 mgkg1d 1) and gentamicin (7.5 mgkg1d 1). His fever subsided within 48 hours after initiation of antibiotic therapy. Cultures from blood, throat swab, and cerebrospinal fluid were negative. The sputum culture from deep tracheal aspiration grew Staphylococcus aureus. A rapid diagnostic protocol was used to detect genes for enterotoxins and TSS toxin-1 (TSST-1) in nucleic acids extracted from the isolated strain of S. aureus [2]. Synthetic oligonucleotide primers SEA1 (5TTGGAAACGGTTAAAACGAA-3), SEA2 (5-GAAC CTTCCCATCAAAAACA-3), SEB1 (5-TCGCATCA AACTGACAAACG-3), SEB2 (5-GCAGGTACT CTATAAGTGCC-3), TSST-1 ( 5-ATGGCAGCATC AGCTTGATA-3), and TSST-2 (5-TTTCCAATAACC ACCCGTTT-3) were designed by computerized analysis [3] and obtained from Life Technologies (Taipei, Taiwan). PCR was performed in a 50-L reaction mixture [4] using 10 ng nucleic acid and the following amplification cycles: denaturation for 2 minutes at 94C, annealing of primers for 1 minute at 58C, and extension for 1 minute at 72C, for 30 cycles. The reference strains were A890638 for SEA, A940340 for SEB, and A960559 for TSST-1, obtained from Dr. G Lina (Laboratorie de Bacteriologie, Rue Gurllaume Paradin, Lyon, France)[5].

All PCR reactions were performed for individual toxin genes using only one primer pair for each tube. The amplification fragments were detected using agarose gel electrophoresis. The sizes of amplified products were SEA, 120 base pairs; SEB, 478 base pairs; and TSST-1, 350 base pairs (Figure) [2]. The infecting S. aureus strain was found to contain enterotoxins A and B. Desquamation over fingers and toes was noted 7 days later. The patient was discharged on the 12th hospital day and was clinically well.

Discussion
When staphylococcal TSS was first described by Todd et al in 1978 [6], it was associated with menstruating women who were using tampons. In the 1980s, most TSS cases were associated with menstruation. More recently, non-menstruation-associated cases of TSS have occurred as often as the classical menstruation-associated cases [7]. However, relatively few confirmed cases of TSS have been reported in children younger than 10 years old [8]. TSS should be considered in the differential diagnosis of patients with fever, rash, and shock. On admission, our patient met the criteria for TSS established by the Centers for Disease Control and Prevention [1]. The child in this report had a multisystem disorder with high fever, hypotension, and skin rash; desquamation developed 7 days after admission. He showed symptoms involving three or more of the following organ systems: lung, gastrointestinal tract, bone marrow, mus-

A
1 2 3 4 1 2

B
3 4 1 2

C
3 4

120 bp 350 bp

478 bp

Figure. Agarose gel electrophoresis patterns showing typical amplification fragments in the polymerase chain reaction (PCR) for staphylococcal enterotoxin (SE) A and B genes, but without the toxic shock syndrome toxin-1 (TSST-1) gene. (A) Lane 1, molecular marker; lane 2, reference strain for SEA; lane 3, Staphylococcus aureus isolated from patient; lane 4, negative control. (B) Lane 1, molecular marker; lane 2, reference strain for SEB; lane 3, S. aureus isolated from patient; lane 4, negative control. (C) Lane 1, molecular marker; lane 2, reference strain for TSST-1; lane 3, S. aureus isolated from patient; lane 4, negative control.

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culoskeletal system, and CNS. There was clinical evidence of seizure, vomiting, conjunctiva hyperemia, thrombocytopenia, and elevated muscle creatine phosphokinase. TSS can be caused by infection from strains of S. aureus that produce certain exoprotein toxins. For patients with clinical TSS, all possible sites of potential S. aureus infection should be evaluated, including the vagina, surgical wounds, lungs, bones, joints, soft tissues, and sinuses. There have been reports of TSS as a complication of primary staphylococcal pneumonia, but it is relatively rare in children [9]. The treatment of TSS remains supportive. Initially, the most important issue is identification and drainage of the focal infection site, which may further minimize uptake of toxins. Aggressive fluid supplementation with appropriate anti-staphylococcal antibiotic therapy may improve the symptoms of TSS. S. aureus exotoxins are thought to be superantigens that stimulate lymphocytes and endothelial cells to produce the endogenous mediators that seem to cause the disease manifestations [10]. TSST-1 is associated with more than 90% of menstrual cases of TSS and approximately half of non-menstrual cases. Non-menstrual cases are more commonly associated with production of SEB than TSST-1 [11]. Three necessary risk factors for non-menstrual TSS include absence of antitoxin antibody, colonization or infection with a toxinproducing strain of S. aureus, and an infected site [7]. Normally, any individual colonized with a staphylococcal strain producing TSST-1 or SEB will have protective antibody to the toxin [12]. Few individuals have antibodies to SEA, particularly very young children. Children are more susceptible than adults to TSS because they are less likely to have antibody to these toxins [13]. The primers used in our PCR protocol [2] specifically detected the genes for SEA and SEB without TSST-1 and were tested against extracted nucleotide from the reference strains [5]. SEB is produced mostly in the absence of TSST-1 in TSS, but SEA is rarely found alone and is frequently coexpressed with TSST-1 [14]. SEA is produced in relatively small amounts compared with TSST-1 and SEB [13], which may explain why SEA is rarely attributed to TSS unless it is associated with another toxin [15]. In conclusion, demonstration of the production of characteristic toxin by S. aureus isolated from the infected patient further supports the diagnosis of TSS. The identification of staphylococcal toxin genes in

strains of S. aureus by PCR offers a very specific and rapid detection method [2].

References
1. Centers for Disease Control: Toxic-shock syndrome: MMWR Morb Mortal Wkly Rep 1980;29:22930. 2. Johnson WM, Tyler SD, Ewan EP, et al: Detection of gene enterotoxins, exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction. J Clin Microbiol 1991;29: 42630. 3. Devereux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acid Res 1984;12:38795. 4. Pollard DR, Johnson WM, Lior H, et al: Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction. J Clin Microbiol 1990;28:5405. 5. Lina G, Gillet Y, Vandenesch F, et al: Toxin involvement in staphylococcal scalded skin syndrome. Clin Infect Dis 1997;25:136973. 6. Todd J, Fishaut M, Kapral F, et al: Toxic shock syndrome associated with phage-group I staphylococci. Lancet 1978; i:1168. 7. Strausbaugh LJ: Toxic shock syndrome. Are you recognizing its changing presentations? Prostgrad Med 1993;94: 1078. 8. Wiesenthal AM, Todd JK: Toxic shock syndrome in children aged 10 years or less. Pediatrics 1984;74:1127. 9. Marchant B, Brown J: Toxic shock syndrome and staphylococcal pneumonia. Lancet 1987;ii:578. 10. Hackett SP, Stevens DL: Superantigens associated with staphylococcal and streptococcal toxic shock syndrome are potent inducers of tumor necrosis factor-beta synthesis. J Infect Dis 1993;168:2325. 11. Bohach GA, Fast DJ, Nelson RD: Staphylococcal and streptococcal pyrogenic toxins involved in toxic shock syndrome and related illnesses. Crit Rev Microbiol 1991; 17:25172. 12. Jacobson JA, Kasworm EM, Crass BA, et al: Nasal carriage of toxigenic Staphylococcal aureus and prevalence of serum antibody to toxic-shock-syndrome toxin 1 in Utah. J Infect Dis 1986;153:3568. 13. Reiser RF, Jacobson JA, Kasworm EM, et al: Staphylococcal enterotoxin antibodies in pediatric patients from Utah. J Infect Dis 1988;158:11058. 14. Crass BA, Bergdoll MS: Toxin involvement in toxic shock syndrome. J Infect Dis 1986;153:91826. 15. Crass BA, Bergdoll MS: Involvement of staphylococcal enterotoxins in nonmenstrual toxic shock syndrome. J Clin Microbiol 1986;153:11389.

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