Jpn. J. Infect. Dis., 60, 2007

Laboratory and Epidemiology Communications

Detection of the Tetanus Toxin Gene by Polymerase
Chain Reaction: A Case Study
Kazuyoshi Nagao, Toshihiko Mori*, Chieko Sawada, Chiiko Sasakawa and Yoshiko Kanezaki1
Division of Public Health Sciences, Tokushima Prefectural Institute of Health andEnvironmental Sciences, Tokushima 770-0941, and 1Division of General Medicine,
Tokushima Red Cross Hospital, Tokushima 773-8502, Japan
Communicated by Yoshichika Arakawa(Accepted March 19, 2007)

Approximately 50 tetanus cases are reported every year inJapan. Although the incidence is low, the mortality rate ishigh (1). We report here a tetanus case in which the tetanus
toxin gene was detected by the polymerase chain reaction(PCR).
A 60-year-old male suffered a laceration injury of 5 cm on
the left parietal region of his head on August 12, 2005, butleft the injury untreated. On August 15, he developed gait
and respiratory disturbances, in addition to head and neck pain.
He was immediately sent to the emergency unit of a hospital.
On admission, he showed clear consciousness but had diffi-culty in speaking. Due to difficulties in respiration, he had atracheal intubation and his respiration was then maintainedby a respirator. He was clinically diagnosed with tetanus andwas treated with anti-tetanus human γ-globulin (total, 6,000units). On August 18, generalized convulsions, trismus, andspastic paralysis of the upper extremities and trunk appeared.
Typical Gram-positive drumstick-shaped bacilli were detectedin samples from the injury site. The administration of penicillin G (20,000,000 units/day) was started together with muscle
relaxing and anticonvulsion therapies. On August 26, since
the tetanus bacilli continued to be detected in the injury site,
the site was washed with oxydol and human γ-globulin (total,3,000 units) was administered.
Beginning on September 10, the convulsions and spasms
lessened and the patient was taken off the respirator on
September 14. He was able to seat himself on September 17.
Because he continued to have difficulty opening his mouth,*Corresponding author: Mailing address: Division of Public HealthSciences, Tokushima Prefectural Institute of Health and Environmental Sciences, 71, 5-Chome Bandaicho, Tokushima 7700941, Japan. Tel: +81-88-625-7751, Fax: +81-88-625-1732, Email: mori_toshihiko_1@pref.tokushima.lg.jp
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he was transferred to another hospital for rehabilitation.
Microscopic examination of the patient’s specimens that
our laboratory received on August 31 showed Gram-positive
cocci and bacilli. Clostridium tetani infection was suspected,
and the specimen was streaked on the periphery (in order totake advantage of its high mobility) of 5% sheep red blood
cell agar that had been incubated under anaerobic conditions.
After anaerobic culture for 24 h, the tips of the colonies wereexamined by Gram and Moeller spore stainings, and were
found to show nearly pure cultures of Clostridium-like bacilli.
For detection of the tetanus toxin (tetanospasmin) gene,colonies on an agar plate were suspended in distilled waterand lysed by incubation at 95°C for 10 min. After centrifugation (15,000 rpm for 60 sec), the supernatant of the lysate wasused for DNA extraction. PCR detection of the tetanospasmin
gene was carried out following the method described by Katoet al. (2). The PCR amplification conditions were 35 cycles of95°C for 20 sec and 55°C for 20 sec, and the PCR productwas separated on 2% agarose gel (TAKARA L0-3; Takara
Bio, Inc., Otsu, Japan). The 331-bp and 229-bp bands expected
for tetanospasmin gene fragment were obtained (Fig. 1).
Although tetanus is usually diagnosed clinically, PCR
detection of tetanospasmin can facilitate the rapid definitive
diagnosis of this infection. In the present case, we obtained
the final results on September 2, only 3 days after receipt ofthe specimen.
We thank Prof. Hiroshi Yoshikura, Emeritus Researcherof the National Institute of Infectious Diseases for advice on
preparing the manuscript.
This article appeared in the Infectious Agents Surveillance
Report (IASR), vol. 27, p. 70, 2006 in Japanese.
REFERENCES
1. Ebisawa, I (1999): Tetanus. Saishin-igaku, 54 (Suppl.), 644-651 (in Japanese).
2. Kato, N., Kato, H., Kobayashi, T., et al. (1993): Identification ofneurotoxin-producing Clostridium tetani and Clostridium botulinum.
Kenkiseikin-kansensho-kenkyu, 23, 86-89 (in Japanese).

Fig. 1. Detection of tetanospasmin gene by PCR. Primer pairs were
GAT1 and GAT2 for S1, and GAT5 and GAT6 for S2. The expected
size for tetanospasmin gene is 331 bp for S1 and 229 bp for S2. M,
molecular weight size marker (Marker 5; Nippon Gene, Tokyo, Japan).

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