Journal of Infection and Public Health (2016) 9, 105—109 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector CASE REPORT Detection of Clostridium tetani in human clinical samples using tetX specific ଝ primers targeting the neurotoxin a a a Madhu Ganesh , Nasira K. Sheikh , Pooja Shah , b b Gajanan Mehetre , Mahesh S. Dharne , c,∗ Basavraj S. Nagoba a Dr. VM Medical College, Solapur 413 001, India b NCIM Resource Centre, CSIR — National Chemical Laboratory (NCL), Pune 411 008, Maharashtra, India c Maharashtra Institute of Medical Sciences & Research, Latur 413 531, Maharashtra, India Received 21 April 2015; received in revised form 22 May 2015; accepted 24 June 2015 KEYWORDS Summary Tetanus resulting from ear injury remains an important health prob- Otogenic tetanus; lem, particularly in the developing world. We report the successful detection of Clostridium tetani using tetX specific primers targeting the Cl. tetani neurotoxin. Cl. tetani; Neurotoxin; The sample was obtained from an ear discharge of a case of otogenic tetanus in Trismus a 2-year-old male child. Based on the culture results of the ear discharge, Gram staining and virulence testing by genotyping, a diagnosis of tetanus was confirmed. This is the first report from India on the successful detection of Cl. tetani in a human clinical sample using tetX specific primers targeting the Cl. tetani neurotoxin. © 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Limited. All rights reserved. Introduction ଝ The GenBank/EMBL/DDBJ accession number for the tetX Tetanus is a potentially fatal muscle spasm dis- gene sequence of Clostridium tetani determined in this study ease caused by Clostridium tetani, a motile, is KM677991. ∗ spore-forming, anaerobic, Gram-positive bacillus. Corresponding author. Tel.: +91 2382227424; Although tetanus resulting from ear injury is fax: +91 2382227246; mobile: +91 9423075786/7588237531. E-mail addresses: dr [email protected], extremely rare, it remains a major problem. [email protected] (B.S. Nagoba). http://dx.doi.org/10.1016/j.jiph.2015.06.014 1876-0341/© 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Limited. All rights reserved. 106 M. Ganesh et al. ◦ Although it is less common in the developed world broth was incubated further at 37 C to study pro- [1], it is a significant disease in the developing teolytic activity [7]. world. Despite a widespread immunization pro- gram, a large number of cases have been reported Sequencing based detection and genotyping from the developing world [2—5]. The most com- of Clostridium tetani directly from clinical mon cause of otogenic tetanus is trauma followed sample by contamination of the wound [6]. Here, we report the isolation of Cl. tetani from a case of oto- DNA extraction genic tetanus and its confirmation by culture and The ear discharge from the patient collected in RCM sequencing based detection and genotyping. To the ◦ broth (stored at 4 C) was used for DNA extraction. best of our knowledge, this is the first report from The total nucleic acid extracted from 1 mL of sam- India on the successful detection of Cl. tetani in a ple using a bacterial DNA kit (Merck-Genei) was human clinical sample using tetX specific primers resuspended in elution buffer. The quality of the targeting the Cl. tetani neurotoxin. extracted DNA was assessed by 0.7% agarose hori- zontal gel electrophoresis in TAE buffer (40 mM Tris, 20 mM acetate, and 2 mM EDTA) and visualized by Subject and methods TM GelRed staining on a Protein Simple gel documen- tation unit. The concentration of the extracted DNA This study reports the microbiological analysis of was ascertained on a NanoDrop Lite spectropho- a clinically diagnosed case of otogenic tetanus for tometer (NanoDrop Biotechnologies). confirmation of the clinical diagnosis and to confirm the virulence of the causative agent and to differ- PCR amplification and sequencing entiate it from morphologically similar Clostridia. The Cl. tetani specific primers targeting a fragment A 2-year-old-non-immunized-male child pre- of the tetX gene (1354 bp) were used to amplify sented with a history of fever for 4 days, trismus and the DNA extracted from the sample [8]. The PCR constant cry, watery, non-foul smelling discharge reaction contained 10 nM each primer (Eurofins), from the ear for 6 days, gradually progressing 200 ␮M each deoxynucleoside triphosphate (dNTP) to breathlessness, and progressively increasing to (Genei), 1 U Taq polymerase (Genei) in the appro- coughing and sneezing. Inability to open mouth, priate reaction buffer, and 50 ng and 100 ng of DNA particularly after coughing and sneezing, and dys- extracts as the templates. The reactions were per- phagia for solid food, were reported by the mother. formed in a 50 ␮L reaction mixture. The cycling Physical examination revealed tautness of the conditions were as follows: initial denaturation of neck muscles with no neck swelling, trismus grade ◦ 95 C for 10 min, followed by 25 cycles each of 1 min 4 and risus sardonicus. The patient had an opistho- ◦ ◦ ◦ at, 94 C 1 min at, 52 C and 1.5 min at 72 C. Pos- tonus posture and a history of trauma to the left ear itive PCR amplicons were documented followed by due to matchstick insertion 6 days prior. His pulse purification using the Exo-rSAP (New England Bio- rate was 180/min, respiratory rate was 28/min and labs) method and sequenced on both strands in an oxygen saturation was 94%. ABI 3500xl genetic analyzer (Thermofisher). The results of the routine investigations showed hemoglobin 9.4 g/dL, total leukocyte count of 3 13,200/mm with a monocyte count of 8%, Sequence analysis 3 platelet count of 367,000/mm and RBC count The obtained sequences were assembled and edited 3 of 2.7 million/mm . The random blood glucose using the sequence analysis software version 5.1 was 50 mg/dL, urea was 21 mg/dL, creatinine was (Thermofisher). Edited sequences were submitted 0.8 mg/dL, serum sodium was 142 meq/L, potas- to BLASTN and BLASTX using the default parame- sium was 4.2 meq/L and calcium was 8.7 meq/L. ters for analysis [9], followed by comparison with the closest homologous sequences retrieved from the GenBank database. Microbiological analysis Based on the results of the microscopy, cul- ture and molecular studies, a clinical diagnosis of Ear discharge was collected in Robertson’s cooked otogenic tetanus was confirmed and the patient meat (RCM) broth and processed for aerobic and was given tetanus toxoid 0.5 mL IM, tetanus glob- anaerobic bacteriological analysis. A smear of RCM ulin 500 IU IM, crystalline penicillin 2.5 lac IV QID, broth was prepared and stained with Gram stain. metronidazole 20 mg/kg/day, and diazepam 1.5 mg A loopful of RCM broth was inoculated onto freshly TDS for 7 days along with oxygen by mask. The child prepared blood agar and incubated aerobically and recovered well and was discharged on the 8th day. anaerobically in a McIntosh and Filde’s jar. RCM Detection of Cl. tetani using tetX specific primers 107 Figure 1 Gram stain smear showing characteristic drum stick appearance on 3rd day. Figure 3 Polymerase chain based amplification of ear Results and discussion discharge using tetX specific primers. (Amplicons elec- trophoresed in an 0.8% agarose gel. Lane 1: 50 ng DNA Microscopic examination of the RCM broth using from ear discharge, Lane 2: 100 ng DNA from ear dis- Gram staining showed Gram-positive bacilli with charge, and Lane 3: 1 Kb DNA ladder (Genei).) round terminal bulging spores (Fig. 1). Further incu- bation of the RCM broth showed proteolytic activity, and anaerobic cultures on freshly prepared blood The tetX gene fragment specific for the Cl. tetani agar showed a thin transparent film of growth with neurotoxin was successfully amplified (approx- swarming typical of Cl. tetani (Fig. 2). Aerobic cul- imately 1354 bp) by PCR (Fig. 3) from 50 ng ture of the ear discharge showed no significant of template DNA. Although we obtained 415 bp findings. sequences toward the 5 end, reverse primer failed Figure 2 (A) Robertson cooked meat broth showing proteolytic activity. (B) Blood agar showing swarming. 108 M. Ganesh et al. Table 1 Homologies of sequences obtained from the ear discharge for the NCBI BLASTN and BLASTX databases. Primer BLASTN BLASTX Reference First match Percent First match Percent homology homology Tet2Fa AJ891297 100% CAJ30283 100% Deposited Clostridium spp. (415/415) Neurotoxin heavy (122/122) in the b RKD partial gene chain 1 GenBank for neurotoxin Clostridium but not heavy chain 1 neurotoxin, published N-terminal receptor binding; pfam07953 Primer BLASTN BLASTX Reference Second match Percent Second match Percent homology homology Tet2F AM076940 100% CAJ28911 100% Dixit et al. Clostridium spp. (415/415) Tetanus toxin (122/122) [10] RKD partial tetX precur- gene for tetanus sor/tetanus toxin b toxin precursor light chain a Binds to 5 end of tetX gene. b Clostridium neurotoxin, N-terminal receptor binding; pfam07953. to yield sequencing data after repeated attempts. Because of the significant decline following The sequence was compared independently for mass immunization, the occurrence of tetanus is homology using BLASTN and BLASTX (Table 1). overlooked, but the occurrence of isolated cases The sequence analysis displayed 100% homologies indicates that this disease remains. In particu- at the nucleotide and amino acid level with the lar, non-immunized and inadequately immunized Cl. tetani RKD strain (Indian origin) neurotoxin individuals carry higher risks, and vaccination in heavy chain 1 and light chain [10]. The tetX gene pregnant women and children should be empha- sequence obtained in this study was deposited in sized.