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  Table of Contents  
BRIEF COMMUNICATION
Year : 2019  |  Volume : 37  |  Issue : 1  |  Page : 116-119
 

Molecular confirmation of the circulating Bacillus anthracis during outbreak of anthrax in different villages of Simdega District, Jharkhand


1 Department of Microbiology, RIMS, Ranchi, Jharkhand, India
2 Rajiv Gandhi Super Speciality Hospital, New Delhi, India

Date of Web Publication16-Aug-2019

Correspondence Address:
Dr. Amber Prasad
Department of Microbiology, RIMS, Bariatu, Ranchi - 834 009, Jharkhand
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_19_111

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 ~ Abstract 


Aims and Objectives: Molecular confirmation of the circulating Bacillus anthracis during outbreak of anthrax in different villages of Simdega district, Jharkhand, India. Materials and Methods: Blood samples with swabs from skin lesions (eschar) were collected from the suspected cases of Anthrax from October 2014 to June 2016 from Simdega district, Jharkhand. All the swabs were inoculated on polymyxin lysozyme EDTA thallous acetate media, nutrient agar media as well as 5% sheep blood agar media. Gamma-phage lysis was done. DNA extraction was done using a QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA) and subjected to polymerase chain reaction (PCR) using anthrax-specific primers. Results: On Gram and acid fast staining, purple rods and pink-coloured anthrax spores were detected. Capsular and M'Fadyean staining was done. Gamma-phage lysed B. anthracis culture. Of 39 suspected cases, 8 were culture and PCR positive and showed gamma-phage lysis. 3 deaths were reported. Discussion and Conclusion: The conventional and real-time PCR methods are suitable for both the clinical and the epidemiological practice.


Keywords: Bacillus anthracis, gamma-phase, outbreak, polymerase chain reaction


How to cite this article:
Kumar M, Seema K, Prasad A, Sharma AK, Sherwal BL. Molecular confirmation of the circulating Bacillus anthracis during outbreak of anthrax in different villages of Simdega District, Jharkhand. Indian J Med Microbiol 2019;37:116-9

How to cite this URL:
Kumar M, Seema K, Prasad A, Sharma AK, Sherwal BL. Molecular confirmation of the circulating Bacillus anthracis during outbreak of anthrax in different villages of Simdega District, Jharkhand. Indian J Med Microbiol [serial online] 2019 [cited 2019 Oct 21];37:116-9. Available from: http://www.ijmm.org/text.asp?2019/37/1/116/264484





 ~ Introduction Top


Bacillus anthracis is a Gram-positive, aerobic, capsulated, spore-forming bacterium that can infect both animals and humans. It causes anthrax, a zoonotic disease that is primarily associated with herbivores and domestic livestock. Humans acquire this infection from close contact with infected animals or contaminated animal products, such as hair, meat and skin.[1] There are three primary forms of the disease in humans – cutaneous, pulmonary and gastrointestinal.[2],[3] Cutaneous anthrax is the most common form of the disease and accounts for 95% of cases worldwide.[4] It is acquired when spores enter through a cut or abrasion in the skin. The most common areas of exposure are the hands, arms, face, back and neck.[5] Because of the ability of its spores to persist in the environment for long periods of time, as well as the potential for spores to be disseminated in a powdered form, which increase its viability and pathogenicity,[6]B. anthracis is regarded as an effective biological weapon.[1],[7],[8] The 2001 bioterrorism-associated anthrax outbreak highlighted the need for validated rapid diagnostics. This outbreak investigation established the use of polymerase chain reaction (PCR) for detection of B. anthracis directly from clinical specimens, a process that established in identifying confirmed cases of cutaneous anthrax.[9],[10] Bacterial culture or direct staining of material from lesion swabs generally establishes the clinical diagnosis of cutaneous anthrax. However, these cultures often yield negative results due to activity of locally used antiseptics as well as local microbiota within the lesion site.[11] With the advancements in molecular techniques, it has become easy to confirm and characterise the epidemiology of disease with this organism.[3],[12],[13]


 ~ Materials and Methods Top


A total of 39 blood samples with swabs in duplicate with smears from skin lesions were collected from the suspected cases of anthrax from October 2014 to June 2016 from Simdega district, Jharkhand, where hospital authorities reported a cluster of suspected human anthrax cases. All the samples were transported in triple packaging system to the Department of Microbiology, RIMS, Ranchi. All the swabs were inoculated on polymyxin lysozyme EDTA thallous acetate (PLET) media, nutrient agar and 5% sheep blood agar for overnight incubation at 37°C. The colonies were tested for biochemical and motility. All duplicate swabs were used for Gram staining, modified acid fast staining for spores, capsular and M'Fadyean stain for capsule detection. Gamma-phage lysis was done on overnight growth in blood agar plate, as it is a bacterial virus that specifically lyses B. anthracis with 96% specificity, as most other strains of the Bacillus group are not susceptible to it. DNA extraction was done using a QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA) and subjected to PCR. The primer sequences used for the study were:

  1. Protective Antigen:


    1. PA 5 3048-3029: TCC TAA CAC TAA CGA AGT CG
    2. PA 8 2452-2471: GAG GTA GAA GGA TAT ACG GT.


  2. Capsule:


    1. 1234 1411-1430: CTG AGC CAT TAA TCG ATA TG
    2. 1301 2257-2283: TCC CAC TTA CGT AAT CTG AG.


The bands obtained were later captured on Gel Imaging System.


 ~ Results Top


Of 39 suspected cases, 8 were found culture positive, PCR positive and also showed gamma-phage lysis for anthrax. 3 deaths were reported. The attack rate was 21% with case fatality rate of 37.5%. All affected people were male with median age 30 years of age group range 18–55 years. On Gram staining from the swabs specimen, it appeared as purple rods [Figure 1]. Acid fast staining with 1% H2 SO4 showed the presence of pink anthrax spores [Figure 2]. Indian ink and M'Fadyean staining were done for the detection of capsule [Figure 3] and [Figure 4]. The colonies on nutrient agar showed irregular, raised, dull, opaque and greyish-white giving it a 'Medusa head appearance' [Figure 5]. On blood agar dry, white and non-haemolytic colonies were seen. PLET medium was used as selective medium to isolate anthrax bacilli from mixtures of other spore-bearing bacilli showing circular, creamy and white colonies with ground glass texture. The bacilli were non-motile. Catalase, indole and nitate reduction test were positive and oxidase, urease negative. The bacilli fermented glucose, maltose and ribose without gas production. Gamma-phage lysis specifically lysed B. anthracis culture which showed clearing in the centre of the blood agar plate [Figure 6]. In PCR, all 8 samples were positive. Samples and positive control had a banding pattern as expected for both protective antigen and capsule [Figure 7]. All affected people belonged to low socioeconomic community, and individuals who had handled, skinned and slaughtered dead livestock were at highest risk of infection.
Figure 1: Bamboo stick appearance on Gram stain

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Figure 2: Pink spores on Ziehl Neelsen staining

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Figure 3: Capsule on India ink staining

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Figure 4: Capsule on M'Fadyean stain

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Figure 5: Medusa head appearance on NA

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Figure 6: Gamma phage lysis on BA

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Figure 7: Gel image of polymerase chain reaction product by conventional polymerase chain reaction

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 ~ Discussion and Conclusion Top


There are very few studies about the epidemiological situation of anthrax in Jharkhand. In fact, epidemiological data of animal anthrax indicated that this disease is more widespread in Simdega district as compared to other districts of Jharkhand State. In this outbreak, anthrax was likely transmitted through unprotected contact with anthrax-affected animal hide and meat. We recommended screening of cattle, sheep and goats for any symptoms and administration of vaccine to all the livestock in that and at its adjacent area. Anthrax is still an important endemic disease of public health importance in several countries of Asia, Africa and Europe. In a country like India, anthrax is a concern of public health as clandestinely encountering in several states such as Andhra Pradesh, Kerala and Karnataka, Orissa, West Bengal and Jharkhand. Although anthrax can be cured by prompt antibiotic therapy, it is fatal in several cases because of lack of proper diagnosis well in time. We recommended ciprofloxacin prophylaxis to close family contacts. Community health education was to be imparted on the dangers of handling, slaughtering and consuming dead or ill livestock. Although cultural methods in microbiology are considered to be the standard methods for identification of B. anthracis in biological materials as well as in materials coming from the environment, these methods are cumbersome. Hence, molecular methods have the greatest advantage that they are able to detect the presence of even one live or dead cell, and even a small amount of the examined sample is enough for the detection of agents and also significantly reduces the time required for determination and confirmation of final result for diagnosis. In conclusion, it is possible to point out that conventional and real-time PCR methods are suitable assistants[14] for both the clinical and the epidemiological practice. The laboratories equipped with these techniques and possibilities really posses a useful and efficient tool for the prevention and therapy of such severe infection disease, as the anthrax really is.

Acknowledgements

The author is thankful to IDSP team for providing support to carry out field investigation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

1.
Inglesby TV, O'Toole T, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, et al. Anthrax as a biological weapon, 2002: Updated recommendations for management. JAMA 2002;287:2236-52.  Back to cited text no. 1
    
2.
Centers for Disease Control and Prevention (CDC). Inhalation anthrax associated with dried animal hides – Pennsylvania and New York city, 2006. MMWR Morb Mortal Wkly Rep 2006;55:280-2.  Back to cited text no. 2
    
3.
Marston CK, Allen CA, Beaudry J, Price EP, Wolken SR, Pearson T, et al. Molecular epidemiology of anthrax cases associated with recreational use of animal hides and yarn in the United States. PLoS One 2011;6:e28274.  Back to cited text no. 3
    
4.
Centers for Disease Control and Prevention (CDC). Human anthrax associated with an epizootic among livestock – North Dakota, 2000. MMWR Morb Mortal Wkly Rep 2001;50:677-80.  Back to cited text no. 4
    
5.
Shieh WJ, Guarner J, Paddock C, Greer P, Tatti K, Fischer M, et al. The critical role of pathology in the investigation of bioterrorism-related cutaneous anthrax. Am J Pathol 2003;163:1901-10.  Back to cited text no. 5
    
6.
Goel AK. Anthrax: A disease of biowarfare and public health importance. World J Clin Cases 2015;3:20-33.  Back to cited text no. 6
    
7.
Rotz LD, Khan AS, Lillibridge SR, Ostroff SM, Hughes JM. Public health assessment of potential biological terrorism agents. Emerg Infect Dis 2002;8:225-30.  Back to cited text no. 7
    
8.
Anderson PD, Bokor G. Bioterrorism: Pathogens as weapons. J Pharm Policy Pract 2012;25:521-9.  Back to cited text no. 8
    
9.
Hoffmaster AR, Meyer RF, Bowen MD, Marston CK, Weyant RS, Thurman K, et al. Evaluation and validation of a real-time polymerase chain reaction assay for rapid identification of Bacillus anthracis. Emerg Infect Dis 2002;8:1178-82.  Back to cited text no. 9
    
10.
Jernigan DB, Raghunathan PL, Bell BP, Brechner R, Bresnitz EA, Butler JC, et al. Investigation of bioterrorism-related anthrax, United States, 2001: Epidemiologic findings. Emerg Infect Dis 2002;8:1019-28.  Back to cited text no. 10
    
11.
Shlyakhov E, Rubinstein E. Evaluation of the anthraxin skin test for diagnosis of acute and past human anthrax. Eur J Clin Microbiol Infect Dis 1996;15:242-5.  Back to cited text no. 11
    
12.
Hoffmaster AR, Fitzgerald CC, Ribot E, Mayer LW, Popovic T. Molecular subtyping of Bacillus anthracis and the 2001 bioterrorism-associated anthrax outbreak, United States. Emerg Infect Dis 2002;8:1111-6.  Back to cited text no. 12
    
13.
Keim P, Van Ert MN, Pearson T, Vogler AJ, Huynh LY, Wagner DM, et al. Anthrax molecular epidemiology and forensics: Using the appropriate marker for different evolutionary scales. Infect Genet Evol 2004;4:205-13.  Back to cited text no. 13
    
14.
Dauphin LA, Marston CK, Bhullar V, Baker D, Rahman M, Hossain MJ,et al. Swab protocol for rapid laboratory diagnosis of cutaneous anthrax. J Clin Microbiol 2012;50:3960-7.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]



 

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