113

Vol. 5. No. 5 May–August 2015

Research Report

Relationship Between CliniCal manifestations and 
antiBody seRum in outBReaks anthRax 

Dhani Redhono
Sub Division Tropical Medicine & Infectious Disease 
Faculty of Medicine, Sebelas Maret University,
Moewardi Hospital
Email of corresponding author : dhani_ipd@yahoo.com

abstract 
Introduction: Anthrax is a zoonotic disease that often affects the grass-eating animals, which occurs due to the entry of spores into 

the bodies of animals and can be transmitted to humans. This disease often appear in certain seasons and occurs in endemic areas, 
including Indonesia. Cutaneous anthrax is the clinical manifestations that often arise on outstanding events in the area. This study aims 
to determine how the relationship between the clinical manifestations of the serum antibodies in people who are exposed to anthrax.  
Material and methods: This study is an observational cross sectional analytic approach, in people exposed to anthrax to assess 
the clinical manifestations and antibody serum Anthrax. Results: Obtained in this study respondents were 101 people with a history 
of contact with animals suffering from anthrax. The number of respondents with the highest age distribution was 31 to 40 years by 
42%, and most were female gender, which is 57.7%, the highest level of education is 74% finished elementary school. Forty-four 
percent of working as a housewife. Risk factors are the most direct contact with and consume the flesh of animals as much as 34.6%. 
Results of Ig G antibody serum showed 50% negative, 15.4 borderline and 34.6% positive. Clinical manifestations that occur in 
the skin as much as 13.5%, that is the eschar on all respondents and 92.8% showed positive Ig G. While 86.5% did not show any 
clinical signs of anthrax, of that number 25.5% with Ig G positive, 16.6% and 57.7% showed borderline negative with p 0.02.  
Conclusion: There was a significant association between the clinical manifestation with antibody serum anthrax. But also found a 
positive Ig G without the appearance of clinical signs in the skin.

Key words: clinical, manifestation, anthrax, serum antibody ELISA, eschar 

abstrak

Pendahuluan. Antraks adalah salah satu penyakit zoonosis yang sering menyerang pada hewan pemakan rumput, yang terjadi 
karena masuknya spora ke dalam tubuh hewan dan dapat ditularkan ke manusia. Penyakit ini sering muncul pada musim tertentu dan 
terjadi di daerah endemi, termasuk Indonesia. Cutaneous Anthrax merupakan manifestasi klinis yang sering timbul pada kejadian 
luar biasa di suatu daerah. Penelitian ini bertujuan untuk mengetahui bagaimana hubungan antara manifestasi klinis terhadap serum 
antibodi pada orang yang terpapar antraks. Bahan dan Metode. Penelitian ini merupakan observasional analitik dengan pendekatan 
Cross Sectional, pada orang yang terpapar antraks dengan menilai manifestasi klinis dan serum antibodi Antraks. Hasil. Pada penelitian 
ini didapatkan responden sebanyak 101 orang dengan riwayat kontak dengan hewan yang menderita antraks. Jumlah responden 
dengan sebaran umur tertinggi adalah pada 31 sampai 40 tahun sebanyak 42 %, dan jenis kelamin terbanyak adalah perempuan, yaitu 
57,7 %, tingkat pendidikan terbanyak adalah lulus SD 74 %. Empat puluh empat persen bekerja sebagai ibu rumah tangga. Faktor 
risiko terbanyak adalah kontak langsung dan mengkomsumsi daging hewan sebanyak 34,6%. Hasil pemeriksaan Ig G antibodi serum 
menunjukkan 50% negatif, 15,4 borderline dan 34,6% positif. Manifestasi klinis yang terjadi pada kulit sebanyak 13,5 % , yaitu adanya 
eschar pada semua responden dan 92,8% menunjukkan Ig G positif. Sedangkan 86,5% tidak menunjukkan adanya tanda klinis antraks, 
dari jumlah tersebut 25,5% dengan Ig G positif, 16,6% menunjukkan borderline dan 57,7% negatif dengan p 0,02. Simpulan. Ada 



114 Indonesian Journal of Tropical and Infectious Disease, Vol. 5. No. 5 May–August 2015: 113-118

hubungan yang bermakna antara manifestasi klinis dengan hasil serum antibodi Antraks. Namun juga didapatkan adanya antibodi Ig 
G positif tanpa disertai munculnya tanda klinis di kulit. Sehingga perlu dilakukan deteksi dini pada orang yang terpapar antraks. 

Kata kunci: manifestasi, klinis, antraks, antibodi serum ELISA, eschar 

introduction

Anthrax is one of the types of zoonotic diseases, 
which can be transmitted to humans, animals suffering 
from anthrax. The disease is caused by bacillus anthracis. 
Anthrax commonly often attacked livestock such as cattle, 
sheep, goats and camels. Transmission to humans occurs 
when there is direct contact of animals or animal products 
that suffer from anthrax, can be skin, blood and flesh.1,2

Anthrax incident in Indonesia in the last ten years has 
occurred five times the plague that is 1996 to 2000 in West 
Java.3,4 

Since the outbreak of anthrax 15 years ago in Indonesia, 
the patient sample should be sent abroad (USA) for the 
diagnosis of anthrax investigation. Based on these events, 
the Moewardi hospital cooperate with Integrated Biomedical 
Laboratory of the Faculty of Medicine, University Sebelas 
Maret has been trying to develop anthrax test-based 
immunoassay using enzyme-linked immunosorbent assay 
(ELISA) for the detection of proteins PA by using the 
Anthrax Protective Antigen Calbiotech (PA) IgG ELISA 
Kit, as catcher agents that are sensitive to the ELISA was 
able to detect PA (≥ 1 ng / ml PA) in the serum of patients 
with suspected anthrax.4,5

This Problem is how is anthrax protective antigen serum 
antibodies (PA) Ig G ELISA in people who are exposed 
to anthrax and its relationship with clinical manifestations 
in outbreak area ?

Diagnosis approach
Anthrax Diagnosis is made through history, clinical 

examination, laboratory and serology : 
1.  History: Early diagnosis of anthrax is difficult to 

know because it does not show the typical signs and 
symptoms, usually preceded by the appearance of 
reddish nodule with pain and swelling. It needs to be 
asked is whether previously had contact with an animal 
that died of anthrax, either direct contact or eating meat 
or contact with animals or their products (skin, bone), 
how the employment status (farmers fields, ranchers, 
RPH, tanners) and whether residence in endemic areas 
of anthrax.2,6

2.  Clinical manifestations: There are 3 clinical 
manifestations that may arise in people is cutaneous 
anthrax, gastrointestinal and inhalation:
a.  Cutaneous anthrax
 Most cases (95%) anthrax is happening in the 

world is cutaneous anthrax. Patients usually have 
a history of contact with animals or their products, 
the anthrax bacteria or spores enter through the skin 

through a lesion on the skin, for example, when 
doing the slaughtering process (cutting, skinning 
or divide meat) cattle infected with anthrax. Then 
came a low germination rate at the location where 
the entry of spores and cause lesions on the skin 
that itch, then papuler lesions arise and develop into 
vesicles accompanied by edema and pain. These 
lesions became necrotic eschar formation and 
accompanied local soft tissue edema. Germination 
occurs within 1-3 hours after inoculation, but 
germination can not cause infection of the skin 
intact. Endospores will undergo phagocytosis by 
macrophages and then be taken to the regional 
lymph nodes, causing lymphadenopathy and 
lymphangitis. Hematogenous dissemination can 
occur, but with the provision of adequate spread 
of systemic antibiotics is quite rare. Several 
case reports of infections caused by insect bites 
suspected of being infected (eating carcasses 
containing anthrax bacillus).4,5

 Common location is on the face, extremities or 
neck. Endospores enter through skin abrasions or 
wounds. One to seven days after entry endospores, 
formed the primary skin lesions that are not painful 
and itchy papules. Twenty-four to 36 hours later 
lesions forming vesicles containing clear fluid or 
serosanguineus, and contains many Gram-positive 
bacteria. Vesicles then undergo central necrosis, 
dry out and cause eschar (necrotic ulcers) blackish 
typical vesicles surrounded by edema and purple. 
Edema usually occurs more severe on the head or 
neck than the body or limbs. Lymphangitis and 
lymphadenopathy that pain can be found following 
systemic symptoms occur. Although cutaneous 
anthrax can heal itself, but still need to be given 
antibiotics (to reduce systemic symptoms). In 80-
90% of cases the lesions healed completely without 
complications or scarring. Malignant edema are 
rare, characterized by severe edema, induration, 
multiple bullae, and shock. Malignant edema 
can occur in the neck and chest area that causes 
difficulty breathing, requiring corticosteroids or 
intubation.5,6

b.  Gastrointestinal Anthrax
 Gastrointestinal anthrax, although it can be fatal, 

has not been reported in the US. Symptoms usually 
appear 2-5 days after eating raw or undercooked 
meat that is contaminated with germs. Some 
cases may occur in the home. On pathological 
examination under a microscope can be found in 



115Redhono: Relationship Between Clinical manifestations and Antibody Serum

the mucosa and submucosa basil lymphoid tissue 
and mesenteric lymphadenitis. Ulceration almost 
always be found. A large number of Gram-positive 
bacteria can be found in the peritoneal fluid. 
Mediastinal widening can also occur.

 Clinical symptoms can include fever, diffuse 
abdominal pain, constipation or diarrhea. If there is 
ulceration of the bowel becomes blackish. Ascites 
can occur with clear liquids until purulent.1,6

c. Anthrax inhalation
 Inhalation anthrax spores began with the entry 

into the alveolar cavity, then macrophages will 
fagocyt spores and some of the spores will lysis 
and broken. Spores that survive will spread to the 
lymph nodes and mediastinal nodes. The process 
of change in vegetative forms occur approximately 
60 days later. The slow process of change in shape 
is not known with certainty, but well-documented 
cases of anthrax in Sverdlovsk that inhalation 
occurs between day 2 to day 43 after exposure. 
Once germination has occurred, the disease will 
arise quickly and replication of bacteria causing 
bleeding, edema and necrosis. The term anthrax 
pneumonia is not used because it turned out 
after pathological examination abnormalities 
were obtained mainly in the form of thoracic 
lymphadenitis and mediastinitis hemorhagis 
without typical bronchopneumonia. However, in 
the event of inhalation anthrax in Sverdlovsk, 25% 
of fatal cases was found bleeding focal necrosis 
and pulmonary lesions.1,2

 Anthrax meningitis
 A complication of cutaneous anthrax, inhalation 

and gastroitestinal, but is most common in 
inhalation anthrax (> 50%). Often addressing 
bleeding and meningoencepalitis. Anthrax death 
rate is over 90%.6,8

3.  Investigations : In the diagnosis of anthrax needed 
routine blood tests, culture swab the wound or blood 
(on the skin), sputum (on inhalation) chest X-ray (on 
inhalation), electrolyte (gastrointestinal) and serology 
using ELISA (Enzyme linked immunosorbent Assay) 
and PCR (Polymerasi Chain Reaction). Samples were 
taken for laboratory examination of the above is the 
blood serum, rub the injured area, sputum and land near 
the cage or a dead animal.9,10

Criteria of Diagnosis
The criteria used in the diagnosis of anthrax consists 

of three types, namely suspected (suspect), Probable 
(possibility) and Confirmed (Confirmation).10,11

Suspected, is clinically shown one form of anthrax and 
there is epidemiological evidence that exposure to anthrax 

environment, but there is no definitive laboratory evidence. 
Probable, is in clinical symptoms of anthrax but do not 
meet the definition of confirmation, but shows one of the 
following : (1) In Epidemiology, there are environmental 
exposures. (2) B. anthracis DNA evidence collected 
from the lesion, usually sterile (such as blood or CSF) 
or lesion of other affected tissue (skin, lung or digestive) 
(3) Positive serology IgG ELISA Anthrax Lethal Factor 
(LF) in the examination of the positive Spectrometry. 
Confirmed, is in clinical symptoms of anthrax with one 
of the following : (1) B. anthracis culture positive (2) 
Demonstrate B. anthracis antigens of the network by 
immunohistochemical staining using the cell wall and 
capsule monoclonal antibody B. anthracis (3) Proven 4x 
increase in antibody titer during the acute period and fixes 
the quantitative examination of anti-PA IgG ELISA testing 
(4) The presence of environmental exposure to anthrax and 
PCR test positive.10,11

method

This study is an observational analytic with cross 
sectional approach, by screening immunoassay based on 
people exposed to anthrax outbreak in the area in 2011. 
The location of sampling is an area of outbreak anthrax, 
Boyolali and Sragen, Central Java.   All the people who 
are exposed to dead animals suffering anthrax, a blood 
sample for examination IgG antibodies in serum by 
ELISA.3,4 Intepretation ELISA will get three categories: 
positive, borderline and negative, with inclusion criteria 
such as direct contact with infected animals anthrax, do not 
suffer from a disease that causes a decrease in the body’s 
immunity, there are currently using Immune suppressant 
drugs, there are pregnant or breastfeeding and not in 
hormone therapy. There were exclusion citeria such as 
not willing to follow research and medium serious illness, 
such as sepsis.

Operational definitions of variables
Dependent variable: clinical manifestations Is the result 

of the interview and physical examination in people who 
are exposed to anthrax animals.

The independent variables: levels of Ig G Anthrax 
Serology examination to assess the titer of anthrax 
protective antigen (PA) Ig G ELISA in order to confirm 
the presence of infection with Bacillus antracis in human 
blood. Interpretation of test results :
<0.9  :  No detectable IgG antibodies against PA protein 

in ELISA.
0.9 - 1.1 :  Borderline
> 1.1  :  Detected the presence of IgG antibodies to the 

protein PA, indicated patients were infected or 
infected with a never Bacillus anthracis.

Scale: nominal



116 Indonesian Journal of Tropical and Infectious Disease, Vol. 5. No. 5 May–August 2015: 113-118

result

In this study, 101 people with a history of contact with 
animals that died of anthrax. Respondents in the youngest 
age is 6 years old (1%) and the oldest 80 years (1%). 
The distribution of the highest age at 21 to 40 years as 
much as 39.6%, and most are women sex, ie 57.4%. The 
education level of respondents at most 74.3% graduated 
from elementary school. Subsistence farmers as much as 
21.8%. The basic characteristics of the study subjects are 
shown in Table 1.

Table 1.  Baseline Characteristics of research subjects (n = 
101)

Variable N %

Gender
• Male
• Female

43
58

42,6
57,4

Age
• 0 - 20 year
• 21 – 40 year
• 41 – 60 year
• 61 – 80 year

2
40
37
22

1,9
39,6
36,6
21,7

Pendidikan
• Elementary
• Junior high school
• Senior high school
• University

75
15
5
6

74,3
14,9

5
6

Profession
• No work
• Farmer
• Civil 
• Private

48
22
5
26

47,6
21,8
5,0
38,8

Of the total sample, showed serum Ig G antibodies 
showed negative 50.5%, borderline 15.8% and 33.7% 
positive. ELISA serology results can be seen in Table 2.

Table 2. Results of ELISA

Variable N %

• Positive
• Borderline
• Negative

34
16
51

33,7
15,8
50,5

In cross-table analysis results between risk factors 
contact with ELISA serology results obtained at the same 
respondents who cook and eat the highest risk on positive 
serologic results 20.8%. Serology results Elisa Risks 
associated with the contact can be seen in Table 3.

Table 3. Results of serology Elisa Risks associated with 
contact

Risk Factors
Elisa

Positif Border line Negatif

•	 Wash	the	meat
•	 Eating
•	 Wash	and	eat
•	 Cooking	and	

eating
•	 Slaughtering	and	

eating
•	 Located	near	the	

cage

1
10
3
11

9

0

0
5
2
6

3

0

1
16
0
19

13

0

Overall there are 11.9% of respondents who showed 
clinical signs of the appearance of the skin in the form of 
vesicles, accompanied by fever and ulcers which ended 
with eschar formation. The skin manifestations can be 
seen in Table 4.

Table 4. Distribution of clinical manifestation

Clinical manifestation N %

•	 Eschar
•	 No Eschar

12
89

11,9
88,1

At respondents with 10.9% positive serology results 
indicate a skin manifestation of the emergence of eschar, 
while only 1.0% with borderline serology showing the skin 
manifestations. There are as many as 22.8% with positive 
ELISA results, but does not cause any skin manifestation 
in the form of eschar or other clinical signs (fever, myalgia, 
stone, spasms, nausea and vomiting). Result of serology 
associated with skin manifestations such as eschar can be 
seen in Table 5.

Table 5. Results of serology Elisha associated with skin 
manifestations in the form of eschar

Eschar
ELISA

Positive Borderline Negative

•	 Yes
•	 No

11 (11%)
23 (23% )

1 ( 1,0 % )
15 ( 14,8 % )

0 ( 0,0 % ) 
51 ( 51% )

P 0.02

Contact risk factor for the emergence of manifestations 
in the skin, especially on the respondents were slaughtered 
at once ate beef (6.0%), followed by the washing and eating 
meat, which is 3.0%, whereas only 1.0% wash and meating. 
The relationship between contact with the manifestation of 
the emergence of the eschar can be seen in Table 6.



117Redhono: Relationship Between Clinical manifestations and Antibody Serum

Table 6. Relationship between contact with the manifestation 
of eschar

Variable
Eschar

Yes No

•	 Wash	the	meat
•	 Eating
•	 Wash	and	eat
•	 Cooking	and	eating
•	 Slaughtering	and	

eating
•	 Located	near	the	cage 

1 ( 1,0 % )
2 ( 2,0 % )
3 ( 3,0 % )
0 ( 0,0 % )
6 ( 6,0 % )

0 ( 0,0 % )

1 ( 1,0 % )
29 (28,7 % )
2 ( 2,0 % )

36 (35,6 % )
19 (18,9 % )

2 ( 2,0 % )

discussion

During the 2011 outbreak of anthrax in Central Java. 
Initially obtained a cow belonging to one of the people who 
had collapsed and accompanied by seizures. The owner 
decided to slaughter cattle meat and sold to residents of 40 
packs. Beef meat and blood samples examined in laboratory 
of Central Java Province and tested positive for anthrax. 
Seven days later, seven people were complaining there are 
small bumps and itching, swelling and lesions accompanied 
wet in the area under the eyes, hands, legs or feet, then taken 
to the health center and declared suspected anthrax. Then in 
May 2011 in Sragen also occur the same thing and be some 
people who show symptoms of anthrax skin contact.

Clinical manifestations in the form of eschar present 
in 11.9% with cutaneous anthrax. Respondents were taken 
from both locations, obtained 101 samples were then 
examined serological Anthrax serum Ig G antibodies. Of 
these 50.5% negative and 33.7% positive, while 15.8% 
borderline. Clinical manifestations in the form of a skin 
disorder that begins their edema or injury which led to 
edema and ends with eschar present in 10.9% of the 
respondents who Ig G antibody positive and 1.0% of 
respondents with Ig G borderline results. This is due to 
the emergence of antibodies against anthrax bacteria on 

respondents who had clinical manifestations in the skin, but 
that can not be explained is the result obtained antibodies 
also borderline clinical manifestations (see Figure 1).

Twenty-two percent of respondents with a positive 
serum Ig G antibody, did not lead to clinical manifestations. 
It might be due to the durability of the respondents, bacterial 
virulence factors and the amount of exposure that occurs 
may not be too much. But this can not be explained further, 
because of the endurance factor all pretty much the same 
condition, which probably is due to the virulence of the 
bacteria and germs that enter the number.

The risk of direct contact, ie cooking and eating meat 
of infected animals showed 30.5% Ig G positive results, 
but does not cause clinical manifestations with the advent 
of eschar (0%). This may be due to immune factors of 
patients as well as virulence B antrhacis that enters the 
body. Risk factors eating only 32% of respondents showed 
positive results. While the risk factors slaughter and eat 24% 
manifested by the appearance of skin eschar.

conclusions

The conclusion of this study is the increase in serum 
antibody titer Ig G anthrax does not occur in all of the 
respondents were exposed to the anthrax outbreak area, all 
respondents were obtained eschar followed by an increase 
in Ig G antibody titers.

Researchers suggest screening anthrax using anthrax 
Ig G antibodies can be done in areas that are outbreaks of 
anthrax and can proceed with dealing with how the eschar 
and its effect on Ig G antibody titer anthrax.

acknowledgements

We thank to Prof. Guntur Hermawan for helpful on the 
study and Paramasari for advice on setting up the Laboratory 
examination and sample analyses with ELISA.

Figure 1. Anthrax manifestations in the skin with the advent of eschar



118 Indonesian Journal of Tropical and Infectious Disease, Vol. 5. No. 5 May–August 2015: 113-118

reference

1. Jeremy Farrar, Peter J. Hotez, Thomas Junghanss, Gagandeep Kang, 
David Lalloo and Nicholas J. White; Anthrax ; Manson’s Tropical 
Diseases; 2014; 31, 395-398.e1.

2. Fred F. Ferri; Anthrax; Ferri’s Clinical Advisor 2015; Mosby, an 
imprint of Elsevier Inc; 2014; 115.e2-115.e4.

3. Redhono, Paramasari. Anthrax Outbreaks in Indonesia. Proceeding 
in APSIC 2011 - The 5th International Congress of the Asia Pacific 
Society of Infection Control. Melbourne ; 2011: 152.

4. Redhono D, Sumandjar T, Hermawan G Pemetaan antraks di jawa 
tengah. Antraks: Sebelas Maret press; 2011: 11–17.

5. Dirgahayu P Pemeriksaan laboratorium deteksi antraks berbasis 
immunoassay. Antraks : Sebelas Maret press; 2011: 18–26.

6. Dixon TC, Meselson BSM, Guillemin J, Hanna PC Anthrax. N Engl 
J Med; (2005) vol. 341 p. 815–826.

 7. Pile JC, Malone JD, Eitzen EM, Friedlander AM. Anthrax as a 
potential biological warfare agent. Arch Intern Med; 2005 vol 158 
p. 429–34.

 8. Shafazand S, Doyle R, Ruoss S, Weinacker A, Raffin TA Inhalation 
anthrax, Epidemiology, diagnosis and management. Chest ; 2005 vol 
116 p. 1369–1376.

 9. John E. Bennett, Raphael Dolin and Martin J. Blaser; Anthrax; 
Mandell, Douglas, and Bennett’s Principles and Practice of Infectious 
Diseases; Saunders, an imprint of Elsevier Inc; 2015; 209, 2391–2409.
e2.

 10. Inglesby TV, Henderson DA, Barlett JG Anthrax as a biological 
weapon medical and public health management. JAMA; 2005 vol 
281 p. 1735–1745.

 11. Holmes RK. Diphtheria, other corynebacterial infection and anthrax. 
In : Fauci AS, Braunwald E, Isselbacher KJ, Wilson JD, Martin JB, 
Kasper DL, et al. Eds. Harrison’s Principles of Internal Medicine. 
16th ed. McGraw-Hill ; New York; 2009: 892–899.