Tropical Medicine System, 2016

Immunodiagnostic in
Infectious Diseases

Yani Triyani
Clinical Pathology
Medical Faculty of Universitas Islam Bandung
 The Use Of “Biomarkers” Released Upon Contact Of Host Cells With
Pathogens To Diagnose Sepsis And Guide Therapeutic Interventions.
(a) Biomarkers are released in response and, thus, indicate contact of pathogen(s) or
pathogen-related compounds with host cells.
(b) Kinetics of release of two virtual biomarkers (marker 1 and 2).

( Bauer M, Kortgen A, and Singer M. Chap 1. Unmet Medical Needs in Life-Threatening Infections – Caring for the
Critically Ill in Modern Techniques for Pathogen Detection-2015 . Wiley-VCH Verlag GmbH & Co)
COURSE STUDY GUIDE of TMS:
1. Bacterial Infections
2. Myco-bacterial Infections
3. Viral Infections
4. Fungal Infections
5. Parasitic Infections
6. Zoonosis
7. Environmental Pathology and diseases
 1.Bacterial Infections
a. Bacterial sepsis
b. Typhoid fever
c. Tetanus
d. Pertusis
e. Diphteri
f. Meningitis
g. Bacterial skin infection (BSI)
h. Anaerobic bacteria
 2. Myco-bacterial Infections
a. Leprosy b. Cutaneous tuberculosis
(Scroffuloderma, cutaneus TB,)
 3.Viral Infections
• Dengue virus infection
• Rotavirus
• Varicella zoster (VZ) virus Infection
• Poliomyelitis
• Measles
• Mumps
• Rubela
• Molluscum contagiosum
• Verruca vulgaris
 4. Fungal Infections
a. Histoplasmosis b. Candidiasis cutis and
unguium
 5. Parasitic Infections
a. Protozoa
b. Soil Transmitted Helminth
. Ascariasis
. Enterobiasis
. Trichuriasis
. Hookworm infections
c. Filaria sp
 GROUPING PROTOZOA
BASED ON HABITAT

 AMEBA
1. Entamoeba
histolyitca
Entamoeba coli
 INTESTINAL (AND (apathogen) - not be
discused
 CILIATA
ATRIAL) PROTOZOA 2. Balantidium coli
 FLAGELLATA
3. Giardia lamblia

 BLOOD AND TISSUE

 SPOROZOA
PROTOZOA 4.
5.
Toxoplasma gondii
Plasmodium sp.

Click “Esc”button
May 2005 (dr. Djaenudin Natadisastra, SpParK) When finished
 6. Zoonosis
• Leptospirosis • Anthrax
 Laboratory diagnosis of infection
is based upon two main methods:
Direct Method Indirect Method
Detection of Serodiagnosis:
microorganism: • Widal test
• Culture: virus, bacteria, • Elisa Ig G and IgM Anti
fungi Dengue
• Staining: Gram, AFB, • Ig M Anti HAV
Neisser etc • Ig M and Ig G anti
• PCR Toxoplasma
• NS1 • Ab detection, etc
• Ag detection, etc
 DIAGNOSIS LABORATORIUM 15,17

15. Prevalence of Hepatitis B Infection in Pregnant Patients using Radioimmunoassay. Department of Obstetrics and Gynecology. Manila. 1999; 28(2):53-58
17. tCenters for Disease Control and Prevention (CDC). Nucleic acid amplification tests for uberculosis. MMWR Morb Mortal Wkly. 2000 Jul 7; 49(26): 593–594
 Modern Techniques
for Pathogen Detection
Identification Methods – An Overview
1. Taxonomy of Pathogenic Organisms
2. Microscopic Methods
3. Culture-Based Methods
4. Nucleic Acid-Based Techniques
5. Serology
 2. Microscopic Methods

Autofluo- Immunofluo-
Stains
rescence rescence

In Situ Dark-Field
Hybridization Microscopy
 3. Culture-Based Methods

Automated
Blood
Differentiation API System
Culture
Systems

Selective Susceptibility Other

Agar Testing Methods
 4. Nucleic Acid-Based Techniques
PCR Sequencing
• Real-Time PCR • Ribosomal RNA Genes
• Multiplex-PCR • MLST
• RAPD and rep-PCR • NG Sequencing
• Microarrays
• RFLP
 5. Serology
1. Antigen–Antibody-BasedMethods
1.1 Agglutination
1.2 Immunodiffusion
1.3 Quantitative Immunoassays
2. Automated Immunoassays
3. Applications of Serological Test
3.1 Types of Human Antibodies and WhatThey Indicate?
3.2 Validity of Antibodies against Opportunistic Species
3.3 Specificity of Fungal Antigen
3.4 Specific Urine Antigen Tests
4. Biomarkers
 Introduction
• Serological methods are used to detect the presence
of specific immune response against pathogenic
organism
• For detecting infectious agents that are either
difficult or impossible to culture
• There are limitations such as low sensitivity,
specificity but with the monoclonal ab and
improvement of the methodology, many serological
testing still be used as a diagnostic tool in microbial
disease
The Function of The Immune System
Phases of Immune Responses
Phases of Immune Responses
 Immunoassay
• A detection methods to localize antigen or
proteins either in cells, tissue section or
soluble proteins using labeled antibodies as
specific reagent through antigen-antibody
interaction that are visualized by a marker
such as fluorescent dye, enzyme or colloidal
gold
Principle of Immunoassay
 Source of Detected Antigen/Protein
• Tissue
• Cell
• Soluble (blood, serum, body fluid)

Serologic Test
 Interpretation of serologic
test
• Acute and convalescent antibody response
• Antibody specificity and cross reactivity
• False negative and false positive serologic
test result
• Value of serologic test
Antibody detection methods
• Agglutination assay
• Precipitation assays
• Gel immunodiffusion
• Complement fixation
• Immunoblotting
• ELISA
• RIA
• Indirect fluorescent ab assay
 METHODE AND PRINCIPLE
(Laboratory diagnosis of Infectious diseases)

• Method :
1. ELISA
Principle : Antigen/ Antibody (labelled with
enzyme)→ + substrate → coloured →
photometer
2. Agglutination
Principle: antigen + antibody  agglutination
 Kompleks
Antigen
antigen-antibodi

Anti IgM manusia yang Ikatan dengan anti

dilabel enzim Horseradish IgM manusia yang
peroksidase (HRP)
dilabel enzim HRP

Substrat kromogen Perubahan warna

substrat kromogen
hasil dari reaksi
enzimatik
 Infectious disease
Bacteri:
Viruses:
• Salmonella typhi
• Dengue
• Treponema Pallidum
• Hepatitis A, B, C
• Leptospira
• HIV
• Streptococci
• Helicobacter Pylori • Herpes viruses
• Mycoplasma • Rubella virus
• Chlamydiae • Cytomegalovirus
• Rickettsiae • Rota virus
Parasite: • Rubeolla (Measles)
• Malaria • Epstein Barr virus
• E. histolytica
• Toxoplasma gondii
 TYPHOID FEVER
S. enterica serovar Typhi (S. typhi) Ag
Culture bacteria is a gold standard
Salmonella typhi infection
 Serological Test for Typhoid Fever
(Widal Test)
Fernand-Isidore • Sample: paired serum (acute &
Widal convalescent)
• Methode WIDAL:
agglutination test
- ab to S. typhii
- O agglutination
- H agglutination
• Interpretation
paired sera/acute and
convalescent ab
response (increased 4X)
Slide method of Widal Test
Interpretation of WIDAL Test

1/20 1/40 1/80 1/160 1/320

O + + - - -
H + + + + -
Vi + - - - -

1/20 1/40 1/80 1/160 1/320

O + + - - -
H + + - - -
Vi + + + - -
 WIDAL : PAIRED SERA

1/20 1/40 1/80 1/160 1/320

O + + - - -
S1 H + - - - -
Vi + - - - -

O + + + + -
S2 H + - - - -
Vi + - - - -

showing increasing titers of O ( 4x )

Salmonella Typhi IgG/IgM
• rapid test is an
immunochromatographic
asay
• rapid qualitative and
differential detection of IgG
and IgM antibodies to
Salmonella typhi in human
serum plasma or whole
blood.
 Salmonella Typhi IgG/IgM

•IgM: Perfect alternative of Widal test

•IgGL Examination of past infection
•Specific screening test for Salmonella typhi
•Test result: 3 lins (IgG, IgM, Control)
•Specimen: Serum, Plasma or Whole blood
•Sensitivity: IgG-70% IgM-69% IgG+IgM-80%
•Specificity: IgG-76% IgM-79% IgG+IgM-81%
•Storage: Room temperature (2~30ºC)
Leptospira

Conjunctiva was icteric

with suffusion

• Diagnosis of Leptospirosis by
the microscopic agglutination
test (MAT)
• The MAT is considered the
reference standard in
serodiagnosis
 DENGUE VIRUS
• Dengue virus (DEN) is a small single-stranded RNA virus,
Flavivirus, family Flaviviridae
• Four distinct serotypes (DEN-1 to -4).
• The mature particle of the dengue virus is spherical with a
diameter of 50nm
• viral proteases in 3 structural proteins (capsid, C, prM, the
precursor of membrane, M, protein and envelope, E) and 7
nonstructural proteins (NS).

© 2011 Nature Education All rights reserved.

 DENGUE INFECTION
Laboratory diagnosis and diagnostic tests

DENGUE GUIDELINES FOR DIAGNOSIS, TREATMENT, PREVENTION AND CONTROL, WHO 2009
Current Concepts Dengue

N Engl J Med 2012;366:1423-32.

N Engl J Med 2012;366:1423-32.
The Stage of Dengue Infection
Advances of Dengue Diagnosis by Decade.
Diagnostic methods, techniques and
clinical samples for dengue.
Interpretation of Dengue Diagnostic Results.
(Adapted from Who/Tdr, 2009.)
Organization of Dengue Laboratory Diagnostic Services.
(Adapted From Who/Tdr, 2009.)
DENGUE GUIDELINES FOR DIAGNOSIS, TREATMENT, PREVENTION AND CONTROL, WHO 2009
Examples of primary and secondary infection in
paired sera tested by HI and IgG ELISA

aSample 1 (S1) collected in the acute phase of illness (days 1–5); sample 2 (S2)
collected at convalescent phase of illness (days 15–21 after S1 collection).
Dengue Infection Detection

• Sample: serum or plasma

• Methode: ELISA or rapid test
Dengue rapid test
 HERPES INFECTION
•Sample : serum or plasma
•Methode : ELISA
•Herpes simplex type l dan 2
• ELISA: Anti HSV 1 & 2 IgG
Anti HSV 1 & 2 IgM
•Interpretation:
HSV 1 IgG or IgM
- Negaive
Interpretation:
HSV 1 IgG or IgM
- Negative: HSV 1 IgG or IgM index ≤ 0.90
- Equivocal : HSV 1 IgG or IgM index 0.91 – 0.99
- Positive : HSV 1 IgG or IgM index ≥ 1.00

HSV 2 IgG or IgM

- Negative: HSV 2 IgG or IgM index ≤ 0.90
- Equivocal : HSV 2 IgG or IgM index 0.91 – 0.99
- Positive : HSV 2 IgG or IgM index ≥ 1.00
 RUBELLA

• Sample: serum
• Methode : ELISA
• Interpretation of Anti Rubella IgG & IgM
- Negative : < 10 IU/ml
- Positive : ≥ 10 IU/ml
 CYTOMEGALOVIRUS

• Sample : serum or plasma

• Methode : ELISA
• Interpretation of Anti CMV IgG & IgM
- Negative: < 90 %
- Gray zone : 90 – 100 %
- Positive : > 100%
Rota virus

Rotavirus & Adenovirus Combo

Feces rapid test kits
 Malaria
• Gold standard laboratory test are thick and
thin microscopic blood smears
Rapid Test for Malaria
 ANTI-AMOEBA
• Sample: serum or plasma
• Methode: Indirect hemaglutination test
• Interpretation:
- < 1 : 32  no invasive infection
- 1 : 32 – 1 : 64  borderline  repeat
- 1 : 64 - 1 : 128  active or recent infection
- > 1 : 256  current infection
 TOXOPLASMOSIS

• Sample: serum or plasma

• Methode: ELISA:
• Interpretation ofAnti toxoplasma IgG & IgM
– Negative < 6 IU/ml
– Positive ≥ 6 IU/ml
1. Apakah prinsip dari metode laboratory
diagnostic pada penyakit infeksi:
a…..
b…..
2. Apa artinya imunoserologi dan apa
perbedaannya dengan pemeriksaan
mikrobiologi?
3. Pemeriksaan serologi apa saja yang anda
ketahui?
 “Setinggi-tingginya Ilmu,
Sepandai-pandainya Siasat Dan
Semurni-murninya Tauhid”
(HOS Tjokroaminoto)

“Mujahid, Mujtahid, Mujaddid”

(Visi dan Misi UNISBA)
 Reference

• CAB International, 2014

• WHO Comprehensive Guidelines for Prevention and Control Dengue and DHF,
2011
• Henry’s Clinical Diagnosis And Management, 2011.
• Cameron P, et al. Current Concepts Dengue. N Engl J Med 2012;366:1423-32.