SEROLOGICAL AND MOLECULAR DETECTIONS
BACTERIAL INFECTIONS
Molecular
Method
Serological Method
Group A
Streptococci
Widely Used/
Common
Method
Lateral Flow
Immunochromatographic
Assays (LFA) –
widely used in
outpatient clinics
Helicobacter
pylori
Mycoplasma
pneumoniae
 atypical
pneumonia
 walking
pneumonia
Ricketssial
Infection
Method of
Choice
Gold Standard
ELISA
Serological Tests
1. Streptozyme Kit Testing
2. Anti-DNase Testing
 Classic Neutralization Method
 Nephelometry Method
3. Anti-Streptolysin O (ASO)
 Slide Agglutination Test: >200 IU/ml is
significant
 Traditional/Classic ASO titer tube
method
 Normal: ≤ 166 Todd units
 Significant: 240 Todd units: ADULT
 Significant: 320 Todd units: CHILD
 Positive: NO HEMOLYSIS
ELISA
Culture
 “mulberry” colony
with typical “fried
egg” appearance
Molecular
Indirect
Immunofluorescence
Assay (IFA)
 Four-fold rise in
antibody titers
Other Methods
1.
2.
3.
4.


ELISA
Immunoblot
LA – rapid test
LFA – rapid test
ELISA – IgM and IgG
Cold agglutinin titer test – no longer
recommended
Weil-Felix Reaction – based on crossreactivity of px antibodies with
polysaccharide antigens present on OX19
and OX2 strain of Proteus vulgaris and
OX-K strain of Proteus mirabilis
 Principle: DIRECT AGGLUTINATION
 Positive titer: 1:320
Real-time PCR
technology
(RT-PCR)
 detect the
presence of
organism
FECAL
samples
 patient’s
bacterial load
1. Urea Breath Test
14
C: radioactive
13
C: nonradioactive
2. CLOtest
 detects urease
activity in GASTIC
MUCOSAL
BIOPSIES
 Positive: yellow
gel will turn hot
pink (20 mins)
 ideal for rapid
diagnosis
Serological Method
Screening Test
Syphilis
(Treponema
pallidum)
Confirmatory Test
Western Blot – congenital
syphilis
1. VDRL (Venereal Disease Research
Laboratory)
 0.03% cardiolipin: main reacting
agent
 0.9% cholesterol: enhances
reactive surface of cardiolipin
 0.21% lecithin: removes
anticomplementary of cardiolipin
1. Treponema Pallidum
Immobilization Test (TPI)
 Positive: ≥50% of treponemes
are IMMOBILIZED
 Negative: fewer 20% are
IMMOBILIZED
 Doubtful: ranges 20-50% are
immobilized
2. RPR (Rapid Plasma Reagin)
Cardiolipin, cholesterol, lecithin +
 EDTA: prevents oxidation of lipid
 Thimerosal: preservative
 Choline Chloride: inactivate the
complement and stabilize the
antigen
2. Fluorescent Treponemal
Antibody Absorption Test
(FTA-ABS)
 Principle: INDIRECT
IMMUNOFLUORESCENT ASSAY
 Instrument: Fluorescence
Microscope
 Reactive: 2+ to 4+
 Negative: No fluorescence
 Minimally Reactive: 1+, test
must be repeated with a 2nd
specimen drawn in 1 to 2 weeks
3. TRUST (Toluidine Red Unheated
Serum Test)
4. USR (Unheated Serum Reagin)
5. RST (Reagin Screen Test)
3. Hemagglutination tests
a. TPHA (T.pallidum
hemagglutination assay)
b. TPPA (T.pallidum particle
agglutination assay)
c. MHA-TP (Micro
hemagglutination assay for
T.pallidum)
d. HATTS (Hemagglutination
Treponemal test for
syphilis)
Molecular Method
Other Serological
Tests
1. Capture EIA –
congenital
syphilis in infants
PCR technology
1.
 Sensitivity is high in
PRIMARY SYPHILIS 2.
 Presence of
treponemes in whole
blood, spinal fluid,
amniotic fluid, various
tissues, swab samples
for syphilis lesions
Other Methods
Dark field
microscopy
corkscrew
motility
Serological Method
Screening Test
Lyme Disease 
(Borrelia
burgdorferi)

IFA (Immunofluorescence Assay)
 Low volume testing
 Positive: 1:256 or higher
EIA (Enzyme Immunoassay)
 High volume testing
*if positive to IFA OR EIA: proceed
to Western Blot (supplemental
testing)
Confirmatory Test
Western Blot
 Positive result in IgG
 Ten proteins used (CDC)
18, 23, 28, 30, 39, 41, 45,
58, 66,
and 93 kDa
 IgM immunoblot positive:
Two o f the following bands
must be present 23(Osp C),
39, and 41 (flagellin) kDa
 IgG immunoblot positive: if
any 5 of the 10 bands are
positive
Molecular Method
Other Serological
Tests
Other Methods
PARASITIC AND FUNGAL INFECTIONS
Aspergillosis

Serological Method
What is detected
Serological test
Galactomannan in serum
 EIA
Β-D-glucan (BDG) in serum
Cryptococcus

Molecular Method
1. Cryptococcal Latex
Agglutination test
 1:2 titer: suggests infection
 1:4 titer: active infection
 Cross reaction with Rheumatoid
factor
Additional Info
Other Methods
 Positive tissue biopsy
demonstrating
hyphae
 Positive culture
 Direct microscopic
examination
 culture

Direct examination
 10% KOH


2. Tube agglutination test for C.
neoformans
 1:2 titer: recent infection
3. India ink
 Detects polysaccharide capsule
 Halo around the yeast cell
4. Indirect Immunofluorescence
Assay (IFA)
 Most valuable when antigen
tests are negative
Candidiasis 
Mannan and anti-mannan
antibodies
PNA-FISH (Peptide
nuclear acid
fluorescent in situ
hybridization)
 Utilizes small PNA
polymer
Differentiate Candida
albicans from other
Candida spp.
Serological Method
What is detected
Coccidioidomycosis 
Molecular
Method
Other Methods
Serological test
1. Complement fixation (IgG test)
 most widely used quantitative for C. immitis
 Early infection 1:2 to 1:4 (test repeated in 3-4 weeks)
 Active infection 1:16
 False negative: Solitary pulmonary lesions
 Cross reactions: Acute histoplasmosis (false positive)
2. Immunodiffusion
3. Latex Agglutination
4. Tube precipitation test (IgM test)
 IgM appear in 1-3 weeks
 Early indication of primary infection
Histoplasmosis
5.
5. ELISA
 (+) test should be confirmedn with CF or TP test
1. Complement Fixation – most common
 1:8 titer to yeast or mycelial antigen: positive
 1:32 titer: indicate histplasmosis

2. Precipitation – most common
 Presence of H and M antibodies
3. ELISA – detect polysaccharide antigen in serum or urine
Serological Method
Toxoplasmosis
Gold standard
Sabin-Feldman Dye test
 Detect IgG antibodies
 Principle NEUTRALIZATION
 Stain: methylene blue
 Positive: not stained, thin and
distorted
 Negative: stain deeply blue
Other Serological Test
Labelled Immunoassays
* ELISA, IFA, and Chemiluminescence immunoassay for
the detection of IgG and/or IgM antibodies
1. EIA – method of choice, more sensitive
2. IFA – widely used
Molecular Method
PCR – method of
choice to detect
T. gondii DNA in CSF
Additional Info
VIRAL INFECTIONS
Hepatitis A
Picornaviridae
Hepatitis C
Flaviviridae

Genus
Hepacivirus
Hepatitis D 
Delta hepatitis
Hepatitis E 
Calciviridae
reclassified to
Serological Method
Confirmatory
Serological test
1. IgM Anti-HAV – detected by
ELISA *most commonly used
2. IgG Anti-HAV – detected by
competitive inhibition ELISA
NAT (Nucleic Acid Testing) 1. Anti-HCV - Anti-HCV IgG
detected by EIA or CLIA
 CDC recommended
2. ELISA
 Reactive: current HCV
 RIBA (Recombinant Immunoblot
infection
Assay) – 2nd answer for
 NR: past HCV infection
confirmatory test
or false (+) antibody
test result
RT-PCR
 RNA viral load
ELISA - screening
 HDV antibodies
1. ELISA
2. Western Blot
3. Fluorescent antibody blocking
assay
Hepeviridae
HBsAg
First serological
marker to appear
Becomes
undetectable in
serum 4-6
months after
onset of
symptoms
HBeAg
(envelope)
 Present
during active
replication of
virus
 High degree
of infectivity
HBcAg
(core)
 Not
detectable in
serum
 Detected only
through
biopsy of
infected liver
Molecular Method
 Infectious Hepatitis
 Short Incubation
Hepatitis
RT-PCR – detect HAV
RNA
1. Qualitative
2. Quantitative
 RT-PCR
 Real-time PCR
 bDNA amplification
Additional Info
Other Methods
Surrogate Test
 Anti-HBc
 ALT
 Most frequent cause of
chronic liver infection
 Leading indicator for
liver transplantation
Treatment
 Pegylated interferon-a
(PEG IFN-a)
 Ribavirin
 Four genotypes (HEV1,
HEV2, HEV3, HEV4)
 Associated with high
rate of mortality in
pregnant women
PCR – detects HEV RNA
 Real-time PCR
 (LAMP) LoopMediated Isothermal
amplification Assa
 both detects HEV
RNA
Hepatitis B
Hepadnaviridae
Anti-HBc IgM
Anti-HBc IgG
 Indicator of
 Persists for
current or
lifetime
recent
 Increased in
infection
chronic
 Useful in
infection &
detection core
recovery stage
window
period
Anti-HBe
Anti-HBs
 Indicates patient
is recovering
from HBV
infection
 Maker of
convalescence
and favorable
prognosis
 Persists for years
and provide
protective
immunity
 Protective titer:
10 mIU/mL or
highers