SEROLOGICA

L DIAGNOSIS
OF BACTERIAL
VH REVIEW CENTER INFECTIONS
DELFIN, RMT,MSMT
 Microbial antigen detection provides
direct evidence of infection, and is
preferred for diagnosis of infection over
antibody detection (indirect evidence of
infection)

 However, not all infectious agents have

available antigen assays or culture
techniques making the detection of
specific antibodies diagnostically useful
Syphilis
 SYPHILIS
 The most commonly acquired spirochete disease in
the U.S.
 A complex sexually transmitted disease that has a
highly variable clinical course
 Over 50,000 cases reported in 1990 in the U.S.
 Causative agent is Treponema pallidum
 No natural reservoir in the environment, requires
living host
 Spiral shaped and motile due to peri-plasmic
flagella
 Variable length
 Three other pathogens in the group Treponema
which are morphologically and anti-genetically
similar to T. pallidum, differences are in
characteristics of lesions, amount of systemic
involvement and course of the disease

 T. pertenue (Yaws)
 T. endemicum (non-venereal syphilis)
 T. carateum (pinta)
 T. cuniculi (rabbit syphilis)
 Mode of Transmission

 Organism is very fragile, destroyed rapidly by

heat, cold and drying
 Sexual transmission most common, occurs
when abraded skin or mucous membranes
come in contact with open lesion
 Can be transmitted to fetus
 Rare transmission from needle stick and blood
transfusion
 Stages of the Disease
 Primary stage
 primary lesion is chancre
 the lesion heals spontaneously after 1-5 weeks
 swab of chancre smeared on slide, examined
under dark-field microscope, spirochetes will be
present
 30% become serologically positive one week
after appearance of chancre, 90% positive after
three weeks
 Secondary Stage

 occurs 6-8 weeks after initial chancre, becomes

systemic, patient highly infectious
 characterized by localized or diffuse
mucocutaneous lesions, often with
generalized lymphadenopathy
 primary chancre may still be present
 secondary lesions subside in about 2-6 weeks
 serology tests nearly 100% positive
 Latent Stage

 stage of infection in which organisms persists in

the body of the infected person without causing
symptoms or signs
 this stage may last for years
 one-third of untreated latent stage individuals
develop signs of tertiary syphilis
 after 4 years it is rarely communicable
sexually but can be passed from mother to
fetus
 Tertiary Stage

 occurs anywhere from months to years after

secondary stage, typically between 10 to 30 years
 gummatous syphilis
 cardiovascular syphilis
 neurosyphilis
 Congenital Syphilis
 Transmitted from mother to fetus
 Fetus affected during the second or third trimester
 40% result in syphilitic stillbirth
 Live-born infants show no signs during first few
weeks
 60-90% develop clear or hemorrhagic
rhinitis
 skin eruptions (rash) especially around
mouth, palms of hands and soles of feet
 general lymphadenopathy,
hepatosplenomegaly, jaundice, anemia,
painful limbs & bone abnormality
 DIAGNOSIS
 Evaluation based on 3 factors
 Clinical findings
 Demonstration of spirochetes in clinical specimen
 Presence of antibodies in blood or CSF
 more than one test should be performed
 no serological test can distinguish between
other treponemal infections
 LABORATORY DIAGNOSIS
 Direct examination of clinical specimen by
dark-field microscopy or fluorescent antibody
testing of sample

 Non-specific or Non-treponemal serological

test to detect REAGIN
 utilized as screening test only
 not diagnostic
 Reagin is an antibody formed against cardiolipin
 Found in sera of patients with syphilis as well as
other diseases
  Non-Treponemal tests become
 positive 1-4 weeks after appearance of primary chancre
 in secondary stage may have false positive due to prozone
 tertiary 25% are negative, after successful
treatment will become non-reactive after 1 to 2 years

 Specific Treponemal antibody tests are used as a

confirmatory test for a positive reagin test
NON-TREPONEMAL SEROLOGICAL TESTS
REAGIN TEST

Venereal Disease Research Laboratory=VDRL

 Flocculation test, antigen consists of very fine particles

that precipitate out in the presence of reagin.

 Utilizes antigen consists of cardiolipin, cholesterol

and lecithin
 serum must be heated to 56 C for 30 minnutes to
remove anti-complimentary activity which may cause
false positive
 reported as Non-reactive, weakly reactive and
reactive
 used primarily to screen CSF
 The CLC
 Cardiolipin
• Alcoholic extract of normal beef heart
 Lecithin
• Helps neutrailize complement
 Cholesterol
• Provides adsorption centers and increase the reacting
surface of cardiolipin
• Increases the complement fixing capacity of cardiolipin
with reagin
Rapid Plasma Reagin – RPR

 General screening test

 Can not be performed on CSF
 The VDRL cardiolipin antigen is modified with
choline chloride to make it more stable and is attached
to charcoal particles to allow macroscopic reading, the
antigen comes prepared and is very stable.
 Serum or plasma may be used for testing, serum is not
heated
 Results are read macroscopically
 Appears to be more sensitive than the VDRL
Syphilis Testing
 VDRL  RPR
 Must heat serum to  Modified
56C for 30 minutes commercially
prior to testing to prepared antigen
inactivate attached to
complement which charcoal.
can cause a false  Serum does not have
positive. to be heated.
 Antigen must be  Plasma can be used.
prepared daily.  Read
 Test read macroscopically.
microscopically.
 Other tests which use modified VDRL Ag

 USR
 unheated serum reagin test
 modified VDRL Ag, uses choline
chloride/EDTA
 microscopic flocculation test
 RST
 reagin screen test
 modified VDRL Ag with Sudan Black
 Sudan Black makes flocculation reaction
macroscopically visible
 SPECIFIC TREPONEMAL TESTS

Treponema pallidum Immobilization Test

TPI
 Live T. pallidum become immobilized by antibody
in serum of infected persons
 Gold standard
 Principle:
 anti-treponemal antibodies in patient serum neutralize
actively motile spirochetes.
 The motile spirochetes (Reiter treponeme, non virulent
strain) are obtained from testicular chancre of rabbits
 Test is reported positive if 50% of the treponemes are
immobilized
 Treponema pallidum Hemagglutination
TPHA
 Micro hemmaglutination:
 Makes use of tanned sheep’RBC’s are coated with T.
pallidum antigen from Nichol’s strain (virulent strain)

 Hemagglutination
 Makes use of glutaraldehyde stabilized turkey RBC coated
with Nichol’s Strain

 positive result: agglutination of RBC’s

 FLUORESCENT TREPONEMAL ANTIBODY
ABSORPTION TEST
(FTA-ABS)
 One of the most used confirmatory test
 Slides are coated with Nichol’s strain of T. pallidum and
add absorbed patient serum
 Slides are washed and incubated with Ab bound to a
fluorescent tag
 After washing again the slides are examined for
fluorescence
 Requires experienced personnel to read highly sensitive
and specific, but time consuming to perform
 NOTE:
 Diluted, heat inactivated serum added to Reiter’s strain of T.
pallidum move cross reactivity due to other Treponemes
 ELISA
 Tubes coated with T. pallidum antigen
 Antibody in serum attaches to antigen
 Following washing, add an anti-antibody
tagged with enzyme alkaline phosphatase
 Detectable color changes occur
SYPHILIS: Biologic False Positives (BFP)
 Collagen diseases such as arthritis, LE, etc.,
sometimes result in increased amount of
reagin.
 Certain infections : IM, malaria, leprosy.
 Other Treponemal infections
SYPHILIS: False negatives
 Very early in disease or latent, inactive
stage
 Immunosuppressed patients
 Consumption of alcohol prior to testing
(temporary)
SYPHILIS: Congenital syphilis

 Non-treponemal tests on cord blood or baby

serum detect IgG antibody, maybe of maternal
origin.
 Detection of IgM lacks sensitivity.
 Western blot has demonstrated high sensitivity
and specificity
 Recommended that all mothers be tested
SYPHILIS: Cerebrospinal Fluid tests
 Used to determine if Treponemes have invaded
the CNS
 VDRL utilized to confirm neurosyphilis
 Lacks sensitivity
 CORRELATION OF TREATMENT WITH TEST
RESULTS
 Treatment at the primary stage, serology tests
become non-reactive after 6 months

 Treatment at secondary stage, tests usually non-

reactive after 12-18 months

 If treatment is not initiated until 10 or more

years, the reagin tests probably positive for life
REMEMBER
 ifa non-treponemal antibody screening test is
positive MUST do specific treponemal antibody
test.
 RPR CANNOT be performed on CSF or cord
blood.
 VDRL can be performed on CSF.
 LYME’S DISEASE
 Disease first recognized in 1977 in Lyme, Connecticut
 Causative organism is Borrelia burgdorferi
 Can be cultured but it is very difficult
 Organism has been isolated from blood, CSF, skin lesions
and joint fluid
 Can be transmitted perinatally, causing intrauterine death
 Vector of transmission is the Ixodes tick
 Must remain attached a minimum of 24-48 hours for
transmission to occur
 STAGES OF THE DISEASE
 Localized rash
 Erythema chronicum migrans
 Dissemination to multiple organ system
 occurs by way of the bloodstream
 may occur weeks to months after infection
 migratory pain may occur in the joints, tendons and
bones
 Neurologic
 Bell’s palsy, peripheral neuropathy, aseptic meningitis
 cardiac include carditis and arrythmia
 Chronic disseminated
 characterized by chronic arthritis
 affects the large joints, especially the knee
 Lyme Disease
 Causative agent: Borrelia
burgdorferi
 Transmitted by Ixodes
scapularis
 Bull’s eye rash
LYME’S DISEASE: Diagnostic criteria
 Isolation of organism from clinical specimen or
 Diagnostic titers of IgG and IgM in serum or CSF or
 Significant change in serum titers of IgG or IgM in paired
acute and convalescent sera
 Significant titer:
 Indirect: ≥1:64
LYME’S DISEASE: LABORATORY DIAGNOSIS
 Diagnosed clinically, confirmed serologically

 Antibodies to antigens of B. burgdorferi can be detected

by latex agglutination, IFA, ELISA, and Western Blot

 Serological tests are often falsely negative during early

weeks.

 Specific IgM Abs usually appear 2- 4 weeks after erythema

migrans, peak after 3-6 weeks of illness, decline to normal
after 4-6 months

 IgG titers appears more slowly (4-8 weeks after the rash),
peak after 4-6 months, may remain high for months or years
 Western Blot is most sensitive
 IgG: ≥ 4 of 9 bands
 IgM: ≥ 2 of 9 bands
 IFA and ELISA are more commonly performed
due to ease of procedure, but are subject to
false positives due to either spirochete diseases
and some autoimmune diseases
Streptococcal Serology
 Streptococci are gram (+), beta-hemolytic,
spherical, ovoid, or lancet-shaped organisms
which are catalase negative and seen in pairs or
chains

 Divided into groups or serotypes based on cell

wall components
 Streptococcus pyogenes belongs to Lancefield
group A

 it is believed the M protein is the chief virulent

factor
 Numerous exo-antigens are produced and excreted as
the cell metabolizes:

 Streptolysin O
 Dnase
 Hyaluronidase
 Nicotinamide
 Adenine dinucleotidase (NADase),
 Streptokinase
 Culture and rapid screening tests detect early infection
 Sequelae include Rheumatic Fever and Acute GN
GROUP A STREPTOCOCCAL INFECTION

 Two major sites of infection

 upper respiratory tract
 skin
 Upper respiratory tract
 sore throat, tonsillar exudate
 Skin
 pyoderma or impetigo
 Suppurative complications
 Erysipelas
 scarlet fever
 septic arthritis
 meningitis
 Non-suppurative complications
 RF
 Post-streptococcal GN
Rheumatic Fever
 Only certain serotypes of S. pyogenes is involved
 Develops as sequelae in 2-3% untreated upper respiratory
infections
 Symptoms occur about 18 days after sore throat
 Group A streptococcus share antigenic determinants with
host tissue, especially heart and even joints
 Inflammation of mitral valve most serious
 30-60% of patients may suffer permanent disability
 Post-Streptococcal Glomerulonephritis

 Follows Streptococcal infection of skin or pharynx

 Occurs about 10 days following initial infection
 Characterized by damage to glomeruli of the kidneys
 Renal function impaired due to reduction in glomerular
filtration rate, results in edema and HPN
 Renal failure not typical
 One theory is damage caused by antigen-antibody
complexes depositing in kidneys
 Complement is activated resulting in low levels
 LABORATORY TESTING

 Most reliable test is culture and identification of the

organism from infected site
 Rapid streptococcal screening tests from the throat exudates
have high specificity but low sensitivity, 60-85%
 Detection of Streptococcal antibodies most useful in
Streptococcal sequelae
 The most useful antibodies are : ASO, anti-DNase B, anti-
NADase, anti-Hyaluronidase
 Serological evidence of disease is based on elevated or
rising titer of Streptococcal antibodies
 Four-fold (2 tube dilution) rise in titer is considered
clinically significant
 Anti-Streptolysin O Titer (ASO Titer)
 Two of the toxins produced are Streptolysin S, which is
oxygen stable, non-antigenic and Streptolysin O (SLO),
which is oxygen labile and antigenic

 SLO is a hemolysin which is toxic to many tissues,

including heart and kidneys

 Evokes an antibody response (anti-SLO) which

neutrolizes the hemolytic action of SLO.

 The test is specific for ASO, it does not test for

antibodies to any other Streptococcal exotoxins
 Reference range will vary

 <125 Todd: adults

 5-125 Todd: children
 recent Strep infections 250 Todd units
• adults, 333 Todd units for children

 a single titer is of little significance unless

extremely elevated, titers performed over a
period of time will give the most information
 Anti-DNase B Testing

 May appear earlier than ASO

 Increased sensitivity for detection of
glomerulonephritis preceded by streptococcal
skin infection
 Macro- and micro-titer, ELISA, and
neutralization techniques are available
 Neutralization technique has advantage of
stability of reagents
 Anti-Hyaluronidase Testing
 Test patient serum for antibodies which inhibit action of
Hyaluronidase.

 After performance of the test, a clot will form into the

tubes where enzyme activity of Hyaluronidase has been
neutralized by patient antibody.

 Hyaluronidase is produced by patients with throat or skin

infections, ASO produced in response to throat infections
only
 Streptozyme Testing
 Hemagglutination procedure to detect antibodies to
numerous Streptococcal antigens

 Sheep RBC’s are coated with Streptolysin, Streptokinase,

Hyaluronidase, DNase, and NADase.

• Patient serum diluted 1 :100, mixed with sheep RBC’s

and observed for agglutination.

• rapid and simple to perform, more false positive and

negative results occur.
STREPTOCCOCUS Mg
 Non-hemolytic gram positive coccus
 Isolated from Mycoplasma pneumoniae infected
individuals
 Antibodies are usually detectable 2-3 weeks after
the onset of the disease
 S. MG titer occur in normal serum is 1:10
 A titer of 1:400 or greater in a single specimen in
PAP is suggestive of infection
 A fourfold rise in S. MG antibodies is of greater
diagnostic value.
Laboratory test
 Agglutination test

 Specimen can be kept in the refrigerator for

antibodies are not absorbed by red blood cells as
with cold agglutinins
Typhoid Serology
 Caused by Salmonella typhi
 Rapid detection is now available in the market
 Typhidot
 a qualitative detection test against a specific antigen of
Salmonella typhi. It can detect both IgG and IgM
separately and simultaneously. Thus, indicating the status
of acute infection, convalescence or previous exposure

 Salmonella typhi IgG/IgM Rapid test

 an immunochromatographic assay for rapid, qualitative and
differential detection of IgG and IgM antibodies to
Salmonella typhi in human serum, plasma or whole blood
Historical Test for typhoid
Widal’s Test
 Specimen of choice:
 First week of infection. Organism maybe isolated
from blood
 Second week and on. Organisms maybe isolated
from stool and urine
 Drainage from the biliary tract is useful in the
identification of carriers
 Third week and on. Antibodies appear in the
serum
Salmonella Antigens:
 Thermolabile flagellar, (Hauch or H antigens)
 Occur exclusively in the flagella. The antigens are
specific for the given species
 Sources:
 Salmonella (Group A, B, and C)
 Typhoid H
 Preparation
 Made by suspending the bacterial growth on agar in saline
containing 2% formalin which destroys the O antigen
 This antigen is good for months if refrigerated.
 Thermostable somatic (Ohne or O antigen)
 Directly associated with bacterial body
 Non-specie specificand can divide the genus into five
groups: A, B, C, D and E.
 Source:
 Salmonella Group A, B, C, D and E
 Preparation:
 Prepared by extracting bacterial cultures with either phenol
of alcohol.
 This extraction preserves the O antigen and destroys the H
antigens
 Good for months if refrigerated.
 Thermolabile capsular antigens (Kapsel or K antigen)
 Occur as capsules or as envelope sorrounding the
bacterial body.
 Several varieties:
 B, L and Vi
 Vi: Virulence antigen, occurs in highly virulent strains
of Salmonella typhi, paratyphi, ballerup
 Suggested as a means of identifying typhoid carriers
with negative O and H titers.
 Febrile Agglutinins
 Agglutinins in typhoid fever appear in blood 7-10 days and
reach the maximum peak in 3-5 weeks.
 Febrile Agglutinins
 O Agglutinins
 Titer of O agglutinins rises earlier in the disease and drops faster than
the H agglutinins
 Not affected by immunization
 H Agglutinins
 H titer are slow to rise in a disease but remain elevated for several years.
 H titer increases after immunization
Serological Tests
 Widal Agglutination Test
 Measures the presence of
anti-O and anti-H antibodies
using bacterial suspensions
of killed S. typhi and S.
paratyphi.
 IgM anti-O are detected
early in the infection
 IgG anti-H antibodies
develop later but persists for
a longer period of time
 Slide Method of Widal
 Performed on large glass plate that is into squares:
 Agglutination is scored from 0 to 4+
 ++++: 100% agglutination
 +++: 75% agglutination
 ++: 25% agglutination
 +: 25% agglutination
 Trace: less than 25% agglutination
 0: Non-Reactive
 Score of ++ or greater is considers positive
 Tube Method
 Requires serial dilution of serum and use to confirm
result of a slide test.
 Expected positive Results:
 Typhoid O: granular type of agglutination
characterized by suspension of fine aggregates
 Typhoid H, Paratyphoid A, and Paratyphoid B:
agglutinate as loose or fluffy clumps
 The highest dilution of a serum that exhibits a 50%
degree agglutination represents the end point titer of the
serum

 A four fold rise in “O” titer occurs in early rapid

typhoid infection (acute infection).

 At least 2 or more test should be performed every 3-5

days after the onset of the disease to demonstrate a
change in antibody titer.
Rickettsiae
 The members of this group are small pleomorphic
coccobacilli.

 They are gram-negative, although they stain poorly with

gram stain.

 They are non-motile obligate intracellular parasites.

 They possess both DNA and RNA

 They posses the characteristics of a bacterial cell wall and

they elaborate bacterial enzymes.
 All rickettsia require living cells for growth usually an
embryonated egg except for Rochalimea Quintana.

 Transmission is generally mediated by arthropods except for

Coxiella burnetti, which can be readily transmitted by
aerosol or by ingestion of contaminated dairy products.

 They are better visualized with Giemsa or the Macchiavelo’s

stain. However Gimenez staining method a modification of
Macchiavelo’s is better. The organism will stain red or pink
when counterstained with malachite green.
 Antibodies against these organisms maybe
detected from patient’s serum by Weil Felix Test.
 This is a bacterial agglutination test using Proteus
derived antigens: OXK, OX-2 and OX-19.
Typhus fever group
 Rickettsia prowazekii var. prowazekii

 Causative agent of epidemic typhus transmitted

through the bite of infected body louse.

 It is also the causative agent of the Brill-Zinsser

disease which is a recrudescent infection appearing
years after the primary typhus infection.
 Rickettsia prowazekii var mooseri

 Also known as Rickettsia typhi

 It is the causative agent of endemic typhus which is

transmitted by the rat flea, “Xenopsylla cheopsis”

 The infection is also called flea-borne typhus or

simply the rat typhus or murine typhus
 Rochalimea quintana

 The causative agent of Trench fever, transmitted by the

body louse.

 It is the only Rickettsia that can be cultivated in an

artificial medium. The organism was successfully
cultured on an enriched blood agar medium.

 The condition is also called as Shinbone fever, Five day

fever , Quintana fever, Wolhynian fever.
Spotted fever group
 Rickettsia rickettsii

 The causative agent of rocky mountain spotted fever (RMSF)

 It is transmitted trough the bite of ticks that is why the

disease is also known as tick-borne typhus
 Rickettsia conori

 The causative agent of the Mediterranean Spotted fever also

known as boutonneuse fever.

 Transmitted trough ticks.

 Rickettsia akari

 The causative agent of rickettsialpox. It is

transmitted trough mites.
Scrub typhus group
 Rickettsia tsutsugamuchi

 Found to cause scrub typhus. Also known as the

Rikettsia orientalis and transmitted trough the bite of
infected chigger mites.
 The condition is also called as chigger borne typhus,
Japanese river fever or simply tropical typhus.
 Coxiella burnetti

 Causes Q fever (Querry)

 Transmitted trough ingestion of contaminated milk
and milk products, inhalation of infectious aerosols,
skin when there is an open abrasion.
Serological Test
 In Typhus and RMSF, agglutinins appear in the
blood 7-10 days and reach the maximum peak by
the 14th day.
 Sources of Antigens:
 OX2 and 19: Proteus vulgaris
 OXK: Proteus mirabilis
 Weil Felix- Agglutination Test:
 Non-specific rickettsial test based on cross reacting
antibodies.
 Antibodies generated in this infection cross react
with polysaccharide O antigens of some Proteus
species
 The single most important aid in the identification of
rickettsial disease is a demonstration of a four-fold
rise in serum antibody titer when paired
 Reaction with Proteus
Strain
Ox2 Ox19 OxK

R. Ricketsii + ++++ -

R. mooseri + ++++ -

R.prowazeki + ++++

R.tsutsugamuchi - - +++

R.akari - - -

R.burneti - - -
Brucellosis
 Ag-Brucella abortus
 Cross – reaction with Abs to Francesella tularensis,
Vibrio cholera
 Diagnosis (in absence of cult. I.D.) based on
historic and clinical data plus sero titer >1:32
 Hemolyzed serum – false positive
 Inactive serum – false negative
Tularemia
 Serologic testing important because organism
hazardous and difficult to culture
 Agglutination test positive during second week of
illness, persist for years
 Diagnosis rests on history and clinical data plus
titer >1:40 (preferably fourfold rise)
 Cross-reaction with B. abortus
Organism Test Clinically Significant Result

S. pyogenes Antistreptolysin O ≥1:240

Anti-DNase B ≥1:240
Antihyaluronidase ≥1:512
S. typhi Widal’s test ≥1:160

L. pneumophila Indirect ≥1:256

Immunofluorescence
T. pallidum RPR +
VDRL +
FTA-ABS +
B. burgdorferi Indirect ≥1:64
Immunoflourescence
EIA +
Western blot IgG ≥4 of 9 bands
Western blot IgM ≥2 of 9 bands
H. pylori EIA +

M. pneumoniae Cold agglutinins ≥1:128

C’ fixation ≥1:32
EIA +
R. rickettsii Weil-Felix ≥1:320
Indirect ≥1:64
Immunofluorescence
E. chaffeensis Indirect ≥1:64
Immunofluorescence
B. henselae Indirect ≥1:128
Immunoflourescence