SCI 8008SEF Medical Microbiology & Virology II

Lecture 4

Advanced laboratory technologies in Clinical Microbiology

– Serology testing in Medical Microbiology

By Dr. Andy YY CHEUNG

cheungyy@hkmu.edu.hk
Communicable Disease Surveillance Case
Definitions
• This document is produced by Communicable Disease Branch, Centre
for Health Protection, Department of Health, Hong Kong Special
Administrative Region, People’s Republic of China
• This document lists the case definitions of notifiable infectious
diseases under the Prevention and Control of Disease Ordinance (Cap
599) (latest revision on 30 January 2023) and communicable diseases
of topical public health concern
• https://cdis.chp.gov.hk/CDIS_CENO_ONLINE/disease.html
Bacillary dysentery
• Description
• An illness of variable severity characterized by diarrhoea (with blood,
mucus or pus), fever, nausea, vomiting, abdominal cramps and
tenesmus
• Laboratory criteria
• Isolation of Shigella spp. from stool or rectal swab specimens
Subgroup and serotype designations of
Shigella

http://narst.dmsc.moph.go.th/manuals/AMRmanual_chapters%207-10.pdf
Cholera
• Description
• An illness that is characterized by acute painless watery diarrhoea
with or without vomiting
• Laboratory criteria
• Isolation of toxigenic Vibrio cholerae O1 or Vibrio cholerae O139 from
stool or rectal swab culture
Vibrio cholerae Antisera

https://www.debendiagnostics.co.uk/wp-content/uploads/2021/12/V.-cholerae-Antisera-IFU-Rev.-1.2-20210914.pdf
Vibrio cholerae Rapid Diagnostic Test
Procedure for Serotypes O1 and O139

• https://www.cdc.gov/cholera/pdf/crystal-vc-eng-p.pdf
Video:
• https://www.youtube.com/watch?v=W_FB_ZWYbh4
Haemophilus influenzae type b infection
(invasive)
• Description
• Invasive disease caused by H. influenzae type b (Hib) can produce several
clinical syndromes including meningitis, bacteraemic pneumonia,
septicaemia, epiglottitis, septic arthritis and osteomyelitis
• Laboratory criteria
• Any one of the following:
• Isolation of H. influenzae type b from a normally sterile site (e.g. blood or
cerebrospinal fluid (CSF) or, less commonly, joint, pleural, or pericardial
fluid)
• Detection of Hib antigen from CSF in a patient with laboratory evidence of
bacterial meningitis
Wellcogen™ Haemophilus influenzae b Rapid
Latex Agglutination Test
• Wellcogen™ H. influenzae b is a rapid latex test for use in the
qualitative detection of antigen from Haemophilus influenzae type b,
present in body fluids as a consequence of infection or in blood
cultures

https://www.thermofisher.com/order/catalog/product/R30858801
Typhoid fever
• Description
• An illness characterized by fever, headache, malaise, cough,
bradycardia, splenomegaly or rose spot on the trunk with or without
gastrointestinal symptoms
• Laboratory criteria
• Salmonella Typhi isolated from any clinical specimen
• Widal test result of a four-fold or greater rise in the titre of
Salmonella Typhi O antibody in paired sera
Paratyphoid fever
• Description
• Patient with paratyphoid fever usually presents with a similar but often
milder clinical picture than typhoid fever
• Symptoms may include fever, headache, malaise, cough, bradycardia,
splenomegaly or rose spot on the trunk with or without gastrointestinal
symptoms
• Laboratory criteria
• Salmonella Paratyphi (excluding S. Paratyphi B variant Java) isolated from
any clinical specimen
• Widal test result of a four-fold or greater rise in the titre of Salmonella
Paratyphi H antibody in paired sera
Typical reactions of Salmonella spp. in
screening biochemicals

http://narst.dmsc.moph.go.th/manuals/AMRmanual_chapters%207-10.pdf
Salmonella ser. Typhi colonies on triple sugar
iron (TSI) Agar
• On triple sugar iron agar (TSI) slants, S. Typhi
characteristically produces an alkaline slant (red,
“K”), an acid butt (yellow, “A”), and a small
amount of blackening of the agar (H2S, +) at the
site of the stab on the slant and in the stab line;
no gas (G) is produced
Antigenic Structure of Salmonella
• Three important antigens on their cell wall
• 1. Somatic antigen (O)
• 2. Flagellar antigen (H)
• 3. Surface envelope antigen (Vi)—found in some species

https://www.jaypeedigital.com/book/9789351523802/chapter/ch39
O antigen
Somatic antigen
-Part of cell wall
lipopolysaccharide
Heat stable, Alcohol stable
Formaldehyde labile
In Widal test-
O antigen of S. Typhi is used
O Ag is less immunogenic
Serogrouping is based on the
O antigen
Smooth colonies - virulent
strains carrying the O antigen
Rough strains - large, rough,
and irregular colonies and are
avirulent; auto-agglutination
in saline suspensions
H antigen
Flagellar antigen
-Made up of proteins –flagellin
-confers motility to the bacteria
Heat labile, Alcohol labile
Formaldehyde stable
In Widal test-
H antigens of S. Typhi, S. Paratyphi A
and B are used
H Ag is more immunogenic
Serogroups are differentiated into
serotypes based on H antigen
H Antigen Phase variation:
A culture in phase 1 can be converted
to phase 2 by passing it through a
Craigie’s tube containing specific
phase 1 antiserum
Vi Antigen
Surface polysaccharide envelope or
capsular antigen covering the O antigen
-Named with belief that Vi antigen is
related to virulence
Heat labile
Renders the bacilli inagglutinable with
O antiserum
After heating at 100°C for 1 hour, Vi
antigen is removed, exposing O antigen
for antisera-agglutination
Vi Ag is poorly immunogenic
Expressed in only few serotypes - S.
Typhi, S. Paratyphi C, etc.
Bacterial Serotyping Guide for Salmonella
• https://www.bio-rad.com/webroot/web/pdf/fsd/literature/FSD_14-
0699.pdf

• Salmonella Typhi and Paratyphi T, A, B, C, and Vi Kit

• https://www.bio-rad.com/en-hk/product/salmonella-typhi-paratyphi-
t-b-c-vi-kit?ID=LS55WA4EH
Salmonella Phase Inversion
• Craigie (1931) described a technique for the selection of motile
organisms from a poorly motile culture by passing them through semi-
solid nutrient agar in a tube containing a narrower, open-ended tube so
that, after inoculation on the agar surface in the inner tube, the motile
organisms moved through twice the depth of the medium to reach the
outer surface
• Using a fine-tipped pipette, 5 ml of the suspension was used to
inoculate a standard Craigie agar tube containing 0.1 ml antiserum to
the primary presenting flagellar phase
• The Craigie tubes were incubated at 37°C for 18–24 hour, after which
the second flagellar phase was determined by slide agglutination

• Another technique:
• https://www.youtube.com/watch?v=ROeJJ47hLuk
Luminex xMAP® Salmonella Serotyping Assay
• Salmonellosis is the most frequently reported cause of foodborne
illness, causing an estimated 1.4 million cases of foodborne illness
and over 400 deaths annually in the US
• The Luminex xMAP® Salmonella Serotyping Assay (SSA) is a multiplex,
nucleic acid-based assay for research or epidemiological use in
identifying serotypes greater than 85% of the most commonly
encountered Salmonella isolates within hours
Luminex xMAP® Salmonella Serotyping Assay
• The xMAP Salmonella Serotyping Assay was developed by the CDC for
fast and comprehensive molecular Salmonella serotyping
• The assay consists of three separate tests that detect O and H
antigens simultaneously, including the ability to identify some
serotype-specific markers in the Additional Targets or AT test

https://www.luminexcorp.com/salmonella-serotyping-assay/#overview
Luminex xMAP® Salmonella Serotyping Assay
®
xMAP SSA Antisera Agglutination
Simultaneous identification of 85% of most commonly Iterations of individual antisera agglutination tests
encountered Salmonella serotypes plus phase inversion
Can serotype rough and problematic isolates Cannot serotype rough and non-motile isolates
cGMP reagents Lengthy individual antisera lab validation due to lot to
lot inconsistencies
Qualitative calls: Subjective calls:
•Set comparative data thresholds •Visual agglutination read
•Four hour turnaround time •Two days to six weeks turnaround time

https://www.luminexcorp.com/salmonella-serotyping-assay/#overview
Widal test
• The bacteria that causes typhoid fever is Salmonella bacteria
• It can spread from person to person or from consuming contaminated food
• The bacteria are of two types:
• S. Typhi- O antigen
• S. Typhi- H antigen
• S. Paratyphi- AH antigen
• S. Paratyphi- BH antigen
• Video:
• https://www.youtube.com/watch?v=dflOQ8hXUbA
The Widal test procedure is carried out in two
steps:
1. Qualitative Widal Test
• For this test, you will use a slide with 6 reaction circles, marked as O, H, AH, BH, PC and NC
To begin with,
• Put one drop of the patient’s serum in four reaction circles, i.e., O, H, AH, BH
• Add one drop of positive control in the PC circle and one in the NC circle
• Next, add one drop of O antigen in the O circle, H antigen in the H circle, AH antigen in the AH
circle, and BH antigen in the BH circle, respectively
• Add any antigen, i.e., O, H, AH, BH in both PC and NC
• Next, mix the serum and antigen in each circle properly so that the mixture doesn’t go out of the
circle and touch the slide
• Also, one mixture should not mix with another, as it can influence the test results
• Finally, rotate the slide in a slow circular motion to ensure a proper mixture of serum and reagent
For example, if O is positive in the qualitative test,
we will further test the O reagent in the semi-
quantitative test
2. Semi-quantitative Widal test:
• Perform serial dilution of patient’s serum
• Add one drop of the patient’s serum in different reaction circles
• In the same way, put one drop of the specific reagent in all circles
• Next, mix the serum and antigen in each circle properly so that the
mixture doesn’t go out of the circle and touch the slide
• The titre of the patient serum using Widal test antigen suspensions is
the highest dilution of the serum sample that gives a visible
agglutination, report this titre
Scarlet fever
• Clinical Description
• Scarlet fever is an illness characterised by fever, sore throat and fine
sandpaper-like rash which blanches on pressure and with a characteristic
distribution
• Strawberry tongue and desquamation may also occur
• Laboratory Criteria
• Any one of the following:
• Detection of nucleic acid of group A Streptococcus in a respiratory
specimen, wound swab or blood specimen;
• Isolation of group A Streptococcus in a respiratory specimen, wound swab
or blood specimen; OR
• An antistreptolysin O titre > 200 in a serum specimen
InstaTest ASO Latex
Principle :
• The ASO latex test contain polystyrene latex particles, coated with
purified and stabilized streptolysin-o (antigen) which reacts with its
corresponding antistreptolysin-o (antibody) in the test sample
resulting in the agglutination of latex particles
Clinical Significance :
• Group A streptococci produces soluble and oxygen labile hemolysin
known as streptolysin 'o’
• This has lethal effects on the human being and especially toxic action
to heart muscle
Typhus and other rickettsial diseases
• Rickettsial diseases are caused by a group of Gram-negative obligate
intracellular bacteria in the family Rickettsiaceae
• They are primarily vector-borne diseases – transmitted by the bite or
faeces of infected arthropod vectors
• The affected patients usually presents with systemic infections often
characterized by fever and rash
• Scrub typhus (Orientia tsutsugamushi) and spotted fever (caused by more
than 30 types of rickettsiae) are common rickettsial diseases in Hong Kong
• Epidemic typhus (Rickettsia prowazekii) is of concern because of its public
health potential; Yet it has not been reported in Hong Kong for the past few
decades
• Urban typhus (Rickettsia typhi) is occasionally reported
Spotted fever
• Description
• The clinical presentation of spotted fever is usually non-specific
• There is a mild to severe febrile illness for a few days to 2 weeks
• Rash is a common clinical feature and may persist for one week
• There may be a primary lesion or eschar at the site of the arthropod bite
• Regional lymph nodes may enlarge
• Laboratory criteria
• Any one of the following:
• Four-fold or greater rise in antibody titre against the “spotted fever group” of rickettsiae
• Polymerase chain reaction demonstrating the presence of the genome of rickettsia of the
“spotted fever group” in the blood specimen
• Probable case
• A clinically compatible case with a single antibody titre against spotted fever group ≥ 512
Epidemic typhus
• Description
• Epidemic typhus is characterized by abrupt onset of headache, fever, chills
and myalgia
• A macular eruption appears on the fifth to sixth day, initially on the upper
trunk, followed by spread to the entire body, but usually not to the face,
palms or soles
• It is transmitted from person-to-person by the body louse (Pediculus
humanus corporis)
• Laboratory criteria
• Polymerase chain reaction assay demonstrating the presence of the
genome of Rickettsia prowazekii in the blood specimen
Urban typhus
• Description
• Urban typhus is characterized by fever, headache, myalgia, rash, vomiting and cough
• It is caused by Rickettsia typhi and transmitted by rat flea (Xenopsylla cheopis)
• Laboratory Criteria
• Any one of the following:
• Immunofluorescence test demonstrating a four-fold or greater increase in antibody titre
against typhus group
• Polymerase chain reaction assay demonstrating the presence of the genome of Rickettsia
typhi in the blood specimen
• Probable case
• A clinically compatible case with supportive laboratory findings:
• Immunofluorescence test demonstrating a single antibody titre against Typhus group ≥
512
Scrub typhus
• Description
• Scrub typhus is characterized by fever, headache, myalgia, eschar, lymphadenopathy and rash
• Though the causative agent of scrub typhus has been reclassified as a distinct genus called
Orientia, it has been conventionally grouped under Rickettsiosis
• Laboratory criteria
• Any one of the following:
• Polymerase chain reaction assay demonstrating the presence of the genome of Orientia
tsutsugamushi in the blood specimen
• Immunofluorescence test demonstrating four fold rise in antibody against Scrub Typhus group
• Probable case
• A clinically compatible case with supportive laboratory findings:
• Weil-Felix Test demonstrating a single Proteus OX-K titre ≥ 320; OR
• Immunofluorescence test demonstrating a single antibody titre against Scrub Typhus group ≥ 512
Weil-Felix Test

OX 19 OX 2 OX K
Spotted fever ++ ++ -
Epidemic typhus ++++ + -
Urban typhus ++++ + -
Scrub typhus - - +++

• Video:
• https://www.youtube.com/watch?v=ilqsQWDvggo
Brucellosis
-Communicable Diseases of Topical Public Health Concern
• Description
• An illness characterized by acute or insidious onset, with continued, intermittent or irregular fever of
variable duration, profuse sweating particularly at night, fatigue, anorexia, weight loss, headache, arthralgia
and generalized aching
• Local infection of various organs may occur
• Laboratory criteria
• Any one of the following:
• Isolation of Brucella species from a clinical specimen
• Four-fold or greater rise in Brucella agglutination titre between acute- and convalescent-phase serum
specimens
• Probable case
• A clinically compatible case with
• Epidemiological linkage to a confirmed case; OR https://rtdiagnostics.net/brucella-antibody-csf-igg

• Supportive serology (i.e., Brucella agglutination titre of ≥ 160 in one or more serum specimens obtained
after onset of symptoms)
Vibrio vulnificus infection
-Communicable Diseases of Topical Public Health Concern
• Description
• Vibrio vulnificus has drawn much concern for causing rapidly fatal necrotizing fasciitis in
some individuals who have suffered from contamination of minor skin wound with salt-
water containing the organism
• It is uncommon but severe involving the subcutaneous soft tissues, particularly the
superficial and the deep fascia
• Most patients present with signs of inflammation such as erythema, swelling, and pain at
the affected site
• Severe pain disproportionate to local findings and in association with systemic toxicity
should raise the suspicion of necrotizing fasciitis
• The organism can also cause septicaemia, cellulitis, and occasionally gastroenteritis
• Laboratory criteria
• Isolation of Vibrio vulnificus from tissue biopsy, blood culture, or the relevant clinical
specimen
Syphilis (Treponema pallidum)
–Diagnosis by RPR, VDRL, FTA-ABS & TPHA
• Tests depended upon T. pallidum demonstration by:
• Darkfield direct microscopy: The patient lesion is abraded and fluid is taken
which is seen immediately directly under the dark field microscope
• It has a sensitivity of 80 %
• Fluorescent microscopy: Where again the sample is taken directly from the
lesion and treated with fluorescently labeled antibody and seen under the
fluorescent microscope
Syphilis (Treponema pallidum)
–Diagnosis by RPR, VDRL, FTA-ABS & TPHA
• Non-treponemal tests measure antibodies against the cardiolipin (antigen)
• These serologic tests are:
• Venereal disease research laboratory (VDRL)
• https://www.youtube.com/watch?v=cFRk6CoupDs&t=184s
• Rapid plasma reagin (RPR)
• https://www.youtube.com/watch?v=RlwykBWQuDA&t=4s
• These tests become start positive after 1 to 2 weeks of infection and are
positive by 4 to 6 weeks
• Titer starts falling after the successful treatment
• These nontreponemal tests are negative in the early stage of the disease,
whereas the darkfield examination could be positive
Syphilis (Treponema Pallidum)
–Diagnosis by RPR, VDRL, FTA-ABS & TPHA
• ELIZA
• Treponemal serologic test detect antibody to the antigen:
• Fluorescent treponemal antibody absorption (FTA-ABS)
• https://youtube.com/watch?v=m8F_-acs_wk&si=EnSIkaIECMiOmarE&t=22
• TPHA (Treponema pallidum hemagglutination assay) is a treponemal antigen serologic
test for syphilis
• Tanned sheep red blood cells coated with antigen from Treponema pallidum are
treated with the serum of the patient
• Sensitivity and specificity are just like the FTA-ABS test
• These two tests are not useful for individuals who have had syphilis in the past
• PCR is recommended for neurosyphilis (CSF)
Syphilis test results interpretations

Disease Dark field RPR VDRL FTA-Abs TPHA

Primary syphilis Positive Positive Positive (False negative) (False negative)
10 to 90 days
Late primary syphilis Positive Positive Positive Positive
Latent syphilis Positive/negative Positive Positive Positive Positive
Secondary syphilis Positive Positive Positive Positive
6 weeks to 6 months
Tertiary syphilis Positive/negative Positive/negative Positive Positive
10 to 30 years
Treated syphilis Negative Positive Positive
False positive Negative Positive Positive Negative Negative