Ma. Christine P.

Atup
Marinela G. Zialcita
1. Obligate Intracellular and
Nonculturable Bacterial Agents

2. Cell Wall-Deficient Bacteria

3. The Spirochetes
Obligate intracellular and nonculturable bacterial
agents
1. Chlamydia
2. Rickettsia , Orientia , Ehrlichia
3. Coexilela

Cell wall-deficient bacteria

1. Mycoplasma pneumoniae
2. Ureaplasma urealyticum

Spirochetes
1. Treponema
2. Borrelia
3. Leptospira
 Chlamydia
 Rickettsia, Orientia, Ehrlichia
 Coexiella
 Unique developmental cycle (48-72hrs)
 Elementary Body (EB)
 Extracellular

 Can’t survive outside the host cell for a

long period metabolically inert
 Infective form

 Reticulate Body (RB)

 Intracellular

 Replicative form
 Were once regarded as viruses, just like
viruses the chlamydia require the
biochemical resources of the eukaryotic host
cell to fuel their metabolism for growth and
replication by providing high-energy
compounds such as ATP
 They resemble gram (-) bacilli in that they
contain LPS in their cell wall but compared
to gram (-) their LPS have less endotoxic
activity. Unlike other gram-negative
bacteria, it does not have a peptidoglycan
layer and has no muramic acid.
 Replication
of RB by binary fission
(phagosomes<a phagosome is a vesicle
formed around a particle absorbed by
phagocytosis> become an inclusion)
 As the numbers of RB increase, the
vacuole expands forming an
intracytoplasmic inclusion. The RB then
revert to EB, and 48 to 72 hours
postinfection, the EB are released from
the host cell.
 No exotoxin nor endotoxin
 Toxic manifestations  attachment and
phagocytosis of the EB
 Tropism for columnar epithelial cells lining
the mucous membranes
 Chlamydia is especially fond of columnar
epithelial cells that line mucous
membranes. This correlates well
 with the types of infection that Chlamydia
causes, including conjunctivitis, cervicitis,
and pneumonia.
 There are 3 species of Chlamydia. Chlamydia
trachomatis primarily infect the eyes,
genitals, and lungs; Chlamydia psittaci and
Chlamydia pneumonia only infect the lungs.
Property C. trachomatis C. psittaci C. pneumoniae
Host range Humans Birds,lower Humans
mammals,
humans (rare)
EB morphology Round Round Pear-shaped
Inclusion morph Round, Variable, dense Round, dense
Vacuolar
Glycogen YES No No
containing
inclusions
Plasmid DNA Yes Yes No
Susceptibility to Yes No No
sulfonamides
 Infertility & ectopic pregnancy
 Asymptomatic
 inadvertent transmission
 high prevalence rates
 Almost exclusively infects humans
 18 serovars  MOMP major outer membrane
protein
• Chlamydiae: major causes of PID-Pelvic
Inflammatory Disease, contributing significantly
to the rising rate of infertility and ectopic
pregnancies in young women.

• May often be asymptomatic resulting in

inadvertent transmission and high prevalence
rates.

• Almost exclusively infects humans

 TRACHOMA
 Major cause of preventable blindness

worldwide
 Endemic Areas:

 Children: main reservoir of infection

 70-90% prevalence of active dss in
preschool kids
 MOT:

 Contact with infected secretions on towels

or fingers or by flies.
 Prevention:Practice of good standard of hygiene
Trachoma is manifested by a chronic inflammation of the
conjunctiva.

Trachoma- starts with itching of eyes/eyelids, presence

of discharge from the eyes then progress to pain in the
eyes, blurred vision and photophobia.

Repeat infxn results in scarring of the inner eyelid that

may lead the eyelid in toward the eye (entropion),
followed by the eyelashes (trichiasis) resulting in
scratching the cornea. In the end, all of the trauma will
lead to loss of vision

(a) Normal conjunctiva, showing area to be examined.

(b) Follicular trachomatous inflammation (TF). (c)
Intense trachomatous inflammation (TI) (and follicular
trachomatous inflammation). (d) Conjunctival scarring
(TS). (e) Trichiasis (TT). (f) Corneal opacity (CO).
  Direct Mx Exam
 Spx: Conjunctival scrapings
 Halberstad-Prowazek bodies
 Compact, clearly defined glycogen containing
intracellular microcolonies or inclusions
 Serologic Testing – more sensitive
 ELISA
 FAT using Monoclonal Antibody
 Treatment:
 Tetracycline
 Sulfonamides
 Antibiotics may be given topically (ointments) or
through IV
 TRIC AGENT
 Trachoma and Inclusion Conjunctivitis

 Newborn

 Sticky exudates
 Premature
 Adults

 Swimming pool conjunctivitis

 The organism can also be acquired from swimming

pools, poorly chlorinated hot tubs, or by sharing eye

makeup.

 Inclusion conjunctivitis is associated with swollen eyes

and a purulent discharge.

 In contrast to trachoma, inclusion conjunctivitis does

not lead to blindness in adults (or newborns).
 CHLAMYDIAL UROGENITAL INFECTION
 20% of males with NGU (non-gonococcal

urethritis)
 Reiter’s syndrome

 Reactive arthritis in which chlamydia may be

the causative agent.
 Sx: Conjunctivitis,polyarthritis,genital inflam.
 White males (20-50 y.o): higher risk
 HLAB27 gene-80% of people w/ Reiter’s synd.
 Prevention:
 No vaccines available
 Avoid multiple sexual partners
 LYMPHOGRANULOMA VENEREUM (LGV)

 Climatic bubo, Tropical bubo, Esthiomene

 Africa, Asia and South America.

 Common among homosexual males

 Blacks>whites ; males>females

 Incubation period: 1-4 weeks

 1° lesion
 Painless, small
 Vesicular
 Males:
 Glans/prepuce

 Females:
 Vaginal wall
 Posterior part of labia
 Cervix
The disease is characterized
by a brief appearance of a
primary genital lesion at the
initial infection site. This
lesion is often small and
may be unrecognized,
especially by female
patients.
 2ND STAGE
 Enlarged matted inguinal and
femoral LN
 Painful, firm, may become
fluctuant

 3RD Stage (Variable)

 Progressive destruction of vulva
and urethra
 Lymphatic destruction
 Elephantiasis of vulva

(Esthiomene)
 Elephentiasis of penis &

scrotum
 Vulvar Cancer

 Rectal stenosis
 DIRECT MICROSCOPIC EXAMINATION
 SPECIMEN: CERVICAL/URETHRAL SCRAPINGS
 STAINING: GIEMSA (TZANCK TEST)
 INCLUSIONS stain dark blue (basophilic)
 Culture in Tissue Culture System
 MOST SENSITIVE AND SPECIFIC DIAGNOSTIC METHOD
 METHOD OF CHOICE
 Common Cell lines used: HeLa cells ; McCoy cells
 Nucleic Acid Amplification Tests (NAAT)
 PCR, SDA, TMA
 SEROLOGY
 Ag detection
 ELISA, DFAb STAINING, RNA-directed DNA probe

 Ab detection
 Complement Fixation Tests

 Microimmunofluorescence method

 Frei test
 Intradermal skin test

 Uses group specific Antigen (not specific LGV)

 May remain + for years

 Antibiotic Susceptibility Testing

 Performed only in few reference laboratories
 Erythromycin
 Tetracyclines
 Fluoroquinolones
 Mostly seen in birds (parrots/parakeets); rarely
in humans
 Parrots- major reservoir for human dse
 Psittacosis – if from psitaccine birds
 Ornithosis – if from nonpsitaccine birds

 MOT: Inhalation of aerosols

 Differs from C. trachomatis in that it is

sulfonamide resistant and morphology of EB &
inclusion bodies

 Variety of symptoms
 Cough, Headache, Encephalitis, Endocarditis,
Convulsion, Coma
 Inclusions: Levinthal-Cole-Lillie Bodies
 Giemsa + Miachiavello stain
 Serology: Dx almost always by serologic means.
 CFT-most freq. used
 Indirect immunofluorescence - more sensi but diff to

perform
 PCR

* ONLY LABORATORIES WITH BIOSAFETY LEVEL 3

BIOHAZARD CONTAINMENT FACILITIES CAN CULTURE
 TWAR
 TW-183 –from conjunctiva of a child in Taiwan
 AR-39 –isolated from college student in the US
 More homogenous
 EB is pear-shaped
 Human Pathogen
 MOT: Inhalation of aerosols
 Assoc. w/ pneumonia, bronchitis, pharyngitis,
sinusitis and flulike ilness
 Specimens:
 Throat swab, Sputum, NP & Bronchoalveolar fluids
 Place swab into chlamydial transport medium &
transport on ice; store 4°c
 Detection:
 PCR – more sensitive than cell culture
 Cell culture: HL cell line; Hep 2 cell line
 Serology:CFT(not specific),microimmunofluorescence
 General Characteristics
 Fastidious bacteria
 Obligate intracellular parasites
 Small, pleomorphic, gram (-) bacilli
 Multiply only intracellularly by binary fission in
the cytoplasm of host cells
 Lysis of host cell – result of the release of mature
of rickettsiae
 Coxiella & Bartonella- former members of the
family
 Infects wild animals
 Humans- accidental hosts
 Transmitted via insect vectors or inhalation of
aerosols
 Genus Rickettsiae/Orientia
 Do not undergo any intracellular developmental
cycle
 Genus Ehrlichia
 Undergo intracellular development following
infection of circulating leukocytes
 two families:
• Rickettsiaceae (Rickettsia and Orientia tsutsugamushi)
• Anaplasmataceae (Ehrlichia, Anaplasma, and Neorickettsia)
 Orientia tsutsugamushi (formerly called Rickettsia tsutsugamushi) was
placed into its own genus
Agent Disease Vector Distribution Dx Test
Spotted Fever Group
R. conorii Mediterranean & Ticks Southern Eu, PCR, Serology,
Israeli spotted fever; ME, Africa Immunohisto
Indian/Kenya Tick
typhus

R. rickettsii Rocky Mountain Ticks N&S PCR, Serology,

spotted fever -Dermacantor America Immunohisto
(Oklahoma)
R. akari Rickettsial pox Mites Worldwide Sero,
immunohisto
Typhus Group
R. Epidemic typhus Lice Worldwide Sero,PCR
prowazekii Brill-Zinsser None
R. typhi Endemic/Murine Fleas Worldwide Sero,PCR
Typhus
Scrub Typhus Group
O.Tsutsugam Scrub Typhus Chiggers S & SE Asia, Sero, PCR
ushi South Pacific
 Ricky the riding Rickettsia loves to travel. He rides
a tick in Rocky Mountain spotted fever, a louse in
epidemic typhus, and a flea in endemic typhus.

 An epidemic is the sudden onset and rapid spread

of an infection that affects a large proportion of a
population. Endemic refers to an infectious disease
that exists constantly throughout a population.
 Rickettsia akari causes rickettsial pox and is transmitted
to humans via mites that live on house mice. Imagine
Ricky, with pox marks, playing Atari (old type of
Nintendo) with his rodent friend matey mouse.
 Ricky is now a South Pacific sumo
wrestler named Ricky Tsutsugamushi. He
is walking in
 the scrub (scrub typhus) being bitten by
chiggers that are on his feet and legs.

Ricky is now a South Pacific sumo wrestler named

Ricky Tsutsugamushi. He is walking in
the scrub (scrub typhus) being bitten by chiggers
that are on his feet and legs.
Agent Disease Vector Distribution Dx Test
Ehrlichia
E. chaffeensis Human Ticks SE, S, Sero, PCR,
monocytic (Ambylomma Central and Immunohisto
ehrlichiosis americanum- mid-Atlantic immunocyto
Lone Star Tick) US

E. ewingii Ticks US PCR

(Ambylomma
americanum-
Lone Star Tick)

Anaplasma Human Ticks US, Eu Sero,PCR

phagocytophilu granulocytic (Ixodes spp)
m anaplasmosis

Neorickettsia Sennetsu fever Ticks SEA (Japan) Serology

sennetsu
 TSUTSUGAMUSHI DSS
 CHIGGER-BORNE TYPHUS
 O. tsutsugamushi- causative agent
 Transmitted by trombiculid mites- larva (chigger)
 1-3 weeks of incubation
 Symptoms: cough, nausea, vomiting, sore throat,
skin rash w/ black scab (eschar),
lymphadenopathy- proximal to the eschar
 Immunity: strain specific
 Direct Examination
 Dye staining: Gimenez stain, Machiavello stain,
Giemsa stain: Use buffy coat of the blood to
test!

 Detect presence of MUROLAE (cytoplasmic containing

enriched organisms)
 Direct immunofluorescent technique
 Isolation:
 Animal inoculation
 Tissue culture
 Embryonated eggs
 Increase risk of laboratory acquired infection
 Serology
 Weil-Felix Reaction
 Ag: Proteus vulgaris 0X-2, OX-19

Proteus mirabilis OX-K

 Significant titer: > 1:160
 Widely available, inexpensive test  provide earlier

clue to certain rickettsial infxn but not specific

 False (+) : Proteus/Borrelia infxn, Liver dss,

Leptospirosis, Sodoku
 False (-) : Rickettsial pox, Trench & Q fever, Brill

Zinsser dss
 DA , CFT, Indirect immunofluorescent test, ELISA
 Although it is not fast, the diagnosis of
rickettsial disease and ehrlichiosis is primarily
accomplished serologically.
 because false-positive and false-negative tests
are a continuing problem, these tests have been
replaced by more accurate serologic methods
such as IFA.
 Some Rickettsia share antigenic characteristics
with certain strains of Proteus vulgaris bacteria
such as OX-2…
 Immunity: Long lasting after recovery from dss
 Treatment: Tetracycline and Chloramphenicol
 Prevention: Control of Arthropod vectors
Disease 0X-19 0X-2 0X-K

Rickettsial pox - - -

RMSF + + -

Epidemic typhus + v -

Brill Zinsser v v -

Murine typhus + v -

Scrub typhus - - v

Q fever - - -

Ehrlichiosis - - -
 Coxiella burnetti
 Causative agent of Q fever
 Gram (-) ccb
 Smaller than Rickettsia spp.
 Can survive extracellularly but only grown in lung cells
 Has sporelike life cycle
 Large Cell Variant Form (Phase I) – isolated from
animals ; infectious
 Small Cell Variant Form (Phase II)- grown from
cultured cell lines; non-infectious
- Acts like a spore which aids in the extracellular
survival of the organism.
 MOT:
 Animals: Urine, Milk, Feces of Cattle, sheep,
goats, etc.
 Humans: Inhalation of of contaminated aerosols
 Incubation Period: 2 weeks- 1 month
 Diagnosis:
 IFA- method of choice
 CFT
 EIA
 Mycoplasma
 Ureaplasma
 Smallest and simplest prokaryotes capable of self-
replication (200-300nm)
 PPLO ; Eaton Agent
 Eaton Monroe Davis –worked on isolation of causative agent of PAP
 Lack true cell wall
 Highly pleomorphic organism
 Coccoid, filamentous, tear drop shaped cells w/ terminal structures
 Facultative anaerobes
 Colonies: “Fried-egg” appearance
 Best studies using the Dienes method
 Mycoplasma colonies retain the methylene blue stain after 15 min
while other colonies are colorless
 Mycoplasma
 Do not hydrolyze urea

 Ureaplasma
 Hydrolyze urea
 Major human pathogen
 Cause Primary Atypical Pneumoniae (PAP)/
Walking pneumonia
 Culture Medium: Modified NYC medium
(mulberry colony)
 M. fermentans: Genital Tract
 M. orale : Oropharynx
 M. hominis: Genital Tratc & Oropharynx
 Associated with postpartum fever & postabortal
fever
 M.genitalium : cause Non-gonococcal
urethritis
 Normal inhabitant of oropharynx and genital
tract
 Associated with venereally transmitted
urethritis (NGU)
 Culture: Edward Hayflick agar
 SP-4 medium (biphasic medium)
 Serology:
 CFT, ELISA – detect antibodies

 Cold hemagglutination test

 Cold agglutinin: nonspecific antibody which

agglutinate human RBC at 4°c
 Patient’s Serum + washed type O cells
(incubate 4°c)
 Result: hemagglutination
 Treponema
 Borrelia
 Leptospira
 Slender, motile, spiral, filamentous org.
 Body consists of
 Outer cell envelope (periplast)
 Axial fibrils
 Inner protoplasmis cylinder
 Helically coiled org: >1 complete turns in the helix
 Axial filaments/Fibrilsendoflagella enclosed w/in outer
sheath + facilitate motility
 The fibrils are attached within the cell wall by platelike
structures(insertion disks) lear the ends of the cells.

 Aka: axial filaments, periplasmic flagella, endoflagella

 Fx: locomotion Makes cell locomotory even in high
viscosity
 Cellular motility includes: rapid rotation around the long
axis; flexation of cells; locomotion along a helical path
 Components: shaft & it’s covering ; insertion apparatus
(proximal hook; insertion discs)

 Inner protoplasmis cylinder: Cell wall, Cell membrane,

Cytoplasmic contents
Pathogenic Genera
GENUS MOVEMENT AXIAL INSERTION
FIBRILS DISCS

Treponema Sluggish w/ a drifting motion 6-10 * 1

and flexous movements
Borrelia Move in forward and 30-40* 2
backward waves w/ a
corkscrew-like motion
Leptospira Undulatory movement and by 2** 3-5
rapid spinning on the long
axis

Multiplication: transverse fission

Susceptible: chemotherapeutic agents & heavy metals
2 groups:
 Pathogenic
 Nonpathogenic

BASES PATHOGENIC NONPATHOGENIC

Capsule-like Present None
Ends of organism Tapered Blunt
Hyaluronidase Yes No
In vitro culture No Yes
 Treponemal species pathogenic for humans

SPECIES HUMAN DISEASE

T. pallidum ssp. pallidum Syphilis

T. pallidum ssp. endemicum Endemic Syphilis

T. pallidum ssp. pertenue Yaws

T. carateum Pinta
 MOT: Sexual contact or Vertical transmission
 8-14 tightly coiled, even spirals
 Microaerophilic
 No exotoxins nor endotoxins produced
 Syphilis: not highly infx
 Acquired
 Venereal syphilis
 Nonveneral syphilis
 Congenital
 Vertically from mother to the unborn fetus
 Venerealsexually transmitted
 Antigenic structure
 Several cell surface CHON Ags &
 Unique class of extracellular CHON Ags
 PRIMARY SYPHILIS
 Inc. period: ave. 3 weeks
 1O lesion : hard chancre; Hunterian chancre
 Single lesion, painless
 Site:
 95%  genitalia
 Male: coronal sulcus
 Females: cervix or vaginal wall
 5%  nipple, mucous membranes of mouth
 Buboes:
 Hard, rubbery, nontender
 Systemic SSx:
 absent
 SECONDARY SYPHILIS
 2-24 weeks after 1O lesion
 SKIN most commonly affected
 Fever, sore throat, headache,Generalized
lymphadenopathy, Rash, Condylomata lata,
Alopecia
 2O lesions of skin & mucous mem.  highly infx
 Other SSx: 2O to generalized immunologic
response
 Nephritic syndrome

 Arthritis

 Arthralgia
 LATENT SYPHILIS
 Subclinical

 Dxserology

 Early latent

 w/in 4 yrs after 1O syphilis

 Relapsescommon ; < 1 year
 Late latent

 Asymptomatic
 Noninfx
 TERTIARY SYPHILIS
 Tissue-destructive phase
 May involve any organ system
 Often asymmetric
 Appears 10-25 years after initial infxn in
 GUMMA:
 Benign 3O syphilis

 Dev. 3-10 yrs following the last evidence of 2O

syphilis
 Nonprogressive, localized lesion of the dermal
elements or supporting structures of the body
 Gummas (Gummy bears) are localized
granulomatous lesions which eventually necrose and
become fibrotic. These noninfectious lesions are
found mainly in the skin and bones. Skin gummas are
painless solitary lesions with sharp borders, while
bone lesions are associated with a deep gnawing
pain. These will resolve with antimicrobial therapy.
 Neurosyphilis
 Cardiovascular syphilis
 Dev. 10-40 years after 1O syphilis
 T. pallidum has a remarkable tropism
(attraction) to arterioles; infection ultimately
leads to endarteritis (inflammation of the lining
of arteries) and subsequent progressive tissue
destruction.
 Great vessels of the heart
 Inflammation of aorta (syphilitic aortits)  aneurysmal
dilatation
 Distortion of aortic valve  aortic insufficiency
 Arteriosclerosis of aorta
 2NDtrimester of pregnancy
 Untreated syphilitic pregnant mother
 1O syphilis: almost all
 2O syphilis: 90%
 3O syphilis: 30%
 Fate of fetus
 Abortion/stillbirth
 Congenital syphilis
 w/o syphilis
2 TYPES OF Ab:
 Treponemal
 Produced against Ag of the organisms
themselves
 Nontreponemal
 aka: Reagin Ab

 Produced in infected px against components

of mammalian cells
 VDRL and RPR
 Reaginic antibodies, although almost always produced
in patients with syphilis, are also produced
 in patients with other infectious diseases such as
leprosy, tuberculosis, chancroid, leptospirosis,
malaria,
 rickettsial disease, trypanosomiasis,
lymphogranuloma venereum (LGV), measles,
chickenpox, hepatitis, and
 infectious mononucleosis; noninfectious conditions
such as drug addiction; autoimmune disorders,
including
 rheumatoid disease and systemic lupus
erythematosus; and in conjunction with increasing
age, pregnancy, and
 recent immunization.
 NONTREPONEMAL Ab TESTS
 Ag: cardiolipin & lecithin in cholesterol
 CFT:
 Ex.: Wasserman test, Kolmer test
 Principle: reagin containing sera fix C’ in the
presence of cardiolipin Ag
 FLOCCULATION TESTS
 Ex.: VDRL, RPR test, Hinton test
 Principle: particles of lipid Ag remain dispersed in N
serum but combined w/ the reagin to form visible
aggregates
 VDRL
 Reactive + clinical Sx  confirmatory for syphilis
 Reactive + no Sx  Latent syphilis, BFP, Tech./clin. error
 Advantages:
 Inexpensive

 Demonstrate rising and falling Ab titer

 Correlate w/ the clinical status of the patient

 Disadvantages:
 High proportion of biologic acute and chronic false (+) rxn

 Increasing proportion of false (-) reaction in the later stages of

untreated syphilis
 Biologic False Positive (BFP)
 Acute BFP: pneumonia, hepatitis, vaccinations

 Chronic BFP: drug addiction, chronic hepapitis, old age,

leprosy, collagen vascular dse

 The VDRL is used as a quantitative test which are useful to follow the patient’s
response to therapy
 and may be performed on serum or CSF in suspected cases of neurosyphilis.
 TREPONEMAL Ab TEST
 EIA
 AGGLUTINATION TESTS:
 TP-PA
 Use gelatin particles sensitized w/ T. pallidum Ag
 MHA-TP
 Passive hemmaglutination assay of sensitized RBC
against px serum
 Technically easy to perform, inexpensive
 TPHA
 Indirect hemmaglutination assay of sensitized RBC
 PaGIA
 FTA-ABS
 Performed after + VDRL and RPR screen
 Expensive and time consuming
 Sensitive and reliable
 Usefulness limited once positive tests tend to
yield positive results througout px life
 T. pallidum IMMOBILIZATION (TPI) TEST
 Principle: in the presence of complement, Abs in the
serum can immobilize living Treponema
 Disadvantages
 Difficult and expensive
 Requires living organisms
 (+) in nonvenereal treponematoses
 Uses:
 Primarily for comparison and evaluation w/ other tests
 Distinguishes bet. Syphilis and BFP reaction in patients
w/ collagen vascular dse w/ abnormal serum globulins
 REITER CHON COMPLEMENT FIXATION
 Detects group Abs
 Frequently nonreactive in late syph
 LABORATORY DX
 DIRECT VISUALIZATION OF THE ORGANISM
 Darkfield microscopy
 Not done on material from lesions w/in the oral cavity
 (+) lesions: highly infectioushandle w/ extreme care
 Fluorescent Ab technique
 Special stains
 Levaditi method: infected tissues
 Fontana-Tribondeau: smears

 DEMONSTRATION OF SEROLOGIC REACTIONS TYPICAL

OF SYPHILIS
 ANIMAL INOCULATION
Most species stain poorly with Gram staining or Giemsa’s
methods(too small to be seen using the light microscope) and are
best observed with the use of dark-field or phase-contrast
microscopy.

 The area around the lesion must first be cleansed with a sterile
gauze pad moistened in saline. The surface
 of the ulcer is then abraded until some blood is expressed. After
blotting the lesion until there is no further bleeding, the area is
squeezed until serous fluid is expressed. The surface of a clean
glass slide is touched to the
 exudate, allowed to air dry, and transported in a dust-free
container for fluorescent antibody staining.

 For dark-field examination, the expressed fluid is aspirated using

a sterile pipette, dropped onto a clean glass slide, and cover
slipped. The slide containing material for darkfield examination
must be transported to the laboratory
 immediately. Because positive lesions may be teeming with
viable spirochetes that are highly infectious, all supplies
 and patient specimens must be handled extreme caution and
carefully discarded as required for
 contaminated materials. Gloves should always be worn.
ROUTINE SCREENING SCHEME FOR EARLY SYPHILIS IN THE US

LESION

YES NO

DARKFIELD OR NONTREPONEMAL
DFA TEST

+ - R NR

TITER REPEA
TREAT T
TREPONEMAL TEST

+ -

TREAT FALSE
(+)
 ORGANISM DISEASE DISTRIBUTION AGE GROUP TRANS
MISSION

T. pallidum ssp. Venereal Worldwide All ages Sexual

pallidum syphilis contact;
Congenital

T. pallidum ssp. Yaws Tropical areas, Children Skin

pertenue (fambresia, Africa, S. contact
pain) America,
Pacific Islands

T. pallidum ssp. Endemic Arid areas, Children to Mucous

endemicum syphilis Africa, MEA adults membrane
(bejel, SEA
dichuchwa)

T. carateum Pinta Semiarid, Children, S kin

(carate, warm areas, adolescents contact
cute) Central and S.
America
 MORPHOLOGY AND PHYSIOLOGY
 Axial fibrils
 Unsheathed
 3-10 loose coils
 Actively motile
 Can be seen w/ light mx
 Stain well with Giemsa (unlike Treponemes)
 Culture
 Microaerophilic; 30-34 C for up to 12 weeks

 All have arthropod vectors

 Louseborne: B. recurrentis

 louse : Pediculus humanus subsp. Humanus

 Reservoir: Human
 Tickborne: B. hermsii. B. burgdorferi

 tick: Ornithodoros/Ixodes (soft tick)

 Reservoir: Human and rodents
 Epidemics: cold season, crowded and poor hygiene
 Transplacental, infected blood (cause of lab
accidents leading to infxn): occasional
 BORRELIOSIS
 aka: RELAPSING FEVER, FAMINE FEVER, TICK
FEVER
 MOT: bite from human-specific body louse or tick
 >15 spp of Borrelia
 Incubation period: 2-15 days
 Sudden onset of: fever w/ chills, Headache,
tachycardia and muscle pain
 Macular rashes: appear near the end of the 1st
paroxysm hepatomegaly, jaundice, nausea and
vomitting: common
As the host produces specific antibody in response
to the agent, organisms disappear from the
bloodstream, becoming sequestered (hidden) in
different organs during the afebrile period.
Subsequently, organisms reemerge with newly
modified antigens and multiply, resulting in
another febrile period. Subsequent relapses are
usually milder and of shorter duration.

Generally, more relapses are associated with cases

of untreated tickborne relapsing fever, but
louseborne relapsing fevers tend to be more
severe.
LABORATORY DX
 DIRECT MX EXAM OF BLOOD
 Stained smear: Giemsa, Wright’s , Gentian Violet
 Darkfield exam
 Best demo in the blood :early febrile period
 SEROLOGIC TECHNIQUES
 Borrelialysin test
 Borrelia immobilzation test
 CULTURE (tickborne)
 Kelly’s medium
 Smibert’s medium
 Although the organisms that cause relapsing fever can be cultured in nutritionally rich
media under microaerobic
 conditions, the procedures are cumbersome and unreliable and are used primarily as
research tools.
 ANIMAL INOCULATION
 Young mice
 LYME DISEASE
 B. burgdorferi
 Tickborne: ixodes, ambylomma
 Natural host: deer and rodents
 3 stages of illness
 ERYTHEMA MIGRANS

 Neurologic or cardiac involvement

 Migrating episodes of arthritis

 Acrodermatitis chronica atrophicans (ACA)

 LABORATORY DX
 SOC: Peripheral blood
 Serologybest means of dx
 Demo of Spirochetes from lesions
 Warthin-Starry silver stain
 Dieterle silver stain
 Indirect immunofluoresece assay
 ELISA
 PCR
 CULTURE
 Barbour-Stoenner-Kelly medium
 Mod. Kelly’s medium
 Numerous fine spirals closely set together
 One or both ends are hooked
 Obligate aerobe
 Optimum temp: 25-30C
 pH:7.2-7.4
 Generation time
 12-16 hr in culture media
 4-8 hr in inoculated animals
 Oxidase (+), catalase (+)
 Resistance
 Susceptible to acid: die w/in few hrs in acid urine
 Survive for > months in neutral/slightly alk. Water
 Susceptible to heat, detergent and dessication
 L. interrogans: pathogenic organism
 L. biflexa: saprophytes/water leptospira
 DETERMINANTS OF PATHOGENICITY
 Mechanism of virulence: uncertain
 Biologic properties of pathogenic strains
 Less sensitive to the leptospiricidal effect of immune
serum
 Some produce a soluble hemolysin
 Thermolabile
 CHON in nature
 Impt. In the manifestations of leptospirosis
 Some clin. Manifestation (conjunctival irritation and
iritis) probably are caused by cell mediated sensitivity to
leptospiral Ag
 Some may contain small amounts of endotoxin
 LEPTOSPIROSIS
 WEIL’S DISEASE
 Zoonotic dse infecting wild & domestic animals
 MOT: ingestion of food & water contaminated w/
leptospirae
 Incidence is more in males than in females
 Inc. period: 2-20 d
 Clinical syndrome:
 Anicteric leptospirosis
 Most common, self-limiting
 Icteric leptospirosis or WEIL’S DSE
 Most severe
 LABORATORY Dx
 Spx:
 Blood, CSF  1st week of illness
 Urine  2nd week of illness and onwards
 Should be incubated soon after inoculation
 Tissues smear or section: Ag impregnation
 Procedure
 DIRECT EXAM
 Darkfield mx, fluorescent Ab tech., Special staining
methods
 CULTURE
 Media: Fletcher’s med., Stewart’s med., EMJH med
 Inc.: in the dark, RT or 30C, 6-8 weeks
 SEROLOGIC TESTS
 MACROSCOPIC SLIDE AGGLUTINATION
 Safe and rapid screening test for the detection of
leptospiral Ab
 MICROSCOPIC SLIDE AGGLUTINATION
 Detect serovar-specific Ab
 OTHER TESTS
 CFT
 FAT
 Hemagglutination
 ANIMAL INOCULATION