Central Philippine University

Department of Medical Laboratory Science

 Spirochetes
General characteristics
• “Spiro” means “coiled,” and “chaete” means “hair”

• Gram negative unicellular organisms

– can be visualized only by darkfield or phase microscopy, silver
impregnation, and immunohistochemical stains in tissue sections

1. Facultatively anaerobic o aerobic

2. Multiply by transverse fission
3. Motile: axial fibrils
– flagella-like organelles
Spirochete
bacteria, coloured
transmission
electron
micrograph
Spirochetes are bacteria
with helically coiled cells.
They contain filamentous
flagella (axial filaments)
that run lengthwise
between the cell
membrane and outer
membrane. The twisting
motion of the flagella
allows the bacterium to
move about. Here
however, the bacteria
have been damaged,
causing the flagella to
break away from the
sheaths. fineartamerica.com
 Spirochetes
• Many commensal and nonpathogenic species of
spirochetes exist, and human disease is limited primarily
to infection by members of three genera:
A. Treponema
B. Borrelia
C. Leptospira

• Diagnosis often relies upon the demonstration of a

patient’s serologic response to the offending agent.
 Spirochetes
• With few exceptions, human disease caused by
spirochetes typically follows a clinical course that reflects
three sequential phenomena:
1. Early, local proliferation of the organisms at the site of
inoculation.
2. Spirochetemia with systemic dissemination.
3. Persistence of small numbers of microbes at various, often
immune, “privileged” sites.
 Varying Characteristics of the Genera
Within the Order Spirochaetales
1. Number of axial fibrils or periplasmic flagella
2. Number of insertion disks
– An insertion disk is a plate-like structure where fibrils are
attached. It is found near the terminal end of the cell.
3. Biochemical and Metabolic features
Scanning Electron Micrograph of Treponema pallidum on cultures of cotton-tail
rabbit epithelium cells.
 Treponema
General Characteristics
• Genus name came from the Greek word: “turning thread”
– “trepein” means to turn
– “nēma” means thread
• Best observed with dark-field microscopy or phase-
contrast microscopy
• Microscopy:
a. Thin, spiral organisms (4 to 14 spirals/organism) with three axial
filaments and one insertion disk)
b. Cells are pointed and covered with a sheath
c. (+) Corkscrew motility
 Treponema
• Genus Treponema contains two species
responsible for disease in humans:
A. T. pallidum
B. T. carateum

• Treponema pallidum is divided into

three subspecies, each of which is the
etiologic agent of a distinct clinical
entity:
1. T. pallidum subspecie pallidum
2. T. pallidum subspecie pertenue
CDC image 2333,
3. T. pallidum subspecie endemicum Treponema pallidum
bacterium
 Treponema species
• Species are serologically & morphologically indistinguishable
• Species are differentiated by type of lesions produced

Causative agent Disease Lesions

T. pallidum subspecie pallidum Venereal syphilis Chancre, etc...
T. pallidum subspecie pertenue Yaws Fambresia
T. pallidum subspecie endemicum Endemic syphilis (Bejel) Scars, papules
Minor lesions @
T. cuniculi Rabbit syphilis
genitalia
T. carateum Pinta Pintid
 Dark Field Microscopy
• Most definite & earliest means
of diagnosis
– Treponema very hard to stain,
best observed by darkfield
microscopy

• DFM can be positive several

weeks before a positive
serologic test
 Dark Field Microscopy
• Procedure:
1. Lesion cleansed with sterile H2O then
gently abraded.
2. Apply pressure & collect exudates

• DFM not recommended for oral

lesions
– Confusion with commensal treponemes
in the mouth
DFM technique reveals the
• DFM should be done 10 minutes after presence of motile
acquiring the sample. spirochetes.
Henry’s 22nd Ed.
In vitro diagnostic modalities for
venereal syphilis detection
Positive FTA-ABS test for antibody
response in patient with syphilis.
CDC/PHIL file photo.
 T. pallidum subspecie pallidum
• Non culturable in artificial medium
– Maintained in testicular chancres of
rabbit

• Causes SYPHILIS
– aka
a. Lues venera
b. Italian disease or French disease
c. Great pox or evil pox
– Disease of blood vessels
– MOT: Sexual, Parenteral, Mother to The earliest known portrayal of patients
fetus suffering from syphilis, from Vienna in 1498.
Photo: Bartholomäus Steber
 T. pallidum subspecie pallidum
• Close-coiled, thin, regular spiral
organism, 6-15μm in length, consisting
of 10 – 13 coils and three
fibrils/periplasmic flagella

• Thin, tightly wound, rigid, spiral

organism

• Sluggish motility
– Exhibits little flexibility and does not move An electron photomicrograph of two
from place to place. spiral-shaped T. Pallidum.
(CDC/PHIL file photo.)
Stages of Syphilis

Tertiary
Latent

Secondary

Primary
 Primary Stage of Syphilis
• Develops after 10 – 90 days after
infection a hard chancre appear
@ site of entrance, which persists
for1-5 weeks

• Lesions: hard chancre or

Hunterian chancre

• Labs: DFM CHANCRE

single, firm, painless, non-itchy skin ulceration
• No systemic signs or symptoms with a clean base and sharp borders between
evident! 0.3 & 3.0 cm in size; evolves from a macule to
a papule, and finally to an erosion or ulcer
 Secondary Stage of Syphilis
• Develops 2 to 12 weeks after the
appearance of the 1° chancre
– Lesions (Condylomata lata)
particularly in the mucous
membranes

• Bloodstream dissemination
– Generalized rash on trunk and
extremities CONDYLOMATA LATA
Rashes that become
maculopapular or postural and
• Labs: DFM & Sero test form flat, whitish, wart-like
lesions
 Latent Stage of Syphilis
No lesions absence of signs &
• It is the period in which the symptoms
disease becomes subclinical
but not dormant

• It occurs within more than a

year of infection

• May last for years or for the

rest of the patient’s life

• Lab tests: Sero tests only!

 Tertiary Stage of Syphilis
• Tissue-destructive phase
• Lesions (Gummatas) seen 10-25 years after
1° stage
✓ Gummas can be found in the
a. CNS (Neurosyphillis)
b. Cardiovascular system (aortic aneurysm)
c. Eyes (blindness)

• Neurosyphilis
1. Incomplete paralysis (paresis) GUMMATA
2. General paralysis (tabes dorsalis) soft, tumor-like balls of
inflammation which may
• Labs: Sero tests only! vary considerably in size.
 Laboratory Diagnosis
• Specimen:
– Skin lesions (cleaned with saline)
• Oral lesions should not be examined
– Non-pathogenic spirochetes will lead to false-positive result

Can be done through the following:

1. Microscopic Examination
2. Serodiagnosis
3. Molecular Tests
 Microscopic Examination
• Direct microscopic examination of exudates
– Recommended
• Diagnostic for Primary Syphilis
– Demonstration of motile treponemes from the chancre
specimen
– Fluorescent antibody staining can be performed
• Definitive test: DFM for motility
• Stains: Levaditi’s Stain and Fontana-Tribondeau
stains
 Serodiagnosis
NON-TREPONEMAL TESTS
• Detects non-treponemal
antibodies or Reagin
• Reacts with lipid Ags (cardiolipin)
• Used to screen for disease, and to
monitor the course of disease after
treatment
• Ex: VDRL, RPR, USR, etc
TREPONEMAL TESTS
• Detects treponemal antibodies
(Abs to treponemal Ags)
• Reacts with T. pallidum and closely
related strains
• Used to confirm a positive
nontreponemal screening test or to
confirm infection in the late latent
which has a negative
nontreponemal test
• Ex: TPI, TPA, FTA-ABS, etc
 Molecular Tests

• Polymerase Chain Reaction

– Used for neurosyphilis detection (AIDS patients)
• Western blot
– Used for the detection of congenital syphilis
 Notes to Remember:

• RPR does not require the heating of the serum and

is not recommended for CSF
• VDRL is recommended for the diagnosis of
neurosyphilis using CSF specimens
• Reagents for the VDRL must be freshly prepared
and the patient’s serum must be heated at 56˚C
for 30 minutes (complement inactivation)
 T. pallidum subspecie pertenue
• Causes YAWS
– Chronic skin & bone disease of the tropics
– Also called frambesia tropica, pian,
parangi, paru & buba
• Most prevalent of the nonvenereal
treponematoses
– MOT: direct contact with skin lesions
(fambresia)
• Approximately 10% develop late yaws, which
shows irreversible, destructive lesions of bone,
cartilage, soft tissue, and the skin
 T. pallidum subspecie endemicum
• aka T. pallidum II
• Causes BEJEL: non venereal endemic syphilis
– MOT: direct contact, sharing of eating or drinking utensils

• Lesions:
a. Primary – oral cavity
b. Secondary – oral mucosa
c. Tertiary – skin/bones/nasopharynx

• Late stage is characterized by tissue destruction of the skin,

bones, and cartilage
Bejel usually begins in childhood as a small mucous patch, often on the interior of the
mouth. immiunology.persianblog.ir
 Treponema carateum
• Causes PINTA
– an ulcerative skin disease of
Central & South America
– also carate, mal de pinto, azul

• Lesions: pintid
– scaly psoriasiform plaques
– commonly occur in hands, feet
and scalp
– skin appears to be the only organ emedicine.medscape.com
affected in this disease
 Treponema denticola and Treponema socranski

• Cause ulcerative
gingivitis and chronic
periodontitis

• Related to T. pallidum

Ulcerative gingivitis
emdocs.net
Epidemiology and Spectrum of Disease of
the Treponemes Pathogenic for Humans
Coloured SEM of Borrelia burgdorferi bacteria, the cause of
Lyme disease.
 Borrelia: Blood Spirochetes
• Flexible twisted organisms
resembling a stretched spiral

• Vectors: lice or ticks

• Slow-growing spirochetes
• Multiply by binary fission

• Actively motile
– Contain 15 – 20 axial filaments
and two insertion disks
– 3 to 10 loose coils
Scanning electron micrograph (SEM) of
Borrelia burgdorferi
Centers for Disease Control and Prevention
 Borrelia: 2 Clades
A. Lyme borreliosis group
1. B. burgdorferi sensu stricto (s.s.)
2. Borrelia garinii
3. Borrelia afzelii

B. Relapsing fever group

1. Argasid (soft) ticks (e.g., Borrelia hermsii,
Borrelia turicatae)
2. Ixodid (hard) ticks (e.g., Borrelia
lonestari, Borrelia miyamotoi)
3. Human body louse (Borrelia recurrentis)
Adult female, black-legged tick
(or “deer tick”), Ixodes
scapularis.
Courtesy James Gathany, CDC/PHIL file photos.)
 Lyme Disease
• It is an acute, recurring inflammatory infection involving the large
joints (e.g. Knees)
• Hallmark of infection: Erythema migrans (bull’s eye lesion on the skin) and
swelling

• Was so named during the 1970s following an epidemic of arthritis in

eastern Connecticut, chief among them the town of Old Lyme

• Caused chiefly by
1. Borrelia burgdorferi sensu stricto
• microaerophilic w/ 7-11 periplasmic flagella
2. Borrelia garinii
3. Borrelia afzelii
 Stages of Lyme Disease
I. Stage I – characteristic skin
rash: erythema chronicum
migrans (ECM)
II. Stage II – neurologic &
cardiac involvement
III. Stage III – chronic stage;
arthritis!

Characteristic “bull’s-eye” lesion, erythema migrans, observed at

the site of spirochete inoculation in ≈80% of patients with Lyme
disease.
Courtesy James Gathany, CDC/PHIL file photo
 Lyme Disease: DIAGNOSIS
• A diagnosis of Lyme disease must be based on a
combination of:
1. Clinicoepidemiologic characteristics
2. Host serologic response (Standard Method)
– ELISA and IFE (First-line tests for ab)
– Western blot
3. Molecular evidence
4. Culture results
– “Gold standard” Borrelia spirochetes
identified on silver stain
– Barber-Stoenner-Kelly medium photo, CDC

– Dieterle’s silver stain

 Relapsing Fever
• An acute infection characterized by febrile episodes that subsides but tend to recur over a
period of weeks (2 to 10 relapses).

A. Epidemic louse-borne relapsing fever (LBRF)

– B. recurrentis
– Occurs worldwide
– Epidemic or European relapsing fever
– Vector: human lice (Pediculus humanus)

B. Endemic tick-borne relapsing fever (TBRF)

– B. parkeri, B. hermsii, B. duttoni, B. turicata, B. anserina
– Narrower geographic distribution
– Endemic or American relapsing fever
– Vector: ticks (Ornithodoros)
 Relapsing Fever: DIAGNOSIS
1. Microscopic examination of peripheral blood
– Obtained during febrile episode
a. Thick blood film
• Traditional laboratory approach to diagnosis of relapsing fever
• only spirochete demonstrated by wright-giemsa (blue-colored) on blood smears
b. Darkfield technique (corkscrew like motion) A spirochete (arrow) in a thin smear of
c. Acridine orange PBS with Giemsa stain.
aafp.org
d. Specific fluorescent antibodies
2. Serologic tests
3. Culture
– Cultivation of the spirochetes is possible,
difficult, and is rarely performed
– Barbour-Stoenner-Kelly medium
and chick embryo
False color transmission electron micrograph of the bacterium Leptospira
sp., which occurs as a long thin spiral
 Leptospira: Infectious Jaundice
• Tightly coiled organisms with one or
both ends bent into a hook
– ”Shepherd’s crook”

• Active rotational (spinning) motion

– Corkscrew like

• Contains diaminopimelic acid

– Differentiate it from Treponema
SEM of Leptospira interrogans
& Borrelia which contain ornithine strain
aapredbook.aappublications.org
 Mode of Acquisition:

A. Entry through breaks in the skin, mucous

membranes, or conjunctiva
B. Direct contact with urine of carriers like rats
C. Contact with bodies of water that are
contaminated with the urine of the carriers
D. Upon the entry, leptospires rapidly invade the
bloodstream and spread throughout the CNS and
kidneys
 Leptospira biflexa
• Includes all the saprophytic strains or water
leptospira

• Nonpathogenic

Saprophytic organism - an organism

that feeds on dead organic matter
especially a fungus or bacterium
 Leptospira interrogans
• Includes all pathogenic strains
• A parasite:
– Rodents, cattle, dogs, etc.

• PCT of vertebrates harbour the

organisms w/c are then passed in
the urine

• Disease: Leptospirosis
– Target organs
1. Kidney
2. Liver
3. CNS
 Leptospira interrogans Serogroups
• most common cause of disease
a. Ictohemorrhagiae • Weil’s disease (severe form)

b. Canicola • Infectious jaundice

c. Automnalis • Pretibial fever, Fort Bragg fever

d. Grippothphosa • Marsch fever

e. Hebdomadis • 7 day fever

f. Mitis & Pomona • Swine herd disease

 Laboratory Diagnosis for Leptospirosis
A. DFM
• Spinning leptospiras with hooked ends

B. Serologic test
• Majority of cases diagnosed serologically
• ELISA, RIA – commonly used
• Microscopic Agglutination (MA) using living cells
– Reference method

C. Culture
1. Fletcher’s
2. Noguchi
3. Stewart medium
4. EMJH (Ellinghausen-McCullough-Johnson-
Harris) medium
 Leptospirosis
• Perhaps the most widespread
global zoonosis
– The organism is carried by many
rodents and feral and domestic
animals

• Contact with urine-

contaminated water along with
entry of the bacteria through
cuts or mucosal surfaces is a
frequent mode of infection
 Leptospirosis
A. Anicteric form
– Mild, influenza-like illness

B. Icteric form or Weil’s disease

– In minority of patients, a more
severe form
– Gastrointestinal/hepatic disease,
meningitis, renal failure, and/or
myocarditis
– 5%–10% fatality rate
 Brachyspira
• Comma-shaped or helical tapered ends with four flagella at each end
• Cultivation
– brain heart infusion (BHI) or tryptic soy agar
• 10% F bovine blood,
• 400 μg/mL of spectinomycin,
• 5 μg/mL polymyxin in anaerobic conditions at 37° C
• Specimen Collection and Direct Detection
– DFM: Fresh stool or rectal swabs
– Tissue biopsy – PAS and H&E stain

• 2 Species of Brachyspira:
1. Brachyspira aalborgi - strong hippurate hydrolysis rxn, weak indole rxn
2. Brachyspira pilosicoli - weak hippurate hydrolysis reaction, indole
negative rxn
 Brachyspira aalborgi

• Requires anaerobic
incubation
• Has not been isolated
from animals

• Transmitted via fecal-oral

contamination
 Brachyspira pilosicoli
• Colonizes the intestine of a variety
of animal species.
• Reside in the brush border within
the intestine
– “false brush border” with H & E
• Infection results from ingesting
water contaminated with feces
from infected animals.
• No pathogenic mechanisms have Intestinal spirochetosis
been identified
 References
▪ Tille, Patricia M., author. (2014). Bailey & Scott's diagnostic
microbiology. St. Louis, Missouri :Elsevier,
▪ Training course on the Laboratory Diagnosis of Medically
Important Bacterial Pathogens, Department of Health, Research
Institute for Tropical Medicine
▪ Mahon, C, Lehman, D. Manuselis, G. (2015), Textbook of
Diagnostic Microbiology, 5th ed. Maryland Heights, Mo.:
Saunders/Elsevier
▪ Rodriguez, M.TT.,(2016) Review Handbook in Diagnostic
Bacteriology. C & E Publishing Incorporated.
▪ Delost, M.D., (2014) Introduction to Diagnostic Microbiology for
the Laboratory Sciences. 2nd ed. Jones and Bartlett Publishers