Bartonella

Finals
Clinical Bacteriology
Bartonella - Giemsa or Diff-Quik stains – for the
detection of morulae during the febrile stage
- Facultative intracellular gram-negative
of Ehrlichiosis
bacilli
Culture
- Do not synthesize acid from carbohydrates
- Can be cultured in embryonated eggs and in
- They live within red blood cells in their
tissue culture
natural mammalian hosts
Serodiagnosis
- They can be cultivated in blood enriched
with 5% CO2 (CAP) or charcoal yeast extract - Weil-Felix reaction – agglutination of
agar (CYEA) certain strains of Proteus vulgaris by serum
from patients with rickettsial diseases;
- Species: B. quintana, B. henselae, B.
presumptive test
elizabethae, B. bacilliformis
- Microimmunofluorescent Dot test –
- Trench fever is transmitted from person to
excellent sensitivity for detecting antibodies
louse (Pediculus humanus corporis) to another
to rickettsia; early diagnosis of RMSF within
person.
7 – 10 days after onset of symptoms
- B. henselae – negative catalase, oxidase and
- Q fever, Ehrlichiosis and Rickettsial pox –
urease
do not induce Weil-Felix antibody in infected
o B. quintana – etiologic agent of trench fever patients

o B. elizabethae – Etiologic agent of Infective - Antibodies to rickettsia (except R.

endocarditis rickettsia) cannot be reliably detected until

o B. bacilliformis – etiologic agent of Oraya at least 2 weeks after the patient has
become ill
fever (chronic verruga peruana) and febrile
acute hemolytic anemia - Other tests: IFA, indirect

Laboratory Diagnosis immunoperoxidase assay, latex agglutination,

EIA, line blot, CF and Western
- Specimen of choice: biopsy of skin tissue, Immunoblotting, Immunocytologic staining
peripheral blood and CSF
Chlamydiaceae
Direct Detection Techniques
Members of the Chlamydiaceae are
- Immunohistology, PCR nonmotile, obligate intracellular prokaryotic
- Polyclonal antibodies bacteria characterized by a unique biphasic
developmental cycle bearing two chlamydial
forms that differ essentially in terms of
morphology and function. According to the
Approved List of Bacterial Names, published
in 1980, the Chlamydiaceae contained one
genus with just two species, Chlamydia
trachomatis and Chlamydia psittaci, which
were separated by their capability to
accumulate glycogen in inclusions and their
susceptibility to sulfadiazine.
In the 1990s, the application of DNA-based
classification methods contributed to the
recognition of the emerging human pathogen
Chlamydia pneumoniae (1) and of Chlamydia
pecorum (2), a pathogen of ruminants, as new
species of the Chlamydiaceae. Phylogenetic
analyses of the 16S and 23S rRNA genes
were the rationale for the proposal of an
emended description of the order
Chlamydiales and a revised taxonomy of the
family Chlamydiaceae in 1999. According to
this proposal, members of the order
Chlamydiales are obligately intracellular
bacteria that have the unique chlamydia like
developmental cycle and more than 80%
sequence identity with chlamydial 16S rRNA
genes and/or 23S rRNA genes. The emended
order now includes four families:
Chlamydiaceae, Parachlamydiaceae,
Simkaniaceae, and Waddliaceae (Gaydos and
Essig, 2015)
CHLAMYDIACEAE
General Characteristics
- Non-motile, small (0.2 – 1.5um), resembles
gram-negative cell wall, obligate intracellular
bacteria that requires living cells for growth
o They stain poorly with gram stain, but they
could be better visualized with the Gutstein
stain
- They contain both the DNA and RNA,
ribosomes and they produce their own
proteins, lipids and nucleic acids
o They were once thought to be large viruses,
but they contain both DNA and RNA
- They multiply by binary fission, replicates I
the cytoplasm of infected cells
- Requires the biochemical resources of the
eukaryotic host cell‘s environment to fuel
their metabolism for growth and replication –
energy parasite
- They have tropism for columnar epithelial
cells
- Possess a heat stable, family specific
antigen that is an essential component of the
cell membrane (LPS)
- Species: Chlamydia tracomatis,
Chlamydophila psittaci and Chlamydia
pneumoniae
Morphologic forms
1. Reticulate body (RB) – replicative form
Intracellular and metabolically active
2. Elementary body (EB) – infective form
Extracellular and structurally resembles a
gram-negative bacillus
Have a rigid cell wall
2 components of the outer membrane
MOMP (major outer membrane protein) and
lipopolysaccharide (LPS) antigen
Chlamydia trachomatis
- Major cause of pelvic inflammatory disease
(PID) and ocular trachoma
- It is divided into 17 different serovars
based on major outer membrane protein
antigenic differences
o Serotypes A, B, Ba, C – endemic trachoma
o Serotypes L1, L2, L3 – lymphogranuloma
venereum (LGV)
o Serotypes D- K – PID, urethritis, cervicitis,
epididymitis, inclusion conjunctivitis
- Humans are the only known host of all
strains
- Associated with infertility and ectopic
pregnancy
Clinical infection
o Primarily spread from human to human by
sexual transmission; mother to infant during
birth
o Intraperitoneal spread may cause
peritonitis or perihepatitis (Fitz-Hugh-Curtis
syndrome)
1. Trachoma
o Chronic inflammation of the conjunctiva
leading to blindness
o It can cause distortion of the eyelids – the
eyelashes become misdirected and turn in
o It is transmitted by contact with individuals
that carries the organism from an infected
eye, or by flies
2. Lymphogranuloma Venereum (LGV)
o A sexually transmitted disease and has
multisystem involvement
o A small painless ulcer or papule appears
initially and nodules (buboes) develop after
several weeks
o Fret‘s test – intradermal skin test
3. Inclusion conjunctivitis
o It is characterized by copious discharge
from the eye, an inflamed and swollen
conjunctiva, and the presence of large
inclusion bodies in the host cell cytoplasm
o Usually affects infants
Laboratory Diagnosis
Specimen of choice: urethra, cervix,
conjunctiva, nasopharynx, rectum, and
material aspirated from fallopian tubes and
epididymis
1. Culture – cell culture (reference method)
o McCoy cells, HeLa 229, buffalo Green
Monkey Kidney Cells, Cycloheximide-treated
McCoy cells
o Centrifugation of the specimen onto the cell
monolayer – growing on a coverslip in the
bottom of a vial. ―shell vial‖ facilitates
adherence of elementary bodies
o After 48 – 72 hours of incubation,
monolayers are stained with iodine or
immunofluorescent stain to examine the
presence of inclusion
2. Cytologic examination
o Cell scrapings from the conjunctiva of
newborns or person with ocular trachoma
o Giemsa stain
3. Antigen Detection and Nucleic Acid
Hybridization
o DFA staining method using the fluorescein-
isothiocyanate-conjugated monoclonal
antibodies – to identify the organism in
infected cells
o ELISA
o Chemiluminescent DNA probe
- Humans acquire the disease by inhalation of
infected aerosols form dried bird excreta or
handling of infected birds
- only laboratories with biosafety level 3
facilities can perform cultivation of samples
Laboratory diagnosis
1. complement fixation – widely used serologic
test to detect psittacosis

o More reliable in patients with symptomatic 2. Direct Microimmunofluorescence –

and shedding large numbers of organisms sensitive method

4. Serodiagnosis 3. Amplification of rDNA using PCR followed

by RFLP – identity and distinguish all 9
o Negative serology can reliably exclude
chlamydial species including C. psittaci
chlamydial infection
Chlamydophila (formerly chlamydia)
Complement fixation
pneumoniae (TWAR strain)
Genus specific antigen can be detected by
- human pathogen
this methd
- transmission is by aerosol droplets via the
Used to diagnose LGV but not trachoma,
respiratory route
inclusion conjunctivitis and neonatal infection
- pear -shaped appearance of EB; has round
Single point titer > 1:64
and dense inclusions
Microimmunofluorescence assay (Micro-IF)
- resistant to sulfonamides
Used for type-specific antibodies of C.
- negative for glycogen staining of inclusion
trachomatis
bodies
Can be used for diagnosis of LGV, trachoma,
- uses HL of Hep-2 cell lines for culture
inclusion conjunctivitis and neonatal infection

IgM titer of 1:32

Chlamydophila (formerly Chlamydia) psittaci

- Etiologic agent of psittacosis/ornithosis

- Endemic pathogen of all bird species –

parrots, parakeets, chicken, ducks

- Outbreaks have occurred among turkey-

processing workers and pigeon aficionados