Transcript Final Case Study - Cal State LA

Case Study
Pathogenic Bacteriology
2009
Case #38
Helen Luu, Maria Navarrete,
& Allen Chiu
Case Summary
 A 12 year old female from Connecticut
developed a fever that lasted for several
days. She also developed a large rash on
her back. She had recently been walking
through tall grass in an area where there
was horseback riding.
Key Information Pointing to
Diagnosis
 Location: Connecticut near New York
 History: Roaming in tall grass where
horses roamed
 Symptoms: Fever & Rash
 Rash has bulls-eye shape
The Diagnosis for Case # 38
 Lyme borreliosis
 Organism: Borrelia burgdorferi
 Classic bulls-eye shaped rash (erythema
chronicum migrans)
 Flu-like symptoms
 Fever
 Played in tall grass area
 Horses had been in tall grass area
 Ticks can be found on horses
Diagnosis of Lyme Disease
•High risk of Lyme disease in Connecticut area
Classification,Gram Stain Results, and
Microscopic Appearance of
Borrelia burgdorferi
 Gram-negative spirochaete
 Order Spirochaetales
 Family Spirochaetaceae
 Seven periplasmic flagella at each cell end and overlap each other
in the central region of the cell
 A multilayer outer envelope surrounds a protoplasmic cylinder
 Cylinder consists of a peptidoglycan layer, cytoplasmic membrane, and the
enclosed cytoplasmic contents.
Microscopic Appearance
of Borrelia burgdorferi
 Stains well with Giemsa and Warthin-Starry
 Visible by darkfield or phase-contrast microscopy
Electron Micrograph
Darkfield Microscopy
Diagnosis/Isolation/Identifica
tion of Borrelia burgdorferi
 Lyme disease is identified by flu-like symptoms
and the classic bulls-eye
rash (erythema migrans).
 Center of rash clears as
it grows causing the bullseye appearance
 Erythema migrans does not
occur in all cases of
Lyme disease
Diagnosis/Isolation/Identification of
Borrelia burgdorferi
without erythema migrans
 Lyme disease can occur without erythema migrans
 Flu-like symptoms with joint aches are a sign of Lyme disease
 Serological testing for IgG and IgM antibodies
are used to verify diagnosis in
later stages of disease
 Sensitive testing with ELISA (enzyme-linked
immunosorbent assay or IFA (indirect
fluorescent antibody) followed by a more
specific Western blot
 Not accurate due to antibodies forming 2-4
or 4-6 weeks after erythema migrans and
antibodies remaining from previous
infections of Lyme disease
Western blot
Isolation Media
 Borrelia burgdorferi is commonly isolated
on two types of media
 Modified Kelly-Pettenkofer (MKP)
 Barbour-Stoenner-Kelly II (BSK-II)
 Kelly media is complex media designed for the
complex nutritional needs of Borrelia spp.
 Rich media includes rabbit serum, serum albumins,
and gelatine
Diseases and Pathogenesis of
Disease Caused by
Borrelia burgdorferi
 Lyme borreliosis
 Organism Borrelia burgdorferi found in
bloodstream
 3 Stages of Lyme Disease (if left untreated)
 Stage 1: Early Localized (1 - 4 weeks)
 Rash (erythema migrans)
 Flu-like symptoms





Lack of energy
Headache and stiff neck
Fever and Chills
Muscle and joint pain
Swollen lymph nodes
2nd Stage of Lyme Disease
 Stage 2: Early Disseminated (1 – 4 months)
 Can affect the skin, joints, nervous system, and
heart if not treated.
 Skin problems including expanding rash at bite site
as well as additional rashes and/or swelling
 Joint problems including redness, swelling, and pain
 Early nervous system problems including numbness
in the arms and legs caused by nerve inflammation
 Heart problems such as irregular heartbeats
(arrhythmia)
3rd Stage of Lyme Disease
 Stage 3: Late Persistent
 Joint problems such as early arthritis (especially in the knees)
 Late nervous system problems such as pain, weakness, and
numbness in the arms and legs due to bacteria spreading to
the nerves and spinal cord
 Can include headaches, fatigue, and problems with vision, hearing,
memory, concentration, and thinking
 Paralysis of nerves in the face (Bell’s Palsy)
 Inflammation of the brain (encephalitis) and tissues surrounding the brain
(meningitis) and spinal cord
 Heart problems such as inflammation of structures surrounding
the heart (pericarditis)
Pathogenesis of Lyme
Disease
 Lyme disease is spread
through tick bites
 Bacteria spreads to salivary
glands of infected tick
 Usually takes 24 – 36 hours
for the bacteria to invade
after tick’s initial bite
 Borrelia burgdorferi is
invasive and attacks the
tissue
Ticks are the vector for Lyme
Disease
 Tick species Ixodes spp. (deer ticks) help
spread the disease
 Ixodes scapularis in Connecticut
Tick Life Cycle
 Ixodes scalpularis can
live on horses in its
nymph or adult form
 Ticks in nymph form are
smaller and harder to see
 Smaller tick bites may not
be felt
Therapy, Prevention and
Prognosis of Patient Infected
with Borrelia burgdorferi
 Antibiotics are given depending on the stage of the
disease and body areas affected
 Early illnesses are usually treated by medicines taken orally
 Doxycycline

A single dose of doxycycline within 72 hours of a tick bite can decrease chances of Lyme
disease by 87%.
 Amoxicillin
 Later illness requires intravenous drugs
 Ceftriaxone
 Penicillin G
 Pain-relief and anti-arthritic medication may be also be used
 Nonsteroidal anti-inflammatory drugs (NSAIDs)
 Ibuprofen
 Swollen joints can be reduced by removing fluid from them
Prevention of Lyme disease
 Avoiding tick bites
 Use insect repellent such as DEET
 Cover up your body using long pants, long sleeves, and hats
especially in wooded areas (Ticks are easier to spot on light
clothes)
 Learn where ticks and their hosts are commonly found and
avoid those areas
 Check for and remove ticks
 Check for symptoms of
Lyme disease
 No vaccine since Feb 2002
 Due to uncertainty of effectiveness
and lack of demand
Primary Research Article Contributing to
the Understanding of the Disease caused
by Borrelia burgdorferi
 Livengood, Jill A., Gilmore Jr, Robert D., 2006, Invasion
of human neuronal and glial cells by an infectious strain
of Borrelia burgdorferi, Microbes and Infection, vol 8:
2832-2840.
 Borrelia burgdorferi’s ability to attach and invade to
human neuroglial and cortical neuronal cells was
examined due to its role in late disorders such as
neuroborreliosis
 Materials
 An infectious and a non-infectious strain of B. burgdorferi was
grown on BSK-II medium.
 Different human neural cells were grown
 Human umbilical vein endothelial cells (HUVECs)
 Human cortical neuronal cell line (HCN-2)
 2 different human neuroglial cell lines (HS-683, H4)
Primary Research Article
Methods
 Methods
 Cell-association assay
 B. burgdorferi was stained one color, while human cells were stained
a second color.
 B. burgdorferi cells were then added to the human cells and
incubated at 35° C with 5% CO2 for 20 hours.
 Gentamicin protection assay
 Used to show bacteria was internalized and viable
 Gentacmicin was added after incubation to kill all extracellular
bacteria
 Cell monolayers were then collected after incubation and grown on
BSK-II to estimate number of B. burgdoferi that were internalized
 Cell viability assay
 Trypan blue staining was used to test for human cell viability after
infection
 Trypan blue is not absorbed by a live cell
Primary Research Article
Results
 Results
 Cell-association assay
 Human cells were dyed
red while B. burgdorferi
cells were dyed green.
 Internalized spirochaete
is seen as yellow due to
the yellow and green combination
Primary Research Article
Results
 Gentamicin protection assay
 Gentamicin does not readily penetrate mammalian
host cells leaving internalized spirochaetes alive.
 All cell lines tested for positive spirochaete growth,
which shows that internalized Borrelia is still viable
Primary Research Article
Results
 Cell viability assay
 No observable adverse effects on
mammalian cells when compared with
uninfected controls
Primary Research Article
 Discussion & Conclusion
 Borrelia burgdorferi is an invasive organism
that can attack human neural cells.
 It can stay within a neural cell without being
detected and remain viable.
 Can be incubated with human neural cells
showing that CNS is a site where B. burgdorferi
can be disseminated.
 Leads to a greater understanding of invasive
mechanism of B. burgdorferi
Take Home Message









Disease Lyme borreliosis involves the organism Borrelia burgdorferi
Typical early symptoms are erythema migrans, flu-like syptoms and joint
pains.

Late symptoms include meningitis, arthritis, numbness, paralysis, and heart problems
Pathogen is Borrelia burgdorferi
Diagnostics include symptom analysis & serologic testing with ELISA,
IFA, and Western Blot (can also be seen using Giemsa stain and darkfield
microscopy
Therapy is based on antibiotics
Prognosis is dependent on what stage of Lyme disease and what areas
are affect


Early antibiotic treatment causes a fast and complete recovery
Late treatment may require a long time to get better
Prevention is avoiding tick bites



Insect repellent
Covering up areas that ticks can bite
Avoiding high tick infestation areas
Transmission is via deer tick bites.
Threat is to younger children and older people.
References









Feder Jr., Henry M., Micha Abeles, Megan Bernstein, Diane Whitaker-Worth, and Jane M.
Grant-kels. "Diagnosis, treatment, and prognosis of erythema migrans and Lyme arthritis."
Clinics in Dermatology 24 (2006): 509-20.
Johnson, Russell C., George P. Schmid, Fred W. Hyde, A. G. Steigerwalt, and Don J. Brenner.
"Borrelia burgdorferi sp. nov.: Etiologic Agent of Lyme Disease." International Journal of
Systemic Bacteriology 34 (1994): 496-97.
Leboffe, Michael J., and Burton E. Pierce. A Photographic Atlas for the Microbiology
Laboratory. Not Avail, 2004.
Livengood, Jill A., and Robert D. Gilmore Jr. "Invasion of human neuronal and glial cells by an
infectious strain of Borrelia burgdorferi." Microbes and Infection 8 (2006): 2832-840.
"Lyme Disease Causes, Symptoms, Diagnosis, and Treatment on WebMD.com." WebMD
Arthritis and Joint Pain Center: Symptoms, Causes, Tests, and Treatments. 11 Mar. 2009
<http://arthritis.webmd.com/tc/lyme-disease-topic-overview>.
"Lyme Disease." Information on symptoms of menopause in women and men as well as advice
on treatments. Health Information for all sexual ages. Menopause,Andropause. 11 Mar. 2009
<http://www.drlera.com/bacterial_diseases/lyme_disease.htm>.
"Lyme Disease Symptoms, Causes, Treatment, Diagnosis and Prevention on
MedicineNet.com." 11 Mar. 2009
<http://www.medicinenet.com/lyme_disease/article.htm#Whatis>.
Mahon, Connie R., George Manuselis, and Donald C. Lehman. Textbook of Diagnostic
Microbiology. Philadelphia: Saunders, 2006.
McQueen, Nancy. "Spirochaetales." California State University, Los Angeles. 2009.