Case StudyCase StudyPathogenic BacteriologyPathogenic Bacteriology20092009
Case #38Case #38
Helen Luu, Maria Navarrete,Helen Luu, Maria Navarrete,
& Allen Chiu& Allen Chiu
Case SummaryCase Summary
A 12 year old female from Connecticut A 12 year old female from Connecticut developed a fever that lasted for several developed a fever that lasted for several days. She also developed a large rash on days. She also developed a large rash on her back. She had recently been walking her back. She had recently been walking through tall grass in an area where there through tall grass in an area where there was horseback riding.was horseback riding.
Key Information Pointing to Key Information Pointing to DiagnosisDiagnosis
Location: Connecticut near New YorkLocation: Connecticut near New York History: Roaming in tall grass where History: Roaming in tall grass where
horses roamedhorses roamed Symptoms: Fever & RashSymptoms: Fever & Rash Rash has bulls-eye shapeRash has bulls-eye shape
The Diagnosis for Case # The Diagnosis for Case # 3838
Lyme borreliosisLyme borreliosis Organism: Organism: Borrelia burgdorferiBorrelia burgdorferi
Classic bulls-eye shaped rash (erythema Classic bulls-eye shaped rash (erythema chronicum migrans)chronicum migrans)
Flu-like symptomsFlu-like symptoms FeverFever
Played in tall grass areaPlayed in tall grass area Horses had been in tall grass areaHorses had been in tall grass area
Ticks can be found on horsesTicks can be found on horses
Classification,Gram Stain Results, Classification,Gram Stain Results, and Microscopic Appearance of and Microscopic Appearance of Borrelia burgdorferiBorrelia burgdorferi
Gram-negative spirochaeteGram-negative spirochaete Order SpirochaetalesOrder Spirochaetales
Family SpirochaetaceaeFamily Spirochaetaceae
Seven periplasmic flagella at each cell end and overlap each Seven periplasmic flagella at each cell end and overlap each other in the central region of the cellother in the central region of the cell
A multilayer outer envelope surrounds a protoplasmic cylinderA multilayer outer envelope surrounds a protoplasmic cylinder Cylinder consists of a peptidoglycan layer, cytoplasmic membrane, and the Cylinder consists of a peptidoglycan layer, cytoplasmic membrane, and the
enclosed cytoplasmic contents.enclosed cytoplasmic contents.
Microscopic Appearance Microscopic Appearance of of Borrelia burgdorferiBorrelia burgdorferi
Stains well with Giemsa and Warthin-StarryStains well with Giemsa and Warthin-Starry Visible by darkfield or phase-contrast microscopyVisible by darkfield or phase-contrast microscopy
Electron Micrograph Darkfield Microscopy
Diagnosis/Isolation/Diagnosis/Isolation/Identification of Identification of Borrelia Borrelia burgdorferiburgdorferi
Lyme disease is identified by flu-like symptoms Lyme disease is identified by flu-like symptoms and the classic bulls-eye and the classic bulls-eye
rash (erythema migrans).rash (erythema migrans). Center of rash clears asCenter of rash clears as
it grows causing the bulls-it grows causing the bulls-
eye appearanceeye appearance Erythema migrans does notErythema migrans does not
occur in all cases of occur in all cases of
Lyme diseaseLyme disease
Diagnosis/Isolation/Identification of Diagnosis/Isolation/Identification of
Borrelia burgdorferi Borrelia burgdorferi without erythema migranswithout erythema migrans
Lyme disease can occur without erythema migransLyme disease can occur without erythema migrans Flu-like symptoms with joint aches are a sign of Lyme diseaseFlu-like symptoms with joint aches are a sign of Lyme disease
Serological testing for IgG and IgM antibodies Serological testing for IgG and IgM antibodies
are used to verify diagnosis in are used to verify diagnosis in
later stages of diseaselater stages of disease Sensitive testing with ELISA (enzyme-linkedSensitive testing with ELISA (enzyme-linked
immunosorbent assay or IFA (indirectimmunosorbent assay or IFA (indirect
fluorescent antibody) followed by a morefluorescent antibody) followed by a more
specific Western blotspecific Western blot Not accurate due to antibodies forming 2-4 Not accurate due to antibodies forming 2-4
or 4-6 weeks after erythema migrans and or 4-6 weeks after erythema migrans and
antibodies remaining from previous antibodies remaining from previous
infections of Lyme diseaseinfections of Lyme disease
Western blot
Isolation MediaIsolation Media
Borrelia burgdorferi Borrelia burgdorferi is commonly isolated is commonly isolated on two types of mediaon two types of media Modified Kelly-Pettenkofer (MKP)Modified Kelly-Pettenkofer (MKP) Barbour-Stoenner-Kelly II (BSK-II)Barbour-Stoenner-Kelly II (BSK-II)
Kelly media is complex media designed for the Kelly media is complex media designed for the complex nutritional needs of complex nutritional needs of Borrelia spp.Borrelia spp.
Rich media includes rabbit serum, serum albumins, Rich media includes rabbit serum, serum albumins, and gelatineand gelatine
Diseases and Pathogenesis of Diseases and Pathogenesis of Disease Caused by Disease Caused by Borrelia burgdorferiBorrelia burgdorferi
Lyme borreliosisLyme borreliosis Organism Organism Borrelia burgdorferiBorrelia burgdorferi found in found in
bloodstreambloodstream 3 Stages of Lyme Disease (if left untreated)3 Stages of Lyme Disease (if left untreated)
Stage 1: Early Localized (1 - 4 weeks)Stage 1: Early Localized (1 - 4 weeks) Rash (erythema migrans)Rash (erythema migrans) Flu-like symptomsFlu-like symptoms
Lack of energyLack of energy Headache and stiff neckHeadache and stiff neck Fever and ChillsFever and Chills Muscle and joint painMuscle and joint pain Swollen lymph nodesSwollen lymph nodes
22ndnd Stage of Lyme Disease Stage of Lyme Disease
Stage 2: Early Disseminated (1 – 4 months)Stage 2: Early Disseminated (1 – 4 months) Can affect the skin, joints, nervous system, and Can affect the skin, joints, nervous system, and
heart if not treated.heart if not treated. Skin problems including expanding rash at bite site Skin problems including expanding rash at bite site
as well as additional rashes and/or swellingas well as additional rashes and/or swelling Joint problems including redness, swelling, and painJoint problems including redness, swelling, and pain Early nervous system problems including numbness Early nervous system problems including numbness
in the arms and legs caused by nerve inflammationin the arms and legs caused by nerve inflammation Heart problems such as irregular heartbeats Heart problems such as irregular heartbeats
(arrhythmia)(arrhythmia)
33rdrd Stage of Lyme Disease Stage of Lyme Disease
Stage 3: Late Persistent Stage 3: Late Persistent Joint problems such as early arthritis (especially in the knees)Joint problems such as early arthritis (especially in the knees) Late nervous system problems such as pain, weakness, and Late nervous system problems such as pain, weakness, and
numbness in the arms and legs due to bacteria spreading to numbness in the arms and legs due to bacteria spreading to the nerves and spinal cordthe nerves and spinal cord
Can include headaches, fatigue, and problems with vision, hearing, Can include headaches, fatigue, and problems with vision, hearing, memory, concentration, and thinkingmemory, concentration, and thinking
Paralysis of nerves in the face (Bell’s Palsy)Paralysis of nerves in the face (Bell’s Palsy) Inflammation of the brain (encephalitis) and tissues surrounding the brain Inflammation of the brain (encephalitis) and tissues surrounding the brain
(meningitis) and spinal cord(meningitis) and spinal cord
Heart problems such as inflammation of structures Heart problems such as inflammation of structures surrounding the heart (pericarditis)surrounding the heart (pericarditis)
Pathogenesis of Lyme Pathogenesis of Lyme DiseaseDisease Lyme disease is spreadLyme disease is spread
through tick bitesthrough tick bites Bacteria spreads to salivary Bacteria spreads to salivary
glands of infected tickglands of infected tick Usually takes 24 – 36 hours Usually takes 24 – 36 hours
for the bacteria to invade for the bacteria to invade after tick’s initial biteafter tick’s initial bite
Borrelia burgdorferiBorrelia burgdorferi is is invasive and attacks the invasive and attacks the tissuetissue
Ticks are the vector for Lyme Ticks are the vector for Lyme DiseaseDisease
Tick species Tick species IxodesIxodes spp. (deer ticks) help spp. (deer ticks) help spread the diseasespread the disease Ixodes scapularis Ixodes scapularis in Connecticutin Connecticut
Tick Life CycleTick Life Cycle
Ixodes scalpularis Ixodes scalpularis can can live on horses in its live on horses in its nymph or adult formnymph or adult form Ticks in nymph form are Ticks in nymph form are
smaller and harder to seesmaller and harder to see Smaller tick bites may not Smaller tick bites may not
be feltbe felt
Therapy, Prevention and Therapy, Prevention and Prognosis of Patient Infected Prognosis of Patient Infected with with Borrelia burgdorferiBorrelia burgdorferi
Antibiotics are given depending on the stage of the Antibiotics are given depending on the stage of the disease and body areas affecteddisease and body areas affected Early illnesses are usually treated by medicines taken orallyEarly illnesses are usually treated by medicines taken orally
DoxycyclineDoxycycline A single dose of doxycycline within 72 hours of a tick bite can decrease chances of Lyme A single dose of doxycycline within 72 hours of a tick bite can decrease chances of Lyme
disease by 87%.disease by 87%.
AmoxicillinAmoxicillin
Later illness requires intravenous drugsLater illness requires intravenous drugs CeftriaxoneCeftriaxone Penicillin GPenicillin G
Pain-relief and anti-arthritic medication may be also be usedPain-relief and anti-arthritic medication may be also be used Nonsteroidal anti-inflammatory drugs (NSAIDs)Nonsteroidal anti-inflammatory drugs (NSAIDs) IbuprofenIbuprofen
Swollen joints can be reduced by removing fluid from themSwollen joints can be reduced by removing fluid from them
Prevention of Lyme diseasePrevention of Lyme disease
Avoiding tick bitesAvoiding tick bites Use insect repellent such as DEETUse insect repellent such as DEET Cover up your body using long pants, long sleeves, and hats Cover up your body using long pants, long sleeves, and hats
especially in wooded areas (Ticks are easier to spot on light especially in wooded areas (Ticks are easier to spot on light clothes) clothes)
Learn where ticks and their hosts are commonly found and Learn where ticks and their hosts are commonly found and avoid those areasavoid those areas
Check for and remove ticksCheck for and remove ticks Check for symptoms ofCheck for symptoms of
Lyme diseaseLyme disease No vaccine since Feb 2002No vaccine since Feb 2002
Due to uncertainty of effectivenessDue to uncertainty of effectivenessand lack of demandand lack of demand
Primary Research Article Contributing to Primary Research Article Contributing to the Understanding of the Disease the Understanding of the Disease caused by caused by Borrelia burgdorferiBorrelia burgdorferi
Livengood, Jill A., Gilmore Jr, Robert D., 2006, Invasion Livengood, Jill A., Gilmore Jr, Robert D., 2006, Invasion of human neuronal and glial cells by an infectious strain of human neuronal and glial cells by an infectious strain of of Borrelia burgdorferiBorrelia burgdorferi, , Microbes and InfectionMicrobes and Infection, , vol 8vol 8: : 2832-2840.2832-2840.
Borrelia burgdorferiBorrelia burgdorferi’s ability to attach and invade to ’s ability to attach and invade to human neuroglial and cortical neuronal cells was human neuroglial and cortical neuronal cells was examined due to its role in late disorders such as examined due to its role in late disorders such as neuroborreliosisneuroborreliosis
MaterialsMaterials An infectious and a non-infectious strain of An infectious and a non-infectious strain of B. burgdorferi B. burgdorferi was was
grown on BSK-II medium.grown on BSK-II medium. Different human neural cells were grownDifferent human neural cells were grown
Human umbilical vein endothelial cells (HUVECs)Human umbilical vein endothelial cells (HUVECs) Human cortical neuronal cell line (HCN-2)Human cortical neuronal cell line (HCN-2) 2 different human neuroglial cell lines (HS-683, H4)2 different human neuroglial cell lines (HS-683, H4)
Primary Research Article Primary Research Article MethodsMethods
MethodsMethods Cell-association assayCell-association assay
B. burgdorferi B. burgdorferi was stained one color, while human cells were stained was stained one color, while human cells were stained a second color.a second color.
B. burgdorferiB. burgdorferi cells were then added to the human cells and cells were then added to the human cells and incubated at 35° C with 5% COincubated at 35° C with 5% CO22 for 20 hours. for 20 hours.
Gentamicin protection assayGentamicin protection assay Used to show bacteria was internalized and viableUsed to show bacteria was internalized and viable Gentacmicin was added after incubation to kill all extracellular Gentacmicin was added after incubation to kill all extracellular
bacteriabacteria Cell monolayers were then collected after incubation and grown on Cell monolayers were then collected after incubation and grown on
BSK-II to estimate number of BSK-II to estimate number of B. burgdoferiB. burgdoferi that were internalized that were internalized Cell viability assayCell viability assay
Trypan blue staining was used to test for human cell viability after Trypan blue staining was used to test for human cell viability after infectioninfection
Trypan blue is not absorbed by a live cellTrypan blue is not absorbed by a live cell
Primary Research Article Primary Research Article ResultsResults
ResultsResults Cell-association assayCell-association assay
Human cells were dyed Human cells were dyed
red while red while B. burgdorferi B. burgdorferi
cells were dyed green.cells were dyed green. Internalized spirochaete Internalized spirochaete
is seen as yellow due to is seen as yellow due to
the yellow and green combinationthe yellow and green combination
Primary Research Article Primary Research Article ResultsResults
Gentamicin protection assayGentamicin protection assay Gentamicin does not readily penetrate mammalian Gentamicin does not readily penetrate mammalian
host cells leaving internalized spirochaetes alive.host cells leaving internalized spirochaetes alive. All cell lines tested for positive spirochaete growth, All cell lines tested for positive spirochaete growth,
which shows that internalized which shows that internalized BorreliaBorrelia is still viable is still viable
Primary Research Article Primary Research Article ResultsResults
Cell viability assayCell viability assay No observable adverse effects on No observable adverse effects on
mammalian cells when compared with mammalian cells when compared with uninfected controlsuninfected controls
Primary Research ArticlePrimary Research Article
Discussion & ConclusionDiscussion & Conclusion Borrelia burgdorferiBorrelia burgdorferi is an invasive organism is an invasive organism
that can attack human neural cells.that can attack human neural cells. It can stay within a neural cell without being It can stay within a neural cell without being
detected and remain viable.detected and remain viable. Can be incubated with human neural cells Can be incubated with human neural cells
showing that CNS is a site where showing that CNS is a site where B. burgdorferiB. burgdorferi can be disseminated.can be disseminated.
Leads to a greater understanding of invasive Leads to a greater understanding of invasive mechanism of mechanism of B. burgdorferiB. burgdorferi
Take Home MessageTake Home Message
Disease Lyme borreliosis involves the organism Disease Lyme borreliosis involves the organism Borrelia burgdorferiBorrelia burgdorferi Typical early symptoms are erythema migrans, flu-like syptoms and joint Typical early symptoms are erythema migrans, flu-like syptoms and joint
pains.pains. Late symptoms include meningitis, arthritis, numbness, paralysis, and heart problemsLate symptoms include meningitis, arthritis, numbness, paralysis, and heart problems
Pathogen is Pathogen is Borrelia burgdorferiBorrelia burgdorferi Diagnostics include symptom analysis & serologic testing with ELISA, Diagnostics include symptom analysis & serologic testing with ELISA,
IFA, and Western Blot (can also be seen using Giemsa stain and darkfield IFA, and Western Blot (can also be seen using Giemsa stain and darkfield microscopymicroscopy
Therapy is based on antibioticsTherapy is based on antibiotics Prognosis is dependent on what stage of Lyme disease and what areas Prognosis is dependent on what stage of Lyme disease and what areas
are affectare affect Early antibiotic treatment causes a fast and complete recoveryEarly antibiotic treatment causes a fast and complete recovery Late treatment may require a long time to get betterLate treatment may require a long time to get better
Prevention is avoiding tick bitesPrevention is avoiding tick bites Insect repellentInsect repellent Covering up areas that ticks can bite Covering up areas that ticks can bite Avoiding high tick infestation areas Avoiding high tick infestation areas
Transmission is via deer tick bites.Transmission is via deer tick bites. Threat is to younger children and older people.Threat is to younger children and older people.
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