Case Study Pathogenic Bacteriology 2009 Case #38 Helen Luu, Maria Navarrete, & Allen Chiu.

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

Diagnosis of Lyme DiseaseDiagnosis of Lyme Disease

•High risk of Lyme disease in Connecticut area

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.

ReferencesReferences

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