Case Study Pathogenic Bacteriology 2009

1. Case StudyPathogenic Bacteriology2009 Case #38 Helen Luu, Maria Navarrete, & Allen Chiu

2. 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.

3. 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

4. The Diagnosis for Case # 38 • Lyme borreliosis • Organism: Borreliaburgdorferi • Classic bulls-eye shaped rash (erythemachronicummigrans) • Flu-like symptoms • Fever • Played in tall grass area • Horses had been in tall grass area • Ticks can be found on horses

5. Diagnosis of Lyme Disease • High risk of Lyme disease in Connecticut area

6. Classification,Gram Stain Results, and Microscopic Appearance of Borreliaburgdorferi • 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.

7. Microscopic Appearance of Borrelia burgdorferi Stains well with Giemsa and Warthin-Starry Visible by darkfield or phase-contrast microscopy Electron Micrograph Darkfield Microscopy

8. Diagnosis/Isolation/Identification of Borrelia burgdorferi • Lyme disease is identified by flu-like symptoms and the classic bulls-eye rash (erythemamigrans). • Center of rash clears as it grows causing the bulls- eye appearance • Erythemamigrans does not occur in all cases of Lyme disease

9. Diagnosis/Isolation/Identification of Borrelia burgdorferi without erythema migrans • Lyme disease can occur without erythemamigrans • 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 erythemamigrans and antibodies remaining from previous infections of Lyme disease Western blot

10. 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

11. Diseases and Pathogenesis of Disease Caused by Borreliaburgdorferi • Lyme borreliosis • Organism Borreliaburgdorferi found in bloodstream • 3 Stages of Lyme Disease (if left untreated) • Stage 1: Early Localized (1 - 4 weeks) • Rash (erythemamigrans) • Flu-like symptoms • Lack of energy • Headache and stiff neck • Fever and Chills • Muscle and joint pain • Swollen lymph nodes

12. 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)

13. 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)

14. 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 • Borreliaburgdorferi is invasive and attacks the tissue

15. Ticks are the vector for Lyme Disease • Tick species Ixodes spp. (deer ticks) help spread the disease • Ixodesscapularisin Connecticut

16. Tick Life Cycle • Ixodesscalpulariscan 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

17. Therapy, Prevention and Prognosis of Patient Infected with Borreliaburgdorferi • 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

18. 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

19. Primary Research Article Contributing to the Understanding of the Disease caused by Borreliaburgdorferi • Livengood, Jill A., Gilmore Jr, Robert D., 2006, Invasion of human neuronal and glial cells by an infectious strain of Borreliaburgdorferi, Microbes and Infection, vol 8: 2832-2840. • Borreliaburgdorferi’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)

20. Primary Research Article Methods • Methods • Cell-association assay • B. burgdorferiwas 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

21. 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

22. 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

23. Primary Research Article Results • Cell viability assay • No observable adverse effects on mammalian cells when compared with uninfected controls

24. Primary Research Article • Discussion & Conclusion • Borreliaburgdorferi 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

25. Take Home Message • Disease Lyme borreliosis involves the organism Borreliaburgdorferi • Typical early symptoms are erythemamigrans, flu-like syptoms and joint pains. • Late symptoms include meningitis, arthritis, numbness, paralysis, and heart problems • Pathogen is Borreliaburgdorferi • 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.

26. References Feder Jr., Henry M., Micha Abeles, Megan Bernstein, Diane Whitaker-Worth, and Jane M. Grant-kels. "Diagnosis, treatment, and prognosis of erythemamigrans 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. "Borreliaburgdorferi 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 Borreliaburgdorferi." 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.