Nonculturable + Spirochetes Flashcards
Chlamydia spp. replication cycle
There are two forms: elementary body (EB) and reticulate body (RB)
1. The EB (infective form) attaches to specific host cell receptors
2. The EB is ingested through endocytosis and resides in host phagosome
3. The EB reorganizes and forms a RB
4. The RBs replicate by binary fission and the phagosome enlarges into an inclusion
5. The RBs reorganize to form EBs and the host cell ruptures, releasing EBs
Chlamydia spp. characteristics
nonmotile, GN, obligate intracellular parasites (rely on host for energy), only grow and replicate inside animal or human cells
Which species of Chlamydia cause disease in humans?
C. trachomatis, C. pneumoniae, and C. psittaci
What are the different serovars of C. trachomatis and what diseases do they cause?
- Serovars A, B, Ba, and C cause trachoma (eye infection) - Major cause of blindness in endemic areas
- Serovars L1, L2, and L3 cause lymphogranuloma venereum (LGV) - STD
- Serovards D-K cause genital tract infections
Trachoma
chronic inflammation of the conjunctiva, resulting from contact with secretions on towels, fingers, or flies
Lymphogranuloma venereum (LGV)
STD that leads to acute lymphadenitis, causing the inguinal lymph nodes to fill with pus and swell (bubo); can lead to chronic LGV, resulting in severe damage to the genital and rectal areas
Symptoms from Serovars D-K
many are asymptomatic; causes nongonococcal urethritis (NGU) in men; can cause epididymitis, prostatitis, proctitis, urethritis, cervicitis, endometritis, PID, infected fallopian tubes, and infertility in women
Can cause inclusion conjunctivitis in adults and newborns
Can cause pneumonia in newborns
What is the difference between trachoma and inclusion conjunctivitis?
Inclusion conjunctivitis does NOT lead to blindness
Specimen for Chlamydia culture
Specimens must have host epithelial cells; – Swabs: endocervical, urethral, and conjunctival can be submitted for culture by removing any secretions and discharge and then vigorously swabbing the mucosal surface; no wood shaft swabs
- Cytological brush of endocervical material
- Biopsy
- Lower respiratory tract secretions
- Aspirates from buboes
Specimen transport and storage for Chlamydia culture
Transport Media with 2-sucrose phosphate (2SP) and sucrose glutamate phosphate;
NO VTM only UTM
Should be REF or Frozen
Chlamydia Culture Set Up
- Vortex clinical specimen w/ 5mm glass beads or use sonication to break up host cells
- Centrifuge the specimen onto the cell monolayer (shell vial)
- After 48-72 hr of incubation at 37C, monolayers are stained with iodine or for DFA test
Nonculture detection methods for Chlamydia (more common)
DFA test, EIA, NAAT, or Giemsa-stained smears for newborns
Recommended testing for diagnosis of Chlamydia infection
Positive culture is a definitive diagnosis
Nonculture methods need to be confirmed with culture or a second nonculture method
It is recommended that asymptomatic individuals, rectal specimen, medicolegal cases, and nasopharyngeal specimens from infants all have cultures done
Serology for Chlamydia
Urogenital tract infections cannot be diagnosed with serology because antibodies can be from a previous infection
Serological tests are good for neonatal pneumonia (C. pneumonia) and for LGV
C. psittaci characteristics
uncommon cause of human infections, generally in birds; “parrot fever”
Infection from inhalation of aerosols from birds, causing pneumonia, headache, mental changes, and hepatoslenomegaly
C. pneumoniae characteristics
common respiratory pathogen that causes flulike illness, pneumonia, bronchitis, pharyngitis, and sinusitis
How are species of Chlamydia differentiated from each other?
C. trachomatis: EB is round, inclusion is round, vacuolar, contains glycogen, and has plasmid DNA; susceptible to sulfonamides
C. psittaci: EB is round, inclusion is dense, w/o glycogen, and has plasmid DNA; resistant to sulfonamides
C. pneumoniae: EB is pear-shaped, inclusion is round, dense, w/o glyogen, and lacks plasmid DNA; resistant to sulfonamides
Rickettsia spp. characteristics
nonmotile, pleomorphic, GNCB, obligate intracellular parasites transmitted to humans by arthropods (no intracellular development unlike Chlamydia)
How are humans infected by Rickettsia spp.?
- The bite from an infected arthropod vector deposites the organism directly into the bloodstream, where endothelial cells from blood vessels engulf it
- Once engulfed, they are carried into the cell’s vacuole cytoplasm
- They multiply, leading to cell injury and death
What are the three groups of Rickettsia spp.?
Spotted-fever group: fever, headache, rash from ticks (except R. akari from mouse mites)
Typhus group: causes typhus
Scrub typhus group: causes scrub typhus
Spotted-fever group species and diagnosis
R. rickettsii: Rocky mountain spotted fever (RMSF) from ticks; diagnosed with serology, PCR, RFLP, or immunohistology
R. conorii: In Europe, Middle East, and Africa; same diagnosis
R. parkeri: mild illness in North and South America; diagnosed with immunohistochemical serology and PCR
Typhus group
Diagnosed with serology, PCR, and RFLP
R. prowazekii: spread worldwide by lice; two types: epidemic typhus (initial infection) and Brill-Zinsser (recurrence)
R. typhi: spread worldwide by fleas; causes murine typhus (endemic typhus)
Scrub Typhus group
R. tsutsugamushi: spread in Asia and Australia from chiggers (mite larvae in rodents)
Diagnosed with serology or PCR
Ehrlichia spp. characteristics
Pleomorphic, GN, obligate intracellular pathogens found in cytoplasmic vacuoles; follows a similar replication cycle as Chlamydia (forms inclusions called morulae); from ticks in the United States
E. chaffeensis characteristics
causes human monocytic ehrlichiosis (HME); symptoms include fever, headache, muscle pain, rash, and malaise; diagnosed with serology, PCR, immunocytology, and immunohistology
E. ewingii characteristics
causes ehrlichiosis; diagnosed with PCR; less common
Anaplasma characteristics
causes Human granulocytic anaplasmosis, from ticks in Europe and the US, diagnosed with serology, PCR, blood smear, phagocytophilum, immunohistology, and immunocytology
Neorickettsia sennetsu characteristics
causes Sennetsu fever in SE Asia from ticks; diagnosed with serology
Coxiella burnetii characteristics
obligate intracellular pathogen that replicates in cytoplasmic vacuoles (can survive extracellularly but will only grow in the lungs); causes Q fever; smaller than Rickettsia and more resistant; it has 2 antigenic states (sporelike life cycle)- large cell variant is infectious while the samll cell variant in noninfectious
Q Fever symptoms and epidemiology
found in cattle, sheep, and goats where it is shed in urine, feces, milk, and birth products; infections are typically from inhalation of infectious aerosols
Usually asymptomatic or mild, but can cause headache, fever, chills, myalgias (no rash), and hepatoslenomegaly; Chronic Q fever can lead to endocarditis and death
Characteristics of Mycoplasma and Ureaplasma
smallest free-living forms, fastidious, slow growing; require nucleic acid precursor mc, fatty acids, and sterols to grow
Pathogenesis of Mycoplasma and Ureaplasma
colonize mucosal surfaces and rarely cause disease in immunocompetent people; ones that cause disease attach to the surface of epithelial cells
M. hominis characteristics
colonizes urogenital tracts of sexually active adults; associated with BV, PID, postpartum fever, meningitis in premature infants, and other infections
M. fermentans characteristics
found in the oropharynx and genitourinary tract; AIDS-associated Mycoplasma found in the tissues of AIDS and non-AIDS patients
M. genitalium characteristics
can cause genital tract infections; associated with cervicitis and PID; causes 15-20% of nongonococcal urethritis in men
Ureaplasma urealyticum
colonizes the urogenital tract; can cause nonchlamydial, nongonoccal urethritis in men and BV in women; has been isolated from the tissues of spontaneously aborted fetuses, stillborns, and infants (may infect chorioamnion)
Collection and Transport of Specimen for Mycoplasma and Ureaplasma
Body fluids: should be REF for up to 24 hr; process with concentrating and then diluting in broth culture media to remove contaminates
Swab: place in media immediately to avoid drying out; REF for up to 24 hr with modified stuart’s, 2SP, Mycoplasma transport media, Shepard’s 10B broth for urea., or SP-4 broth for myco.
Tissues: prevent drying out and process by mincing and diluting in transport media
Identification of Mycoplasma and Ureaplasma
M. fermentans: glucose +, arginine +, and urease -
M. genitalium: glucose +, arginine -, and urease -
M. hominis: glucose -, arginine +, and urease -
M. pneumoniae: glucose +, arginine -, and urease -
U. urealyticum: glucose -, arginine -, and urease +
Cultures for Mycoplasma and Ureaplasma
Needs special media like SP-4, U broth, E agar, or NYC; grows at 37C w/ CO2; Mycoplasma colonies have a “fried egg” appearance
ELISA can be used for diagnosis and PCR
Spirochete characteristics
helically curved GNRs, with axial fibril (flagella-like organelles that wrap around the cell wall, enclosing within the outer sheath, and facilitate motility) and an outer sheath; axial fibrils attach to insertion disks
What are the different types of Spirochetes?
Treponema, Borrelia, Leptospira, and Brachyspira
Characteristics of Treponema spp.
slender with tight coils, 6-10 fibrils and 1 insertion disk
T. pallidum subsp. pallidum disease
Transmitted sexually or congenitally worldwide, causing venereal syphilis
What are the different stages of syphilis?
Primary: hard chancre and regional lymphadenopathy
Secondary: chancre heals and dissemination occurs; fever, fatigue, polymorphic rash, alopecia, ocular syphilis
Latent: Can become latent at the secondary stage
Tertiary: infection/inflammation of the blood vessels in the CNS (general paresis, tabes dorsalis, optic atrophy) and cardiovascular systems (aortic aneurysm and aortic insufficiency); gummas: destructive lesions of soft tissue, cartilage, internal organs, and bone
All stages can have neurosyphilis
Congenital Syphilis
Transmitted from mother to fetus; 40% are stillborn; can lead to bone deformities, blindness, deafness, deformed faces, skin rashes, anemia, and death
T. pallidum subsp. pertenue characteristics
causes Yaws in children; located in Africa, Pacific Islands, South and Central America; spread through person-to-person skin contact, leading to papules, nodules, and ulcers that can progress to bone and cartilage destructive lesions
T. pallidum subsp. endemicum characteristics
Causes endemic nonveneral syphilis in all ages from mouth to mouth contact in SE Asia, Middle East, and North Africa, leading to papules, macules, and ulcers
T. carateum characteristics
Primarily in children and adolescents from person-to-person skin contact; causes Pinta in Mexico, Central and South America, leading to papules, macules, lesions, and lymphadenopathy
Laboratory Diagnosis of syphilis
Direct detection with dark-field exam or fluorescent antibody staining
Serodiagnosis
What is the serodiagnosis for syphilis?
Measure the presence of treponemal (EIA or agglutination tests) and nontreponemal (reagin) antibodies (VDRL and RPR tests)
2 Methods:
Positive Quantitative test (RPR or VDRL) then run a specific test (ex: EIA) to confirm
Run a specific test (ex: EIA) and then confirm positive with first pathway
Venereal Disease Research Laboratory (VDRL) Method
Serum is heated for 30 min at 56C to remove any anti-complementary activity that can lead to a false positive; if it is not tested within 4 hours, it must be reheated for 10 min
1. Spread 0.05 mL of serum to a circle on a ceramic slide
2. Add one calibrated drop of antigen to the circle (calibrated to drop 60 drops/mL)
3. Rotate at 180 rpms for 4 minutes
4. Read microscopically at 100x
5. Perform titer on positive samples
Usually used to screen CSF; Antigen consists of cardiolipin, cholesterol, and lecithin
Rapid Plasma Reagin Test (RPR)
The cardiolipin antigen from VDRL is modified with choline chloride to make it more stable; can be read macroscopically due to an attached charcoal particle
Note: Cannot be performed on CSF, only serum or plasma
1. Serum/Plasma is added to the RPR card circle and spread
2. One drop of antigen from a calibrated needle is added
3. Rotate at 100 rpms/minute for 8 min
4. Read macroscopically to see clumping with charcoal
Which test should be used to see if treatment is successful when treating syphilis?
Nontreponemal tests (VDRL or RPR) because they are quantitative unlike treponemal tests (can also remain positive after treatment)
Fluorescent Treponemal Antibody Absorption Test (FTA-ABS)
- Diluted, heat inactivated serum is added to Reiter’s stain of T. pallidum to remove cross reactivity from other Treponemes
- Slides are coated with Nichol’s strain of T. pallidum and absorbed patient serum is added (If there are antibodies against T. pallidum, they will bind to the spirochete)
- Slides are washed and incubated with anti-human gamma globulin (anti-HGG), which will bind with human IgG antibodies bound to the T. pallidum on the slide
- After washing, slides are examined for fluorescence
Borrelia burgdoferi characteristics
thicker, 3-10 looser coils, 30-40 fibrils and 2 insertion disks; actively motile; stain well with Giemsa’s stain unlike treponemes; anaerobic or microaerophilic
Borrelia Diseases
Borreliosis
Lyme Disease
Borreliosis
relapsing fever that is transmitted by a tick or louse bite; 2-15 days are infection, fever, headache, and myalgia start and last 4-10 days. Then, the organism disappears from blood and hides in organs until it has newly modified antigens, then symptoms occur again
Lyme Disease
Stage 1: Erythema migrans (EM)- red, ring-shaped skin lesion with a central clearing appears at the bite site; can have a headache, fever, malaise, and muscle/joint pain
Stage 2: weeks to months after infection; may include arthritis, carditis, but mostly neurological disorders from spread into organs
Stage 3: can continue for years; characterized by acrodermatitis chronica atrophicans (ACA) and diffuse rash
Diagnosis of relapsing fever
Peripheral blood should be used; direct observation is mostly used by viewing blood under dark or bright field illumination (see spirochetes moving/pushing RBC around) or Wright’s or Giemsa’s stain
Serodiagnosis is not used because of antigenic shifts
Diagnosis of lyme disease
Can collect blood, biopsy, and body fluids for testing
Direct detection: tissue sections can be stained with Warthin-Starry Silver Stain
PCR testing can be used for body fluids (not standardized yet)
Serodiagnosis is mostly used, typically with a 2 step appraoch to avoid false positives (especially in those with autoimmune disorders)
Culture is not commonly used but possible with Modified-Barbour-Stoenner-Kelly medium
Different serodiagnosis tests for lyme disease
ELISA, IFA, and Western blot can be used (typically 2 step with Western blot used to confirm positives)
Treatment of Lyme Disease
Early localized infection: doxycycline or amoxicillin orally for 2-3 weeks
Late disease: 2-4 weeks of oral treatment or parenteral therapy with ceftriaxone
Brachyspira characteristics
tapered ends with 4 fibrils at each end; reside in the brush border within the intestine (submit stool, biopsy, or rectal swab); can grow on BHI or tryptic soy agar with additives in an anaerobic environment at 37C; can view with dark field microscopy or histologic exam with PAs or hematoxylin teosin stain
How are the two species of Brachyspira differentiated from each other?
B. aalborg: hippurate positive and weak indole
B. pilosicoli: indole negative and weak hippurate
Leptospira characteristics
right-handed helices with hooked ends, 2 fibril and 3-5 insertion disks; spinning or rapid motility
Disease and Diagnosis of Leptospira
Leptospirosis: worldwide; caused by contact with infected animal urine or blood, resulting in anicteric leptospirosis (septicemia stage and immune stage) and Weil disease if severe (rapidly spreads to CNS and kidneys
Diagnosis: test body fluids with PCR, broth culture at room temp or 30C for 6-8 weeks (can grow at 10C), then view broth with microscopy, or serology MA
