Chlamydiae, Mycoplasma and Rickettsiae

Chlamydiae
Obligate intracellular parasite Ubiquitous organisms Generally highly infectious easily transferred to new hosts remarkable ability to escape host immune mechanism Nonmotile and nonpiliated but possess unusual cylindric surface projections arranged in a hexagonal array

 All share a common genus-specific or group antigen and multiply in the cytoplasm of their host cells by a distinctive development cycle All require living cells for growth Rapidly inactivated by heat and lose complete infectivity after 10 minutes at 60C

 All species exhibit two morphologically distinct forms: 1. elementary body (EB) small, dense spherical body which is the infectious form 2. reticulate body (RB) intracellular, metabolically active form that divides by binary fission

Developmental cycle
Phase 1 Attachment and penetration of the EB >EB attaches to the surface of a susceptible host cell >then induces endocytosis and is enclosed within cytoplasmic vesicle, a phagosome Phase 2 Reorganization of the EB to a more metabolically active form, the RB Phase 3 Growth and division by binary fission of the RB

 Phase 4 Maturation of the RBs into infection EBs a. newly formed reticulate and elementary bodies are still enclosed within the vesicle, which is visible microscopically as an inclusion b. inclusions of C. trachomatis (Halberstadter Prowazek bodies) are rigid, compact and consist primarily of glycogen, hence can be stained brown with Lugols iodine; dark purple with Giemsas stain c. inclusions of C. psittaci (Levinthal-Cole-Lillie bodies) are diffuse, irregular in shape and contain no glycogen; the microcolonies strain with the Giemsa and Machiavello stains

 Phase 5 release of EBs from the host cell

Differential characteristics from

viruses
1. Possess both RNA and DNA 2. Multiply by binary fission 3. Possess a cell envelope similar to that of gram-negative bacteria 4. Possess ribosomes and synthesize their own proteins, nucleic acids and lipids 5. Susceptible to a wide range of antibiotics, especially tetracycline and erythromycin

Antigens Produced
1. Genus-specific or group antigens

these are heat-stable lipopolysaccharides (LPS) shared by all chlamhydia

2. Species-specific antigens these are mainly outer membrane proteins shared by all members of a chlamydial species

 There are three described species of chlamydiae that commonly infect humans: i. Chlamydia trachomatis causes trachoma (eye disease) and chlamydia (sexually transmitted) ii. Chamydia pneumoniae causes pneumonia iii. Chlamydia psittaci causes psittacosis

Differentiation among Chlamydia Species

Characteristic 1. Host range 2. Inclusion
3. Elementary body morphology 4. Glycogen in inclusions 5. Number of serovars 6. Folate biosynthesis 7. Susceptibility to sulfonamides & Dcycloserine 8. Plasmid DNA

C.trachomatis Humans, mice Oval, vacuolar

Round (+) 15 (+) (+) (+)

C. psittaci Birds, humans lower mammals Variable, dense

Round (-) NA (-) (-) (+)

C. pneumoniae Humans Oval, dense

Pear-shaped (-) 1 (-) (-) (-)

C. trachomatis
Biovars and Serovars a. biovar LGV consists of 3 serovars (LGV1-LGV3) b. biovar trachoma have 12 serovars i. serovars A,B,Ba, &C isolated primarily from eyes of trachoma patients in endemic areas ii. Serovars D to K isolated most often from genital tracts and eyes of trachoma patients in non-endemic areas

C. trachomatis
Diseases Produced: a. endemic trachoma i. a chronic keratoconjunctivitis that begins with acute inflammatory changes in the conjunctiva and cornea progressing to scarring and blindness ii. Caused by serotypes A, B, Ba or C iii. Seen in poverty-stricken families (overcrowding) iv. Transmitted by droplets, hands and contaminated clothing from one eye to another

b. inclusion conjunctivitis i. caused by serotypes D through K; historically referred to as the TRIC (trachoma inclusion conjunctivitis) agent ii. Infants acquire the infection from passage through an infected birth canal; does not lead to blindness iii. Neonatal pneumonia may also be seen iv. Adults acquire the infection via selfinoculation of genital secretions or following contamination of unchlorinated swimming pools (swimming pool conjunctivitis)

c. Sexually transmitted chlamydial disease i. caused by oculogenital serotypes D through K

ii. Includes nongonococcal urethritis, cervicitis, bartholinitis, epididymitis, proctitis, salpingitis, etc
iii. Associated with Reiters syndrome

d. Lymphogranuloma venereum LGV i. other names are lymphrogranuloma inguinale, climactic bubo, tropical bubo and esthiomene ii. Sexually transmitted disease caused by serotypes LGV1, LGV2 and LGV3 iii. Presents a painless, small, inconspicuous and vesicular primary lesion with painful, firm and enlarged matted inguinal and femoral lymph nodes iv. Elephantiasis of the vulva (called esthiomene) may occur 2 to lymphatic obstruction

Laboratory Diagnosis
>>Specimens: scrapings of epithelial cells from urethra, cervix, vagina, conjunctiva, rectum and nasopharynx; throat specimens; salpinx and epididymis aspiration biopsis; pus and buboes a. cytological examination of cell scrapings for the presence of inclusion bodies which are stained with iodine, fluorescent antibody and Giemsa stains (Tzanck test) dark blue b. rapid antigen detection methods i. direct FAT staining (Micro Trak DFA) ii. ELISA (Chlamydiazyme assay) iii. RNA-directed DNA probe conjugated to a chemiluminescent marker (PACE, Gen-Probe)

Laboratory diagnosis
i. Inoculation into embryonated eggs (chick embryo) ii. Inoculation of experimental animals (usually mice, inoculated intracerebrally) iii. Selected tissue culture cell lines like McCoy, HeLa 229, BHK-21, L929 and Buffalo green monkey which are inoculated by centrifugation; a. most sensitive and specific diagnostic method b. cells are pretreated with cycloheximide, cytochalasin B or idoxuridine to enhance chlamydial replication and allow easier recognition of inclusions

c. inclusions are visualized using the above mentioned stains, immunofluorescence being most sensitive d. cycloheximide-treated McCoy cells have been shown to produce higher inclusion counts than other cell lines

Serologic testing
i. Complement fixation - detects antibodies to a genus-specific antigen used commonly for LGV Microimmunofluorescence (micro-IF) technique - detects type specific antibodies - used mainly in ocular and genital infections and neonatal pneumonia - may also be used in LGV

ii.

iii. Frei test - an intradermal skin test used in the diagnosis of LGV which detects a delayed hypersensitivity response to chlamydial antigen

 Treatment susceptible to tetracycline, sulfonamides and erythromycin

C. psittaci
Disease Produced : Psittacosis or Ornithosis >>Primarily a disease of birds especially the psittacine birds (parrots, parakeets, cockatoos, etc.) which is occasionally transmissible to humans a. mode of transmission i. inhalation of organisms from infected birds (aerosols) and their droppings ii. Person to person transmission (rare)

b. clinical manifestations i. pneumonia ii. Severe headache and changes in mentation iii. hepatosplenomegaly

Laboratory Diagnosis: >>Specimens: blood, sputum and biopsy tissue specimens a. isolation i. yolk sacs of embryonated eggs ii. Mice, inoculated intracerebrally or subcutaneously iii. Grows best in L 929 cells b. FA test using monoclonal antibody -used primarily as research tool

c. Serologic testing i. Complement fixation test most commonly used ii. Micro-IF technique Treatment: tetracycline and erythromycin

C. pneumoniae
History: >>initially considered to be a psittacosis strain since its inclusion bodies produced in cell culture resemble that of C. psittaci >>Strain was named TWAR, an acronym reflecting the history of the first two isolates, Taiwan and acute respiratory

Diseases produced a. asso. with pneumonia, bronchitis, pharyngitis, sinusitis, and a flulike illness b. pneumonia is transmitted from human to human c. severe pneumonia in elderly or in IC pxs

General characteristics >>more homogenous than C. trachomatis and C. psittaci all isolates tested are immunologically similar >>EB is pear-shaped

Epidemiology and Pathogenesis >>a human pathogen >> infections are both endemic and epidemic >> little is known as pathogenesis >> Ab prevalence to C. pneumoniae starts to rise in school age children; 30-45% in adolescents; >50% in adults a. mode of transmission: i. inhalation of aerosolized droplets

Laboratory Dianosis >> Specimens: throat swab, sputum, nasopharyngeal and bronchoalveolar fluids >>Methods: a. Direct Detection: assays to detect C. pneumoniae Ag: have poor sensitivity PCR using C. pneumoniae-specific primers: more sensitive than cell culture

b. Cultivation: specimens for isolation: swabs from oropharynxn(place into chlamydial transport medium; transport on ice -> store at 4C HeLa 229 cells and heteroploid cell line are more sensitive than McCoy cells for C.pneumoniae

c. RA test using monoclonal antibody > used as research tool d. Serologic testing i. complement fixation ii. Micro-IF test > more reliable

Treatment: tetracycline and erythromycin

Mycoplasma
Pleuropneumonia- like organisms or PPLO 2 genera of Mycoplasmataceae: i. Mycoplasma: does not hydrolyze urea i. Ureaplasma: hydrolyze urea General Characteristics: >>Class Mollicutes, free living saprophytes or as parasites in many animal and plant species >>smallest and simplest procaryotes that are able to survive extracellularly and capable of self replication

>>highly pleomorphic, lacks a true cell wall and bounded by a trilaminar cell membrane >>morphologically, Gram (-), spherical or pear-shaped structures to filamentous cells with branching or with terminal structures

>>most are facultative anaerobes but growth is better in an aerobic condition

1. M. pneumoniae
Eaton agent 10 accepted human species of Mycoplasma, only few produce disease such as Mycoplasma pneumoniae known as Primary atypical pneumonia (walking pneumonia)-> major human pathogen

Not a normal flora

General Characteristics:

a. Morphology: >>small, coccoidal to short, branched filamentous cells >>bulbous enlargement with a differentiated tip structure
- most distinctive feature seen in young filamentous cells - grown in broth culture

>> has the ability to attach glass surfaces, red cells and respiratory epithelial cells >> exhibits a gliding motility on liquidcovered surfaces
b. Cultural characteristics: >>mulberry-colony appearance (modified NYC as culture medium suitable for growth) and a typical fried egg appearance after repeated transfer to artificial media

Mode of transmission: >> person to person via aerosol droplets Determinants of Pathogenicity >>organism attach to respiratory epithelial cells with a predilection for the lower tract (attachment is mediated by a specific adhesin, designated protein P1 >>its gliding motility facilitates penetration and its minute size and plastic nature enables it to adapt its shape to conform to the contours of the host cell surface

Clinical Manifestations: 1. tracheobronchitis (most common syndrome) 2. pneumonia 3. pharyngitis and rhinitis 4. ear infection including bullous myringitis 5. meningitis and encephalitis 6. myocarditis and pericarditis

Laboratory Diagnosis: (usually serologic) >> Specimen: sputum (best), nasopharyngeal and throat swabs and washings, tracheal aspirates and lung biopsy specimens i. phase contrast and darkfield microscopy ii. Isolation: ->selective biphasic medium containing broth over agar with addition of penicillin or thallium to inhibit overgrowth of other organisms a. SP-4 Mycoplasma medium- for primary isolation b. Edwar Hayflick agar

b. Edward Hayflick agar >> inoculated with subculture coming from SP-4 medium >> colonies are spherical, grainy, yellowish and embedded in the agar with a thin outer layer >> growth confirmed with by a small zone of beta hemolysis around colonies after a thin layer of sheeps BA was overlaid on the medium

iii. Serologic tests >>cold hemagglutination reaction >>complement fixation >>growth inhibition test >>immunofluorescence >>passive hemagglutination >>RNA-directed DNA probe test >>ELISA >>hemadsorption >>Radioimmunoprecipitation tests

iv. Dienes staining method for PPLO colonies a. uses azure II and methylene blue b. colon stain light blue at the periphery and bright deep blue at the center Treatment: tetracycline and erythromycin

2. Genital Mycoplasma
Pathogenic species: a. Ureaplasma urealyticum i. a cause of nongonococcal urethritis in males ii. May possibly contribute to low birth weight of newborns and perinatal mortality iii. Originally referred to as T. strain mycoplasmas (T for tiny) iv. Urea for growth

b. M. hominis i. associated with pospartum fever, postabortal fever, pelvic inflammatory and pyelonephritis
c. M. genitalium i. associated with NGU ii. Shares extensive serologic crossreactivity with M. pneumoniae iii. Possesses an attachment mechanism and surface protein similar to that of M. pneumoniae

Laboratory diagnosis >> Specimens: urethral or genital discharges collected on swabs and inflammatory exudates a. microscopic examination: useless b. Culture: i. transport media such as modified Stuarts, 2-SP or trypticase soy broth with 0.5% albumin and 400 U/ml PCN ii. Shepards a7-B agar iii. Ureaplasma agar plate and broth

c. Serologic tests i. ELISA ii. RNA-directed DNA probes (PACE system, Gen Probe) iii. Indirect hemagglutination iv. Growth inhibition v. Nucleic acid dot-blot hybridization methods- for M. genitalium
Treatment: tetracycline and erythromycin

Rickettsiae
Family Rickettsiaceae (3 tribes) Rickettsiae, Ehrlichieae and Wolbachieae Rickettsieae Ehrlichia

3 genera infect man Rickettsia, Rochalimaea, Coxiella

Ehrlichia

2 species E. sennetsu and E. canis

General characteristics: >>filterable size, pleomorphic, Gram (-) bacilli, obligate intracellular parasite and multiply by binary fission, singly or in pairs, in short chains or in filaments

>>fastidious organism
>>stain poorly with Grams stain and appear blue in Giemsas stain and red with Machiavellos stain

>>Rickettsia: do not undergo any type of development cycle >>Ehrlichia: undergoes intracellular development cycle enters WBC EB initial body morula release

Rickettsieae
Clinical Infection:

a. Scrub Typhus or Tsutsugamushi disease or Chigger-borne typhus Etiologic agent: R. tsutsugamushi( R. orientalis) Vector: larva of trombiculid mites Transmission: a. humans are only accidentally infected b. transmitted by bites of an infected chigger (larva)

Clinical manifestations:

a. resembles epidemic typhus clinically b. skin rasheschar c. generalized lymphadenopathy and lymphocytosis are common

b. Spotted Fever Group Etiologic agents: R. ricketsii >> Rocky Mountain/American spotted fever R. conorii>>Mediterranean spotted fever R. akari>> Rickettsial pox vectors: ticks and mites Transmission: bites/accidentally C. manifestation: rash from extremities to trunk, eschar

c. Typhus group etiologic agents: R. prowazekii (Epidemic typhus, Brill Zinsser), R. typhi (Murine typhus) >>human body louse, oriental rat flea d. Trench/Shinbone fever etiologic agents: Rochalimaea quintana >>human body louse e. Q fever Coxiella burnetii >>inhalation of infected aerosols, consumption of infected/contaminated milk, wool or hides

Laboratory Diagnosis: Specimens: primarily blood sputum and urine for Q fever vesicular fluid for Rickettsial pox Stained smear using: Giemsa stain Machiavello stain Gimenez stain (the best)

Isolation a. embryonated hens eggs b. laboratory animals (guinea pigs and mice) c. tissue culture Serologic tests a. Neutralization test b. Weil Felix test c. ELISA d. CFT and etc Treatment: tetracycline and chloramphenicol

Ehrlichia
E. sennetsu Sennetsu rickettsiosis other names: infectious mononucleosis, glandular fever, hyuganetsu, kagaminetsu Transmission : unknown (probably by ticks) Clinical Findings: fever, lymphadenopathy, atypical lymphocytosis

 Laboratory Diagnosis: Specimen: blood, lymph nodes and bone marrow a. intraperitoneal mice inoculation b. specific immunoserologic techniques Treatment: tetracycline

E. Canis Canine ehrlichiosis/Tropical canine pancytopenia tick bites Clinical findings: a. leukopenia and thrombocytopenia b. resembles RMSF few cases have rashes Lab. Diagnosis: a. demo of intracytoplasmic inclusions in leukocytes b. serologic test tetracycline

Reactions in Weil Felix test

Disease
Epidemic typhus Murine typhus Scrub typhus

OX-19 OX-2 OX-K

++ ++ + ++

RMSF
Boutonneuse fever Queensland tick typhus North Asian tick typhus Rickettsial pox Q fever Trench fever Ehrlichiosis

+
+ + + ? -

+
+ + + ? -

? -

Weil Felix Reaction (Serology) i. Ag: P.vulgaris OX-2, OX-19, P. mirabilis OX-K ii. Significant titer: >1:160 iii. Provides early clue for rickettsial infection but not specific and not reliable test long lasting immunity after recovery from disease Control of arthropod vectors