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Nonmotile, gram negative, obligate, intracellular bacteria; possess DNA and RNA with restricted
metabolic capacity, cannot produce energy; ATP scavenged from host cell. These organisms
possess a gram negative type of cell wall. Recent taxonomic changes have lead to the creation
of two genera in the Order Chlamydiales: Chlamydia (C. trachomatis) and Chlamydophila (C.
pneumoniae, C. psittaci).

Unique life cycle
The infectious, extracellular form, called the elementary body (EB, 0.25 - 0.35 m), is
phagocytized by host cells; primarily nonciliated, columnar or transitional epithelial cells lining
the conjunctiva, respiratory tract and urogenital tract. The chlamydiae promote phagocytosis.
The organism prevents phagolysosomal fusion and replicates within phagosomes. The entire
life cycle takes place within the cell. After entry, EB reorganizes into the reticulate body (RB,
0.5 - 1.0 m) [also called the initial body - IB] which begins replicating via binary fission
approximately eight hours later. Within 18 - 24 hours, RBs begin reorganizing to form EBs.
This stage is visible as a vacuole within infected cells when stained appropriately. Forty-eight to
seventy-two hours after entry, the cell lyses releasing EBs to initiate next cycle of replication.
The RBs can be visualized as inclusions within the cytoplasm of infected cells. The presence of
the inclusions can be used for diagnostic and differential purposes. Only the inclusions of C.
trachomatis can be stained using iodine; all inclusions can be stained with Giemsa stain.
Characteristics useful for distinguishing among the species of Chlamydia are found in textbook.

Organisms can only be cultured on living cells/organisms.
   1. Embryonated hens’ eggs
   2. Tissue culture, McCoy cells or HeLa cells

Chlamydophila psittaci
Common pathogen in many avian and mammalian species
Human infections predominantly result from exposure to infected birds. Transmission is via
inhalation of aerosols containing infectious bird droppings. For 2004, two cases of psitticosis
have been reported to the CDC through November 20.

Psittacosis: After 5 - 15 days incubation period, initial symptoms include pneumonia like
    presentation. Following dissemination from the lungs, the patient may exhibit altered mental
    state and hepatosplenomegaly. Diagnosis usually relies on history of bird contact, clinical
    presentation and specific serology using complement fixation or other procedures. The
    organism can also be cultured from clinical material including blood, sputum, liver or spleen.
     According to the CDC, a confirmed case is culture positive or clinical evidence plus at least
    a fourfold increase in antibody titer to >1:32; presumptive case is clinical evidence plus a
    single titer of >1:32. NOTE! Some patients may show no increase in antibody titer or a
    delayed increase as a result of antimicrobic therapy. In addition, patients infected with other
    species of Chlamydia will demonstrate genus specific complement fixation antibodies.

Chlamydia trachomatis
Almost exclusively a human pathogen; rodent reservoir is possible. Diagnosis of the various
manifestations of C. trachomatis infection involves review of clinical picture and patient history,
demonstration of inclusions in clinical material (cytology), direct fluorescent antibody staining, or
cultivation of the organism on McCoy cells. Detection of antibody is of little diagnostic value for

Chlamydia & Rickettsia                                                                              1
most chlamydial infections since these infections tend to be localized and do not promote a
strong antibody response.

  1. Trachoma - chronic keratoconjunctivitis which can progress to scarring and blindness
  over a period of years; a leading cause of blindness worldwide; seen predominantly in Asia,
  Africa and Mediterranean area. Caused by serogroups A, B, Ba, C

   2. Inclusion conjunctivitis - milder form of conjunctivitis seen mainly among infants as a
   result of contact with sexually transmitted organisms in the birth canal. Blindness is not a
   sequela to this infection.

   3. LGV - lymphogranuloma venereum; VD involving specific serotypes (L1, L2, L3 of C.
   trachomatis). Following a 1-4 week incubation period there is development of an initially
   small painless lesion. Spontaneous healing occurs after a few days followed by regional
   lymphadenitis which may develop draining sinuses (bubose); also fever, headache, joint
   pain and nausea. This infection is uncommon in the U.S and Europe, but is reported in
   Africa, Asia and South America. Diagnosis is generally established by clinical picture and
   the isolation of the organism from buboes material. The Frei's test is a skin test that can be
   used to aid in the diagnosis. However, the Frei's test lacks sensitivity during the early
   stages of the disease and specificity in the later stages. Sequelae include meningitis,
   meningoencephalitis, pneumonia, polyarthritis, and keratoconjunctivitis.

   4. NGU/PGU - STD with symptoms that mimic gonorrhea; seen in 30 - 50% of females and
   20 - 60% males after penicillin therapy for GC (25% NGU Ureaplasma). Chlamydial
   urethritis has become the leading sexually transmitted bacterial infection in the U.S (768,
   148 cases reported to CDC through 11/20/04; 15,583 cases in Miss [highest incidence];
   estimated 3-5 X 106 cases/yr occur). A significant number of the cases are asymptomatic
   (25% of males, 70-80% of females). Sequelae: epididymitis, proctitis, cervicitis, salpingitis
   (30% of PID [major cause] and acute salpingitis) and infertility (10% risk after one episode).
   In Mississippi the peak age for males is 20-24, for females 15-19. For babies of infected
   mothers, inclusion conjunctivitis (25-50% risk) and chlamydial pneumonia (10-20% risk) are
   common perinatal infections, accounting for 30 - 50% of infant pneumonia in the first six
   months postpartum.

   5. Reiter’s Syndrome - a complex of symptoms consisting of urethritis, conjunctivitis,
   arthritis and mucocutaneous lesions; recurrences and chronic disease are seen in one half
   of infected individuals. Serologic evidence links RS to infection with C. trachomatis
   serotypes D-K.

Data indicate that different serotypes of C. trachomatis are involved in each manifestation

Chylamydophila pneumoniae (TWAR Strain)
This organism has been associated with cases of pneumonia, bronchitis, pharyngitis, and
sinusitis. The disease is usually mild and self-limiting (90% of cases are asymptomatic or mildly
symptomatic). Most commonly C. pneumoniae infections present as a mild pharyngitis or
bronchitis. However, it may also present as a pulmonary infection mimicking mycoplasmal
pneumonia. The typical clinical picture involves a college age individual presenting with a
prolonged episode of pharyngitis with hoarseness. This is usually followed by flu-like lower
respiratory tract infection. Among debilitated patients, the infection may be severe resulting in
respiratory failure and death. It has been estimated that approximately 10% of nosocomial and
community acquired pneumonia are due to C. pneumoniae infections. The organism is
Chlamydia & Rickettsia                                                                              2
distributed worldwide; most adults (40 - 50%) demonstrate antibody to the organism and are
subject to more than one infection during their lifetime. Antibody response is generally not
observed in children under five years of age. The highest attack rate is in the segment of the
population aged six to 20 years. The infection is transmitted person-to-person. Diagnosis is
currently dependent on culture, cytology and/or specific serology.

Recent studies have linked C. pneumoniae to blood vessel damage. Individuals with higher-
than-normal levels of antibody to C. pneumoniae have a higher prevalence of severe coronary
artery disease. These individuals are 2-3 times more likely to experience a heart attack than
individuals with a low antibody response to this organism. The organism has been
demonstrated in arterial plaques and among individuals who have suffered myocardial
infarctions. Clinical trials evaluating the effects of treating C. pneumoniae infections among
patients at risk for heart attacks have demonstrated some beneficial effects.

Specimen collection
Psittacosis - sputum, blood, biopsy material
LGV - buboes aspirate
Conjunctivitis - swab or scrapings of conjunctiva and cornea
NGU/PGU - anterior urethral swab, cervical swab/scrapings
C. pneumoniae - bronchial washings, pharyngeal swabs, sputum

   a. Direct Examination - demonstrate inclusions in clinical material; can be non-specific
         staining to demonstrate inclusions or specific identification with fluorescent antibody
   b. Cell culture - demonstrate typical intracytoplasmic inclusions
         Species differentiation - history plus sulfonamide sensitivity - C. trachomatis sensitive, C.
         psittaci and C. pneumoniae are resistant to sulfonamides; Iodine stain of inclusions - C.
         trachomatis positive, C. psittaci and C. pneumoniae do not produce iodine-staining
         inclusions; EB and RB morphology. NOTE! Cultivation of C. psittaci requires a BSL 3
         facility. This technique is mainly used for medicolegal purposes.
   c. Serology (CF).
         Psittacosis - useful technique look for a 4X increase.
         LGV - single titer greater than 1:64 is considered positive.
         TRIC/NGU CF antibody not elevated significantly.
         Neonatal chlamydial pneumonia - look for IgM
         ELISA becoming test of choice for chlamydia
         FA - for Ab/Ag detection for NGU; FA may also be used to identify isolates grown in cell
   d. Nucleic acid amplification tests (NAAT) are becoming the favored testing methodology;
         90-95% sensitivity with 98-100% specificity

Chlamydia & Rickettsia                                                                               3

General Characteristics
The family Rickettsiaceae, Order Rickettsiales, contains at least three genera of medical
importance to humans; Rickettsia, Ehrlichia and Coxiella. This probably represents an
oversimplification of the situation and future reclassification will occur. While the family contains
a diverse group of organisms, a few morphologic and biochemical characteristics are shared.

Members of the family are highly fastidious pleomorphic coccobacilli that are obligate
intracellular parasites. In contrast to Chlamydia, these organisms possess enzymes for energy
production. Members of the family induce phagocytosis by host cells. Once inside the host cell,
the organism escapes from the phagosome into the cytoplasm prior to fusion with the lysosome.
 Replication is via binary fission followed by lysis of the infected host cell. Members of the
genus Ehrlichia appear to remain within the phagosome and follow a replication cycle similar to
that for Chlamydia. Cultivation requires animal inoculation or tissue culture as well as a
biological safety cabinet.

Rickettsia and Ehrlichia are transmitted primarily by arthropod bites or contact with arthropod
feces. Coxiella burnetii is transmitted via aerosols. Small animals (mainly rodents) serve as

General disease pattern
Entry through the skin (RESPIRATORY TRACT for Q fever) leads to localized replication within
the endothelial cells of blood vessels (pulmonary lymph nodes for Q fever). Rickettsemia
follows with the abrupt onset of clinical symptoms including chills, fever, malaise, variable
prostration, rash (generally not present in Q fever and Ehrlichiosis), toxicity, myalgia, arthralgia,
photophobia. The three common features of rickettsial disease are headache, fever and rash.
Hepatitis and endocarditis are common sequelae. The severity of the disease depends to a
large extent on the infectious agent and ranges from mild and self-limiting to severe and life
threatening. Diagnosis generally depends on the clinical picture, geographic location of patient
and serology. Rickettsiae are highly infectious and cultivation of the organism from clinical
material is not attempted by routine laboratories.

In the U.S., the predominant rickettsial diseases are: Rocky Mountain Spotted Fever (90%), Q
Fever, murine typhus, rickettsialpox and Ehrlichiosis.

Members of the genus are divided into three antigenically related subgroups; spotted fevers
(ticks and mite), typhus (fleas & lice) and scrub typhus (mites). The disease manifestations
within each subgroup are similar, but vary with regard to severity and rash pattern. The various
diseases also have somewhat different geographic distributions.

Spotted Fever Group (SFG)
RMSF - Rocky Mountain Spotted Fever: R. rickettsii (wood/dog tick) Approximately 1000
   cases/year (1,330 cases reported through 11/20/04; 34 in Mississippi) are reported with an
   overall mortality rate of 3% when treated, <25% without treatment. Rodents, dogs and foxes
   serve as reservoirs. Almost 95% cases are diagnosed from April - September with 60% of
   cases involving people less than 20 years of age. More common in rural areas; sporadic

   Diagnosis - fever, history of tick exposure and rash by third day after onset. The rash begins
   at the palms and soles of feet, spreading to the trunk; face is not involved. This pattern is
Chlamydia & Rickettsia                                                                              4
   opposite to that of meningococcemia, which must be considered in the differential diagnosis.
    Serology may be helpful.
Rickettsialpox - R. akari: transmitted to man by the bite of the mouse mite. The rash is
   significantly different from other spotted fevers in that it is a papulovesicular type of rash that
   often forms sloughing scabs (eschars). The clinical manifestations are generally milder than
   that for the other spotted fevers. The epidemiology is similar to that for murine typhus.

   Diagnosis - clinical picture and evidence of contact with mice; serology is not very helpful.

American Boutonneuse Fever – R. parkeri: work by Drs Goddard and Norment at MSU
  demonstrated that R. parkeri, could infect guinea pigs. A recent case demonstrates that this
  organism can infect humans. A 40 yo male in Virginia is one of the first reported naturally
  occurring human infections. Patient developed papules on lower leg. Two days prior to
  admission he developed fever (39.2C) headache, malaise, diffuse myalgias and arthralgias.
   The papules ulcerated and developed into eschars. In addition, the patient developed a
  faint, diffuse, salmon-colored rash primarily on the abdomen with a few lesions on hands,
  feet and face. Initial diagnosis was rickettsialpox. Molecular analysis of an isolate from a
  lesion biopsy confirmed the identification of the isolate as R. parkeri. Patient did not recall
  any tick or mite bites after walking his dog in a grassy field. The disease has been named
  American Boutonneuse Fever because it clinically resembles a disease in Europe and Africa
  called boutonneuse fever caused by R. conorii.

Typhus Group
Murine/endemic typhus - R. typhi is transmitted by introduction of flea feces into skin lesions.
   Rash appears 6-7 days after onset, appearing initially on the trunk and spreads to face;
   rarely involves soles and palms. More common in crowded urban areas where poor
   sanitation and overcrowding allow rodents and people to come in contact. Rats and mice
   serve as reservoirs.

   Diagnosis - clinical picture and a history of flea bites. Rash pattern is helpful. Serology may
   be useful.

Epidemic typhus - R. prowazekii - historically responsible for much morbidity and mortality;
   transmitted by human body louse. Rash, which develops later than in RMSF and murine
   typhus, appears on the trunk first and spreads to the extremities but the palms and soles are
   not involved. Disease is characterized by severe headaches that do not respond to
   analgesics as well as very high fever. While not historically found in the US, recent reports
   have found an association between patients with serological evidence of infection and
   proximity to flying squirrels (Glaucomys volans). These squirrels are found in the
   southeastern US. R. prowazekii has been recovered from the squirrels but not from the
   patients. The mode of transmission between human and squirrel is unknown.

   Diagnosis - clinical picture and history of infestation by the human louse; serology can
   confirm the diagnosis. Sporadic cases in the US are more difficult to diagnose and depend
   on the ruling out of other possibilities in conjunction with serologic evidence.

   Brill-Zinsser Disease - recrudescence of typhus (reactivation of previous disease).
   Symptoms are milder and the disease course is shorter. Diagnosis is based on history of
   prior typhus, clinical picture and serology.

Scrub Typhus Group

Chlamydia & Rickettsia                                                                               5
Scrub Typhus - Orientia/Rickettsia tsutsugamushi is transmitted to humans via chigger bites.
   The disease is currently limited to areas of the Pacific Rim. Disease ranges from mild to
   severe and has an associated macular rash that may produce eschars.

Chlamydia & Rickettsia                                                                         6
Ehrlichia species are small, gram negative, obligate intracellular bacteria that are related to
rickettsia. The organisms reside and replicate within the phagosome of host white cells.
Ehrlichia species replicate via binary fission. Depending on the species, the intraphagosomal
dividing forms may form a cytoplasmic inclusion referred to as a morula (resembles a mulberry
in appearance). These organisms are responsible for a variety of animal infections and have
recently been described as the causative agents for at least two human infections. As with
many rickettsial diseases, ehrlichiosis is transmitted through tick bites; the majority of individuals
report tick exposure within three weeks of disease onset. Reported cases are predominantly
rural and occur during late spring and summer. Various wild animals (deer, dogs and/or small
rodents) are believed to serve as reservoirs. The two most commonly reported diseases share
some clinical features but differ with regard to causative agent, diagnostic features and
geographic distribution.

Common clinical features of human ehrlichiosis include; fever, headache, myalgia, nausea, and
vomiting. A small percentage (< 20%) of patients demonstrate confusion. Complications can
include respiratory and pulmonary insufficiency. Mortality rate for both forms of human
ehrlichiosis has been reported to be less than five percent. Clinically, human ehrlichiosis
resembles rash-negative Rocky Mountain Spotted Fever (RMSF).

Human Monocytic Ehrlichiosis (HME) Several 1000 cases have been reported in the U.S. since
  the first case was described in 1987 (283 reported through 11/20/04). HME has been
  confirmed in 30 states, the majority of those being in the southern Atlantic and southeastern
  US. In some areas, the incidence of HME can exceed RMSF. The causative agent is
  Ehrlichia chaffeensis. Morulae are generally not observed within peripheral blood
  monocytes but may be detected in aspirates of spleen or bone marrow. HME should be
  suspected in those individuals from endemic areas who have a history of tick exposure,
  fever, leukopenia and thrombocytopenia and have negative RMSF serology. It should be
  noted that up to 36% of infected individuals demonstrate a rash. Diagnosis is confirmed by
  serology, however the titers are generally not elevated until late in the course of the disease.

Human Granulocytic Ehrlichiosis (HGE) A few 1000 cases of HGE have been reported since
  the index case was identified in 1994 (299 cases reported through 11/20/04). HGE has
  been reported mainly from the upper midwestern (Wisconsin and Minnesota) and
  northeastern states (primarily southeastern New York) with a few cases reported from
  northern California, Arkansas and Florida. Unlike HME, only about 2% of individuals
  experiencing HGE demonstrate a rash. Another distinction between HME and HGE is that
  circulating PMNs may demonstrate morulae. The causative agent has not been fully
  characterized. Evidence to date indicates that the agent of HGE is closely related to two
  veterinary pathogens E. equi (horses and dogs) and E. phagocytophila (goats).
  Immunologic studies show that the agents of human, canine and equine Ehrlichiosis are
  closely related and antigenically distinct from E. chaffeensis. Recent reports indicate that an
  additional agent of HGE has been identified: E. ewingii which may be transmitted by the
  black legged tick Ixodes scapularis. This tick is also the vector for Lyme disease. Not
  surprisingly, the geographic distribution of E. ewingii infection overlaps with Lyme disease.
  Anitbodies against E. ewingii crossreact with E. chaffeenesis. Diagnosis is confirmed by
  serology, however the titers are generally not elevated until late in the course of the disease.

Q fever - Coxiella burnetii is transmitted via the inhalation of infectious aerosol and manifests as
    a lower respiratory tract infection without rash. This is an occupational disease for farm
    workers, slaughter house workers, vets, etc. since rodents, cattle, sheep and goats serve as
Chlamydia & Rickettsia                                                                               7
   reservoirs. Unlike rickettsia and clamydia, Coxiella can survive (but not replicate) for
   extended periods outside a host.

   Diagnosis - Clinical picture and patient history including potential occupational exposure.
   Specific serology can be helpful. Sixty four cases have been reported through 11/20/04.

   The taxonomic status of this genus is currently under review. Analysis of the nucleic acid of
   members of this genus indicate that these organisms are not significantly related to
   Rickettsia prowazekii, the type species for this genus. Taxonomists have proposed that
   members of the genus Rochalimaea be removed from the order Rickettsiales and be placed
   in the genus Bartonella. Some studies suggest that Bartonella species may be most
   closely related to Brucella species.

   Trench Fever - B. quintana is believed to be transmitted in the feces of the body louse. The
   disease is relatively mild with a low mortality rate. It occurs in areas of Central and South
   America as well as Africa. Transmission requires poor personal hygiene, high population
   density and louse infestation. Humans appear to be the only reservoir. Recent evidence
   suggests that a form of trench fever, Urban Trench Fever, is beginning to appear among the
   homeless in large US cities.
   Cat Scratch Disease - (CSD) is a lymphadenopathy transmitted via the scratch of a cat. The
   identification of the causative agent is somewhat controversial. Several agents have been
   proposed including several viruses, Chlamydia species and a new agent Afipia felis.
   Several authors have proposed Bartonella henselae as the agent of CSD. This proposal is
   based on the evidence that approximately 85% of patients clinically diagnosed as having
   CSD demonstrate antibodies to B. henselae. Furthermore, B. henselae was isolated from
   10/19 patients with clinical evidence of CSD. B. henselae has been isolated from a
   significant percentage of cats (< 40%).

   CLINICAL: It is estimated that approximately 24,000 cases/year of CSD resulting in over
   8,000 hospitalizations. The classic presentation of CSD is a self-limited lymphadenopathy
   generally restricted to the region around the site of the scratch; a papule often develops at
   the inoculation site. Because of the difficulty in recovering the organism from clinical
   material, biopsy of the affected nodes is usually required in order to rule out lymphoma and
   related diseases. In less than 1% of infected individuals the disease progresses to
   encephalitis with a 10% mortality rate.

   DIAGNOSIS: Conventional diagnosis has depended on clinical picture (including history of
   animal contact), demonstration of the organism in clinical material and/or ruling out of other
   causes for the lymphadenopathy. There is a skin test that employs extract of material from
   lesions of confirmed cases. The endpoint is a typical wheal and flare reaction. The
   organism can be demonstrated using the Warthin-Starry silver stain. B. henselae can also
   be recovered from clinical material by using enriched media (CDC anaerobic blood agar,
   CAP) incubated at 35              2 for up to six weeks. Best growth is observed on media
   containing human blood.

   CHARACTERISTICS OF AGENT: B. henselae grows on enriched solid media following at
                                                   -hemolytic, dry, rough and yellow to gray
   in color. When Gram-stained the organisms are small, curved, pleomorphic gram negative
   rods (Figure 38-1B). The organism requires X-factor for growth; it is catalase and oxidase

Chlamydia & Rickettsia                                                                              8
   OTHER DISEASES: B. henselae is considered to be the causative agent of bacillary
   angiomatosis (multiple angiomas - tumors composed of dilated blood and/or lymph vessels)
   and bacillary peliosis hepatitis (subcutaneous bleeding resulting in purple macules with
   associated hepatitis) in patients with AIDS or other severe immunodeficiency. Other studies
   have implicated B. henselae as a causative agent of neurologic problems in AIDS patients.
   Infection is characterized by acute confusion, progressive dementia with pronounced
   hallucinations. Cat ownership among HIV positive patients is associated with a 22-fold
   increase of neurological problems. On rare occasion, B. henselae has been recovered for
   cases of bacteremia and meningitis in both immunocompetent and immunodeficient

Diagnosis of Rickettsial Diseases
The organisms extremely dangerous to work with, therefore diagnosis is based on serologic
tests in conjunction with careful evaluation of the clinical picture and patient history.

Weil-Felix test – (Table 49-5) classic protocol depending on agglutination of Proteus vulgaris
strains OX-19, OX-2 and OX-K by antibodies produced against various rickettsiae of the typhus
and spotted fever groups.

       DISEASE                       OX-19                 OX-2                 OX-K

   Epidemic typhus                   ++++                  +                    0
   Murine typhus                     ++++                  +                    0
   Spotted Fevers                    ++++/+                +/++++               0
   Q Fever/Ehrlichiosis              0                     0                    0
Note this protocol can not readily distinguish RMSF from murine typhus; Rickettsialpox does not
induce antibodies that react with any of the P. vulgaris strains used in the protocol. The test
also yields many nonspecific positive reactions.

Currently tests for the detection of species specific antibody are unavailable commercially.
Specific serologic tests are available from the CDC and a limited number of reference labs.
Complement fixation - antibodies appear within 8-10 days with 1:8 titer considered significant.
Immunofluorescence - can detect specific antibodies or detect antigen in clinical specimens, can
also distinguish between IgM and IgG.

Agglutination Procedures - latex or hemagglutination; not routinely utilized due to lack of
availability of antigens.

Chlamydia & Rickettsia                                                                           9

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