Rickettsia Chlamydia And Mycoplasma Notes

Rickettsia Chlamydia And Mycoplasma

General Properties

Members of Rickettsiae possess the following common characteristics:

• They are obligate intracellular organisms
• Cannot grow on artificial media, but can grow on cell line/egg/mice inoculation (except Bartonella)
• Transmitted by arthropod vector (except Coxiella, transmitted by inhalational mode)

The order Rickettsiales comprises of genera, such as Rickettsia, Orientia and Ehrlichia

Read And Learn More: Micro Biology And Immunology Notes

Former members, such as Coxiella and Bartonella are now excluded from the family, because

Coxiella is not arthropod-borne; infection is transmitted by inhalational mode; and Bartonella is not an obligate intracellular parasite; capable of growing in cell-free media.

Pathogenesis

• Spread: Rickettsiae spread through the lymphatics from the portal of entry, multiply in the regional lymph nodes and then spread via bloodstream
• Target sites: For all rickettsiae, the final target site is the endothelial cells (in addition, R. akari and O. tsutsugamushi, attack the monocytes)
• Phagocytosis: Adhesion to the endothelial cells is mediated by OmpA and OmpB and are phagocytosed and remain inside a vacuole.
• Rickettsia and Orientia produce phospholipase A that lyses the vacuoles are found free in the cytoplasm (spotted fever rickettsiae are also found free in the nucleus)
• Coxiella and Ehrlichia continue to multiply in cytoplasmic vacuoles
• Coxiella vacuole fuses with lysosome, but it is able to survive inside the acidic environment of the phagolysosome
• Ehrlichiae are maintained inside the vacuoles.
• Endothelial cell injury: Occurs via lipid peroxidation of host-cell membranes.

Geographical Distribution

• Epidemic typhus: It is endemic in Africa (notably Burundi, Rwanda and Ethiopia) and South
  America (Peru, Bolivia and Ecuador).
• Endemic typhus: It is endemic worldwide. Recent days, it is increasingly reported from South
  East Asia and Western Pacific and from India, it has been reported from Shimla, Kashmir, Mumbai, Jabalpur, Lucknow, and Pune.
• Rocky mountain spotted fever (RMSF)-endemic in high tick population areas of USA,
  Central and South America; common during tick season (summer in tropics) and among children and males.

Clinical Manifestation

• Most severe form with systemic involvement: Rocky mountain spotted fever
• Mildest form: R. pox
• Eschars are seen: Rickettsial Pox, African Tick bite fever, Indian tick typhus (ITT) and Scrub typhus
• Distribution of Rash:
  • Epidemic typhus (all over, except palm and sole)
  • Endemic typhus (trunk followed by extremities)
  • RMS, Indian tick typhus (ITT) (palm and sole)
  • Vesicular/vericelliform rash: Rickettsial pox, African Tick bite fever
  • No rash: fever
• Q fever:
• Acute fever: Fever, pulmonary feature, hepatitis
  • Chronic fever: Endocarditis
• CNS involvement like mental confusion and coma: Epidemic and endemic typhus
• Recrudescent illness (Brill-Zinsser disease)-R. prowazekii
• Tick and mite are transmitted by (i) bite, (ii) transovarial transmission
• Louse and flea borne rickettsiae are transmitted by:
• Autoinoculation following rubbing the insect
• Aerosol (by inhaling dried louse or flea feces in the laboratory or as part of bioterrorism).

Scrub Typhus

• Agent: Orientia tsutsugamushi. It differs from Rickettsia by both genetically as well as lacking LPS in cell wall.
• Vector: Trombiculid mites of genus Leptotrombidium (L.akamushi in Japan and L.deliensis in India). The larval (called chiggers) stage of mite are the only stage that feed on humans.
  Hence, scrub typhus is also called chiggerosis.
• Clinical manifestations: Classic presentation consists of triad of an eschar (at the site of
  bite), regional lymphadenopathy and maculopapular rash. However, the classical triad is
  seen only in 40–50% of cases.
• Antigenic diversity: Three major antigenic types have been identified: Karp, Gilliam and
  Kato. Because of this remarkable antigenic diversity exhibited by the organism, immunity wanes over 1–3 years.
• Zoonotic tetrad: Four elements are essential to maintain O. tsutsugamushi in nature:
• Trombiculid mites
• Small mammals (e.g. field mice, rats, shrews)
• Secondary scrub vegetations or forests (hence named as scrub typhus)
• Wet season (when mites lay eggs).
• World scenario: Scrub typhus is endemic to a part of the world known as the “tsutsugamushi triangle”—Various countries included are Japan, China, Philippines, and South-East Asia, including India, Pakistan, Afghanistan, tropical Australia, New Guinea, and Pacific Islands
• Indian scenario: It is the most common rickettsial disease in India; outbreaks reported from sub-Himalayan belt, from Jammu to Nagaland, Himanchal Pradesh, Sikkim and Darjeeling (West Bengal), Puducherry, Karnataka, Tamil Nadu, Kerala and occasional reports from Bihar, Rajasthan, Maharashtra.

Laboratory diagnosis

1. Serology (antibody detection): IgM (by the end of the 1st week), and IgG (by end of the 2nd week)
   • Weil-Felix test: Nonspecific, detects high titers of heterophile antibodies to Proteus OXK antigens
   • Indirect immunofluorescence antibody (IFA): It is specific, considered as the gold standard
   • ELISA using 56-kDa recombinant major surface protein antigens derived from Gilliam, Karp, and Kato strains.
2. Culture: O. tsutsugamushi can be isolated using- egg (yolk sac), cell culture (Vero cells, MRC 5 cells, BHK21, L929 mouse fibroblast cells).
3. Molecular test: PCR, nested PCR, LAMP (loop mediated isothermal amplification) targeting 56-kDa gene, 47-kDa gene, 16S rRNA gene and 60-kDa heat shock protein (groEL) gene.

Ehrlichiosis

Family Anaplasmataceae comprises of four obligatory intracellular organisms named Ehrlichia, Wolbachia, Anaplasma, and Neorickettsia

Common to all three Species:

• Obligate intracellular parasite, Cannot be cultivated in artificial media
• Grow in cluster inside the phagosome as mulberry-like inclusions called as MORULA
• DOC: Doxycycline.

Laboratory Diagnosis of Rickettsiosis

Weil Felix Test

It is heterophile agglutination test; where rickettsial antibodies are detected by using certain Proteus strains (OX 19, OX 2, and OX K strains) due to sharing cross-reactive alkali stable LPS antigen.

• Procedure: It is a tube agglutination test; serial dilutions of patient’s serum are treated with nonmotile strains ofP. vulgaris OX 19, and OX 2 and P. mirabilis OX K.
• Results:
  • In epidemic and endemic typhus- Sera agglutinate mainly with OX 19 and sometimes with OX 2.
  • In tickborne spotted fever-Antibodies to both OX 19 and OX 2 are elevated.
  • In scrub typhus- Antibodies to OX K are raised.
  • The test is negative in rickettsial pox, fever, ehrlichiosis and bartonellosis.
• False positive titer may be seen in presence of underlying Proteus infection.
  Hence, fourfold rise of antibody titer in paired sera is more meaningful than a single high titer.
• False negative result may occur due to excess antibodies in patient’s sera (prozone phenomena). This can be obviated by testing with serial dilutions of patient’s sera.
• Weil Felix test being a nonspecific test should always be confirmed by specific tests

Specific Antibody Detection Tests

• Indirect immunofluorescence assay: It is the most common serologic test used for confirmation of diagnosis
  • Titer of ≥1:64 is considered as significant
  • The sensitivity and specificity are 94–100% and 100% respectively.
• CFT (specific, but less sensitive)
• IgM capture ELISA: It is useful in early diagnosis (< 1 week) with excellent sensitivity and specificity
• Latex agglutination test.

Other Methods of Diagnosis Include

• Histological examination of a cutaneous biopsy sample from a rash lesion can be done even during acute illness.
• Isolation: Rickettsiae cannot be cultivated in cell free media.
  Isolation can be done by cell lines (Vero, primary chick embryo, WI-38, HeLa), egg (yolk sac inoculation), or animal inoculation (guinea pig).
• Neil Mooser reaction: Specimens are inoculated intraperitoneally into male guinea pigs.
  The changes observed in the animal (over 3–4 weeks), varies among various rickettsial species.
  • R.rickettsii: Produces scrotal necrosis
  • R.prowazekii: Produces only fever without any testicular inflammation
  • R.conori, R. akari and R. typhi, (code-CAT): Produce fever and positive tunica reaction (testicular inflammation).
• Molecular tests: PCR and real time PCR
• They are rapid and specific; can detect specific rickettsial DNA [56 kDa or 47 kDa], 16S rRNA or Omp genes) and 60-kDa heat shock protein (groEL) gene.
  • Useful specimens are: Whole blood, buffy coat fraction, skin rash biopsies, lymph node biopsies or tissue specimen.

Treatment of Rickettsiosis

Doxycycline is the drug of choice for treatment of most rickettsial illnesses. Chloramphenicol is used as alternative.

Bartonellosis

Bartonella species are fastidious, intracellular, gram-negative bacteria that have ability to invade RBCs. They differ from other rickettsiae being capable of growing on blood agar.

Chlamydia

General Properties

• Obligate intracellular gram-negative bacteria.
• Cannot grow on artificial media, but can grow on cell line/egg/mice inoculation
• They are filterable and produce inclusion bodies like viruses.
• But they differ from viruses being possessing both RNA and DNA.
• Possess modified peptidoglycans.
• Cannot produce their own ATP: They are called Energy parasite as they depend on host cell ATP.
• Shows tropism for squamous epithelium and LN.
• Life cycle: They exist in two distinct morphological forms: Elementary body (EB) and Reticulate body.

C.Trachomatis Serovars D–K

C.Trachomatis Is The Mc Cause Of:

• STD, nongonococcal urethritis (NGU-30-59%), and post-gonococcal urethritis (PGU)
• Pelvic Inflammatory Disease (PID) and acute epididymitis
• Inclusion conjunctivitis: Swimming pool conjunctivitis (adult) and ophthalmic neonatorum or inclusion blennorrhea (neonate).
• Infant pneumonia: It is an interstitial pneumonia that develops within 3 weeks to 3 months of birth; Infection spreads from conjunctiva to pharynx via the nasolacrimal duct.

Complications:

Reiter syndrome

• • Characterized by: CUP (conjunctivitis + urethritis + polyarthritis) and mucocutaneous lesions
  • Associated with people with HLAB27.
  • MC cause of peripheral inflammatory arthritis in young men, MC site: Large joints of the legs
• Fitz Hugh Curtis syndrome: Perihepatitis in, sexually active women
• Urethral Syndrome in Women: Dysuria and frequency, urethritis, pyuria, and no bacteriuria.

C. trachomatis Serovars A, B, Ba and C (Trachoma)

• Mode of transmission: Transmitted through direct contact (fingers and fomites) with discharges from the eyes of the infected patients or indirect contact through contaminated clothes or flies
• Age: Infection is acquired by 2–3 years of age and active disease is most common among young children
• Chronic conjunctivitis: (follicular hypertrophy + papillary hyperplasia + pannus +cicatrization)
• Stages: Trachoma dubium, protrachoma, established trachoma I –IV
• Inclusion body (HP- Halberstaedter – Prowazek) seen only in, established trachoma stages I–IV.
• Worldwide, the hyperendemic areas of trachoma include sub-Saharan Africa, Middle East, and Southeast Asia including India
• Trachoma still continues to be a leading cause of preventable infectious blindness worldwide.

C.trachomatis Serovar (L1, L2, L2a, and L3)

LGV is an invasive STD, caused by C.trachomatis Serovar L1, L2, and L3, characterized by:

• MC Serotype by L2 > L1, L3
• LGV serovars are more invasive than others
• Incidence is falling, Male: female: 3.4:1; it is still endemic in Southeast Asia (including India), South America and Caribbean.
• Clinical features:
  • Painless ulcer + painful lymph nodes ↑ (enlarged inguinal LN called bubo),
  • Esthiomone (elephantiasis of vulva) rectal stricture, proctitis
• Skin test positive → Frie test.

Laboratory Diagnosis of Chlamydial Infection

• Microscopy:
• Gram staining: Often reveals sterile pyuria (↑ neutrophils, no organisms) as they are poorly gram-negative.
• Other stains, such as Castaneda, Machiavello or Gimenez stains are better methods to detect chlamydiae from samples. The inclusion bodies can also be detected in cytoplasm; which are known by different names:
• LCL body (Levinthal-Cole-Lillie) body: Psittacosis
• Miyagawa corpuscle: LGV
• HP (Halberstaedter – Prowazek) body: trachoma Lugol’s I2- used only for C. trachomatis (stains the glycogen inclusion body)
• Direct immunofluorescence test (DIF) for direct detection of inclusion bodies in clinical material.
• Antigen detection: Enzyme Immunoassays detects chlamydial group specific antigens (LPS).
• Culture:
  • Mice inoculation (infective by only C.psittaci and LGV biovars)
  • Yolk sac inoculation
• Cell culture inoculation
  • McCoy Cells and HeLa
  • HEp2 for C. pneumoniae
• Serology:
  • Micro-IF test: It is the serological test of choice, detects serovar specific antibody by
    using outer membrane protein (OMP) antigen
  • CFT (genus-specific) detects Genus specific antibody by using LPS antigen
  • High antibody titer seen in LGV, infant pneumonia, salpingitis.
• Nucleic acid amplification tests (NAATs), i.e. PCR: Diagnosis of choice, most sensitive and specific assay, almost replacing, the so-called gold standard culture.

Treatment of Chlamydial Infections

• Chlamydia Trachomatis
  • For uncomplicated genital infection or trachoma or adult conjunctivitis: azithromycin, single dose of 1 gram tablet, per oral or Alternatively doxycycline, tetracycline, erythromycin or ofloxacin for 7 days; and both the sex partners should be treated
  • For complicated genital infection: Doxycycline (100 mg twice daily), or erythromycin (500 mg four times daily)
  • For neonatal infections (ophthalmia neonatorum and infant pneumonia): Erythromycin for 2 weeks and Topical erythromycin ointment
• Chlamydophila psittaci: Tetracycline for 3 weeks and Erythromycin is given as alternate.
• Chlamydophila pneumoniae: Tetracycline or erythromycin for 10–14 days.

Mycoplasma

Mycoplasma are the smallest free living organism known.

General Properties

• Filterable (Hence known as Eaton’s agent)
• Formerly called PPLO- Pleuro pneumonia-like organism.
• Lack rigid cell wall. Peptidoglycan layer is absent; replaced by cholesterol.
• Hence they are resistance to cell wall active antibiotics like beta-lactams.

Clinical Features

• Incubation period is 2–4 weeks, person to person spread by respiratory droplets.
• MC manifestation-upper respiratory illness
• MC cause of community acquired atypical pneumonia in adults.
• Pneumonia is called Primary atypical pneumonia (PAP) or ‘walking’ pneumonia or Eaton agent pneumonia (Atypical pneumonia can be caused by M. pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila and Agents of viral pneumonia).
• Extrapulmonary Manifestations-neurologic, dermatologic, cardiac, rheumatologic, and hematologic.

Note: Ureaplasma urealyticum cause NGU, epididymitis, vaginitis, and cervicitis.

Laboratory Diagnosis

• Poorly gram-negative, shows pleomorphism, resemble like L forms
• Staining with Dienes stain: Block of agar containing Mycoplasma colony added to methylene blue is observed under microscope.
• Show gliding mobility (however, they lack flagella, pili)
• Culture medium: PPLO broth and PPLO agar. Culture of respiratory specimens has a sensitivity of ≤60% and specificity of 100%.
• Produces Fried egg colonies.
• Antigenic detection: By Direct immunofluorescence, antigen capture ELISA
• Molecular methods: Detects 16S rRNA and P1adhesin gene.
• Detection of Antibody:
  • Heterophile antibody:
    • Cold agglutination test: Detects Mycoplasma antibody by using human ‘O’ RBC antigen.
    • Streptococcus MG test: Detects Mycoplasma antibody by using Streptococcus MG antigen.
  • Specific antibody:
    • CFT (complement fixation test)
    • ELISA for IgM, IgG and IgA detection
    • The combination of PCR for respiratory tract secretions and serologic testing constitutes the most sensitive and rapid approach to the diagnosis of M.pneumoniae infection.

Treatment

• Mycoplasma pneumoniae and Ureoplasma urealyticum: DOC—Azithromycin and doxycycline alternate drug
• M. hominis: DOC—Doxycycline.