Rickettsial Diseases

 Rickettsiae - heterogenous group

• small obligatory intracellular, gram negative coccobacilli and short bacilli
• most of which are transmitted by a tick, mite, flea or louse vector.

The family Rickettsiaceae is named after Havard Taylor Ricketts who discovered Rocky Mountain spotted fever (1906) and died of typhus fever contracted during his studies.

The family currently comprises three genera- Rickettsia, Orientia and Ehrlichia

Former members of the family, Coxiella Burnetti, which causes Q fever and Rochalimaea quintana causing Trench fever have been excluded because the former is not primarily a arthropod borne and later is not an obligate intracellular parasite being capable of growing in cell free media besides being different in genetic properties

Some Rickettsiosis, such as epidemic typhus, have been described since the 16th century.

Some of these diseases are benign, others may be potentially fatal.

Morphology

• Pleomorphic-coccobacilli,
• Aerobic, nonmotile, non-capsulated
• Gram negative (don’t stain well)

Cultivation

• Obligate intracellular parasite- Unable to grow in cell free media
• Grows best in cells which are not metabolizing actively- incubation at 32-35^oC
• Grown in yolk sac of developing chick embryos, mouse fibroblast, HeLa, Detroit 6 and other continuous cell lines
• Maintained in animal (Guinea pigs, mice) and arthropod reservoirs

Pathogenesis:

Rickettsia –transmitted to humans by  Arthropod vectors- through their bite/feces –multiply locally, enter the blood- invade vascular endothelial cells which enlarge, degenerate, and cause thrombus formation - Phospholipases cause damage to vascular endothelium -destroy endothelial cells - Inflammatory cells accumulate and blood leakage resulting in spots, rashes

Rickettsial Family

3 genera - obligate intracellular parasites:

– Rickettsia – Coxiella – Ehrlichia

Typhus Fever Group

• Epidemic typhus
• Recrudescent typhus (Brill-Zenser disease)
• Endemic Typhus

Epidemic typhus (Louse borne typhus, Classical typhus, Gaol fever)

• caused devastating epidemics in times of war/famine
• Napolean’s retreat from Moscow was forced by typhus fever
• In India, Kashmir is the endemic spot
• Caused by R. prowazekii: named after von Prowazek, who died of typhus fever while studying it
• Humans –only natural vertebrate host-guinea pigs, mice can be  infected experimentally
• Human body louse –Pediculus humanis corporis is the vector- head louse also transmits the infection
• Lice become infected when they feed on rickettsiaemic patients- Rickettsiae multiple in the gut of lice-appear in feces in 3-5 days- Lice eventually die, but remain infective till death-transmit the infection-
• Lice may be transferred from person to person-they defecate while feeding-feces enter the body of host through minor abrasions caused by scratching-infection also transmitted by aerosols of dried louse feces by inhalation or through the conjunctiva
• Incubation period-5 to 15 days-fever and chills-characteristic rash on trunk and extremities on the 4th or 5^th day, by the second week-patient is stupurous/delirious -Case fatality 40 percent-increases with age
• Typhos=cloud/smoke- cloudy state of consciousness in the disease
• Disease spread in crowded, unhygienic conditions 

 Recrudescent typhus/Brill-Zinsser disease

• For those who recover from Typhus fever, the rickettsiae may remain latent in lympoid tissues/organs-reactivated causing recrudescent typhus (Brill-Zenser disease)
• Brill described the disease and Zinsser isolated R. prowazekii in such patients and showed they are recrudescences of past infections
• Shows that, Rickettsiae survive without extra-human reservoirs- can initiate epidemics, even in louse ridden communities

Endemic Typhus

• Murine typhus or flea borne typhus -Rickettsia typhi (R. mooseri)
• common in endemic areas-maintained in nature as mild infection of rats, transmitted by the rat flea, Xenopsylla cheopsis
• Rickettsia multiply in the gut of flea and is shed in its feces-flea is unaffected by remains infectious throughout life
• Ingestion of food contaminated with infected rat urine/flea feces also can transmit the disease
• Human infection is  a dead end- man to man transmission does not occur
• Endemic typhus-worldiwde in appearance but mild, sporadic, easily controlled
• R. prowazekii & R. typhi are similar but can be differentiated by biological & immunological tests like IFA, ELISA and PCR-based DNA tests

Neill-Mooser reaction/Tunica reaction- Male guinea pigs when inoculated intraperitoneally with blood/culture develop fever and scrotal inflammation-testes cannot be pushed back into the abdomen because of inflammation of the pouch that covers the testis-tunica vaginalis-

Neill-Mooser reaction is shown by R. typhi. Absent in R. prowazekii

 

Spotted Fever Group – Rickettsia rickettsii

R. rickettsii -– Spread by tick bite; rodents are the reservoir

R. akarii – mite borne

Tick typhus

Symptoms- fever/severe headache and skin rash = wrists and ankles to trunk/palms of hands, soles of feet

Rickettsia rickettsii causes “tick typhus”, also known as Rocky Mountain spotted fever (RMSF)

R. conori – Indian tick typhus - first observed in the foothills of Himalayas. The tick Rhipicephalus sanguineus is the most important vector

Haemaphysalis leachi, Amblyomma and Hyalomma ticks can also transmit the infection

R. japonica – Oriental spotted fever

R. africae – tick bite fever (sub-Saharan Africa)

Rocky mountain spotted fever or “tick typhus”,

·         caused by Rickettsia rickettsii

·         Identified by Ricketts in 1906- first insect transmitted bacterial pathogen to be recognized

·         Transmitted transovarially by ticks- both vectors and reservoirs. Ticks are not harmed so remain infected for life. Shed in tick feces but transmission to humans is primarily by bite

·         Infection may be transmitted to vertebrate hosts by any of the larval stages or by adult ticks

·         Rocky mountain spotted fever is the most serious type of spotted fever- first to be described

·         R. rickettsii causes 95% of all modern typhus.

·         If untreated mortality is ~20%.

·         Most cases occur in children during the spring or summer.

·         CNS symptoms include headache, delirium and coma.

·         Circulatory damage includes coagulation, edema and collapse..

Rickettsial pox

The mildest rickettsial disease of humans –selflimited, nonfatal, vesicular exanthema- Resembles chicken pox- also called vesicular or varicelliform rickettsioisis

Caused by R. akari (akari=mite)

Reservoir of the infection-domestic mouse, Mus musculus and vector is the mite Liponyssoides sanguineus, transovarial transmission

First observed in New York- now reported in Korea, Eastern Europe

 

Genus Orientia

Scrub Typhus

·         Incubation period is 1-3 weeks

·         Patients typically develop a characteristic eschar (dead tissue after ulcer) at the site of the mite bite, with regional lymphadenopathy and maculopapular rush

·         Disease starts with fever/severe headache and conjunctival injection- Encephalitis and pneumonia in severe cases

·         Caused by Rickettsia tsutsugamushi / Orientia tsutsugamushi

·         First observed in Japan, transmitted by mites

·         The disease was called tsutsugamushi (tsutsuga = dangerous, mushi = insect/mite)

·         It is found in areas with a suitable climate, plenty of moisture and scrub vegetation

·         Scrub typhus by mite bite -The vectors are Leptotrombium akamushi in Japan, and L. deliensis in India

·         Human beings are infected when they trespass into the areas inhabited by the mites and are bitten by the mite larvae (chiggers)

·         The mite feeds on the serum of warm blooded animals only once during its cycle of development and adult mites feed only on plants

·         The microbes are transmitted transovarially in mites.

·         Various rodents and birds act as reservoirs and help in spreading the Orientiae to fresh areas.

 

Genus Ehrlichia

Ehrlichiae are small, Gram negative, obligately intracellular bacteria with an affinity towards blood cells- form mulberry like clusters called “morula” inside infected phagocytic cells.

Three human infections caused by

1. Ehrlichia sennetsu – ingestion of fish carrying infected flukes

2. E. chaffeensis – Human monocytic ehrlichiosis, transmitted by Amblyomma tick. Deer and rodents are the reservoirs. Human disease associated with leucopenia, thrombocytopenia and elevated liver enzymes. Multisystem involvement and fatality may occur

 3. Pathogen similar to equine pathogen, E. equi = Human granulocytic ehrlichiosis, transmitted by Ixodes ticks. Deer, cattle, sheep could be reservoirs. Leucopenia and thrombocytopenia may occur.

Rickettsial diseases

Group	Species	Disease	Vector	Vertebrate reservoir	Symptoms
Typhus	R prowazekii  R prowazekii     R typhi	Epidemic typhus Brill Zinsser disease   Endemic typhus	Louse   Louse   Rat flea	Human beings   Human beings   Rat	fever and chills-characteristic rash on trunk and extremities; stupur/delirium
Spotted fever group	R rickettsia   R conori     R akari	Rocky mountain spotted fever Indian tick typhus, Fever Boutonneuse,   Rickettsial pox	Tick   Tick     Mite	Rabbit, dog, small rodents   Rodents    Mouse	CNS symptoms -fever/severe headache Circulatory damage- skin rash   vesicular exanthema- like chicken pox
Scrub typhus	O tsutsugamushi	Scrub typhus	Trombiculid mite	Small rodents, birds	Eschar, lymphadenopathy &maculopapular rash, fever/severe headache, conjunctival infection
Ehrlichia	E. chaffeensis	Human monocytic ehrlichiosis	Amblyomma tick		leucopenia, thrombocytopenia, elevated liver enzymes- Multisystem involvement & fatality
Ehrlichia	E. equi	Human granulocytic ehrlichiosis	Ixodes ticks	Deer, cattle, sheep	leucopenia and thrombocytopenia

Diagnosis of Rickettsial Diseases

Either by isolation of rickettsiae or by serology

As rickettsiae are highly infectious and cause several serious and fatal infections among laboratory workers, Rickettsial isolation done with utmost care and appropriate safety provisions and equipments

Isolation of Rickettsia

• Done in male guinea pigs or mice - from patients in the early phase of the disease
• Blood clot ground in skimmed milk or any suitable suspending medium is inoculated intraperitoneally. The animals should be observed for 3-4 weeks and their temperature recorded daily.

1. In rocky mountain spotted fever, guinea pigs develop fever, scrotal necrosis and may even die
2. With R typhi, R conori and R akari, they develop fever and tunica reaction. R prowazekii produces only fever without testicular inflammation

• Smears from the peritoneum, tunica, spleen of infected animals may be stained by Giemsa/Gimenez methods.

Cell culture techniques

Rickettsiae grow well in the yolk sac of chick embryos, but this method and tissue culture is not suitable for primary isolation from clinical specimens.

Cellculture is faster and more sensitive for isolation- Continuous cell culture on Verocell MRC (cell line from monkey cells) can be used to isolate rickettsiae from clinical samples in 3 – 5 days. Growth is identified by immunofluorescence using group- and strain- specific monoclonal antibodies.

These methods require high end facilities and expertise which may not be feasible for all diagnostic laboratories.

Serology

Serological methods like Weil Felix test, Immunoflourescence and ELISA are used to detect the presence of IgM or IgG antibodies against rickettsial infections.

Weil Felix test is a heterophile antibody test using the O antigens of Proteus strains OX 19, OX K and OX 2,  for diagnosing the antibodies against rickettsial infections. Proteus and some rickettsiae share an alkali stable carbohydrate antigen, which forms the basis of this test. Weil and Felix (1916) observed this first. Tube or slide agglutination test may be performed.

Antibodies are usually positive by 7 – 10 days after infection. This test is not very specific as it can be positive in case of other related infections. But in countries like India, this is the test which is easily available and affordable. This test can be used as a screening test and correlated with the clinical symptoms. It also helps the physician in diagnosis and prognosis of the disease.

Interpretation of Weil Felix test for diagnosing the antibodies against rickettsial infections.

Rickettsial infection	OX 19	OX 2	OX K
Epidemic typhus/Endemic typhus	++++	+	0
Brill Zinsser disease	++++	+	0
Scrub typhus	0	0	+++
Rocky Mountain spotted fever -RMSF	++++	+	0
Rickettsial pox (R. akari)	0	0	0
Indian tick typhus (R.conori)	+	++++	0

 ELISA test to confirm the IgM antibodies against Scrub typhus can be used once the OX K antigen is positive by Weil Felix test

False positive reaction obtained in case of urinary/Proteus infections, typhoid fever/liver diseases. Testing of paired sera to demonstrate the rising titre is confirmatory in case of any serological tests as antibodies may persist in the body after an initial exposure to the organism. Testing of paired sera to demonstrate the rising titre is confirmatory in such cases

Complement fixation test may be performed using group specific or type specific Rickettsial antigens.

Other serological tests include agglutination of rickettsial suspensions, passive haemagglutination of RBC’s, toxin neutralization, immunofluorescence, and radioisotope precipitation. They are preferable to the nonspecific and insensitive Weil-Felix test based on the cross-reactive antigens of Proteus vulgaris strains(OX19). It usually takes 10-12 days for serologic data to become positive.

Immunoflourescence is the reference serological test which can be considered as the gold standard. But this test is not available in India.

Detection of rickettsiae in tissue samples is attempted by immunohistochemistry. Intracellular rickettsiae can be visualized even after 48hours of initiation of appropriate treatment.

Polymerase chain reaction: Detection of rickettsial DNA by molecular methods is more rapid and specific but this test is not widely available. Polymerase chain reaction (PCR) to detect rickettsiae in blood or tissue provides promise for early diagnosis.

No rapid laboratory tests are available to diagnose rickettsial diseases early in the course of illness. Rise in serum antibody/often do not develop in early stages. Serology remains the backbone of diagnosis because these other tests are expensive and less available to clinicians

Immunoprophylaxis

Prevention – Control of vectors (Tick Removal) and animal reservoirs (Limiting exposure to ticks)

Immunisation – Killed and live vaccines against epidemic typhus

Inactivated yolk sac vaccines developed by Cox and live vaccine using attenuated strain E

But some vaccines develop a mild disease, so no satisfactory vaccine yet.

Treatment - Doxycycline is a drug of choice for treating suspected Rocky Mountain Spotted

Despite effective treatment and advances in medical care, approximately mortality is still 3- 5%