Orientia tsutsugamushi is a gram-negative bacterium that causes scrub typhus, a disease transmitted through the bites of infected chigger mites. The disease is endemic in parts of Asia and the Pacific, including Nepal. It causes non-specific fever and symptoms like eschar, rash, and organ dysfunction. Diagnosis involves serology, culture or PCR on samples. Doxycycline is the treatment of choice. Prevention focuses on avoiding chigger bites through proper clothing and repellents in endemic areas. Scrub typhus outbreaks have increased in Nepal in recent years.

1. Manisha Shrestha
M.Sc. Third Semester( Medical Microbiology)
Central Department of Microbiology, TU, Kirtipur

2. INTRODUCTION
 A rickettsial disease, caused by Orientia tsutsugamushi
 Also known as tsutsugamushi disease/bush typhus
 A zoonosis primarily affecting rodents, rabbits, and marsupials
 Earliest record of O. tsutsugamushi infection was in the 3rd
century (313 AD) in China
 First described as tsutsugamushi(1899) and isolated(1930) in
Japan
 Scrub: Refers to the type of vegetation harboring the vector
Typhus: Greek word meaning ‘fever with stupor’ or smoke

3. EPIDEMIOLOGY
 Most common reemerging rickettsial infection in South-East
Asian countries
 Endemic to a part of the world known as tsutsugamushi triangle;
which includes Bhutan, India, Indonesia, Maldives, Myanmar,
Nepal, Srilanka, and Thailand
 An occupational disease commonly found in particular
geographical locations conductive to the survival and
multiplication of mites as well as reservoir host
 Prevalent in the areas such as sandy beaches, mountain deserts,
and equatorial rain forest
 Caused the deaths of over 36,000 soldiers in the far east during
world war II
 An estimated one billion of people are at the risk for scrub
typhus and an estimated one million cases occur annually

5. Scrub Typhus in Nepal:
 First reported in 1981, as a cause of FUO
 Very few attempts were made before 2014 to determine the prevalence of
scrub typhus in Nepal.
 In 2015, following the devastating earthquake and aftershocks, the number of
recurrent outbreaks of scrub typhus increased.
Since then several episodes of outbreaks of scrub typhus have been reported.
 Epidemiology and Disease Control Division (EDCD) developed an interim
guideline on Prevention and Control of Scrub Typhus in Nepal in 2015.
 Next outbreak was recorded in 2016, affecting 47 districts
 In 2019, highest cases of scrub typhus was recorded affecting as many as 63
districts
 Recently, in this year, scrub typhus and dengue case were reported in Myagdi
on February. A total of 325 cases have been reportted in 2020 until August 10
 The agro-climatic conditions, socio-economic situation, poor sanitation, low
hygiene and health standards are major contributing factors to the emergence
of scrub typhus in Nepal.
 In the current days of Nepal huge burden of scrub typhus have been
found, mainly Southern Nepal.

6. CAUSATIVE AGENT AND RESERVOIR
 Caused by Orientia tsutsugamushi, an obligatory intracellular
gram negative proteobacterium
 O. tsutsugamushi has five major serotypes
 Chigger mites act as the primary reservoir as well as the vector for
O. tsutsugamushi
 The mite is very small (0.2–0.4mm) and can only be seen
through a microscope or magnifying glass.
 A number of small rodents, particularly wild rats, are natural hosts
Fig. Chigger Mite

7. TRANSMISSION
 It is a zoonosis (an animal disease that can spread to humans)
primarily affecting rodents, rabbits, and marsupials
 This rickettsial infection is transmitted to humans through the bite
of infected Leptotrombidium mite larvae (chiggers)
 The mites are found in grasslands, forests, bush areas, wood piles,
gardens, and beaches
 Can also be transmitted through unscreened blood transfusions and
unhygienic needles
 It does not spread from person to person
 It does not transmit through the bite of infected rodent

8. LIFE CYCLE
oHuman gets infection by
the bite of infected chigger
(only form in lifecycle of
mite that bites warm
blooded animals)
o Chiggers feed on lymph
and tissue fluid
o Large number of O.
tsutsugamushi are present
in salivary glands of larvae
and are injected to host
while feeding
o Transfer of the causative
agent in between different
stages of the vector as well
as transmission of agent
from adult to offspring
maintains the infectivity for
prolonged time period.

9. PATHOGENESIS
 Endothelial cells of most organs including skin, heart, lung, brain,
kidney, pancreas have been presented as the target cells
of O. tsutsugamushi.
 The pathophysiological hallmark of scrub typhus is disseminated
vasculitis with subsequent vascular injury.
 The organism multiplies at the site of inoculation that progresses
on to necrosis and evolves into an eschar with regional
lymphadenopathy.
 Within a few days, patients develop rickettsemia with infection of
vascular endothelium resulting in vascular injury in several organs.
 The injury causes disseminated intravascular coagulation (DIC)
with platelet consumption, vascular leak, pulmonary edema, shock,
hepatic dysfunction and meningoencephalitis.
 Mortality rate ranges from 6.1% to 25% in Southeast Asia.

10. CLINICAL ASPECTS
Incubation period: 5–20 days (average 10–12 days)
Scrub typhus presents as an acute undifferentiated fever; and the signs and
symptoms may include:
 A dark, scab-like region at the site of the chigger bite (also known as
eschar),which is painless and may be noticed as a transient localized itch,
often found on the groin, armpits, genitalia or neck
 High grade fever (>104ºF) and chills
 Headaache
 Body aches and muscle pain
 Rashses
 Enlarged lymphnodes
 Complications: Pneumonia, Myocarditis, Meningitis, Encephalitis,
Hepatitis, Pancreatitis, Acute respiratory distress syndrome(ARDS),
Acute kidney injury, Subacute painful thyroiditis
People with severe illness may develop organ failure and bleeding, which
can be fatal if left untreated
Fig. An Eschar at the site
of Chigger Bite

11. Case Definition:
Suspected/clinical case:
 Acute undifferentiated febrile illness (UFI) of 5 days or more
with or without eschar should be suspected as a case of
Rickettsial infection. (If eschar is present, fever of less than 5
days duration should be considered as scrub typhus.)
Probable case:
 A suspected clinical case with an IgM titer > 1:32 and/or a four-
fold increase of titers between two sera confirm a recent
infection.
Confirmed case:
 The one in which Rickettsial DNA is detected in eschar samples
or whole blood by PCR or Rising antibody titers on acute and
convalescent sera detected by Indirect Immune Fluorescence
Assay (IFA) or Indirect Immunoperoxidase Assay (IPA)

12. BACTERIOLOGY
Scientific Classification
Domain: Bacteria
Phylum: Proteobacteria
Class: Alphaproteobacteria
Order: Rickettsiales
Family: Rickettsiaceae
Genus: Orientia
Species: O. tsutsugamushi
Initially, categorized under genus Rickettsia, but now classed in a
separate genus - Orientia.
Based on the antigenic variation of the major outer membrane
protein (56-kD), a type-specific antigen (TSA56), several strains
have been described.
The six classic strains: Karp (accounts for 50% of all infections),
Gilliam (25%), Kato (<10%), Shimokoshi, Kuroki and Kawasaki.

13.  Obligate intracellular pathogen which needs to infect
eukaryotic cells in order to multiply
 Gram negative short rod or coccobacillus,
 Measures about 0.5 to 0.8 µm wide and 1.2 to 3.0 µm long.
 Aerobic, non-motile, non-capsulated
 Requires growth factors provided by host cell (acetyl-CoA,
NAD, ATP )
 Do not grow on artificial media
 With a genome of only 2.0–2.7 Mb, it has the most repeated
DNA sequences among bacterial genomes sequenced so far
 O. tsutsugamushi does not possess lipopolysaccharides,
peptidoglycan, a slime layer, or other T-independent antigens
 Cell wall consists of proteins linked by disulfide bonds

14. LABORATORY DIAGNOSIS
Diagnosis of rickettsial diseases relies on clinical recognition and is
aided by such epidemiological consideration as residence or recent
travels in endemic areas, recent exposure to relevant arthropods
Scrub typhus can mimic other acute febrile illnesses common in the
tropics, especially when pathognomonic eschars are absent.
Diagnostic methods available for laboratory confirmation include:
 Identification of the organism in cell culture
 Detection of the antigen by immune-histochemical methods or the
antibodies by the indirect immunofluorescence assay (IFA)
 Finding specific nucleic acid targets using molecular methods

15. Specimens:
Heparinized blood: Conserve at -80°C; ship in dry ice for culture
EDTA blood: Conserve at +4°C; ship at room temperature for PCR
Serum: Conserve at +4°C, room temperature; collect two serum
specimens 10 days apart
Skin or lymph node biopsy
Direct Microscopy:
 Smears of infected tissues maybe made and examined directly for the
rickettsiae after Giemsa staining
 Gram’s staining is not preferred but if
done may appear as Gram negative
Fig. O. tsutsugamushi
(Giemsa staining)

16. Isolation of O. tsutsugamushi :
 Can be performed using cell culture
 Cell lines like HeLa cells, L929 cells, Vero cells, BHK-21 are used
 Not routinely done as it requires a cell culture facility, trained personnel,
strict bio-safety precautions and a BSL-III facility
 As the organism doubling time is 9-18 h, it takes an average of four
weeks for identification by culture
Serology:
 Suspected infections are confirmed with serological tests
 Serological tests like Weil–Felix test, RICT, IFA, ELISA, RDTs are used
 IFA uses cell culture-grown O. tsutsugamushi as antigens, and can be
used to differentiate IgM and IgG antibodies
 Currently most diagnostic laboratories use ELISA for the detection of
IgM antibodies in scrub typhus as it provides an objective result and has
sensitivity similar to that of IFA
 4-fold increase in antibody titer between two consecutive samples is
diagnostic

17. Molecular Methods:
 PCR assays, either conventional or real-time, targeting the 56
kDa gene, 47 kDa gene, 16 S rRNA and groEL gene have also
been explored and reported to have specificity approaching 100%
 Loop isothermal amplification (LAMP) targets groEl and 47 kDa
gene
Supportive laboratory investigations:
 Total leucocytes count during early stages may be normal but
may be elevated to more than 10,000/cu mm later in the course of
disease.
 Thrombocytopenia (low platelet count), usually <1,50,000/cu mm
 Elivated liver transaminses

18. TREATMENT
 Doxycycline is the preferred drug in the treatment of scrub
typhus.
 Doxycycline is used orally (100 mg twice daily for 7 days) or
chloramphenicol in more severe cases (500 mg every 6 hours for
7 days or 50-75/mg/kg/day in children).
 Azithromycin has been used in resistant cases and may be better
than doxycycline – especially in children and pregnant women.
Supportive treatment:
 Patients with mild disease presenting with fever without organ
dysfunction may require only antipyretics along with antibiotics.
 However, patients with organ dysfunction would need organ
support depending on the nature and extent of organ dysfunction

19. PREVENTION AND CONTROL
Individuals:
 In endemic areas, wear full-length clothing, socks and shoes.
 Apply, as necessary, insect repellents to the skin and clothing to
prevent chigger bites.
 Do not sit or lie on bare ground or grass; use suitable groundsheet
or other ground cover.
 Clear vegetation and do chemical treatment of the soil.
Community:
 Rodent control and improved living conditions
 Rapid case identification by health-care workers
 Public education on case recognition and personal protection help
in the identification and prompt treatment of cases
No vaccine is available to prevent scrub typhus.

20. BIBLIOGRAPGY
 Tille PM. (2014). Bailey and Scotts Diagnostic Microbiology.
Thirteenth Edition. Elsvier, St. Louis, Missouri 63043.
 Chakraborty S and Sarma N. Scrub Typhus: An Emerging
Threat. Indian J Deer, 2017 Sep-Oct; 62(5): 478–485
 Shirai A. Laboratory diagnosis of scrub typhus. Malaysian J
Path., 1:11-14 1978
 Acharya KP, Adhikari N, Tariq M. Scrub typhus: A serious
public health issue in Nepal. Clinical Epidemiology amd
Global Health 8 (2020) 815-817
 https://apps.who.int/iris/handle/10665/204722
 https://www.cdc.gov/typhus/scrub/index.htmlmatol
 http://nhrc.gov.np/projects/descriptive-epidemiology-of-scrub-
typhus-in-nepal-2/
 http://www.edcd.gov.np/resources/download/scrub-typhus-
guideline-on-prevention-and-control

21. THANK YOU