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Indian Journal of Basic and Applied Medical Research; September 2015: Vol.-4, Issue- 4, P. 394-401
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Review article
Scrub typhus: An emerging scourge
M. Chauhan¹ , S. Mahajan², Manish S3, R K. Abrol4
Assistant Professor1, 2, Senior Resident3, Associate professor4
*Corresponding Author: M Chouhan
Dept. of Microbiology, Dr Rajendra Prasad Govt. Medical College ,Kangra , at Tanda (H.P) , India
*Corresponding Author: M Chouhan
Abstract:
Scrub typhus is a public health problem causing severe morbidity and mortality. Clinical picture consist of high grade fever,
severe headache, apathy, myalgia and generalised lymphadenopathy. A maculopapular rash may appear first on the trunk a nd
then on extremities. Black eschar may be seen at the site of inoculation.Patients may develop complication like interstitial
pneumonia, meningoencephalitis and myocarditis. Diagnostic approaches for scub typhus are based on several aspects. We can
diagnose them clinically based on sign & symptoms or can be diagnosed serologically. Molecular methods can be used for their
rapid identification as well as for epidemiological purposes. Most cases of fever were treated with drugs like chloromphenicol
and tetracycline which effectively treat scrub typhus also. No vaccine is available for Scrub typhus but many vaccines using
Sta47 and Sta56 antigens are under trial as a recombinant vaccine.
Key words: scrub typhus, Eschar, Rickettsial diseases
Introduction:
Scrub typhus is an acute, febrile, infectious illness
that is caused by Orientia (formerly Rickettsia)
tsutsugamushi. It is also known as tsutsugamushi
disease. Mite borne t yphus fever, tropical typhus and
various other local names. Scrub typhus was first
described from Japan in 1899. Humans are accidental
hosts in this zoonotic disease. The term scrub is used
because of the type of vegetation (terrain between
woods and clearings) that harbours the vector. The
infection is found in wide range of ecological
conditions primary jungles, semi-desert, mountain
desert, alpine meadows of Himalyas (1). scrub
typhus is known to be prevalent in foot hills of
Himalayas viz. Jammu & Kashmir, Sikkim, Manipur,
Nagaland, Meghalaya, Himachal Pradesh etc. The
disease has also been reported from Tamilnadu and
Kerala. However, currently samples are being tested
positive from Delhi, Haryana, Rajasthan,
Maharashtra, Uttrakhand and Chhattisgarh. During
2012, outbreaks of scrub typhus were reported from
many states in India. Over the years, the numbers of
samples and areas which detected scrub typhus have
also increased. The National Centre for Disease
Control at its Zoonosis Division received 742
samples from suspected cases for scrub typhus from
11 states and 202 ( 27%) were found positive. In
2011, the number of samples received was 484 and in
2010, these were 204(2).
Clinical symptoms of scrub typhus: The chigger
bite is painless and may become noticed as a
transient localized itch. Bites are often found on the
groin, axillae, genetalia or neck (3). An eschar is
often seen in humans at the site of the chigger bite.
The illness begins rather suddenly with shaking
chills, fever, severe headache, infection of the
mucous membrane lining the eyes (the conjunctiva),
and swelling of the lymph nodes. A spotted rash on
the trunk may be present. Eschars are rare in patients
in countries of South-East Asia and indigenous
persons of typhus-endemic areas commonly have less
severe illness, often without rash or eschar (4).
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Symptoms may include muscle and gastrointestinal
pains. More virulent strains of O. tsutsugamushi can
cause haemorrhaging and intravascular coagulation.
Complications may include atypical pneumonia,
overwhelming pneumonia with adult respiratory
distress syndrome (ARDS)–like presentation,
myocarditis, and disseminated intravascular
coagulation (DIC). Patients with scrub typhus often
exhibit leucopenia.
Chigger mite Eschar
Etiology:
O tutsugambushi belongs to the typhus group. In
Greek the word typhos means ‘’ stupor caused by a
fever’’ there is a high degree of antigenic
heterogeneity among the different strains several
serotypes coexist in an endemic area, one of them
may predominate .The different strain vary in their
virulence.Its distribution is uneven, since it depends
upon the presence of the agent and the
vector/resevoir complex, later consisting of trobiculid
mites and the small mammals, especially rodents,on
which they feed,when all these elements come
together they form “typhus islands(1) It is an obligate
intracellular gram-negative bacterium that has a large
number of serotypes. Five major serotypes: Boryon,
Gilliam, Karp, Kato and Kawazaki . Differentiation
of serotypes is important for laboratory diagnosis. (5)
This pathogen does not have a vacuolar membrane;
thus, it grows freely in the cytoplasm of infected
cells. Because they are intracellular parasites, they
can live only within the cells of other animals. Even
though it is recognized as one of the tropical
rickettsioses diseases, O. tsutsugamushi has a
different cell wall structure and genetic composition
than that of the rickettsiae. Scrub typhus is
transmitted to humans and rodents by some species
of trombiculid mites (“chiggers”, Leptotrombidium
deliense and others). Most notably L akamushi, L
arenicola, L fletcheri,L pallidum and L pavlovsky.the
vector species differs depending on the particular
ecosystem. e.g L akamushi is found in partially
cultivated fields that flood in spring and early
summer, whereas L deliense is associated more with
jungle(1). The adult mites have a four-stage lifecycle:
egg, larva, nymph and adult. The larva is the only
stage (chigger) that can transmit the disease to
humans and other vertebrates, since the other life
stages (nymph and adult) do not feed on vertebrate
animals. Both the nymph and the adult are free-living
in the soil.
Global scenario
Geographic distribution of the disease occurs within
Afghanistan and Pakistan to the west; Russia to the
north; Korea and Japan to the northeast; Indonesia,
Papua New Guinea, and northern Australia to the
south; and some smaller islands in the western
Pacific. It was first observed in Japan where it was
found to be transmitted by mites. The disease was,
therefore, called tsutsugamushi (from tsutsuga
meaning dangerous and mushi meaning insect or
mite). This is found only in areas with a suitable
climate, plenty of moisture and scrub vegetation.
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Recently, rickettsioses has been an emerging disease
along the Thai Myanmar border. There are reports of
emergence of scrub typhus in Maldive Islands and
Micronesia. (6)
Indian scenario
In India, scrub t yphus has been reported from
Rajasthan, Jammu & Kashmir and Vellore. In
addition, few cases have been tested positive for IgM
antibodies for scrub typus in Sikkim , Darrjeeling,
Nagaland & Manipur . In a study conducted from
July through October 2004 in Himalayas, among
several cases of acute febrile illness of unknown
origin, O.tsutsugamushi was identified as causative
agent by microimmunofluorescence and PCR (7) . In
an entomolgic study in Himachal Pradesh, vector
species Leptotombidium deliense and Gahrliepia
(schoengastilla) spp. were recorded.(8)
Map showing Distribution of scrub typhus in india
Collection, storage & transportation of specimen
The collection, transportation and storage of
specimens are extremely vital steps in laboratory
diagnosis and hence, must be undertaken with utmost
care
Specimen
Serum
Blood collected in tubes containing EDTA or
Sodium citrate
Blood clot
Blood collection in tubes and vials
Aseptically collect 4-5 ml of venous blood.
Allow blood to clot at room temperature, centrifuge
at 2000 rpm to separate serum.
Collect the serum in sterile dry vial.
Fix the cap with adhesive tape, wax or other sealing
material to prevent leakage during transport.
Use adhesive tape marked with pencil, indelible ink,
or a typewritten self adhesive label to identify the
container. The name of the patient, identification
number and date of collection must be indicated on
the label.
Precautions while collecting specimen:
Collect sufficient quantity of specimen
Avoid contamination by using sterile equipment and
aseptic precautions.
Despatch the specimen immediately to laboratory at
2-8ºC (ice box) as soon as possible.
Don’t freeze whole blood as haemolysis may
interfere with serology test results.
In case the delay is inevitable, keep the specimen at +
4ºC in a refrigerator.
Label all specimens accurately and send all pertinent
information to laboratory which will help in better
interpretation of the laboratory findings.
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Isolation of the organism
As rickettsiae are highly infectious and have caused
several serious and fatal infections among laboratory
workers, it comes under Risk Group 3 organisms.
Isolation should be done in laboratories equipped
with appropriate safety provisions preferably
Biosafety level-3 laboratory following strict biosafety
precautions.
Rickettsia may be isolated in male guinea pigs or
mice; yolk sac of chick embryos; vero cell line or
MRC 5 cell lines from patients in early phase of the
disease.
Isolation of Rickettsiae: only research laboratories
that had biosafety level 3 are able to isolate
rickettsiae from clinical specimens but,. Since
different rickettsial diseases may have
indistinguishable clinical manifestations, the isolation
of new isolates followed by their molecular
characterization is critical for the discovery of new
rickettsial diseases. The isolation of rickettsiae may
be attempted with several samples: buffys coat of
heparinized blood, defibrinated whole blood,
triturated clot, plasma, necropsy tissue, skin biopsy,
and arthropod samples.
(A) Embryonated chicken egg yolk sacs: have
been widely used in the past, but they are now being
replaced by cell culture systems. The mouse is the
species of choice for the isolation of R. akari,
Rickettsia australis, and especially O. tsutsugamushi.
Blood clot ground in skimmed milk or any suitable
medium is inoculated intraperitoneally. The animals
have to be observed for 3-4 weeks. Smears from
peritoneum, tunica and spleen of animal, stained by
Giemsa or Gimenez methods demonstrates
rickettsiae.
(B) Cell cultures: Cell culture, is now the most
widely used method for isolating rickettsiae from
clinical samples. Verocells, MRC 5 cells are being
used frequently but L929 mouse fibroblast cell
monolayer in tube culture is best suited for the
isolation of R. rickettsii and O. tsutsugamushi from
blood (9).
(C) Shell Vial Assay: More recently, the shell vial
assay, detection of the microorganism being possible
in 48 to 72 h in most cases. Inoculation should be
made onto two types of cells. Vero or L929 cells
have been shown to allow better and faster isolation
(10).
Serological diagnosis
Diagnosis of the etiology of rickettsial diseases can
be accomplished most easily and rapidly by
demonstrating a significant increase in antibodies in
the serum of the patient during the course of infection
and convalescence. Several serological tests are
currently available for the diagnosis of rickettsial
diseases like Weil-Felix Test (WFT), Indirect
Immunoflourescence (IIF), Enzyme linked
Immunosorbent assay (ELISA) etc. Although many
techniques have been used successfully for rickettsial
serodiagnosis, relatively few are used regularly by
most laboratories. BSL-3 Lab is not required for
performing serology, when the test is to be used for
seroepidemiologic studies, it should be highly
specific to prevent false-positive results due to cross-
reacting antibodies. In primary infection with O.
tsutsugamushi, a significant antibody titer is observed
at the end of the first week, concomitant with e
detection of IgM antibodies, whereas IgG antibodies
appear at the end of the second week. In the case of
reinfection with O. tsutsugamushi, IgG antibodies are
detectable by day 6, with IgM antibody titers being
variable(11) . Following tests can be used for
diagnosis : -
1. Weil-Felix Test:The cheapest and most easily
available serological test . The Weil-Felix test is
based on the detection of antibodies to alkali based
carbohydrate antigen which are shared by some
rickettsiae and certain strains of Proteus species,
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P.vulgaris OX19, and OX2 and P.mirabilis OXK.
The OX-K strain of Proteus mirabilis was
demonstrated to agglutinate with sera from scrub
typhus patients. .By the Weil-Felix test, agglutinating
antibodies are detectable after 5 to 10 days following
the onset of symptoms, with the antibodies detected
being mainly of the immunoglobulin M (IgM) type
(12).
2. Complement Fixation (CF) Test: It is a
serological test to detect specific antibody or specific
antigen in a patient's serum. Each patient's serum is
systematically tested against five O.tsutsugamushi
serotypes. An IgM titer >1:32 and/or a four-fold
increase of titers between two sera confirm a recent
infection. However, due to cross-reactions among
serotypes, it is difficult to identify accurately a
specific serotype.
3.Indirect Hemagglutination Test:The indirect
hemagglutination test detects antibodies to an
antigenic erythrocyte-sensitizing substance (ESS)
used to coat human or sheep erythrocytes that are
either fresh or fixed in glutaraldehyde. The ESS is
rickettsial group specific with cross-reactivity among
Rocky mountain spotted fever (RMSF) and
rickettsialpox .This test detects both IgG and IgM
antibodies, but agglutination is more efficient with
IgM antibodies.
4.Latex Agglutination Test: In the latex
agglutination test, ESS is used to coat latex beads.
The reactivity is not exactly the same as that of the
indirect hemagglutination test, because the ESS on
latex beads probably contains more antigenic
fractions than the ESS adsorbed onto erythrocytes.
This test is rapid (15 min) and does not require
elaborate instrumentation.This test allows the
demonstration of antibodies within 1 week after the
onset of illness. Significant antibody titers disappear
after 2 months.
5.Enzyme-linked Immunosorbent Assay (ELISA):
ELISA was first introduced for detection of
antibodies against Rickettsia t yphi and Rickettsia
prowazekii. The use of this technique is highly
sensitive and reproducible, allowing the
differentiation of IgG and IgM antibodies.
6.Immunofluorescence Antibody (IFA):IFA is the
gold standard and is used as a reference technique in
most laboratories. . Detection of rickettsiae by using
immunofluorescence allows the confirmation of
infection in patients prior to their seroconversion.
Samples can be tested fresh or after formalin fixation
and paraffin embedment. Biopsy specimens of the
skin with a rash around the lesion, preferably
petechial lesions are the most common samples used.
In animals or patients with fatal cases of infection,
bacteria are detectable at autopsy in the tissues of
numerous organs.(13)
7.Indirect Immunoperoxidase (IIP): IIP is a
modification of the standard IFA method that can be
used with a light microscope, The procedure is the
same as IFA, but fluorescein is replaced by
peroxidase. The advantage of the immunoperoxidase
assay is that the results can be read with an ordinary
light microscope. In addition, it provides a permanent
slide record.(14)
8.Microimmunofluorescence.The micro-IFA has
the advantage that it can simultaneously detect
antibodies to a number of rickettsial antigens (up to
nine antigens) with the same drop of serum in a
single well containing multiple rickettsial antigen
dots. IFA allows the detection of IgG and IgM
antibodies or both. This technique is, furthermore,
affected by RF, thus requiring the use of a RF
absorbent before IgM determination(15.)
9.Western Immunoblot: Western immunoblot
assay with sodium dodecyl sulphate
gelelectrophoresed andelectroblotted a ntigens is a
powerful serodiagnostic tool for seroepidemiology
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and confirmation of serologic diagnoses obtained by
conventional tests. It is especially useful in
differentiating true-positive from false-positive
results created by cross-reacting
10.Line blot Assay:The line blot assay allows the
testing of more than 45 antigens simultaneously. It
is a useful test for large-scale screening of sera when
quantitative titers are not needed might be considered
for patients with nonspecific or atypical clinical
presentation.(16).
11.Molecular Biology-Based Identification: The
first proposed molecular biology-based identification
method was based on PCR-restriction fragment
length polymorphism (RFLP) analysis of the gene
encoding the OmpA protein. Molecular detection
using polymerase chain reaction (PCR) is possible
from skin rash biopsies, lymph node biopsies or
ethylenediaminetetraacetic acid (EDTA) blood.O.
tsutsugamushi can be demonstrated by standard and
by nested PCR. Realtime PCR assays are as sensitive
as standard PCR but are more rapid and can give
quantitativeresults.PCR-based detection in published
reports has been based on amplification of the gene
encoding the 56kDa antigen for O.tsutsugamushi.(17)
Treatment
Prompt institution of effective antibiotic therapy
against rickettsiae is the single most effective
measure for preventing morbidity and mortality due
to rickettsial diseases. Anti rickettsial therapy
improves the outcome of all rickettsioses. If the
illness is severe, the cardiac, pulmonary, renal, and
central nervous systems should be assessed and
additional measures instituted to prevent
complications.
Tetracyclines and chloramphenicol remain the only
proven therapy for the rickettsial diseases.
Doxycycline in a dose of 100 mg twice daily for 7-15
days or Chloramphenicol 500 mg four times a day
PO for 7-15 days (for children 150 mg/kg/day for 5
days) is recommended. , tetracycline should not be
used for children under 8 years of age and for
pregnant women.
Prevention and control
The mite vectors of scrub typhus are especially
amenable to control because they are often
found in distinct areas (Typhus Island).
These foci can be eliminated by treating the ground
and vegetation with residual insecticides, reducing
rodent populations, and destroying limited amounts
of local vegetation.
Persons who cannot avoid infested terrain should
wear protective clothing, impregnate their ing and
bedding with a mitecide (e.g. benzyl benzoate) and
apply a mite repellent, diethyltoluamide, to exposed
skin. Chemoprophylaxis should also be considered.
In a controlled trial, the weekly administration of 200
mg doxycycline decreased the incidence of clinical
illnesses but not of inapparent infection.
An effective vaccine for humans has not been
developed till now, mainly due to serotypic
heterogeneity of the organism.(18)
Vaccines:
Scrub typhus vaccine were tried earlier during
World War II in Britain using cotton rat.. Formalin
killed volner vaccine-prepared from rat lung-spleen
extracts and Inactivated Karp vaccine trials were
also unsucessful .Then a polyvalent Gamma
irradiated vaccine which elicited protection against
heterologous serological types of scrub typhus was
developed(19) . Recent advances in molecular
biology and immunology have lead to detailed
antigenic structure of Orienta tsutsugamushi which
includes proteins with molecular masses of 70, 58,
56, 47,110 and 22KD. 47kd and 56kd protein are the
major surface antigens and are called as [Sta 47, Sta
56] scrub typhus antigens. These proteins have now
become the focus of modern research for the
development of scrub typhus vaccine. The 47kd
protein [Sta47] is found in outer membrane of
Orienta tsutsugamushi and contains both group
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reactive and strain specific epitopes .Sta-56 protein
has an immense capability to induce CMI against
Orienta tsutsugamush.In addition, Sta-56 also plays a
vital role in the adhesion and internalization of
Orienta tsutsugamushi into host cells(20, 21,22,23,)
, the gene for Sta-56 was cloned into the DNA
vaccine vector pvR1012 as a vaccine candidate
(pkarp 56(24). , the Sta-47 and Sta-56 proteins were
fused together by ligating their genes and the fusion
product was expressed in E.Coli cells.In this way
immune system was effectively stimulated to
generate a high level of humoral and cellular immune
responses against the disease . Future vaccines are
focusing more on immunodominant protein
combinations which can provide long term,
effective and heterologous protection against scrub
typhus. newer adjuvant-Vaccine combinations
(titermax+Kpr56,liposome+pKarp110,
FIA+pKarp47) are also tried. Sta-56-47 fusion
product is also looked upon as a suitable candidate
for recombinant vaccine against the scrub typhus.
(25)
Conclusion:
A high index of clinical suspicion, prompt diagnosis
and early institution of appropriate antimicrobials can
decrease the morbidity and mortality. Scrub typhus is
easily treatable disease if we focus on case
identification, public education, rodent control and
habitat modification to control the impact of Scrub
typhus on public health. Vaccines are under trial
sooner we will be able to control this scourge of
mankind.
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