EPIDEMIOLOGICAL SIGNIFICANCE OF IMMUNE STATUS OF COMMUNITIES IN KALA-AZAR ENDEMIC AREAS
Item
- Title
- EPIDEMIOLOGICAL SIGNIFICANCE OF IMMUNE STATUS OF COMMUNITIES IN KALA-AZAR ENDEMIC AREAS
- extracted text
-
TITLE
EPIDEMIOLOGICAL significance of immune
STATUS OF COMMUNITIES IN KALA-AZAR
ENDEMIC AREAS.
I
I
I-
A CROSS SECTIONAL AND COHORT STUDY.
I
Dissertation submitted in partial fulfillment
of the requirement of the Dr. MGR Medical
university for the
degree of M.Sc Epidemiology.
MARCH 2001.
1
CERTIFICATF
I
This is to certify that the thesis
enlilled "EPIDEMIOLOGICAL SIGNIF
OF IMMUNE STATUS OF
'?
»
bonafide W°rk by Dr Raian R Pa"1
I"
in
Partia, fulrainwnt
reouralions for MSc. Epidern,o,ogy
Medical Universlly Chennai Io be held In March 2001.
1
i
Thesis guide:
i I
a
ICANCE
COMMUNITfES lN KALA-AZAR ENDEMIC
Christian Medical College,
Vellore-632 002.
•’’iW
77
AREA" Is a
of the rules and
Tamllnadu Dr. MGR
acknowledgements
lamgreatfut to the follotolng forgMng me
their consent and approval to
carry out my thests mark on Kula „ar
Er. JAYPRAKASH
in Jharkhand & West Bengal
MULntlLL ICMC.Venore)-
My
personal,., for humility & dadty in teaching and patience in
an incredible
guiding.
Er. RAVI NARAYAN ( CHc n
»
lor his s
~
men,°r’ “ inSPirinS —"ality.
h,s support, encouragement and guidance.
MY FAMILY- My strength and vulnerability, for
unwillingly agreeing to send
me.
Veto by any of the three
stage, and this study in
above would have
had the idea aborted at conceptual
Jharld.and state would have never taken off.
Mi
Dr'
to heli
Den,,). for r,nandal
ABRAHAM <
0nme.oStlckloU,eideaofdoingmydissertat.
contribution and prevailing
on working with tribals
Dr' ',RAB'R CHA™™ (Bihar)- for being involved
I
!
f
/.
the stud
r
through th e nitty gritties
of ihe study process & being ray fosler family t
ly in Bihar, to help, ,
over come my
home sickness.
Dr. A NANDYf Calcutta) -
to visit the study
V
for preparing required antigens,
nnd taking time off
area during vital leishmanin skin test
survey.
!
I am indebted
to technical
collecting the
specimens
assistance given by Md. Anish
and helping me with the field
W.Bengal respectively.
1
and
i
P- Patro in
work in Bihar and
r
i.
T/\BLE OF CONTENTS
S.No.
CONTENTS
1.
INTRODUCTION
3
2.
JUSTIFICATION OF THE STUDY
6
3.
OBJECTIVES
7
4.
LITERATURE REVIEW
8
5.
MATERIALS AND METHODOLOGY
33
6.
RESULTS
37
7.
DISCUSSION
57
8.
SUMMARY AND CONCLUSIONS
65
9.
LIMITATIONS
68
10.
RECOMMENDATIONS
69
II.
BIBLIOGRAPHY
70
12.
APPENDIX
75
PAGE
INTRODUCTION
Kala azar is a disease
of public health problem predominantly in the
third world countries. India Bangladesh and Nepal account
for 90%
of the disease burden of the world. It comes under the so called 'orphan
diseases' owing to its neglect both at national and international level.
Kala-azar is an endemic disease in parts of India. It is a serious problem
in the states of Bihar and West Bengal. Annual reported number of cases
is 24665 with frequency of 36.8/100,000 in 1996 in Bihar. In West
Bengal total reported cases for the same time period is 1987. UP
occasionally is in news, with sporadic outbreaks. The case fatality rate of
the disease in untreated cases is close to 90%, which with treatment
reduces 15% and is 3.4% even in specialized hospitals1 .
Planning for Kala azar is hindered by conflicting reports of official and
actual figures.Just to give an instance, 54650 cases were reported from
Bihar In 1990, but an expert team constituted by government of India in
1991chaired by CP Thakur, put the actual numbers closed to 250000.
The latest annual figures were 15485 and 6750 cases in 1998(till May) of
19972
I
Kala azar
has re-emerged from near eradication all over the world. Its
annual estimate for incidence and prevalence worldwide is 0.5 million
and 2.5 million, respectively'
i
India too showed the same trend, an epidemic of kala-azar in Bihar.
Qudi;i)zm 1950s died out within a. decade,
as a collateral effect to the DDT
spraying which was part of the National Malaria Control Programme.
Sand flies disappeared from the house
spiaycd with DDT to eliminate
Anopheles moscpiitoes. The disease became rate. When spraying was
discontinued however, sand Hies surviving in cattle shed returned to the
*
houses and some became icinfected with Leishmania,
possibly by ■
J
feeding on the remaining Post kala- azar Dermal Lcirhmaniasis (PKDL)
cases 3
For prevention ■'aid coniiol '.'t •!;•? uiscase?epidemiological works at field
level is a must to understand li-c but den of the disease. The diagnosis
and treatment arc important aspect of kala
azar control programme.
I
I
Reliable and valid diagnostic tools arc required for the success of control
programme.
'i
l
WHO has laid emphasis on bevel
!
as screening pigtanunes. The aldehyde and bonemarrow aspirate tests
I.
r?
I
g
ing simple tests for diagnostic as well
presentiy used are seen wanting in sensitivity and specificity. Morever
they can the employed for the screening programmes/
I
More studies are recommended to assess the utility of newer serological
tests like Direct agglutination test for diagnostic as well as screening
progammes5
Leishmanin skin test is valuable tool for epidemiological works to
measure past and possibly future incidence in the world®
c
JUSTIFICATION OF THE STUDY
♦
Information on the
magnitude of kala-azar in endemic areas and
among tribal communities is limited.
♦
Uncertainty regarding the usefulness
of serological tests used in the
screening as well as diagnosis of kala-azar.
♦ Need for
developing indicators for assessing factors responsible for
the disease transmission, which would be in
turn help in assessing
I
J
OBJECTIVES
1. To estimate the prevalence of Kala
past) in Pahadias and Santhal tribal
azar infection (present and
communities of Jharkhand
state.
2. To study the dynamics
Of the antibodies in cured cases of kala-
azar for its implication on the validity serological test.
J
/
I
ST I •
4.1 HISTORY OF KALA-AZAR
Kala-azar has been occurring in India in
i epidemic and sporadic form
over the past centuries. The earliest epidemics of Kala- azar were
confused with those of malaria. However, the first recorded epidemic
which could be attributed to the manifestations of the disease in India
could be in 1824-25 in Jessore (Elliot). Burdwan fever of 1854-75 was
also attributed to Kala-azar (roger). The first epidemic in Assam is
supposed to have occurred in 1870 in Garo Hills. However, Carke first
described the disease in Assam in 1882 in his Sanitary report of Assam
based on the report on MC Naught , the then Civil Surgeon of Turf.
Kaladukh in Purnea (Brown 1898) and the Kala -azar in Darjeeling
(Rossl899) are attributed to Kala-azar. The epidemic fever dinaz pur and
Rungpur Between 1871-76 and iin Patna during 1856-59 could also be
attributed to Kala -azar.7
Leishman in 1903 reported peculiar bodies in
1
the spleen of a soldier who
died of Dum Dum fever in Nctley Hospital. Donavan in the same
year
reported aetection of similar bodies in the spleen of the patients in
Madras suffering from prolonged fever with splenomegaly. Therafter,
conclusive evidence was obtained and the causative
agent i.e., the
parasite was named an after Leishman and Donovan 8
i
8
h
i
M
I
4.2 kala-azar distribution -place and person.
Apart from severa! states in mdia, the disease is present in East and
Africa, Sudan, Bangladesh, pans of china and USSR, South Iran
Arabia, Mediterranean countries of Europe. Ethiopia. Kenya and South
America, mcludmg Brazil and Venezuela. In India, the disease is most
endemic in eastern half, but isoiated pockets exist in almost all regions
cases and even small outbreaks have
been reported
from time to time.
In Mediterranean region, infants and children are almost exclusively
affected. In other endemic areas including India, though these
are more
frequent in
children
and
the young, older
age groups are also
susceptible. Males are affected
more than females. Kala-azar is more
prevalent in rura| setting .here conditions exist for tnultipiication of the
rnatn vector sand ny Ph.eobtonrous argentipes. OnIy fenra.es
act as
vectors.9
4.3 RESERVOIRS OF INFECTION IN INDIA
No anima, reservoir has been found in .ndia and transmission of ka.aazar
occurs
from
P.argentipes.The
man
m
to
through
man
(he
recognized
cutaneous or dermal leshnraniasis
vector
is caused by
is
L-troprca and this is restricted to Rajasthan where it is zoonoticjo
In some regions such
as northeastern India, human
appear to be their
own reservoir and several factors serve
to facilitate person-to-person
n
transmission. The vector P
argentipes, has a preference for human
Wood. The parade is found in eiceuiating monocytes in indian cases of
Kala-asar more frequently than usual. Dermal lesions
that develop after
the initial disease in Indian patients’ may be
an additional source of
parasites for the vector.
4.4 RESERVOIRS OUTSIDE INDIA
cycle involving man and animal is
humans and dogs which
wild canines such
seen between
act as canine reservoir. The domestic d
as the fox, develop
similar to that of humans
a chronic
systemic disease very
when infected with L. donovani.
additional unique feature of leishmnaial in
°g, as
But an
canines is the frequent
presence of organisms in the skin including the
nose and ears, which are
favorite feeling sites of s;lll(| Hies. An
e|>i<lemioloKi,:„| cyc|,, „(■ p„rul)ilc
involving wild foxes, domestic dogs, and human via the
vector
Lutzokmyia longipapis has been documented in
northeastern Brazil. The
domestic dog has also
been incriminated
for visceral leishmaniasis of the
as an important reservoir host
Mediterranean region and in certain
areas of China.
Rodents are the likely reservoir in the Sudan, and
activity of the vector,
Phlebotomies
onentalis, is high in clumps of
villages. In Kenya, transmission of disease is
hills, which serve as
acacia woodland near
associated with termite
resting places for the
vector P.martini, and around
which village men gather in the evening. However, the animal reservoir
in Kenya has not been identified.11
4.5 VECTORS
Sinton originally suggested Sandfly as a possible vector in 1922.
There are about 600 species of the phlebotomine sandflies distributed
through out the world and among these 70 are proven or suspected
vectors of leishmaniasis. In India, the only proven vector of kala-azar is
phlebotomus argentipcs. The biology of this
been worked out in details. It breeds in
vector in West Bengal has
cowsheds. They can attack man.
The vector can invade the clean biotype from the
attacking man out
nearby resting places,
doors, hence acquiring out door infection is also
possible. It was revealed that they could take 5 blood meals in nature,
especially in rainy season, indicating high vector potentiality.
i
In this
connection it may be mentioned that
period of peak transmission
usually corresponds to the time when
vector population is relatively old
(as in rainy season June to September).
1
P. argentipcs may disperse 500 m from the place of release. The flies can
invade the hving rooms and both exit and entry activities concerning
cowshed and human habitation. 12
I
f
The conditions conducive to the development of the vectors are;
1. altitude below 700 ft.
2. high rainfall and humidity
!
3. alluvial soil
4. abundant vegetation,
5. rural settings.
Average life span of vector '
in nature is 10 days.
I
i
i
4.6 SPECIES OF LEISHMANIA13
SPECIES
L.donavanl
I
L. Major
L. tropica
L. infantum
L.chagasi
A
L.ethiopica___________
L. mexicana complex
L.m.mexicana
L.m.amazonensis
L.m.venezuelensis
h
I
.s*.
•:
?.
L braziliensis complex
L. b.braziliensis
L. b.panamensis
L. b. guyanesis
L.b. peruviana
______ DISTRIBUTION
Old world - East Africa and
south of Sahara, South Asia
including India and Iran
N Afiica, Middle east, central
Asia, a»id southern Asia
Middle east and S. Asia.
Old world -N Afiica, and
southcin Europe________
New world Brazil, Venezuela
and Colombia,
Ethiopia
New world- from southern US
through Central America,
northern and central south
America, Dominican republic.
New world from Central
America through various
parts of South America,
including Brazil, Venezuela,
Bolivia, Peru to N.Argentina
DISEASE
Vliceral liohmanlaiia
Cutaneous ulcer
Cutaneous ulcer and chronic
relapsing cutaneous disease.
Visceral leishmaniasis
Visceral leishmaniasis
Cutaneous ulcer, rarely DCL.
Cutaneous ulcers, small
proportion of Cases may
develop diffuse
cutaneous(DCL) or
mucourtaneous (MCL)
leishmaniasis
Cutaneous ulcers some
cases may later develop
(MCL)
4.7 POSSIBLE MODES OF INFECTION OTHER THAN VECTOR
Maleness and Brooks discovered that cases of Kala-azar in sandfly free
area in persons with no history of contact with any known case of Kalaazar or of visiting any endemic area. Further it can be taken note of that
L.D bodies have been recovered in
i
the secretions of nose, skin,
conjuctiva, urine faeces and mouth of infected persons. On the basis of
these observations the following modes of entry of infection were
suggested.14
1. direct infection from man to man by contact with skin lesions,
i-
2. through bites of an infected insect
3. nasal discharge
4. through flies and other insects mechanically contaminated through
■I
various discharges from patients
t
■
As to the available findings in respect of the above hypotheses certain
i
observers have noted cases among contacts in the absence of vectors.
I
The Bed bugs can be experimentally infected but not found infected i~
in
nature. The question of spread by nasal discharge has not been
explored. But if the large number of cases, as reported now, did actually
crop within comparatively short period of time an ultimate mode of
transmission other than by sanddys has to be thought of. Also, human
experiments to eliminate man-to-man transmission by contact and
i
exploration of reservoirs other than man along with their ectoparasites if
any have not been done adequately. These are some of the possibilities
that can be examined afresh in connections with the outbreak At least it
13
l;;
would be an advantage of the
sand fly theory if jt js
fully confirmed by
detecting natural infection among them.
4.8 MODES OP SPI!EAD Af)D cHARECTERIST,cs Of,
kala-azar
epidemics
The s,oW spreading nature of the epidemic travel at a rate of I0 miles
-Year and aitvaysaiong the routes of traffic.
2. When it attacks
a particular viUagc. it clings to particular houses and
'hen spreads from one h„„„ ano(b h
and
one house to
V means of communication.
smaller foci.
4. The disease ding to the fam,lies in the same house.
‘
margins of
larger cjties are common.
6. It occurs in houses, which
are damp and
Surrounded with vegetation.
7. Poorer class of all races is
equally liable
to infection. As sand Hies
Prevail in ill-ventilated houses,
Poor classes who live in insanitary
houses arc usually affected
14
■
j
I
8. Age and sex have no influence on the
definite decline in cases of Kala -azar
epidemic pattern. There was a
in India from 1961. In
1960
there was 3916 number of cases and in 1961 only 196
cases were
recorded. In thof time-4 Kala azar was not at all
a public health
problem. The factors responsible for the decline were associated to’ :
e
Effective treatment of cases,
•
DDT spraying under NMEP during 1953-57 and under NMEP
from 1958
•
Increased immunity inthe population
4.9 INCUBATION PERIOD
It has been difficult to estimate the incubation
period of kala-azar.
Usually it is between 2-6 months but could be as short as
10 days or as
long as 9 years
The ratio of clinical to subclinical infection
may range from 1:5 4 12 15
4.10 IMMNUNITY IN KALA AZAR
Kala-azar unlike other diseases (paras,tic) is characterized by
(parasitic) is
near
absence of a second attack after successful cure of the first.
Leishmania
parasites
are
able
to
persist
very
successfully
immunocompetent individual and they are also able to
in
suppress the
immune response suggesting that relationship of the parasite with man
is very old.12
Visceral leishmaniasis
has always been thought to be unique, in that
infection of man with L.donovani resulted in kala-azar. syndrome with
little or no immune response, poor cellular tissue
response, many
parasites and no delayed hypersensitivity
. However it was found that
delayed hyersensitivity and a positive leishmr<nin skin developed after 6
cases were immune to reinfection.6
Resistance to Leishmania depends
upon the development of specific cell
mediated immunity. The capacity of the individual
to mount such
response varies, and consequently Leishmaniasis presents a spectrum of
disease in the same way that leprosy does. At
lie
visceral
leishmaniasis
and
diffuse
one end of the spectrum
cutaneous
Leishmaniasis,
characterized by abundance of parasites, absence of lymphocytes in
lesions, insensitivity to leishmanin, and
poor prognosis. At the other end
lie the self-healing sore, with relatively scanty
parasites, marked
lymphocytic innitration, and leishmanin sensitivity. Accompanying cell
medicated immunity is delayed hypersensitivity to numerous parasite
antigens, which causes the destructive pathology of leishmanial ulcers,
especially in chronic conditions of espundia and Leishmaniasis recidivia
in which the normal balance between
been iJt. The mechanism
immunity and hypersensitivity has
underlying these abnormal immune response
arc not understood.10
16
The major biological properties of viscero-tropic leishmaniasis is its
invasive character which permits escape from the cellular defenses of the
host at the portal entry. The rapidly multiplying parasites within the
cells of mononuclear phagocytic system disseminate widely in the
tissues of the host particularly in the haemopoietic and lymphopoietic
system. .
T he initial response to Kala-azar seems to stimulate both specific and
non specific increase of immunogloblins. The specific response to
leishmania antigcns'jiot pherhaps protective. The non-specific increase
of immuonglobulins in Kala-azar may be the result of deviation to
systemic lymphorcticular system of antigens,
which are normally
mopped up by the kupffer cells of liver.
I
Spontaneous cure as well as ■premunition' or 'sterile' resistance due to
sub clinical infection in endemic zones is more frequently encountered in
Indian Kala-azar . It appears that in Kala-azar, there i
is ^exaggerated
I
stimulation
r/the production of immunoglobulins some specific and
other non-specific. On the other hand, it fails to stimulate requisite CMI
necessary for spontaneous cure and sub clinical infection, suggests that
the human host can mount up sufficient immunological reactions both
humoral and cell medicated, to protect the individual.
CMI probably
plays the crucial role but the T cells are suppressed during active phase
I
of the disease. 17
17
The parasite stimulate
group and
I
several antibodies producing B-cell including
genus-specific (polyclonal)
as well
as
species specific
(monoclonal) cells. Smce the antibodies elicited are more of a reactionary
than protective in nature.
The seropositivity in kala-asar is a long tasting (few
years), during any
serological assessment
a positive test, is an indication of recent
infection and not active disease.
There are several hypotheses
or past
on why the antibodies linger very long after cure .-
1. Agglutination antibody have longer life span
2. a
clinical
antibodies,
infection
could
treatment
having
have
persisted,
not
been
eliciting
sufficient
to
circulating
result in
parasitological cure
3. cured patients had been re-exposed to Leishmania in endemic area-
These circulating antibodies of humorai immunity offers ve^ ,itUe heip
in
protecting
against
re-infections.
The
The
Coii
Cell
mediated
immunity
(CMI)which is
suppressed during the active disease,
develops well
following treatment
or self resolulion. The leishmanin skin test is '
always negative during the aetire phase of the disease
positive after six months of initiation of treatment.
18
and becomes
i
With development CMl/patients
*
i ecover from infections and achieve
HfCong immunity. No.record of second attack of viscera! leishmaniasis is
I
there. Posit.ee leishmanin test, acquired naturahy from inapparent
infections also protects from infection.>2
In the absence of recovery in Leishmaniasis, there is
suppression, impairing the immune
bonemarrow
response to host of infections like
tuberculosis, pneumococcal
pneumonia etc. Leishmaniasis has been
recognized as one of the infection that
may complicate the course of
AIDS.‘s
4.11 CLINICAL MANIFESTATIONS OF LEISHMANIA DONAVANI
INFECTION
UNAPPERENT INFECTION.
At this stage (he infec.ion by .be parasite although is unatte to produee
chsense, results in stimulation of immune system.
OLIGOSYMPTOMATIC/SUBCLINICAL INFECTION
> n this condition there mieht be transient ciinica! expression in the Iorm
ot fever and hepatospienotnegaly. Many of these individuals might cure
A small proportion
of these cases might progress to deve.op Iranh Ka.a-azar and /or dermal
letshmaniasis within a per.od ranging from 4 weeks to few months.
lymphatic LEISHMANIASIS
This is condilion orgen„a,ised
involvement by the
ie.sh^an.a donavan,>ithout cIinica, signs
or
spleen.
kala-azar.
Kala-azar is the
severest form of clinical expression^infectionjeshmania
donavani in man and is a result of
uninhibited multiplication of the
parasites because of cell
i
i
mediated immunosuppression. If not
treated,
up fatally.7
Clinically it presents with fever of
years. The onset is
over 80
varying duration and
types often for
usually insidious, especially in indigenous peoples
percent of cases, however, the fever
charecterisc
eventually develops a
Pattern with twice daily elevations
brucellosis. Splenic enlargement is not
i
and may undulate as in
necessarily rapidly progressive,
nor does the size of the spleen correlate with duration of the di
sease. In
many instances, however, it reaches the
I
right iliac fossa.
enlarges more slowly and becomes palpable in
likewise firm and not tender.19
I'
The liver
20% of the cases and is
11
- around malnr boncs at)J (cmpJcs Qnd
mouth (hence iaheied btack sickncss,
and brittle.
20
around the
Haemorrhagic manifestations- epistaxis
(
seen often, haemorrhages of
skin, mucous,membranes and retina are rare.
m lh= most acute cases fever and toxemia may be the only signs
Tongue is always nearly clean.20
POST KALAZAR DERMAL LEISHMANIASIS (PKDL)
PKDL is perhaps second to no other disease in regard to it,
•
■
legara to its enigmatic
eitiophathogenesis.
Although the oumc
same parasite
parasite causing kalazar is
P sible, very httle is known about the mechanism of development of
PKDL. This is a condition, which primarily affects skin and in late cases
may involve mucus membranes of
eye, respiratory tract. GI tract and
genitalia, In majority of cases there would be
a past history of Kala-azar
however a sizeable
proportion do not provide such
past histories,
although there are evidences of prior visceralisation.
Some Kala-azar cases
may develop post Kala-azar
dermal leishmaniasis
J this ‘•I’parent change ,n v.scerotropic properties of L donovani to
be
indu^a
duced
mechanism in cured Kala-azar patients.
dermatotropism
PKDL shows
is
believed
believed
to
important differences
from
important in terms of epidemiology.
•
by lommunoregulatoiy
k
Kala-azar,
which
are
♦ About 20% Of Indian palicms devclop a
one to two years after
treatment or spontaneous
recovery^ The lesions develop slowly and
may last for several, 20
years. I n Africa the rash develops in 2
percent of cases, usually during treatment.17
♦ Maximum number of
cases have been described from India and
Bangladesh, ln other endemic regions PKDL is rare or absent..
♦ Unlike kala-azar, PKDL i
. ------ is never fatal and
can remain active
uuuve and
ana a
potentia, source of sand ny infec.io,, i,, unlrcatcd podcnt for at
35 years. Unlr«
treatment
ami
free
Uniess free ...........
„.cc „.
n,lsp(
„.( (o
Offered^,oor patients, they se.dom report for treatment of disease with
relatively mild symptoms.3
♦ Possibility of PKDL cases, as
a potential source of reservoirs of
infection in a community,
except for theoretical basis has
never been
verified.12
4.12 diagnosis of kala-azar
In Kala-azar endemic areas, all the fever cases of more than
duration, which do
two weeks
not respond to anti-malarials, should be
suspected
for Kala-azar
Laboratory confirmation
donavani in Stained
smears from bone marrow,
glands or blood and by
materials on NNN media
i
diagnosis is by demonstrating
Leishmania
spleen, liver, lymph
recovery of the parasites by culture of these
1. Examination of peripheral blood
smear, leukopenia with relative
neutropenia is seen
2.
There is marked
-
production globulin by the
g oouim uy ine plasma cells in Kala-
-
in
globuIin
is
detected
by aMehyde
tMt
”” P-itive only af[er 2.3 mon(hs of
test: one ml of clear
kept at room
■ Napier's aldehyde
serum from the patient and
a drop of fromaline
temperature. A positive
reaction is gellification
and
within 3-30minutes.
3. Serological tests such as hy indirect immUnonUorescence (IFT , &
nsj-me hnked immunosorbent Assay (Elisa)
4.13 DIAGNOSTIC TESTS
direct evidences.
This method invo.ves demonstration of the parasite ,n the
atnast.go.es) cuiture (to demonstrate promastigote)
smear (for
BONE MARROW examination
Bone marrow
aspirations is a painful
technique and also
needs
experienced medical
persons. However the
greatest disadvantage of the
technique is its Poor sensitivity, which
ranges from 55%-7O% depending
upon the duration
3"^ severity of the disease. Because of its pain. the
people have not
accepted this method very well.7
SPLENIC ASPIRATION
Although splenic aspiration gives a
this method is totally unsuitable for
positivity rate of about 85%-90%,
many rural situations because of
the danger of iintraperitoneal
■
hemorrhage leading to death
due to
abnormality in blood
coagulation during Kala -azar
The other
disadvantage of the method is that it
cannot be done in smaller sized
spleen (which should be more than 4-5
cms. Below the left costal
margin) thereby failing to diagnose early
cases when the spleen size
would be very small.?
aldehyde test
One drop of 30% fonnaidebyde or connnercial formal is added b
1 ml. •
Of serum of patient's blood. The tube is
well shaken and placed at room,
temperature. Jellification with
opacity like the whiEe, of parboiled egg
occurring within 60 minutes is
a positive reaction. This test becomes
positive within 1 -2 months of development of Kala-azar.
4.14 SEROLOGICAL TEST.
Serological tests derive there
main advantage from the fact that the
antibodies appear much before the parasites are detnonstrabie in the
clinical materials al a signifies,nly early stage, so much so
that by using
these methods onecan identify prospective kala
-azar cases 21
24
Advantages 7
1. diagnosis of active cases of all forms of leishmaniasis
2. diagnosis of infection, before clinical presentation
3. screening a population for endemicity of the disease
4. screening a population for mass diagnosis
5. as a surveillance tool to monitor intervention activities.
In patients with kala -azar antibody production i
----- is vigorous and rapid.
Various techniques of serodiagnosis of kala
azar are based on polyclonal
stimulation of B-cell (non -specific) or clonal stimulation
of B-cell
(specific tests) 22
4.15 NON-SPECIFIC TESTS
Infection by L.donavani stimulates production of i
-------- immunolobulins by Bcells. In eonlrasf, production of albumin is
humpcred leading to reversed
albumin-globulin ratio s This increased production of immunoplohnlin.
immunoglobulins
is used quite frequently as diagnosis of Kala-azar at lessequipped,
peripheral laboratories. Some of these
tests are Napier's aldehyde test
and Chopra's antimony test. These tests
high false-positive rate due to
are easy to perform but have
over production of irnmunoplnhotimo
immunoglobulins in
many other diseases. Since these tests fail lo aetect cases of early
to detect
leishmaniasis, the sensitivity of these tests is around 85% only.
25
4.16 SPECIFIC TESTS.
Specific serological techniques are based on demonstration of antibodies
produced against the circulating parasitic antigens. The specificity of
various tests depends on the antigen or its epttome used in the test, as
the parasite will stimulate several antibodies producing B-cell including
group and genusspecific (polyclonal; as well as
(monoclonal) cells. Therefore,
species specific
the sensitivity may depend on the test
and its methodology by the speelfidty „i» depend on the antigen
rather than method used.
Some serological tests are:
DAT - Direct agglutination test.
This test has been found to have a
sensitivity of 96.5-100% and
specificity 91-95%. Hs strength is its high sensitivity and specificity in
early diagnosis of ka.a-asar pai.en.s. The draw back is ihai it eentains
positive for more than 5 years after complete cure.
ELISA -. The sensitivity of ELIS/X i
-------is nearly 100%, but
specificity is not
very high as cross-reaction with
were from patents with TB and
toxoplasmosis has been recorded.
Dot-ELISA
is another method that has been
developed in which
inierpreiaiion can easily>de by visual inspection of
reaction end
points, obviating the need for ELISA reader. >9
26
4.17 STUDIES ON VALIDITY OF DAT TESTS.
<
♦ A sensitivity of 100% and specificity of 99.3% and 100% were
reported by HARITH et al (1986).23
In one study on direct agglutination test for visceral leishmaniasis, IFAT
and ELISA were applied to sera of patients with visceral leishmaniasis,
African, American trypanosomiasis other parasitic infection and healthy
controls. The sensitivities of 3 tests were comparable (96.3% to
100%): excluding patients with African and American tiypanosmiasis.
the specificities of DAT and IFAT were 100% and ELISA 87.3% 5
Following studies recommend different cut off values :
Study done in India showed, serum dilution of 1:800 differentiates a
case of visceral leishmaniasis from healthy controls. Therefore any
scrum specimen with titre of > = 1:800 was considered to contain antileishmanial agglutinating antibodies, Fifty-six of 58 sera (96.5%) from
confirmed
cases
of
visceral
leishmaniasis
had
anti
leishmanial
antibodies, while none of the clinically suspected cases, apparently
healthy control subjects, tuberculosis or malarial cases had significant
titres i., e >. 1:800. The mean titre of agglutinating antibodies in visceral
leishmaniasis cases was significantly higher than that of the control
subjects. Although the mean titres for patients with malaria and
tuberculosis where higher than those of both the endemic and non-
27
endemic control subjects how ever the highest titres in these
groups
were below the cut-off value 1:800.
Relatively high levels of agglutinating antibodies in
apparently healthy
subjects from an endemic area, compared to these in
subjects living in
non-endemic areas might be due to previous exposure of the former to L.
Donavani, through the bite of an infected vector, resulting in
some
degree of humoral response. The relatively high titres in sera from cases
of tuberculosis may be due to the presence of antigenic determinants of
L.donavani
promastigotes
cross
react
with
mycobacterium
tuberculosis24.
*
In patients with recent kala-azar,titres of 1:5200 or higher were
found. Cured Kala-azar patients treated 4-14 months before testing,
showed the titres in tiie range of 1:3200 to
> 1:5200.Healthy and
diseased controls had titres below 1:1600 with the
exception of
African trypansomiasis patients who showed titresl:200
overlapping with the titres of cured
tol:12800,
Kala-azar patients. Where
trypanosomiasis is not a consideration, a titre of
1:1600 could
considered indicative of vi
visceral leishmaniasis, the sensitivity and
specificity were then 100%. 23
A titre of 1:3200 or greater in an area considered To be free of African
Trypanosomiasis is highly predictive of visceral leishmaniasis (recent or
Past).. In areas where trypanosomiasis occurs, further serum dilution
beyond
1:12800 would be required. Active kala-azar and African
trypanosomiasis could not be separated.5
COMMENT
In above cited studies we see
1. discrepancies in reporting the validity of DAT,
2. different cut-off values have been adopted.
It is obvious since validity of any given test is not constant or uniform
every where. However such indices are known to be affected by standard
used to classify subjects into those with and without the disease
(VALENSTEIN, 1990). Performance of the DAT varies depending on the
whether smears are made of lymph nod; bone marrow or splenic
aspirate, or whether the diagnosis is based on the clinical grounds. 25
4.18 DAT AS SCREENING TEST.
High discrepancies exist in the results obtained by various investigators
with IFAT and ELISA. Though HOMMEL et al. (1978) doubted that the
reason was the choice of conjugate, in variations in ELISA readings
using the same antigen and serum samples were due to the differences
in batches of plate, conjugate, or both. Standardization . of these factors
are tedious and time consuming. Because of its high sensitivity and
specificity, and the ease and low cost of its application compared to IFAT
and
ELISA,
the
newly developed
DAT is
recommended for mass
screening programmes and sero-epidemiological
leishmaniasis 5
■>Q
studies of visceral
I
The DAT appears to be a useful tool for screening, with a high sensitivity
and a high predictive value of negative test. However, the DAT
cannot
differentiate between past kala-azar, sub-clinical infection, and active
disease. 25
Anti-leishmanial antibodies have been demonstrated in clinical cases of
visceral leishmaniasis by variety of immuno diagnostic tests but these
?'■
tests may give false positive results with case of typhoid, malaria
and
tuberculosis. Thus attempts have been made to develop a simple but
specific diagnostic serological tests for the early identification of cases of
Visceral leishmaniasis. The DAT has been
shown to be not only highly
sensitive but also a specific and simple test for confirming the diagnosis
of visceral leishmaniasis. However, further investigation is required to
assess the specificity and sensitivity of DAT in different geographical
areas. 24
Since DAT has a h.gh negative predictive value the absence of antibodies
detectable by DAT could tend to rule out active kala
suspected cases. Furthermore
-azar in clinically
assessing the titres of agglutinating
antibody could monitor effectiveness of therapy. However, investigation
on larger number of cases is required before a final conclusion can be
drawn.
4.19 LEISHMANIN AS SCREENING TEST.
Leishmanin is a suspension of 10 to the power of 6 promastigotes in 1
ml of .5 percent phenol saline. The test is an index of delayed
hypersensitivity. It has been also used as important epidemiological tool
to assess the immune status of population against leishmanial infection
and the spread of disease within and from an epidemic focus. It is not
species specific. It is used to map out the extent of past infection in
1
community and may be of help in diagnosis iin individual patients.16
Interpretation of the results of a leishmanin test survey requires
consideration of factors such as the time during the epidemic when the
survey was conducted, past experience of the community of such
epidemics, the age of the community at that particular geographical
location and the frequency of population exchange.26
True second attack of Kala-azar after successful recovery from the first is
almost
unknown although there arc some isolated reports needing
verification.27 The sensitivity of leishmanin test is reported to be 93%28
The leishmanin test is specific for leishmaniasis but is not species
specific. Leishmanin positivity increases with age in endemic areas of
cutaneous leishmaniasis and in endemic kala-azar areas leishmanin
positiWly is acquired without any clinical evidence of infection
and
varies inversely with incidence of kala-azar indicating population
immunity. Leishmanin positivity has also been shown to develop without
clinical signs of infection in Northern Italy during an outbreak of kalaI?
az ar.6
7
boi
31
Leishmanin positivity deontes an immune state that prevents further
infection provided no immunosuppressive factors intervene and this •
immunity has been demonstrated. In old endemic foci where there are
frequent contacts with infected sand Hies only new generation of children
are likely to contact the infection. si
since the older generation are already
immune. This phenomenon is aided^the fact that foci often
remain
constant, strictly limited to the same district or to the same group of
houses or even the same house for years. It could be used as indirect
diagnostic reaction. If someone is suspected of having kala-azar and the
leishmanin test is positive in one or more members of household,
this
observation along with negative reaction in the patient can suggest the
diagnosis.
These observations suggest that the leishmanin skin test is valuable tool
to measure the past present possibly future incidence of kala-azar in all
areas of the world.6
Interpretation should be done carefully inter observer variation is
significant.29
I
5 MATERIALS AND METHODOLOGY
5.1 STUDY AREA
Litipara block (Jharkhand)
LiUpara block is the most predominant tribal block in Pakur district of
Jharkhand state with 25% Pahadias and 50% Santhal tribes?" It is
2
among the six blocks of Pakur district of
■ ’
./
A’
•?
newly created Jharkhand State,
Jharkhand literally means ‘Jhar’ (cluster of thick
forest) and ‘khand’
(tract of road.3i).Tribal population in this area is
mainly dependent on
the forest produce, which they either sell in the market
or exchange for
the food grains.
Out of four villages chosen for the study, one
‘1
<1
J]
J
4
1*11
7
one
tribe and rest three villages
-
is inhabited by Santhal
t by the Pahadia tribe
Pahadias have B dravidian background, speak dravidian language who
migrated from south mdia."" The total number of this tribe is less than a
lakh, there is concern that they might become
extinct. Government has
banned family planning programmes in this community.
5.2 NORTH 24 PRAGANAS DIST.
Residents of north 24
(West. Bengal)
Parganas district of West Bengal district.
•4
Predominently rural area. The paddy and fisheries form
’ z|
lifeline of the region. Muslims are in
significant numbers in the
•I
I
ji
population
4
I
33
the economic
<
5.3 TESTS USED
DAT - After cleaning the thumb, the skin
lancet, a large drop of blood
was punctured with a sterile
was allowed to fill the circles on the DAT
filter paper. It was allowed to air dry .25
LEISHMANIN SKIN TEST - After antiseptic
antigen intradermally into the volar
measures, Injecting .1 ml of
surface of the forearm . A palpable
nodule, 5 mm or more in diameter after 48-72 hours,
positive.16
is considered
methodology
5.4 For Community based - cross sectional study
>• Census of entire
entire popuiation of the four tribal study
conducted for sampling purpose. Eeety individual in each
according to hierarchy Wthe family was
2. Population in the
sampling technique,
enlisted as the study sample.
3- The sampled individuals
family
enumerated.
census record formed
Systematic Random
was
sampling frame. Through
every fourth individual was
'VCre ViS“ed at their Place of residence, and
purpose of the study was explained
4. Voluntary informed
clinical informatio
5.5 Hospital based
1.
consent was obtained before taking
n and subjecting them to the
necessaiy
screening tests.
-cohort study.
Data of treated Kala-
a^ar cases from Government Hospital in 24
paraganas district of West Benga., was obtained for the
- a
*2000.
the penod of 1999
2.
.
completion of treatme
cases were selected on the basis of
(treatment cohorts) months from
the reference month August 2000 (110 days) was short-listed
3.
The cases were traced and explained the purpose of the study.
After obtaining the voluntary informed
consent, they were subjected to
DAT.
4.
The same treatment cohorts
were repeat tested again after three
months (Nov.2000) by applying DAT test.
5.
I
The titres of phase one and phase two tests
were analysed fox
dynamics of the antibodies for the period.
C
ir
CO *
cn CJ
7
^•r
a
0
’
J"
I
I
I
I
,'n-
i
7) "
(1
I
I
q4
©0n2j|
GT
*
"*
t
■;-^t
I
K’SBMi jfl
■^It-IAGL
1/
F>[t-
I
I
■ tm
TW
(P
o
ti
rr
”3
'■•■CH
■5.A
1
3>
/
Z
>
J
o
<c ’
-J
n f ?
(2 -
11J
r>
/•
J
J
£
z
t
>
X
I I
I
rr
5
;• cp
r
<3
• £-
__O
d
h£»H yy*
V-.-f,
5
V
r
r
- »
V?'
fa
\p
c
c
’
®
j-vckj
-;
c
.£
<—
4=
iI i 7-: !1 -PC
■
-
I-
r ii i.?i J -i I
-?
<□ pototaQ^j
a
i
•
|
i
|
"r
—
<
I
I
Mak BHI7A
@®S50
L •
T
tei.
®
I
{
\
i*' •
A**
5-‘- ■'
;
p*.:,. :
ViltfS
results
6.1AGE AND SEX DISTRIBUTION
OF STUDY POPULATION*
Table -1.
age group
0-.9
10-19
26
(41.3%)
12
20
{56.6%)
12
20-29
30-39
40-49
Above 50 year
■ about
MALE
8
[88.9? y_____
8 '
(80o/o)
FEMALE
37
_______ (58.7%)
63
13
________ (52%)
16
25
(44.4%)
36
13
_______ (52%)
1
______ (11.1%)
2
_______ (20%)
peop,e eou.d n0[ bc incIudcd jn t,,is (ab|e
not available.
TOTAL
25
9
10
ICE
6.2 PREVALENCE OF
leishmanin skin test positivity
In Paharias and Santhals community
Prevalence - 44.4% (95%CI = 33%-55%)
6.3 PREVALENCE OF DAT SEROPOSIVITY (> 1:800)
In Paharias and Santhals community
Prevalence - 44% (95%CI=35%-53%)
38
6.4 ANNUAL RATE OF INFECTION IN AGE GROUPS
Table No-2.
AGE
GROUPS
CUMULATIVE
INCIDENCE
1-10 yrs
19.4%
ANNUAL
RATE OF
INFECTION
4%
11 -25 yrs
61.1%
5%
26-40 yrs
66.7%
3%
S 40 yrs
36.4%
.8%
CALCULATION OF ANNUAL RATE OF INFECTION
e“ = 1- CI
I =
-In (1-CI)
t
where,
CI- Cumulative Incidence.
I = Incidence.
t = Time (median age)
i r<
Graph No. 1
LEISHMANIN TEST POSITIVITY RATES AMONG AGEGROUPS
.V 70
LEISHMANIN SKIN TEST IN AGEGROUPS
60 ■
‘v5
o 50-
CL
C 40’c
£ 30-
£2
0} 20.f
10__
1-10
11-25
26-40
age groups
40
>40
6.5 EFFECT OF SEX ON LEISHMANIN SKIN TEST
Table -3.
MALE
(48)
45.8%
FEMALE
(51)
43.1%
no. in parenthesis is no. of people examined
% is percentage positivity
X 2 = .67 ( P>.O5 )
OR = 1.12
[ 95% CI .47 to 2.66
6.6 EFFECT OF AGE ON LEISHMANIN SKIN TEST
Table - 4.
1-10 yrs
(36) I 19.4%
(18)
61.1%
26-40 yrs
(33)
66.7%
>40 yrs
(H)
36.4%
no. in parenthesis is no. of people examined
% is percentage positivity
X 2 for linear trend =5.287 (P< .05 )
41
]
c
6.7 EFFECT OF DAT TEST ON LEISHMANIN SKIN TEST
Table -5.
DAT >800
(35)
48.6%
DAT < 800
(43)
44.2%
no. in parenthesis is no. of people examined
% is percentage positivity
X2=
.15
(P>.O5 )
OR =1.19
( 95% Cl=.44 to 3.22 ]
6.8 EFFECT OF FAMILY SIZE ON LEISHMANIN SKIN TEST
Table -6.
KAM!LY>6
(30)
73.3%
FAMILY<6
(59)
37.3%
no. in parenthesis is no. of people examined
% is percentage positivity
X2 =
3.03
(P=.O8 )
OR = 2.06
42
[ 95% CI = .84 to 5.06
]
6.9 EFFECT OF SPLEEN SIZE ON LEISHMANIN SKIN TEST
Table -7.
PALPABLE
(43)
32.5%
NON
PALPABLE
(30)
53.6%
no. in parenthesis is no. of people examined
% is percentage positivity
X 2 =
4.35
(P> .05 )
OR = .42
( 95% CI= .17 to 1.03 ]
6.10 EFFECT OF FEVER ON LEISHMANIN SKIN TEST
Table -8.
FEBRILE
(20)
35%
AFEBRILE
(79)
46.8%
no. in parenthesis is no. of people examined
% is percentage positivity
X 2 =
.91
(P> .05 )
OR = .61
43
[ 95% CI = .19 to 1.87 ]
6.11 EFFECT OF SEX ON DAT POSITIVITY
Table -9.
MALE
(77)
46.75%
FEMALE
(69)
40.5%
no. in parenthesis is no. of people examined
% is percentage positivity
X2=
.56
(P> .05 )
OR = .78
[ 95% CI= .38 to 1.58
6.12 EFFECT OF AGE ON DAT POSITIVITY
Table -10
110 yrs
26-40 yrs
>40 yrs
(43)
39.5%
(29)
48.3%
(40)
50%
(17)
41.2%
no. in parenthesis is no. of people examined
% is percentage positivity
X 2 for linear trend = .197 ( P>.05)
44
]
6.13 EFFECT OF FEVER ON DAT POSITIVITY
Table -11.
FEVER
(29)
44.8%
AFEBRILE
(H7)
43.6%
no. in parenthesis is no. of people examined
% is percentage positivity
X 2 = .01
(P> .05 )
OR = 1.05
[ 95% CI= .43 to 2.56 ]
6.14 EFFECT OF LEISHMANIN SKIN TEST ON DAT POSITIVITY
Table -12
LST +VE
(36)
47.2%
LST - VE
(42)
42?o
no. in parenthesis is no. of people examined
% is percentage positivity
X 2 =
.15
(P> .05 )
OR =1.19
45
[ 95% CI= .44 to 3.22 ]
c
6.15 EFFECT OF SPLEEN SIZE ON DAT POSITIVITY
Table -13.
PALPABLE
(69)
36.2%
NON
PALPABLE
(77)
50.6%
no. m parenthesis is no. of people examined
% is percentage positivity
X 2 =
3.07
(P=.O8 )
OR =
.55
[ 95% CI= .27 to 1.13 ]
6.16 EFFECT OF FAMILY SIZE ON DAT POSITIVITY
Table -14.
FAMILY >6
(54)
55.5%
FAMILY < 6
(91)
56%
no. in parenthesis is no. of people examined
% is percentage positivity
X 2 =
• 00
(P>.O5 )
OR =.98
[ 95% CI= .47 to 2.04 ]
46
c
6.17 EFFECT OF CUT -OFF TITRE > 1; 800 ON VALIDITY OF
DIRECT AGGLUTINATION TEST
Table-15.
D +
D-
DAT
>800
74
51
DAT<800
8
67
82
1 18
Sensitivity
Specificity -
.97-
56 7-
Likelihood ratio-
-17
2.04
6.18 EFFECT OF CUT -OFF TITRE > 1 ; l600
ON VALIDITY OF
direct agglutination test
Table-16.
D+
D-
DAT >1600
69
21
OAT<1600
13
97
82
118
Sensitivity-
84 •/.
Specificity-
82/
Likelihood ratio- 4.6
6.19 EFFECT OF CUT -OFF TITRE > I: 3200 ON VALIDITY OF
direct agglutination test
Table -17.
D+
D-
DAT >3200
52
20
OAT<3200
30
98
82
1 18
Sensitivity- f>3 7-
Specifictj- -
83/.
Likelihood ratio- 3.7
49
6.20 EFFECT OF CUT-OFF TITRE > 1: 6400 ON VALIDITY OF
DIRECT AGGLUTINATION TEST
Tablc-18.
D+
D-
DAT >800
38
2
DAT<800
44
1 16
82
1 18
Sensitivity-
46/
Specificity -
98/
Likelihood ratio- 23
50
6.21 MEAN TITRE LEVELS OF ALL TREATMENT COHORTS IN
PHASE-1
Table-19.
TREATMENT
COHORTS
UNDER
LOG MEAN
TITRE
GEOMETRIC
MEAN TITRE.
4.05
11220
3 MONTHS
3.51
3235
6 MONTHS
3.60
3981
9 MONTHS
3.46
2884
12 MONTHS
3.60
3981
TREATMENT(0)MONTH
Note : Tluii < v< n ;ificr 12 months of treatment the titre remain very high above
the seropositive cut off of >1:800 within the treatable range.
6.22 MEAN TITRE LEVELS OF ALL TREATMENT COHORTS IN
PHASE-2
Table-20.
TEATMENT
LOG MEAN
GEOMETRIC
COHORTS
TITRE
MEAN TITRE.
3 Months
3.97
9332
6 Months
3.26
1819
9 Months
3.17
. 1479
12 Months
2.72
524
15 Months
2.96
912
note that The Nine month group has become sero negative < 800 in the
12th month in 2nd phase.
52
c
6.23 COMAPARISON OF MEAN TITRES OF ALL THE TREATMENT
COHORTS.
Table-21.
TREATMENT
GEOMETRIC
GEOMETRIC
COHORTS
MEAN TITRE.
MEAN TITRE
IN PHASE-1
IN PHASE-2.
0 Month
1 1220
9332
3 Month
3235
1819
6 Month
3981
1479
9 Month
2884
524
1 2 month
3981
912
note the differences increasing widely with increasing time
53
Graph-2
INDIVIDUAL LOG TITRES OF TREATMENT COHORTS IN
PHASE ONE AND PHASE TWO
5
Individual logtitres in phase-1
4
co
0)
k_.
3
p
2
Ft
1
Q
CD
O
0
0
3
9^
6
12
15
Treatment duration
Graph-3.
5
Individual Logtires in Phase- 2
4.
CM
(D
CO
(0
Q.
2
3.
9.
.
a
1<
Q o.
o
0
9
6
9
12
TREATMENT DURATION PHASE - 2
54
:15
PHASE2
18
Graph-4.
DECLINING TITRES IN PHASE 1 AND PHASE 2 WITH TIME
40
3.8
36
34
F; 3 2
Q 30
O 28
26,
PHASE
<5
03
6
9
TREATMENT DURATION
I
i
55
12
15
2.
1
Graph-5.
MEAN OF DIFFERENCES IN LOGTITRES OF TWO PAHASES
.8
<D
CD
O
c
ro
e
6
4
.2
00
Mean of differences in logtitres of two pahses
_.lll
0
3
6
9
Treatment duration
1
12
DISCUSSION
I
Kala-azar is one of the major public health problems in India.
Bihar
accounts for the lion's share with nearly 75% of the total cases reported
from India. Tribals in Bihar are worst affected with the disease.
In present study we attempted
to assess the magnitude of the
leishmanial infection in the tribal area by using two screening tests
namely
Direct agglutination
test
and
Leishmanin
skin
test.
The
leishmanin skin test has been around since 1940s.. Direct agglutination
test is relatively a newer test since last 15 years.
Prevalence of the Leishmanin Skin test positivity in our study area was
44.4% (95%CI= 33%-55%) . Our prevalence figure was similar to the one
reported from Mcdiicrrancan
region 44.2%( in valley zenaf From India
prevalence figures are available from West Bengal (19.2)% reported by
nandy et al.[ 1987]
J
i
1
The significance of the prevalence of this magnitude is in the fact that,
kala-azar epidemics do not occur in communities when more than 40%
of the population is Leishmanin skin positive i.e., immune to the
infection.33
t
c
57
/The prevalence of leishmanin skin test positivity depends on
1) the endemicity of the disease
2) The immune status of the population
against the leishmanial
infection and
'
3) the transmission pattern of the disease i
in and out of an epidemic or
endemic focus.
Interpretation of the leishmanin
test survey requires consideration of
factors such as time during epidemic when the
survey was conducted,
past experience of the community of such epidemics and
age of the
community at that particular geographical location and the frequency of
the population exchange.
Leishamanin. skin test gives a good indication of past infection in the
community. Since Leishmanin skin test is a life long phenomenon,
specific point prevalence is an indication of
that specified age groups,
Cumulative Incidence in
which in our study were
66.7% and 36.4% in children, youth, middle
age
19.4%, 61.1%,
age and old population
respectively.
The knowledge of Cumulative Incidence for
a given disease helps to
compulc lhe annual rale of infection
for that disease in a given
community In our study area the annual rate leishmanin infection was
almost same in all the age groups ranging from3% to 5%.
58
This was an
interesting finding, especially so when compared annual rates of
infection for disease like TB which is about 1%. in India.
While Leishmanin test is a good indicator of the past infection in the
community shown by
hypersensitivity to the leishmanin antigen by cell
mediated response, it does not give any information- about recent
infection which mainly elicit humoral response in the body. Since our
objective was
to
study the prevalence of leishmania infection in the
community irrespective of whether it was past or present , we had to
employ another serological test called Direct Agglutination test,
which
reacts to the circulating antibodies. Leishmanin skin test in recent
infection or active disease will be negative due to suppression of cell
mediated immunity there by not picking up active infections.
With Leishmanin picking up the past infection and DAT picking up the
recent infection and active disease we could have a better assessment
disease burden in the community.
In our study area prevalence of sero positivity ( titre
> 1:800) to
leishmania infection was 44% ( 95%CI=35%-53%). By employing DAT
test we could capture extra 18 (23.1%) cases in the community in whom
the leishmanin skin test was negative.
Having both serological and leishmanin test result to our disposal, our
interest was to look for any correlation between between the two results
59
I
and especially to see whether leishmanin skin test
positivity was higher
in seronegative as compared to the seropositive. No negative correlation
was seen.
Earlier studies have shown male preponderance in kala
-azar
34 a
retrospective study conducted on the Bihar epidemics i
in 70s by CP
Thakur 3,showcd the male : h inale ratio was 5.5 :1. In
such phenomenon was not obscn’ccl. In
our community
our hospital study there was
male preponderance, however this raises
two key issues whether the
probablity of disease given infection is lower for females or is it due to
health seeking behavior difference between sex.
The young and
Middle age groups showed higher rates of leishmanin
skin test positivity 61% and 66.7 %
respectively compared to the
extremes in d,e age speeirum 19.4% in children and 36.4% in above 40
years groups. This is ntiribuied lo (he high transmission of infection in
1
I
kala-azar endemic areas, where the population get infected early in
childhood, consequently by noddle age, half of the population
would
have experienced leishmanial infection and hence be immune to disease.
I
Middle aged group showing
higher infection is also reported by CP
Thakuru. One possible reason for the low leishmanin skin
i
in old age groups could be due
test positivity
to fading immunity. Nandy et al 2*
reported 19.2% in 1-10 years group 25.6% after 40
year. Studies from
Mediterranean region6 showed age group 50-70 year with
maximum
leishmanin positivity. Badaro et al reported
predominantly higher higher
60
infection in children under 15
in Brazil, where adults get rarely
infected.1'
In sharp contrast to leishmanin skin test, there was little difference in
DAT sero positivity in different age groups.
The absence of association between Family size and kala-azar can be
explained on the fact that, over crowding has no bearing on the
transmission of leishmania infection, unlike in diseases like tuberculosis
or measles.
We looked for any association between DAT sero positivity with fever and
splenomegaly as surrogate variables for kala-azar, since the probability
of the kala-azar is higher in cases with these signs in endemic area, but
no association could be found. Reasons for absence of correlation
between
seropositvity
with
surrogate
variables
like
fever
and
splenomegaly could not be explained since DAT is a test for recent
infections.
High titre of antibodies can be elicited in subclinical and aymptomatic
cases. Although the mean level in such cases will always be lower than
clinical cases.15
Incidentally DAT posed more questions in our study than it could solve,
l.what is the status of the individuals who are apparently healthy, being
negative to the leishmanin
skin test but seropositive to DAT.
61
2.Why there was no difference in seropositivity rates, between different
age groups as in leishmanin skin test.
We attempted to find the answers for the above questions by conducting
a hospital based study with the objective of studying dynamics of the
sero titre in kala-azar cases after the cure. We began studying the
dynamics ol the antibody level in cured kala -azar cases, by measuring
the antibody levels in two phases. In the first phase blood samples
were
collected 82 kala azar cases based on their completion of treatment like
0( just completed),3,6,9,1 2 months prior to the reference month (June)
when our community study was conducted,
15 cases were recruited in
each treatment cohort. Same cases were repeat tested after 3months.
The results of the hospital based study showed declining pattern in (he
antibody levels in 82 cases successfully treated for kala-azar, but did not
show any definite trend in relation to time. The titre
with time but the consistency was absent. The
mean titre level during
the treatment was 1 1220, which went down to 3235
i
seem to decline
at 3rd month then
increased to 3981 at 6 months, dipping to 2884 i
in 9 months again. The
j
upsurge to 3981 at 12 months could not be explained, which in effect
meant the antibody titres returning
back
to 6
level.
back to
6 month
month level.
The
1
fluctuations especially the upsurge towards the later months could
be accounted for. Since
not
< >iilv one• cohoi t (9 month ) showed its mean
titre dipping from 2884 to529 wc could not tell with conviction, how long
do the
kala azar cases take to convert into
sero-negative status
—<a
which needs an elaborate* cohort study which is out of scope for our
present study.
We could show the means of differences between the logtitre values of
phase one and phase two was significant F statistics=7.41 (p<.05) The
differences increased with the time after completion of the treatment.
This implied that the fall in antibodies is slower iin the beginning and
gradually accelerate with the time.
The variation and inconsistency in the antibody dynamics lowers validity
as diagnostic test, which explains misclassification of many normal
people in the cornmunitj' as diseased through seropositvity with higher
titres, seriously undermining the validity of DAT as diagnostic test.
Since the validity of any given test is dependent on prevalence of the
disease, we were prompted to look into the validity of DAT with different
cut-off titre. For this reason we recruited (n=82) treated kala-azar cases
from referral hospital of West Bengal into case series and study subjects
from community without fever (n=118)were included into control series.
We looked at the effect on the validity pn DAT with different cut off titres.
The cut off titre of 1:1600 gave better validity (sensitivity=.84 and
specificity^ 82)
than
ciiiimtlv used
sensitivity=.63 and spccificitv=.82j
t
cut off titre of
1:3200
(
8. SUMMARY Xnd CONCLUSION
Kala-azar is a major public health problem In our country. We have to
tackle it by
giving due priority. Our study showed 44.4% leishmanin
skin test positivity
prevalence
and 44%
DAT seropositivity , indicating
rates of leishmania infection in
the
high
tribal community
. The middle age groups showed higher rates of positivity.
Leishmanin skin test showed significant association with age. The male
to female ratio for disease was similar, we did not find the male
preponderance.
Leishamnin
skin
test
had
clear
cut
results
for
population as to past infected or not.
DAT was found to be good epidemiological tool for community survey
but its validity as diagnostic test was not very high. There was no
association
between DAT seropositvity and other variables like age, sex
or surrogate variables for kala azar like splenomegaly and fever. DAT
results showed ambiguity in classifying the population into recent and
past infection.
Our hospital based study showed declining trend iin the antibody levels
with no definite trend in relation with time. The variation in declining
antibody level was high. The means of differences of antibody levels in
!
the phase-1 and phase- 2 was statistically significant. The effect on the
.7
1:
DAT Validity was measured by adopting different cut-off titres of
.-•k.
antibody levels. The titre of 1:1600 was found to have the best diagnostic
validity for DAT in Santhal parganas area with sensitivity of 84% and
specificity of 86%. The likelihood ratio for 1:1600 cut-off titre was 4.6.
This was much better than the 1:3200 cut-off titre
presently used in the
area which had specificity of 83% and a low sensitivity of 63%.
Our study gives the baseline information of the
magnitude of the disease
and infection in the tribal communities in Santhal parganas area. Clear
i
Idea of the magnitude is necessary in any area for taking on intervention
■
1
programmes. The impact of the intervention programme should be
measurable in any evaluation.
For disease control and prevention, test/s used
and
screening programs should conform
repeatability,
i
acceptability,
administration and low cost
simplicity,
to
for community surveys
criteria like Validity,
rapidity,
safety,
case
of
Both Aldehyde and Bone marrow tests
which are the most widely used test for diagnosing kala
azar, are not
■<
»
feasible for screening purposes.
I
Sternal bone marrow puncture and aspirate the definitive test for Kalaazar diagnosis is very painful procedure, intimidating
the patients.
Moreover it involves technical expertise and therefore is not feasible in
in
i
i
field conditions.
Aldeyhyde test is a very simple but has low sensitivity and specificity. It
continues to be mainstay for lab diagnosis of Kala-azar infection in rural
<
and semiurban
areas where the disease burden
maximum.Aldehyde test is not positive until a
onset thus further missing the disease in
of kala-azar is
few months after disease
undetected cases of Kala-azar
DAT test is a very simple test to
carryout, where in collection of
specimen is very simple, involving collection few drops blood
sample
from finger prick on the filter
paper, which could then be sent to
concerned laboratory', even by
post. Thus, ideally suited for Seroepidemiological work
The
DAT
and
Leishmanin
epidemiological tool
skin
tests
are
most
widely
used
all over the world for conducting field studies
assess the magnitude of the disease in the community
to
c
9. LIMITATION
i
Bone marrow test could not be carried out for definitive diagnosis of
kala-azar .
The Hospital cases could not be followed for longer time due to time
constraints.
Many of the cases of kala-azar in referral hospital were diagnosed on the
basis of DAT
10. RECOMMENDATION
Use of multiple test in series or parallel to increase sensitivity and
specificity, for diagnosis, in the absence of Bone marrow test.
An elaborate Cohort study to understand the long term dynamics of
antibodies.
12. BIBLIOGRAPHY
1.
i
Bora D. Epidemiology of visceral leishmaniasis in India. NMJI
1999;12(2):
2.
Is the Alaram Justified, Down to Earth, April 30,1999:37-40
3.
Thakur CP, Kumar K. Post
leishman.asis a
neglected aspect of kala-azar control programmes. Ann Trop Med
Parasitol 1992 Aug; 86(4):355-9
4.
WHO. Control of leishmaniasis. Technical report series: 1990.
5.
1 lartth AE, kolk, Al Id, Eager PA. Leeuwenburg J, Faber JF, Muigai
R. K.ugu S. Laarman JJ. Evaluation of a newly developed direct
agglutination test (DAT) for serodiagnosis and sero-epidemiological
studies of Visceral leishmaniasis comparison with IFAT and
ELISA. Trans Roy Soc Trop Med Hyg 1987;81:603-6
6.
Pamptghone S. Manson-Bhar PEC, Placa La. M, Borgatli MA and
Musumeci
S.
Studies in
Mediterranean
kishmaniasis' 3 the
Letshmanm skin test in Kala-Azar. Trans Roy Soc Trop Med Hyg
♦1
1975; 69(1): 60-7.
7.
AC 1 ION AlD,Kala-Azar in India.Thcmes in
i Development Series-2,
Banglaore : Development support division.
1995.
8.
Chatterji KD. Parasitology protozoology and Helminthology
in
relation to Medicine. 12 th ed. Calcutta
Calcutta : Chatterjee Medical
Publishers, 1980.
9.
Krishna das KV, A Short text book of Medicine. New Delhi: Jaypee
brothers, 1986: 92-99 [viscral leishmaniasi]
10.
Prasad LSN. Kala-azar. Indian J Pcdiatr 1999;66:539-46.
11.
Neva A Franklin. Leishmaniasis. In: Wyngarden, Smith [editors],
Ccsil
Text
Book
of
Medicine, 18
th
ed.
Jourich
:WB
Saunders, 1988,: 1869-75.
12.
Hati AK. Kala-azar-an enigmatic disease,Ind J Pub Hcalh 1989
April-June;33(2): 45-54.
13.
Plorde James J. Leishmaniasis. In: Issebacher Kurth J, Adan
Raymond D.Braunwal Eugone, Robert G, Dorf Peter S, Wilson
Jane D [editors].Harrison's principles of internal medicninc,9th ed
Tokyo: Me Graw Hill: 873 - 876.
14.
Seal SC, Return of Kala-azar[editorial], Ind. J Pub Health. 1977
July Sept ;2 I (3).
15.
Soulimni HL, Moxham J [editors]. Text Book of Medicine. New York
:Churchill
livingstone,1990
:305-7.[tissue
blood
protozoa][leishmaniasis]
16.
Bryceson Anthony DM. Lishmaniasis. In : Beeson Paul B,Mc
Dermott Walshfeditors). Text Book of Medicine. 14,h ed. Toronto
:WB Saunders, 1975: 490-495.
17.
Aikat BK.Pathania AGS.Sehgal Shobha.Bhattacharya PK.Dutta
Usha,Pasricha Neelam, et.al. Immunological responses in Indian
Kala-azar. Indian J Med Res 1979 Oct; 70:583-91.
'
I
18.
Hailu
A.Pre and
leishmaniasis.
post treatment antibody levels in visceral
Trans
Roy
Soc
Med
Trop
Hyg
1990
Sept-
Oct;84(5):673-5.
19.
Bryceson Anthony DM. Visceral leishmaniasis. In : Wayngarden
James B, Smith Lloyd H,Cesil[editorsj. Text Book of Medicine Vol2.Mexico: WB Saunders, 1982 :1869-75.
20.
Vakil Jal Rustam. Text Book of Medicine.2 nd rev.ed.Bombay : The
Assn. Of Physicians of India, 1973 : 182-9.[kalaazar]
21.
Thakur CP. Leishmaniasi. In : Sha Shanti J[eclitor].APl Text Book
of Medicine. 4 th ed. Bombay : Association of Physicians of
India,1988:131-3
22.
Aggrawal Praveen.Handa Rohini,Singh Sarman and Wali Prakash
Jyothi. Kala-azar- new developments in diagnosis and treatment.
Ind J Pcdialr 1999;66:63-71.
23.
Harilh E.Kolk AHJ,Eager PA.Leeuwenburg J,Muigai R.Kiugu S,
agglutination
direct
Simple
and
economical
scrod iagnosis
and
sero-epidemiological
cl.al.
studies
of
test
for
visceral
leishmaniasis. Trans Roy Soc Trop Med Hyg 1986; 80:583-7.
24.
Singh Neena,Singh GS, Sundar Shyam, Vinayak VK.Evaluation of
direct agglutination test as an immunodiagnostic tool for kala-azar
in India. Trans.Roy Soc Trop Med Hyg 1993;87:276-8.
25.
Zillestra EE.Ali Siddig M, El-Hassan AM,E1-Toum Islam A,Satti
Maria,Ghalib
Wil,
Eager
PA.
Direct
agglunination
test
for
diagnosis and sero-epidemiological of kala-azar in Sudan. Trans
Roy Soc Trop Med Hyg 1991; 85:474-6.
7">
26.
Nandy A.Neogy AB.Chowdhary AB, leishmanin test sruvej' in an
endemic village of Indian Kala-azar near Calcutta. Ann Trop med
Parasilol 1987;81 (6) :693-9.
27.
All A, Ashford RW, Visceral leishmaniasis in Ethiopia II.Annual
Leishmanin transformation .Is Positive leishmanin reaction a
lifelong phenomenon?. Ann.Trop Med Parasitol; 87(2): 163-7.
28.
Sinval P.Pilho Brandao.Diarmind CL,Brito Maria EF, Shaw Jeffeiy
J,Davies Clive R, Epidemiological surveys confirm an increasing
burden of cutaneous leishamanin. Trans Roy Soc Trop Med Ilyg
1999;93:488-94.
29.
Sokal Joseph E,Measurement of delayed skin testfeditorial], New
Engl J Med 1973 Scpt;1293( 10):50 1-2.
30.
Chatterjee Prabir. Health medicine and Pakur - a monograph
31.
RTC
Tarunoday.
Tribal
unity
a
political
perspective.Annual
seminar.Feb Ranchi. 1999
32.
Bain bridge.The saorias of Rajamahal hills memoires of the asiatic
society of Benal volume2,No. 1:1906.
33.
Kala azar Rides again.Assn. Phys Ind. Vol.26. Junel978;529-31.
34.
Badaro R,Jones Tc, Lorenco R, Cerf BJ.Sampaio D.carvalhoEM, et
al. A prospective study of visceral leishmaniasis in endemic area of
Brazil. The journal of infectious diseasesl986 oct;154(4):639-649
!I
35.
Badaro R,Jones Tc, Lorcnco R, Cerf BJ,Sampaio D,carvalhoEM, et
al. A prospective study of visceral leishmaniasis in endemic area of
Brazil. The journal of infectious diseasesl986 oct;154(6):10051011.
36.
Park. K. Park’s
text
book
of preventive
and
social
medicine.
Jabalpur. Banarasi das. 1997
37.
Thakur CP. Epidemiological, clinical and therapeutic feature of
Bihar kala-azar (including post kala azar dermal leishmaniasis).
Trans Roy See Trop Med Hyg 1984;78:391-98.
t
11. APPENDIX I
PROFORMA FOR COMMUNITY BASED STUDY
ID NO.
NAME
GUARDIAN NAME
AGE
SEX
FAMILY SIZE
TRIBE
VILLAGE
H/o KALAAZAR
C/O OF FEVER
SPLEEN SIZE
DAT TITRE
LEISHMANIN SKIN TEST
11.2 APPENDIX II
PROFORMA FOR THE HOSPITAL
BASED STUDY
ID NO
NAME
AGE
SEX
VILLAGE
TREATMENT MONTH
DAT
- Media
318.pdf
Position: 1804 (5 views)