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Rajendra Memorial Research Institute of Medical Sciences, Patna Research Projects
Reports of completed projects
Nucleic acid probe
for diagnosis, detection and epidemiology of
kala-azar.(DBT supported) Principal
Investigator : Dr. S. K. Kar,
RMRIMS Co-Investigator : Dr. V. M. Katoch, CJIL, Agra Funding
Agency : Department of Biotechnology, Govt. of India. Keywords : rRNA, rDNA, RFLP, SAG Study
type code : LAB Objectives
: The
objectives of this study are as
follows: 1.To
develop probes and assays for rRNA finger printing of Indian status of L.donovani and L.tropica for species variation/ strain heterogeneity. 2.To
identify specific gene sequence in rRNA gene region (specially the NTS) of
prototype Indian strains. 3.To
design probes and PCR primers for detection / strain variation. 4.To
attempt to develop prototype kits after initial evaluation specially those
using non- radioactive markers for easy application in the field. Introduction
: The
study was initiated with an aim to develop nucleic acid based method, which may
be suitable for use in the field and to utilize it for diagnosis, detection and
characterization of leishmania so as to gain an insight on disease epidemiology
and the problem of the unresponsiveness to SAG. It was proposed to sequence the
non-transcribed spacer region of ribosomal gene as well as other relevant
stages with rRNA and develop probe/PCR primer. Methodology : Establishment
of culture Field survey
was carried out to identify cases of kala-azar. Patients attending OPD and
admitted to indoor were subjected to splenic/bone marrow aspiration and skin
snips of PKDL for diagnosis. These cases were from different geographical regions.
These specimens were cultured in modified NNN biphasic medium (commercially
available Brain heart infusion Agar medium with 30% rabbit blood) and in
Grace’s / Schneider’s Drosophila Insect Tissue Culture media for primary
isolation of leishmania promastigotes. The promastigotes were tried for
adaptation. At present, 38 isolates are being maintained in biphasic media and
mass cultured in insect tissue media supplemented with 15-20% FCS. Log phase
promastigotes culture were collected by centrifugation in a refrigerated
centrifuged at 3000 RPM for 20 minutes. Apart from these clinical isolates, WHO
reference strain of L.donovani i.e. DD8 was also grown and processed as above.
Later, the different isolates of leishmania promastigotes were cryopreserved in
liquid nitrogen. Extraction of Nucleic acids (DNA/RNA) Altogether 38 isolates are in in-vitro maintenance which
are isolated from different geographical regions. DNA and r-RNA were isolated
by step wise isolation procedure after lysing the parasite (1 gm. App.) in 6 M
Guanidium Hydro-chloride buffer as described by Katoch and Cox,1986. Isolation
of DNA was also done by “Physico-chemical Method”. Nuclear DNA was extracted
from these isolates obtained from clinical specimens . For this equal volume of
chloroform - isoamyl alcohol (24:1) were added to lysed and ethanol preserved
supernatants and were separated by centrifugation. From the aqueous phase ,
0.3M DNA was precipitated with 0.75 volume of iso-propanol and was separated by
centrifugation at 4000G for 10 min. The pellet was washed with 70% ethanol.
Air-dried DNA was dissolved in TE buffer and stored at -20 degree C.
Subsequently, total RNA was isolated from WHO reference strain of L.donovani
(MHOM/IM/80/DD8). Probe preparation Probe was prepared from WHO reference strain of DD8 using
Chemiluminiscent assay with ECL kit and DIG kit later on. Typing of leishmania isolates with
reference strain rRNA probe The genomic nuclear DNAs from the promastigotes of 21 out
of 38 different isolates were restriction digested using ECORI, HindIII and
HaeIII enzyme which was subsequently electrophoresed in 1% agarose gel. The
resolved DNA pattern was blotted on to nitro-cellulose paper (SIGMA,13x13
sq.mts.) . The labeled RNA was used as a probe for southern hybridization at 65
degree C over night, which was subsequently auto- radiographed. Of the 21
samples screened, result was obtained
for five samples. Results : 1. The
purified DNA was found with sufficiently high proportion of DNA (Purity 1.8 -
2.0 & Yield 104 - 220 micro gm. per ml) which was electrophoresed on 1%
agarose gel to about the same extent as L DNA. 2. Treatment
of DNA preparation with restriction endonuclease resulted into production of a
range of DNA patterns which could be resolved as clear bands on 1% agarose gel. 3. On Southern
blotting, distinct bands were detected by autoradiography corresponding to
fragments of DNA with rRNA probe. This reflected that DNA isolated by the above
procedures is suitable for use in leishmania typing. 4.Digestion
pattern was compared between KA and PKDL isolates and also among Kala-azar
isolates both responsive and unresponsive to SAG therapy. It was observed that
: a. Five restriction sites obtained with
Kala-azar isolates, DNA digested with HaeIII. b. Two restriction sites obtained with
PKDL isolates, DNA digested with HaeIII. c. HaeIII pattern was similar at one,
different in one restriction site of DNA in KA and PKDL. The difference at restriction sites so observed in KA and
PKDL isolates are being analyzed. 5. The
comparison among the Kala-azar isolates reflected the following: a. Responsive
and unresponsive cases differed at EcoRI and Hae III restriction sites. EcoRI- 4
restriction sites obtained in isolates of responsive cases as compared to 3
restriction sites in isolates of unresponsive cases (instead of 6 digestion
sites).Among unresponsive isolates, difference was also observed with single
similar digestion site. Reason for such variations is being looked into in
context of possibility of strain variation . HaeIII- Five
restriction sites were observed in isolates of both unresponsive and responsive cases however, the responsive
and unresponsive isolates differed at four sites . Base pair analysis with EcoRI was made in
Kb which reflected that responsive
isolates had the base pair in the range of 3.73 - 34.59 and unresponsive
isolates in the range of 2.77 - 39.15(Isolate1-95/97) and 5.6 - 28.3 (Isolate2
- 29/98). 6. A PCR
system for DNA finger printing amplifying the NTS gene sequence has been
designed to find out the relation of NTS gene sequences with different
leishmania isolates 7.The RFLP
pattern had shown differences in PKDL strain as well as in one responsive and
four unresponsive to SAG from the nucleotides base pairs of reference strain
(MHOM/IN/80/DD8) of leishmania donavani, where as two responsive and 8
unresponsive to SAG had shown similarity. The strains belong to Patna,
Vaishali, Muzaffarpur, Begusarai and Buxur. Field trial of DAT in the diagnosis of kala-azar Principal
Investigator : Dr. M. C.
Sharma Co-Investigator : Dr. S. Bimal Funding
Agency : Intra-mural project Study type
code : FOR Objectives
: To
assess the efficacy of DAT in diagnosis
of kala-azar and its use in epidemiological situation at peripheral level of
health facility. Introduction
:
The
diagnosis of disease is usually made parasitologically by microscopic
examination of leishmanin stained smears of bone marrow or splenic aspirates.
These procedures are not only difficult to perform under field conditions but
often complications such as bleeding can be encountered. Serological tests to
detect specific antibodies by direct agglutination test (DAT) which is
non-invasive has been reported to compare well with parasitological result .
Furthermore, it shows its feasibility in field and infective status of host
even in very early stage. Methodology:
Eleven
batches of antigen from different strains of Log phase promastigotes of
L.donovani including WHO reference strain (DD8) were made. These
promastigotes were cultured in biphasic rabbit supplemented NNN media.
Viability of the parasites was more than 90%. Quality check of the antigen was
performed with different sera at AIIMS, New-Delhi , CDRI, Lucknow, RMRI, Patna
and CSTM, Calcutta. For standardization , filter paper blood
samples collected from 108 confirmed leishmania patients (BM/ splenic aspirate
positive) were obtained from ward of the Institute (96) and from endemic area
(12). Sera from two non-endemic area, Lohardagga and Nalanda districts were
collected(n=452) while Lohardagga situated in south Bihar and kala-azar is not
known and Nalanda district is close to Patna and recently reporting only
occasional case of kala-azar. Sera from endemic area collected from Karja PHC
of Vaishali district (n=189). Blood samples of patients suffering from other
diseases were also collected (n=60). These sera belonged to Typhoid (n=8),
toxoplasmoid (n=10), Leprosy (n=10), Malaria (n=7), Filaria (n=7), STD (n=3) and
Tuberculosis (n=15). (Table-1). Diluted samples were screened at 1:100 to
1:12800 test dilution. The test was performed as per the method of Harith et.al
(1986). Results
: For
test standardization, sera from 108 confirmed VL patients and 452 non-endemic
controls were screened using serial dilution. There was clear difference
between kala-azar sera and non-endemic sera at serial dilution of 1:800 and
above(figure-2). A total of 99 confirmed VL patients had anti-bodies titre in
the dilution range of 1:800 - 1:12800. On the other hand, 387 non-endemic sera
had antibodies detected in the dilution
range of 1:100 - 1:400 while rest 65 had <1:100 titre. None of the
non-endemic sera was positive beyond 1:400 test dilution . DAT titre obtained with sera collected from healthy control subjects, VL confirmed cases and some other parasitic diseases like Malaria, TB, Leprosy etc. The frequency distribution of titre for each category of subject is plotted(Figure-1) . This showed different mean titre for each category of the subjects. The frequency distribution of titre for VL confirmed cases and non-VL cases are plotted(Figure-2). The cut-off titre is shown at 1:800 dilution. The sensitivity and specificity of the test at each titre is estimated. The sensitivity and specificity of the test were 91.66% and 100% respectively and positive and negative predictive value were 100% and 98.7% respectively at 1:800 dilution .
Receiver Operator Characteristic (ROC) curves
are another way to determine cut-off titre (Figure-3). It is a graphic way of
portraying the trade-off involved between improving either a test’s sensitivity
or its specificity. It graphs sensitivity as a function of 1-specificity. Here,
at dilution 1:800, the curve reaches at the upper left corner of the graph
which indicates a cut-off titre. Conclusions
: It
can be stated that for purpose of diagnosis in mass screening of the population
, 1:800 cut-off titre can be taken as yardstick. For validation of DAT, the
sera from confirmed Kala-azar detected by bone marrow aspiration technique can
not serve as gold standard as a yardstick for comparison since the technique
itself has sensitivity of 80%. Hence follow up of both DAT positive and negative for disease development is also
needed. Hence the population are being followed up prospectively by clinical & DAT survey at an interval of
3 months for a year. Antigen characterization for diagnosis of Visceral Leishmaniasis Principal
Investigator : Dr. M. C.
Sharma Co-Investigator : Dr. S. Bimal, Mr.A. K. Bagachi Funding
Agency : Intra-Mural Project Study
type code : LAB Objectives : Characterization
of different antigenic fractions of
L.donovani promastigotes and to identify the specific antigenic fractions that
can be reactive to sera of kala-azar and PKDL. Methodology : Culture of 14
different isolate of Leishmania parasite from different clinical spectrum of
kala-azar including WHO reference
strain MHOM/IM/80/Dd8, were harvested from monophasic media M199 with 10% FCS.
These isolates include 5 fresh kala-azar , 8 SAG unresponsive and 1 responsive
cases. These isolates were sonicated at 150
prep/60w/20 minutes and their protein contents were estimated by Lowry’s
Method. These were electrophoresed in SDS_PAGE at 12-10% resolution in order to
get the different antigenic protein fractions using standard protein marker
(8600 Da-207,000 Da; Biorad, Cat. No. 161-0324). Electrophoresed protein fraction in SDS-PAGE
were electro-transferred into the surface of nitrocellulose paper (NCP ) for
immunobolization and then identified based on their immunological reaction with
IgG antibody and enzymed labeled second antibody probe (HRP). Immunoblot analysis was performed on sera
obtained from cases of different clinical spectrum of Kala-azar against
different antigenic fractions of 8 isolates. These sera comprised of fresh
KA, responsive, unresponsive, PKDL , TB, Leprosy and
healthy control subjects. Based on the above, diagnostic potential of antigenic
fractions were evaluated. Results : Protein
contents measured in MHOM/IM/80/Dd8 WHO
strain was found to be 1000 mg/ml .
Isolates obtained from patients ,the protein contents ranged between 300-950 mg/ml . Total 7 bands were obtained with Dd8 strain
which were 35Kda, 27Kda, 19 Kda, 15Kda, 10Kda, 9.5Kda and 8 Kda . Of these 7
bands, 8.0 Kda protein fraction was shown to react with all kala-azar sera.
However, sera from cured cases of kala-azar and other disease sera did not show
any reaction with this 8.0 Kda protein fraction. This
8.0Kda protein fraction was observed in all 14 isolates tested including DD8 .
Antigenic fraction of 8 of these isolates were electro-transferred on to the
surface of NCP and subsequently
immunobloted . Experiment was performed with different sera. The 8 Kda
protein antigenic fraction of 4 isolates (Dd8-a;37/98, 27/98, 134/98) were
shown to react only with sera from all active kala-azar cases. In while isolate
no. 53/98 having 70 Kda protein was reacted to sear obtained from fresh and
unresponsive kala-azar but not reactive to cured kala-azar and other disease
sera. Conclusions : It was
observed that sera of active and unresponsive Kala-azar cases reacted to 8 Kda. It can discriminate with fresh and
cured cases since the antigenic fraction of less molecular weight seem to have
diagnostic potential, further attempt is taken to confirm its diagnostic
ability and gene expression of the fraction Immuno- histochemical staining of dermal lesions in the diagnosis of post kala-azar dermal leishmaniasis Principal
Investigator : Dr. Neena
Verma Funding
Agency : Intra-Mural Project Study
type code : LAB Objectives : 1. To study the efficacy of immunohistochemical method using rabbit polyclonal antiserum in
the diagnosis of PKDL in comparison to the conventional methods like tissue
smear and culture. 2. To demonstrate and localize the leishmania parasite
antigen in relation to cellular infiltrates in histological section of PKDL
lesions. Introduction
:
For
the diagnosis of PKDL, methods like microscopic detection of parasite from
tissue smear and culture are not very sensitive and specific.
Immuno-histochemical staining method may prove to be more sensitive and
specific that may assist in confirming diagnosis in different forms of PKDL. Methodology
: Skin
biopsy from PKDL lesion (n=12) and sixteen control cases were collected and
imprint smear and paraffin sections of the tissue prepared. These tissues were subjected to culture in culture
media . Microscopic study of the imprint smear and H/E stained histological
sections were done for parasite detection. L.donovani promastigotes (DD8 strain)
in medium 199±10% FCS was
cultured and washed with PBS
thrice, cell counts adjusted then lysed
with distilled water and freeze thawed. After lysis of promastigotes, protein
contents of the supernatant was assayed. Two rabbits aged eight months each
were immunized subcutaneously with parasite protein antigen and FCA at multiple
sites. Subsequently five booster injections were injected at 7 days interval.
Before primary inoculation and after 3 days of each booster dose, blood from
ear vein were tested for the antibody titre by DAT & DOT ELISA. Antibody
titre was negative before antigen inoculation and it rose to >1,28,000 after
5th booster. These sera have been used as primary antibody in the IHC staining. Formaline fixed paraffin embedded tissue
sections from PKDL lesions and control cases had been deparaffinised and
rehydrated and then processed for immuno peroxides staining by peroxides
-antiperoxidase technique. Microscopically in PAP stained sections from PKDL
lesions (papulo-nodular, erythematous) many leishmania amastigotes or its
antigenic products have been seen as brown stained structure or granular
deposits beneath epidermis in the upper dermis amidst many histocytes and
plasma cells. They were sparse in the macular lesions with few scattered
lymphocyte and histocytes. None of the control samples demonstrated the brown
deposits in dermis. Results : The result of
various methods used to show parasite antigen positivity are presented in
Table-1. Table-1:
Results of various tests in diagnosis of PKDL. ---------------------------------------------------------------------------------- Tests IHC Biopsy Smear Culture Types +ve -ve +ve -ve +ve -ve ---------------------------------------------------------------------------------- PKDL(12) 10 2 8 4 4 8 CONTROL(16) 0 16 0 16 0 16 ---------------------------------------------------------------------------------- Positivity(%) 83 66.6 33.3---------------------------------------------------------------------------------- Conclusion : IHC technique
was found to be 83% sensitive as compared to 66% by tissue smear and 33% by
culture. Establishment of Leishmania bank Principal
Investigator : Mr. A. K. Gupta Co-Investigator : Dr. C. S. Lal,
Mrs. Raina Singh Funding
Agency : Intra-Mural Project Study type code : LAB Objectives
: To elicit any
species or strain variation amongst leishmania parasites isolated from patients
of different regions of Bihar by A.
Analysis of
isoenzyme pattern. B.
Evaluation of growth kinetics of different isolates of
leishmania promastigotes in liquid
culture media. C.
Evaluation of the infection pattern of leishmaniasis in
BALB/C mice. D.
Cryopreservation of different isolates. Introduction :
Different
isolates of leishmania parasites are needed for various research work and for
use in future. Leishmania parasites isolated from different KA/PKDL patients
showing same or different clinical picture/severity of disease, belonging to
different geographical region of Bihar, need their characterization ,
evaluation of variations in their growth kinetics and infection pattern and
cryopreservation. Hence, efforts are being made for establishment of leishmania
bank. Methodology : Bone-marrow
aspirates / splenic aspirates of
suspected/treated kala-azar and skin snips of PKDL patients were cultured in
biphasic media for primary isolation of leishmania promastigotes . Newly
isolated promastigotes were tried for culture adaptation. At present, 45
different isolates (KA-36 and PKDL-9) of leishmania promastigotes are being
maintained in-vitro in rabbit blood(10% approx.) based brain heart infusion
agar biphasic media with Locke’s overlay by weekly transfer and 33 isolates are
under adaptation in above media with 20% (approx.) rabbit blood. These isolates
are being utilized in different studies such as characterization of parasites
by isoenzyme pattern of using nucleic acid probe, growth kinetics of different
isolates. Twenty five (20 VL and 5 PKDL) clinical
isolates were characterized through isoenzyme profile. These isolates were D1,
D2, D3, 155 & 158 (PKDL) and 93, 95, 96, 101, 112, 7, 11, 21, 26, 29, 40,
47, 52, 53, 134, 92, 94, 156, 5 & 6 (VL). These isolates belong to
different geographical distributions. The enzyme studied for characterization
were GPI, LDH, MDH, SOD, ASAT, ALAT, 6-PGDH, G6PD, ME & PGM on poly
acrylamide gel. All the above isolates were compared with reference strain
L.donovani (MHOM/IN/80/DD8). For
evaluation of growth pattern of different isolates in Mitsuhashi-Maramorosch’s
Medium (mm medium) , at first promastigotes were sub-passaged 7 times at the interval of 3-4 days. MM
medium was supplemented with 10% (v/v) F.B.S. Gentamycin was used at the
concentration of 40/mg/ml. 5 ml of complete medium was taken in 30 ml culture
vials. Each culture vials were inoculated with 1x10^6 promastigotes/ml for each
isolates. Then the culture vials were incubated at 24 degree C in slanting
position. At every 24 hours interval after mixing well, 0.02 ml culture samples
were asceptically withdrawn from the experimental culture tubes until a decline
in numbers of viable promastigotes was apparent. Parasites were counted using
haemocytometer after making the required dilutions in 1% formalin in phosphate
buffer saline (PBS) . Growth pattern of 16 different isolates (14 KA & 2
PKDL) was studied. To
see the infection pattern of L.donovani in BALB/C mice, newly isolated
promastigotes were tried for culture adaptation and mass cultured (within 10
passages) . The promastigotes of
stationary phase culture of 33 leishmania parasite isolates were harvested by
centrifugation at 3000 rpm for 20 minutes at 4 degree C and was washed with
sterile phosphate buffer saline (pH 7.2). Promastigotes were suspended in PBS
and were inoculated in BALB/C mice (male, 6 to 8 weeks old) for establishment
of infection. The BALB/C mice with well established infection with different
isolates will be used as a source of amastigotes in future experiments. Animals
were sacrificed after 2-3 months. Splenic smears and culture were examined for
presence of parasites. Splenic homogenates were centrifuged at 500 rpm for 5
minutes at 4 degree C to remove heavy extraneous materials. Supernatant was
re-centrifuged at 2500 rpm for 30 minutes. The pallet was suspended in PBS and
was injected intraperitoneally in BALB/C mice. Infected animals were sacrificed
and 3 were found positive. Results : The
electrophoretic mobility observed of different enzymes is presented in Table-1. Table-1 : Electrophoretic mobility observed of different enzymesEnzymes Test Control(Ref.Strain) LDH 0.94 0.94 MDH 0.91 0.91 ME 0.82 0.82 SOD 0.93 0.93 GPI 0.92 0.92 G6PD 0.63 0.45 PGM 0.85 0.85 ALAT 0.94 0.94 ASAT 0.93 0.93 6PGDH 0.90 0.90 The difference was observed only with G6PD,
however in all other enzymes the zymodeme pattern was similar to DD8 pattern.
The enzyme selection criteria was based on Chance et. al. (1978), Schnur et.
al. (1981) . The isoenzyme profile difference in only G6PD mobility has also
been observed by Bichichi et. al.(1999) and Chicharro et. al. (1999). Total
16 isolates are in cryopreservation.
The optimum growth varies from 6 to 14 days and survival time varies between 16
to 27 days in different isolates studied. The study of DNA finger printing , zymodeme
pattern and growth kinetics of isolates (no.7/96) revealed significantly low
optimum growth pattern and variant in G6PD and the bands observed in finger
print was different than other isolates studied. Conclusions : The study is
continued. Trial of SAG in fresh cases of Visceral Leishmaniasis with a view to assess unresponsiveness Principal
Investigator : Dr. V. N. R. Das Co-Investigator : Alok Ranjan Funding
Agency : Intra-Mural Project Study
type code : CLN Objectives
: The
objective of this study is to assess association of various factors in relation
to the rate of unresponsiveness to SAG treatment in fresh visceral leishmaniasis cases in Bihar in open clinical
trial. Introduction
: Currently
SAG is being used as a first line drug in the treatment of Kala-azar. In last
few years, unresponsiveness to SAG have been reported. The magnitude of such
unresponsiveness shows a wide range of variability. Whether such variation
reflects variability in geographical distribution of disease, duration of
illness, level of immunological competence, Hb% level of host at the time of
reporting or initiating treatment reflects the degree of unresponsiveness need
to be ascertained. Hence, an attempt was taken to standardize such factors that
can possibly influence drug response and ascertain the therapeutic response of
SAG in treatment of fresh cases of confirmed kala-azar. Methodology
: Based
on the unresponsiveness rate ascertained through a pilot study conducted last
year, sample size of 100 fresh cases of Kala-azar was estimated for this study.
All the cases attending OPD of the Institute were assessed initially for
diagnosis .The initial assessment included X-ray chest (PA view), detailed case
history, clinical examination, haematological (TC,DC,Hb%), biochemical (SGOT,
SGPT, Serum Biliribuin, CKMB, Urea, Serum Creatinine) and parasitological
investigation (bone marrow/ splenic aspiration
for parasite detection and culture) to confirm diagnosis. For case
recruitment, the inclusion criteria was followed as per study protocol. So far,
64 fresh cases have been included in this study . All subjects recruited were administered SAG in the dose of 20
mg/kg body weight IM daily for 30 days. Investigations were carried out on 0 ,
15 and after 30th day of
initiating treatment. The cases, free of symptom and parasitologically negative, were termed as apparently cured.
Discharged cases were followed up on 1st, 2nd, 3rd,
and 6th months. Patients were termed as ultimate cured if no relapse
occurred till last follow up. Results of 64 cases are presented here as an
interim analysis. Out of 64 cases , 62 cases have completed full course
and and only 2 cases developed toxicity
hence withdrawn from the study. Results
: The
apparently cured subjects were 30(48%) and unresponsive at the end of the
treatment were 32(52%)(Table-1). The duration of illness of cases
associated with other diseases like UTI, TB, Enteric fever , hemoglobin level
of the subjects at the intake did not affect SAG responsiveness. The
geographical area has shown a significant variation of SAG responsiveness. It
was significantly higher amongst patients coming from hyper-endemic zone as compared to meso-endemic and hypo-endemic
areas .The immunity level of the patients before treatment was observed to be
significantly associated with unresponsiveness (Table-2) . Out of 64
cases admitted in the study, 2 cases developed adverse reactions like dyspnoea associated with severe anemia
and oedema during treatment which were reversible. Other laboratory parameters
were within normal limit. Table-1:
Age-sex distribution of cases completed treatment and responsiveness Age Male Female Total group Trt. Unres.(%) Trt. Unres(%) Trt. Unres(%) 5
- 9 5 2(40) 3 3(100) 8 5(63) 10-14 9 4(44) 9 4(44) 18 8(44) 15-24 10 8(80) 6 3(50) 16 11(69) 25-34 5 2(40) 4 2(50) 9 4(44) 35-44 6 2(33) 1 0(0) 7 2(29) 45>= 3 1(33) 1 1(100) 4 2(50) ----------------------------------------------------------------------------------------------- Total 38 19(50) 24 13(54) 62 32(52) ----------------------------------------------------------------------------------------------- Table-2 : Association of unresponsiveness with various characteristics------------------------------------------------------------------------------ Characteristics Unrespon. N p-value -------------------------------------------------------------------------- A.
Duration of illness >0.05 2weeks-3mon. 19(54) 35 4 mon.-6mon. 11(52) 21 7mon.>= 2(33) 6 B.
Other Diseases >0.05 Yes 4(57) 7No 28(51) 55 C.
Hb% >0.05 6 - 8
19(51) 37 8.1 - 10
13(52) 25 D.
Endemicity <0.01 Endemic 32(54) 59 Low- endemic 0 (0) 3 E.
MIF <0.05 Depressed(<30%) 27(57) 47 Elevated(>30%) 5(33) 15 ------------------------------------------------------------------------------------ Conclusions:
It is observed that unresponsiveness is not significantly associated with age
and sex .Factors like duration of
illness, association of other diseases, Hb% level have shown no association
with rate of unresponsiveness but distribution of disease from endemic zones
showed associated with unresponsiveness. The finger print analysis of the
parasite strains isolated from patients from different geographic regions are
also analyzed simultaneously to indicate any possibility of variation in the strain. A pilot study to assess efficacy of SAG in the treatment of post kala-azar dermal leishmaniasis Principal
Investigator : Dr. V. N. R. Das Co-Investigator : Dr. N. Verma, A.
Ranjan Funding
Agency : Intra-Mural Project Study type
code : CHM Objectives : To
assess efficacy of SAG ,in the dose of 10 mg/kg body weight continuously for 90
days giving rest to patients in case of any intoxication, in the treatment of
PKDL cases in Bihar. Introduction
: SAG
is being used as a first line of drug in the treatment of kala-azar as well as
PKDL. Several reports indicate variation in response to the dosages and
duration of SAG in the treatment of PKDL. Variation in the therapy of PKDL has
been observed. One report indicates that SAG in the doses of 10 mg/kg body
weight given for 40 doses by deep intra-mascular route gives a very
satisfactory cure rate (Rai, R. N. et. al.,1989). There is a need to establish
a standard drug schedule for the treatment of PKDL cases by SAG. Methodology
: For
the pilot study, 30 cases of PKDL were taken as sample size. So far, 13 cases
of PKDL have been included in the study between date of initiation till
November’99. Results
: Out
of 13 cases admitted to the study, 8 were males and rest females with age
ranged from 5-44 years. The clinical form of cases were distributed in form of
macular (7) nodular(5) and papular(1). Ten had past history of kala-azar . The
duration of onset of PKDL lesion following cure ranged from 2-12 years . Three
cases had no past history of kala-azar but had family history of disease. Skin
biopsy was carried out in all the cases prior to treatment and 10 were found
positive by microscopy or culture or both. All cases were treated with SAG in
dosage of 10mg/kg body weight daily for 90 consecutive days.The size , number,
colour of lesion and its distribution pattern were monitored every month for
three months during treatment and periodically for six months there after. So far, 8 cases completed 3 months treatment.
All were found to be parasite negative at the end of 3 months. All cases had
reduction of dermal lesion, but did not show complete clearence even at 2
months follow up following treatment. Conclusion : The study is continuing. Clinico-pathological changes in post kala-azar dermal leishmaniasis lesions Principal
Investigator : Dr. Neena Verma Funding
Agency : Intra-Mural Project Study
type code : CLN Objectives : 1. To confirm
diagnosis of different clinical forms of PKDL by imprint smear study of dermal
lesions . 2. To
correlate histopathological features
and clinical response to treatment with
parasite load in different forms of PKDL . Methodology
: PKDL
cases either attending the outdoor or admitted to the research ward of the
Institute have been studied for their clinico-pathological changes and MIF
response at intake and during different stages of treatment. So far, 165 skin
biopsies from PKDL cases have been collected
including 33 studied this year. Skin lesions were assessed for its duration, distribution, type of coalescing
with maximum size and sensation. Tissue smears were examined microscopically
for detection of leishmania parasite and cytological changes in different
lesions. H/E stained sections were examined under microscope for histology to
ascertain cellular changes and parasite load. PKDL
patients were given treatment with SAG ,10mg/kg body weight IM daily for 90
days. The histo-pathological, cytological and immunological parameters were
studied before, during and after treatment . Results
: Past history of kala-azar was present in 78.5% of PKDL cases with duration
ranging from 1 to 11 years. Leishmania
parasite was demonstrated in 65% of all
cases, where as in macular type parasite could be detected in 44% cases by
using both techniques like microscopy
and culture. Haematological parameters studied were within normal
limits. Data of 6 patients who completed full course of treatment were analysed
for parasitological cure rate, lesion persistence following full course of treatment and their sequential changes
of dermal lesion observed (Table-1). Table-1:Clinico-pathological
changes in response to 90 days SAG treatment(n=6) Before After Treatment Treatment Clinical
Nodulo-papular Absent
or papular lesion Changes
Erythematous(I) over face erythematous
on chin only Macular all over body
Macular lesion fainter Leishmania Parasite Positive-65% cases Negative in all No.-0-50/OIF intracellular Cytological Changes Mononuclear cells(+++) Number decreased, scattered in
cluster Mostly
Histiocytes 25-250/OIF Histiocytes
20-40/OIF Lymphocytes-
a few(5-10/F) Lymphocytes(15-20/F) Plasma
cells-0-2/OIF Plasma cells - nil Many
activated macrophages with
vacolated appearance. Immune Response MIF- Mean-33.7% Mean-37% Range-(17
- 54%) Range-(23 - 50%) DAT-
75%+ve 66.6% -ve As
dermal pathology persisted in the skin
lesions after full course of treatment, these PKDL cases are followed up
clinically and pathologically in prospective fashion till complete recovery or
reappearance. Conclusions
: As
dermal pathology persisted in the skin
lesions after full course of treatment, these PKDL cases need to be followed up
clinically and pathologically to observe any redevelopment of lesions or
complete recovery. More number of PKDL cases are required for further study. Action plan for the control of kala-azar in Bihar Principal
Investigator : Mr. Narendra
Kumar, Mr. A. Palit Co-Investigator : Dr. V. N. R. Das,
Alok Ranjan Funding
Agency : Intra-Mural Project Study type
code : EPI Objectives : The
first phase of the study was initiated in four blocks of Muzaffarpur with 4012
sampled population with following objectives: 1. To ascertain
disease prevalence, incidence, population infectivity , vector distribution and
density in the defined population.. 2. To find out the clinical pattern of disease
manifestation and treatment modalities
currently used. Introduction
: This
study was initiated by RMRI in collaboration with NICD, Patna, ROHFW, Patna and
state Health Department. To stipulate the appropriate control strategy, attempt was taken to review the current
scenario of disease in most affected district of Muzaffarpur. Methodology
: The
population were re-surveyed yearly for two consecutive years with same parameters.
Door to door census, clinical examination of population and DAT test of each
study subject were conducted. The questionnaire was made to collect
sociological, clinical and entomological information. Entomological survey was
carried out in houses and cattle sheds in the study area by aspiration
technique with aid of torch light .Data on geographical and ecological set up
of the area was also collected. Case reports of those patients
attended/admitted to local PHC hospitals were also collected to obtain
treatment data. During survey any suspected case of Kala-azar / PKDL
encountered were referred to local hospital/RMRI, for necessary investigation
and treatment. A total of 4110 sampled
population from 4 villages in 4 most affected PHCs of Muzaffarpur were studied.
During first survey 2431 individuals were covered by both clinical and DAT
examination while in second survey only 2044 could be covered. The age-sex wise
sero-prevalence is presented in Table-1. Comparative sero-prevalence of the
same individual covered on both survey
is presented in Table-2. All 622 household along with cattle sheds were
searched for vectors in both surveys (Table-3). Results
: Out
of 2044 subjects screened for DAT during second survey, 422 (20.65%) were found
sero-positive . This was higher than the sero-positive rate observed in first
survey (10.29%). The frequency of sero-positives in all the age groups and both
sexes were observed to be higher during second survey except in the males in the age group of 20-29 years. Children in
the age group 0-14 years had highest sero-positive rate. During the survey of
population, only three active kala-azar
cases were encountered. However, the total number of cases reported in the
study area between period of first and second survey was 22 that included 16 fresh and 6 relapsed cases. During second survey , man-hour density of P.argentipes
and P.papatasi, insecticide
tolerance ,bio-assay test, age-grading and natural infection in vector were
assessed. It appears from the Table-4 that PMH density of P.argentipes
was higher as compared to P.papatasi and the overall PMH density during
second survey was slightly higher as compared to first survey. The density of P.argentipes
was higher than the critical density level. Table-4 presents the presence of
significant number of P2 parity status of P.argentipes (36.22%). Sandflies with
higher parity status have higher potential of transmitting infection. More
importantly, in Paranti village (PHC-Bochaha) , two specimens of argentipes were
found positive for promastigote by mass detection method. The result is more
interesting in view of rarity of coming across naturally infected P.argentipes
by mass dissection. Bio-assay test was conducted in the study area and
control sites to assess vector survival time but no significant difference was
noticed . Both varied between 10-20%. This indicates lack of residual
insecticidal effect in study villages. No DDT spray operation was conducted
during the year. Mean vector density was as high as 8-11 MHD i.e. much
higher than critical density estimated earlier during epidemics (8 MHD). The result
indicates a high transmission of infection in the study area requires
urgent attention. The man-hour density of vector species maintained almost
static pattern in selected foci of Bochaha (Village Paranti) and Dholi (Village -Mirapur) and with minor
variations in the two foci, Gaighat (Village-Berua) and Minapur
(Village-Raghopur) respectively. However, due to lack of insecticidal
intervention, for the last two years, there has been an increase in
P.argentipes density in all the foci in 1999 as compared to 1998.
Interestingly, simultaneously density of P.papatasi has decreased more than 50%
in 1999 in comparison to 1998. Dissection of wild caught sandflies for age
gradation revealed that during end of summer and on set of rainy season,
propensity of vector population of longer life expectancy was found to be high
(114/184 i.e.62% app.) , indicating chances of increased transmission during
this period. Altogether 298 flies were dissected out of those collected during
the study period. Conclusion
: The second survey revealed that the population infection rate in the study area has increased two fold as
compared to data of initial survey conducted in the year 1998. The increase is
observed in all age groups and both sexes. Incidence of kala-azar reported
during second survey in the study area has also increased . No DDT spray was
undertaken in study area during this period. Mean vector density was as high as
8-11 MHD i.e. much higher than critical density estimated earlier during
epidemics (8 MHD). The result indicates a high transmission of infection
in the study area requires urgent attention. Table-1
: Age-sex wise distribution of sero-prevalence(%) --------------------------------------------------------------------------------- Year MALE FEMALE TOTAL Age 1998 1999 1998 1999 1998 1999 --------------------------------------------------------------------------------- 0-9 9 21 9 21 9 21 10-19 9 22 11 19 10 20 20-29 11 19 11 22 11 21 30-39 14 23 11 13 12 17 40>= 14 25 9 21 11 23 ----------------------------------------------------------------------------------------- Total 11 22 10 19 10 21 ---------------------------------------------------------------------------------- Table-2 : Age-wise transition frequency of
sero-conversion of same population(n=1405) Age-group 0-9 10-19 20-29 30-39 40>= Total Loss
of 1998 +ve
DAT 46 42 27 27 42 184 Infection 1999 -ve
DAT 37 34 20 20 29 140 1999 +ve DAT 9 8 7 7 13 44 % of Loss of Infec. 79 77 76 74 69 77 Gain
of 1998 -ve DAT 434 272 123 140 252 1211 Infection 1999 +ve
DAT 86 58 25 20 52 241 1999 -ve DAT 348 214 98 120 200 980 % of Gain of Infec. 20 22 18 16 20 20 ------------------------------------------------------------------------------------ Table-3:Vector
density of P.argentipes and papatasi in four PHCs. -------------------------------------------------------------------------------- 1998 1999 PHC No. of MHD of MHD
of house. P.arg. P.papa. P.arg. P.pa. -------------------------------------------------------------------------------- 1.Bochaha 233 10 5 10 1 2.Gaighat 143 16 5 21 2 3.Dholi
138 6 3 7 1 4.Minapur 108 9 2 11 1 -------------------------------------------------------------------------------- Total 622 10 4 12 1 -------------------------------------------------------------------------------- Table-4 : Number of sandflies dissected for age grading----------------------------------------------------------------- No. N P P1 P2 P3 185 37 34 42 67 5 ------------------------------------------------------------------ A longitudinal study for the estimation of infection dynamics of
L.donovani in an endemic population of kala-azar in Bihar Principal
Investigator : Dr. Neena Verma Co-Investigator : Intra-Mural
Project Study type
code : EPI Objectives
: The
objectives of this study are as follow: 1. To estimate
the background infectivity and immuno-reactivity of a defined kala-azar endemic
population. 2. To study
infection dynamics in the cohort periodically by prospective follow up in
relation to disease occurrence. Introduction
: Study
of leishmanin skin test (LST) in combination with Direct agglutination test
(DAT) in endemic cohort population in prospective fashion can serve useful
epidemiological information in determining infection status of population. Methodology
: Before screening of the population clinically and serologically, village mapping, numbering of houses and door to door census of the population were carried out in the study area. Attempts were taken to screen all the individuals residing in the households. The population of the study area was 611 out of which 67 were not residing in the village at the time of survey. A total of 544 subjects were present in which 433 (80%) were screened by clinical examination, DAT and LST. Two rounds of vector search in each household was carried out to assess its morphology , density and infection status.
Results
: The age-sex distribution of population exhibiting LST and DAT positivity
respectively are presented in Fig.-1. Distribution of LST positivity and DAT
positivity in various category of population is presented in Table-1. Out of
433 subjects screened, 88(20%) were found LST positive. No significant
difference of leishmanin positivity was observed between male(21.65%) and
female(18.98). The frequency of leishmanin positives increases with advancement
of age reaching peak at age group of 35-44 years. Sero-positivity was found in
186(42.75%) population screened . No
significant difference of sero-positivity noticed between male(46.08%) and
female(39.81%) . The age group showing peak positive rate for leishmanin was
preceded by age group showing the peak positive rate of DAT. Both
LST and DAT value in population is presented in Table-2. Table-1: Leishmanin and DAT Positivity
in various category of subjects Clinical Gr. N LST(+ve) LST(%) DAT(+ve) DAT(%) a.
Active VL 4 0 0 4 100 b.
Active PKDL 1 0 0 1 100 c.
VL(1-3Mon.) 5 3 60 3 60 d.
VL(4-6Mon.) 5 2 40 1 20 e.
VL(7-12Mon.) 1 0 0 0 0 f.
VL>12Mon. 11 9 82 6 54 a.
VL contact 166 38 23 75 45 b.
VL non-contact 267 50 19 111 42 Total 433 88 20 186 43 Table-2.-LST and DAT status in
population with various level of infection. ----------------------------------------------------------------------------------------- LST +ve -ve Total DAT ----------------------------------------------------------------------------------------- +ve 37(9%) 143(33%) 180(42%) -ve 51(12%) 202(46%) 253(58%) ----------------------------------------------------------------------------------------- Total 88(20%) 345(80%) 433 ----------------------------------------------------------------------------------------- VL
contacts were defined as the individuals residing in houses having a subject
with current or past history of kala-azar in the family. The frequency of
leishmanin positives was not significantly associated with VL contacts(22.8%)
as compared to non-contacts (18.7%) . Patients with Kala-azar or PKDL did not
show reactivity to LST. Similarly, sero-positivity was also comparable between
VL-contacts(45.1%) and non-contacts(41.5%).
Out of 67 households searched for vector,
only 8(12%) and 5(7.4%) houses were having P.argentipes during first and second
round of search made respectively within 30 days of spacing. The man-hour
density of sandflies is presented in Table-3. In first round of vector survey,
24 sandflies of different species were caught, out of them 58.3% were
P.argentipes, 37.5% were P.papatasi and 4.2% were Sergentomia. In second round
of vector survey, MHD of P.argentipes increased to 4.2 with total number of
sandflies caught was 30 indicating high rate of vector generation or
maturation. The vector infectivity of above sandflies so collected were tested
by dot blot hybridisation technique in total of five pools . Each pools
containing 2 to 7 number of P.argentipes.
All the pools showed positive for parasite indicating high transmission zone.
The ratio of mean MHD for P.argentipes : P.papatasi : Sergentomyia was observed
to be 1: 0.30 :0.08. Table-3: Man-hour density of sandflies
species in the study area Round P.argentipes P.papatasi Sergentomyia First 4.2 0.5 0.3 Second 2.3 1.5 0.2 Mean 3.25 1.0 0.25 The
man-hour density of P.argentipes was observed to be higher as compared to other
species of sandflies. Other than above found was identified as P.sergenti. The prevalence of kala-azar in
the study area was 37 per 1000 indicative of high transmission of disease. The
cohort population are being followed up every six months to study infection
dynamics of population in the endemic area. Conclusions
: The
longitudinal follow up of the same cohort seasonally will reveal the changes in
infectivity status of the cohort . Feasibility of application of remote sensing for prediction of kala-azar epidemic in selected foci in Bihar Principal
Investigator : Mr. A. Palit Funding
Agency : Intra-Mural Project Study type
code : EPI Objectives : The
objective of this study is to evaluate
the feasibility of use of satellite data
for monitoring the macro-ecosystem,
specific vegetation cover and human settlements and to relate them with the
changing sandfly-genic conditions to evaluate its applicability as an epidemic
prediction. Introduction:
Disease
epidemiology investigations on Kala-azar suggest that case multiplication or increasing trend is more pronounced in regions with a poor
health infra-structure and areas under intense developmental activities related to agriculture. Further
studies on Kala-azar transmission
dynamics highlighted the importance of focal epidemiology of the disease.
Upholding the limitations of available technology in establishing cause and
relationship of spread of the disease, it has become imperative to address new
vistas for possible prediction of the disease epidemic. With increasing
accessibility to new technologies viz. remote
sensing it has become possible to
monitor land-use features on Earth's surface over various time interval to
develop methods for rapid
stratification of high susceptive areas and for the design of remedial
measures. Methodology
: Study
area : The study is being continued in the different types of eco-system, one
in hyper endemic Vaishali district
and another in non-endemic Lohardagga
district. The study has been
limited to "Block " level at
the initial phase of the study.
Study
design : Five tested villages were selected randomly in each
block using the center point co-ordinate of a village to ensure 2-3 Kms.
distance between the two selected villages. This distance requirement minimizes
the risk of non-unique observations due to overlap of surrounding land.
However, few such villages were also included that did not meet the 2 Kms.
criteria, due to problem of access to during the rainy seasons. Ground
data (entomological & land cover variables) : The changes
in sandfly density are being analyzed with seasons, in both types of foci, for
its subsequent co-relation with eco-environmental parameters to evaluate
influence of specific parameters, if any. Vector population are being analyzed with three main
variables, namely vegetation, waterbodies and settlements. Other criteria
remain the same . Results
: Comparative
analysis of vector density in two study sites in different season for last two
years, showed an almost consistent
pattern as presented in Figure-1 The trees/plants, edible peri-domestic
vegetation (<50 mts. Radius) and grasses in the positive sites and negative
sites in relation to P.argentipes in the two study foci is presented in
Table-1. It depicts that banana, bamboo, sugarcane and maize has association with
distribution of P.argentipes. The same
is true in distribution of peridomestic edible vegetations and grasses. It
appears that vector thrive well in endemic foci with their significant
association in contrast to non-endemic foci. Comparison of landscape composition of two
study foci has been presented in Figure-2. There is a distinct difference in
terms of relative proportion of the landscape elements. P.argentipes has been found to have an
association with the presence of particular tree/plant species (eg. Banana,
bamboo, sugarcane etc.) and landscape features (eg. Water bodies, vegetation,
swamps, annual crops etc.). Conclusions
: The
preliminary analysis of both ground data and data sources included the
construction of digital map to show location of landscape elements and probable
sandfly breeding sites in relation to settlements. The base maps of the some of
the foci which were generated were subsequently scanned, digitized and plotted
through the use of a plotter (Calcomp Plotter). These were developed after
taking into consideration all parameters of base maps. After procurement of analyzed satellite data of selected foci and
its comparison with digitized map of same foci, more conclusive evidences of
sandflygenic conditions could be achieved for delineation of environmental
determinants. Table-1: Association of P.argentipes with common vegetation typesPlant species Chi-square
p value Sites-> endemic non-endemic area area Banana +ve for P.arg. 20 2 -ve for P.arg.
3 38 39.63 0.0000001 Bamboo
+ve for P.arg. 16 1 -ve for P.arg. 4 24 24.17 0.0000009 Sugarcane +ve for P.arg. 18 3 -ve for P.arg. 2 21 23.25 0.00000014 Maize +ve for P.arg.
17 2 -ve for P.arg. 2 24 26.84 0.0000002 Peridomestic +ve for P.arg. 18 4 Edible -ve for P.arg.
4 36 28.92 0.0000001 Vegetation Grasses
+ve for P.arg. 45 4 -ve for P.arg.
5 45 53.03 0.0000001 Principal
Investigator : Dr. K. Kishore Co-Investigator : Dr. D. S. Dinesh Funding
Agency : Intra-Mural Project Study type
code : LAB Objectives : 1. To find out
intra-species variation among and to the population of P.argentipes , if
so , collected from different endemic zones of India. Identify and
characterize sub-species and sibling
species of P.argentipes . 2. Vector
incrimination. Introduction :
Morphological
variation of P.argentipes has already been reported in various part of the
world. In India also, the morphological and biological variation of
P.argentipes has been confirmed. In Bihar, there may be some morphological
variation in P.argentipes from one endemic foci to another. The conventional
methods for detecting infection in parasite is not very accurate. DNA probe
method is highly sensitive method for identification of infection in the
vector. Methodology
: Preliminary
work has been conducted in different kala-azar endemic districts like Patna,
Vaishali, and Samastipur. Sandflies of different species P.argentipes (1.66 -
45.0 MHD), P.papatasi ( 0.3 - 20.16 MHD) and sergentomyia species (0.3 - 20.16 MHD) were collected.
Besides these, two new species were also identified using the key of Lewis like
P.sergenti and P.major. Morphological variations were observed in specimens of
P.argentipes in the measurement of genital filament (0.124 - 0.294 mm),
Aedeagus (0.017 -0.139 mm) , Coxite (0.077 - 0.124mm) and style (0.093 - 1.7
mm). Pools of wild sandflies were used for
extraction of DNA by physical methods using Lysozymes, Proteinase K, SDS,
CTAB/NaCl, Chloroform :isoamyl (24:1) and precipitation by isopropanol. Total
DNA probe was prepared from reference strain of leishmania donovani
(MHOM/IN/80/DD8) labelled with non-radioactive isotope digoxigenin (DIG kit,
Boehringer-Mannheim Germany). Dot blot hybridization was made by using
‘Hybridot Manifold , USA” apparatus on nylon membrane. Results
: The
result reveals that out of 92 pools, 59.78% had shown positive result.
P.argentipes had shown 65.8% positivity, The sensitivity of the probe was
tested in 10 folds dilution. Positive signal was found up to DNA concentration
of 0.2 Pg/ml . Laboratory
bred negative controls were found negative by the test. It will possibly cross
reaction with other flagellates like trypanosome which need to be tested in the sandflies. Conclusions
: DNA
probe technique may proved to be an useful test for establishing infectivity in
the wild caught sandfly. Reports of completed projects Objective
:
To
ascertain larval biology of sandflies Introduction
:
One
of the limitations of vector biology study of Kala-azar lies in its paucity of
knowledge on larval biology and detection of its early stages of life cycle.
Detection of such stages will assist detecting the exact breeding sites and
transmission studies. Hence, an attempt was taken to study the breeding habitat
of vectors in human and cattle dwellings by screening large numbers of soil
samples from expected breeding sites. Methodology
:
Soil
samples were collected from the three endemic villages i.e. Runisaidpur in
Sitamarhi district, Azampur in Vaishali district and Bahapur in Patna district.
Collection of soil samples were made from human dwellings and cattle sheds by
scrapping mud plaster of wall by 1.5 cm. Collected samples were numbered and
kept in inter locked plastic bags and transported to the laboratory. Collected
samples were put into petri dishes and examined under 40x resolution of
binocular microscope. Soil samples were examined by Sugar Floatation Technique.
Sample was mixed with 3 parts of sugar and 5 parts of water in a enamel tray
and the powdered soils were mixed into it. After settlement of soil, larvae and
pupae floats on the surface of water which can be separated by sieving method. Results
: Table-1: Larval Biology of sandfliesType
of No. of total Observation Immature Exuvae Rem. Habitat soil
sample Micro. Float. Lar. Pup. Lar. Pup ------------------------------------------------------------------------------------------------ CS-105 251 183/27
nil nil nil nil 27 HD-39 MD-107
68/0 nil nil nil nil CS-211 368 211/197
nil 41 29 28 99 HD-48 MD-109 157/81 7 23 15 36 619 48 52 43 162 Table-2 : Positive for Immature stage---------------------------------------------------------- Live Dead Total Larvae 34 14 48 Pupae 39 13 52 ------------------------------------------------------------ Total 73 27 100 ------------------------------------------------------------ Male-29,
Female-44 . All are P.argentipes. CD-2+ T cell distribution and IL2 and MIF response in kala-azar Objectives : To evaluate
distribution pattern of CD2 + T cells among total T-cells in peripheral and
bone marrow suspension of active kala-azar patients during course of treatment
with SAG enabling to compare results between responsive and unresponsive cases.
The study was extended further to find out if any association exist with
differentially expressed CD-2 +T cells with immunological profile (IL-2 &
MIF) of Kala-azar patients under treatment with SAG. Introduction : The characteristics feature associated with Kala-azar is known
to be suppression of CMI at T-cell level. Recovered patients regain their
normal CMI function which makes them usually immune to further infection. Lack
of development of immune protective mechanism in drug unresponsive cases of
kala-azar might be due to poor early activation of T cells where regulatory
activity of receptors present on naïve memory subset might dictate functional
expression of helper T cells in Kala-azar. Methodology : Mononuclear
cells (5x10^6) were isolated from peripheral and bone marrow suspensions of 10
confirmed , 4 SAG unresponsive and 3
cured cases of kala-azar. MNC (5x10^6) obtained from 4-6 ml of blood collected
from 5 apparently healthy subjects were also taken as control. Mononuclear
cells thus obtained were stimulated in-vitro with Leishmania donovani
antigen (1x10^5 ml) for 7 days. With re-stimulation with antigen (day 7) ,
cells were further co-incubated with UV-irradiated autologus MNC (1x10^5) for 5
days. Response was measured by phenotype analysis of CD-2 +T cells by panning
and cytokine production (IL-2 and MIF). Results : Leishmanial
antigen failed to induce significant CD-2 + T cells in peripheral blood
(35.07%) and bone marrow (49.85%) of kala-azar cases during active infection
(Table-1) . This was observed associated with low immunological profile, as
both MIF and IL-2 level in such cases were maintained at baseline (MIF 17-30%,
IL-2 0.085) (Table-2) . On the other hand, leishmanial antigen induced
significant number of CD-2 + T cells
in-vitro in kala-azar cured subjects
(78.26%) and the healthy controls (65%). This significant association between
CD-2 + T cells in protected subjects was associated with significant MIF (36.85%) and IL-2 (0.73) release. On
contrary CD-2 induction of T cells in SAG unresponsive cases was 43.64% in
peripheral blood and 46.87% in bone
marrow which was associated with low MIF (22.33%) and IL-2 level(0.05) shown by
T cells of such cases. Conclusions : The results
indicate that CD-2 + T cells might proliferate in response to Leishmanial
antigen and are possibly involved in influencing immunological profile in
Kala-azar patients. +- Table-1 : Phenotype
characterization with respect to CD-2 of Lymphoid & peripheral Lymphocytes
in kala-azar. -------------------------------------------------------------------------------------------------------------- Peripheral
blood Bone-marrow Subjects
n Total
CD-2+ %CD-2 Total CD-2
% *T-cells mean T-cells mean CD-2 mean mean -------------------------------------------------------------------------------------------------------------- 1.Fresh
4 3460.00
1213.75 35.07
2800.5 1396.25 49.85 KA(untreated) (2240-6400)
(400-1855) (1000-9000) (800-1855) 2.During
trt 3 1818.33 838.33 46.10 1846.66 900.0 48.73 (1555-2200) (675-1240) (1640-1800) (700-1200) 3.Drug un-
3 3513.33 1533.33 43.64 3413.33 1600 46.87 responsive (2240-5800) (600-1500)
(2000-6000) (700-2500) cases. 4.Cured
kala- 3 2300 1800.00 78.26 - - - azar (2000-2700) (1700-1900) 5.
Healthy 5 2380 1560 65.54 - - - Control (2000-3200) (1200-2500) ------------------------------------------------------------------------------------------------------------- *T cells are expressed in number per ml of blood Table -2:Relation
between CD-2 + T-cell and MIF and IL-2 response in KA (12th day of culture). --------------------------------------------------------------------------------------------------------- Subjects No. MIF(%) IL-2(570
nm) %CD-2
cells --------------------------------------------------------------------------------------------------------- 1.Fresh KA 4 21.00 (17-30) 0.085 (.07-.09) 35.07 2.During trt 3 25.33 (20-31) 0.37 (.07-.97) 48.73 3.Drug unrespon- 3 22.33 (17-30) 0.05 (.07-.15) 43.64
sive KA. 4. Cured KA 4 36.85 (28-50) 0.73 (.07-1.0) 78.26 5. Healthy control 5
--- ---
65.54 -------------------------------------------------------------------------------------------------------- Study
on transmission of kala-azar through artificially infected P.argentipes Objective
: For
vector incrimination, the parasite should harbor frequently in the gut. Introduction
: Phlebotomine sandflies are the vectors of ‘new’ and ‘old world’ species of
leishmania. Incrimination of sandfly species as vectors requires the
identification of the parasite in infected flies. Routine dissection and
microscopic examination are used to demonstrate parasite in the guts. Methodology : To
demonstrate parasite in gut, Forud experiment was conducted. Results
: Study of the table I,II and III indicates that percentage of feeding varies
from 7.8% to 8.9% which is very low. For control experiment, laboratory bred
P.argentipes were used. After artificial membrane feeding with 1x10^7/ml promastigotes, 80% (24 out of 30 P.papatasi)
took feed blood meal but none of them harbored promastigotes. In the case of P.argentipes, 7%(2 samples)
showed positive for promastigotes. This indicates low detection rate in
transmission in this condition. Table-I : Artificial membrane feeding with the help of promastigotes------------------------------------------------------------------------------ No.
of P.arg. No. of the
fed/ Result exposed per centage ------------------------------------------------------------------------------ 145 13 (8.9%) 2 slides positive for
promastigotes after
2 post feeding day ------------------------------------------------------------------------------ Table-II : Feeding with the bone
marrow aspirate --------------------------------------------------------------------------------- No. of P.arg. No.
of fed/ Result exposed percentage ---------------------------------------------------------------------------------- 131 10
(7.6%) All were
negative ---------------------------------------------------------------------------------- Table-III :
Result of Xeno-diagnosis ------------------------------------------------------- No. of
P.arg Result Exposed ------------------------------------------------------- 130 One slide positive
when dissected
after 14th post feeding done. ------------------------------------------------------- Twenty years of visceral leishmaniasis in Bihar - an epidemiological assessment Objective
: The
objective of this study is to assess the magnitude of the current problem of kala-azar in Bihar state, based on
retrospective analyses of epidemiological data reported by State Government for
last twenty years i.e. from 1977 to 1996. Introduction
: Kala-azar has been a major health
problem of Bihar for last many decades.
After 1977 there has been an increased
trend of morbidity and mortality. During the period two epidemics
appeared in the year 1978 and 1992 causing very high morbidity and mortality (Table:1).The total number of cases and
deaths reported during period in the state from 1977 to 1996 seem to be an
under estimation. It is well known that some cases directly report to private practitioners for the treatment. The
actual number of Kala-azar cases may be twice or more than that of the cases
actually recorded at PHCs and district
level hospitals. Hence, the burden of the disease in the community may be very
high and has increased significantly over a period of twenty years . The
reports of cases and deaths of
Kala-azar by State Government agencies is ,so far remain the only source of data on Kala-azar generated through established registration
system.
Methodology
: The
reported cases and deaths of Kala-azar are the crude estimate of disease. It is
made more meaningful by applying suitable statistical techniques to convert
into rates and ratios. For statistical analysis , the number of cases and
deaths have been converted into Morbidity-Incidence-rate(MIR),
Mortality-rate(MR) and Case-Fatality rate(CFR) and the Figures 1,2, & 3
present the bar diagram of these rates. Time-Series technique has been used to
assess the trend and cyclic movements of the Morbidity-Incidence
-Rate(MIR).Age-specific prevalence/ incidence was not available in the record. In order to measure trend and cyclic movements of
incidence-rate over a period of twenty years, time-series method has been
applied. The principle of least square for fitting an appropriate equation has
been used to measure trend by fitting second degree polynomial of logarithm of
trend value. The cyclic movement of the incidence-rate has been estimated by
using "Periodogram Analysis" which measures the maximum intensity
corresponding to given trial periods. Results
:
During
the first ten years of reported data i.e. from period 1977-86, the average
annual incidence of cases and deaths of VL recorded were 17,660 and 688, with
an average incidence-rate of 32.51 per 100,000 population and mortality-rate of
0.125 per 100,000 population respectively. During the second ten years i.e.
1987-96, the average annual incidence
of cases and deaths of VL reported was 37,184 and 5491,with an average incidence-rate of 54.73 per 100,000 population and
mortality-rate of 0.802 per 100,000 population respectively. These results
indicate a two-fold increase in Kala-azar cases and eight fold increase in
deaths, registered during 1987-96 as compared to 1977-86, which accounts for
nearly 70% increase in Kala-azar cases and around 500% increase in deaths due
to VL in the second decade as compared to the first decade of the study period.
Similarly, the case-fatality rate due to VL was an average of 4.41 per 1000
Kala-azar cases during 1977-86 as compared to 14.04 during 1987-96, showing a
three fold increase. Trend analysis of incidence rate
showed an increasing trend over twenty years during 1977-96 with a slight
downward concave curve between two peaks of 1978 and 1992 (Figure:4). The cycle
of maximum incidence repeats at every 12 to 14 years (Figure:5) i.e. if the
same situation prevails in future, another epidemic may occur between 2004 to 2006
AD. The geographical distribution of Kala-azar
shows that out of 54 districts at present in Bihar, 32 districts were endemic
for Kala-azar during 1977-86 whereas 40 districts becomes endemic for Kala-azar
during 1987-96 i.e. the disease has spread to 8 districts which were earlier
free from the risk during 1977-86. Conclusions
: The
data indicates significant increase in disease burden in the community during
1987-96 as compared to 1977-86.It suggests for adoption and implementation of a
suitable integrated control strategy i.e. evolution of a simple early
diagnostic tool, suitable therapeutic measures to reduce the fatality, vector
control and social-awareness programme to educate the masses. Study on focal epidemics of kala-azar in Bihar - epidemiological aspect Objective:
To
study the epidemiological parameters of disease transmission during epidemic
period and compare it with that of epidemic free period in endemic areas of Bihar that can assist to
get insight in to causation of such focal epidemics. Introduction
: Preliminary
studies on focal outbreak of Kala-azar were undertaken in three districts of
North Bihar namely, Darbhanga, Samastipur and Madhubani in the year 1996 and
1997 during pre-monsoon season to ascertain case incidence, infectivity status
of host and vector density. The epidemiological variants observed during
epidemic period were compared with that of same variants observed during
non-epidemic period in similar affected zone like Muzaffarpur district in the
year 1998 during pre-monsoon period. Methodology
: Cross-sectional
studies were carried out in the epidemic affected villages of the endemic PHCs in three districts . During 1996 and
1997, three epidemic sites were surveyed with cluster sampled population of 424
and 1167 respectively. Out of these, total population of 205 and 607
respectively were examined clinically and tested for DAT in two consecutive
years. In 1998, survey of Muzaffarpur
district during pre-monsoon period comprised
a population of 2182 individuals
of both sex in all age groups. They
were subjected to clinical and DAT. During this period the village was free of
any epidemic where as disease endemicity was observed perennially in this
region. Results:
Age-sexwise
distribution of serological positives during epidemic and epidemic free period is presented in Table-1. Table-2
indicates epidemiological variables like prevalence rate, infectivity rate and
vector density during epidemic period and epidemic-free period. Table-1:
Age-wise distribution of sero-positivity epidemic and endemic areas. Age- Epidemic Area* Endemic
Area** group 1996 1997 1998 n +ve(%) n +ve(%) n +ve(%) 0 - 9 64 43.75 188 35.1 794 8.81 10 -19 59 44.06 145 36.5 538 10.22 20 -29 31 38.70 67 30.0 281 11.00 30 -39 26 42.30 106 28.3 319 12.00 40>= 25 24.00 97 31.0 499 11.02 Total 205 41.00 603 33.00 2431 10.24 *Chi-square=5.47
at 4d.f.,p=0.24 **Chi-square=3.25
at 4d.f, p=0.52 Table-2:
Epidemiological parameters in epidemic and endemic areas. Parameters Epidemic Endemic 1996
1997 1998 p
value 1.Prevalence(Infectivity%)
41 33 10 * 0.00001 2.GMRT 1600±82 1460±74 978±42 **0.001 3.Incidence
rate(cases per 1000) 34 23 4.5 *0.00001 4.Vector
Density(MHD) 26 33
10 5.Household
Index(%) 66 60 48 *0.00008 *Yates
corrected chi-square statistic **
Z-statistic During epidemic period, there is considerable increase in all
epidemiological variables as compared to that of endemic period .The
infectivity rate of L.donovani during
epidemic situation is three times
higher as compared to endemic situation. The prevalence rate of the disease in
the community is five times higher during epidemic period as compared to
endemic period. The man-hour density of vector population in the community increases by nearly three
times during epidemic period . Conclusions
:All these variables indicate that during epidemic period the rate of
transmission of the disease increased significantly leading to an outbreak.
Period coincides with vector-breeding. Open sleeping habits of rural population
in summer increases man-vector contact. However , any change in pattern of host
preference by vector during epidemic need to be ascertained. Hence,
intervention programme in terms of vector control measures are stressed during
March-April to curtail vector population. The reservoir host, PKDL cases need
also be searched by active surveillance and adequately treated. Adequate timely
intervention of vector control measure, appropriate dose of SAG instituted in
early phase of the disease, use of simple diagnostic technique like DAT and
population awareness programme are required to avert such outbreak in future. Study on mother's perception in relation to post kala-azar dermal leishmaniasis (PKDL) Objective
: To
assess the perception of mothers in relation to occurrence of PKDL and the social stigma attached to it. Introduction
:Post Kala-azar dermal leishmaniasis(PKDL) is known to occur following lapse of
period from cure of kala-azar that manifest as hypo-pigmented skin patches or
nodules all over body including face and extremities. The morbid condition
persists for prolonged period without accompanying any fatality. The dermal
manifestation makes the look ugly and resembles leprosy. Because of fear of its
social implication, the cases are usually not reported for long period or are
not adequately treated. Since PKDL is presumed to act as reservoir host of
kala-azar infection and implicated in kala-azar transmission, early detection
and prompt treatment is mandatory. To achieve above, it is imperative to
understand social awareness of disease and stigma attached to it that need to
be identified for taking steps for educating people and take measure for early
reporting. Since children are commonly affected and mother's play significant
role in decision making in households activities, mother's role in family is
stressed. Methodology
: Two
endemic villages of Patna district were
selected for this study. Complete enumeration was done in both villages
covering 371 households. One woman from each household was interviewed through
questionnaire. Results
:
Analysis
revealed that around 70% houses were kutchha with no accessibility of natural
light. Drainage facility was found poor with water logging observed in 68%
houses. Knowledge about PKDL amongst mothers revealed that only 15% describe
PKDL with symptoms like white spots on body and 10% describe it as nodular
manifestation on face . Around 10% consider PKDL as suspected reservoir in
spreading Kala-azar, 9% felt PKDL as a very serious disease that the duration of treatment requires beyond 60
days was told by 8% of the respondents .That
the treatment of PKDL is very expensive and private hospitals be the
appropriate place for treatment was viewed by eleven percent of the
respondents. As regards treatment is
concerned, the problems conceived were
hospitalization for a long duration (5%), high cost involved (6%) and away from
work for a long time 4.3%. Response was elicited on stigmas attached to
the disease like disfigurement and its social consequences. To this only 15%
respondents considered it as a serious problem in negotiating marriages for
their son(9%) and daughters(15%). Conclusions
: The
result depicts the poor knowledge about the disease which not only affects the
individual physically but psychologically also. There is a need to launch an
awareness programme in the rural areas in form of group discussion, slide show,
pamphlet distribution and by convincing people on benign nature of disease with treatment modalities, specially for
the women as mother can plays a vital role in early detection of the disease. Evaluation of long acting
malathion paint formulation in the control of sandflies in an endemic area of
kala-azar in Bihar - ( WHO/TDR FUNDED) Objectives: 1.To
evaluate the efficacy of Malathion paint formulation as an insecticide as
compare to DDT. 2.To
assess its tolerability among population in the community. Introduction
: Kala-azar
has been a major health problem in Bihar for last many decades. Various efforts
have been taken by the state health agencies to control the disease. During 1960's,
indoor residual spraying of DDT under NMEP had proved to be very effective in
reducing the vector population i.e. P.argentipes. Based on this experience, two
round of DDT spray in a year has been adopted as one of the control measures
during 1980's and 1990's. In last few years, DDT application had been banned by
most of the developed countries because of its toxic effect on flora and fauna.
Also, if applied for a long time, it
develops resistance in vector population. In this circumstances, an alternative
insecticide has to be evolved which is equally effective like DDT. Malathion
Paint Formulation has been tried against triatomid bugs which proved to very
effective in controlling Chagas disease in Brazil. Use of slow release
formulation of Malathion paint may be feasible proposition since this
formulation of Malathion is expected to give desired effect for a long duration
as sandflies exhibit preferential resting on wall surface. Methodology
: Two
villages in endemic district of Patna were selected for this pilot study. The
village , Gulmahiyabagh ,having 203 household and without DDT intervention ,was taken as experimental
village for Malathion spray. Another village, having same socio-economic,
demographic, ecological and vector density and adjacent to experimental
village, was selected as a control village. Initial vector survey was done in
both villages before spray of DDT and Malathion. Malathion paint formulation
was sprayed in all the 203 houses of experimental village in July,94 and two round
DDT spray was done in control village by the state health department. Both the
villages were monitored for the vector
and resurgence quarterly with monthly
recording of temperature and humidity . Plasma cholinesterase level was
evaluated in sample of individuals in
the test village .
Results
: Pre-spray
entomological evaluation revealed that in May'94, the man hour density (MHD)
was 0.30. Post-spray entomological monitoring was done from March'95 onwards
till July'97 on twelve occasion during study period. Figure-1 indicates that
MHD remains very low till February'96 and crosses the pre-spray MHD in
June'96.It shows that resurgence started after 22 months of Malathion spray in
test village. The small number of sandflies collected during 22 months period
cannot be considered as a cases of resurgence because of some inherent chance
causes which are beyond the control of human hand at the time of spraying of
Malathion. So it appears that Malathion lethal potential remains effective for
22 months which can also be correlated with the data obtained through bio-assay
of Malathion in laboratary.Figure-2 indicates household index (HI) of the study
area during pre- and post-spray of Malathion. Pre-spray HI was 13.3 and after
spray it remains very low till Feb'96 and a very slight increase in June'96
i.e.4.4. These finding depict that Malathion paint formulation proved to be
effective in controlling vector population in test village for nearly two
years. The data on DDT sprayed
(control) village reveal that resurgence of vector started after one year with two round of DDT spray carried out in
that year . Malathion
paint was quite effective for two
continuous years. However, it requires
social awareness amongst the population not to paint/wash the sprayed surface.
In this case, even it was washed every month, the result was satisfactory. HPLC
analysis of 130 samples for (wall scrapping) has been done to know the residual
efficacy of the formulation on the wall surfaces. To evaluate toxicity amongst exposed, blood samples were
collected from the sampled population that included all age groups and both
sex, prior to spraying operation. Besides samples were collected from spraymen,
following one and twelfth month of spray. Plasma cholinesterase was evaluated in
the samples(Sigma diagnostics). There was a significant drop of the mean plasma
cholinesterase level (p<0.05) from base level amongst the exposed population
as observed after one month of spraying. However all the individual's value
were within the normal range. It was not accompanied by any clinical change
amongst exposed. To test the residual effect of Malathion
(SRES) formulation, Bio assay test was also conducted in the field and laboratory condition in Bio assay chamber 49
times in spaced intervals. In field condition, Malathion was effective on
cement surface upto 18 months, mortality higher than that on Mud surface, where
mortality was recorded only upto 15 months . However, this was higher than the mortality observed in the
control. When the control mortality was 5% - 20%, the experiment was corrected
by applying Abotts method, when the control mortality was more than 20%, the
experiment was repeated. After 18 months on cement surface and after 15 months
on mud surface, the mortality values matches with that of control. In the
laboratory condition, the result was slightly different from that observed in
field condition. On the cement surface, the mortality was observed upto 20
months and higher as compared to
control (40% and 25% following Abotts method) . On mud surface the mortality
recovered upto 16 months and higher than the control mortality. When it came
between 5% - 20% the experiment was corrected by applying Abott method. Conclusions
: Spraying
of Malathion paint formulation can help in reducing vector density in the
endemic area of Kala-azar. It is less
toxic as compared to DDT. Study on social acceptability of long acting malathion paint formulation in the control of kala-azar Objective
: To
assess the social acceptability of long acting Malathion paint formulation in
the rural community. Introduction
: For
the successful implementation of insecticide as a vector control measure in the
community, it is imperative to assess its acceptability as well as awareness in
the community. It is well known that social acceptability of such strategy
plays a determinant role in its feasibility for further application. Methodology
: The
study was conducted in an endemic village of kala-azar, where Malathion paint
was sprayed to assess its efficacy as a vector control. Another village was
sprayed with 2 rounds of DDT . The spray operation was conducted on the walls
of all rooms of houses and cattlesheds. A well designed questionnaire was
prepared to record the response of the head of the household to assess the
knowledge of the respondents on kala-azar vector, its prevention, acceptability
to spray operation, odor, adjustment with living conditions of households, mud
plastering of walls following spray . Questionnaires were filled up after
initiation of spray operation and 2
years after . Results
: The
result reveals that all the respondents were satisfied with the Malathion spray
and they feel that the smell of Malathion to be different from that of DDT. The
tolerance to such smell was reported from 96%
of the respondents. Around 70% of the respondents are of the view that
it has no adverse effect on the day to day working, but rest of the respondents
told that precautions like extra care for children and food stuffs for few days
after the spray are required . Ninety two percent of the respondents are
of the opinion that it has no adverse effect on the health of the family
members or the domestic animals. Only few of them (5%) reported breathing
problem, nausea etc. after few days of the spray. So far the role of the spray
in controlling sandfly / mosquito is concerned, 995 of the respondents
responded positively. Around 87% of the respondents told that they did not paint their house with mud / lime after
the spray. Out of the rest of the
respondents, 24%, 48% and 28% of
respondents replastered their house with mud / lime after a lapse of 1 , 2 and beyond 2 years respectively . On inquiring, whether Malathion spray which is required once in a span of three years is more acceptable than DDT, the response was cent percent in the favor of the Malathion. A large proportion of the respondents (92.5%) were of the opinion that as a custom, social or religio |
