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.



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 enzymes

Enzymes                          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)              7

No                                  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



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



+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.




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 types

Plant 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



Grasses              +ve for P.arg.          45              4

                           -ve for P.arg.            5              45                    53.03                 0.0000001
















Identification and characterization of sibling species of P.argentipes relevant in transmission of kala-azar



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






Study on breeding habitat and density of immature status  of sandflies for ascertaining its larval biology



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 sandflies

Type 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


MD-107                                         68/0            nil         nil         nil         nil


CS-211        368             211/197     nil               41        29        28        99


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 naďve 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)


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



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




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.



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)



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 religious functions are not possible without fresh white-wash or mud-plastering of the house.


Conclusion :

It was observed that Malathion paint was well accepted in the community.














Use of cell wall membrane antigenic preparation and CD-2 memory T-cell receptor in kala-azar vaccine against  kala-azar



Introduction :

Attempts to control Kala-azar by means of vector control and anti-leishmanial drugs has not shown satisfactory result as yet. Anti-Kala-azar vaccine can act as alternative tool for disease control. Membrane antigens on surface of Leishmania parasite have been implicated in  evasion of host immunity, that help parasite to establish in macrophages (King and Chang,1987). As such these antigens may be potential candidate to design future vaccine protocol. The membrane antigens when used alone does not seem to confer  complete protection, since there might be loss of immunogenicity of vaccinating organism arising out of  lack of immuno-competency  or Leishmania reactive T-cells. Earlier we have shown presence of CD-2 receptors on the cells of memory T-cells that shows a predominance of Th1 cytokines expression, IL-2 and MIF . Since, there are co-stimulatory molecules present on memory subset, their persistence with membrane antigen preparation in the immunogen construct can help T-cells of vaccinated hosts to effectively maintain their memory against Leishmania antigens and this may help them to generate significant number of antigen reactive T-cells, resulting into protection of the vaccinated hosts.


Methodology :

Keeping above , a preliminary study was undertaken. Whole cell of LD promastigote and its membrane antigenic components  were linked separately to CD-2 mAb and natural immuno-potentiator(FIA). These antigenic preparations as well as immuno-potentiator and CD-2 receptors were studied in 8 post-immunization changes in macrophage- HLADR interaction in inbred strain of BALB/C mice .The impact of this interaction on cytokine (IL-2, MIF) generation response of T-lymphocytes was assessed.

Whole cell promastigotes (2.5*10^8/ml) of DD8 strain and cell wall of Leishmania antigen were obtained. Immunogens were prepared in either saline (0.85% NaCl), FIA or with CD-2 supplemented with FIA. Inbred BALB/C mice (30-35 g) bred at RMRI (n=25) were immunized with different Immunogens sub-cutaneously on 3rd, 5th and 7th week. On day 10 post immunization , animals were challenged with 1*10^6 LD promastigotes given intravenously. Antibody level (DAT) were measured on day 0, 14, 28 and 40. On 30th day of  challenge, mononuclear cells were isolated and cultured in presence of monocytes of KA patients .The number of macrophages with HLA-DR molecules were assessed by staining the macrophage by ABC immunoperoxide staining (Madhabnanda,1985). Change in T-cell function following immunization was examined through estimation of IL-2 and MIF level.


Results :

1. Mice immunized with membrane components linked to CD-2 induced higher antibody response (1:6400 in 3/5 mice).Antibody titres obtained against whole cell antigen linked to CD-2 was lower.

2. Following Leishmania challenge, membrane immunogen with CD-2 supplement triggered strong macrophage response for HLA-DR expression (40-45%) in patients macrophage incubated with macrophage of immunized mice. Corresponding response with whole cell immunogen was 20-22%.

3. The high expression of HLA-Dr on macrophages as induced in mice following membrane immunization linked to CD-2 enabled the latter to present antigen in such a way that T-cells became stimulated and were able to generate significant level of IL-2(1.10-1.56) and MIF(45-50%).