Regional Medical Research Centre
Dibrugarh, Assam

Research Projects

 

 

 

Studies on bionomics of Anopheles dirus, the major vector of forest malaria in NE region of India

Investigator:                       Dr. Anil Prakash

Co-Investigators:           (i) Dr. D.R. Bhattacharyya (ii) Dr. P.K. Mohapatra

Subject keywords :   

Subject key words:     

Forest malaria
Vector bionomics
Anopheles dirus
North-east India
Mosquito ecology

Objectives:

(i) To study adult biology of An. dirus in relation to the transmission of malaria.
(ii) To study the larval ecology of An. Dirus
(iii) To ascertain sibling species status of An. dirus in NE India.

Introduction: Malaria is the most pressing mosquito borne public health problem of the north-eastern region of India. Malaria burden in this region accounts for 8-10% of all cases and 10-20% of the total P. falciparum cases reported in the country per annum. Malaria, in NE region, is mostly concentrated in forested and forest fringed hill and foot hill regions. Anopheles minimus and An. dirus are the primary vectors of malaria in this part of the country along with An. fluviatilis playing a secondary role. Initially, An. minimus was accorded the importance of the only major vector present in the north-eastern region so much so that the role of An. dirus was overlooked in malaria transmission. It was the persistence of malaria in NE region even after achieving good control of An. minimus with the DDT that led to the recognition of the role of An. dirus as the efficient vector. An. dirus is a very efficient vector species maintaining malaria transmission in forest areas of the region. In spite of well known vectorial status of this species no systematic work has been carried out on the ecology of An. dirus in the north-eastern region and as such entomological information on various aspects of its bionomics relevant to the transmission of malaria remain unexplored. This prompted us to take up the present project to investigate various facets of bio-ecology of An. dirus in a broken forest area of the tropical rain forest range of district Dibrugarh,upper Assam.

Methodology:

Area -Study was conducted in an isolated, topographically plain, forest fringed village named Soraipung (27.350 N and 95.410 E, 152 meters above MSL) under Tengakhat primary health center of district Dibrugarh, Assam (India). Population of the village was 401 and comprised of ex-tea garden laborers and marginal farmers living in mud plastered thatched huts, sleeping indoors. The village had 1.0: 0.9 human to cattle ratio. Mean annual rainfall of the study area was about 2800 mm. Pre-monsoon rains begin in March and wet hot season last till October with peak rainfall occurring during July/August. November to February was the cold dry period. Relative humidity always remained over 70% throughout the year.

Period- The data given here were generated during 1995-96 at monthly frequency. Part of the study is still continuing.

Seasonal And Relative Prevalemce Of An. Dirus- To study the prevalence of house frequenting mosquitoes and seasonal density pattern of An. dirus, dusk to dawn mosquitoes were collected with the help of battery operated CDC miniature light trap in 2-3 human dwellings (1 fixed and 1 or 2 random).

Indoor Biting Behaviour Of An. Dirus- Indoor, human baited, whole night landing catches on a local volunteer were carried out once in a month inside a village hut by two pairs of experienced collectors from 18 00 to 06 00 hour to record mosquito biting densities. Observations were also recorded on biting behaviour of An. dirus.

Anthropophilic Index Of An. Dirus- Host preference of An. dirus was investigated by trapping it with CDC light traps from a mixed dwelling where cows, dogs, hens, ducks and goat stayed together in the night with the occupants of the house. Blood meal of An. dirus was identified by gel diffusion method against antisera of human, cow, pig, dog, goat and fowl.

Sporozoite And Parous Rates Of An. Dirus- Salivary glands and ovaries of suitable specimens of An. dirus collected in CDC light traps and human baited collections were dissected for sporozoites and parity status respectively.

Breeding Habitats Of An. Dirus- All types of temporary, semi-permanent and permanent breeding habitats within the village and in the adjoining forest area were searched regularly and mosquito larvae from positive habitats were collected, reared in laboratory till emergence to identify the preferred breeding habitats of An. dirus.

Day Resting Behaviour Of An. Dirus- Day resting collections from the human dwellings, cattle sheds in the village and out doors in village and forest areas were carried out between 06 00 and 09 00 hours to investigate the day resting behaviour of An. dirus.

Insecticide Susceptibility Of An. Dirus- Susceptibility to commonly used insecticides in public health of An. dirus adults was investigated by standard WHO bio-assay method - Prevalence of Malaria in the Study Area- It was recorded by carrying out door to door active fever survey once in a month and collecting blood smears from cases with fever or fever history and examining under the microscope for malaria parasite. Malaria positives were treated by standard regimen of chloroquine only.

Results:

Seasonal And Relative Prevalence Of An. Dirus - Overall mosquito fauna of the study area comprised of 11 genera and 43 species. A total of 986 mosquitoes belonging to six genera and thirty species were captured in 25 trap nights. The percentage of anophelines ( 9 species) and culicines ( 21 species) in light trap collections were 42.4 and 57.5% respectively. An. dirus was the only malaria vector constituting 15.4% of all mosquitoes and 36.3% of total anophelines collected. The unfed and fed An. dirus in light trap collections were 32 and 68% respectively. Mean density of An. dirus per trap per night was 6.1 and the density varied with the season. Its population with the onset of pre-monsoon rain (March onwards) started building up to reach the peak in July (39 per trap per night), remained high till October and thereafter with the cessation of rains it came down and became zero in the month of December (Table-1).

Table-1: Seasonal Prevalence Of An. Dirus

Month

Trap collection (No.)

Mean density / trap / night
An dirus                      All                             Al
                                 anophelines            mosquitoes

% of An. dirus to all mosquitoes

1995

August

2

3.5

18.0

29.0

12.1

September

3

12.7

21.0

83.6

15.2

October

2

10.0

13.0

22.0

45.5

November

3

2.0

3.0

7.0

28.6

December

2

0.0

0.0

1.0

0.0

1996

January

2

0.0

0.0

0.5

0.0

February

2

0.0

0.0

11.0

0.0

March

2

1.0

1.5

13.0

7.7

April

2

2.0

4.5

14.0

14.3

May

2

8.0

19.5

41.0

20.0

June

2

10.0

13.5

31.5

31.7

July

1

39.0

207.0

400.0

9.8

Total

25

6.1

16.8

39.4

15.4

Table-2 : Indoor Man Biting Densities Of An. Dirus

Month

No. of mosquitoes collected on human bait

Anopheles dirus

Other mos- quitoes

All mos- quitoes

I Qtr           II Qtr          III Qtr        IV Qtr

Total

1995

August

1

16

11

8

36

60

96

Sept.

6

14

25

7

52

482

534

Oct.

2

11

15

32

60

45

105

Nov.

3

4

0

2

9

8

17

Dec.

0

0

0

0

0

0

0

1996

January

0

0

0

0

0

0

0

Feb.

0

0

0

0

0

1

1

March

0

0

0

0

0

10

10

April

0

6

9

4

19

148

167

May

2

17

8

3

30

81

111

June

4

16

13

7

40

175

215

July

8

53

49

9

119

341

460

Total

MBR*

26

2.2

137

11.4

130

10.8

72

6.0

365

30.4

1351

112.6

1716

143.0

 

* Mean man biting rate

Indoor Biting Behaviour Of An. Dirus - In human baited indoors whole night collections, proportions of anophelines and culicines was 21.6 and 78.4% respectively. Biting anopheline population was predominantly (99%) of An. dirus. The mean man-biting rate of An. dirus was 30.4 with highest biting density (119) recorded in the month of July (Table-2). Biting rhythm showed that it was mainly the mid night biter. Its biting practically started after 19 00 hour and maximum biting occurred between 21 00 and 04 00 hours in a wave like pattern with minor fluctuations. In premonsoon months (March-May) the mean peak biting (47%) was recorded in the II quarter of night ( 21 00 - 24 00 hrs), which was almost similar (40%) to the III quarter ( 24 00 - 03 00 hrs) biting in the monsoon months (June-September) and shifted to the IV quarter ( 03 00 - 06 00 hrs) in the post monsoon months of October and November. Overall, least amount of biting (7.1%) in the I quarter (18 00 - 21 00 hrs), high biting in II (37.5%) and III (35.6%) quarter and moderate biting in IV quarter (19.8%) of the night was observed. Observations on house frequenting behaviour revealed that about 25% unfed females took a brief rest for a mean period of 19± 23.5 minutes (range 2 to 95 minutes) on the walls after entering the house and prior to biting. The landing of hungry females was significantly higher (p<0.05) on legs as compared to face, trunk or arms. The mean parous rates in biting population of An. dirus in different quarters of night were 53.8% (I Qtr), 63.9% (II Qtr), 59.6% (III Qtr) and 61.5% (IV Qtr), the difference being statistically non-significant (p>0.05).

Anthropophilic Index Of An. Dirus - Mosquito blood meal analysis of 181 specimens of An. dirus was undertaken. A total of 167 were found to have fed on human giving anthropophilic index of 92.3% (Table-3). Two mosquitoes had mixed blood of human and bovine in their stomach while source of blood meal of 7 mosquitoes could not be identified. Not a single dirus female was found to feed on pig, dog, fowl or goat.

Table-3 : Sporozoite Rate, Parity And Blood Meal Analysis of An. Dirus

Month

Dissection results

Blood meal analysis

 

Number            Sporozoite         Parous
Dissected           rate (%)            rate (%)

Number       Anthropophilic
MBM tested   index (%)

1995

 

August

8

0.0

60.0

85

97.6

 

September

66

1.5

100.0

32

84.4

 

October

36

0.0

83.3

20

100.0

 

November

13

7.7

66.7

06

100.0

 

1996

 

March

02

0.0

-

02

100.0

 

April

39

2.6

73.3

-

-

 

May

47

0.0

46.6

04

75.0

 

June

88

1.1

78.0

18

66.7

 

July

195

2.1

48.7

14

100.0

 

Total

494

1.6

64.7

181

92.3

 

 - =Not done

Sporozoite And Parous Rates Of An. Dirus - A total of 494 females of An. dirus were dissected in different months of the study, which yielded 8 sporozoite positives (sporozoite rate 1.6%). Parity ranged from 46.6% (May) to 100% (September) with overall parous rate of 64.7% (Table-3).

Breeding Habitats Of An. Dirus - Breeding of An. dirus was always detected in the forest area but never in the village near human habitations. The nearest breeding site of this mosquito was detected at a distance of about 200 meters away from the village at the forest fringe. Rain water filled, transient, shady/part shady ground pools/puddles usually on jungle paths/elephant foot prints were the preferred breeding sites of An. dirus in most part of the year with the highest species diversity (Table-4). Most of these pools with clear to muddy water were shallow (15-20 cm deep), frequently had rotting leaves at the bed and usually became dried up during a dry spell of 6-8 days. During non-monsoon months, however, when such ground pools were not available, the breeding of An. dirus was detected in pools connected with the margin of streams. The breeding of An. dirus was found to occur in association with 11 mosquito species. Of all species breeding in association with An. dirus, Aedes caecus had the highest degree of association as well as the index of association (Table-4) in jungle pools.

Table-4 : Species And Habitat Association Of An. Dirus With Other Cohabiting Species
 

 

Species  

Breeding habitats in forest area  

1        2       3        4

In forest ground pools

Degree of interspecific association (CAB)  

Index of association (I)

Aedes caecus

 

 

+ 0.238 0.174

+ 0.428

 

Anopheles aconitus

 

 

 

+ 0.048 0.038

-0.422

 

An. hyrcanus gp.

 

 

+ 0.048 0.038

-0.826

 

An. kochi

 

 

+ 0.002 0.091

-0.700

 

An. vagus

 

 

 

-  0.032 0.682

-0.760

 

Culex bailyi

 

 

-  0.025 0.381

-0.668

 

Cx. fuscocephala

 

 

 

-  0.032 0.682

-0.820

 

Cx. pseudovishnui

 

 

 

+ 0.048 0.038

-0.860

 

Cx tritaeniorhynchus

 

 

 

+ 0.048 0.038

-0.648

 

Orthopodomyia anopheloides

 

 

 

+ 0.048 0.038

-0.424

 

Uranotaenia  spp.

 

 

 

+ 0.048 0.038

-0.424

 

1 = Shady, transient puddle/ground pools                   2 = Elephant foot prints
3 = Drying pools in seasonal streams
4 = Pools connected with margins of slow moving perennial streams

Day Resting Habitats Of An. Dirus - In 27 man-hours of indoor collections from human dwellings and 9 hours from cattle sheds during the study period, not a single day resting An. dirus adult could be collected. Drop net collections, both in forest and village, also failed to capture any day resting An. dirus adult from the ground level vegetations. However, extensive scanning of various above ground day resting niches in the forest yielded 22 (8 males and 14 females) resting adults. The majority (20 out of 22) were collected from the moist, dark corners of the large tree trunks, avoiding direct sunlight, up to the height of 1.5 meters from the ground level. Only 2 females were found resting on a bunch of intermingled creepers at a height of 1.8 meters. The nearest day resting An. dirus was found at a distance of about 150 meters from the village in the forest. Of the 14 day resting females, 8 were unfed, 4 full fed and 2 semi gravid with 57.1% parity and none of them was sporozoite positive.

Susceptibility To Insecticides Of An. Dirus - When fully fed females of An. dirus were exposed to diagnostic dosage of DDT 4%, Dieldrin 0.4% and malathion 5% in the laboratory following WHO bio-assay method, cent percent mortalities were obtained at 1 hour exposure and 24 hours post exposure period. This indicated the complete susceptibility of tested population of An. dirus to the common insecticides used in public health.

Malaria Prevalence In The Study Village - Monthly prevalence of malaria in the study population in given in Table-5. Malaria was endemic in the study village throughout the year. Distribution of the 264 fever cases, detected during the study, showed that 171 persons suffered at least once, 60 twice, 19 thrice and 11,2 and 1 person four, five and six times respectively. Overall slide positive rate (SPR) was 47.0% with predominance of P. falciparum infection (83.1%). Overall malaria prevalence was significantly higher (p<0.05) in females (SPR 53.5%) than males (SPR 39.2%). SPR in infants and children up to five years of age was significantly higher ( p< 0.01) as compared to children of 5-15 years and people above 15 years of age.

Table-5: Malaria Prevalence In The Study Village

Month

BSE

POS

Pv

Pf

Mix

SPR

SfR

Pf %

1995

August

30

18

0

18

0

60.0

60.0

100.0

September

15

8

1

7

0

53.3

46.7

87.5

October

21

11

1

10

0

52.4

47.6

90.0

November

26

17

3

13

1

65.4

53.8

82.4

December

17

16

3

12

1

94.1

76.5

81.1

1996

January

11

5

0

5

0

45.5

45.5

100.0

February

19

9

0

9

0

47.4

47.4

100.0

March

8

2

1

1

0

25.0

12.5

50.0

April

7

1

0

1

0

14.3

14.3

100.0

May

16

11

4

7

0

68.8

43.8

63.6

June

39

14

4

10

0

35.9

25.6

71.4

July

55

12

4

8

0

21.8

14.5

66.7

Total

264

124

21

101

2

47.0

39.0

83.1

BSE = Blood slides examined             POS = Malaria positives                         Pv = Plasmodium vivax
Pf = Plasmodium falciparum             Mix = Pv + Pf                                             SPR = Slide Positive Rate
SfR = Slide falciparum rate               Pf % = P. falciparum percentage

Conclusions:

(a) An. dirus depicted distinct seasonality. In forest fringe areas, its density built up started from March onwards with the onset of Pre-monsoon rains, attained peak in July/August (Monsoon months), remained high till October (Post monsoon period) and dropped to very low level in cool dry months (November- February). The density was positively correlated (r =0.72) with the amount of rainfall occurring 2 weeks prior to the collections.

(b) An. dirus exhibited considerable degree of endophagy.

(c) An. dirus was predominantly a late night biting mosquito with intense biting between 21 00 and 03 00 hours showing seasonal differences in the peak biting hours.

(d) A proportion of host seeking unfed females, after entering the dwelling and before feeding, rested on the walls for some time whereas the majority alighted for feeding as soon as they entered the dwelling.

(e) Legs were the most preferred part of the human body for feeding by the hungry An. dirus females.

(f) An. dirus was highly anthropophilic mosquito preferring human blood more for feeding.

7(g) An. dirus was exclusively a forest breeding mosquito. Small, shallow, rain filled, transient, partly shaded puddles/ground pools on jungle path/elephant foot prints having muddy water and leaf litter were the preferred breeding habitats of An. dirus during the rainy season. However, during non-monsoon months, breeding of An. dirus was shifted to pools at the margins of the perennial jungle streams.

(h) Aedes caecus was the most common cohabiting mosquito species breeding in association with An. dirus in the jungle pools.

(i) An. dirus was completely exophilic, always day-resting in the forest mostly on tree trunks in dark moist niches avoiding direct sun light.

(j) High sporozoite rate as well as high parity of An. dirus indicated its high potential of transmitting malaria in forest areas. Malaria transmitted by it was prolonged and intense between May and December.

Funding agency code  Intramural

Study Type code

NON

 

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Epidemiological observations on malaria in some parts of Tengakhat PHC, Dibrugarh district, Assam.

 

Investigator :        Dr P Dutta.
Co investigator:           Dr D.R.Bhattacharyya.

Subject key words:     

Epidemiology
Malaria
Mosquito vector
Bionomics

Objectives:

(i) to know the true incidence of malaria, seasonal prevalence of parasite species

(ii) to survey the anopheline fauna and to detect the vector(s) responsible for the current transmission of malaria and to know their bionomics.

Introduction: Many parts of Northeast India are highly afflicted with the problem of malaria. When P falciparum infection in relation to resurgence of malaria is considered, there is a steady increase of it in the total cases in the Northeastern Region. Although P vivax was the predominant species earlier in recent years, a greater prevalence of P falciparum malaria is observed. The present study was conducted in some parts of Tengakhat PHC of Dibrugarh district, Assam during 1987-89 undertaking a longitudinal study.

Materials and methods: Some of the areas of the PHC which are not far away from the Arunachal border were selected for the study. It has a heterogeneous terrain with foothills, river beds, tea gardens, paddy fields and on one side there is a forest range.44 villages with a population of 17,938 were divided into 4 sectors and in each sector , one surveillance worker was recruited. The SWS enumerated the houses and collected information in a predesigned proforma. A weekly house to house survey was carried out by them to record the fever cases. Blood smears were collected from fever cases and presumptive treatment was given to them. After confirmation of parasites, radical treatment was given according to species of parasite. The anopheline mosquitoes were collected in cattle sheds.outdoors, indoors and by operating CDC light traps for whole night.These were dissected for gland or gut infection. Human bait collections were made using 3 different bait types-(i) outdoor human bait(ii) indoor human bait (iii) a cattle bait. Blood meals of vector species were collected on Whatman no 1 filter paper to know the host feeding index of the vector by using different antisera in gel diffusion technique.

Results: Results of surveillance conducted in the study area for three consecutive years (1987-89) are given in Table 1. It can be seen that malaria cases were maximum in the year 1988, SPR ( Slide Positivity Rate) being 28.59 which is significantly higher in comparison with that of 1987 (16.05) and 1989 (12.90). The active surveillance reveals that the area is endemic for P.falciparum malaria as in 3 years of study, P.falciparum has been observed to be the dominant species over P.vivax. Maximum malaria cases were recorded in the months of September, October and November. No separate outbreaks of these species had been observed, P.vivax cases were found almost in association with P.falciparum. It can be seen that persons of all age groups and sex were affected and the attack rate is highest in the age group 2-9 years. It is interesting to note that the villages near to the forest reserves are more prone to malaria than that of non forested areas. It is evident from our study that the estimated attack rate in the villages of non forested areas was 58.87 as against the observed value of 3.34, thus disclosing the fact that the forest fringes are more prone to malaria than the rest of the population. The entomological survey reveals that a total of 16 Anopheline sp have been encountered (Table 3). An. dirus which constituted 2.6 % of the total Anopheline collection was incriminated as vector for current transmission of malaria. The vector density was found high in the period from June to October and during this period high transmission of malaria took place as evident from the high malaria positivity in this period. The vector, An. dirus disappears on commencement of winter. A total of 137 samples of blood meal were tested to identify the host preference of the vector and out of these 124 (90.5 %)samples were positive for human blood, 3 (2.2 %) for bovine blood, 2 (1.5 %) for avian blood and 8 (5.8 %) did not show any reaction. It is evident from the result that An. dirus prefers human blood, the anthropophilic index being 90.5. Results of bait collections of vector reveal that almost equal prevalence of An. dirus using outdoor human bait (46.5 %) and in indoor human bait (44.2 %) was observed whereas its presence on cattle bait was not so pronounced. The feeding of An. dirus on human bait was observed throughout the night and its biting activity was prominently recorded between 2000 - 2100, 2300 - 2400 and 0200 - 0300 hours. The peak of biting activity was high in between 2000 - 2100 hours. It was observed that An. dirus females just after coming from their day resting abodes took a rest in outdoor vegetation around catching stations very early in the evening. They were found to remain inactive for some time before seeking a blood meal and to start their biting activity from 1900 hours.

Table 1: Result Of Active Surveillance In The Study Area Under Tengakhat PHC Of Dibrugarh District During 1987-89  

____________________________________________________________________

Population  Year   No.of          No. of      Pf     PV    Mix    SPR    SFR     API 

                             blood          total

                              slides          positive

                              examined
__________________________________________________________________

                   1987       529             85         60     22      3      16.06    11.34    6.45
17,938        1988     1435           409       359     46      4      28.50    25.01  31.06
                   1989       612             79         66     13      0      12.90    10.78    6.00
___________________________________________________________________

SPR = Slide positivity rate; SFR =Slide falciparum rate ; API = Annual  parasite index.

 

Table 2: Age And Sex Specific Attack Rate Of Malaria In The Study Area

___________________________________________________________

Age group    Population    No. suffering from malaria          Attack rate
                                         --------------------------          per 1000
                                           Male      Female    Total
__________________________________________________________

0-1 & > 1        771                 5             2             7              9.07
  2-9              4483             128         114         242            53.98
10-20            4578               71           29         100            21.84
21-30            3580               69           57         126            35.19
31-50            3190               51           30           81            25.39
  > 50            1336                 9             7           16            11.97
______________________________________________________

 

Table 3:  Anophelines Collected By Light-Trap And Suction Tube From Different 
                Sources During 1987-88

------------------------------------------------------------------------------------------------------
Sl.No. Species                   Light-trap collection                Suction tube collection  
                                   ________________________     _______________________
                                  Cattle shed    Human dwelling          Cattle shed       Outdoor rest.  

                                  -------------     ----------------       -----------       -------------- 

                                   No.       %        No.       %            No.     MHD         No.   MHD 
                            

1. An.aconitus           153      3.11        7          2.0            140       0.17        8      0.009
2. An. annularis          87       1.77        0          0.0             95        0.11       7       0.008
3.An.barbirostris        45       0.91        1         0.29            35        0.04       0       0.000
4.An. dirus                  36       0.32      94       27.50             6       0.007     97       0.11
5.An.culicifacies           0       0.00        1         0.29             0         0.00       0       0.00
6.An.nigerrimus        941     19.14      11         3.22         955         1.05     20       0.02
7.An. jamesii                 1       0.02        0         0.00            2        0.002      0        0.00
8.An. karwari          1413     31.65        9         2.34        615          0.75    45        0.05
9.An. kochi              1556     28.74        8         2.63        220          0.27    14        0.01
10.An.maculatus         31       0.63        1         0.29        105           0.12     0        0.00
11.An.philippi           447       9.09      16         4.69        448           0.55    40       0.04
     nensis
12.An.minimus            68       1.38        3         0.87          20           0.02      0       0.00
13.An.splendidus          1        0.02        1         0.29           9            0.01      0       0.00
14.An.tessellatus        16        0.32        0         0.00           1          0.001      0       0.00
15.An. vagus            135        2.74     189       55.42      643            0.79    14       0.01
16.An. gigas                 5        0.10         0         0.00          0            0.00      0       0.00
------------------------------------------------------------------------------------------------------
* Collected in 126 trap nights ; MHD= Per man hour density.       

Conclusion: From this study, it is evident that P. falciparum malaria is the predominant species. The vector incriminated is An. dirus which is highly exophilic and therefore, DDT spraying did not interrupt transmission in the study areas. In such a situation, personal protection measures by the use of mosquito nets may be an effective measure in reducing the chances of man - mosquito contact while sleeping as our observation in the study area reveal that the risk of acquiring malaria by the mosquito net users are 5 times less than the non bednet users.The quality of surveillance should be improved in the endemic region along with chemoprophylactic measures for the forest workers so as to reduce the parasite reservoir among them. As the people of the backward areas are of lower socioeconomic status and are less conscious about maintaining hygienic conditions, the health education will be essential to gaining community participation so that the containment programme of malaria through integrated approach can be implemented successfully in this region.

Funding Agency 

Intramural

Study type code

EPI

 

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Malariometric studies in some parts of Assam and
Arunachal Pradesh

Investigator:                            Dr P Dutta
Co investigator:                      Dr D.R. Bhattacharyya

Subject key words:     

Malaria
Chloroquine resistance
P falciparum
Malaria vector


Objectives:

(i) to determine the prevalence of malaria
(ii) to know the magnitude of chloroquine resistance
(iii) to identify the vectors.

Introduction: Resurgence of malaria, particularly its virulent form of chloroquine resistant P falciparum has created a formidable problem. The Northeastern Region of India has not escaped from this global problem. When P falciparum infection in relation to resurgence of malaria is considered,there is a steady increase in the number as well as its ratio with total cases in the N.E region of India. Among the Anophelines, An balabacensis (An dirus) had been considered in transmission of malaria and An minimus was thought to have disappeared from N.E region after application of DDT as residual insecticides in malaria control program. Keeping these views into consideration, malariometric studies were made in some selected areas of Assam and Arunachal Pradesh during the period from 1988-94).

Materials and Methods: The study was made in some parts of Assam and Arunachal Pradesh. A house to house survey was carried out in the investigation areas to detect the fever cases. Blood smears were collected from the patients and after JSB staining examined under microscope for identification of parasite species. All the fever cases were treated with presumptive treatment and after confirmation of parasites, radical treatment was given to the patients. Suitable P falciparum cases were selected for further studies to know the status of chloroquine resistance by WHO in vivo 7th and 28th day field tests. The excretion of chloroquine in the urine was monitored to ensure absorption of chloroquine in the blood. The Anopheles mosquitoes were collected with the help of suction tubes between 1800-2400 hrs. mostly from cattlesheds adjacent to human dwellings ,indoors and outdoors and also by CDC light traps from dusk to dawn ie. from 1800-0500 hrs. The collected mosquitoes were transported to the laboratory for identification and dissected to detect gland or gut positivity for malaria infection.

Results: The study revealed that malaria is a major health problem in this region. High prevalence of P falciparum malaria about 70-97% was detected in the study areas(Table-1). All the age groups and sex are affected by malaria and lower age groups (2-9 yrs) are more vulnerable to malaria infection (attack rate 54.0) in comparison to other age groups (9.07-35.i9). Males and females are equally affected. Table-2 shows the status of chloroquine resistance in P falciparum cases detected in different areas. Except in Boko area of Kamrup district, the other areas, the magnitude of chloroquine resistance was remarkably high (more than 70%). RII level of resistance was very common. RIII level of resistance was also detected and considerably high as observed in Tengakhat area followed by Digboi area and Nampong area. Entomological collections reveal that Anopheline fauna is very rich in foothill and forest ecosystems. The abundance of An minimus was more pronounced in foothills without much forests having streams and paddy fields. On the other hand, the preponderance of An dirus was observed in forested/ forest fringe areas. The collected mosquitoes were dissected and Table-3 shows the vector species incriminated from different areas during the study period.

Table 1: Record Of Malaria Cases In Different Study Areas Of Assam & 
Arunachal Pradesh

_____________________________________________________________ 
Study area          Period      B.S.      Positive     Pf     Pf%     SPR     SfR
                                             Exam.
_____________________________________________________________

Namsamg area    Sept./Oct.   190          51        39     76.4     26.8      21.0
(Arunachal)          1986 
Nampong PHC   1987/88     2664       614      440      71.6    23.0      16.5
(Arunachal)
Tengakhat PHC  1987/88     2057       458      391      85.3    22.2      19.0
(Assam)
Boko area         November      120         61       51       83.6   50.83     42.5
(Assam)            1991
Khotkhoti area  1991/92         703        122       98       80.3    19.0     13.9
Karbi Anglong,
(Assam)
Digboi area     Sept.-Nov.        286       113      110      97.3    39.5     38.4
(Assam)            1993
_____________________________________________________________

Table 2:  Chloroquine Resistant P.Falciparum Cases Detected From Different
Study Area

________________________________________________________________
Study area           No.of   Test         Level of resistance        Total          Rate of
                            cases   Method    ________________    Resistant    Resistance
                                       (in vivo)     S R1/S R1 R11 R111   cases    
_________________________________________________________________
Namsang area         11   7th day      3     -     -     8       8           8             72.7
(Arunachal)                    field test
Nampong PHC       36   28th day     4    6   12    10     4         26             72.2
(Arunachal)                    field test
Tengakhat PHC      36    28th day    7    3     5      7    14        26             72.2
(Assam)                          field test
Boko area               39    7th day     33    -     -      5      1          6            18.1
(Assam)                          field test
Digboi area            50    28th day     8    5    15    15      7        37             74.0
(Assam)           field test
_________________________________________________________________

    

Table 3: Incrimination Of Malaria Vector From Different Study Areas

_____________________________________________________
 Study area                      Species                    Year of incrimination
_____________________________________________________
Tirap District                   An. minimus              1987(Arunachal)
Changlang District           An. dirus                   1992
(Arunachal)
Dibrugarh District            An. dirus                   1989
(Assam)
Kamrup District               An. minimus             1994
(Assam)
KarbiAnglong District     An. minimus              1995
 (Assam)
Goalpara District            An. minimus               1997
 (Assam)
_____________________________________________________    

Conclusion: P falciparum malaria along with chloroquine resistant status of P falciparum cases has been found remarkably high. Therefore, malaria is creating a havoc by causing several deaths due to sudden outbreaks in recent years. As the physiography of N.E Region is totally different from the rest of the country, the vector prevalence with ecology also vary. An dirus and An minimus are the major malaria vectors found to be responsible for transmission of malaria. Their distribution pattern also vary according to the ecosystems of the region. An minimus although was thought to have disappeared from Northeast,is still present in the areas of poor DDT spray coverage. The increasing trend ofP falciparum malaria along with chloroquine resistant P falciparum foci and the specific breeding and resting habits of these vectors, the control/management of malaria in this region is becoming not only a formidable problem but also a great challenge to the field of malaria research.

Funding Agency

Intramural

Study code

 EPI

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 Mosquito fauna of Northeast India with special reference to the medically important vectors

Investigator:               Dr P. Dutta

Coinvestigator:             Dr S.A. Khan

Subject key words:     Mosquito Fauna
                                            Northeastern Region
                                            Disease vectors
Objectives:
(i) To identify the mosquito fauna in different states / geographical regions of Northeast and their medical significance
(ii) To study the larval ecology of different species.

Introduction: The Northeastern Region of India, the physiography of which is totally different from the rest of the country is endowed with very rich flora and fauna. The high hilly terrains,foot hills,deep forestation,high rainfall,high humidity,moderate climate in plains and valleys and cold climate in high altitudes are the main ecological components of this region. This region is very rich in mosquito fauna. Many areas of this region are endemic for different mosquito borne diseases like malaria, filariasis, JE, dengue. The studies on mosquitoes in this region are limited and quite old. In India,most of the work on mosquitoes was previously carried out by Barraud and Christophers in 1930"s. However, the inadequate material and insufficient descriptions in the past have led to confusion and misidentification of mosquitoes particularly in this region. Thus, there is a great scope to explore the detail mosquito fauna of this region which is very essential in relation to the epidemiological point of view in transmission of mosquito borne diseases.

Methodology: Attempt will be made to cover all the seven states of Northeast viz. Assam, Arunachal Pradesh, Meghalaya, Nagaland, Tripura, Manipur and Mizoram in a phased manner. The survey will be conducted in different locations representing different ecotypes like foothills, hills, high hills, forests, valleys, plains etc. Adult mosquitoes will be collected by operating CDC light traps. More emphasis will be given to collect the immatures from different habitat types. These will be individually reared to adult stage so as to trace out all the stages of the mosquito for confirmed identification. Informations about their breeding habitat types, water characteristics will also be noted. Identification of species will be done by following standard keys.

Results: Survey work of mosquitoes was initiated in the year 1995 and uptil now 5 states have been covered.

Assam: A total of 66 species of mosquitoes belonging to 9 genera were recorded as Anopheles (20), Culex (18),Aedes (23), Armegeres (4), Mansonia (5), Toxorhynchites (2), Malaya (1), Uranotaenia (1) and Tripteroides (2).

Arunachal Pradesh: From immature and adult collection 65 different species of mosquitoes under 11 genera are recorded which are as follows - Anopheles (25), Culex (18), Aedes (7), Mansonia (2), Armegeres (3), Malaya (2), Toxorhynchites (2), Uranotaenia(2),Ficalbia (2), Tripteroides (1) and Heizmania (1).

Meghalaya: A total of 59 mosquito species under 9 genera were recorded from this state as Anopheles (23), Culex (15), Aedes(7),Mansonia (4),Armegeres (2), Malaya (2), Toxorhynchites (2), Uranotaenia (3), Tripteroides (2).

Nagaland: 55 mosquito species of 9 different genera were recorded from Nagaland state which are as follows- Anopheles (21), Culex (16), Aedes (9), Armegeres (2), Mansonia(3), Toxorhynchites (1), Coquillitidae (1), Malaya (1), Uranotaenia (1).

Mizoram: In one survey conducted during monsoon season, 48 species of mosquitoes of 9 genera which are as Anopheles (19), Culex (12), Aedes (9), Armegeres (2), Mansonia (3), Toxorhynchites (2), Coquillittidia (1), Malaya (1), Tripteroides (1) were detected.

Conclusion: Among ground water habitats, richest diversity of mosquito species was found in jungle pools followed by irrigated paddy fields, animal hoof marks, fallow fields, rain water pools, stagnant roadside canals and mud pools. Among container habitats, treeholes harboured more species followed by plantain leaf axils and bamboo stumps. Anopheline fauna is rich in hilly and foot hill ecosystems breeding mostly in fresh water pools,slow moving streams,irrigation canals,jungle pools,animal hoof marks etc. Major malaria vectors of the Northeast viz. Anopheles dirus and An minimus are characteristically the species of hills and foothills. The former breeds in jungle pools, animal footprints in forest ecosystem. The later breeds in slow moving stream water, irrigated canals in foothill ecosystem. Species under genus Culex and Mansonia are ground water habitat breeders. Most of the potential vectors of JE are found to breed in mud pools, fallow fields, rainwater pools, drains near paddy field etc. Mansonioides are found breeding in pond water, large swampy areas in association with water plants. The species of Aedes which includes mostly the potential dengue vectors viz. Aedes aegypti and Ae albopictus are found to breed in waste tyre dumps, waste battery boxes and other solid waste pollutants which can store rain water.

Funding Agency

Intramural

Study type code

NON

 

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A study on prevalence and pattern of substance use in North-East India

Investigator Code:     H. K. Chaturvedi

Co-Investigator:          R. K. Phukan

Funding Agency:        Internal Fund of Institute

Subject Keywords:     Epidemiology
                                           Substance use
                                           North-East India.
Study Type Code:       EPI

Objectives:
(i) To study the prevalence of substance use in different locations(rural/urban/hilly areas)
(ii) Types of substance use, its consumption pattern and association with socioeconomic, demographic, religion and culture of people.

Introduction: Millions of people all over the world consuming one or multiple substances which cause severe damage to their health and society at large. Many scientific literatures and reports, described the magnitude of drug abuse problem and associated cause of increasing substance use, have been published from different parts of world. According to published scientific reports, excessive use of tobacco and alcohol are the major cause of cancers, cirrhosis and many other diseases. Despite of increasing knowledge about the harmful effect substance use (tobacco, alcohol, opium, cannabis, etc.), the progressive increase of substance use has been reported in many studies. Oral cancer rates are highest in India and trend is still increasing. As per WHO report, the estimated prevalence of bidi and cigarette smoking in India is about 40% among men and 3% among women in the 15 year of age and over. In India, the basic drugs like opium, cannabis and alcohol (somras) has been known and consumed by the people since ancient time. Substance use has deep relation with religious faith, cultural and traditional beliefs of people. Ancient knowledge about the medicinal properties of substance are still attracting people towards their use especially in remote areas. A special emphasis to study the type of substance use in North-East India is becoming priority to monitor the situation, because the long international border with Myanmar and proximity across the border. Heroin use in Nagaland and rapid spread of HIV/AIDS infection due to intravenous drug use in Manipur has created alarming situation of drug use problems in this region.

Methodology: Extensive study on substance use in North-East India needs sufficient funding and proper plan of work involving trained personals to collect information directly from community of the entire region. Considering the importance of the study, the project work was initiated with limited available internal resources and man power of this Institute. Accordingly, the study was confined to some selected areas of North-East states based on operational and feasibility of the study.

Study Area: Selected study areas for this study are a) Changlang district of Arunachal Pradesh, b) Aizawl district of Mizoram, c) East Khasi Hill district of Meghalaya and d) Dibrugarh and Tinsukia district of upper Assam. Literature review on epidemiological study of substance use reveals that the scientific study on substance use from these selected areas was not reported earlier. However, study on heroin use in Manipur and Nagaland was reported. Basic information regarding the types of substance use and their prevalence and pattern are essential inputs to formulate and plan extensive study to acquire knowledge about the over all situation in this region. With this view, two PHC areas in all four locations (a,b,c,d) were selected in such a way to represent rural and urban population of the region. Due to manpower limitations, data collection/survey in the selected district was carried out one after another jointly by scientists of RMRC, Dibrugarh and staffs of Primary Health Centres of the concerned area.

Sample Survey and Data Collection: Household survey was carried out in the randomly selected villages/locations of the PHC areas of the district by using method of systematic sampling. Head of the household or the senior most person was interviewed to collect general information, whereas habits of substance use(like tobacco, alcohol, opium, cannabis, heroin etc.) were recorded from all the family members(age 10 year and above) in the pretested structured questionnaire.

Duration of survey:

a) Changlang: Survey was carried out in Changlang district to collect basic information about the type of substance use among tribal population during October to November,1996. Total 165 households were selected and 702 persons were interviewed to collect information about their habits of substance use. The results of this study was useful to prepare a plan of comprehensive epidemiological study in the district covering the entire population. Further, a comprehesive study was also carried out in the entire Changlang district covering all the 10 PHC areas. A required sample size was worked out using the population of the district(10 years age and above) and prevalence of opium (5%). Total 5135 respondents selected radomly covering all the PHC areas of the district were interviewed and information about their habits of substance use were recorded. Survey has been completed during 1997-1999. Data analysis and report preparation is under progress.

b) Aizawl: Survey on types of substance use was carried out in two Sub-Health Centres of Aizawl district namely Dinthar and Sakawrtuichhun during June to September,1997. Total 105 households were selected and 375 persons were interviewed to collect information about their habits of substance use.

c) East Khasi Hills: Survey in Mawlai and Pomlum area of East Khasi Hill district was carried out in the year 1998 (June-October,1998). Total 114 households were selected and 448 persons were interviewed to collect the information about their habits of substance use.

d) Dibrugarh and Tinsukia: Survey in the two selected PHC areas namely Lahowal in Dibrugarh and Kakopather in Tinsukia district was carried out during the year 1997-1998. Total 303 households (166 from Dibrugarh and 137 from Tinsukia) were selected and 1383 persons were interviewed to collect the information about their habits of substance use if any.

Results: The results of the study carried out in Changlang district of Arunachal Pradesh and Aizawl district of Mizoram are published in Scientific Journals and the detail results are presented below. Study carried out in East Khasi Hills district of Meghalaya, Dibrugarh and Tinsukia district of upper Assam were completed and results are communicated for publication.

a) Changlang, Arunachal Pradesh:

Part-I. Results of the study (October-November,1996)
Use of substances (tobacco,alcohol and opium) among the 702 respondents are presented in Table-1.1. Prevalence of tobacco use was 24.3 percent among all the respondents. However, 40.5 percent were found among males and 10.8 percent among females. Among tobacco users, 67 percent were only tobacco chewers, 20 percent were only smokers and 13 percent were practicing both the modes (chewing and smoking). Further, among males there were 25.8 percent only tobacco chewers, 9.2 percent only smokers and 5.4 percent both. Though the tobacco use among females was found low (10.8%), among them 8.7 percent chew tobacco, 0.9 percent smoke and 1.2 percent practice both the modes. Prevalence of alcohol use was 25.1 percent among the tribal people (Table-1.1). Alcohol use among males was very high (34.8%) as compared to females (14.4%) with 3:1 male and female ratio among alcohol users. Tobacco and alcohol use has significant association with sex. Of the 702 respondents, 29 (4.1%) were found taking opium. Distribution by sex shows that 7.3 percent male and 0.6 percent female were opium users (Table-1.1). Overall 256 (36.5%) persons were in habit of taking at least one substance and among them 50.4 percent were multiple substance users. Distribution of respondents by religion was 43% Hindu, 32% Christian and 25% Buddhist. Tobacco and alcohol use was high among Hindu (33.7% & 46.7%) followed by Buddhist (33.5% & 12.1%) and Christian (10.8% & 5.4%). In case of opium, it was highest among Buddhist (9.8%). Religion has significant association with substance use(Table 1.1). Among educational groups, tobacco users were high among illiterates (32.3%) followed by Middle pass( 22%). In the group of higher education (i.e. Secondary and above), it was found to be low (12.3%). Alcohol use was 32% among Middle pass followed by 27.7% among Secondary and higher. The alcohol use was found comparatively higher among educated group of people. There was significant association of education with tobacco and alcohol use. However, the trend was significant only for tobacco. Opium use was mostly confined among illiterates (6.2%) and Primary level literates (0.7%). In higher educational group, opium use was not found (Table1.1). Distribution of substance use by age are given in Table-1.1. Maximum tobacco users (59.7%) were found in the higher age group (60 year & above) followed by 55.6% in 50-59 years age. It was recorded low 2.5% in adolescent (10-19 year) and 20.8% in young age (20-29 year). Above 40 year of age, differences in prevalence was insignificant. Use of alcohol among adolescents (10-19 years) age group was low (8.3%) whereas in the higher age groups, it varies from 27.2 to 43.9 percent. Chi-square test of association and trend of tobacco and alcohol use by age were found highly significant. Pair wise comparison indicates that there was no significant difference in the prevalence of alcohol usage among adult (30 and above). Percentage of opium users by age also showed a trend in increasing order with the highest (27.8%) users recorded in higher age group (60 year & above).

Part-II. Results of Epidemiological study in Changlang(1997-2000)

Though results are under preparation, some important findings of analysis are presented without table. Types of substance use among the 5135 respodents were tobacco - 30.9%(22.8% chewer and 12.1% smokers), alcohol - 30% and opium - 4.8%. Prevalence of opium use was high in Khimiyong(16.6%) and Bordumsa(14.4%) Circles whereas it was below 5%( 0 - 4.4%) in the remaining Circles of the district. High prevalence of opium use was also recorded among Singpho(15.5%) and Khamti(13.5%) tribes. The detail results of the study will be communicated later.

Table-1.1 : Distribution of substance users by age, sex, religion and education.

Variables

No. of Respondents

                                     users (%)
Substance

Tobacco

Alcohol

Opium

SEX
Male
Female
Chi-square(A)

     
368
334
D.F. 1

        
40.5
10.8
79.63**

       
34.8
14.4
38.83**

     
7.3
0.6
-

RELIGION
Hindu
Christian
Buddhist
Chi-square(A)

 
306
223
173
D.F. 2


33.7
10.8
33.5
40.93**


46.7
5.4
12.1
137.85**

  
2.6
1.8
9.8
19.02**

EDUCATION
Illiterate
Primary
Middle
Secondary & higher
Chi-square(A)
(T)

    
449
138
50
65
D.F. 3
D.F. 1

       
32.3
15.2
22.0
12.3
24.08**
17.43**

      
26.9
15.2
32.0
27.7
9.49*
0.02 NS

    
6.2
0.8
-
-
-
-

AGE (Year)
10-19
20-29
30-39
40-49
50-59
60+
Chi-square (A)
(T)

     
240
173
104
  74
  57
  54
D.F. 5
D.F. 1

      
2.5
20.8
39.4
51.4a
55.6a
59.7a
162.36**
149.42**

       
8.3
27.2a
33.7ab
35.1ab
42.6b
43.9b
63.80**
53.37**

     
-
0.6
3.8
9.5
10.5
20.4
-
-

Total

702

26.3

25.1

4.1

* Significant at 5%;         ** Significant at 1%;         NS - Not significant;

D.F.- Degrees of freedom;     Chi-square A- Association,             T- Trend;

a,b Values marked by a or b show no significant difference.

b) Aizawl, Mizoram:

Types of substance use recorded in Aizawl was mainly tobacco. Though alcohol users were not detected in this study, but it was possibly due to the prohibition of alcohol use by the state Government. However, illegal use of alcohol was possible. During the survey in the randomly selected households in the town area, one youth was found using proxyvan by injecting the drug. Prevalence and pattern of tobacco use in different sociodemographic class of peoples are presented in Table-2.1. Percentage of tobacco user was 48.7 percent in Dinthar and 53.7 percent in Sakwrtuichhun. The difference was not significant. In Dinthar tobacco was consumed by smoking (30.5%), chewing(17.7%) and using as "Tuibu" (3.7%), whereas tuibu user was not found in Sakawrtuichhun. Distribution by sex shows that smokers were higher (42.3%) among males than females (16.1%), but just reverse in case of chewers. Tobacco use was found significantly associated with sex. In different age groups, 64.3 percent tobacco users was found in the higher age( 30 year and above) and 38.9 percent among lower age(10-29 year). Percentage of smokers was found increasing by age, but no such trend was found for chewers. Among different educational groups, the use of tobacco was varying from 60.3% to 41.6% from Primary level to higher level of education respectively, but again low (46.4%) among illiterate. Association of educational level was not significant. Distribution of tobacco users by occupation was 44.1 percent chewers among housewives followed by 25.4 percent among employed respondents. It was very low (10.4%) among students. High percentage of smokers was found among unemployed(44..4%) and self-employed(44.6%) respondents whereas it was low among students(20%) and housewives(20.6%). Overall, tobacco users were less (29.6%) among students, but it was very high ( 57.8 to 67.7%) among other occupations. Association of occupation with tobacco use was highly significant. Of the 192 tobacco users, most of them (96.9%) were single mode user (chewer or smoker or tuibu user). Only four males and two females were found taking tobacco in multiple mode. Female tobacco chewers were higher(26.0%) than males (14.1%) whereas among smokers, females were less(15.1%) compare to males(39.6%), presented in Table-2.2.

Table 2.1: Distribution of tobacco users by age, sex, education and occupation
                  in Aizawl district, Mizoram.

Variables

No. of
Respondents

                                    users (%)
Tobacco

Chewer

Smoker

Total #

LOCATION

Dinthar

187

17.7

30.5

48.7

Sakawrtuichhun

188

26.1

28.2

53.7

Z - test

 

1.85 NS

0.37 NS

0.88 NS

SEX

Male

189

15.9

42.3

56.6

Female

186

27.9

16.1

45.7

Chi-square(A)

D.F. 1

8.01*

31.04**

4.47*

AGE (Year)

10-29

193

19.2a

20.2

38.9

30-49

112

29.5

35.7a

64.3a

50 & above

70

17.1a

44.3a

64.3a

Chi-square(A)

D.F. 2

5.52 NS

8.89*

24.24**

(T)

D.F. 1

0.09 NS

2.74 NS

19.55**

EDUCATION

Illiterate

28

14.3a

32.1a

46.4ab

Primary

58

29.3b

27.6a

60.3b

Middle

91

24.2ab

32.9a

56.0b

Secondary

109

20.2ab

30.3a

51.4b

Degree +

89

19.1a

24.7a

41.6a

Chi-square(A)

D.F. 4

3.69 NS

1.73 NS

6.35 NS

OCCUPATION

Student

135

10.4a

20.0a

29.6

Unemployed

36

16.7ab

44.4b

61.1a

Self-employed

65

21.5b

44.6b

67.7a

Employed

71

25.4b

33.8b

57.8a

House-wife

68

44.1

20.6a

66.2a

Chi-square (A)

D.F. 4

31.23**

20.16**

40.95**

# Total includes seven `Tuibu' users with all chewers and smokers.

* Significant at 5%;       ** Significant at 1%;           NS - Not significant;

D.F.- Degrees of freedom;         Chi-square A- Association,         T- Trend;

a,b Values marked by similar letter (a or b) indicate no significant difference.

 

Table 2.2 : Percentage frequency of male and female tobacco users.

Categories

Percentage of  user

 

Male

Female

Total

Tobacco use
Single mode

Chewer(C)

14.1

26.0

40.1

Smoker(S)

39.6

15.1

54.7

Tuibu (T)

-

2.1

2.1

Multiple mode

C & S

1.6

-

1.6

C & T

-

0.5

0.5

S & T

0.5

-

0.5

S & C & T

-

0.5

0.5

Tuibu preparation and use: Tuibu is a liquid preparation and its processing is technically similar to water pipe (hookah). Tobacco is burned in a container on the head of a pipe and smoke is drawn by the pipe and passed through water by sucking mechanically. Approximately, smoke of 1 kg tobacco required for two liters water (tuibu). This addicting substance (liquid form of tobacco) is used by keeping in the mouth for few seconds, which gives instant stimulation to the user.

c) East Khasi Hills, Meghalaya:

Prevalence of tobacco use among the people of Khasi Hills was 41.7% (20.8% chewers and 22.5% smokers). Female tobacco chewers were higher(33.0%) compare to males(7.8%) whereas smokers were higher(45.4%) among males.Of the 448 respondents, alcohol users were 11.8 percent. Detail results of the study will be given after the publication of findings. However, comparative results of East Khasi Hills, Meghalaya and upper Assam(Dibrugarh and Tinsukia) has been prepared and communicated for publication.

d) Dibrugarh and Tinsukia, Assam:

Prevalence of tobacco use was 36.6% (29.7% chewers and 14.8% smokers) in Dibrugarh and 15.6% (12.3% chewers and 5.0% smokers) in Tinsukia. Alcohol use was higher in Dibrugarh(20.0%) than Tinsukia(6.2%). Besides tobacco and alcohol, opium and cannabis users were also recorded. Except one cannabis users from Dibrugarh, 85 (11.6%) opium and 8 (1.1%) cannabis users were recorded in Tinsukia. Among the 85 users, 65 stopped taking opium currently due to family pressure and poor economic condition, though they were consuming opium regularly for many years.

Conclusion: Based on the study carried out in different locations of North-East India, findings of these studies highlights the contribution of regional, social, demographic and other factors on type of substance use in the community level. Wide differences on types of substance use was observed in different parts of North-East India. It is essential to develop a continuous monitoring system to study and collect the information from different sources directly and indirectly like private agencies, social workers of community and Government agencies involved in health care and control of narcotic substance abuse. Identification of high risk areas based on direct and indirect sources of information. Evolving a comprehensive programme to control the use of addictive substance and providing treatment facility and rehabilitation care to the addicts. Considering the drug addiction as a disease which can be cured by proper care, but certainly not as a evil or curse of life.

Funding Agency

Internal Fund of Institute

Study Type Code

EPI

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Studies on prevalence of filariasis in some tea garden population of Assam

 

Investigator :               Dr. A.M. Khan

Co-Investigators :     (i) Dr. P.Dutta (ii) Dr. S.A. Khan

Funding agency :         Intra-mural

Subjects Keywords :    Lymphatic filariasis
                                                Prevalence
                                                Diethyl carbamazine citrate
                                                Culex quinquefasciatus
                                                Northeastern India
Study type :                     Prevalence and Control

Objectives:

(i) To find out prevalence of filariasis.
(ii) To determine whether infection is indigenous or imported
(iii) To identify vector and to study their prevalence, biting and breeding behaviour and susceptibility to different insecticides.
(iv) To extend the prevalence study in non tea garden (local) population.
(v) To find out microfilaria periodicity
(vi) To assess chemotherapeutic efficacy of diethyl carbamazine citrate in microfilaria carriers
(vii) To suggest effective control measures.

Introduction: India alone accounts for 58% of the total global infection of lymphatic filariasis. The disease is endemic in more than 18 states of the country and of these, 7 states viz. Andhra Pradesh, Kerala, Orissa, Tamil Nadu, Uttar Pradesh, West Bengal and Bihar are worse affected. Unlike other eighteen filaria endemic states which have got due recognition in terms of controls and management of lymphatic filariasis, Assam state is still considered almost free from filarial infection (WHO, 1996), although the disease has been documented as back as in 1957. According to an estimate, in Assam about 9.25 million people are at risk, 0.35 million are microfilaraemic and 0.09 million possess chronic disease (Health Information of India, 1990). Filariasis has already been identified as the second leading cause of permanent and long term disability. Direct and indirect losses associated with management of acute and chronic manifestations of the disease and from diminished productivity or incapicitaion can be enormous and constitute a severe drain on the economy both at local and National level. Tea industry in Assam is one of the most important and viable sector spread over to more than 848 tea estates and employ >10 lakhs of active labour force. These workers are the descendant of migrants from various highly filaria endemic states of India during pre independence era and are at higher risk of getting infection. In order to assess the present situation of the disease among the tea worker population and to map the affected area for strategic control measures.

Methodology :

Ethnic background of study population: Ethnically tea garden population consist of migrant people from various part of India such as Orissa, Bihar, Uttar Pradesh, Tamil Nadu, Madhya Pradesh, and Kerala, who had shifted to Assam in pre independence era and were engaged in tea industry during British period. Now they had passed more than two-three generations in the study area and most of them do not have any link with their original homes and are living in close and densely established labour lines. The non tea worker population belongs to the original native of the area and descendent of the Ahom, Muttak and Mikir group and lived more than 20 generations in Assam often residing nearby the tea worker population and share common ecological and mosquitogenic condition. Both the groups maintain their own identity through different religious, social, professional and behavioral practices.

Methods: Areas for sampling were randomly chosen covering each and every corner of the labour lines and villages. A detailed history of each subject was recorded on a predesigned proforma. From each individual 20 cmm finger prick sample of blood was taken and thick smear prepared during the high mf density of periodic microfilaraemia i.e. in between 20.00 hr to 24.00 hr . The smear was dehaemoglobinised, fixed and examined for the presence of microfilaria. Microfilarial periodicity was studied in volunteers (having low, moderate and high mf counts) at 12 equally spaced intervals in 24 hr by taking finger prick blood samples. For vector mosquito, morning in door resting collection in between 6.00-9.00 hr and dusk collection in between 18.00-20.00 hr by using suction tubes and flash lights, whole night human bait collection (hourly) and light trap collection in human dwelling and cattle shed were done. Caught mosquitoes were counted, identified, grouped and dissected for presence of larval stages of the filarial parasite (L1,L2,and L3). For chemotherapeutic studies, aims and objectives of the study was told among the workers and informed verbal consent were taken. The workers were registered during house to house survey. The microfilaria carriers were provided with DEC treatment as per the WHO recommended dose. Treated microfilaraemic individuals were re-examined with same sampling methods after 5th year of the post treatment for the presence of microfilaria. The microfilarae were counted and the number of microfilarae per ml of blood was calculated. DEC tablets (50 mg ; BanocideR manufactured by Burroughs' Wellcome (India) Limited, in tablet forms in the dose regimen of 72 mg/ kg body weight were given. The drug was administered in presence of the member of the research team and doctor of the garden hospital. Individuals complaining minor side effects were treated symptomatically. The geometric mean microfilaria density (GMI) was calculated as antilog [log(x +1)/ n) ] where x is the number of microfilarae (mf) per ml in the blood and n is the total number of microfilaraemic subjects examined. Subjects becoming amicrofilaraemic on post treatment observation were also included when calculating the geometric mean. Those who did not complete the full treatment or missed follow up examinations due to refusal, migration or death were excluded.

Results:

Prevalence of filariasis: A total of 6092 tea garden workers population were surveyed. Overall microfilaria (mf) rate (Wuchereria bancrofti) was 5.6 % and ranged in between 0.46 -10.3. Sex-wise mf rate was significantly (P value <0.01) higher in males (7.23%) than females (4.03%). Mf rate increases with the age in both the sexes and it was higher in-between the age of 20 to 50 yr. Mean mf density ranged in between 12.2 to 36.3. However, difference in mean mf density was insignificant in both the sexes. Chronic disease rate ranged from 0.44 -7.0 and was found higher in males than females. Among the local population, out of the total 1113 blood samples examined only 11 subjects were found positive (0.98% mf rate). Difference in the mf rate in both the sexes were insignificant. Chronic disease rate was higher in the females (0.88%) than males (0.77%). Remarkable difference in the mf rate and vector abundance has been noticed in the tea gardens located on slope (foot-hill areas) than those of planes.

Studies on Vector mosquito: Culex quinquefasciatus was incriminated as the vector mosquito and infection and infectivity rates were in between 0-7.5 and 0-4.8 respectively. Culex quinquefasciatus comprised of 96.87% of the total mosquitoes collected resting indoors during day-time and the remaining 3.13% comprised of Anopheles vagus. In whole night human bait collection also, Cx quinquefasciatus was dominant accounting for 92.26 % of the total catch and remaining 7.74 % were Cx vishnui, Cx tritaeniorynchus, Cx pseudovishnui and An vagus. The per man hour density ( MHD ) in in-door resting collection was 20.6. On dissection, only Cx quinquefasciatus was found harboring larval stages of filarial parasite. The parity was 48.7 %. The mean L3 load per infective mosquito was 5.1. Abundance of the vector species (Cx quinquefasciatus) was recorded high during March to April and September to November and the lowest was in the month of January. Day resting behaviour was found highest inside the houses preferably in the dark places like corners and empty utensils, hanging objects rather coming from the thatch-ceiling and in the vegetation of surrounding area. The breeding habitats of the Cx quinquefasciatus were domestic sewage water, small ditches around water hand pumps, low land fields and drains with domestic waste water near the houses. Mosquito larvae collected from the water bodies of the survey areas were reared in the laboratory and Cx quinquefasciatus predominated. Biting density were found high during March-April and lowest in the month of January. High biting activity were from 7pm to mid night. Cx quinquefasciatus was found susceptible to Malathion, Proxpoxur and resistant to DDT.

The periodicity of microfilaria was found nocturnal. Chemotherapeutic Efficacy: Diethyl carbamazine citrate in the dose schedule of 72 mg/kg body weight cleared 51.6% microfilaraemic individuals on 5th year of post treatment examination. Of the 121 treated mf carriers 85 subjects were successfully followed and among these 10 (11.8%) had shown increase in mf counts, 31 (36.4%) reduction but remain microfilaraemic and 44 (51.8%) become amicrofilaraemic. No significant effect of age and sex of the host on the clearance of mf was noticed.

Conclusions: Earlier studies conducted in Assam had shown presence of lymphatic filariasis among tea garden workers and local population. In present study, the infection rate remains steady/or higher compared with the earlier findings in tea workers population but in adjoining villages inhabited by local population, infection remain significantly low in spite of the presence of the potential infective mosquito vector (Cx quinquefasciatus). Positivity in subjects of tender age of both sexes (as low as 4yr of age) emphasizes that the active indigenous transmission is going on in tea workers population. Positive cases among the non tea workers group being in older age suggests infection might have been imported from tea workers population area. Among tea workers the mf positivity was more in males than females probably because of the differential exposure rate to the infective mosquito bite due to behavioral, clothing, occupational difference and influence of female hormone. It is well documented that the proportion of the normal healthy individuals in an endemic area getting infected depends upon factors like transmission potential, force of infection, exposure rate, immunological status and susceptibility of the human host. In our study the possible reason of very low incidence among the local population may be due to any of these factors operating in the area. On the other hand, the high infection rate among the tea garden workers is possibly due to the fact that they are the descendent of a high susceptible population migrated from high filaria endemic areas like Orissa, Bengal, Bihar, Madhya Pradesh, Uttar Pradesh and south Indian states. Secondly, in local population, it may be due to genetic resistance as in filaria endemic areas not all individual acquire patent infection. Experimental studies on filariasis in animals also support that genetic variation of the host play a direct role in the control of microfilarae and disease out come. Familial clustering among microfilaraemic cases of tea garden workers was well evident and suggests involvement of host susceptibility. However, further studies covering genetic and immunological factors is needed to support these view. Although DEC is considered the drug of choice, many basic question remains about its effectiveness and how best to use it for controlling filariasis in a population. In our study, three types of subjects were observed in respect of response to the drug and mf clearance. In the first pattern individuals showing increase in mf count, secondly subjects showing reduction in mf count but remain microfilaraemic and thirdly individuals become amicrofilaraemic. DEC kills parasites indirectly by activating host immune response and it seems that individuals with varying degree of immune status may be responsible for differential action of the drug. The individuals showing increase or static mf count on post treatment observation as compared to pretreatment level were probably due to very low pretreatment mf count and thus a slight increase in the post treatment count give rise to an unusually large percentage increase or due to reinfection, although this can not be positively ascertained as no concerted efforts have been taken to interrupt reinfection in the study area. Moreover, when all the 85 followed mf carriers were grouped in low, moderate and high mf counts on the basis of pre treatment counts it is seen that there is a progressive decrease in efficacy of DEC in clearing the microfilarae. However, reduction in mean mf intensity in all the three groups were noticed and is more prominent in high density group. Reduction in mean mf density in post treated individuals suggests prolong effects of DEC on microfilaraemia status. The adulticidal action of the DEC is now well established, however, total clearance of adult worm from the treated individuals is difficult and probability of surviving one or few adult female worm is fairly high. The persistence of a relatively stable low number of mf over a period of several years is more likely due to survival of one or more female worms which mate and periodically release new microfilarae. Thus use of DEC could provide long term benefit in the control of bancroftian filariasis. On the basis of our findings, control of lymphatic filariasis seems feasible in tea garden set up by applying mass chemotherapy along with vector control measures.

Funding Agency code

Intramural

Study type code

Prevalence and Control

 

 

 

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Mosquito fauna of Dibrugarh district with special reference to Mansonia annulifera, a reported vector of Japanese encephalitis from the Dibrugarh district of Assam

Investigator:                 Dr.Siraj Ahmed Khan

Co-Investigators:        Dr.Prafulla Dutta

Objectives:

(i) To conduct extensive surveys of mosquito fauna of Dibrugarh district and adjoining areas (study area) with special reference to JE vectors.

(ii) To study larval habitats of mosquitoes and their distribution in the study area and also to find the interrelationship between larval habitats on the basis of mosquito larval community structure and patterns of association.

(iii) To determine the seasonal abundance of adult mosquitoes with special reference to JE vectors, including Mansonia annulifera (Theobald) in JE prone areas and to study the relationship between ecological factors and seasonal abundance of mosquitoes using statistical tools in order to get an insight into epidemiological aspects of JE.

(iv) To study the biting behaviour of mosquitoes off human and animal baits over whole night period.

(v) To study the daytime resting habits and susceptibility status of some Mansonioid mosquito species to different insecticides in the study area.

Introduction: Mosquitoes are small and slender dipterous flies and are currently the most extensively studied group of haematophagus insects because of their premier role in the transmission of diseases to man and other animals For the past few years, Japanese encephalitis (JE), a mosquito borne viral disease caused by a flavivirus has been causing large scale epidemics in Assam, especially Dibrugarh district. JE is a serious disease because of its high case-fatality rate with grave clinical sequelae resulting in neuropsychological disorders. JE with almost annual recurrences in Dibrugarh is a source of distress and concern from the public health point of view because of high case fatality and morbidity coupled with unpredictable pattern of epidemics. Preliminary studies on the mosquito fauna of Dibrugarh district has shown that out of 14 mosquito species known to act as vectors of JE elsewhere in India, 11 species are found in Dibrugarh. Mansonia annulifera for the first time has been implicated in the transmission of the JE virus from Dibrugarh and that too on a single occasion. However, no virus transmission study by this mosquito has yet been conducted. The study area lying between 93o45'east to 96oeast longitude and 26o45' north to 28o north latitude on the banks of the river Brahmaputra in Assam has a subtropical climate with moderate temperature and heavy rainfall. This region has extensive tea garden plantations, bamboo forests, reserve forests, paddy fields and is frequently affected by floods. The ecological peculiarities of this area provide ample mosquito breeding sites for mosquito proliferation and diversification.

However, no extensive and systematic study has been conducted earlier in Dibrugarh district regarding mosquito fauna and their breeding habitats which is essential not only for understanding the epidemiology of JE and other mosquito borne diseases but is also a sine qua non for vector control. Therefore, an extensive survey of mosquito fauna and their breeding habitats in Dibrugarh was undertaken keeping in view the greatly changed ecology during the last few decades of socio-economic developments such as deforestation, urbanisation, exploitation of natural resources like oil and coal mining, etc., local biotope changes and various other factors. The consequences of these changes is evident from the sudden upsurge of vector borne diseases including JE in this region.

8.Methodology : The study area was divided into five major zones viz., Dibrugarh, Dhemaji, North Lakhimpur, Tinsukia and Jonai. The study was undertaken in 1989 and different facets of the project were studied upto 1992. Immature mosquitoes were collected from twenty two different habitat types namely, bamboo stumps, mud pools, ponds, irrigation channels, spring pools, rainwater pools, manure pits, tree holes, stream pools, slow flowing streams, sandy pools, plantain leaf axils, fallow fields, paddy fields, elephant foot prints, springs, pineapple leaf axils, split bamboos, wells, plantain stumps, papaya stumps and an unused boat. Standard size plastic dippers of 11 cm diameter and 350 ml volume were used for collecting mosquito immatures from surface habitats. Collection from tree holes, bamboo stumps and leaf axil were made by glass pipettes. The larvae and pupae thus collected were individually link reared to adult stage being fed on powdered dog biscuit and yeast mixture (ratio of 1:1). The 4th stage larval and pupal skein were mounted on glass slides (in Hoyer's mounting medium). Emerged adults were killed in chloroform vapours and dry preserved in plastic vials containing a few grains of creosote beneath a piece of filter paper. The mosquitoes were identified using standard keys for both immature and adult characters. Altitude of the collection sites were recorded using portable altimeter. Adult mosquito collections were made by suction tubes and flash lights during dusk hours, whole nights (off human and cattle baits) and during daytime (outdoor and indoor) resting. For studying composition and structure of mosquito communities in different aquatic habitats in the study area, different ecological indices (Cf. Odum, Eugene P., Fundamentals of ecology; W.B. Saunders Company, Philadelphia,1971) were used as follows: (A)index of Dominance was used to measure the degree of dominance of component species of mosquito communities in different aquatic habitats,(B)index of species diversity was used to measure species richness of mosquito communities in larval habitats,(C) Equitability index was used to measure evenness (i.e. the number of individuals of mosquito larvae distributed among different mosquito species) in mosquito communities in different aquatic habitats,(D)Shannon's index of general diversity was used to measure overall diversity (both species richness and equitability) in different larval mosquito communities. Species abundance pattern in different mosquito habitats was studied graphically by using dominance diversity curves which gave an indication to the nature of niche relationship in a group of species in a particular habitat. For this, graphs of log abundance of individuals per species (ordinate) were plotted against rank of species (ie., species sequence (abscissa) in each type of larval habitat). Similarly, absolute abundance of individuals per species (ordinate) was plotted against log of rank of species (abscissa). Cluster analysis was used to study the interrelationship between habitats and also interrelationship among mosquito species. Two types of hierarchichal clustering models have been used - unweighted arithmetic average clustering (also known as group average sorting or UPGMA) and Ward's clustering method. For studying interrelationship between larval habitats, a binary data matrix was generated from the presence/absence data of different mosquito species in a particular habitat. Similarly, for studying the relationship between different mosquito species, a binary data set was constructed showing presence/absence of a particular mosquito species in different habitats. From the above data sets, a correlation co-efficient matrix and a distance matrix based on Squared Euclidian Distance was generated. The above association matrices were used as starting points for UPGMA and Ward's clustering algorithm respectively. To get a clearer picture of the interrelationship between the aquatic habitats on the basis of the larval mosquito fauna they harbour, nearest neighbour linkage analysis was performed. In the linkage analysis, larval habitats were grouped on the basis of Jaccard's coefficient. Vector surveillance was conducted regularly from May 1991 to April 1992 in four villages of Dibrugarh namely, Madhupur Mishing village, Jokai Panitola village, Padumoni village and Lepetkota Tea Estate. These villages were selected from amongst a list of villages in Dibrugarh endemic for JE. Adult mosquitoes were collected for one hour during dusk at fortnightly intervals from each locality. In all, 137 man hours were spent. Mosquitoes were collected by two trained persons from in and around cattle sheds by mechanical aspirators using flash lights. Monthly collections of mosquitoes were pooled species-wise for analysis. Human JE cases data for Dibrugarh was collected from the Directorate of Health Services, Guwahati, Assam and meteorological data and Brahmaputra river flood information for Dibrugarh was collected from Toklai Tea Research Centre, Dikom branch and Brahmaputra Board Flood Monitoring Department, Dibrugarh, respectively. Various statistical techniques were employed to examine the interrelationship amongst entomological and meteorological variables and JE cases which are as follows a)Pearson's correlation coefficients were calculated between log abundances of each pair of mosquito vectors in order to get an estimate of temporal relationship among mosquito abundances. Similarly, correlation coefficients were calculated between some environmental variables and log abundance of mosquito species, b)cross correlation was studied between environmental variables and mosquito abundance with a one month's time lag as it was observed that there was a time gap before cause and effect relationship was established between them. Therefore, the time series for log of (JE cases+1) were cross correlated with minimum temperature lagged by one month. Similarly, time series for the abundance of Mansonioid mosquito species were cross-correlated with flood level of Brahmaputra river lagged by one month, c) simple regression analysis was used to estimate the relationship between JE cases (dependant variable) and one month lagged minimum temperature (independent variable). The index of fit i.e. goodness of fit of the model to the observed data was estimated using simple correlation coefficient computed between the observed JE cases (epidemic and post epidemic period) and estimated JE cases. The sample correlation coefficient thus obtained was squared and the index R2 obtained is the proportion of the total variability explained by the model. T-test was used to examine the significance of R2 at 0.01 and 0.001 probability levels. Finally, the structure of residuals and data pattern was investigated through graphs in order to check for gross model violations. For this, the standardized residuals were plotted as the ordinate against the independent variable as abscissa, d) multiple regression analysis and the associated significance tests (t-test and ANOVA) were carried out using SPSS/PC. Multiple regression analysis was used to understand the role of selected environmental factors on the seasonal abundance of each species of the potential JE vector mosquito. The main purpose of employing multiple regression analysis was to clarify the nature of complex interactions between some environmental variables and mosquito abundance using the principle of parsimony. Therefore, procedure STEPWISE was used to select a subset of predictor variables in the multiple regression that best explained variations in the seasonal abundance of JE vector mosquitoes. Multiple correlation coefficient was estimated to assess the goodness of fit to the data. The square of the multiple correlation (R2) can be interpreted as the proportion of variance in the mosquito abundance of a particular species (dependant variable) accounted for by the selected environmental factors (independent variables). For studying man-vector contact and biting behaviour of mosquito vectors, whole night biting collections were carried out from two localities - Madhupur Mishing village and Tarazan Tea Estate. Collections were made off human baits outdoors and indoors and off cattle bait outdoor during August 1991- July 1992. Human bait collections indoors were performed in the living rooms of huts at ground level using the method `stationary direct bait technique' in which a man acts both as bait and collector. The collector used to sit on a stool exposing his legs from knee downward. After mosquitoes landed on the exposed part of his leg, they were caught by test-tubes as far as possible before the mosquitoes could bite. The test-tubes were plugged with cotton and hourly catches were separately recorded. The outdoor biting collection off human baits were also performed in a similar manner outside the house. Mosquitoes off cattle bait were collected by one person with the help of suction tube. Hourly catches of mosquitoes were pooled separately as above. The source of light in all the above collections both indoors and outdoors was dry cell operated torch. A total of six persons participated each night in continuous bait collections. A second group of three persons replaced the first group of three collectors during the second half of the night. In order to reduce the individual bias as far as possible, there was rotation of catchers from night to night and from locality to locality. Finally, the data was pooled species wise in order to study the biting behaviour and host preference of individual mosquito species. Periodicity index of the mosquitoes was computed using co-efficient of variation of hourly pooled mosquito counts collected during 14 nights. The above index was used to classify six most predominant species of mosquitoes into nocturnally periodic and nocturnally subperiodic forms. .Daytime resting habitats of mosquitoes were studied in Madhupur village of Dibrugarh. outdoor resting mosquitoes were collected from undestroyed ground vegetation and shrubs from peridomestc areas and near edges of riparian vegetation using dropnet cages. Indoor resting mosquito collections were made during 0900 to 1100 hours from the living rooms using aspirator tubes. Insecticide susceptibility status of Ma. annulifera and Ma. uniformis was tested against 3 insecticides using papers pre-impregnated (WHO kits) with 4% DDT (p,p'-isomer), 0.4% dieldrin and 5% malathion following the WHO standard procedure. Abbot's formula was used to correct percentage mortality of mosquitoes when the mortality in the control experiment exceeded 5%.

Results: A total of 2,407 immature mosquitoes were collected during this study. These represented 77 different species belonging to 15 genera. The genus Anopheles was most dominant represented by 23 species followed by Culex (17 species), Aedes (13 species), Armigeris (5 species), Uranotaenia (3 species). Mimomyia, Malaya, Topomyia, Tripteroides, Toxorhynchites and Orthopodomyia represented by 2 species each and Aediomyia, Heizmania, Udaya and Ficalbia represented by 1 species each. Different habitat types harboured one to 24 species of mosquitoes. Bamboo stumps harboured maximum number of larval mosquito species (24 species) followed by mud pools (18 species), ponds (15 species) and other habitats harboured less than 12 species. Forest areas were more species rich and 58 species were recovered from them whereas non forest areas yielded 45 mosquito species. Mosquitoes were collected from altitudes ranging between 100 meters to 650 m above mean sea level. Maximum species were found from altitudes between 300-400m.Besides larval collections, adult mosquitoes were also collected during dusk hours, whole nights (off human and cattle baits) and during day time. A total of 31,423 adult mosquitoes were collected representing 39 species under 6 genera. Thus a total of 96 species of mosquitoes were collected out of which 20 species of mosquitoes are reported for the first time from Assam including three species reported for the first time in India (Table-1). Among the species of mosquitoes encountered, twelve are potential vectors of JE reported from elsewhere in India and eight species are known potential malaria vectors. In order to study the organisation of mosquito community in different habitats, composition and structure of the mosquito communities in various aquatic habitats in the study area was analysed using index of dominance, variety index, equitability of species index and Shanon-Weaver's general diversity index. Of the 13 mosquito habitats examined, bamboo stumps had the highest mosquito species richness (d = 3.49). Maximum equitability of species (e = 0.91) was found in sandy pools and minimum in tree holes. Conversely,, maximum concentration of dominance was in tree holes (c = 0.67) and lowest concentration of dominance was recorded from irrigation channels (c = 0.15 ). The maximum general diversity index (H = 4.85) was in pond and minimum in plantain leaf axil (H=1.25). The graphical analysis of rank abundance relationship revealed that mosquito communities in different habitats follow log normal distribution.

To study the interrelationship of aquatic habitats and mosquito fauna, an attempt was made to classify aquatic habitats on the basis of presence or absence of mosquito larvae of a particular taxon and conversely to classify mosquito species on the basis of similarities in their larval habitats. Bamboo stumps emerged as a unique group of habitat because of their specialised larval fauna they harbour. Four groups of larval habitats were identified using nearest neighbour linkage analysis which was found to give a meaningful classification of larval habitats (Fig.1). The first group represented ground-water habitats, the second group represented container habitats, the third group represented running-water habitats and the fourth type of habitat was represented exclusively by split bamboos. Although split bamboo is a container type of habitat, yet it was not linked with any other habitat because two mosquito species, namely, Aedes (Finlaya) albotaeniatus and Ae. (Fin.) harveyi which were found to breed in split bamboos were not found in other type of habitats. Dendrogram showing interrelationship among mosquito species was constructed. Four major groups of mosquitoes were recognized. Among the medically important mosquito species, JE vectors were found to breed mostly in temporary ground water habitats such as mud pools, rain water pools, manure pits, irrigation channels, etc. It is interesting to note that about 34 mosquito species (comprising 44% of the total species collected as immatures) were collected from natural container habitats. This fact gives support to the idea of some workers that culicidae as a whole originated as container breeders.

A total of 19,789 female mosquitoes were collected during dusk hours comprising of 26 species in 5 genera. Ma. uniformis was the most dominant species (19.27% of the total catch) followed by Ma. indiana (14.35%), Culex vishnui (13.14%), Cx. ritaeniorhynchus (11.86%), Cx. fuscocephala (11.6%), Cx .pseudivishnui (10.32%)and rest of the mosquito species comprising less than 5%. Among potential JE vectors, the most abundant mosquito was Cx. vishnui (22.32%). Maximum per man hour density (MHD) of mosquito collection was recorded in April and minimum in the month of January. Two seasonal peaks of mosquito abundance were evident during the months of September and April. Seasonal variations of abundance of individual potential JE vectors is reported. The monthwise mean data of JE vector MHDs, environmental variables and JE cases were subjected to multiple regression analysis. Mean monthly minimum temperature was found to have a significant relationship with JE cases in the epidemic and post epidemic period. However, highly significant correlation was obtained between one month lagged mean minimum temperature and JE cases (r=0.82; p<0.01).

A total of 9972 adult host seeking mosquitoes representing 26 species in 5 genera were collected off human baits indoors and outdoors and cattle bait outdoors during whole night mosquito collections. Man:cattle biting ratios indicated that all mosquitoes collected were primarily zoophilic although significant number of mosquitoes (2093) were collected while biting men outdoors. The hourly biting activity of important JE vectors throughout the night upon the three bait types along with periodicity index of the more abundant mosquito species have been reported with graphs. Biting cycles of mosquitoes vary in different species and also in different races of the same species in different geographical areas. In the present study, an attempt has been made to classify biting patterns of mosquitoes. Five basic types of biting patterns were noticed (Fig.2). In type one, unimodal mosquito biting activity was observed during the midnight as in Cx. bitaeniorhynchus and An. hyrcanus s.l. biting on outdoor human bait. Second type was characterised by early night biting behaviour of mosquitoes represented by An. hyrcanus s.l. and Cx. quinquefasciatus on outdoor cattle bait. In the third type, mosquitoes were active during the late night hours. This type included Ma. annulifera biting human bait both outdoor and indoor as well as on outdoor cattle bait. In type four, bimodal activity was noticed which included Cx. pseudovishnui upon all bait types both outdoors and indoors, An. hyrcanus s.l. on indoor human bait, Cx. tritaeniorhynchus on outdoor human bait and Cx. bitaeniorhynchus on outdoor cattle bait. Type fifth was characterised by biting activity of mosquito almost throughout the night. This pattern could further be subdivided into three types, subtype first showing a declining trend as the night proceeded including Cx. vishnui s.s. and Ma. uniformis (on outdoor and indoor human bait as well as outdoor cattle bait) and Cx. tritaeniorhynchus on outdoor cattle bait. Subtype second showed increasing trend in biting activity as night proceeded as examplified by Cx. bitaeniorhynchus upon indoor human bait and subtype three showing an irregular biting trend throughout most parts of the night including Cx. quinquefasciatus on outdoor and indoor human, Cx. triaeniorhynchus on indoor human and Cx. fuscocephala on outdoor cattle baits. Different types of biting rhythms exhibited by the mosquitoes in a given locality may be probably an adaptive behaviour for reducing intra-specific and inter-specific competition by resource partitioning in time. It is pertinent to point out here that An. hyrcanus s.l. shows three types of biting behaviour on outdoor and indoor human baits and outdoor cattle bait suggesting its heterogeneous nature (probably a mixture of sibling species). It is very interesting to note that the biting rhythm of Cx. on the indoor human bait in the present study area showed a marked deviation from the typical unimodal pattern with peak activity around mid-night as observed by some other workers from Mysore city. This deviation may be due to different ecological factors or most probably due to strain variations.

A total of 3402 mosquitoes were collected from various resting habitats during day time which represented 20 species in 6 genera. Majority of mosquitoes were collected resting outdoors. Cx. tritaeniorhynchus was the most predominant species resting outdoors constituting 25% of the total outdoor resting catch. The insecticide susceptibility tests revealed that both Ma.annulifera and Ma.uniformis were susceptible to DDT, malathion and dieldrin.

Conclusion: Mosquito fauna of Dibrugarh (Assam) shows considerable diversity and richness because of ample mosquito breeding sites resulting in mosquito proliferation and diversification. Twenty species of mosquitoes are reported for the first time from Assam including 3 species reported for the first time in India. Forest areas are more species rich than non-forest areas. Both forest and non-forest areas are risk zones for contracting Japanese encephalitis. Mosquito fauna shows similarities and dissimilarities with respect to geographical zones. North Lakhimpur area has dissimilar mosquito fauna from rest of the study area (based on UPGMA hierarchical cluster analysis). Eleven species of potential JE vectors are present in the study area. However, further studies are required to prove their virus transmission capability. Mosquito communities exhibit a predictable pattern of community composition and structure in different aquatic habitats due to different processes organising these communities. All species in mosquito community in different aquatic habitats are not equally dominant, instead, only a few species are dominant by virtue of their number, etc. Mosquito communities in different habitats follow log normal distribution. The biological reasons for such a distribution pattern can be explained if we assume that individuals are distributed between species in accordance with Gaussian distribution and population growth is geometric. Linkage analysis gives a meaningful classification of aquatic habitats on the basis of the mosquito larval community they harbour. Some mosquito species prefer a restricted habitat for breeding whereas other species exhibit a certain degree of plasticity with respect to breeding preferences. Ward's clustering method is an appropriate technique in grouping mosquitoes on the basis of similarities in their breeding preferences. This study supports the hypothesis that mosquitoes probably originated as container breeders. Complex interactions among environmental factors like minimum and maximum temperature, rainfall and flood level are involved for modulating the seasonal abundance of mosquitoes. A time lag is involved before a cause and effect relationship is established between environmental parameters (independent variables) and entomological variables (dependant variables). Minimum temperature is the most limiting factor affecting JE virus activity. Circumstantial evidence indicates that Culex vishnui s.l. plays a dominant role in JE epidemics. Inundations caused by flooding is favourable for Mansonia species whereas it has an adverse effect on non-Mansonioid mosquito species. High abundance of potential JE vectors is not a sufficient factor by itself to trigger a JE epidemic if mean monthly minimum temperature is below 17oC. No mosquito species was found to be exclusively zoophilic or anthropophilic. There is increased risk of contracting JE outdoors than indoors. As majority of mosquitoes were found resting outdoors, therefore, indoor spraying of insecticides will be of little value for controlling JE vector abundance. Mansonia annulifera and Ma.uniformis are susceptible to DDT, malathion and dieldrin.

Table -1 - Checklist of mosquito taxa recorded for the first time from Assam.

Serial No. Species

  1.  Aedes (Stg.) pseudoalbopictus (Borel), 1928
  2.   Armigeres (Lei.) digitatus (Edwards), 1914
  3.   Culex (Cui.) fragilis Ludlow, 1903
  4.   Culex (Lop.) bengalensis Barraud, 1934
  5.   Culex (Lop.) cintellus Edwards, 1922
  6.   Culex (Lop.) halifaxii Theobald, 1903
  7.   Mimomyia (Eto.) luzonensis (Ludlow), 1905
  8.   Anopheles (Ano.) hodgkini Reid, 1962
  9.   Anopheles (Ano.) lasteri paraliae Sandoshan, 1958
  10.   Anopheles (Cel.) pseudowillmorei Theobald, 1910
  11.   Aedes (Adm.) pipersalatus Giles, 1901
  12. Aedes (Fin.) albotaeniatus (Leicester), 1904
  13. Aedes (Fin.) harveyi Barraud, 1923
  14. Aedes (Fin.) prominens Barraud, 1923
  15. Culex (Cux.) fuscitarsis Barraud, 1924
  16. Culex (Eum.) khazani Edwards, 1922
  17. Heizmannia (Hez.) chandi Edwards, 1922
  18. ** Culex (Cux.) alienus Colless, 1957
  19. ** Culex (Lop.) variatus (Leicester), 1908
  20. ** Tripteroides (Trp.) tarsalis Delfinodo and Hodges, 1968

* New record from North eastern region of India
** New record from India.

 

Fig.2 flow diagram showing different types of biting cycles exhibited by mosquito species over the twelve hours dusk to dawn period

Sl.          Types of biting                 Representative species exhibiting different
No.         patterns                            biting patterns.

1.            Unimodal peak activity     i. An.hyrcanus s.l. on outdoor human bait
               during midnight                 ii. Cx.bitaeniorhynchus on outdoor human bait

2.             Early night peak                i. An.hyrcanus on cattle bait
                                                           ii. Cx.quinquefasciatus on cattle bait

3.             Late night peak                 i. Ma.annulifera on all bait types

4.             Bimodal activity                 i. An.hyrcanus on indoor human bait
                                                            ii. Cx.pseudovishnui on all bait types
                                                           iii. Cx.tritaeniorhynchus on out-door human bait
                                                           iv. Cx.bitaeniorhynchus on outdoor cattle bait

5.             Almost uniform activity
   
             throughout the night
                 _______|_______________________________________
                |                                        |                                                    |
                5a                                         5b                                           5c
Cycle showing                                     Cycle showing                 Cycle showing irregular
declining trend                                     increasing trend                 biting activity

i. Cx.vishnui s.s. on                       i. Cx.bitaeniorhynchus         i. Cx.tritaeniorhynchus on
   all bait types                                   on indoor human bait              indoor human bait

ii. Cx.tritaeniorhynchus                ii. Cx.fuscocephala on
   on outdoor cattle bait                      outdoor cattle bait

iii. Ma.uniformis on all                   iii. Cx.quinquefasciatus on
    bait types                                        outdoor and indoor human bait.

Fig. 1 linkage diagram of mosquito larval habitats on the basis of jaccard's co-efficient analysis

GROUP - III - RUNNING WATER HABITATS

Spring = Slow flowing stream

GROUP - I - GROUND WATER HABITATS


                                   ___                     ____
                Spring pool          Fallow field          Stream pool
                  |                               |  |
                  |                        Sandy pool 
Elephant --Rainwater pool
foot print    |
                  |                                            
                Manure pit  -- MUD POOL    ===  Stream pool
                                        |               |
                                       Pond         |
                                                        |
                                               Irrigation channel
                                                        |
                                                        |
                                                Paddy field

GROUP - II - CONTAINER HABITATS (NATURAL)

Papaya stump      Bamboo stump        Tree hole      Plantain stump

        Pineapple leaf axil     ====          Plantain leaf axil

 

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Studies on paddy-field dermatitis (Panikaint) among workers in rice fields of upper Assam

Investigator:             Dr. Kanwar Narain

Co-Investigators:        Dr. J. Mahanta

Subject key words:    Paddy field dermatitis
                                           Occupational health problem
                                            Animal schistosomes
                                            Cercarial dermatitis
                                            Plant molluscicides

Study type: EPI & LAB

Objectives:

(i) To identify the aetiological factor/s responsible for paddy field dermatitis locally known as "Panikaint" and study its epidemiology

(ii) To suggest preventive measures against paddy field dermatitis.

Introduction: People of Assam mostly live in villages where paddy cultivation is the main source of livelihood. About 70.1% (4.4 million people) of the rural population of Assam are cultivators and agricultural laborers and about 2.563 million hectares of land in 1993-94 was under paddy cultivation (Statistical handbook of Assam, 1994). Paddy field dermatitis locally known as "Panikaint" or "Oopersukwa" is an important occupational health problem in Assam. Hundreds of farmers are affected each year by this disease. It has been observed that when paddy field workers get exposed to water in rice fields during paddy cultivation they often develop severe dermatitis on the exposed parts of their skin mainly legs and arms. Initial exposure to water in affected paddy fields cause prickling sensation after about 30 minutes followed by, after a lag period of 12 to 24 hours, severe itching and papular eruption. These eruptions usually get pustular with secondary infection within a week. Due to intense pruritic eruptions, paddy field dermatitis seriously interferes with the productive activities of the afflicted people resulting in loss of working capacity. Paddy field dermatitis appears to be a sensitization phenomenon because repeated exposures to water in rice fields leads to more intense dermatitis. Therefore, a study was undertaken with the objectives given below.

Methodology :

Study area: Two districts of upper Assam, namely, Dibrugarh and Tinsukia were chosen for undertaking investigations where more than 41,000 agricultural laborers are working (Primary census abstract, 1991).

Selection of Paddy fields: Information was gathered from local farmers regarding the occurrence of "Panikaint" in their area and accordingly paddy fields were selected for further investigations.

Collection of snails: From preliminary observations we suspected animal schistosome cercarial involvement (known to invade human skin and causing dermatitis) because firstly, the distribution of the disease in the affected paddy fields is patchy/clumped (this indicated an organismal involvement) and secondly, the clinical course of the disease was suggestive of cercarial involvement. Therefore, numerous gastropod snails were collected from snail infested paddy fields. These snails were transported alive to the laboratory and kept in trays until used for further investigation.

Identification of snails: Standard keys for identification of fresh water snails were used (Danish Bilharziasis Laboratory, 1983, A field guide to fresh water snails in countries of the WHO Eastern Mediterranean region, p. 1 - 45).

Examination of snails for cercarial infection: Individual snails were cleaned and placed in glass tubes or beakers containing distilled water for detecting cercarial infection. Either alternate exposure to darkness and light was used as a stimulus for cercarial release or the snails were kept overnight in the containers and were examined the next day for schistosome cercarial infection.

Experimental infection of laboratory animals: Laboratory animals were exposed to schistosome cercariae: 1) for development of adult flukes for confirmation species diagnosis 2) to demonstrate the capability of the schistosome cercariae to invade intact mammalian skin and 3) for histopathological studies and development of suitable animal models. Experimental animals used were Swiss albino mice, Mastomys natalensis, hamsters, rabbits, ducklings and chickens. Experimental animals were exposed percutaneously with freshly emerged schistosome cercariae.

Histopathological studies: Livers of infected animals were fixed in 10% neutral buffered formalin. Before fixation, the livers were sliced at 5mm interval in coronal plane and 3 to 4 tissue blocks were selected randomly. The fixed tissues were dehydrated in graded series of ethanol, cleared in two changes of xylene and embedded in paraffin wax with cresin (congealing point 60 0 C). Finally, 7 m m thick sections were cut using rotatory microtome. For staining, the deparaffinized and hydrated sections were stained using routine haematoxylin & eosin.

Scanning electron microscopy: For SEM studies some adult flukes were fixed in 2% cold (4o C) glutaraldehyde buffered with 0.1M phosphate buffer (pH 7.3) for 24 hours, rinsed in fresh 0.1 M phosphate buffer 3 times and post fixed in 2% osmium tetraoxide. Finally, the specimens were dehydrated in graded ethanol series, dried using tetramethylsilane, sputter coated with gold, mounted on stubs and viewed under Jeol 35 SEM at 15 kV accelerating voltage.

Search for plant based molluscicides: This study was carried out to find locally available plant/(s), which has molluscicidal potential and can be used for control of snails in dermatitis affected paddy fields of Assam. Uninfected Indoplanorbis exustus snails were used for conducting bioassay. Snails were collected from paddy fields of Mahakali, Tinsukia and maintained in our laboratory with vegetable food. The aqueous extract was screened for molluscicidal activity at various concentrations, ranging from 0.025 to 0.013 g/L. Five snails were placed in a 500ml beaker containing 200ml of each concentration in de-chlorinated tap water. Sixteen replicates for each concentration were used to reach 80 snails per concentration. Exposure period to test solution was 24 hours. After 24 h, the snails were carefully rinsed with water and transferred to fresh water without molluscicide for further 24 h for recovery. Criterion of death of snails was lack of response to mechanical stimulus. The dead snails were immobile and either retracted into or were hanging out of the shell with the body and shell discoloured. LC50 and LC90 values with 95% confidence limits were estimated from the percentage mortality and the logarithm of concentration by probit analysis. Abbot's correction for mortality was not used since control mortality was nil. The above experiment was again repeated to study the stability of aqueous extracts stored for one year at room temperature.

Results: The paddy field dermatitis among farmers' of Assam was found to be cercarial dermatitis caused by penetration of animal schistosome cercariae. Three types of schistosome cercariae were found infecting Indoplanorbis exustus snails, in the dermatitis affected paddy fields of Assam during the present investigation. These are: 1) Non-oculate brevifurcate (species 1), 2) Non-oculate brevifurcate (species 2) and 3) Oculate brevifurcate (species 3).

Non-oculate brevifurcate (species 1)

These cercariae were identified as cercariae of Schistosoma spindale and were found in those paddy fields where cattle were used for ploughing fields. These cercariae were found to have the ability to cause dermatitis in humans and were often isolated from snails in dermatitis affected paddy fields. Exposure of the skin of the working scientists and human volunteers to these cercariae resulted in typical papular eruptions, which were highly pruritic. Cercariae of Schistosoma spindale were found responsible for causing dermatitis in most of the paddy fields as compared to the other species of schistosomes detected during the study period. Prevalence of infection in snails (Indoplanorbis exustus) was most often found to be less than 1% (range 0 to 25%). However, the intensity of infection of the infected snails was high and about 100 to 8000 cercariae were shed per snail per day. The cercariae, which were shed in water, were able to survive for 24 hours under laboratory conditions. These cercariae were found to have the ability to invade intact mammalian skin and develop into adult flukes in liver of experimental animals. Experimental hosts, which were successfully infected in the present study, were Swiss albino mice and rabbits. However, hamsters were found refractory to infection. Natural definitive hosts of Schistosoma spindale are cattle, goat and wild rodents.

Non-oculate brevifurcate (species 2)

In some paddy fields of Assam, it was found that Indoplanorbis exustus snails also liberated the cercariae of Schistosoma nasale, a natural parasite of water buffaloes. Circumstantial evidence indicated that these cercariae may also be involved in causing farmers' dermatitis in Assam where water buffaloes are used for ploughing fields. The ability of these cercariae to invade the intact mammalian skin was proved using Swiss albino mice and Mastomys rats (Praomys (Mastomys) coucha) (new experimental hosts). Overall, prevalence of infection was found less than 1% (range 0 to 2%).

Oculate brevifurcate (species 3)

The details pertaining to these cercariae will be given after publication of the results. In brief, Indoplanorbis exustus snails collected from several paddy fields in two localities (where dermatitis was prevalent) were found infected with an unique, hitherto unknown schistosome cercariae, these cercariae were brevifurcate, oculate and had distinct horizontal striations on their tail. These cercariae are capable to penetrate intact mammalian skin and develop into adult schistosomes in the hepatic portal system and intestinal mesenteries of Swiss albino mice. These cercariae were also capable of causing dermatitis. The overall prevalence of infection in the snails was less during 1998 (0.32 %; n = 1546) and ranged from 0 to 0.46%. The intensity of infection was high as 2000 to 8000 cercariae were shed per snail per day for 7 to 9 days.

Snail intermediate hosts and farming practices

The snails Indoplanorbis exustus which were found to act as intermediate host of S. spindale, S. nasale and the new schistosome in Assam were found in considerable number in rice fields, ditches, ponds and irrigation channels. The density of the snail hosts in some dermatitis affected paddy fields was very high and as many as 2000-3000 snails could be collected within an hour by one person in some paddy fields. However, the distribution of the snails was not uniform in the paddy fields and they showed clumped distribution. The times when farmers in relation to farming and other activity contract "Panikaint" disease are given in table one. During the ploughing of rice fields from mid May to July there is some dermatitis, although, the intensity of dermatitis seems to vary somewhat from year to year depending upon the weather. At the time of transplanting, the paddy fields are flooded with water, so the conditions are favorable for shedding the cercariae from infected snail hosts. The peak period of cercarial shedding occurs during the de-weeding in the month August. Gradually the infections diminishes (late November) and at the time of rice harvest there is practically little dermatitis when the paddy fields are relatively dry. All rice farmers are exposed to this disease irrespective of age and sex. Since the paddy sapling plantation and de-weeding is done by hand so both arms and legs are exposed to the cercariae and are affected by dermatitis.

Prevalence and severity of paddy field dermatitis

The prevalence and severity of paddy field dermatitis was found to vary from place to place and from time to time. It closely follows the population density of Indoplanorbis exustus, the intermediate snail host that also showed high variability in their population density, spatially and temporally. In addition to paddy fields, cercarial dermatitis is also associated with large perennial ponds, locally known as "Pukheri", and wetlands near riverbanks. The persons affected in addition to farmers are persons engaged in fishing, swimmers and bathers. Severe form of dermatitis was found associated with oculate schistosome cercariae, which were found in some paddy fields of Tinsukia district.

Liver histopathology in experimental animals

Liver histopathology in experimental animals due to Schistosoma spindale

Pathology due to unisexual infections by male flukes only (in absence of eggs):

In mice, the colour of the liver appeared to be normal. There was mild eosinophilic infiltration towards the presence of flukes in the hepatic portal vessels. Phlebitis and peri-phlebitis was evident in the infected portal triad. Kupffer's cells showed hyperplasia and contained black pigment. There was no evidence of thrombo-phlebitis, septal fibrosis or pipe-stem fibrosis.

Table 1: Occurrence of cercarial dermatitis in relation to farming and other practices in Assam, India

Activity                     Condition of fields        Time              Extent of dermatitis 

Preparation of paddy-        Partially flooded with             Mid May              Some
fields using bullocks          water                                         to mid June 

Paddy seedling                   Flooded                                     Mid June             Much
plantation                                                                                to August

Manual de-weeding            Flooded                                    Mid July             Much 
                                                                                                  mid August
Harvesting 
        Wet Paddy                   Partially flooded                      June to                Some  
                                                                                                  August 
         Dry Paddy                   Relatively dry                          November           Practically None 
                                                                                                  to December  

Fishing                                  Flooded paddy fields             Any time             Some   
                                               and perennial ponds
  
Marry time swimming          Perennial ponds                     May to Nov.       Some 

Rabbit liver showed similar liver pathology except there was acute allergic reaction with profuse eosinophilic infiltration towards the presence of adult flukes in the hepatic portal vessels.

Pathology due to schistosome eggs and bisexual infection:

In our earlier experiments, we always got unisexual infections. However, recently in one batch of experimental animals we were able to get bisexual infection with heavy deposition of eggs in liver. The livers of infected mice were pale, had nodular appearance and the surface of the liver had "irregular tortoise shell appearance". The liver was firm in consistency and was deeply pigmented with black and yellow spots. The histopathological studies are under progress.

Liver histopathology in experimental animals due to Schistosoma nasale

The liver of mice infected with S. nasale flukes showed macroscopic haemorrhagic lesions due to the presence of dead flukes. There was profuse eosinophilic response towards the presence of worms in liver. The work is in progress.

Liver histopathology in experimental animals due to new schistosome flukes

The new schistosome appeared to be more pathogenic and caused extensive liver damage. The liver of infected mice was gray in colour. There was extensive egg granuloma formation and septal fibrosis.

Scanning electron microscopy of Schistosoma spindale flukes

The male flukes were 3.6 to 4.9 mm in length and 0.312 to 0.365 mm in width. The gynaecophoral canal was well developed and extends from just below the ventral sucker up to the posterior region of the body. The posterior end of the body was drawn out into a characteristic peg-like projection. The oral sucker was triangular and subterminal. The ventral surface of the oral sucker was completely covered with numerous spines measuring about 2.5 - 3 m in length. Irrespective of their position on the oral sucker, all the spines were directed towards the aperture of the oral cavity. The spines were more abundant towards the anterior part of the oral sucker. An aspinose muscular wall surrounded the oral sucker, which was about 18 m high. The ventral sucker was larger than the oral sucker and was pedunculated, round and thick rimmed. The rim of the ventral sucker was about 22.5 m in width. The inner side of this rim was covered with numerous pointed spines measuring about 2-2.5 m in length and were pointed towards the center of the ventral sucker, whereas towards the distal side of the rim the spines tended to be blunt and small in size. The central zone of the ventral sucker was provided with numerous blunt spines 1.4 m in thickness. There was an aspinose area approximately 6.5 m in width lying between the rim bearing conical spines and central region bearing blunt spines. Under low magnification, the body appeared to be smooth. However, under higher magnification the tegumental surface appeared ridged bearing minute bosses approximately 3 - 4 m in diameter and were more numerous in the posterior part of the flukes. These bosses were aspinose and some were provided with central raised projection. There was also no spination in between tubercles. However, numerous small, blunt cuticular spines were present on the ventrolateral surface of the flukes towards the anterior part of the gynaecophoral canal but no bosses were present in this region. The tegumental surface of the worm had numerous uniciliated papillae, which were more numerous on the ventro-lateral margin and just below the ventral sucker. These uniciliated papillae measured 3.6 m in diameter. Some of these papillae were devoid of a cilium, which gives them pitted-appearance.

Studies on plant based molluscicides:

This study was carried out to find a locally available plant, which has molluscicidal potential and can be used for control of snails in dermatitis affected paddy fields of Assam. An indigenous, locally available wild growing plant was found to possess considerable molluscicidal activity. The aqueous extract of dried fruits of this plant cause 100% mortality in Indoplanorbis exustus snails at 21 PPM (LC50 = 14.3 PPM; LC90 = 20.3 PPM). The molluscicidal activity of the dried aqueous extract of the fruits of this indigenous plants, was stable for one year at room temperature without much loss of activity (LC50 = 17.3 PPM; LC90 = 21.3 PPM). Preliminary studies revealed that aqueous neem leaf extract also has molluscicidal properties. Neem leaf extract was also found to be lethal to cercariae.

Table 2. Molluscicidal activity of aqueous extract of fruit of
              indigenous plant against lndoplan rbis exustus 

 ______________________________________________________________________________________
Tested material          Concentration                   Mortality                           LC50(g/1)           LC90(gli) 
   
                             gli                 ( n= 80 for each concentration)         (95% Cl)            (95% Cl) 
                                                        _______________________________ 
                                                        observed     |    (Exact binomial 95% Cl) 
 _____________________________________|_________________________________________________ 
 Fruit                                                                                                                 0.0174               0.0204
(aqueous extract)             0.025            100                (95.5 - 100)              (0.0153 - 0.0183) (0.0196 - 0.0216) 
                                        0.023            100                (95.5 - 100)
                                        0.021            97.5               (91.3 - 99.7) 
                                        0.019            81.3               (71.0 - 89.1)
                                        0.017            68.8               (57.4 - 78.7) 
                                        0.015            56.3               (44.7 - 67.3) 
                                        0.013             40                 (29.2 - 51.6) 
control                                0                   0                   (0.0 - 3.6) 
 ______________________________________________________________________________________

Conclusions: Paddy field dermatitis in Assam is cercarial dermatitis. Cercariae of  Schistosoma spindale was incriminated in most paddy field were cattle are used for ploughing fields. Circumstantial evidence indicates cercariae of Schistosoma nasale may be involved in causing cercarial dermatitis where water buffaloes are used for ploughing fields. In addition to the above schistosome cercariae, a new schistosome cercariae has been found whose cercariae are oculate and are capable of developing in mammals and caused extensive liver damage in experimental mice. These cercariae were also associated with dermatitis. A new plant based molluscicide has been found during the present study.

Control measures suggested: Control of paddy field dermatitis in Assam can be achieved by eliminating the intermediate snail hosts, which are present in large numbers in the affected paddy fields. This can be achieved using appropriate molluscicides thus interrupting the life cycle of the parasite. Currently, Niclosamide is the only safe and acceptable molluscicide according to WHO Pesticide Evaluation Scheme (WHO 1995). Due to high cost of the synthetic molluscicides, many workers have recently tried to find plant-based molluscicides for snail control programs. The aim is to identify cheap, effective and environmentally acceptable alternatives (Clark et al., 1997). Since the use of organic solvents for extraction of molluscicidal compounds from plants are costly, it has been suggested that crude aqueous suspensions are ideal for use in rural communities and can be directly applied to the snail infested focal sites (Clark and Appleton, 1997). Treatment of infected cattle, water buffaloes and goat with anti-schistosomal drugs will also be effective. Topical application of 1% Niclosamide has been found to give some protection against cercarial invasion (Abu-Elyazeed et al., 1993). However, the use of such lotions will be less effective when the exposure to water is for longer periods as in the case of farmers working for up to 6 to 7 hours in paddy fields. Therefore, in farmers' use of, water resistant, cercariae repellent creams can be used for preventing cercarial dermatitis. Treatment of cercarial dermatitis is mostly symptomatic. Anti-histamine and anti-pruritic medications are usually sufficient (Baird and Wear, 1987). Occasionally antibiotics are used when bacterial infection develops.

Zoonotic importance of Indian schistosomes: The question of true systemic infection caused by Indian animal schistosomes is still wide open. Our experiments using laboratory animals clearly indicated that wide range of mammalian hosts are susceptible to infection. Moreover, it was seen that in spite of hepato-biliary involvement in laboratory animals, we were unable to detect eggs in repeated stool examination by formol-ether concentration method or egg hatching technique for miracidia detection in infected experimental animals. Besides, perusal of literature revealed some case of S. spindale systemic infection in man. Therefore, further studies are required to generate some insight into the poorly understood problems of animal schistosomiasis and their public health impact in northeastern region of India where cercarial dermatitis is an occupational health problem. During a preliminary survey, 108 urine samples and 141 stool samples of villagers of dermatitis affected places were screened for schistosome eggs during 1999. However, no eggs of schistosomes were detected but 18.5% people had microscopic haematuria. It is planned to carry out comprehensive survey using immuno-diagnostic techniques to detect circulatory antigens of schistosomes in high-risk population of Assam.

Funding Agency

Intramural project of ICMR

Study type code

EPI & LAB

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Food-borne parasitic zoonosis: status of metacercarial infection in fishes of Assam

Investigator:                      Dr. Kanwar Narain
Co-Investigator:                Dr. J. Mahanta
Funding agency:                         World Health Organisation
Subject key words:           Food-borne tematodes
                                                     Parasitic Zoonosis
                                                     Food fishes
                                                     Metacercaria
Study type code: EPI & LAB
Objectives:

(i) To identify different species of fish serving as intermediate hosts of food-borne trematodes.

(ii) To study the prevalence and intensity of infection due to metacercariae of food-borne trematodes in common food fishes of Assam.

Introduction:

Statement of the Problem: Food-borne trematode infections have been recently identified as an important public health problem having considerable economic impact in terms of morbidity, loss of productivity and health care costs. Fish serve as second intermediate host of food-borne trematode infections and serve as direct source of infection in humans. Earlier studies in northeast region of India show that food-borne trematode infections also endemic in Assam where fish serve as a major protein source. However, no information is available on which species of food fishes of Assam are infected with trematodes of zoonotic importance and what is the prevalence of infection in them. In the proposed study the main objective is to identify commonly used food fishes serving as intermediate host of trematode infections in Assam and to find what is the prevalence of infection in different species of fishes. It is known that there is a direct correlation between the severity of infection and the number of metacercariae per gram of infected fish flesh (Dixon and Flohr, 1997). Therefore, it is intended to study the intensity of metacercarial infection also, in the fish hosts.

Present knowledge relating to the problem: It has been estimated that over 40 million people throughout the world are infected with food-borne trematodes (WHO, 1995). Majority of food borne trematodes results from consumption of raw or undercooked fish containing infective metacercariae (WHO, 1995; Dixon B.R. and Flohr R.B., 1997). Depending upon the parasite species infections caused by food-borne trematodes may affect liver, lungs, small intestine and other organs producing disease ranging in severity from mild to debilitating (WHO, 1995; Manson-Bahr 1982). Recently, preliminary studies conducted by our Centre (Narain et al., 1997; Mahanta et al., 1995 a, b) indicate that fish-borne trematode infections are endemic in Assam. The public health importance of these parasites is due to severe morbidity caused by them. Clonorchiasis/opisthorchiasis is an important zoonotic infection of man causing gastrointestinal disturbances and heavy infections are known to cause recurrent attacks of cholengitis, pancreatitis etc. It is also considered an important risk factor for primary adenocarcinoma of liver in man. The intestinal flukes cause inflammation, ulceration, haemorrhage, persistent diarrhoea and other enteropathic conditions leading to malabsorption and protein loss (WHO, 1985).

Usefulness of the study: This study will be useful to public health authorities for planning preventive and intervention strategies for controlling fish-borne trematode infection in Assam.

Methodology:

A) Study area - upper Assam

B) Collection of samples

Different samples of fish will be collected from numerous water bodies of upper Assam and from the local fish markets

C) Detection of infection

Fishes will be examined for metacercariae of Opisthorchis /Clonorchis and heterophyids by compression method and digestion technique.

E) Identification of metacercariae

The metacercariae will be identified using standard keys. Confirmation of identification will be aided with experimental infection of laboratory animals to obtain adult worms if possible

F) Techniques:

Experimental techniques: The fishes will be examined for metacercarial infection using tissue crush method and pepsin digestion technique. The recovered metacercariae will be fixed and stained with carmine for identification. Some metacercariae will be fed to laboratory-reared rats or mice for developing adult flukes for confirmation of species identity.

Statistical methods: Descriptive statistical techniques will be used to describe prevalence and intensity of metacercarial infection in different species of fishes along with their 95% confidence intervals. Differences in intensity and prevalence of metacercarial infection in different fish species will be evaluated using both parametrical and non-parametrical statistical methods as appropriate.

G) Calendar: Two years

Results: Work on this project will start from July 2000.

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Study on Coronary Heart Disease and Hypertension (in Mizoram, Assam and Tea garden of Upper Assam)

Investigator:         Dr.N.C.Hazarika. 

Co-investigators:       (i) Dr.D.Biswas (ii) Mr.R.K.Phukan (iii) Dr.H.C.Kalita,

Subject keywords:          Cardiovascular diseases 
                                                  Coronary Heart Disease
                                                  Hypertension
                                                  Epidemiology
                                                  Risk factors

Study type code :     EPI

Objectives:

(i) To estimate the prevalence of CHD and Hypertension in a defined urban/ rural population of Mizoram, Assam and in a selected Tea garden population of Upper Assam.

(ii) To study the distribution of Risk factors for CHD and Hypertension in selected population.

Introduction: Cardiovascular disease specially Coronary Heart Disease (CHD) and Hypertension are major cause of morbidity and mortality in the developed countries. There is ample evidence from several sources that the problem is increasing in magnitude in the developing countries as well including India. There are reasons to believe that the historical experience of the developed countries in terms of non communicable disease gaining prominence with the control of communicable and nutritional disease, will also be the course of epidemiological transition in India. If preventive measures are initiated early specially at a community level this course need not be termed inevitable. There have been a few epidemiological studies addressing the issue of prevalence of hypertension and coronary heart disease in urban as well as rural population groups in India. The report of an excess risk of cardiovascular disease mortality in the developing countries calls for precise contemporary estimates of cardiovascular disease prevalence in Indian population and its associated risk factors.

The north-eastern state of India is famous for its Ethno-Cultural diversity. It is said that incidence of hypertension and coronary heart disease is less in some of the states specially Mizoram. Data available with us from hospital sources also supports this view. But no estimate of prevalence has so far been done in the community. It is necessary to validitate the hypothetical assumption of less occurrence of CHD and Hypertension and other ethnic groups of this region and to compare it with other ethnic groups of Assam, Arunachal and Tea garden community of Assam. The study will highlight the prevalence of CHD and Hypertension in the different ethnic population with distinct life style. As expected if low prevalence is established in hill tribes like Mizos the study is expected to bring out the factors associated with such prevalence. A long term evaluation of registered cases of CHD and Hypertension is to be incorporated in this study.

The present study proposes to estimate the prevalence of the said diseases in a defined urban and rural area of Assam, Arunachal and Mizoram as well as in a selected Tea arden community in Upper Assam.

Methodology:

Study Design: The study will be conducted in a defined urban and rural population of Assam, Arunachal and Mizoram as well as in three Tea gardens of Upper Assam.

For the states of Assam, Arunachal and Mizoram 1 urban centre in a city or town and 2 PHC areas for rural population will be selected.

For rural areas: A stratified random sample of households from all villages under the 2 PHCs of the states as mentioned will be selected.

For Tea tribals: No. of households and population to be studied will be selected after calculating by relevant statistical method.

Diet as a risk factor for CHD and Hypertension is important. However a detailed dietary survey using the standard acceptable weighment method is not feasible. Salt & oil consumption frequency measure will be attempted.

Sample size: The sample size will be estimated based on the expected prevalence. The population in the study will include persons aged 30+ years. The sample size will also include an allowance for 10% to non responders or dropout during the survey.

Sampling design:

For Tea tribal: A representative cluster sample will be selected.

For Rural area: A stratified random sample of households from all village under PHCs will be selected.

Data collection: The project involves collection of data related to the demographic profile, individual characteristics, selected dietary items, clinical profile and biochemical parameters.

Data will be collected using two proformas :

a) Family schedule: The schedule includes details of family members, demographic profile, socioeconomic status and type of oils/ fats used and their consumption.

b) Individual schedule: This proforma will be used to elicit informations on the individuals occupation, education, physical activity, diet, tobacco use, alcohol, tea, coffee consumption and in case of female use of oral contraceptives. This proforma will also include detailed information on the current and past health status of individuals with specific reference to the cardiovascular system will be noted. Rose & Blackburn's angina questionnaire will be used to elicit the angina status. Family history of C.V. disease will also be recorded. Clinical records viz, B.P. measurements, anthropometric measurements will also be maintained in the proforma. For B.P. two readings will be obtained and the average will be taken for the purpose of analysis. Anthropometry will include height weight and waist hip ratio. Signs of congestive heart failure will be specially looked for. ECG will be obtained using a 12 lead electrocardiograph. At least 2 records will be kept - one for the Centre and other to be sent for coding. Blood samples will be collected from 10% of the sample population by laboratory technician and will be carried to the Centre for estimation observing the necessary precautionary measures.

Results: Initially a pilot survey was conducted in 50 households in each of the study area. Prevalence of hypertension was found 2.04%, 11.8%, 30.1% in order.

Actual study has been initiated in a tea garden of upper Assam and a population above 1000 in the age group 30 years and plus has been covered. ECG was recorded in all the persons clinically examined and samples were collected for laboratory estimation of lipid profile and Blood sugar.

Conclusions: The study is not yet completed and is progressing.

Funding Agency

Intramural

Study type code

EPI

 

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