NATIONAL INSTITUTE OF CHOLERA AND ENTERIC DISEASES, CALCUTTA

Research Projects

 

 

Ř     Studies on the nature and significance of invasive diarrhoea in a rural community amongst children below 4 yrs. of age

Ř     Observational study on infant feeding practices in a rural community of West Bengal


Ř     Observational study on behavioral practices of rural mothers which might have an influence on the incidence of diarrhoea of under-five children

 

Ř     Epidemiology of V.cholerae O139 infection with special reference to transmission

 

Ř     Studies on use of "home available fluids" by mothers: Development of a three-tier implementation strategy for National Diarrhoeal Diseases Control Programme

 

Ř     Non-membrane Damaging Cytotoxin (NMDCY) of Vibrio cholerae

 

Ř     Molecular Epidemiology of Vibrio parahaemolyticus

 

Ř     New Phage Typing scheme for Vibrio cholerae

 

Ř     Multiple Antibiotic Resistance among Clinical Strains of Vibrio cholerae Isolated from 1998 to 1999 in Calcutta, India

 

Ř     Sequential studies of Vibrio cholerae 0139 and some of its defined virulence phenotypes on intestinal pathology in rabbit ileal loop model

 

Ř     Comparative evaluation of DNA amplification method with existing microbiological techniques in the detection of Shigella and Enteroinvasive Escherichia coli from children with dysentery

 

Ř     Immunoregulatory functions of porin of Shigella dysenteriae type 1

 

Ř     Antigenic recognition of Shigella dysenteriae outer membrane proteins using human convalescent sera and to evaluate their role in cell-mediated immune response in shigellosis

 

Ř     Genetic studies on virulence mechanisms in Shigella dysenteriae 1

 

Ř     Search for virulence traits and determination of the mechanism of pathogenicity of Klebsiella pneumoniae isolated from childhood diarrhoea cases

 

Ř     Studies on the role of enteroaggregative Escherichia coli as a cause of diarrhoea with special reference to its virulence propertie

 

Ř     Studies on the binding of Escherichia coli heat-stable enterotoxin to intestinal epithelial cell and brush border membranes of different animals

 

Ř     Detection and molecular characterization of rotaviruses

 

            CLINICAL STUDIES

 

        All clinical studies were carried out either at Infectious Disease Hospital or B.C. Roy Children Hospital, Calcutta. The following is a brief account of these studies.

 

Ř     STUDIES ON ORAL REHYDRATION SOLUTIONS

 

Ř     DRUG TRIALS ON CHOLERA

 

Ř     STUDIES ON TYPHOID FEVER

 

 

 

 

 

 

 

 

 

 

 

 


 

 

Studies on the nature and significance of invasive diarrhoea in a rural community amongst children below 4 yrs. of age

 

Investigator:                      D.N. Gupta

 

Co-investigators:             (i) B.K. Sircar  (ii) P.G. Sengupta  (iii) S.K. Mondal

                                                (iv) S. Ghosh    (v) S.N. Sikder  (vi)  B. Manna

 

Subject key words:      mucoid diarrhoea,

                                                dysentery,

                                                faecal leucocyte count

                                                 

Study type:                          EPI

 

Objective:

 

To determine the clinico-epidemiological and aetiological profile of invasive diarrhoeal episodes having only mucus or both blood and mucus in the stool and to find out differences and similarities, if any, between the two.

 

Introduction: An earlier study conducted in a rural community near Calcutta by the institute revealed that around 50% of all diarrhoeal episodes were dysenteric in nature with or without blood and therefore attention must be given to this subset of diarrhoeal episodes for effective control. This study was undertaken to investigate the aetiological and epidemiological aspects of these episodes with special reference to mucoid diarrhoea at the community level.

 

Methodology: A cohort of rural children below 4 years of age was subjected to bi-weekly surveillance using locally resident workers to detect diarrhoeal episode. Once detected an epidemiological team recorded details of the episodes. Faecal samples from active diarrhoea cases were collected before administration of antimicrobials and processed in the laboratory for established enteric pathogens. Examination of faecal leucocytes, RBC and occult blood tests were also undertaken.

 

Results: Overall incidence of diarrhoea was 1.7 epi./child/year and that of mucoid and blood dysentery was 0.8 and 0.2 episodes/child/year respectively. Children aged 6-11 month had a higher incidence of mucoid diarrhoea (1.3 episodes/child/year) and the peak season was in June and July. Multivariate analysis using logistic regression showed that mucoid diarrhoea and blood dysentery were closely similar in both clinical and laboratory findings, including raised faecal leucocyte count (>10/hpf). However, abdominal pain occurred more frequently in blood dysentery than mucoid diarrhoea.

 

Conclusion: This study identified "mucoid" episodes as a distinct entity and described it's clinical, epidemiological and laboratory profiles. This study also suggested that raised faecal leucocyte count >10/hpf as a useful indicator for invasiveness in absence of culture facility.

 

 

 

 

 

 

 

 

 


 

 

Observational study on infant feeding practices in a rural community of West Bengal

 

 

Investigator:                      S.K. Mondal

 

Co-investigators:             (i) B.K. Sircar  (ii) P.G. Sengupta (iii) D.N. Gupta

                                                (iv) S. Ghosh    (v) S.N. Sikder   (vi) B. Manna

 

Key words:                         Exclusive breast feeding

                                                Diarrhoea

                                                infant feeding

                                                lactoferrin

 

Study type:                          EPI

 

Objectives:

 

To find out the feeding practices of infants including BF and to elicit possible role of contamination of mothers' hand, weaning food, left over milk and utensils for causation of diarrhoea. The study further aimed to assess the quality of breast milk through measurement of Lactoferrin content in breast milk.

 

Introduction: Exclusive Breast Feeding (EBF) protects children from diarrhoea. But data is lacking about exact duration of EBF in West Bengal, where climatic condition may not be suitable for continuation of only breast feeding (BF) without any intake of water.

 

Methodology: Rural infants aged 0-2 months were followed up longitudinally during the first year of life. The study families were visited once a week to collect detail information through observation followed by interview regarding the feeding practices, feeding container, method of cleaning and hand washing practices of mothers. Occurrence of diarrhoea in preceding week was also recorded.Handwashings of mothers and children, swabs from used utensils and leftover food/milk were sampled periodically. Breast milk was collected from sub-samples of mothers and estimation of lactoferrin was made.

 

Results: Survival analysis showed that Exclusive Breast Feeding (EBF) dropped by 75% at 4 months of age. Study indicated a changing habit of EBF to complementary feeding during early life. Early weaning was associated with increased risk of diarrhoea. Incidence Rate Ratio was 3.02 (95% CI. 1.043-8.802).

 

Lactoferrin level of study mothers were comparable to the mothers of other area. Mean lactoferrin content in age group 17-20 yrs. (1.13+-0.19 mg/ml) was similar to that of older age group i.e, 21-26 yrs. (1.08±0.21 mg/ml). Mean lactoferrin level of mothers having 0-3 months children were significantly higher compared to mothers having children >= 4 month irrespective of age of mother.

 

Escherichia coli(as an indicator of faecal contamination) was isolated from 17% of children's and 40% of mother’s hands, also from 59% of leftover food/drink and 30% of utensil's swab.

 

Conclusion: This study clearly established the role of EBF in reducing the incidence of diarrhoea among infants. However there is a tendency of early switch over from EBF increasing the risk of diarrhoea. It was observed that infants are highly exposed to faecal contamination by their mothers and also by other sources. Lactoferrin content in breast milk is high during first 3 months after childbirth irrespective of age of mother, which protects children from the diarrhoea in early infancy.

 

 

 

 

 

 

 

 

 

 

 

 

 


 

 

Observational study on behavioral practices of rural mothers which might have an influence on the incidence of diarrhoea of under-five children

 

Investigator:                      S. Ghosh

 

Co-investigators:             (i) P.G. Sengupta (ii) S.K. Mondal (iii) D.N. Gupta

                                                (iv) S.N. Sikder (v) B. Manna (vi) B.K. Sircar

 

Objectives:

 

To identify the mothers’ behavioral practices having influence on occurrence of childhood diarrhoea, and to describe relationship between these diarrhoeagenic behavioral practices and mothers’ existing knowledge.

 

Introduction: Earlier community-based studies carried out by this institute indicated that a sizable proportion of children do not suffer from diarrhoea although they live in the same environment as that of diarrhoeal children. The present study was initiated to identify the  diarrhoeagenic behavioral practices of mother which might explain occurrence of diarrhoea in a child in one family and not in others.

 

Methodology: A nested case control study was carried out in a rural area where rural children were kept under active surveillance for detection of diarrhoea through longitudinal follow up. Information on exposure to mother’s behavioral practices as risk factors were recorded by direct observation. These were followed by interview of mothers to record their knowledge on a structured proforma. Families having a diarrhoeal child were selected as ‘case family’. Controls were selected from neighborhood families with an age-matched child without diarrhoea within preceding 7 months. Multivariate analysis was used to identify the most significant behaviors associated with diarrhoea. Finally we compared the incidence of childhood diarrhoea amongst the mothers having knowledge but do not practice these risk behaviors and mothers without knowledge and practice these (five) risk behaviors.

 

Results: Out of twelve risk behaviors studied closely, five showed significant association with presence of diarrhoeal child in the family. These five risk behaviors could predict 83.0% of diarrhoeal families. Five risk behaviors are bottle feeding (OR-2.87), non use of soap for cleaning feeding container (OR-2.61), water storage in wide-mouthed container (OR- 2.75), use of pond water for the same (OR-2.36) and indiscriminate disposal of children’s stool (OR- 1.99). A high proportion (66.9-78.9%) of mother had knowledge about these risk practices excluding the use of pond water for cleaning utensils.  Children suffered significantly less where mothers had utilised their knowledge to avoid risk practices.

 

Conclusion: The revealed that only educating mothers about the risk of diarrhoea may not have the desired impact possibly because of other factors controlling these risk practices. So apart from boosting knowledge, other factors i.e. attitude of mothers, family tradition, old customs and beliefs, lack of required facilities, and how they are related, need to be considered to alter mother’s risk behavior.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

Epidemiology of V.cholerae O139 infection with special reference to transmission

 

Investigator:                      P.G. Sengupta

           

Co-investigator:               (i) S.K. Mondal (ii) D.N. Gupta (iii) S. Ghosh

                                                (iv) S.N. Sikder (v) B. Manna (vi) B.K. Sircar

 

Key words:                         V.cholerae O139

                                                Transmission carriers

 

Objective

 

To determine the mode of transmission of V.cholerae O139 in the community of Calcutta.

 

Introduction: With the emergence of a CT producing V.cholerae non-O1 strain, later on designated as V.cholerae O139, synonym Bengal, in Calcutta in the form of an explosive outbreak during March 1993, an epidemiological study was undertaken to determine the transmission of this organism so that effective control may be instituted.

 

Methodology: The study was undertaken with hospitalised bacteriologically confirmed cases of cholera caused by V.cholerae O139. Acute cholera like cases above 5 years of age admitted to Infectious Diseases Hospital were selected and screened for V.cholerae O139. Positive cases were termed as "index cases" and their families as "index case families". The index case families were followed prospectively for consecutive 4 days. Stool samples and finger washings of contacts, drinking and domestic water families at source, stored drinking water, left over food and flies were collected daily. A family living in close proximity to index case family was subjected to similar follow up like that of index cases and acted as a control.

 

Results: Twenty-seven (52.0%) of the 52 index hospitalised acute diarrhoea cases, bacteriologically  confirmed for V.cholerae O139 were considered for study. Another fourteen neighborhood families consented to the study served as control. V.cholerae O139 was isolated from faeces of 14.6% healthy contacts in index case families as compared to none in control families (P=0.0002). The organism could be recovered from 3.7% of handwashing of contacts of index cases and also from stored drinking water (8.0%), open well water (28.6%), flies (3.8%) and pond water (25.0%) used by the index case families and none from the neighborhood families.

 

Conclusion: Large number of asymptomatic infected cases indicates an epidemiological similarity to that of eltor cholera. The organism may be carried on hands and may act as a potential source of infection to other inmates through contamination of stored drinking water, open well water etc. The results may be useful in formulating strategies for intervention of transmission of V.cholerae O139 at the community level.

 

 

 

 

 

 

 

 

 

 

 

 

 


 

Studies on use of "home available fluids" by mothers: Development of a three-tier implementation strategy for National Diarrhoeal Diseases Control Programme

 

Investigator:                      P.G. Sen Gupta

 

Co-investigators:             (i) B.C. Deb (ii) B.K. Sircar (iii) S.K. Mondal

                                                (iv) D.N. Gupta (v) S. Ghosh (vi) N.C. Saha (vii) S.C. Pal

Objectives:

 

To determine the proportion of acute diarrhoea cases that could be managed by mothers with Home-available-fluids (HAF) reducing referrals to Village Health Workers (VHW) /Health Facilities and eventually leading to development of a three-tier strategy for implementation of Diarrhoeal Diseases Control (CDD) Programme.

 

Introduction: Epidemiological studies conducted by this institute during late 1970s in Calcutta slums conclusively showed that over 90% of  diarrhoea cases in children at the community level was mild in nature and do not develop dehydration. A hypothesis was developed that these mild cases did not require administration of ORS but could be easily managed by mothers themselves if adequate amount of any fluids available in the home (later on termed as ‘Home Available Fluids’or HAF) which has a Carbohydrate base and little salt in it is used for preventing dehydration from occurring. Thus the concept of Oral Rehydration Therapy (ORT) including use of HAF came into existence. The hypothesis was tested through an operational research in a number of villages near Calcutta during 1983-84.

 

Methodology: A number of villages near Calcutta with a population around 10200 were selected for this study. Home fluids, which were locally available and culturally acceptable, were identified through a baseline survey involving mothers. Mothers were educated through interpersonal communication, posters and leaflets about the usefulness of early administration of HAF in the home management of  diarrhoea of their children. The locally resident VHWs were trained to recognise signs of dehydration, preparation of ORS solution, treatment of cases referred to them by mothers with ORS and refer intractable cases to nearest health facility. Evaluation was made every month to determine the number of diarrhoea cases occurring in the area, number that could be managed by mothers with HAF and the proportion of cases referred to VHW for ORS and also to health facilities.

 

Results: HAF usage rate was 76.6% amongst those who used HAF 48.2% did not require to be further treated with ORS. A number of locally available and culturally acceptable and affordable HAFs could be identified having a Carbohydrate base and some salt. These are sugar-salt-lemon solution (sarbat), barley water, puffed-rice soaked water, pressed-rice soaked water, green coconut water, buttermilk (lassi) etc.

 

Conclusion: This study made a notable contribution in identifying strategy for management of diarrhoea cases at the community level. The institute was involved in formulation of the National Diarrhoeal Diseases Control Programme (NCDD) for the country. The programme was launched by Government of India in a comprehensive manner on a high priority basis. It is amply satisfying that NCDD Programme is primarily based on the three tier implementation strategy developed as a result of this study where mothers were found to play a pivotal role in prevention of dehydration with early institution of Home-available fluid therapy (first tier), VHWs acted as depot holders for ORS (second tier) who treated the dehydrated child brought by mothers and the health facilities served as the third tier who tackles the severely dehydrated cases who were referred by the VHW. This was the key strategy in both Global and National Programmes for Control of Diarrhoeal Diseases (CDD).

 

 

 

 

 

 

 

 

 

 

 

 


 

Non-membrane Damaging Cytotoxin (NMDCY) of Vibrio cholerae

 

Investigator:                      G.B. Nair

 

Co-investigator:               T. Ramamurthy

 

Key words:                         Cytotoxin

                                                V.cholerae

 

The residual diarrhoea caused by candidate vaccine strains has been the main drawback against the development of a safe live oral cholera vaccine. One hypothesis states that this residual diarrhoea is due to additional secretogogues produced by strains of V.cholerae apart from the well characterized virulent determinants which are all thought to act in coordinated manner in precipitating  the secretogenic effect. In pursuit of such elusive factors, a secretory factor from non-O1 non-O139 Vibrio cholerae (serogroup O26) as well as from nontoxigenic clinical V.cholerae O1 Inaba strain was purified to homogeneity by a three step procedure. The factor was found to be monomeric in nature with a molecular mass of about 35 kDa and induced dramatic rounding of cultured eukaryotic cells and was thus designated as non-membrane damaging cytotoxin (NMDCY) or cell rounding factor (CRF). NMDCY was heat labile and proteinaceous in nature. Western blot analysis showed that the antiserum raised against NMDCY recognized only a single band and no other protein in the whole cell lysate of the V.cholerae strain thereby establishing NMDCY as a secretory product having no cell associated form.

 

The determination of first 15 amino-terminal amino acids of purified NMDCY showed complete homology to that of soluble hemagglutinin protease (HA/P) reported earlier (Finkelstein et al., 1982). Despite of the amino terminal homology, NMDCY did not exhibit hemagglutinating activity although it has a strong metalloprotease activity which was not related to the cell rounding activity. The interesting finding about the activity of NMDCY was its enterotoxic potential both in vivo and in vitro. Rabbit ileal loop assay demonstrated that 100 mg of purified NMDCY from both V.cholerae O26 and nontoxigenic V.cholerae O1 elicited nonhemorrhagic fluid accumulation and was similar to that elicited by live nontoxigenic V.cholerae cells.

 

Using chambers study showed that NMDCY induced a concentration dependent increase in short circuit current indicative of CL- secretion. A gradual increase in tissue conductance was also observed with an increase in time interval which is a direct reflection of variation in short circuit current. Interestingly, NMDCY also elicited an antitoxin serum IgG response in patients infected with V.cholerae O139 which directly proves the association of NMDCY with the disease cholera at least when O139 is the etiologic agent.

 

Both monoclonal and polyclonal antisera raised against NMDCY reacted with the antigen in Ouchterlony immunodiffusion to form a fused precipitin arc. Polyclonal antibody inhibited both the cytotoxin as well as the enterotoxic activity. A monoclonal-polyclonal based sandwich ELISA was developed to detect the prevalence of this toxin among wild strains of V.cholerae and other enteric bacteria. Fifty-six percent of the V.cholerae strains from diverse sources examined produced this toxin. Among the clinical strains of V.cholerae O1, 81% produced NMDCY. Additionally, 76% V.parahaemolyticus, 38% Aeromonas spp. and 16% Shigella spp. also produced this toxin.

 

Because of its enterotoxic and cytotoxic activities as well as its serum antibody response in V.cholerae O139 infected cholera patients and widespread distribution among V.cholerae, NMDCY has been incriminated as a covert virulence element in the cascade of events which enable the organism to precipitate the disease and may also be responsible for the residual diarrhoea evoked by recombinant live oral candidate vaccine strains deleted of all known putative toxin/haemolysin.

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

Molecular Epidemiology of Vibrio parahaemolyticus

 

 

 

Investigator:                      G.B. Nair

 

Co-investigator:               T. Ramamurthy

 

Diarrhoea is the most common clinical infection caused by the members of the genus Vibrio and majority of the illness are attributed to either Vibrio cholerae or Vibrio parahaemolyticus. V.parahaemolyticus has been observed to be responsible for acute gastroenteritis cases in Calcutta. V.parahaemolyticus, discovered by Fujino et al. (1951), is a halophilic gram negative bacterium that is a part of the normal flora of marine and estuarine waters making them potential pathogens in a widening spectrum of infections. V.parahaemolyticus infections are usually acquired by persons who eat raw or undercooked seafood or whose wounds are exposed to warm seawaters. The common clinical manifestation is self-limiting gastroenteritis, but infections may result in septicemia that can be life threatening. It was reported in the late 1960s that almost all clinical strains, but very few environmental strains, manifest Kanagawa phenomenon (KP), b-type hemolysis on Wagatsuma agar. KP is caused by high level production of thermostable direct hemolysin encoded by the tdh gene. Construction and examination of the tdh-deficient mutant of a KP-positive strain demonstrated the role of thermostable direct hemolysin in enterotoxigenicity. Investigation of an outbreak in the Maldives in 1985 revealed that some clinical strains do not possess the tdh gene but carry the tdh-related hemolysin (trh) gene. The trh sequence was approximately 70% identical to the tdh sequence. There is much greater strain-to-strain divergence among trh sequences than among tdh sequences. The trh sequences in different strains, however, can be clustered into two groups represented by the trh1 and trh2 genes, which have 84% sequence identity. Strains possessing either the tdh gene, the trh gene, or both were shown to be strongly associated with gastroenteritis.

 

Surveillance for V.parahaemolyticus infection was initiated in January 1994 in Calcutta, India. Generally, infections caused by this organism were found to be associated with diverse serovars. However, a group of strains belonging to serovar O3:K6 and possessing the tdh gene but not the trh gene appeared suddenly in February 1996 and was shown to be responsible for the high incidence of V.parahaemolyticus infection since then in Calcutta. In addition, the O3:K6 strains isolated in Calcutta were shown to exhibit unique profiles in arbitrarily primed PCR (AP-PCR) analysis. Strains belonging to the same serovar, i.e., O3:K6 strains having similar genotypic traits and showing the unique AP-PCR were also detected in 8 different countries including the United States. Thus, the Calcutta O3:K6 strains and those isolated from different countries were considered to belong to a single clone. These results suggested that this unique clone, referred to as a new O3:K6 clone, might have emerged recently and become prevalent not only in Calcutta, India, but also in other parts of the world. Analysis of the toxR region of the new and the old O3:K6 strains was done on the assumption that variation in the toxR sequence may be found in phylogenetially distinct clusters of V.parahaemolyticus. The difference in the sequence between the new and the old O3:K6 strains ranged from 11 to 14 bp within the 1,364 –bp region covering 95.4% of the toxRS coding regions, and the sequences differed invariably at 7 base positions. Based on these results a PCR method, referred to as the group specific PCR (GS-PCR), was designed to distinguish the new from the old O3:K6 strains. GS-PCR of non-O3:K6 strains presented interesting data with two other serovars O4:K68 and O1:K untypable (KUT) producing the 671-bp amplicon with the GS-PCR specific primers. Strains belonging to both these serovars were not only genotypically similar to the new O3:K6 strains but they also exhibited AP-PCR profiles indistinguishable from that of the new clone. The results indicate that the GS-PCR-positive strains belonging to the O4:K68 and O1:KUT serovars are genetically very close to the new O3:K6 clone. The O4:K68 serovar has never existed in the list of known O:K serovars before. The strains of this serovar were first isolated in 1997 from international travelers and were subsequently detected in India (March 1998), Bangladesh and Japan. Although strains of serovar O1:KUT have been detected since 1980, GS-PCR-positive O1:KUT strains first appeared in India in 1997 and were subsequently detected in Bangladesh and from an international traveler. Therefore, GS-PCR-positive O4:K68 and O1:KUT strains may have diverged from the existing new O3:K6 clone by alteration of the genes associated with the O:K antigens and followed a spreading pattern similar to the new O3:K6 clone. Hence, the infection caused by the 3 pandemic serovars, O3:K6, O4:K68 and O1:KUT, may be categorized as an emerging infectious disease, considering the extent of their geographical spread thus comprising the first described pandemic in the history of this organism.

 

Why the new clone, circumscribing 3 different serovars, is responsible for the current pandemic is not yet understood. The level of thermostable direct hemolysin production of the pandemic clone is not very different from that of classical KP-positive strains, and the pandemic clone is sensitive to representative antibiotics. It might be hypothesized that the new clone may be more potent than classical KP-positive strains in persisting in the environment or establishing infection. However, this alarming acquisition of pandemic traits by V.parahaemolyticus calls for a closer monitoring of the epidemiology of this bacterium.

 

 

 

 

 

 

 

 

 

 

 

 


 

 

New Phage Typing scheme for Vibrio cholerae

 

Investigator:                      B.L. Sarkar

           

Co-investigators:             (i) A.N. Ghosh (ii) S.K. Niyogi (iii) M.R. Saha (iv) G.B. Nair

 

Key words:                         ElT
Phage

                                      Typeable

                                                Biotype

 

Introduction: The conventional phage typing scheme proposed by Basu & Mukerjee has been used routinely for identification of the strains at the Vibrio Phage Reference Laboratory since 1968. Over the years, most of the strains restricted to only two phage types - phage type 2 and 4 with untypeable strains representing 10-20%. This limitation prompted us to develop a new phage typing scheme for V.cholerae O1 biotype ElTor.

 

Subsequently, the scenario of cholera that existed previously changed in 1992 and 1993 with the emergence of toxigenic V.cholerae O139 in India. The genesis of the new serogroup formed the impetus to search for O139 phages in and around the country.

 

Material and Methods: Five out of ninety eight phage isolated from endemic areas through out India were selected. A series of tests were performed for characterization of these phages include SDS-PAGE, restriction enzyme analysis, electron micrograph and antiphage antiserum. All of these phages were different from each other as well from the existing phages of Basu & Mukerjee. A different definition of routine test dilution (almost confluent lysis) was found to be more useful than the one previously used (confluent lysis). A total of thousand strains of V.cholerae O1 biotype ElTor were included in the phage typing study on soft agar overlay method.

 

Results: Thousand strains of V.cholerae O1 were phaged typed with five newly isolated phages. Almost 100% strains were found to be typeable and the strains were differentiated into 27 distinct phage types. The strains were found to be equally distributed and the maximum number of strains grouped in one type was not more than 22.4%.

 

Conclusion: The scheme comprises only five phages provides 27 phage types which are convenient and significant for the discrimination of strains of V.cholerae O1. The most important of this scheme is 100% typeability. The untypeable strains with Basu & Mukerjee scheme could be typed with this new scheme. The phage typing pattern is also important because from adequate epidemiological analysis, a laboratory can indicate those strains that could reasonably be considered to have a common source and a common mode or route of infection. The present scheme was found to be highly effective and applicable for phage typing of V.cholerae O1 biotype ElTor strains.

 

Important announcement: The genesis of the new serogroup V.cholerae O139 formed the impetus to search for O139 phages in and around the country. Five newly isolated phages lytic to V.cholerae O139 strain different from each other and also differed from O1 phages in lytic pattern, morphologic restriction endonuclease digestion profile and immunological criteria. A total of 500 strains of V.cholerae O139 could be clustered into ten different phage types. The most important is that O1 phages do not react with O139 strains and O139 phages do not react with O1 strains. Except the serology, the phage specially O139 phages are the marker to differentiate between O1 and O139. The scheme comprising five newly isolated phages would be another useful tool in the study of the epidemiology of cholera caused by V.cholerae O139.

 

 

 

 

 

 

 

 


 

Multiple Antibiotic Resistance among Clinical Strains of Vibrio cholerae Isolated from 1998 to 1999 in Calcutta, India

 

Investigator:                      G.B. Nair

 

Co-investigator:               T. Ramamurthy

 

Key words :                                    Vibrio cholerae

                                                 multiple antibiotic resistance

 

Introduction: The definition of emerging infectious diseases in the Institution of Medicine report includes drug-resistance infections, which have been on the upsurge for the past several years. Recent examples includes multi drug resistance in Mycobacterium tuberculosis in USA, Shigella dysenteriae type I infection in Africa, Salmonella typhi in India, and Vibrio cholerae in Ecuador. Vibrio cholerae O1 and O139 serogroups are the well-known etiologic agents of epidemic cholera. It is important to ascertain the variations in resistance and to relate these variations to mechanisms of resistance. We have been monitoring different serogroups of V.cholerae among hospitalized cholera patients for the past several years in Calcutta, India. The major objective of this study was to analyze the trends in multiple antibiotic resistance among clinical strains of V.cholerae isolated from 1998 to 1999 in Calcutta.

 

Methods: Three hundred and fifty nine strains of V.cholerae isolated between 1998 and 1999 from cholera and cholera-like patients admitted in the Infectious Diseases Hospital, Calcutta were included in this study. All the V. chlolerae strains were isolated and identified by standard laboratory methods, and further confirmed by serology using antisera prepared in our Institute. The 359 strains of V.cholerae strains included 150 V.cholerae O1 Ogawa, 104 V.cholerae O139 and 105 V.cholerae non-O1 non-O139 strains. Antimicrobial susceptibility analysis of V.cholerae was performed by the disk diffusion technique, with commercial discs (Hi Media, Bombay, India). The following antibiotics were used, ampicillin (A, 10 mcg), chloramphenicol (C, 30 mcg), ciprofloxacin (Cf, 5 mcg), co-trimoxazole (Co, 25 mcg), furazolidone (Fz, 50 mcg), gentamicin (G, 10 mcg), nalidixic acid (Na, 30 mcg), neomycin (N, 30 mcg), norfloxacin (Nf, 10 mcg), streptomycin (S, 10 mcg) and tetracycline (T, 30 mcg). Characterization of strains as susceptible, intermediately resistant, or resistant was based on the size of the inhibition zones according to the manufacturer’s instructions, which matched the interpretive criteria recommended by World Health Organization. In this study, strains showing intermediate zones of growth inhibition were interpreted as resistant on the basis of previous MIC studies with V.cholerae.

 

Results: Majority of the V.cholerae O1 strains were resistance to co-trimoxazole, furazolidone, nalidixic acid and streptomycin throughout the study period. V.cholerae O1 strains were mostly susceptible to gentamicin, norfloxacin and tetracycline. A perceptible increase (39%) in the isolation of ciprofloxacin resistant strains was noticed during 1999. Like V.cholerae O1, O139 strains were resistant to ampicillin and furazolidone and mostly susceptible to co-trimoxazole, norfloxacin, gentamicin and tetracycline. Frequency in the isolation of co-trimoxazole resistant strains of V.cholerae O139 was highest during 1994 to 1995 and thereafter declined sharply in the succeeding years. Ampicillin, co-trimoxazole, furazolidone, neomycin and streptomycin resistant strains of V. cholerae non-O1 non-O139 strains were generally high between 1998 and 1999. In contrast to V.cholerae O1 and O139, the non-O1, non-O139 strains were more frequently resistant to ciprofloxacin, norfloxacin and tetracycline.

 

Discussion: Even though the therapy for cholera is principally supportive, antimicrobial therapy can be useful in decreasing the volume of stools and length of illness. While tetracycline has been the mainstay of therapy, chloramphenicol, furazolidone, and co-trimoxazole are the other reported alternatives. Since cholera is a non-invasive disease, drugs such as co-trimoxazole, which is not absorbed from the gastro-intestinal tract, was widely used for the treatment. Resistance of an ElTor strain of V.cholerae to trimethoprim, streptomycin and the vibriostatic agent O/129 (2, 4-diamino-6, 7-diisopropylpteridine) is due to a transposon inserted into the chromosome, whose transfer is being enhanced by pretreatment with these drugs for which the markers encode resistance. This phenomenon may, in large part, be responsible for the rapid dissemination and high incidence of co-trimoxazole and streptomycin resistance among V.cholerae isolated from 1989 in Calcutta. Almost all the V.cholerae O1 strains were resistance to co-trimoxazole versus none of V. cholerae O139 strains isolated during 1998-99. The higher incidence of V.cholerae non-O1, non-O139 strains resistant to tetracycline compared to O1 and O139 strains in this study could be a prelude to the possible emergence of tetracycline resistant strains of V.cholerae O1 and O139. Reservation about promotion of ciprofloxacin as a first line drug for the treatment of cholera in developing countries has been expressed since it is an important substitute drug for treatment of multi-drug resistant enteric and other pathogens. Extensive use of this drug and empirical therapy for treating diarrhoeal infection might have promoted incidence of ciprofloxacin resistance V.cholerae, which has emerged for the first time in Calcutta during 1992 among V. cholerae non-O1 non-O139 and during 1995 among V.cholerae O1 and O139 strains. Since tetracycline resistant V.cholerae O1 strains have been responsible for major epidemics of cholera in Latin America, Tanzania, Bangladesh and Zaire, norfloxacin is widely used as an alternative to tetracycline for the treatment. Even though the incidence level of norfloxacin resistant strains among V.cholerae O1 is less in the present study, the non-O1 non-O139 strains exhibited higher level of incidence. Early studies conducted in India showed that the prevalence of multidrug resistant strains of V.cholerae non-O1 was a rare event. In the current study, we have observed that like O1, non-O1 and non-O139 isolates exhibited expanding resistance to furazolidone and nalidixic acid. It is amply clear that long-term surveillance programs are essential to identify changes in the spectrum of microbial pathogens causing serious infection and to monitor trends in antimicrobial resistance patterns. The information gleaned from the surveillance efforts is integral to the designing approaches to the therapy of serious infection and also to defining appropriate control measures for antimicrobial-resistance pathogens.

 

 

 

 

 

 

 

 

 

 

 

 

 


 

 

Sequential studies of Vibrio cholerae 0139 and some of its defined virulence phenotypes on intestinal pathology in rabbit ileal loop model

 

Investigator:                      Dr. D.R. Saha

 

Co-investigators:             (i) Dr. A.N. Ghosh       (ii) Dr. G.B. Nair

 

Subject Key Words:      Vibrio cholerae O139

                                                Pathology

                                                Virulence

                                                Rabbit ileal loop

                                                Ultrastructure

 

Subject type code:                  LAB, CLN

 

Objectives:

 

To determine the nature and location of lesion in the rabbit intestine caused by V.cholerae 0139 and few other virulent strains at different time intervals using histological and ultrastructural methodology.

 

Introduction: Since November 1992, a new epidemic strain of V.cholerae was detected in India; it spread to other countries of Asia quickly and was named V.cholerae O139 Bengal. This strain was shown to produce toxins like (CT, ZOT) etc. but was different from the epidemic causing V.cholerae O1 strains in that it had a capsule.

 

Based on human volunteer studies, the RITARD challenge experiments, histologic and ultrastructural features of epithelial damage, it has been suggested that invasion is probably a factor of importance in mechanism of diarrhoeal diseases caused by V.cholerae non-O1. To further investigate about the invasion and intestinal pathology, ileal loop model will be used in our study.

 

Methodology:

(a) Selection of strains:- Strains of V.cholerae 0139 and two of its virulence traits from our culture collection were used in the study.

(b) New Zealand white rabbits of age 6-8 weeks weighing 1.75 - 2.00 Kg were used in all experiments. Proper gut treatment was done with metronidazole and sulphaquinoxaline before starting the experiment to free the animals from Giardia and Coccidia infection.

(c) The animals were treated with different strains at different time intervals along with a control at each time period of inoculation.

(d) The intestinal tissues were taken out, kept in 10% formalin as well as in 3% chilled cacodylate buffered glufaraldehyde. The tissues after proper fixation stepwise were processed, embedded in paraffin for light microscopy and in resin for electron microscopy. Sections after cutting, were stained separately and examined by light as well as electron microscopy.

 

Results: During the period under report tissues treated with 3 different virulent strains were studied at 2, 6, 10, & 14 hourly intervals. Swollen mitochondria, hypertrophic endoplasmic reticulum were detected as early as 2 hours indicating hyperactivity of the cells. The goblet cells showed mucus depletion at places. Degenerated epithelial cells, congested lamina propria, presence of inflammatory cells were noted in different strains from 10 hours onwards. These changes were not uniform in all the strains. Comparative studies among the strains along with a stored cholera toxin are in progress.

 

Conclusion:          From the study carried out so far, it can be concluded that some degree of inflammatory changes is present in some cases of non-O1 Vibrio cholerae diarrhoeal diseases.

 

 

 

 

 

 

 

 

 

 


 

 

Comparative evaluation of DNA amplification method with existing microbiological techniques in the detection of Shigella  and Enteroinvasive Escherichia coli from children with dysentery

 

Investigator:                      Dr. S. Dutta

 

Co-investigator:               (i) S. Chakrabarti (ii) S.K. Niyogi (iii) P. Dutta (iv) B. Manna

 

Subject key words:       Shigella

                                                PCR

                                                ELISA

                                                Entero Invasive E.coli

                                                Dysenteriae

 

Subject type:                 EPI, LAB

 

Objectives:

 

(i)                  To determine the sensitivity, specificity of IpaH DNA amplification based PCR method as compared to diagnosis by DNA probe assuming culture technique as gold standard.

(ii)                To evaluate the use of the PCR technique to detect the prevalence of shigella and EIEC infection among children with diarrhoea and healthy controls.

 

Introduction: Bacillary dysentery is caused primarily by Shigella species and Enteroinvasive Esch. coli (EIEC). Isolation of shigella and EIEC by culture methods, is time consuming and requires prompt processing of specimens by well trained  microbiologists; often it remains obscure from cases of bacillary dysentery due to inappropriate sample collection (i.e., sample collected after antibiotic use, delay in transportation and processing).

 

It is a consensus that a case of shigellosis should be treated with apropriate antimicrobial agents to prevent further complications. So missing the diagnosis of shigellosis by culture method may render the infected individual to become a chronic carrier or to develop some complications

 

Other methods e.g., enzyme linked Immuno Sorbent Assay (ELISA) was developed for the detection of virulent Shigellae and EIEC. Colony hybridisation with DNA probes derived from invasion associated locus (ial) was also used to identify potentially invasive isolates. But all these test procedures are laborious and lengthy because organisms need to be grown on media O. Sethabutr et al detected shigellae and EIEC in general by amplification of the invasion plasmid antigen H DNA (IpaH) sequence from stools of patients with dysentery in Thailand. The test was reported to be more sensitive than existing microbiological methods and may be useful for diagnosis even after initiation of treatment with antibiotics. The advantage of using IpaH gene sequence as the target DNA is that IpaH sequences are present at multiple sites on both the large invasive plasmid and on chromosomes. So chances of unidentified cases of shigellosis remain negligible, even if the large plasmid is lost. However, specificity was not evaluated in defined population, where shigellosis is endemic. N.G. Tornieporth et al also identified EIEC by Polymerase Chain Reaction (PCR), which amplified IpaH target gene, among brazilian children with diarrhoea.

 

Though EIEC have been reported as an aetiological agent of acute diarrhoea from North India by culture methods, it was not recovered from this part of the country.

 

In view of Shigella isolation rate of 5.1% from paediatric population and recently few identified strains of EIEC from children with diarrhoea attending B.C. Roy Memorial Hospital for Children, by culture methods, we propose to undertake the study.

 

Methodology:

 

i)       Specificity of the PCR assay will be determined by investigating a number of known Shigella strains (including ATCC standard strains and local isolates) and other reference strains not belong to Shigella species, with the PCR method using primers and PCR conditions as described previously.

ii)      Sensitivity will be assessed by conducing PCR with tenfold dilution of the organisms in order to determine the detection limit.

iii)      Clinical samples: children, irrespective of previous antibiotic therapy, suffering from blood mucus, mucoid diarrhoea and watery diarrhoea attending B.C. Roy Hospital for Children either O.P.D. or inpatient department will be selected as cases and age, sex, matched controls from non-diarrhoea patients will be included in the study.

 

         Sample size calculation: Assuming power of the test to be 80%, sample size has been calculated using the formula:

 

              {mÖP1(1-P1)+(1-P2)P2 + vÖ2P(1-P)}2

         n = -------------------------------

                        (P2-P1)2

         m = Power

         u = 1.96

         P1            = Proportion of control exposed (assuming 1%)

         P2            = proportion of cases exposed (assuming 5%) 13

 

         n = 284

        

         So at least 300 children with diarrhoea will be enrolled as cases and for each two cases one control will be included.

 

         Sample collection: Stool samples or rectal swabs in transport media will be collected from cases and control, allowed to grow in laboratory at 37°C/24hr. in Luria broth. The Broth culture will be tested by PCR and blotted on to nylon membrane to be tested by EIEC probe.

 

iv)     All samples will be examined for presence of Shigella by culture method and then confirmed by agglutination with commercially available antisera (Murex Diagnostics, UK). Ten lactose fermenting and 10 non lactose fermenting colonies from Mac.Conkey Plate will be screened for EIEC by culture method. These tests (conventional) will be referred as gold standard in the present study.

 

v)      From each stool sample, 10ml of overnight culture will be blotted onto Hybond N+ membrane filters (Amershan Life Science). The membrane will be tested by DNA hybridization with available EIEC probe following standard technique.

 

vi)     PCR method will be standardized to be performed with stool samples, using primers which will amplify ipaH gene at 424 bp region (8). The amplified band will be confirmed by hydridization with IpaH probe, if available.

 

vii)     The results of each test procedure will be compared and statistically analysed.

 

Results: Specificity of the PCR was determined by using a number of Shigella strains (both reference and local isolates) and other strains, not belonged to Shigella species (EPEC, ETEC, V.cholerae, Salmonella); as template DNA. The method was found to be highly specific for Shigella species and EIEC strains.

 

Sensitivity of the method was determined by using tenfold dilution of the culture of Shigella strains. PCR was found to be highly sensitive test being positive at 3x102 CFU/ml.

 

Conclusion: The PCR was also found sensitive when done directly with the stool samples. So the results suggest that PCR may be used alternative to culture of shigellosis in children in India. The method is simple, rapid and efficient even when stool samples are collected after antibiotic therapy and there is delay in transportation of the samples.

 

 

 

 

 

 

 

 

 

 


 

Immunoregulatory functions of porin of Shigella dysenteriae type 1

 

Investigator:                      Dr. T. Biswas

 

Subject key words:       Porin

                                                Immunology

                                                S.dysenteriae

                                                Antigen

                                                Envelope

 

Subject type code:                  LAB

 

Objectives:

The aim of this project was to isolate envelope proteins of Shigella spp., study its antigenic response, then purify porin of S.dysenteriae type 1 and characterize its immunobiological activities.

 

Introduction: The envelope proteins of Shigella spp. was found to generate antibody response in patients as well as in experimental animals such as BALB/c mice. We, therefore, isolated the envelope proteins of S.dysenteriae type 1 and purified porin to homogeneity. Besides possessing pore-forming ability by forming diffusion channels, it was found to be exposed on bacterial surface and antigenically related among the four species of Shigella.

 

Methodology:

a.   Purification of porin by gel filtration and PAGE.

b.   Western blotting to determine cross-reactivity of porin.

c.   ELISA and immunoelectron microscopy to determine surface-exposure of porin.

d.   Determination of pore-forming activity by liposome swelling assay.

e.   HeLa monolayer culture and crystal violet staining to assay for bacterial penetration of HeLa cells.

f.    MTT assay to demonstrate porin mediated murine splenocyte proliferation.

g.   Assay for nitrite was done by taking 100ml of culture aliquots and incubating with an equal vol. of the Griess reagent at R.T. for 10 min.

 

Results: The process of cellular invasion and multiplication of Shigella spp. can be studied by in vitro assays using mammalian cell monolayers, such as HeLa. Infected cells may eventually be killed by intracellular Shigella, developing an area of dead or dying host cells or a plaque. As we found the porin to be exposed on cell surface of S.dysenteriae type 1, we studied if murine anti-porin antibody blocked the HeLa cell penetration by the bacteria. This blockade of bacterial entry through HeLa cell by anti-porin antibody as found by the reduction of plaque-formation in HeLa cell monolayers by 45% suggested the importance of the porin in the pathogenicity of Shigella.

 

Lipopolysaccharide (LPS) was purified from S.dysenteriae type 1 and found to mediate murine splenocyte proliferation. However, this LPS response of splenocytes could be completely abolished by addition of polymyxin B. Incubation of murine splenocyte with varying concentrations of porin of S.dysenteriae type 1 profoundly stimulated proliferation of the cells. The proliferative response of splenocytes to porin increased in a dose-dependent manner between 1x10-4 and 1 mg of protein. Proliferation of splenocytes reached a plateau at 1 mg of protein or above. To exclude the possibility that the observed stimulation of murine splenocytes by porin was contributed by traces of LPS, polymyxin B was added over wide range of porin concentrations, prior to stimulation. Addition of polymyxin B had no significant effect on splenocyte proliferation by porin. This data indicated that the porin-mediated stimulation of murine splenocytes was specific for protein rather than LPS. Also, preincubation of S.dysenteriae type 1 with anti-porin antibody reduced the bacterial plaque formation in HeLa cell monolayers by 45%. The two immunobiological activities indicate that porin might be important in the induction of protective immunity against shigellosis.

 

Conclusion: Porin of S.dysenteriae type 1 released nitric oxide from murine macrophages. It also modulated LPS-mediated nitric oxide release by the macrophages.

 

 

 

 

 

 

 

 

 

 

 

 

 


 

 

Antigenic recognition of Shigella dysenteriae outer membrane proteins using human convalescent sera and to evaluate their role in cell-mediated immune response in shigellosis

 

Investigator:                      Dr. A. Sinha

 

Co-investigators:             (i) Dr. M.K. Bhattacharya (ii) M.K. Chakraborti.

 

Subject key words:       IFN-t

                                                IL-2

                                                Shigella

                                                Immunity

 

Subject type code:                  LAB    

 

Objectives:

(i)                  Antigenic recognition of the outer membrane protein (OMP) from Shigella dysenteriae type 1, using acute and convalescent sera from human.

(ii)                Studies on cell-mediated immune (CMI) response to evaluate the role of T-cell function in host immunity.

 

Introduction: In Shigellosis, serum antibody responses against bacterial lipopolysaccharides (LPS) and outer membrane protein (OMP) antigen have been reported earlier from our laboratory.

 

Besides LPS, in many bacteria, the outer membrane protein (OMP) plays an important role in attachment, invasion, serum resistance, chelation of iron and resistance to phagocytosis. The hypothesis that OMPs have potential in immunoprophylaxis against infections caused by gram-negative bacteria. Protective role of OMPs from other Shigella spp. have been reported earlier but no such information is available on the antigenic components of the OMP in the induction of host immunity against Shigella dysenteriae type 1 infection. Recently, we have reported their role in the host defence of shigella infected guineapigs.

 

With this objective, we are initially planning to analyze the recognition of the antigenic component from OMP of S.dysenteriae 1 and to identify the major immunodominant components. So far it has been reported that OMP have some role in induction of the cell-mediated immunity by using skin hypersensitivity test in animals. We have also reported the role of the eluted 57 kDa component from OMPs of S.dysenteriae 1 in adhesion to cultured HeLa cells. Later on, it was also reported murine antiserum directed against S.dysenteriae 1 OMPs was found to be highly cross-reactive with the OMPs isolated from heterologous species, but the mechanism of host immunity in shigellosis still remains unclear.

 

Methodology:

i)                    Recognition of the antigenic components of outermembrane protein (OMP) extracts from Shigella dysenteriae type 1 will be done by using immunoblotting technique.

ii)                   Determination of the major immunodominant components by elution of the antigen from SDS gel/nitrocellulose strip.

iii)                 Evaluation of the role of cell-mediated immune response by determining the functional assay of T lymphocytes using delayed skin hypersensitivity test in animal and in vitro mitogenic stimulus of the lymphocytes from infected Shigella cases as well as asymptomatic controls.

 

Results: Earlier, we have reported that the proliferation of IL-2 dependent CTLL-2 cell line against PBMC culture fluids after exposure to eluted major OMP, reflected the participation of active T-lymphocytes in shigellosis. The quantitation of IL-2 concentration is serum of naturally infected patients was also done, which also co-related our previously established DTH responses against EOMP. However, with reflection of the above study of enhanced cellular immunity may indicate EOMP have some role in host defence in shigellosis. We have also studied the correlation of passive adoptive transfer and specific suppression experiments by transfering immunocompetant cells from infected animals to normal recipients. As their interaction in the effector phase of DTH reaction as regarded as a hallmark may confirm precisely about the regulation of CMI responses in shigellosis. For studing such effector DTH functions, we had prepared shigella susceptible mice model as descried earlier. DTH was elicited by injecting the major antigenic protein in doses mentioned earlier. The kinetics of the responses was monitored. The spleen cells and peritoneal exudate (PE) cells were collected from sentized mice, washed twice with HBSS and assessed for viability by trypan blue dye exclusion test.

 

After different step of the experiments by calculating percentage of suppression of DTH, it was noted, at our priliminary examinations, the spleen cells as a whole renders marked suppression of DTH reactivity. Later on, it was observed that almost identical level of suppression for transfering immunocompetent cells in the range of 1x107-5x107. It was also observed that at higher splenic cells doses, the effect was marked, whereas, in case of peritoneal exudate (PE) cells, no such suppression was noted.

 

Conclusion: Studies was carried out to evaluate the role of cytokines (INF-t  and IL-2) in host immunity in Shigella dysenteriae type 1 in natural infection. It was observed that serum  IFN-t levels were depended during acute phase of the disease and increased gradull during the convalescent phase, which indicate the selective down regulation of IFN-t. It was also found the proliferation of IL-2 dependent CTLL-2 cell line against PBMC culture fluids after exposure to major antigen reflected the participation of functionally active T-lymphocytes in Shigellosis patients.

 

 

 

 

 

 

 

 

 

 

 


 

Genetic studies on virulence mechanisms in Shigella dysenteriae 1

 

Investigator:                      Dr. R. Kumar

 

Subject Key words:       Shigella dysenteriae

                                                Salmonella

                                                Typhimurium

                                                Plasmid

                                                Vector

                                                Transconjugants

 

Subject type code:                  LAB, CLN

 

Objectives: In this study our primary objectives are:

i)                    Construction of a strain without manipulating the chromosomal and plasmid gene(s).

ii)                   To find out the synthesis and secretory pattern of virG and Ipa BC gene products.

iii)                 Stability of the constructed strain.

iv)                 Regain of the virulence after elimination of rfb and rfc gene clusters.

v)                  To find out whether human convalescent serum can recognize virG and Ipa proteins of the transconjugants.

 

Introduction: Genetic characterization of the major virulence factors in S.dysenteriae 1 should help in designing vaccine strains. The primary step in pathogenesis of bacillary dysentery caused by S.dysenteriae 1 is adhesion to and invasion of the human colonic mucosa. Besides the chromosomal gene products, the invasive phenotype is encoded by a large 120 to 140 MDa, non-conjugative plasmid. The invasive plasmid antigens (Ipa) genes encoded four highly immunogenic polypeptide. Among the four Ipa proteins, IpaB and IpaC are the principal immunogens which induce strong humoral immune response during Shigellosis. Our stragegy is to construct a hybrid vaccine strain against shigella.

 

The LPS somatic antigen plays a critical role in the pathogenesis of shigella. It has been known, e.g. that rough strains of this organism loose virulence. We have investigated the changes in virulence produced not by the removal of the side chains of the O antigen, but by the change in their type. We have introduced into the Shigella dysenteriae type I strain a plasmid encoding the complete operons of rfb and rfc from Salmonella typhimurium (pPR1347). The resulting strain was noninvasive and showed positive reaction with both shigella and salmonella specific antisera.

 

Methodology: Plasmid vector (pPR 1347) carrying both rfb gene cluster and the rfc gene of Salmonella typhimurium was transferred to an invasive, virulent S.dysenterie 1 by triparental cross with a frequency of 1.5 x 10-6.

 

Results: Five stable transconjugants were unable to produce keratoconjunctivitis in guineapigs and were cross reactive with both Shigella/Salmonella antisera. The strains were 100% stable.

Transconjugants carrying all the plasmids of S.dysenteriae 1 along with pPR 1347 showed strong cross reactivity with Shigella antisera but weak reaction with the antisera of S.typhimurium. Transconjugants without 120-Kb (virulent) or 10-Kb plasmid showed strong reaction with Salmonella antisera but weak reaction with S.dysenteriae 1 antisera. Human convalescent serum was able to recognize Ipa proteins from the culture supernatant and whole cell lysate of the transconjugants by immunoblot analysis. Transconjugants failed to produce keratoconjunctivitis in guineapigs. Secretion of Ipa H protein (60-kDa) was not detected in the culture supernatant of the transconjugants. However, synthesis of IpaH protein was detected in whole cell lysate of the transconjugants. Protein secretion of non-invasive and invasive strains was increased in presence of Ca++ ion at 2mM concentration. Optimum secretion in presence of Ca++ ion was observed within 6 hrs. However, adhesion to the HeLa cells of the transconjugant or wild type strain was not affected by Ca++ ions.

 

Conclusion: We have constructed a bivalent hybrid strain of highly virulent Shigella dysenteriae type 1, after introducing a plasmid vector pPR1347 containing rfb and rfc gene cluster of Salmonella typhimurium. The resulting strain became avirulent and does not produce keratoconjunctivities in guineapig. This constructed strain may have the possibility of use as a vaccine strain against shigellosis.

 

 

 

 

 

 

 

 

 


 

Search for virulence traits and determination of the mechanism of pathogenicity of Klebsiella pneumoniae isolated from childhood diarrhoea cases

 

Investigator:                      Dr. S.K. Niyogi

 

Co-investigators:             (i) Dr. G.B. Nair (ii) Dr. S. Dutta (iii) Dr. P. Dutta

                                                (iv) Dr. A. Pal.

 

Subject key words:       Virulence

                                                Pathogenicity

                                                Klebsiella pneumoniae

                                                PCR

                                                Diarrhoea

 

Subject type:                 LAB

 

Objectives:

 

i)                    To determine if Klebsiella pneumoniae produce any of the known enteric toxin/s by Polymerase Chain Reaction and by tissue culture assay using established cell lines.

ii)                   To study the effect of various cultural conditions in the elaboration of toxin by enterotoxigenic Klebsiella pneumoniae

iii)                 To establish the immunological relatedness of Klebsiella pneumoniae enterotoxin with other known enteric toxins.

iv)                 To determine the diarrhoeagenic ability of strains of Klebsiella pneumoniae with different virulent traits in removable intestinal tie adult rabbit diarrhoea (RITARD) model.

 

Introduction: Enterotoxin producing bacteria have been shown to be associated with acute episode of diarrhoea in children. Klebsiella pneumoniae is a member of the family Enterobacteriaceae and is reportedly associated with watery diarrhoea. Nosocomial intestinal infections with Klebsiella were reported by Orskov et al. Subsequently, Klebsiella has been isolated from stool of infants during outbreak of acute diarrhoea in nurseries in USA and India, among children with sporadic episodes of diarrhoea in Brazil, Ethiopia and South Africa. In these studies enterotoxigenicity of Klebsiella strains were examined by fluid accumulation in rabbit ileal loops and tissue culture assays but the nature of the enterotoxin was not fully elucidated.

 

In the present study, Klebsiella pneumoniae strains isolated from children suffering from acute diarrhoea will be studied for enterotoxin(s) production and characterized using molecular methods.

 

Methodology:

(a)  Klebsiella pneumoniae strains isolated as a sole pathogen from acute diarrhoea cases admitted to the B.C. Roy Memorial Hospital for Children, Calcutta will be screened for enterotoxin production. Stool samples from matched controls will also be screened for Klebsiella pneumoniae. Klebsiella pneumoniae strains will be identified by conventional techniques.

(b)  The different strains of Klebsiella pneumoniae will be examined for known toxins like CT, LT, ST and VT using appropriate sense and antisense primers by the Polymerase Chain Reaction.

(c)  If toxigenic, then the Klebsiella pneumoniae strains will be cultivated in different media, cultural conditions, and incubation temperatures to determine optimal production of enterotoxin(s).

(d)  The removable intestinal tie adult rabbit diarrhoea (RITARD) model will be used to demonstrate the diarrhoeagenic potency of the strains. New Zealand white rabbits, 6-8 wks of age, weighing 1.70 - 2.00 kg. of either sex will be used in the experiments. All animals used in this assay will be treated with metronidazole (125 mg per rabbit per day) and sulfaquinoxaline sodium (464 ng per rabbit per day) for 5 days, to clear the gut of giardias and coccidie respectively before the experiment.

(e)  Finally, if enterotoxin is detected in any strain of Klebsiella pneumoniae by the above methods, then the immunological relatedness of the toxin will be compared using antitoxins by immunological methods.

 

Results: During the period under study, 275 faecal samples were collected and were screened for the presence of Klebsiella pneumoniae.

 

Of these patients, 242 were suffering from acute diarrhoea and 33 paitents who served as controls were admitted over the same period in the same hospital for diseases other than gastro-intestinal disorders.

 

During the study period Klebsiella pneumoniae was isolated from the faeces of 11(4.5%) of the 242 patients with acute diarrhoea. In the 33 nondiarrhoeal controls Klebsiella pneumoniae was isolated from 3(1.2%).

 

From our collection of Klebsiella strains, nine Klebsiella pneumonae strains gave a band greater than 1kb in size when PCR was done using STc primers. Control strain of E.coli gave 100bp and 708bp amplified products for ST and LT respectively. Strains which gave a 1kb PCR fragment were tested for heat stable enterotoxin using suckling mice assay. None of the strains were positive in suckling mice assay. Further testing of the strains after concentrating the toxin will be done.

 

Conclusion: It is concluded from this study that diarrhoea-producing strains of K.pneumoniae do not produce either LT or ST but possess some other virulence mechanism.

 

 

 

 

 

 

 

 

 

 

 

 

 


 

Studies on the role of enteroaggregative Escherichia coli

as a cause of diarrhoea with special reference to its

virulence properties

 

 

 

Investigator:                      Dr. S. Dutta

 

Co-investigators:             (i) G.B. Nair (ii) M.R. Saha (iii) S.K. Niyogi

                                                (iv) S.K. Bhattacharya (v) P. Dutta

 

Subject key words:       Enteroaggregative

                                                E.coli

                                                Diarrhoea

                                                Virulence

                                                Markers

 

Subject type:                 EPI, LAB

 

Objectives:

i)                    To determine the age specific prevalence of enteroaggregative Escherichia coli among childhood acute diarrhoea cases and healthy controls.

ii)                   To identify virulent markers of EAggEC isolates.

 

Introduction: Though undiagnosed Escherichia coli still remains an important cause of diarrhoeal diseases especially in children in developing countries. On the basis of distinct virulence properties and syndromes diarrhogenic Escherichia coli have been classified into five distinct types. E.g. EPEC, ETEC, EIEC, EHEC and EAEC.

 

Previously referred to as EAEC, but recently termed as EAggEC are now recognised as a new category of diarrhoeagenic E.coli causing secretory as well as bloody persistent diarrhoea.

 

From studies on pathogenesis of EAggEC infections in genotobiotic piglets and human intestinal mucosa, a well defined pattern of colonization has emerged, which is consistent with the characteristic stack brick pattern of aggregation. A putative fimbrial antigen, aggregative adherence fimbriae 1 (AAF/1) coded by the 60 MDa plasmid also confers the aggregative phenotype. EAggEC have been shown to elaborate a heat stable toxin which stimulates net anion secretion in mammalian intestinal mucosa. Another heat labile enterotoxin has been reported to elevate intracellular calcium levels and stimulate calcium dependent protein phosphorylation of actin.

 

Methodology:

(1)               Study population:

Diarrhoea cases admitted to the B.C. Roy Memorial Hospital were examined. The age, sex and other parameters of subjects with diarrhoea were recorded and matched controls from nondiarrhoea out patients were included in the study.

(2)               Faecal samples or rectal swabs in transport media collected from subjects under study were streaked on Mac Conkey Agar plate (Difco) and incubated overnight at 37°C. A pool of 5-8 LF colonies, identified biochemically as E.coli, were taken from each plate and stored in Nutrient Agar stab for further testing.

(3)               First, the Escherichia coli isolates from each patient were screened by the PCR method, using oligonucleotide primers, which amplified a 630 bp region.

Secondly, all E.coli isolates were tested for strain identification with HeLa or HEp-2 cell culture for typical aggregative pattern. The results of the two methods were compared and analysed.

In the third step, species were identified by biochemical characterization of positive colonies followed by O and H antigen determination.

The EAggEC strains, isolated from aforesaid study, will be examined for following virulence properties:

 

a)   Haemagglutination activity :  Erythrocytes of human, rabbit, guineapig, chicken, sheep will be used for performing Haemagglutination test to detect presence of any fimbriae on its surface.

b)   Haemolysin Production  :  Blood Agar media containing Sheep/Rabbit blood will be used for zone of haemolysis around the colony to detect diffusible haemolysin. Attempts will be made to test contact haemolysin also.

c)   Plasmid analysis : To examine the plasmid profiles of the isolated strains.

d)   Presence of EAST toxin gene : astA gene segment will be amplified by using suitable primers.

e)   Salt aggregation test using various concentrations of ammonium sulphate.

f)    Other reported virulence markers e.g. Pet (Plasmid encoded toxin) and any other toxin e.g. LT, ST and VT.

g)   Any unidentified toxins or adhesins.

h)   Invasiveness - by HeLa cell invasion assay.

 

Results: E.coli isolates that adhered to HeLa cells in an aggregative pattern were the sole isolates significantly more often in 254 cases (9%) than in 134 control (2%) children. (p<0.05) Age stratification showed that EAggEC were isolated more frequently from children aged below 36m. The EAggEC belonged to several O serogroups and showed multiple drug resistance. Both methods (PCR and culture) were positive for 26 samples, nine samples were positive by PCR alone and seven samples were positive by culture alone, indicating 78% sensitivity and 97% specificity of PCR assay.

 

Conclusion: The results suggests that EAggEC plays an important role in the aetiology of acute diarrhoea among children below 36m in India and the PCR system may be useful in epidemiological studies to identify EAggEC infection, although the definitive test for EAggEC remains the cell adherence assay.

 

 

 

 

 

 

 

 

 

 

 


 

Studies on the binding of Escherichia coli heat-stable enterotoxin to intestinal epithelial cell and brush border membranes of different animals

 

Investigator:                      Dr. M.K. Chakrabarti

 

Co-investigator:               Dr. Dr. A.K. Sinha

 

Subject key words:       Enterotoxin

                                                E.coli

                                                Receptors

                                                Guanylate cyclase

                                                Cyclic GMP

 

Subject type code:                  CLN, LAB

 

Objectives:

i)                    To determine the presence and density of STa receptor in intestinal epithelial cell and brush border membranes of different animals.

ii)                   To determine association of radio-iodinated STa to epithelial cell and brush border membrane receptor.

iii)                 To characterise the purified receptors of STa from a high density receptor system.

 

Introduction: In developing countries enterotoxigenic Escherichia coli that produce heat stable toxins (STa) may be responsible for 50% to 80% of the reported cases of diarrhoea4. STa are also a major cause of diarrhoea in laboratory and domestic animals. The heat stable toxin bind to cell surface receptors in the intestine, which subsequently leads to an activation of guanylate cyclase. There are controversies whether guanylate cyclase and STa receptor are same protein or two different proteins. Kuno et al, 1986 showed that STa receptor is a distinctly different protein from guanylate cyclase. Although guanylate cyclase coupled STa receptors have been reported to exist only in intestinal epithelial cells of mammals, the research of Forte and colleagues, 1989 has shown that guanylate cyclase coupled STa receptors are expressed in epithelial cells throughout the body of the north American opossum. Katwa et al reported that receptors for the E.coli STa are present throughout the digestive tract of chicken, with binding activity present not only in the intestinal epithelium but also in the intestinal smooth muscle. It is, therefore, possible that guanylate cyclase coupled STa receptors are also expressed in the epithelial cells of other animals. Studies by Frantz and Robertson, 1984 showed that STa binds to a single class of high affinity receptors present on rat intestinal epithelial cells and brush border membranes. Most of the studies on STa-receptors were done with rat brush border membranes and epithelial cells. However, the STa receptors are present in low numbers in rat enterocytes.

 

Methodology:

(a)                E.coli STa will be purified by the method of Drefus and Robertson, 1984.

(b)               Purified, salt free STa will be radio-iodinated enzymatically using enzymobead procedure.

(c)                Enterocytes will be isolated from small intestine of different animals by the method of Weiser, 1973 and brush border membrane vesicles will be prepared by the method of Robertson et al, 1984.

(d)               Binding of radio-iodinated STa to receptors of enterocytes and brush border membranes will be assayed according to the method of Cohen et al, 1987.

(e)                Guanylate cyclase activity of epithelial cell and brush border membranes will be quantitated by radio-immuno assay.

(f)                 Attempts will be made to purify the STa receptor by using different purification techniques.

 

Results: Initially, STa was purified to homogeneity by HPLC and tested for its biological activity in suckling mice. STa was conjugated to HSA and antisera to STa was raised in rabbits. By ligand blotting technique, STa receptor could be detected in brush border membranes of different animals and crude membranes of human colonic cells. In order to measure the density of receptors, STa was radiolabelled with Na125I by enzymobead method. 125I-STa was tested for its biological activity in 2-3 days old suckling mice. It was found that the iodinated STa possessed full biological activity like native STa. The binding studies with 125I-STa was done with intestinal brush border membranes of 14-21 days old rats, guineapigs, rabbits and hamsters. The receptor binding studies were done with 30-50 µg of brush border membrane (BBM) proteins at 37°C. It was found that the rate of binding of 125I-STa to BBM of all species were maximum at 15-30 min and pletaeus were obtained after 50-60 min of incubations. Competitive binding assays were done with different molar concentrations of unlabelled STa. It was found that about 90-95% 125I-STa were displaced in competitive binding by 100-1000 fold molar excess of unlabelled STa. It was also noted that at 1 : 1 molar ratio of both hot and cold STa, cold STa replaced 40-50% of hot STa.

 

Next, equilibrium bindings were done to determine the kinetics of binding of 125I-STa to intestinal BBM of different species. Scatchard plots of the stoichiometric binding revealed a single class of receptors in rats, guineapigs, rabbits and hamsters with association constants of 1.17 x 1012M-1, 1.04 x 1012M-1, 0.85 x 1011M-1 and 0.31 x 1012M-1 respectively. The STa receptor densities in these species were also calculated from the Bmax (maximum binding at equilibrium) values and it was found that rats, guineapigs, rabbits and hamsters have following numbers of STa receptors respectively; 1.3 x 1012. mg-1 BBM, 1.5 x 1012. mg-1 BBM, 1.7 x 1012. mg-1 BBM and 4.1 x 10-9. mg-1 BBM.

 

In the next step, 125I-STa was crosslinked to BBMs of each species, in order to know the molecular masses of STa receptor. The crosslinking was done with disuccinimidyl suberte (DSS), a homobifunctional crosslinker and the crosslinked BBMs were run in 5-20% gradient SDS-PAGE under reducing conditions. The gels were stained, dried and autoradiographed censequently. It was found that 125I-STa was incorporated to BBM of rats with molecular weight corresponds to 160 kDa, that of 115 kDa in guineapigs, 140 kDa and 38 kDa in rabbits and 65 kDa in hamsters.

 

So, it suggested that the number of STa receptors present in rabbits and guineapigs were comparable to that of rats, but in hamsters, lower number of receptors were found in comparison to other tested animals.

 

We then extended our study on a human colonic carcinoma cell line COLO 205. To see whether there exist any cell surface STa receptors on COLO 205 cells, STa was allowed to bind with COLO 205 cells and probed subsequently with STa specific antisera. The indirect immunofluorescence was obtained using FITC-conjugated goat antirabbit IgG. Using intact cells, indirect immunofluorescence study revealed the localisation of intensely stained areas mostly at the cell surface which indicated that STa bound only to the cell surface regions. A competition of binding between unlabelled STa and 125ISTa to COLO 205 cell membranes were done in a dose-dependent manner. About 95% 125ISTa was displaced by 1000 fold molar excess of unlabelled STa. Time course study revealed that maximum binding of 125STA to COLO 205 occurred at 50 minutes.

 

Conclusion: It has been shown for the first time that STa can bind specifically to its receptor on the cell surface of a human colonic cell line COLO 205 and subsequently raises intracellular cyclic GMP accumulation, inositol triphosphage level and Ca2+ mobilization. It was found that STa not only binds and accumulates cyclic GMP, it also raises intracellular calcium in COLO 205 human colonic cells unlike to that observed in other cell lines. This cell line may be helpful in studying the mechanism of action of STa in detail.

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

 

Detection and molecular characterization of rotaviruses

 

Investigator:                      Dr. T.N. Naik

 

Co-investigator:               Dr. T. Krishnan

 

Subject key words:       Rotavirus

                                                Serotype

                                                Sequence

                                                Epidemiology

                                                ELISA

 

Subject type:                 EPI, LAB

 

Objectives:

Detection of rotavirus to be followed by complete molecular characterization of serotype/subgroup-specific genes by sequence analysis.

 

Introduction: Rotaviruses are a major causal agent for mild to severe gastroenteritis among children and young ones of animals all over the world. There are seven different groups [A-G] of rotaviruses which can be readily distinguished by their characteristic dsRNA electropherotypes or PAGE profiles and other antigenic properties. The outer capsid antigens viz VP7 and VP4 are responsible for the G-Serotype and P-serotype specificity of the Group A rotaviruses; the wide diversity of Group A rotaviruses encompasses 14 different G-serotypes and 20 different P-types, widespread among humans and various species of animals. According to the nature of the inner capsid antigen [VP6], Group A rotaviruses have been subgrouped into (a) Subgroup I, (b) Subgroup II, (c) Subroup I&II, and (d) non Subgroup I - non Subgroup II. The Group-specific nature of non Group A viruses [Group B and others] is clearly defined and it is seen that rotaviruses belonging to different groups share antigenic properties only specific for those particular viruses [Estes et al 1996].

 

Methodology:

Detection of rotavirus from faecal specimens

Rotavirus was detected from faecal specimens according to the method of Herring et. al (1982) by [a] extraction of dsRNA and [b] electropherotyping of the dsRNA genome. The eleven dsRNA segments are found to have a characteristic [a] 4-2-3-2 profile in Group A rotaviruses, [b] 4-2-1-1-1-1-1 profile in ADRV or Adult Group B rotavirus and [c] 4-(2+1)-2-2 profile in Group C rotavirus where the 7th segment if found to be migrating closer to the 6th genomic segment in polyacrylamide gels.

Subgrouping of Group A rotavirus [inner capsid antigen, VP6]

The nature of the inner capsid antigen [VP6] was determined in a monoclonal antibody based ELISA according to the method of Follett et al [1984] to differentiate between Group A viruses having either one of the following VP6 subgroups viz Subgroup I, Subgroup II, Subgroup I&II, Non subgroup I - Non Subgroup II [reactive only towards the Group A specific MAb] or Non reactive [no reaction towards any of the Group A specific MAbs].

G-Serotyping of Group A Rotavirus [outer capsid antigen, VP7]

(a)  The nature of the G-serotype of the outer capsid antigen [VP7] was determined in a monoclonal antibody based ELISA to study the prevalence of the four major human G-serotypes [G-1 to G-4]. The G1-G4 serotyping of VP7 was carried out according to the method of Coulson et al [1987] and Taniguchi et al [1987] to differentiate between Group A rotaviruses with G-1, G-2, G-3 & G-4 types of VP7.

(b)  The nature of G-serotype of the VP7 gene of rotaviruses [Group A, Group B, or Group C] were also confirmed by Reverse Transcription Polymerase Chain Reaction with gene specific primers according to the method of Gouvea et al [1990].

 

Results: Rotavirus of Group A nature was detected among diarrhoea cases that consisted of (a) children (aged below five years), (b) Older children (aged above five years), and (c) adults (aged above 18 years). The relative percentage values of Group A rotavirus positive cases in the three categories was 79%, 6% and 15% respectively.

 

Rotavirus of Group B nature was also detected among adults (4%) during the study period. The Group B rotavirus resembled the Chinese ADRV (Adult Diarrhoea Rotavirus strains which was responsible for large outbreaks of adult gastroenteritis in several different provinces of China during 1982) and has been shown to cause adult diarrhoea sporadically outisde China for the first time.

 

The nature of VP6 inner capsid antigen of Group A rotaviruses could be determined in 86% of the diarrhoea cases; it was observed that 33% were of SGI nature, 40% were of SGII nature, 3% were of SGI & SGII nature and 10% were non SGI or SGII nature respectively. There was no reaction towards any of the Group A specific monoclonal antibodies among 14% of rotavirus; these nonreactive (NR) viruses however showed the characteristic 4-2-3-2 Group A rotavirus genome profile of their eleven double stranded RNA segments in silver stained PAGE gels; the nature of the VP6 genes from SGI, SGII, SGI & SGII, non SGI or SGII and NR will be studied further by reverse transcription-polymerase chain reaction (RT-PCR) and sequence analysis to compare sequence homology between different types.

 

The nature of one of the outer capsid antigens, VP7 (G-serotype specific) of the Group A rotaviruses was also studied to determine the prevalence of the major human G-serotypes (G1-G4) in a monoclonal antibody based ELISA for 76% diarrhoea cases. The relative percentage value for G1, G2, G3, & G4 type of Group A rotavirus was 23%, 2%, 16% and 46% respectively. The preliminary data suggested that some of the rotaviruses were reactive towards different G-serotypes, in 13% cases. The G-serotyping of rotaviruses will also be carried out by RT-PCR and sequence analysis to study the relative prevalence of different VP7 genes in circulation and thereafter compare the sequence homology with existing G-serotypes (G1 - G14).

 

The dsRNA of Group B human rotaviruses [ADRV] was extracted by using RNaid kit [BIO 101, USA] according to the instructions of the manufacturer. The ADRV genes 4,5,6,7,8,9 and 11 were amplified by reverse transcriptase polymerase reaction [RT-PCR] using gene-specific primers designed from published sequences of the Chinese ADRV strain. Sequencing of gene 4 & 9 coding for VP4 and VP7 have been completed and sequening of other genes will be taken up shortly.

 

Conclusions: The prevalence of rotaviruses of widely diverse nature pose a serious challenge for the development of successful control measures for them. Hence the continued surveillance of newly emerging strains and their complete characterization at the molecular level is highly essential for knowing the variants that are responsible for causing diarrhoea at any given point of time.


 

 

 

 

 

 

 

 

STUDIES ON ORAL REHYDRATION SOLUTIONS

 

 

 

Introduction

 

            Oral rehydration therapy has revolutionised the treatment of acute dehydrating diarrhoea. However, in the early days of development of oral rehydration solution (ORS), it was considered suitable for only maintenance therapy of acute dehydrating diarrhoea. Later on standard ORS recommended UNICEF/WHO became the effective therapy in all age groups and for all etiologic of diarrhoea throughout the world. The clinical trials in national perspective was still essential and also for development of newer formulations of ORS which might have capacity to reduce the duration, frequency and volume of diarrhoea.

 

Objectives

 

            Studies on ORS were conducted (i) to find out the efficacy of standard ORS recommended by UNICEF/WHO in our national scenario, (ii) to compare the efficacy of standard ORS and different ORS formulations (amino acid fortified ORS, cereal based ORS and hypo-osmolar ORS) in the management of acute dehydrating diarrhoea.

 

Methods

 

            Male children suffering from acute watery diarrhoea of less than 3 days duration with "some" dehydration were chosen for ease of collection of urine and stool separately. They received "trial ORS formulation" or standard ORS according to the study design. Admission criterias of study children were noted and compared. Children were followed up 8 hourly till recovery and outcome variables (stool output, frequency and duration of diarrhoea, intake of ORS, intake of other liquids) were noted on daily basis in pre-designed proforma and analysed. Stool samples were collected to detect the established enteropathogens. Blood samples were also collected to estimate serum electrolytes.

 

Results

 

            A study conducted in 1972-73, showed that if repeated small volume of `ORS' were administered orally at frequent intervals, nearly 91% of diarrhoeal children with moderate dehydration could be successfully hydrated by standard ORS alone. Further studies documented that neonates and young infants could also be safely and effectively hydrated by standard ORS, provided breast feeding is continued throughout the course of treatment. ORS formula recommended by UNICEF/WHO is also safe and effective in the treatment of dehydrating acute diarrhoea is severely malnourished (marasmic) children.

 

            A comparative study of standard ORS and glycine fortified ORS showed that glycine fortified ORS does not have any therapeutic advantage over standard ORS. Similarly, studies on cereal based ORS formulations (pep rice powder and rice powder ORS) failed to document any significant reduction of duration of diarrhoea. However, another study using hypo-osmolar ORS showed significant reduction of stool volume and duration of diarrhoea in small children with diarrhoea of diverse etiology as well as adult cholera patients.

 

Conclusion

 

            Results of these studies documented that standard ORS recommended of UNICEF/WHO is safe and effective for the treatment of diarrhoea of all age groups and all etiologies. ORS is also safe and effective in the treatment of severely malnourished (marasmic) children. "Rice based ORS" and "glycine fortified ORS" doubt have any added advantage over standard ORS. However, hypo-osmolar ORS have some beneficial effects.

 

 

 

 

 

 

 

 

 

 

 

                       DRUG TRIALS ON CHOLERA

 

 

 

 

Introduction

 

            Acute watery diarrhoea caused by Vibrio cholerae is an important cause of hospitalisation in Calcutta. Antibiotic is needed to shorten the course of illness. Tetracycline is useful and proved to be the drug of choice in cholera for all age groups. However, clinical evaluation of alternative drug(s) is always preferred for fear of less efficacy of existing drug due to development of resistance.

 

Objectives

 

            Clinical trials were conducted to evaluate the efficacy of doxycycline and norfloxacin in the treatment of adult cholera patients.

 

Methods

 

            In one study, doxycietine was compared with tetracycline in the treatment of bacteriologically confirmed cholera in adults. Four types of treatment were compared : Group A was given 200 mg doxycycline on admission and 100 mg on 2nd day; Group B was given 200 mg doxycycline on admission only; Group C received 300 mg doxycycline on admission only; Group D received 500 mg tetracycline every 6 hours for 48 hours.

 

            Another double blind, randomised, controlled clinical trial of norfloxacin was conducted with bacteriologically confirmed adult cholera patients. In adjunct with rehydration therapy, one group of patients received norfloxacin, another group of patients received trimethoprim-sulphamethoxazole (TMP-SMX) and another group received placebo.

 

Results

 

            Tetracycline showed a slight advantage in respect to duration of diarrhoea and vibrio excretion compared with dexycycline given in a single dose of 300 mg; but fluid intake and stool output were about the same in those two groups. The other two doxycycline treatment schedule did not compare well with tetracycline treatment schedule.

 

            Norfloxacin trial showed that norfloxacin was superior to trimethoprim. Sulphamethoxazole (TMP-SMX) and placebo in reducing stool output, duration of diarrhoea, fluid requirement and vibrio excretion.

 

Conclusion

 

            Doxycycline 300 mg single dose and norfloxacin 400 mg in a twice daily dosage schedule can be used as alternative drug for the treatment of cholera in adults.

 

 

 

 

 

                                     STUDIES ON SHIGELLOSIS

 

 

Introduction

 

            An epidemic of multidrug resistant Shigella dysenteriae type 1 occurred in the State of West Bengal and also in the state capital Calcutta, India in the year 1984. Since then West Bengal including Calcutta became the endemic foci for Shigella. A large number of shigellosis cases were admitted in different hospitals of Calcutta which provided an opportunity to study the different aspects of shigellosis including drug trials.

 

Objectives

 

            Studies were conducted to observe the species differences of shigellae strains isolated from children and adults in different periods. Changes in susceptibility pattern of the shigellae strains were also studied. Several clinical trials were also conducted to evaluate the efficacy of drugs like trimethoprim-sulphamethoxazole, nalidixic acid, norfloxacin, ciprofloxacin and furazolidone for the treatment of bacteriologically confirmed shigellosis patients.

 

Methods

 

            Representative stool samples of all acute hospitalised diarrhoea cases (including dysentery) were collected to detect Shigella spp. as sole pathogen and the suspected colonies were biochemically confirmed and were finally serotyped with Shigella polyvalent and monovalent antisera, using standard techniques. Isolates were also tested for their antimicrobial susceptibility pattern using dry disc diffusion techniques.

 

            Drug trials were conducted in adult and pediatric shigellosis patients to compare the efficacy of "trial drug" to that of standard drug.

 

Results

 

            During the epidemic of shigellosis in 1984 Shigella dysenteriae type 1 was the predominant serotype. However, during the post epidemic period (1985 to 1997). Shigella flexneri was the predominant serotype. Isolation of Shigella sp was possible all throughout the year with high isolation rate during summer and early monsoon months.

 

            During 1984, isolation strains of Shigellae were susceptible to norfloxacin, ciprofloxacin, furazolidone and gentamycin but were resistant to tetracycline, trimethoprim-sulphamethoxazole, chloramphenicol, ampicillin, amoxycillin, kanamycin, neomycin. After 1990, nearly 35% strains of Shigella became resistant to nalidixic acid and 6% strains became resistant to furazolidone. While comparing the clinical efficacy of nalidixic acid and trimethoprim-sulphamethoxazole (TMP-SMX), nalidixic acid was found superior to TMP-SMX as the patients belonging to nalidixic acid group had significantly shorter duration of fever, abdominal pain and presence of blood and mucus in the stool than those who received TMP-SMX. However, no significant difference in clinical responses were observed in shigellosis patients who received either nalidixic acid or norfloxacin. Single dose (1 gm) of ciprofloxacin in adult shigellosis patients was effective and safe. In another clinical trial, it was observed that nalidixic acid treated group had significantly higher cure rate as compared to furazolidone treated group. However, 85% cure rate in furazolidone treated group may be potentially useful.

 

Conclusion

 

            After 1984 epidemic of MDR Shigella dysenteriae type 1, whole West Bengal including the capital city of Calcutta became the endemic foci  Shigella. Shigella flexneri was the predominant serotype isolated during the post epidemic period. Nalidixic acid became the drug of choice for Shigellosis, norfloxacin, ciprofloxacin and furazolidone were considered to be the other alternative of nalidixic acid.

 

 

 

 

 

 

 

 

 

 

 

 

 

                                STUDIES ON TYPHOID FEVER

 

 

Introduction

 

            Several epidemics of typhoid fever caused by multi-drug resistant Salmonella typhi have been reported from different parts of India including Calcutta. A large number of typhoid fever cases were admitted in different hospitals of Calcutta and also received treatment from outpatients department (OPD) which gave us an opportunity to study the different aspects of MDR typhoid fever in children.

 

Objectives

 

            Studies were conducted to know the bacteriological, clinical and epidemiological profiles of MDR typhoid fever in children. Clinical trials were also conducted to evaluate the efficacy of ciprofloxacin in several ill and furazolidone in less severe cases of typhoid fever.

 

Methods

 

            Irrespective of sex and severity of illness, children with clinical diagnosis of typhoid fever were included in the study. Thorough physical examination was done and history was taken from each patient or from the parents. History related to socio-economic status, epidemiological information, domestic and personal hygiene and use of the pre-designed proforma. Blood samples of these patients were screened for isolation of Salmonella typhi. Susceptibility patients of isolated strains of S.typhi were also determined.

 

            Several typhoid fever cases (having abnormal state of consciousness) were treated with intravenous ciprofloxacin, however, the therapy was changed to tablet form when the patients were able to take orally. Clinical cure with eradication of MDR S.typhi was observed. Efficacy of furazolidone and chloramphenicol for the treatment of less severe typhoid fever cases was also evaluated in a randomised clinical trial.

 

Results

 

            Clinically suspected typhoid fever cases admitted to hospital were screened for isolation of S.typhi by blood culture. S.typhi was isolated from 37% patients. Majority of the strains (92%) showed multidrug resistance (MDR). They were resistant to chloramphenicol, ampicillin, tetracycline and trimethoprim-sulphamethoxazole but uniformly (100%) susceptible to gentamycin, amikacin, furazolidone, norfloxacin and ciprofloxacin. Majority of the cases came from low socio-economic class with poor personal and domestic hygiene. Except fever other common clinical presentations were headache, chill and rigor, diarrhoea, anorexia, vomiting, cough and abdominal pain. Hepatospenomegally was observed in 42% cases.

 

            Bacteriologically confirmed severe typhoid fever cases were initially treated with ciprofloxacin intravenously and followed by tables. Children regained consciousness within an average period of 2 days. The temperature of the children returned to normal within 3.3 days. S.typhi could not be detected by blood culture after 14 days drug therapy.

 

            Efficacy of furazolidone and chloramphenicol was compared in a randomised clinical trial which showed that cure was achieved in 86.2% of furazolidone recipients and 90% chloramphenicol recipients who were infected with the strains of S.typhi susceptible to both the drugs. The difference in cure rate was statistically significant (86.2% vs 44.8%, p = 0.000003) when the two treatment groups infected with the strains of S.typhi susceptible to furazolidone but resistant to chloramphenicol were compared. There was no relapse or carrier in either of the group.

 

Conclusion

 

            An epidemic of typhoid fever caused by MDR strains of S.typhi occurred in Calcutta during 1990-1991. However, after that Calcutta and its suburbs became endemic for typhoid fever. Furazolidone appears to be a satisfactory alternative to chloramphenicol in the treatment of typhoid fever caused by chloramphenicol resistant strains of S.typhi. However, severely ill patients should receive ciprofloxacin.