NATIONAL
INSTITUTE OF CHOLERA AND ENTERIC DISEASES, CALCUTTA
Research
Projects
Ø Observational study on infant feeding practices in a rural community of West Bengal
Ø Epidemiology of V.cholerae O139 infection with special reference to transmission
Ø Non-membrane Damaging Cytotoxin (NMDCY) of Vibrio cholerae
Ø Molecular Epidemiology of Vibrio parahaemolyticus
Ø New Phage Typing scheme for Vibrio cholerae
Ø Immunoregulatory functions of porin of Shigella dysenteriae type 1
Ø Genetic studies on virulence mechanisms in Shigella dysenteriae 1
Ø 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.
Ø STUDIES ON ORAL REHYDRATION SOLUTIONS
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
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.
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
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.
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.
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
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.
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
To determine the mode of transmission of V.cholerae O139 in the community of Calcutta.
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.
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.
Investigator: G.B.
Nair
Co-investigator: T.
Ramamurthy
Key words : Vibrio
cholerae
multiple antibiotic resistance
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.
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.
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.
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.
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.
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
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.
Clinical trials were
conducted to evaluate the efficacy of doxycycline and norfloxacin in the
treatment of adult cholera patients.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.