during the past few decades , dengue fever has gradually become one of the leading causes of morbidity and mortality in tropical and subtropical areas throughout the world . the dengue virus ( denv ) ,
a mosquito - borne member of the family flaviviridae , circulates as four distinct serological types denv 1 , denv 2 , denv 3 , and denv 4 .
over all , two - fifth of the world population are living in areas , at risk for dengue [ 24 ] .
infection with any of these leads to a mild self - limiting febrile illness ( dengue fever , df ) .
a more severe form of the disease , dengue hemorrhagic fever / dengue shock syndrome ( dhf / dss ) , is responsible for high mortality rate , especially in children .
it has been estimated that about 50 million cases of df occur annually , with 10,000 infant deaths due to dhf / dss .
dhf / dss has been postulated to result from immune enhancement after a second heterologous denv infection . in india ,
denv was first isolated in 1946 and many outbreaks have been reported [ 710 ] .
dhf was first reported in calcutta ( kolkata ) , west bengal in 1963 , again in 1964 . since then , there are numerous studies from the indian subcontinent investigating dhf in various parts of the country [ 1322 ] .
but there are no studies investigating the overall prevalence of the dengue serotype circulating in the endemic zone , apart from the epidemic outbreak .
the purpose of this paper is to present a comprehensive report on the diagnosis of dengue infection amongst the febrile cases , available from january to december , 2010 , in the city of kolkata and also to identify the serotype presently circulating in this region .
the incidence of df in the rural west bengal is on the increase and is spreading to geographic regions not previously affected [ 23 , 24 ] .
antibodies against group b - arthropod - borne viruses in more than 80% of the kolkata population have been recorded almost fifty years back , and that is too possibly due to the infection by dengue viruses . the present study aimed to identify the serotypes of denv in the population of kolkata as well as to study the sociodemographic status in relation to denv infection .
the present population of kolkata is 44,86,679 of which 23,62,662 are males and 21,24,017 are females .
the city has an international sea and airport and one rail way station ( sealdah ) which is busiest in the world .
the rail road of this station covers a number of districts , situated at the border of bangladesh .
adjacent to the city , there is a thickly populated town , howrah , which has one of the biggest terminating railway stations of the eastern india .
cases were mainly referred from outpatient department ( opd ) and indoor of i.d & b. g hospital , attached to this unit , from different medical colleges as well as other hospitals in kolkata along with a short history of the patients .
a good number of cases were referred to us by the private practitioners also . in the matter of selection of dengue fever ( df ) cases ,
the following criteria were initially considered : ( 1 ) high fever ; ( 2 ) head ache ; ( 3 ) retro - orbital pain ; ( 4 ) nausea / vomiting ; ( 5 ) malaise / joint pain ; ( 6 ) generalized skin rashes . in the present study two or more of these criteria , apart from fever , were fulfilled .
the possibilities of bacterial and prokaryotic etiology in the collected samples were excluded through investigations at the respective hospitals . the case history and the investigations of the patients were compiled . in the case of dhf
, the history of illness was revealed by the sudden rise of high fever ( 38.3c39.4c ) , headache , retro - orbital pain , conjunctival congestion , and facial flashing .
in addition to that , some cases had the history of hemorrhagic manifestation either with petechiae or with gum bleeding or malena .
in such cases , intermittent or biphasic course of fever was recorded , where the first phase of fever persisted for 27 days and the second bout of fever persisted for 23 days .
no cases with the history of plasma leakage were observed . in the admitted cases , we found that fever was also accompanied by generalized malaise and lumbosacral pain .
those patients represented rashes , appeared on 25 days of illness in limbs , sparing palm , and souls .
sera were separated from the collected blood samples , were stored at 80c , and were tested within 1 month from the date of collection .
a total of 378 samples were thus received from the suspected cases and analyzed for the detection of dengue viruses , if any . to study the sociodemographic status in relation to denv infection ,
all the 378 samples were screened for the presence of dengue igm antibodies by igm capture enzyme - linked immunosorbent assay ( elisa ) : using a kit , prepared by the national institute of virology , pune , india , following the prescribed protocol . optical density ( od )
was measured at 492  nm using an elisa reader ( titertek multiskan plus , lab systems finland , type-314 ) . to study the molecular typing of denv
, attempts were made to isolate the rna from all the samples as well as from four different denv serotype strains , which were used as positive control .
viral rna was isolated by using qiagen viral rna isolation kit ( qiagen , gmbh , hilden , germany ) according to the manufacturer 's protocol . in this study ,
, viral rna was converted to a dna copy ( cdna ) prior to enzymatic dna amplification by the use of reverse transcriptase ( rt ) and the denv downstream consensus primer d2 - 5-ttgcaccaacagtcaatgtcttcaggttc-3 homologous to the genomic rna of the four serotypes .
subsequent taq polymerase amplification was performed on the resulting cdna with the upstream dengue virus consensus primer d1 - 5-tcaatatgctgaaacgcgcgagaaaccg-3. target rna was amplified in 25  l volumes containing the following components : 800  mm deoxynucleotide triphosphates ( dntps ) , 8  mm dithiothreitol , 0.24  m each of primers d1 and d2 , 0.5  u of amv rt ( promega , madison , wi , usa ) , and 0.625  u of dreamtaq dna polymerase ( fermentas inc . ,
the reactions were allowed to proceed for 1  h at 42c and then to proceed with 95c for 3 minutes for initial denaturation followed by 35 cycles of denaturation ( 95c for 30  sec ) , primer annealing ( 55c for 1  min ) , and primer extension ( 72c for 2  min ) along with final extension ( 72c for 5  min ) .
denv serotyping was conducted by second - round amplification ( nested pcr ) initiated with 10  u of diluted material ( 1  :  100 in sterile distilled water ) from the initial amplification reaction .
the total 20  l of reaction mixture was prepared using 2  l of diluted first pcr products , 0.8  mm dntps , 0.5  u of dreamtaq dna polymerase and 0.3  m of primer d1 and 0.3  m of dengue virus type - specific primers : ts1 5-cgtctcagtgatccggggg-3 , ts2 5-cgccacaagggccatgaacag-3 , ts3 5-taacatcatcatgagacagagc-3 , and ts4 5-ctctgttgtcttaaacaagaga-3. dithiothreitol and amv rt were eliminated .
the samples were subjected to initial denaturation ( 95c for 3  min ) followed by 20 cycles of denaturation ( 95c for 30  s ) , primer annealing ( 55c for 1  min ) , and primer extension ( 72c for 1  min ) along with final extension ( 72c for 5  min ) .
the pcr products were analyzed by running a 1.5% agarose gel stained with ethidium bromide .
out of 378 samples collected , only 173 samples were reactive to dengue igm antibody by elisa method .
maximum numbers of igm positive cases were observed in the age group of 010 years in both male and female patients ( figure 1 ) .
females were more affected ( 46.5% ) than the males ( 45.1% ) . as regards the seasonal prevalence ,
it is evident from the result that although sporadic cases obtained throughout the year , dengue cases started from the month of july and attained maximum number of cases in the month of november ( figure 2 ) . out of 378 samples ,
seventy - four samples ( 68.5% ) had the denv infection by single different serotypes , of which 7 samples ( 9.5% ) had the monotypic infection with denv-1 , 45 samples ( 60.8% ) had the monotypic infection with denv-2 and 22 samples ( 29.7% ) had the monotypic infections with denv-3 serotype .
only one sample had all the three serotypes , that is , denv-1 , denv-2 , and denv-3 .
out of 22 dhf cases , 3 were found with single - serotype infection and 18 cases with dual infections of denv 2 and denv 3 .
only a female patient of 24 years of age had the infection with denv-1 , denv-2 , and denv-3 serotypes at a time and also suffered from dhf .
clinical data regarding the death of the dhf cases was inadequate to reach any conclusion about the severity of illness .
the specimen containing denv-1 , denv-2 , and/or denv-3 is identified by the detection of a dna band of 482 , 119 , or 290  bp in size , respectively , on 1.5% agarose gel , loaded with nested pcr products along with positive controls , stained with ethidium bromide ( figure 3 ) .
the monitoring of denv activity is required for public health importance , as the dengue fever and dhf / dss are increasing worldwide and are spreading in the places , where it was previously not reported .
the first isolation of denv serotype 1 and 4 was reported from india in 1964 [ 31 , 32 ] and serotype 3 in 1968 . although concurrent infection with more than one serotype of denv in the same individual is uncommon , high percentage of concurrent infections with different denv serotypes had been detected at an outbreak in delhi , india , in 2006 .
kolkata is a dengue endemic zone ; frequent outbreaks of df and dhf have been occurring since last centuries . in kolkata ,
dengue was first documented in 1824 and several epidemics took place in the city during the years 1836 , 1906 , 1911 , and 1972 , affecting 40% of the city people . the last large - scale dengue outbreak has been recorded in the year 2005 . in the rural areas of west bengal , dengue is gradually spreading and establishes new reports [ 23 , 24 ] .
no continuous monitoring of the molecular detection of the dengue serotype has yet been attempted in the city of kolkata , either in epidemic or in sporadic dengue outbreaks . out of 378 blood samples ,
agewise distribution revealed that the highest number of dengue cases were detected in the age group of 010 years , followed by 1120 years and above ( figure 1 ) . in the highest age group ( above 50 years ) , the number of positive cases were too small and only 16.6% dengue igm positivity was found in the male individuals . in all the age groups , females were more affected than the males ( figure 1 ) .
are domestic and peridomestics in nature , the females get more exposure than the males , as most of the time they reside inside the house . during the year - round study , although small number of samples from suspected dengue cases were referred to us during the period from january to may , only a few were positive to dengue igm antibody by elisa method .
it is evident from our study ( figure 2 ) that the dengue cases actually started from the month of june and attained its peak in the month of november during this year , which is the post - monsoon period .
it may be explained by the fact that the stagnant fresh water during the rainy seasons ( june to october ) favoured the breeding of the vector mosquitoes . for the molecular detection , rt - pcr
was performed with all samples , of which 108 samples were positive by that method .
a total of 74 samples had the monotypic infections , involving denv 1 , denv 2 , and denv 3 , of which denv 2 was predominated .
conversely , 33 samples had the dual infections with denv 2 and denv 3 , of which all the samples were igm reactive .
only one sample produced prominent band against denv1 , denv 2 , and denv 3 and also contained igm antibody .
the detection of the viral rna in presence of the igm antibody may be explained by the fact that , due to the consecutive infection , the igm detected in those samples possibly appeared due to the initial infection , which is evident by the intensity of the bands in gel electrophoresis ( figure 3 ) .
the hemorrhagic manifestation was found mainly among the patients , infected with multiple dengue serotypes .
dhf cases were observed mainly among the young and young - adult age groups ( 030 years ) which might be due to the absence of immunity against all serotypes of denv in them .
although the increasing trend of cocirculation of multiple denv serotypes suggests that kolkata is becoming a hyperendemic state , a large - scale monitoring on the circulating strains of denv is highly required to draw a definite conclusion .
 dengue is one of the major public health threats in kolkata . 
 every year , blood samples with dengue - like illness are referred to us from different medical colleges and hospitals in kolkata for the detection of dengue infection in them . in 2010 , 
 a total of 378 samples were referred to us for that purpose . 
 all the samples were tested for the detection of igm antibodies by elisa method , followed by rt - pcr test for the detection of serotypes . 
 only 173 samples were elisa positive . 
 out of 378 samples , 108 were rt - pcr positive . 
 out of 108 samples , 74 samples had monotypic infection with different serotypes of denv and 33 samples had dual infections with denv-2 and denv-3 . 
 only one sample had the infection with denv-1 , denv-2 , and denv-3 . 
 dhf was found mainly among the patients , infected with multiple dengue serotypes . 
 only 3 dengue monotypic infected patients had suffered from dhf . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion