Dengue is one of the most of import arthropod borne viral diseases caused because of infection of one or more dengue viruses ( DENV ) . Harmonizing to World Health Organization ( WHO ) , two fifth of the universe ‘s population is at hazard from dengue disease and every twelvemonth 50 million DENV infections are suspected worldover1. In India, the disease is prevailing and all four serotypes are known to be go arounding either singly or in combination2,3, ensuing in several eruptions over the old ages. The disease is more terrible in kids than in adults4. Classical dandy fever febrility ( DF ) characterized by high class febrility and mild roseolas that may take to terrible dandy fever haemorrhagic febrility ( DHF ) to dengue daze syndrome ( DSS ) and is the major cause of hospitalization and mortality chiefly among children5,6, particularly in tropical and semitropical countries7. However, due to miss of any diagnostic marker and any specific clinical symptoms to place instances who will hold terrible disease outcome8, early diagnosing and close monitoring with diagnostic intervention is necessary.
492 INDIAN J MED RES, september2012
Therefore, we undertook this survey for diagnosing of dandy fever utilizing IgM and IgG ELISA on serum samples of hospitalized patients suspected to hold dengue infection at Jabalpur, MP, India, and to find the go arounding serotype nested rearward written text polymerase concatenation reaction ( nRT-PCR ) was done.
Material & A ; Methods
Sample aggregation: This survey was carried out during the period of August – December 2010. Children ( n=86 ) and striplings ( n=3 ) in and around Jabalpur metropolis ( 23o9’38 ” N 79o56’19 ” Tocopherol, 1.348 foot above sea degree ) suspected of dandy fever and admitted and/or treated at authorities infirmary ( Seth Govind Das Hospital ) , Jabalpur and from bordering Narsinghpur territory following WHO instance definition9 were included in the survey. The clinician at the infirmary after thorough scrutiny and recording symptoms collected 89 blood samples which were sent for dandy fever diagnosing to virology research lab of Regional Medical Research Centre for Tribals, Jabalpur. The ethical clearance for the survey was taken from centre ‘s ethical commission and informations presented in this survey of the take parting patients is unbroken anon. .
Mosquito aggregation: Mosquito studies were besides carried out in different parts of the metropolis during the survey period ; both mature and immature phases of mosquitoes were collected and identified. The breteau index ( BI ) , container index ( CI ) and for house index ( HI ) were calculated.
ELISA and nRT-PCR: The serum was separated from all 89 samples and was subjected to MAC-ELISA for testing DEN specific IgM and IgG antibodies utilizing Pan Bio IgM and IgG ELISA kits ( Inverness Medical Innovations Pvt. Ltd. , Australia ) following maker ‘s instructions. All IgM ELISA positive ( n=18 ) and samples collected between 0-5th twenty-four hours of oncoming ( n=6 ) were subjected to nRT-PCR for finding the serotype of the DENV10 with minor alterations. Briefly, viral RNA was isolated utilizing QIAamp viral RNA Mini kit ( QIAGEN, USA ) harmonizing to the maker ‘s instructions in 40 I?l extraction buffer. Human RNase P ( RNP ) RNA amplification11 was used as internal quality control for RNA extraction and RT-PCR elaboration. Super Script III one measure RT-PCR with Platinum Pfx DNA polymerase was used for the elaboration harmonizing to the maker ‘s instructions ( Invitrogen, USA ) . The amplified fragment of first RT-PCR ( 511 bp ) was diluted in nuclease free H2O ( 1:100 v/v ) and was subjected to nested-PCR for sensing of serotype/s. The PCR merchandises were analyzed in agarose gel ( 2 % ) and extracted from the gel and sequenced straight utilizing Big Dye Terminator Cycle Sequencing kit ( Applied Biosystems, USA ) . The resulting sequences were analyzed for their homologies utilizing Basic Local Alignment Search Tool ( BLAST ) 12.
Of the 18 IgM positives, seven were females. The thrombocyte counts were available for 14 patients merely ( run 50 A- 103 to 150 A- 103/I?l ) , and none required transfusion. Seven patients had rash and/or petechiae either on arm/limbs/chest, three had mild bleeding ; nevertheless, no DSS instance was noted. Seven patients had hepatomegaly. All patients recovered within 7-12 yearss of oncoming and were discharged.
In all, 47 samples received from rural countries bordering Jabalpur. Out of these 10 samples were found IgM positive whereas out of 28 samples received from Jabalpur metropolis, 7 were IgM positive. One IgM positive sample was from bordering Narsinghpur territory.
The mosquito aggregation done in the country demonstrated presence of the vector mosquito Ae. aegypti. The H2O storage containers such as cement armored combat vehicles, clay pots and metal membranophones were chief beginning of vector genteelness sites. Breteau index ( 26.8 ) container index ( 9.0 ) house index ( 25.5 ) were high.
ELISA and nRT-PCR: Of the 89 screened samples, 18 ( 20.22 % ) were positive for DEN IgM and 7 were positive for IgG, of which 4 were positive for IgM every bit good ( Table ) .
Twenty four samples subjected to nRT-PCR showed RNP elaboration, and one sample showed presence of DENV, in which the coveted merchandise of 511 bp was observed. On nested PCR for designation of serotype, the DENV 4 was identified ( Fig. ) . Sequencing and BLAST analyses confirmed the individuality of DENV 4 ( Gen bank accession no. JF929180 ) . When analyzed farther, utilizing blast tree/distance tree ( Neighbor Joining method ) 13, this virus showed 99 per cent base homology and 100 per cent protein homology with the virus isolated from Andhra Pradesh, India in 2007 ( ND 110 ) belonging to clade C of South East Asian genotype I14,15.
Dengue virus infection is known to be endemic in India3. It is now good established that different
genotypes belonging to four DENV serotypes are go arounding, either singly or in combinations2,3,16. These serotypes and genotypes are known to replace each other17,18. Rodrigues et al19 established the aetiology of eruption of feverish unwellness of 1966 as DENV at Jabalpur, Madhya Pradesh. During the same period, Sehgal and colleagues18 reported entomological groundss for the presence of DENV vectors20. Although a few other surveies have demonstrated presence of DENV vectors21 and serological groundss of dandy fever infection22,23, yet for a long period dandy fever remained a ignored disease in the cardinal state of India and virus sensing or serotyping was non done.
Of the samples tested, 20.2 per cent yielded IgM positiveness, which is notable, since the samples were non collected during any epidemic or outbreak state of affairs. Further, seven samples were positive for dandy fever IgG, of which 4 were positive for IgM bespeaking secondary infection24 and regular circulation of dandy fever virus in the country. The survey although based on little sample size, reconfirms the presence of DENV activity in and around Jabalpur metropolis after a long letup.
In this survey, we observed that patients from both urban and rural scenes were admitted with intuition of dengue infection. Out of 89 samples, approximately 50 per cent and out of 18 DEN IgM positive samples 10 ( 55 % ) were from rural scenes clearly bespeaking constitution of DEN in the rural country neighbouring Jabalpur. The serological surveies on the samples collected by door-to-door studies in 2006-2007 besides have demonstrated high ( 37 % ) IgM positivity23. The entomological studies conducted in the wards holding DENV activity in the metropolis and in the rural countries have demonstrated that, the H2O storage containers were the chief beginnings of vector genteelness sites.
Detection of DENV 4 in this survey corroborates active DENV circulation in this country. The DENV 2 activity has been reported from cardinal India earlier25. It was noted that in Delhi DENV 2 and 3 are replaced by DENV 117. The outgrowth of DENV 4 has been reported from Andhra Pradesh, south India also14, during the eruption of 2007, late Cecilia and others15 besides reported sensing of DENV 4 from Maharashtra, western India, nevertheless, this is possibly the first study of sensing of DENV 4 from cardinal India. South East Asiatic genotype I of DENV 4 is known to be go arounding in southern and western India, and the DENV 4 grouped in this bunch are extremely diverse15. The clinical manifestations of the DENV 4 from Jabalpur were non every bit terrible as seen at Pune15, nevertheless, it will be worthwhile to farther qualify this DENV 4 virus soon go arounding in the cardinal India so as to understand the epidemiology of the disease and apportion it to a peculiar clade.
One of the purposes of this survey was to give IgM based diagnosing of dandy fever infection, hence samples were sooner collected after 7th twenty-four hours of oncoming of unwellness. That could be a ground for acquiring merely one RT-PCR positive as it is good known that virus can be detected up to 5th twenty-four hours of illness9.
Presence of vectors and mosquitogenic conditions in the country are dismaying and name for strict
BARDE et Al: DENGUE 4 IN CENTRAL INDIA
Table. Age distribution of the suspected and confirmed dandy fever instances
Age group ( year )
IgM positive ( % )
Merely IgG positive ( % )
& gt ; 0-5
11 ( 23 )
1 ( 2 )
5 ( 17 )
1 ( 3 )
2 ( 16 )
1 ( 8 )
16 and above
0 ( 0 )
0 ( 00 )
18 ( 20.22 )
3 ( 3.4 )