V olume 40, December 2018 Dengue Bulletin
Dengue Bulletin Volume 40, December 2018
I S S N 0 2 5 0 - 8 3 6 2
W H O
The WHO Regional Office for South-East Asia publish the annual Dengue Bulletin.
The objective of the Bulletin is to disseminate updated information on the current status of dengue fever/dengue haemorrhagic fever infection, changing epidemiological patterns, new attempted control strategies, clinical management, information about circulating DENV strains and all other related aspects. The Bulletin also accepts review articles, short notes, book reviews and letters to the editor on DF/DHF-related subjects. To provide information for research workers and programme managers, proceedings of national/international meetings are also published.
All manuscripts received for publication are subjected to in-house review by professional experts and are peer-reviewed by international experts in the respective disciplines.
2018
There is about a 30-fold increase in dengue incidence over the past 50 years and it is now regarded as one of the most important arboviral infections in the world. There are many reasons for this increase, including faster spread of the virus through global travel, spread of the vectors to new geographical locations, rapid urbanization, global warming and climate change. About 52%
of the population in the WHO South-East Asia Region is estimated to be at risk for dengue with 10 out of the 11 Member States (with the exception of the Democratic People's Republic of Korea) being endemic. All four serotypes of the virus are circulating in the Region. The Region has seen larger outbreaks of dengue in the past two years, with Sri Lanka experiencing the largest outbreak ever recorded in 2017. Many other countries, including India, Indonesia and Myanmar, have also seen focal outbreaks of increasing magnitude. All these resulted in researchers engaging in the clinical features, management, vector biology and control of dengue. In line with this priority, the Dengue Bulletin is published every year, encouraging researchers to explore different aspects of the disease and contribute to the knowledge gap and evidence base for combating the rapid spread of this deadly disease.
The 40th volume of Dengue Bulletin is in your hands. It consists of papers on a new GIS surveillance tool, clinical management, vector behaviour, and the role of knowledge, attitudes and practices in dengue control.
We now invite contributions for volume 41. The deadline for the receipt of the manuscripts is 31 October 2019. Contributors are requested to kindly follow the instructions given at the end of the Bulletin during the preparation of their manuscripts. Contributions should either be accompanied by flash drives and sent to the Editor, Dengue Bulletin, WHO Regional Office for South-East Asia, Red Fort Capital Parsvnath Tower 1, Bhai Vir Singh Marg, Gole Market Sector 4, New Delhi 110 001 India, or by email as a file attachment to the Editor at [email protected]. Readers who want copies of the Dengue Bulletin may write to the same address or the WHO Country Representative in their country of residence. The pdf version will be available on the WHO Regional Office website.
Dr Ahmed Jamsheed Mohamed Regional Adviser
Neglected Tropical Diseases Control and Editor, Dengue Bulletin
World Health Organization Regional Office for South-East Asia New Delhi, India
Dengue
Bulletin
Volume 40, December 2018ISSN 0250-8362
© World Health Organization 2019
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Acknowledgements ... iii 1. Risk factors of mortality among children with dengue in a
tertiary care centre, Thiruvananthapuram, Kerala ...1
Gayatri Lekshmy Kumar, Zinia T Nujum, Lalitha Kailas, Vijayakumar K
2. Utility of CDC DENV 1–4 real-time RT-PCR assay for the
diagnosis of dengue ...15
Mohan K. Shukla, Pradip V. Barde, Neeru Singh
3. Detection and serotyping of dengue virus in a tertiary care centre
in Thrissur, Kerala: a cross-sectional study ...19
Heera Hassan, Reena John, Prithi Nair, MA Andrews
4. Scenario of dengue and chikungunya in Pune district, Maharashtra,
India during 2016: a retrospective study at an apex referral laboratory ...33
Alagarasu K, Jadhav SM, Bachal RV, Bote M, Kakade MB, Ashwini M, Singh A, Parashar D
5. Circulation of dengue serotypes in four provinces of northern Thailand
during 2013–2017 ...44
Punnarai Veeraseatakul, Jarurin Waneesorn, Kongphob Thilaogam, Yuddhakarn Yananto, Kotchakorn Intamul, Somkhid Thichak
6. Molecular identification of dengue virus and erythrovirus B19 in
three towns of the State of Amazonas, Brazil during 2013–2018 ...53
Regina Maria Pinto de Figueiredo, Thiago Serrão Pinto, Kelry Mazurega de Oliveira Dinelly, Wellyngton do Nascimento Lopes, Luzia de Souza Granjeiro, Carlene Barroso Caripuna, Naylê de Oliveira Alves Mendes, Maria Itelvina Rodrigues de Souza,
Anete Jane Cavalcante da Silva, Valcinei Silva Amorim, Victor Costa de Souza, Valdinete Alves do Nascimento, Felipe Gomes Naveca
7. Potential breeding sites of Aedes aegypti in Maldives ...63
B. N. Nagpal, K. Vikram, S. K. Gupta, Sana Saleem, Nishan, Sushil Pant, Arvind Mathur, Ahmed Jamsheed Mohamed
Mrigenedra P. Singh, Sunil K. Chand, Arvind Jaiswal, Ramesh C. Dhiman
9. Biology and bionomical aspects of invasive Aedes albopictus
circulating in Odisha ...83
Animesha Rath, Rupenangshu K Hazra
10. Association between entomological indices, breeding of Aedes mosquitoes and container types in Delhi for the prevention and control of dengue ...100
Babita Bisht, RoopKumari, Himmat Singh, BN Nagpal, AK Bansal, NR Tuli
11. Neem-and karanj oil-based nanoemulsion for control of larval stages
of the dengue vector Aedes aegypti...114
Nisha Sogan, Smriti Kala, Neera Kapoor, BN Nagpal
12. Study of dengue vector breeding habitats and entomological indices in
Nagapattinam district, Tamil Nadu, India ...124
Rajalakshmi Anbalagan, Arpita Shukla, Kaviyarasan, Jayalakshmi Krishnan
13. Knowledge, attitude and practices for prevention and control of dengue fever among community members in
North Delhi Municipal Corporation ...137
Babita Bisht, Roop Kumari, BN Nagpal, Himmat Singh,Kumar Vikram, Sanjay Sinha, NR Tuli
14. A geostatistical study to prioritize dengue-affected areas for implementation of effective control by municipal corporations
of Delhi, India ...153
Sanjeev Kumar Gupta, Poonam Saroha, Kumar Vikram, NR Tuli, Himmat Singh, Rekha Saxena, Aruna Srivastava, BN Nagpal, MC Joshi
15. Reading levels of selected USA Federal Government dengue webpages ...164
Jeffrey L. Lennon, Christopher M. Seitz
16. Unusual complications of dengue fever ...168
Rajeev Upreti, Monica Mahajan, Ram Shankar Mishra
17. Instructions for contributors ...173
The Editor, Dengue Bulletin, World Health Organization (WHO) Regional Office for South-East Asia, gratefully thanks the following for peer-reviewing the manuscripts submitted for publication.
1. Barde, Pradip Scientist ‘E’
National Institute of Research in Tribal Health Jabalpur, India
2. Biswas, Ashutosh
Professor, Department of Medicine All India Institute of Medical Sciences New Delhi, India
3. Chand, S.K.
Research Scientist
National Institute of Malaria Jabalpur, India
4. Das, Aparup Director
National Institute of Research in Tribal Health, Jabalpur
5. Dash, A.P.
Vice Chancellor
Central University of Tamil Nadu Thiruvarur, India
6. Hazra, R.K.
Scientist ‘E’
Regional Medical Research Centre Bhubaneswar, Odisa
7. Joshi, P.L.
Retd. Former Director
National Vector Borne Disease Control Programme
Sham Nath Marg Delhi-110054, India 8. Kumar Vikram
Technical officer
National Institute of Malaria Research Center Dwarka Sec 8, New Delhi, India
9. P.S. Indu
Professor and Head
Department of Community Medicine Thiruvananthapuram, Kerala
10. Ranjit, Manoranjan Scientist ‘F’
Regional Medical Research Centre Bhubaneswar, Odisha
11. Savargaonkar, Deepali Scientist ‘D’
National Institute of Malaria Research Center Dwarka Sec 8, New Delhi, India
12. Sharma, R.S.
Retd. Additional Director
National Centre for Disease Control New Delhi, India
13. Shrivastav, Aruna Retd. Scientist ‘F’
National Institute of Malaria Research Center Dwarka Sec 8, New Delhi, India
14. Singh, Himmat Scientist ‘D’
National Institute of Malaria Research Center Dwarka Sec 8, New Delhi, India
15. Srivastava, P.K.
Retd. Additional Director
National Vector Borne Disease Control Programme
Delhi, India 16. Tuli, N.R.
DHO South Delhi Municipal Corporation New Delhi, India
The quality and scientific standing of the Dengue Bulletin is largely due to the conscious efforts of the experts and also to the positive response of contributors to comments and suggestions.
The manuscripts were reviewed by Dr B.N. Nagpal and Dr Mohammad A. Jamsheed, with respect to format; content; conclusions drawn, including review of tabular and illustrative materials for clear, concise and focused presentation; and bibliographic references.
a tertiary care centre, Thiruvananthapuram, Kerala
Gayatri Lekshmy Kumar,# Zinia T Nujum, Lalitha Kailas, Vijayakumar K Government Medical College, Thiruvananthapuram
Abstract Background
The state of Kerala is hyperendemic for dengue. Infants and children are more likely than adults to develop severe clinical disease due to dengue. Few studies have been done to elucidate the risk factors for mortality among dengue-affected children in the state. We undertook this study to identify the risk factors for mortality in dengue-affected children admitted to Sri Avittom Thirunal (SAT) Hospital, Government Medical College, Thiruvananthapuram.
Methods
We conducted a retrospective study based on the case records of patients admitted with dengue fever to the paediatric wards of SAT Hospital during the period 2003–2013. It was an unmatched case–control design. The cases were children admitted with dengue fever who had died. Controls were children admitted with dengue who had been discharged from the hospital after recovering from their illness. A total of 661 cases of dengue fever were admitted in the years2003–2013 in the paediatric wards of SAT Hospital. Among those admitted, we studied 17 cases and 39 controls.
Results
Among children with dengue who had died, a higher percentage were boys. The mean age of cases was higher (7.77years) than that of controls (4.2[SD 3.7]years). The mean serum creatinine level in cases (0.8 [SD 0.3]mg/dL)was significantly higher than in controls (0.6 [SD 0.3]) mg/dL). The factors associated with mortality in dengue were the presence of comorbidities, being a referred case, history of drug intake and previous history of dengue. Following multivariable analysis, the factors independently associated with dengue mortality among children were the presence of comorbidity (75.9 [5.68−1014]), anorexia (20.81 [1.1−392.6]), altered sensorium(59.6 [4.4−808]) and raised serum creatinine(0.09 [0.01−0.79]).
Conclusion
Children with comorbidity, anorexia, altered sensorium and raised creatinine levels are at a higher risk of mortality from dengue. Such children should be prioritized for care in order to save their lives. More area-specific research is required in this direction.
Keywords: Comorbidity; creatinine levels; dengue mortality; paediatric; Thiruvananthapuram.
#E-mail: [email protected]
Introduction
Dengue is an emerging tropical viral disease that causes widespread morbidity and mortality among affected individuals in high-risk areas. The majority of dengue viral infections are self-limiting, but complications result in high morbidity and mortality. Today, severe dengue has become a leading cause of hospitalization and death among children in high-risk areas.
The diagnosis of dengue viral infection is essentially clinical, although confirmation requires laboratory tests, including serology, non-structural protein 1 (NS1) antigen detection, polymerase chain reaction (PCR) and viral cultures. There are no specific anti dengue drugs and treatment is basically supportive; it consists of early recognition of complications and appropriate fluid therapy.
Burden of dengue
Globally, there were an average of 9221 deaths due to dengue per year between 1990 and 2013. Considering fatal and non-fatal outcomes together, dengue was responsible for 1.14 million (0.73–1.98 million) disability-adjusted life years in 20131.
The WHO South-East Asia Region contributes to 52% of dengue cases annually. India is one of the seven identified countries in this Region that regularly reports outbreaks of dengue fever/ dengue haemorrhagic fever (DF/DHF)2. Based on data from the National Vector Borne Disease Control Programme (NVBDCP), the number of cases reported in 2013 was 74 454 for dengue with 167 deaths. Paediatric cases of DHF have a high mortality. In 2015, Delhi recorded its worst outbreak since 2006 with over 15 000 dengue cases reported2. In 2013, Kerala had the largest number of cases (7938) and 29 deaths3. Kerala is now hyperendemic for dengue, with the presence of all four serotypes, high rates of co infection and local genomic evolution of viral strains4.
Infants and children 4–6 years of age are significantly more likely than adults to develop DHF/dengue shock syndrome (DSS) or manifestations of severe clinical illness. There have been isolated reports of mortality in paediatric DHF/DSS cases, which show that mortality is much higher in these cases than the cumulative mortality reported by the NVBDCP.
Secondary prevention to reduce mortality through improved clinical case management has substantially lowered the mortality rate for severe dengue over the past two decades from 10–20% to <1%5,6. The first objective of the WHO Global Strategy 2012–2020 is to reduce the mortality due to dengue by 50% from 2010 levels7. The number of deaths is determined not only by factors that facilitate transmission but also by those that influence the severity of the disease and the ease of access to health care8. Organ involvement, shock, bacteraemia, comorbidities, haemorrhage and certain biochemical parameters have been identified as useful predictors of mortality in studies conducted in dengue-endemic countries, but there may be regional differences.
Few studies have been done to elucidate the risk factors for morbidity and mortality among dengue-affected children in Kerala. Hence, we undertook this study to identify the factors associated with mortality due to dengue among children admitted to SAT Hospital, Government Medical College, Thiruvananthapuram.
Methods
We conducted a retrospective, unmatched case–control study based on the case records of patients admitted with dengue fever to the paediatric wards of Government Medical College, Thiruvananthapuram from 2003 to 2013. Data collection from case records spanned a duration of 4 months, starting in July 2013. We included all completed laboratory-confirmed case records from 2003 to 2013. The inpatient departments, intensive care units (ICUs) and records library of the Department of Paediatrics formed the study setting. Cases were children admitted with dengue fever during 2003–2013 who had died. Controls were children admitted with dengue who had improved and had been discharged from hospital after any duration. A total of 661 cases of dengue fever were admitted from 2003 to 2013.
We identified 17 children who had died as cases and randomly included 39 children with dengue who had been admitted and recovered as controls.
Study variables
The sociodemographic variables studied were age, residence and sex of the patients. The weight of children in the control group was taken from the case records.
Disease related variables
The disease-related variables studied were duration of fever, presence of any comorbidities,* referral status, history of any drug intake and previous history of dengue. The symptoms and signs of dengue were fever, conjunctival congestion, retro-orbital pain, frontal headache, myalgia, backache, arthralgia, mucosal bleeding, lethargy, bleeding manifestations, type of bleed, rash, flushed face, anorexia, nausea, abdominal pain, restlessness, vomiting, cough, breathlessness, diarrhoea, periorbital oedema, pedal oedema, constipation, seizures and altered consciousness.
* Comorbidities obxserved were febrile seizures, diabetes insipidus, obesity, asthma, ventricular septal defect, hand, foot and mouth disease, protein–energy malnutrition and recurrent epistaxis.
Investigation-related variables
We diagnosed dengue using anti dengue IgM, NS1 antigen and anti-dengue IgG antibody.
We also did routine blood examination, platelet count, liver function tests and renal function tests. Other investigations included random blood sugar (RBS), reverse transcription polymerase chain reaction(RT-PCR), prothrombin time and international normalized ratio (PT/INR), activated partial thromboplastin time(aPTT), packed cell volume/haematocrit(PCV/
HCT) (and rise in PCV). Serum electrolytes tested were calcium, sodium, bicarbonate, potassium and creatine phosphokinase-muscle/brain (CPK-MB). We also estimated total protein and albumin, and did a routine urine examination. Other investigations were ECG (electrocardiogram),Widal and rapid malaria tests, as the case demanded.
Ethical clearance
We obtained clearance from the Institutional Ethics Committee. We sought permission from the heads of departments of the concerned institution and from the head of the records library. Confidentiality was maintained throughout the study.
Statistical data analysis
The clinical and laboratory data of cases and controls were entered in an Excel file and analyzed using the SPSS software. All qualitative variables were expressed as proportion and quantitative variables as means with standard deviations; the strength of association was studied using the odds ratio and confidence interval. The chi-square test and t-test were used to test for significance. After bivariable analysis, multivariable analysis was done using logistic regression to find the independent predictors of mortality.
Results
The mean time from the onset of symptoms to hospitalization among cases was 4.1 days (SD=1.67 days). The mean time from hospitalization to mortality was 3 (3.02) days. Most of the deaths occurred at night. Two of the children who had died had a history of dengue among relatives.
Comparison of baseline characteristics of cases and controls
Among the cases (n=17), 11 (64.7%) were boys and 6 (35.2%) were girls, whereas among the controls (n=39), 18 (46.1%) were boys and 21 (53.8%) were girls. The mean (SD) age
of children who had died due to dengue was higher (7.77 [3.88] years) compared to that of children who had recovered from dengue (4.2 [3.7] years). Most commonly affected age group was 2-3 years. Most of the deaths occurred among children in the age group of 5–10 years followed by those in the age group of 10–15 years (Figure 1). Dengue with warning signs was noted in 1 (5.9%) case and 17 (43.6%) controls. The mean weight of the children who had died due to dengue was higher (25.3 [12.1] kg) and fell into the unhealthy body mass index (BMI) range compared to the children who had recovered (12.7 [1.5] kg).
Figure 1: Age categories of cases and controls
11 3 2 6 5
24
2
9
3
0 5 10 15 20 25 30
0–1yrs 2–3 yrs 3–5 yrs 5–10 yrs 10–15 yrs
Number of children
Age of children cases controls
Comparison of clinical features of study participants
The mean duration of fever in cases was 4.7 (1.5) days and in controls it was 4.6 (2.1) days.
Arthralgia and mucosal bleeding were noted in 11.8% of those who had died. Symptoms such as lethargy, anorexia, restlessness, abdominal pain and breathlessness predominated in dengue-affected children who had died compared to those who had recovered. Bleeding manifestations, seizures and altered sensorium were also more frequent in children who had died than in those who had recovered (Table 1). Severe dengue was observed in 16 (94.1%) cases and 2 (5.1%) controls. The presence of comorbidities, anorexia and altered sensorium were found to be significant predictors of death in children with dengue.
Table 1: Baseline characteristics and clinical features associated with dengue mortality in children
Variables Cases
(n=17) Controls
(n=39) P value Odds ratio (OR;
95%CI ) Adjusted
OR(95%CI)
Boys 11 (64.7%) 18 (46.25%) 0.16 2.1(0.66–6.94) -
Clinical features
Arthralgia 2 (11.8%) 0 0.03 - -
Mucosal bleed 2 (11.8%) 0 0.03 - -
Lethargy 11 (64.7%) 4 (10.3%) 0.0001 16.0 (3.82−67.38) -
Bleeding
manifestation 12 (70.6%) 4 (10.3%) 0.0001 21.0 (4.8−91.2) -
Anorexia 7 (41.2%) 1 (2.6%) 0.0001 26.6 (2.9− 242.1) 20.81 (1.1−392.6)
Restlessness 5 (29.4%) 0 0.0001 - -
Abdominal pain 7 (41.2%) 6 (15.4%) 0.04 - -
Breathlessness 3 (17.6%) 1 (2.6%) 0.04 8.14 (1.0−84.93) -
Seizures 6 (35.3%) 3 (7.7%) 0.01 6.5 (1.4−30.58) -
Altered
sensorium 9 (52.9%) 1 (2.6%) 0.0001 42.7 (4.72−386.7) 59.6( 4.4−808) Comorbidity 8 (47.1%) 1 (2.6%) 0.0001 33.7 (3.7−305.5) 75.9 (5.68−1014) Drug intake 11 (64.7%) 4 (10.3%) 0.0001 16.3 (3.82−67.38) - Referred cases 13 (76.5%) 9 (23.1%) 0.0001 10.8 (2.8−41.61) -
The mean pulse rate (119.41 [25.9]/min) was higher in those children who had died compared to those who had recovered (103.8 [19.4]/min). Urine output was relatively lower (1.85 [2.45] mL/kg/h) among cases compared to controls (4.38[10.6] mL/kg/h)]. The complications seen among cases were disseminated intravascular coagulation (DIC) in 8 (47.1%), acute respiratory distress syndrome (ARDS) in 5 (29.4%), renal failure in 3 (17.6%) and liver failure in 9 (52.9%). Ventilator support was given for 16 cases and 1control. The mean time to intubation was 12.9 (19.9) h and the mean duration of intubation was 37.5 (55.2) h.
Comparison of the findings of study participants
NS1 antigen positivity was significantly more common in cases than in controls (Table 2).
Dengue IgG positivity was also more frequent in cases (7 [41.2%]) than in controls(11 [28.2%]).
Dengue IgM was positive in 8 (47.1%) cases and 24 (61.5%) controls. Haemoconcentration (haematocrit >20) was seen more often in cases than in controls with a mean haematocrit value of 24. More cases had significantly greater thrombocytopenia than controls, with the lowest platelet count being significantly low (P<0.04). Among the haematological
investigations carried out, the mean blood urea level was significantly higher in cases (33.2 [15.7] mg/dL) compared to controls (15.7 [10.8]mg/dL), as was the mean serum creatinine level (Table 2). The mean serum sodium level in cases (128.8 [36.1]mg/dL)was significantly higher than in controls (99.6 [59.4] mg/dL). Cases had a significantly higher mean haemoglobin (13 [2] mg%) than controls (11.4 [1.7] mg%). The comparison of PT, INR andaPTT is shown in Table 2. Mean aspartate aminotransferase (AST) among cases was 784.53 mg/dL (1736 mg/dL) and among controls 225.18 (581) mg/dL. Mean alanine aminotransferase (ALT) values of cases and controls was 210 mg/dL and 89.31 mg/dL, respectively.
Table 2: Investigation and treatment-related characteristics associated with dengue Investigations
done
N (%)
P value OR (95%CI) Adjusted OR (95%CI)
Cases Controls
NS1 antigen
positive 9 (52.9%) 4 (10.8%) 0.003 - 2.13
(1.29−3.5) Dengue IgG
positive 7 (41.2%) 11 (28.2%) 0.74 - -
Dengue IgM
positive 8 (47.1%) 24 (61.5%) 0.17 - -
Blood urea 33.2 (15.7) mg/dL 15.7 (10.8) mg/dL 0.007 - -
Serum
creatinine 0.8 (0.3) mg/dL 0.6 (0.3) mg/dL 0.03 - 0.09
(0.01−0.79) Serum
sodium 128.8 (36.1) mg/
dL 99.6 (59.4) mg/dL 0.03 - -
Haemoglobin 13 (2) mg% 11.4 (1.7) mg% 0.009 - -
Clot formation
time (CFT) 1.53 (0.5)s 2 s 0.002 - -
Treatment given Crystalloids used
0.5% normal
saline 4 (23.5) 0 0.002 0.25
(0.15−0.40) -
3%normal
saline 4 (23.5) 0 0.002 0.25
(0.156−0.40) -
Hydroxyethyl
starch (HES) 14 (82.4) 2 (5.6) 0.001 0.013
(0.002−0.084) - Fresh frozen
plasma (FFP) 8 (47.1) 0 0.001 0.200
(0.111−0.359) -
Platelets 6 (35.3) 0 0.001 0.234
(0.140−0.393) -
Investigations done
N (%)
P value OR (95%CI) Adjusted OR (95%CI)
Cases Controls
Packed RBCs 12 (70.6) 1 (2.6) 0.001 0.012
(0.001−0.112) -
Sympathomimetics
Dobutamine 5 (29.4%) 0 NA NA -
Dopamine 13 (76.5%) 0 NA NA -
Noradrenaline 10 (58.8%) 0 NA NA -
Adrenaline 16 (94.1%) 1 (2.6%) 0.001 608
(35.78−10330) -
Steroids 4 (23.5%) 1 (2.6%) 0.01 11.69
(1.19−114.3) -
Antibiotics 14 (82.4%) 3 (7.7%) 0.001 56
(10.07−311.28) -
Antivirals 2 (11.8%) 0 NA NA -
The presence of any comorbidity, referred cases, history of drug intake and previous history of dengue were factors that were significantly associated with dengue mortality in children. The significant symptoms and signs in cases that had died were arthralgia, mucosal bleeding, lethargy, bleeding manifestations, anorexia, restlessness ,abdominal pain, breathlessness and altered sensorium (Table 2).
Treatment characteristics of the study participants
The patients were treated with crystalloids and colloids, as their condition required. Four cases (23.5%) received normal saline (0.5% and 3%). Hydroxyethyl starch (HES), fresh frozen plasma (FFP), platelet concentrate and packed red blood cells (RBCs) were required significantly more often by cases than controls (Table 2). Children who had died due to dengue were also administered steroids and antiviral drugs during the course of their treatment (Table 2).
Determinants of dengue mortality
Following multivariable analysis, the factors independently associated with dengue mortality among children were the presence of any comorbidity (P=0.001), anorexia (P=0.04), altered sensorium (P=0.003) and raised serum creatinine (P=0.03).
Discussion
Few studies have examined the clinical features and outcomes of children affected by dengue in Kerala. We undertook an unmatched case–control study to identify the determinants of dengue mortality in children based on the case records of patients admitted with dengue fever in the paediatric wards of GMC hospital, Thiruvananthapuram from 2003 to 2013.
We included a total of 56 study subjects of which 17 were cases (children who had died) and 39 were controls (children who had recovered).
We found that more boys had died than girls (Figure 1), as in some other studies11–13. However, one study found that girls had a higher risk of mortality (OR 1.57, 95% CI 1.1–2.2)14, and in another, adult women constituted 90% of those who died due to dengue15. The observed predominance of women in these studies may have been due to their relatively robust immune response, making them more prone to developing a greater inflammatory response or higher susceptibility to capillary permeability (15,16). This disparity in findings points to the need for more studies to elucidate an association between gender and dengue.
The mean age of the cases was 7.77years (SD=3.88). Though the most common age group affected by dengue was 2–3 years, mortality was highest in the age group of 5–10 years.
Another study in Mumbai reported three deaths in 38 DHF/DSS cases with a mean age of 4.9 years17. In a study done in a reference hospital in north-east Brazil to study dengue infection in children and adolescents, the majority of children with dengue who required hospitalization were in the 10–15 years’ age group18.
We diagnosed dengue using anti dengue IgM, NS1 antigen and anti-dengue IgG antibody.
Performing concurrent assays for dengue virus NS1, anti-dengue IgM and anti-dengue IgG along with platelet enumeration in the “dengue package” is immensely beneficial for patients, clinicians and public health officials19. NS1 antigen was positive significantly more often in cases (9 [52.9]%) than controls. The NS1 antigen circulates uniformly in all serotypes of the dengue virus at high levels during the first few days of illness20. Hence, it helps in early detection of the illness, especially within 4 days of onset compared to IgM, which reaches adequate levels for detection only in 5–10 days21. Studies claim that in addition to early diagnosis, the NS1 antigen may also be an indicator of disease severity22. Secondary dengue and severe dengue were more common among them compared to controls, as evidenced by the variation in positivity of dengue IgM and dengue IgG.
The presence of any comorbidity is usually associated with higher rates of mortality in dengue. The comorbidities observed in our study were febrile seizures, diabetes insipidus, obesity, asthma, ventricular septal defect (VSD), hand, foot and mouth disease (HFMD), protein–energy malnutrition (PEM) and recurrent epistaxis. Other studies have also found dengue patients with comorbidities to have a higher incidence of mortality22–25.
Common reasons for referral among patients were decreased platelet count, poor general condition and seizures. Most patients usually seek primary medical care in private hospitals and are referred to government hospitals only when their condition worsens. This time lag results in loss of treatment opportunity, which is critical to their life.
The bleeding manifestations observed were haematemesis, melaena, bleeding from the gums, haematochezia and haematuria. Melaena was the most common symptom.
Haemorrhagic complications such as epistaxis, gingival bleeding, gastrointestinal bleeding, haematuria and hypermenorrhoea, although rare, are important causes of death in dengue25.
Significant clinical signs associated with dengue mortality in children were neurological manifestations (lethargy, anorexia, restlessness, seizures and altered sensorium), respiratory manifestations (breathlessness), joint manifestations (arthralgia) and gastrointestinal manifestations (abdominal pain). Abdominal pain is one of the warning signs of dengue, according to the WHO 2009 case definition26. In another study, the clinical manifestations that were most significantly associated with dengue fever were myalgia, headache, skin rash and anorexia27.
In our study, complications noted in cases of mortality were ARDS, DIC, liver failure, renal failure and cardiac complications. These were similar to the findings of another study done in south India, in which severe refractory shock, DIC, ARDS, hepatic failure and neurological manifestations, singly or in combination, were the most common causes of death28.
In our study, blood urea and serum creatinine levels were higher in cases compared to controls. These abnormal renal function tests could be attributed to the failing renal function in severe dengue. Serum sodium level was also higher in cases than in controls. This observation was contrary to the findings of another study done in north India, in which out of 5 patients with significant hyponatraemia, 3 had a poor outcome29. The wide variation in the standard deviations of AST and ALT was due to extremes of values such as the high value of AST (7161mg/dL) among cases.
PT, INR and aPTT were raised in cases compared to controls. This finding points to activation of the coagulation system. In one study, PT and aPTT were significantly prolonged and fibrinogen levels significantly lower in patients with DSS as compared to patients with non-shock DHF30. The mean pulse rate of cases was higher than that of controls. The mean capillary filling time, respiratory rate and the urine output were lower in cases than in controls. The mean pulse pressure at the time of admission was significantly higher in cases (37.1 [18]mmHg) than controls (22.15 [10]mmHg). This could signify the stage prior to haemoconcentration following plasma leakage since a progressive decline was noted in the levels of pulse pressure following admission.
The mean haemoglobin was significantly higher in cases than in controls. This was contradictory to the findings of a study in which a haemoglobin value of less than 9 mg/
dL was associated with a fourfold higher risk of mortality and/or severity31. This could be
attributed to the small sample size of the study. Haemoconcentration and thrombocytopenia were greater in cases compared with controls in our study. The main pathogenic feature of dengue is an increase in vascular permeability leading to loss of plasma from the blood vessels, which causes haemoconcentration, low blood pressure and shock32. This may also be accompanied by haemostatic abnormalities such as thrombocytopenia, vascular changes and coagulopathy32.
Most of the deaths occurred at night. This could be due to the disproportionately less emergency care received at night compared to that during the day.
In our study, the mean time of onset of disease to hospitalization was 4.1 days (SD=1.67 days), and from hospitalization to mortality it was 3 days (SD= 3.02 days). Other studies found that the time from onset of fever to hospital admission ranged from a mean of 2.9 days to 4.7 days33–35. The median time from admission to a positive test result was 2.5 days and the median time from the onset of illness to death was 12 days15. The duration of stay of dengue patients from hospitalization to death can be considered as the window of opportunity available for the health professional to prevent death.
In a hyperendemic state such as Kerala, where prevalence among vulnerable groups such as children is high, control measures should be aimed at the key determinants of dengue mortality. Though the aggregated data were analyzed, the unique factors that could have played a role in the pathogenesis of deaths due to dengue could not be studied as it was beyond the scope of this study. Further, the study was retrospective and based on records.
Since the sample size for the study was small, some results had wide confidence limits.
Conclusion and recommendations
The presence of any comorbidity (pre-existing disease) can augment the severity of dengue fever and hence should be addressed with equal importance in affected patients to prevent mortality. Raised creatinine levels can be a valuable biomarker of the risk of mortality. Deaths are commonly seen at night, and strengthening emergency health facilities could avert such deaths. More area-specific research in larger samples is required for this. Such studies also need to be replicated in other dengue-endemic areas for better decision-making and prioritization to prevent childhood mortality due to dengue.
Acknowledgements
We thank the house surgeons of the 2008 batch of Government Medical College, Thiruvananthapuram for their valuable help in data collection for the study in 2013. Above all, we thank and pray for those patients whose information was shared with us as a part of the study.
Funding sources
We did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Conflicts of interest None.
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for the diagnosis of dengue
Mohan K. Shukla, Pradip V. Barde,# Neeru Singh
National Institute of Research in Tribal Health (NIRTH), Jabalpur, Madhya Pradesh, India
Abstract
Prompt diagnosis is essential for patient care, and early outbreak warnings are important for activating epidemic control systems. The Centers for Disease Control and Prevention (CDC) has developed the DENV 1–4 real-time reverse transcription polymerase chain reaction (RT-PCR) multiplex assay (qRT-PCR) for the detection of dengue virus (DENV). We compared this assay with conventional RT-PCRs (cRT-PCR) and found the CDC real-time RT-PCR kit to be better than conventional RT-PCRs in meso-endemic and epidemic areas. The CDC DENV 1–4 real-time RT- PCR multiplex assay can be performed in lesser time (223 [+8.4] min) than conventional RT-PCRs (499.5 [+7.7] min). Serotype identification is important for surveillance and the CDC kit detects the serotype simultaneously with DENV. Thus, this test would be a useful tool for both DEN surveillance and serotype monitoring.
Keywords: Dengue; real-time RT-PCR; conventional RT-PCR.
Introduction
Dengue (DEN) is a re-emerging infection. An estimated 390 million cases are reported globally every year1. Dengue virus (DENV) has four serotypes – DENV 1–4, which are classified into several genotypes that differ by 5–6% at the nucleotide level2. All four serotypes are capable of causing an array of symptoms, ranging from mild disease (classical dengue fever) to severe disease (haemorrhagic fever/shock syndrome)3. DENV viraemia is relatively high at the onset of symptoms, which makes reverse transcription polymerase chain reaction (RT-PCR) a reliable test for the early diagnosis of dengue. In addition, RT-PCR may allow serotype identification, which has implications for surveillance. Detection of IgM antibody and NS1 DENV soluble antigen by enzyme-linked immunosorbent assay (ELISA) in serum samples has the limitations of delayed diagnosis (fifth day after onset) and inability to identify the serotype. Conventional RT-PCRs (cRT-PCR) developed for the detection of DENV-specific nucleic acid in the acute phase can detect the serotype, although with limitations on the number of samples that can be handled owing to problems such as cross-contamination,
#E-mail: [email protected]
multiple reactions and longer time (6–8 hours) for serotyping. The US Centers for Disease Control and Prevention (CDC) has developed the DENV 1–4 real-time RT-PCR multiplex assay (qRT-PCR) to overcome these hurdles, as it can diagnose DENV and its serotype4. We tested this assay for DEN diagnosis in Madhya Pradesh (MP), India and compared its utility for early diagnosis with cRT-PCR.
Methods
Serum samples collected from patients suspected to have DEN from different districts of MP and Chhattisgarh were referred to the Virology Laboratory of the ICMR-National Institute of Research in Tribal Health (NIRTH), Jabalpur, for serological and molecular diagnosis of DEN5. One hundred and two serum samples collected during the acute phase of illness, received between April and November 2014 from various districts of MP, were subjected to molecular tests by cRT-PCR as described by Lanciotii et al. with a few modifications, and qRT-PCR was done as per the manufacturer’s instructions4,6. The samples collected during outbreak(s) within the study period were also included in the study7. Two trained technicians performed both the tests following standard operating procedures; the tests were monitored and recorded using a stopwatch.
RNA samples used for both the tests were extracted using the viral RNA extraction kit (Qiagen, Germany Cat. No. 52906) following the manufacturer’s protocol. For cRT-PCR, SuperScript® III (Cat. No. 12574-026, Invitrogen, USA) and for qRT-PCR, SuperScript® III system (Cat. No. 11732-020, Invitrogen, USA) were used.
The timings were noted for all the steps. The cRT-PCR was performed in steps, i.e.
reaction set-up for one-step RT-PCR, run time in thermal cycler (ABI GeneAmp® PCR System 9700), electrophoresis, nested PCR, run time in thermal cycler, and electrophoresis and gel analysis. On the other hand, the steps performed in qRT-PCR were qRT-PCR set-up, run time in thermal cycler (ABI 7500 PCR System) and data analysis. In general, at a time, four samples were subjected to cRT-PCR and 10–13 samples for qRT-PCR. The data were analysed using appropriate statistical tests.
The analysis of time taken for the test up to serotyping by both the technicians for qRT- PCR was 223 (+8.4) min compared to 499.5 (+7.7) min for cRT-PCR. This difference was statistically significant (P<0.005). Although setting up of the qRT-PCR reaction took 43.5 (+4.9) min compared to 23 (+1.4) min for cRT-PCR, it may be noted that the cRT-PCR (without serotyping) time (302 [+2.1 min]) was longer than the qRT-PCR time (with serotyping) (223 [+8.4 minutes]) (Figure 1).
All 94 DENV-positive cases (DENV 1 [n=8], DENV 2 [n=8] and DENV 3 [n=78]) were detected using these tests, which included three cases of coinfection of DENV-2 with DENV-1 and DENV-3. The results of serotyping by both the methods were similar.
Results and discussion
our findings show that both qRT-PCR and cRT-PCR can detect serotypes with equal efficiency, but the former has a distinct advantage due to its shorter detection time. Early diagnosis of DENV along with detection of the serotype is crucial as the infecting serotype is an important factor in determining severity8. Further, in an area where more than one serotype is circulating, there is a possibility of multiple serotype infection, which is more severe8. Thus, a test that can diagnose dengue along with the serotype in a relatively short time helps in better patient management. It also helps in curbing outbreaks by quick and reliable detection of etiology along with the cause of transmission.
We suggest that the qRT-PCR test be standardized for detection of viral RNA from mosquitoes caught from the field. This will help in incriminating the vector, which will allow health authorities to control specific vector(s).
The CDC assay has an internal control mechanism to detect human ribonucleoprotein complex (RNP); it is able to distinguish non-reactive samples from those with PCR inhibitors.
If the chances of contamination are minimized, this assay can give a reliable diagnosis4. Some limitations of this test include the inability to quantitatively detect DENV and the absence of chikungunya virus (CHIKV) detection primer probes in the panel. Despite these limitations, the CDC DENV 1–4 RT-PCR assay enables the diagnosis of DEN with DENV serotyping in a shorter time and permits handling of a larger number of samples, giving it an advantage over cRT-PCR. The simplicity in performing the test in an equipped laboratory makes the CDC DENV 1–4 RT-PCR assay beneficial for DEN surveillance and serotype monitoring.
Figure 1: Graph depicting the time taken for conducting two assays. The X-axis denotes the steps involved in the test and the Y-axis denotes the time in minutes. The standard
deviation in the operators is given in brackets.
Acknowledgments
The authors are thankful to the Secretary, Government of India, DHR, MoH&FW, and the Director General, ICMR for financial support under the Viral Diagnostic Network project (no. VIR/43/2011-ECD-1). The authors also thank CDC, Puerto Rico, USA for providing CDC DENV 1–4 real-time RT-PCR assay kits gratis, and Dr Jorge Munoz, CDC, Puerto Rico, USA for a critical review and suggestions on the manuscript. The authors thank Dr S Rajasubramaniam, Scientist “E”, NIRTH, Jabalpur for critical comments on the manuscript and assistance with language.
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care centre in Thrissur, Kerala: a cross-sectional study
Heera Hassan,a# Reena John,b Prithi Nair,b MA Andrewsc
aDepartment of Microbiology, Govt Medical College, Thiruvananthapuram
bDepartment of Microbiology, Govt Medical College, Thrissur
cDepartment of Medicine, Govt Medical College, Thrissur
Abstract
Dengue is endemic in India and exacts a high economic burden on both governments and individuals. The objective of this study was to detect dengue virus in clinically suspected fever cases as per WHO dengue case definition by reverse transcriptase polymerase chain reaction (RT-PCR), and to serotype the virus to find out the currently circulating serotypes of dengue virus in Thrissur district of Kerala. Additionally, a commercially available non-structural protein 1 (NS1) rapid card test was also evaluated. Serum samples from 102 probable dengue cases were collected during the pre-monsoon showers and subjected to NS1 rapid card test, RT-PCR and IgM enzyme-linked immunosorbent assay (ELISA). NS1 rapid card test was found to have a sensitivity of 63.38%
and a specificity of 83.87%. Samples positive by RT-PCR were further serotyped using the same method. Seventy-one samples were confirmed to have dengue infection; 40 were positive for dengue virus (DENV). All the four serotypes of DENV were found. DENV-2 was the most prevalent serotype. This is the first case report of DENV-4 infection from Kerala. Coinfection with DENV-1 and DENV-4 was detected in one patient. Co-circulation of all the DENV serotypes provides a favourable environment for micro-evolution of new serotypes. The study finding can help the National Vector Borne Disease Control Programme (NVBDCP) and the state health department of Kerala to prioritize efforts to control dengue by effective vector control tools.
Keywords: Dengue virus; RT-PCR; IgM ELISA; NS1antigen; coinfection.
Introduction
Member States of the WHO-South East Asia Region and Western Pacific Region bear nearly 75% of the global disease burden due to dengue. Of the 11 countries in the South-East Asia Region, 10 countries, including India, are endemic for dengue1. In 2012, the Region reported 0.29 million cases, out of which India contributed 20%. Circulation of several serotypes has also been reported from these countries1,2. Co-circulation of multiple dengue serotypes coupled with increased human activity increases the likelihood of genetic changes,
#E-mail: [email protected]
leading to diversity in virus populations. Genetic recombination, natural selection and genetic bottlenecks have been implicated as factors that may lead to the emergence of new serotypes, such as the dengue virus serotype 5 (DENV-5), which follows a sylvatic cycle and was isolated in October 2013. Discovery of newer sylvatic strains in future may impede the dengue vaccine initiative3. The role of host immune status in disease severity with respect to host immune response and host genetics requires more research at the molecular level. More epidemiological studies are required to study disease severity with respect to the infecting serotypes and genotypes, sequence of infecting serotypes during primary and secondary infections, and time interval between primary and secondary infections4. Repeated infections with multiple serotypes of the virus lead to complications and hence circulation of multiple serotypes is associated with an increased risk of complications of dengue in susceptible individuals. Estimation of the geographical distribution and corresponding burden of dengue contributes to its global burden in terms of morbidity and mortality, and provides an idea of how to control dengue with the limited resources that may be available and to evaluate the impact of such activities.
Currently, these data can also be used for reliably scoping vaccine demand and delivery strategies. All the four serotypes of DENV have been reported from India5. Cyclic dengue epidemics have been reported mostly from the central and southern districts of Kerala since 20016–9 and are on the increase. Most of the reported cases are patients admitted with complications and hence represent only the tip of the iceberg. We aimed to bridge the gap in the literature regarding the circulating DENV serotypes in Thrissur, Kerala. This area’s climate, vegetation and other favourable conditions (coconut shells used for latex collection in rubber plantations, cocoa pods) could account for the increased vector survival and multiplication. Local human movement10 allows virus spread to urban areas. As expected, most of the cases that would otherwise be treated as mere fever cases and dismissed as outpatients (due to lack of severity) proved to be dengue. The efficiency in detecting cases of a commercially available NS1 rapid card test that was being used in the hospital was also evaluated. The presence of 102 cases in a tertiary care centre just during the pre-monsoon showers indicates the possibility of an ongoing dengue endemic in the area in the form of a sylvatic cycle or an epidemic cycle with periodic remissions and exacerbations.
Materials and methods
We conducted a cross-sectional study for a period of 1 year. Sample collection commenced in the month of April 2015. Though the monsoon showers were expected in June, the area witnessed pre-monsoon showers in the first week of April 2015. The population selected for study were paediatric and adult patients who were clinically diagnosed as probable cases of dengue or who presented with complications of dengue as per the WHO 2009 case definitions or 1997 case definitions, but within 7 days of fever. Patients presenting with viral fever-like symptoms (probable dengue fever or classical dengue fever) to the Medicine and Paediatric OPD were treated as outpatients. Patients who presented with complications such as decreased platelet counts, hemorrhage or shock were admitted in the Medicine or Paediatric ICU and treated as inpatients. Patients with fever for more than 7 days, those
classified as having fever of unknown origin who did not satisfy the WHO dengue case definition, those with fever of known etiology and neonates less than 28 days of age were excluded from the study.
The sample size was calculated from a previous study8 using the formula 4pq/d2, where p was the proportion of cases that were positive by reverse transcriptase polymerase chain reaction (RT-PCR)(49.3%), q was 100–p and d was 20% of p. A minimum of 102 fever cases was studied. Blood samples of outpatients were collected in the blood collection centre and in person from patients admitted to the ICU. The samples were subjected to centrifugation and the separated serum was used for further tests. The tests used were (see colour plate 1):
NS1 rapid card test (CTK Biotech – CE OnSite; catalogue no: R0063C10 ), IgM capture enzyme-linked immunosorbent assay (ELISA) (National Institute of Virology kit) for dengue and RT-PCR. The first two tests were done in our institution and RT-PCR was done at the National Institute of Virology unit in the Alappuzha district of Kerala. The minimum sample size was completed in June 2015, in a matter of just 3 months.
Results
The sample size of 102 cases, intended to be reached over a period of 1year, was completed in a period of 3 months. This highlights the heavy incidence of dengue cases in the area.
Eleven patients were <15 years of age, 32 patients were in the age group of 16–30 years, 28 patients were in the age group of 31–45 years, 21 were in the age group of 46–60 years and 10 were above 60 years (Figure 1).
Figure 1: Age group-wise distribution of probable dengue cases
11
32
28
21
10
0 10 20 30 40
<15 years 16–30 years 31–45 years 46–60 years >60 years
Number of cases
Age group ranges
Of the 102 cases, 40(39%) were hospitalized due to complications, the rest were outpatients (Figure 2).
Figure 2: Outpatient and inpatient distribution of probable dengue cases
39%
61%
Inpatients=40 Outpatients-62
Table 1 shows that 42.2% of the total cases were outpatients without severe disease but diagnosed as dengue, in contrast to the usually reported severe dengue cases who get admitted and form only 27.5% of the total.
Table 1: The percentage of laboratory-confirmed cases among inpatients and statistically missed/uncounted outpatients who met the WHO clinical diagnostic criteria for dengue
Types of cases Dengue positive Dengue negative Total
Number % Number % Number %
Outpatients 43 42.2 19 18.6 62 60.8
Inpatients 28 27.5 12 11.8 40 39.2
Total 71 69.6 31 30.4 102 100.0
Out of the total 102 cases,32 cases presented within1–4 days of illness, of whom 18 were RT-PCR positive (56.25%). The remaining70 cases (68.6%) presented within 5–7 days of illness, out of whom 22 were RT-PCR positive (31.43%). A total of 40 cases were found to be positive for DENV with RT-PCR (Figure 3).