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Clinical Infection & Immunity

Original Article

Volume 6, Number 1, March 2021, pages 11-17

Pattern of Dengue Fever in Three Centers in Three Consecutive Years: An Evaluation of 2,098 Patients

Dengue virus (DENV) is a single-stranded ribonucleic acid (RNA) virus which was first isolated from Japan in 1942 by Hotta [1]. This flavivirus is transmitted to humans by infective female Aedes genus, mainly by Aedes aegypti, Aedes albopictus, Aedes polynesiensis and several species of the Aedes scutellaris complex [2]. Dengue has four antigenically distinct serotypes, DENV-1, 2, 3 and 4, which are evolved from a common ancestor [3, 4]. Three point nine seven billion people live at risk of counteracting the infection worldwide. Three hundred and ninety million infections and 96 million cases and 24,000 deaths are estimated to occur in 100 endemic countries every year. Treated dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) is associated with 1% mortality rate, whereas the mortality rate increases to 20% in untreated cases [5]. Report of dengue dates back to 1940s in India and at present it is one of the seven countries in Southeast Asia reporting regular incidence of dengue. All the four serotypes are documented in different states of India [6]. Incidence of dengue has increased 30-fold in the last five decades [7]. The disease is mostly found in tropical and subtropical regions. Aedes aegypti has been the most important epidemic vector in the tropical and subtropical regions. Secondary vectors like Aedes albopictus, Aedes polynesiensis members of Aedes scutellaris and Aedes niveus all play an important role in the spread of DENV [8]. Aedes albopictus is an important vector which can adapt to new environments including temperate regions, so the virus can be spread to Aedes aegypti free countries, opening newer horizons for the DENV [9]. But so far it is only a minor contributor.

A recent report highlighted the prevalence of DENV-2, DENV-3 and DENV-1 serotypes in a hospital-based study in Ernakulam city, Kerala, India [10]. A study by Dash et al (2005) showed that the DENV-3 has emerged as the major serotype in different parts of the country. There is no study before 2008 in Kerala, India to show the evolutionary trends of different serotypes in Kerala, India. The study by Anoop et al showed that all four serotypes are prevalent in the state and serotypes 2 and 3 are the major serotypes involved in the outbreaks. The large scale movement of people to and from Kerala for a range of activities from pilgrimage to employment might have contributed to the prevalence and spread of all the four dengue serotypes in the state. In Kerala, the Aedes aegypti is mainly restricted to urban settlements and coastal areas [10]. The rest of the area composed of semi-forested plantation areas where a variety of crops are cultivated. Aedes albopictus has been found abundant in this semi-forested plantation sector of Kerala [10]. When the population density of this vector species was at a peak level, dengue outbreaks occurred at Kerala [10]. Co-circulation of all the serotypes of DENV in a region as shown in Kerala State can be considered as an important contributing factor to the severity of dengue fever [10]. Aedes albopictus has wide spread prevalence in the state. Even though Aedes albopictus has less vector capacity than Aedes aegypti, the chance of DENV undergoing crucial mutations to overcome this disadvantage makes it much more alarming [10]. On account of nucleotide sequence variability, dengue serotypes may be further classified into distinct genotypes within a single serotype. The variation in severity of the disease during outbreaks has been attributed to emergence of such new genotypes [11].

Variability in presentations and manifestations of dengue viral infection is quite well known. Adaptability of the virus according to change in the vector may be responsible for this variation. Another possibility is the emergence of newer serotypes of the virus. The trend and pattern of dengue viral infection in three centers in two different districts of Kerala, India are studied from data available at these centers. The data analyzed 2,098 patients with dengue fever over three years in these three centers.

The total number of dengue cases in three institutions in Kerala, India, in 2017, 2018 and 2019 were analyzed using the identity (ID) number obtained from the infectious disease control register of these hospitals. The ID number was used to retrieve all the data from the hospital information system (HIS). The discharge summary, investigations, details, complications, mode of presentation and similar data were retrieved to chart the details of 2,098 patients from the three centers (center A: Baby Memorial Hospital, Calicut, Kerala, India; center B: Aster MIMS, Calicut, Kerala, India and center C: VSM Hospital Mavelikkara, Alappuzha, Kerala, India.). The first two centers (A & B) are at the same district head and the third center (C) is a much smaller center in another district. The first two centers are 1 km apart and the third center (C) is 311 km away from the first two centers. All these three centers belong to the same state, Kerala, in India. The tabulated data in tables are used to assess the trend of infection in these institutions over 3 years from 2017 to 2019 (Tables 1, 2). The data collected are of patients, who were treated in three institutions according to international standards. There was no procedure or treatment done for this study. The data were analyzed retrospectively. So there is no conflict with ethical committee or institutional review board. The data were analyzed with the permission of the head of the institute.

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Table 1. Observations and Results

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Table 2. Number of Cases in Each Month

The chart shows the maximum number of cases in in May, June, July and August, the monsoon season of all the 3 years in all the three centers. Among these, the number of cases was biggest during 2017 compared to 2018 and 2019. During May, June, July and August 2017, there were 804 cases from center A, 450 cases from center B and 372 cases from center C. Of the 2,098 cases reported in the three years in the three centers, 1,626 cases were reported in four months (May to August) in 2017. So, 77.5% of cases in the three years in the three centers happened during four months in 2017. The surge of infection looked episodic when comparing the rest of the data, which showed a handful of cases spanned across most of the months in 2018 and 2019 and the rest of 2017. No case reports were available at center C during 2018.

The age group at center A ranged from 1 to 90 years old. The age group at center B ranged from 1 to 93 years old.

At center A, 513 were males and 424 were females. At center B, 270 were males and 448 were females. At center C, 202 were males and 241 were females (Table 3).

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Table 3. Salient Clinical Features

An interesting finding is that at center A, total white cell count (WBC) increased in 41 patients. Thrombocytopenia occurred in 305 patients. Bicytopenia (WBC and platelet) occurred in 30 patients and pancytopenia in 1 patient, but red blood cell (RBC) count increased beyond 5.5 ×10⁶/mm³ in 138 patients at center A and 115 patients in center B, even though DHF and DSS were only in 16 patients. Hemoconcentration would increase all the cell counts, but there was isolated elevation of RBC count up to 5.6 - 7.5 × 10⁶/mm³ in 134 patients, 7.5 - 10 × 10⁶/mm³ in three patients and more than 10 × 10⁶/mm³ in one patient in center A. This isolated increase in RBC count has to be further investigated, whether it is secondary to capillary leak syndrome or not. At center B, the data showed elevated WBC count more than 10.6 × 10³/mm³ in 52 patients and decreased WBC count, less than 4 × 10³/mm³ in 371 patients (Tables 4, 5).

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Table 4. Center A - Chart Showing RBC Count

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Table 5. Center B - Chart Showing RBC Count

Platelet count became less than 20,000 /mm³ in 96 patients at center A, 21 - 50,000/mm³ in 185 patients and 51 - 100,000/mm³ in 273 patients. No patients had platelet count beyond 450,000. At center B, 225 patients had platelets less than 20,000/mm³, 160 patients had platelet count between 21 and 50,000/mm³, and 180 patients had platelet count between 51,000 and 100,000/mm³. Five patients at center B had platelet count more than 4.50,000/mm³. Seven hundred patients presented with fever, myalgia, headache and vomiting. Three hundred and five patients presented with fever and thrombocytopenia. Some patients presented with abdominal pain and loose stools. One patient presented with leaking polycythemia vera (PV) at 31 weeks gestation. She was found to have dengue fever. Fever, myalgia, headache and vomiting were the most common way of presentation.

All the patients were diagnosed mostly with nonstructural proteins 1 (NS1) antigen positivity, NS1 antigen and immunoglobulin M (IgM) antigen positivity. A very small number of patients was diagnosed with IgM positivity. The trend was infection peaked from May to August in 2017 compared to 2018 and 2019 in all the three centers.

Other systems involved in patients at center A were gastrointestinal (GIT) (193), genitourinary (16), cardiovascular system (CVS) (7), respiratory (68), no other system involvement (619), and multi-system involvement (0). Some patients presented with bleeding PV (17), menorrhagia (2), and sepsis (15).

Other systems involved at center B were genitourinary (5 - 2 renal failure), respiratory (lower respiratory tract infection - 1, acute respiratory distress syndrome - 1), glycemic index (GI) (1), no other system involvement (705) and multi-system involvement (2). Three patients presented with sepsis.

Neural predilection was seen in the form of encephalitis (14 patients) and seizure (14 patients) at center A only. Post dengue fever immunosuppression and cases related to that are not included in this study.

All these patients were treated symptomatically, along with appropriate medication depending on other system involvement. At center A, platelet transfusion was given to 64 patients, fresh frozen plasma (FFP) was given to 18 patients and combined FFP and platelet transfusion was given to 24 patients. At center B, platelet transfusion was given to (78) patients, combined FFP and platelet transfusion was given to two patients. FFP and platelet transfusion was given when the platelet was below 20,000/mm³ [1].

All the patients treated at center A and center C recovered completely. At center B, there were five deaths, one due to multi-organ dysfunction syndrome (MODS), septic shock and renal dysfunction. The second patient died of sepsis and intra-abdominal bleed. The third patient died of acute respiratory distress syndrome (ARDS), MODS and renal failure. The fourth patient died of hepatic failure and sepsis. The cause of death of the fifth patient was not available from the records.

Dengue viral genome constitutes a positive RNA, which is translated to a single polyprotein. This polyprotein encodes three structural proteins, capsid (C), pre-membrane (prM) and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4 A, NS4B and NS5). The pathogenesis of dengue viral infection is not well understood. There is no appropriate animal models of infection and disease. Non-human species do not exhibit more severe forms of dengue disease that mimics human dengue fever, dengue hemorrhagic fever and DSS. Non-human primates (e.g. rhesus macaques) are the only vertebrates apart from humans that get naturally infected with DENV. But the strains isolated from non-human primates are genetically distinct from those infecting humans [10]. Non-human primates develop only mild infection. They do not develop severe forms of dengue infections like DHF, nor they develop abnormalities in hematocrit or prothrombin time, but some animals displayed a mild decrease in platelet count. Mouse models also got infected with DENV, but do not exhibit disease similar to humans. This lack of proper animal model impaired further developments in the treatment and prevention (vaccine) of dengue viral infection in humans. Vector control remains the sole modality to contain the most important mosquito-borne viral disease which affects two thirds of the world population.

The life cycle of Aedes aegypti lasts for 8 - 10 days depending on the extent of feeding at room temperature, in two phases, aquatic and terrestrial.

The clinical manifestations of dengue fever vary from asymptomatic flu-like infection to severe dengue [12, 14]. The clinical manifestations depend on the immunity and nutritional status of the patients. The incubation period of dengue fever is on an average four days after the mosquito bite. It may manifest with fever and a discrete maculopapular or macular rash, making it indistinguishable from other viral fevers. Usually it is self-limited. In severe dengue fever, fever is high (39 °C or more) lasting for 5 - 6 days and is biphasic in nature (fever reaches normal in the middle of the febrile period), so the temperature chart looks like a saddle back. This may be associated with headache, arthralgia, myalgia, retro-orbital pain, severe backache, sore throat or abdominal pain. The fever may last up to 6 days, during which period the macular rash becomes erythematous leaving clear areas in between. Hepatomegaly and elevation in liver enzymes (aspartate aminotransferase in particular) and thrombocytopenia may also be present. Usually patients recover from dengue fever in 7 - 10 days. DHF is characterized by acute onset high fever, drowsiness, lethargy, increased vascular permeability and abnormal hemostasis leading to hypovolemia, hypotension and shock associated with severe internal bleeding [15]. Hemorrhagic manifestations occur usually by the third day with petechiae over the trunk, axillae and limbs. As a rule it bleeds at venipuncture sites, and hemorrhage from GI tract, nose and gums may follow. There can be pleural effusion and ascites associated with these symptoms. This phase can be resolved within 24 - 48 h with appropriate treatment. The clinical manifestations are similar to the standard way of presentation in this analysis.

Leakage of plasma into extracellular compartment can lead to DSS. Plasma leakage may be due to disseminated vasculitis and disseminated intravascular coagulation may ensue. Plasma leakage can result in hemoconcentration and increased hematocrit, but here even though DHF and DSS developed only in 16 patients, elevated RBC count was found in 253 patients. Hemoconcentration would also increase the WBC levels. The decrease in platelet in dengue is a different mechanism altogether. This isolated increase in RBC has to be further investigated.

Neurological manifestations are increasingly noticed as a separate entity, not as a sequelae of hemorrhage. It may be attributable to immune-mediated reaction on the neural tissue or direct invasion by the virus [16]. DENV causes immunosuppression [16]. Virus may be isolated from the cerebrospinal fluide (CSF) and brain tissue which indicates infectious encephalitis. DENV neurotropism was considered an opportunistic characteristic [16]. It was found that DENV is highly neurotropic in Aedes aegypti [16]. Neurotropism may be for monocytes, macrophages and dendritic cells [4]. Increased neural predilection with epidural abscess with or without spondylodiscitis was noted in 2017 [17].

There is leukopenia and thrombocytopenia reported in dengue. The exact reason for bone marrow suppression is unclear. Bone marrow shows hypocellularity and arrest of megakarocyte maturation [18]. The major pathogenetic mechanism of dengue infection is development of coagulation disorders. Clinical science is mostly due to endothelial cell damage, enhanced vascular permeability and increased plasma leakage [15].

Vector interactions determine the transmission of DENV infections. Aedes aegypti is less susceptible to infection by DENV than Aedes albopictus, which would act as a selection mechanism for more virulent strains of DENV. Lower susceptibility would require a higher viral load to get infected [19].

First infection of an individual with any of the four serotypes is called primary dengue infection. This results in high titers of IgM (3 - 5 days) and immunoglobulin G (IgG) (6 - 10 days) in the patients’ blood. IgM is transient (persist only for 2 - 3 months), but IgG remains for life and provides lifelong immunity against that serotype, but not against other serotypes [20]. A secondary infection with a different serotype causes a severe infection. Here the antibodies to the previous serotype binds to the virus and forms a complex which enhances the access of the virus into monocytes via Fc receptors. This phenomenon is called antibody dependent enhancement (ADE) [20].

Dengue infection is confirmed by identification of the viral genomic RNA, antigen or antibodies it elicits, so a NS1, IgG and IgM testing can be made.

Till date no anti-viral drug is available for dengue. Treatment is symptomatic and supportive [20]. Vaccines are developed against dengue, but further researches are on it.

A few interesting facts are revealed in the analysis of these data. Among the 2,098 patients with dengue fever, 1,626 patients were reported in four months’ time spanning from May to August in 2017. The reason for the surge of infection is unknown, maybe the monsoon season in Kerala, which is the breeding season of Aedes causing higher prevalence of infection during these months. During the rest of the months in 2017, 2018 and 2019, there were only very few cases in all the three centers, which was also evenly distributed during all the months in a year. There was no outbreak during this period. More than 99.7% of the patients recovered after the infection. Of the patients, 0.24% died of complications related to dengue fever at center B. There were no death reported at Center A and Center C. This shows excellent outcome after dengue fever infection during this period. ADE and severe dengue are possible when reinfection occurs with another serotype. The absence of such severity would point to the existence of a single serotype of DENV around the centers studied in Kerala. The correlation between competence of domestic mosquitoes and human virus transmission could be adaptation of the virus to the mosquito. Once a mosquito called Aedes aegypti evolves to use humans as blood meals, there would be pressure on human arbovirus to adapt to this species of mosquito for transmission, in particular to the particular mosquito genotypes feeding on humans [21].

Another article published by Debashish Biswas et al [22] in current urban studies 2014, “A note on distribution of breeding sources of Aedes aegypti in the city of Kolkata during an outbreak of dengue during 2012,” have shown that an Aedes aegypti larval survey and vector control measures by Vector Control Department of Kolkata Muncipal Corporation have decreased the case load from 1,852 (with two deaths) in 2012 to 238 (with no death) in 2013. But here without any vector control measures, the case load was very low in 2018 and 2019. The surge of cases were from May to August in 2017 even in a center 311 km away from another two centers. This might point out to the episodic nature of dengue infection, even though the infection rate has increased about five-fold across the world.

Much variation exists within a single species of Aedes aegypti [20]. Many insect vectors show the variation [20]. In Aedes aegypti such variable traits are color and pattern of scaling, host choice for blood meal, oviposition choice, larval sites, egg dormancy, development time and competence to vector viruses [20], so categorizing this species of Aedes into two or three sub species is a folly. Similarly, new viral serotypes might emerge for adaptation to these changing vectors.

The exact mechanism of DENV-mediated bone marrow suppression is not known. Pancytopenia, bicytopenia (WBC and platelet) and thrombocytopenia are all documented in this analysis. It might be secondary to bone marrow suppression or anti-platelet activity of these viruses. Thrombocytopenia was present in 814 patients from center A and B. Pancytopenia occurred in 161 patients at center B and one patient at center A. An interesting finding is an elevation of RBC count more than 5.5 × 10⁶/mm³ occurred in 138 patients at center A and 115 patients at center B. Even though the number of DHF and DSS patients was only 16, the reason for isolated RBC count increase up to 1,000,000/mm³ needs to be further investigated.

Neurotropism of DENV is well documented, but evaluation of 2,098 patients from three centers showed only 14 patients with encephalitis and another 14 patients with seizure at center A. Post dengue fever epidural abscess with or without spondylodiscitis was reported from center A and B. At center A, there were five cases of post dengue epidural abscess with or without spondylodiscitis in 2017 during the surge of infection between May and August. At center B, there were eight cases of epidural abscess with or without spondylodiscitis during the same period [4]. Post dengue immunosuppression and problems related to that are not analyzed in the present study.

Analysis of dengue fever data of 2,098 patients from three centers in two districts of Kerala, India over three years from 2017 to 2019 shows certain important findings about the manifestation and trend of this most common vector-mediated viral infection in the world. Most of the patients presented with fever, myalgia and headache. The incidence of severe dengue, dengue hemorrhagic fever and dengue shock syndrome is very low. Even without vector control measures, the dengue fever cases were much fewer during all the months in 2017, 2018 and 2019 except from May to August in 2017. This points to the possible presence of single serotype around areas studied. The change of characters of the vector might change the virus for its adaptability to the vector. This might extend this virus infection to even temperate countries. Another important finding of this study is the isolated elevation of RBCs even up to 1,000,000/mm³, even in the presence of decrease in other cells, which needs further studies. The cases secondary to dengue-mediated immunosuppression is not addressed in this study.

Acknowledgments

None to declare.

Financial Disclosure

No financial assistance received for this study in any manner.

Conflict of Interest

No conflict of interest.

Informed Consent

Informed consent was obtained.

Author Contributions

Dr. Suresh Sivadasan Pillai was responsible for conceptualization of the idea, writing the article, data collection and analysis; Dr. Sonia Suresh, Dr. Moidu Shameer and Dr. Shameem G Mohammad were responsible for data collection and contributed in analysis of the data; Dr. Swaroop Sujath contributed in data collection and analysis of the data, and provided technical support; Ranjit Menon P.K. provided technical support.

Data Availability

The authors declare that data supporting the findings of this study are available within the article.

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