Comparison of dengue and other febrile illnesses in patients admitted to Teaching Hospital- Peradeniya

Background: Dengue is a severe and notifiable infection in Sri Lanka and other tropical countries. There are other febrile illnesses,which have the same symptoms as dengue. Identifying early changing markers in blood and their changing patterns in dengue and other febrile illnesses will help to differentiate dengue patients from other febrile illnesses.It is important for the early recovery and management of the dengue patients. Methods: Dengue suspected 288 patients were selected from adult medical wards in Teaching hospital-Peradeniya, after initial screening process. Out of whole population, 56% were RTPCR positive for dengue and RT-PCR negative population (44%) was named as other febrile illnesses. Results of variations in liver enzymes, full blood count (FBC), demographic and hospitalization details in both groups were obtained from hospital records. Results: Male predominance and highly affected young population were detected in both groups. Number of days from onset of fever to admission to the hospital is lower and hospitalization period is higher in dengue than other febrile illnesses. On admission, higher level of aspartate transaminase, significant lymphopenia and higher mean cell volume (MCV) were noticed in dengue patients. On discharge, lower total white blood cell count, lower absolute lymphocyte count, lower platelet count and increased levels of transaminases were detected in dengue compared with other febrile illnesses.Conclusion: From dengue suspected admissions, 44% was due to other febrile illnesses which was very difficult to distinguish by symptoms of dengue. Young patients with male predominance is common in dengue as well as in other febrile illnesses. Recovery of cytopenia in FBC and normalization of liver transaminases were lower in dengue.


Introduction
Dengue is an epidemic disease present in more than 100 countries in the world. Especially as tropical countries, Sri Lanka, India and Pakistan have better environmental conditions which are more preferable for dengue vector 1 . Annual Health Bulletin has shown that in 2012, 44 456 infected dengue patients were reported in Sri Lanka 2 . During first 42 weeks of 2016, 43 895 cases of dengue fever were reported 3 . When considering the whole population in Sri Lanka, dengue affected amount is highly remarkable.
Dengue is categorized as asymptomatic infection, dengue fever (DF), DHF and dengue shock syndrome (DSS) according to the WHO criteria 1 . Dengue infection is caused by four serotypes as DEN 1 -4 4 .
Real time Polymerase chain reaction (RT PCR), enzyme-linked immunosorbent assay (ELISA), IgM, IgG, Nonstructural protein (NS1) antigen rapid strip test are used to diagnosis of dengue. As supportive parameters, platelet count, PT (Prothrombin Time) and APTT (Activated Partial Thromboplastin Time) are used. Hematocrit and total WBC count are mostly used for the monitoring of dengue 5 . It is shown that false positive results for dengue found in some diagnostic methods 6 . Detection of viral nucleic acid is vital to prevent false positive results 7 .
Similar dengue symptoms as fever, headache, joint pain, muscle pain and rash are observed in other febrile illnesses as Chikungunya/Zika viral infections 8 . These infections are also transmitted by the dengue vector, Aedesaegypti and Aedes albopictus 9 .
This study was based on changes in demography, hospitalization details and blood parameters (Liver transaminases, FBC) among patients with RT-PCR positive dengue and RT-PCR negative OFI. Dengue and OFI express similar clinical symptoms. In Sri Lanka, more patients are admitted into hospitals in dengue epidemics by suspecting dengue on symptoms. Because of the no availability of reliable investigations like RT-PCR in more hospitals, all patients are considered as dengue. Because of that, more hospitals are packed with patients indengue epidemics and the real dengue patient is not getting special attention and treatments.
There are facilities in most hospitals including rural to perform FBC and liver function tests. By following recent research studies, liver transaminases and some FBC parameters are believed as more preferable, benefited investigations as markers for the management of dengue patients. Therefore, early finding of significant differences in blood parameters between these two categories were more important for the patient management and monitoring. It will be also helpful for the identifying vital effects of dengue and early recovery. This is more useful for clinicians and other health professionals to prevent wastage of sources for unnecessary investigations.

Ethical clearance
Ethical clearance for the study was obtained from the Ethical Review Committee of Kotelawala Defence University. Hospital permission was obtained from the Director, Teaching Hospital-Peradeniya. Dengue suspected 288 patients were selected from adult medical wards of Teaching Hospital-Peradeniya from 01 st of May 2016 to 31 st of August 2016 and categorized as RT-PCR positive dengue (162 patients) and RT-PCR negative other febrile illnesses(126 patients).

Inclusion and exclusion criteria
Adult patients (14years<) with any two dengue suspected symptoms as headache, high fever (more than 3 days), fatigue, joint pain and muscle pain, abdominal pain with a low platelet count (≤ 100,000 / mm3) were included in dengue suspected population. Screening process was done by the medical officer in outpatient department (OPD).

Planned subgroup analysis
For each suspected dengue patient, following tests were performed routinely (daily in hospitalization period). All information about age, gender (demographic data), days from fever onset to admission and hospitalization period of whole study population were obtained from hospital records. Study didn't affect to any treatment of patients.
Real time RT-PCR test was performed in Analyzer-Applied Biosystems Quant Studio 6flex Real Time PCR. ALT and AST were done in fully automated analyzer -Beckman Coulter AU 480 by using kinetic method. Full blood count (FBC) was done by 7-part analyzer -Mindray 6800.

Methods of statistical analysis
Collected data was analyzed statistically by using SPSS software by dividing the study population into two groups as patients with RT-PCR positive dengue and patients with RT-PCR negative other febrile illnesses. The mean values of investigated parameters were used to compare changes in both groups. Distribution of age, gender, mean number of days from fever onset to admission and hospitalization period of both groups were obtained by using descriptive statistics.
By using descriptive statistics and p value by independent samples t test, mean values of ALT, AST and FBC parameters of patients were compared between both groups. For each group, all parameters were analyzed on admission and on discharge to detect whether they are normalized within hospitalization period.

Results
Out of the 288 suspected dengue population, 162 (56%) were real time RT-PCR (Reverse transcriptase -polymerase chain reaction) positive dengue patients and 126 (44%) were real time RT-PCR negative patients named as other febrile illnesses. Most dengue patients in population were belonged to DEN-1(59.6%) serotype (Table 01). P < 0.05 *, p < 0.01 **, p < 0.001 ***  Dengue and OFI have shown a lower neutrophil count near to lower limit of normal range on admission and neutropenia condition on discharge. Similar changing patterns were revealed equally. But speed in normalization of neutrophil count is slower in dengue than OFI and 3 rd day from the admission indicated the lowest neutrophil count in dengue.
When considering the lymphocyte count mutually, dengue patients have shown a lymphopenia condition on admission and it is resolved on discharge (Table  03). No remarkable change was happened in lymphocyte count of OFI patients on admission and discharge. Lowest lymphocyte count in dengue patients was detected in 2 nd day of the admission.
Platelet count was lower than normal lower limit (150 * 10 9 / L) in dengue and OFI on admission and on discharge. Individually, groups have shown a thrombocytopenia condition at discharge ( Table  03). Count of Platelet changing indicates same patterns and the lowest value during hospitalization was shown in 6 th day of the admission in both groups. Mean Cell Volume (MCV), Hemoglobin (Hb), Packed Cell Volume (PCV) are not shown a significant change (Table 03).

Discussion
Dengue fever was very difficult to separate from other febrile illnesses which had similar characteristics and features as dengue 7,8 . Current study was based on 162 RT-PCR positive dengue patients and 126 RT-PCR negative (for dengue) patients (with other febrile illnesses) ( Table 01). Wastage minimizing for unnecessary investigations is an essential requirement for hospitals in Sri Lankan government setting. Viral RNA was detected by RT-PCR with a high sensitivity and specificity (confirmatory test) which was used to divide suspected dengue population into 02 groups and DEN-1 serotype has shown the highest percentage from the dengue population. (Table 01).
The study conducted in Bangladesh mentioned that DEN 2 serotype was most prevalent in their population 9 .
Significant differences and similarities in demographic characters, liver transaminases and main full blood count parameters were detected between two groups of this study.
Significant differences were detected between two similarly categorized groups in a study conducted by using 111 of adult male patients 10 . In India, degree of hepatic dysfunction was detected among patients with serologically positive dengue and serologically negative OFI 5 . It seems that several studies have conducted to compare dengue from OFI. In this study, both categories of dengue patients and patients with OFI had male predominance. Male category has shown higher percentages while lower percentages were shown by female category in dengue and OFI (Table 02).
It was found that 82.1% of dengue patients and 79.3% of patients with OFIwere belonged to the 18-60 years (working population) age group. Current research was based on 288 suspected dengue patients. The mode of age for dengue patients was 24 and the mean value was 31.28 years. Similar findings were observed in OFI as mode of age was 22 and mean was 32 years (Table 02). It seems that most of the members in study population were belonged to young age group. In 2012, health statistics have shown 20-29 and 30-39 were the mostly affected age groups by dengue 2 . It was compatible with age which has shown a higher sensitivity to infection in younger. These findings were generally agreed with some studies who have found the male predominance in confirmed dengue patients 11,12,13 . As current finding, 3:2 ratios for male to female was observed in study conducted by using 425 confirmed dengue patients 14 . Significant differences were identified by analyzing average number of days from fever onset to admission and hospitalization period of patients in both groups in this study (Table 02). To confirm the finding, clinical history of these patients needs to be analyzed. Average of hospitalization period for dengue patients was higher in dengue. It has shown that patients with OFI were discharged earlier than dengue patients (Table 02). This finding was approximately correlated with a study who has observed 5.65 days as the hospitalization period 15 .
The detection of changes in liver function between two groups was a main objective in current study. It was found that the mean and mode ALT and AST values on admission were increased in both dengue and OFIthan normal values. Mean, median and mode values of AST of dengue patients were higher than OFI. At discharge, average of both AST and ALT values were high in 02 populations and mean, median values were higher in dengue patients than OFI. Even though significant differences weren't detected on admission and discharge between dengue and OFI, difference between mean AST values on discharge have reached more towards to the p-value of 0.05. When comparing AST and ALT, mean value of AST in both admission and discharge were higher than mean value of ALT. Therefore, AST was believed to be taken as more prominent measurement in dengue patients on admission (early appearance) and discharge than ALT in the study (Table 03).
More literature has shown higher values of AST and ALT in dengue positive patients. It is confirmed that median values of AST and ALT were increased than the normal values in dengue patients 10,16,17,18,19 .
Full blood count was the main routine investigation which was performed to detect differences occurred by fever conditions. It seems that some parameters of FBC have shown significant differences between dengue and OFI. Significant lymphopenia and leucopenia were seen more in dengue patients than OFI while higher percentage of patients with OFI had neutropenia than dengue (Table 03).
Dengue patients have shown a lower percentage of thrombocytopenia (< 100X10 3 /mm 3 ) than OFI. On discharge, average of total WBC count (p < 0.01), absolute lymphocyte count (p < 0.001) and platelet count (p < 0.00), were reduced significantly and mean of absolute neutrophil count was reduced in dengue than OFI (Table 03). Leucopenia, neutropenia, lymphopenia and thrombocytopenia were seen in more dengue patients than OFI at discharge. Considerable amount of patients in dengue had severe neutropenia. It was also found that lymphocytosis was detected in low amount of patients in dengue than OFI. Mean values of some blood parameters have shown changes in a pattern with the day of fever onset. Leucopenia was detected earlier in dengue. The lowest value of total WBC was shown in 3 rd day in dengue and it was 4 th day in OFI. Minimum neutrophil count (with neutropenia) was detected in 3 rd day from the onset in both populations. Pattern of platelet changes was same for dengue and OFI. Therefore, platelet was not a better parameter which characterizes the dengue.It was found that the platelet count is not a better parameter to describe the severity of patients withdengue hemorrhagic fever 20 .
Current findings of FBC were compatible with several previous studies. A significant difference was detected in the decreasing of mean WBC count for PCR positive patients than RT-PCR negative group 9 . It is found that significantly decreased value of total WBC as early marker in dengue patients on admission than for patients with OFI 18,21 .
This research was a model study with a simple comparison of multi-parameters for adult dengue patients. Recognition of clinical symptoms on admission needs to be required for differentiation of paediatrics and adult patients with dengue and OFI. Correlation of blood parameters (PCV, MCV) with fluid management of patient is important to get an exact analysis of them. In addition to AST and ALT, total liver profile needs to be performed to validate current findings. The causative agents for OFI need to be found to differentiate from dengue.

Conclusion
RT-PCR is very suitable for early diagnosis of dengue. Lymphocyte count was most significantly changed and mean, mode and median values of AST were increased in both admission and discharge of dengue patients than Other Febrile Illnesses. Recovery of cytopenia is lower in dengue patients than OFI.