38 Journal of The Association of Physicians of India Vol. 63 May 2015

Catching Dengue Early: Clinical Features and Laboratory Markers of Dengue Virus InfectionPrakash Babaliche1, Darshan Doshi2

1Professor, 2Post Graduate, Department of General Medicine, JNMC, KLE University, Belgaum, KarnatakaReceived: 07.11.2013; Revised: 07.04.2014; Re-revised: 08.07.2014; Accepted: 10.07.2014

O r i g i n a l a r t i c l e

AbstractBackground and Objectives : Dengue fever is one of the most common tropical diseases worldwide. Early diagnosis of dengue helps in patient triage and timely management of dengue virus infection. This study was undertaken to note the early clinical features supported by detection of NS1 antigen for diagnosis of dengue virus infection.

Methodology : In this study a total of 100 adult patients presenting with clinical features of dengue infection from January 2012 to December 2012 were studied. The diagnosis was confirmed with NS1 rapid diagnostic test (RDT) whose efficacy was later tested with IgM ELISA.

Results : In this study young males were predominantly affected. NS1 positivity was 68%. The sensitivity of NS1 in predicting dengue infection compared to IgM was 92.86% and specificity was 90% with strength of agreement considered to be very good based on Kappa statistics. Clinical features like retro-orbital pain, myalgia, arthralgia, rashes and bleeding manifestations were significantly associated with NS1 positivity. Similarly icterus, oedema, hypotension, altered sensorium, thrombocytopenia and raised creatinine were significantly more in NS1 positive patients. Anomalously, SGOT was more than SGPT, which can help in differentiating dengue virus infection from other viral infections early in the course.

Conclusion : We conclude that dengue infection, which possesses serious public health problem, can be diagnosed early with the help of clinical features like retro-orbital pain, myalgia, bleeding manifestations, thrombocytopenia and anomalously SGOT greater than SGPT that is supported by detection of NS1 antigen.

Background

Dengue fever is an important a r t h r o p o d - b o r n e v i r a l infection of humans. Worldwide, an estimated 2.5 billion people are at risk of infection.1 It is estimated that more than 50 million infections occur each year, of which 500,000 hospitalisations are of dengue haemorrhagic fever, mainly among children, with the case fatality rate exceeding 5% in some areas.1-4

Diagnosing dengue early is challenging because the initial symptoms of dengue infection are

often non-specific and serological tests , which are the mainstay of current laboratory diagnosis, confirm dengue late in the course of illness.5

Before day 5 of illness, during t h e f e b r i l e p e r i o d , d e n g u e infections may be diagnosed by virus isolation in cell culture, by detection of viral RNA by nucleic acid amplification tests (NAAT), or by detection of viral antigens

by ELISA or rapid tests. Virus isolation in cell culture is usually performed only in laboratories with the necessary infrastructure and technical expertise. The isolation a n d i d e n t i f i c a t i o n o f de n g u e viruses in cell cultures usually takes several days. Nucleic acid detection assays with excellent performance characteristics may identify dengue viral RNA within 24-48 hours. However, these tests require expensive equipment and reagents.6

Clinical features of dengue virus infection are usually nonspecific ranging right from self-limiting d i sease l ike fever , headache , myalgia, rashes, and arthralgia to dengue haemorrhagic fever and dengue shock syndrome.

Hence there is a lacunae in diagnosing dengue virus infection early both in relation to clinical features and laboratory tests . Hence, this study was undertaken to address this issue and help clinicians to diagnose dengue fever early with help of clinical features and laboratory investigations.

Editorial Viewpoint The s tudy i s a c ross -

sectional one. As per this study SGOT

can be more elevated than SGPTindengue.

The study reiterates theknown facts about dengue.

39Journal of The Association of Physicians of India Vol. 63 May 2015

Material and Methods

The present study was a one year cross-sectional study comprising of 100 patients conducted in the Department of Medicine, KLES Dr. Prabhakar KoreHospital andMedical Research Centre, Belgaum from January 2012 to December 2012.

Patients with age more than12 years, history of documented fever of more than 38 C of less than seven days p lus two or more signs and symptoms from the following: headache, retro-orbital pain, myalgia, arthralgia, rash, hypotension and bleeding manifestations, were included in the study. Patientswith localisedsource of infection were excluded from the study.

Ethical clearance was obtained prior to the study and written informed consent was obtained from the participating patients.

Patients were interviewed fordemographic data such as age, sex and occupation were noted. Histories of similar complaints in past and current treatment were noted. Patientswere subjected toa thorough physical examination, vitals (pulse rate, blood pressure and respiratory rate) and other clinical signs and symptoms of dengue fever. Systemic examination wascarriedout.Patientsunderwentroutine laboratory investigations, electrocardiograms in addition to NS1 Rapid detection test using SD Bioline Dengue Duo kit and establishing the efficacy of this t es t aga ins t IgM ELISA done on the 8th day of fever. Patientswith suspected co-infections like malaria , leptospirosis , enteric fever or other infect ions were investigated and those who were found to have a specific cause were excluded from the study.

T h e c a t e g o r i c a l d a t a w a s expressed as rates , rat ios and proportions and comparison was done using chi-square test. The continuous data was expressed

as mean s tandard deviat ion (SD). The diagnostic accuracy of NS1 antigen testing, in predicting dengue infection was determined by sensitivity, specificity, positive predict ive value and negative predictive value. Kappa agreement w a s u s e d t o c o r r e l a t e t h e agreements. A p value of less than or equal to 0.05 was considered as statistically significant.

Results

A total of 100 patients fitting the inclusion criteria were selected for the study. 68% of the patients tested NS1positive.Of the 100patients,70 patients tested posit ive for IgMELISA.Outofwhich65werepositive for NS1 antigen test while five were negative. The sensitivity of NS1 in predicting dengue infection compared to IgM ELISA was 92.86% and speci f ic i ty was 90% with 95.59% positive predictive value and 84.38% negative predictive value (p < 0.001) with kappa value of 0.813 and strength of agreement considered very good (Table 1).Majority of themweremales(62%). 60% of the patients aged less than 30 years, The presenting complaintsaregiveninTable2.Ofthese, myalgia, rashes, retro-orbital pain , ar thralgia and bleeding manifestations were significantly more in NS1 posit ive patients (Table 3). Bleeding manifestations included melaena, epistaxis, gum bleeding, pervaginal bleeding and haematuria. Of thesemajority ofthem had melaena (8 patients). T h e p r e s e n t s t u d y r e ve a l e d , bradycardia was present in 31% of the patients and hypotension in 4% of patients all of whom were NS1 positive. Icterus and oedema was observed in 16% and 9% of patients respectively both of which were significantly associated with NS1 positivity(p < 0.05). Systemic examination revealed abdominal tenderness in 49% of the patients, reduced air entry in bilateral lung bases in 16% and bilateral coarse crepitations in 5% of the patients. Neurological involvement was

present in 5% of the patients, all of whom were NS1 positive (Table 4).

L a b o r a t o r y i n v e s t i g a t i o n s revealed thrombocytopenia in 87% of the patients (mean 37120.57 32304.99 /mm3) with significantly more in NS1 positive patients (p 0.002). Leucopenia was present in 16% of the pat ients (mean leucocyte count 5500 2489.66 /mm3). Haematocrit was raised in 11% of the patients (Table 5). Serum creatinine was deranged in 17% of the patients (mean 1.19 1.07 mg/dl) and was significantly deranged in NS1 positive patients (p 0.011). In this study, total bilirubin was raised in 25% of patients and the mean total bilirubin was found to be 1.04 1.17 mg/dl. The SGOTand SGPTwere abnormal in 76%and 51% of patients with mean values being 122.04 172.27 and 91.81 123.60 IU/L, respectively. Interestingly, the study showed, SGOTmorethanSGPT.Thealkalinephosphatase was raised in 43% of patients with mean values being 144.39 91.99 (Table 6). Serum sodium and serum potassium were found to be normal in 95% and 97% of the patients with mean values of 136.16 4.33 and 4.04 0.55 meq/L respectively. Hyponatraemia and hyperkalaemia were present in 3% patients each while hypernatraemia was noted in 2% of the patients. ECGrevealedsinusbradycardiain31% of the patients.

Discussion

Dengue i s one o f the most underreported tropical diseases and during epidemics, overreporting can occur at some hospitals. The lack of laboratory resources and the nonspecific clinical presentation of non-severe cases greatly contribute to this situation.

In the present study slight male preponderance was seen that is, 62% of patients were males and the male to female ratio was 1.63:1. In this study most of the patients (60%) were aged less than 30 years followed by 30 to 40 years (15%),

40 Journal of The Association of Physicians of India Vol. 63 May 2015

Table 1: Diagnostic accuracy of NS1 in comparison to IgM ELISA after seven days

NS1 testIgM

TotalPositive Negative

Positive 65 3 68Negative 5 27 32Total 70 30 100p < 0.001; Kappa = 0.813 SE of kappa = 0.063 95%confidenceinterval:From0.689to0.937

The strength of agreement is considered to be very good.Sensitivity: 92.86%; Specificity: 90%PPV: 95.59%; NPV:84.38%

Table 2: Presenting complaints

HistoryDistribution (n=100)

Number (%)Fever 100 (100)Headache 82 (82)Myalgia 72 (72)Retro-orbital pain 62 (62)Painabdomen 49 (49)Rash 36 (36)Arthralgia 31 (31)Bleeding manifestation 22 (22)

Vomiting 20 (20)Diarrhoea 2 (2)

Table 3: Clinical features associated with dengue diagnosis using NS1 antigen test in patients evaluated up to 7 days of fever onset

Presenting complaints

NS1 positive NS1 negativep value

No. (%) No. (%)Headache Present 53 (64.6) 29 (35.4) 0.124

Absent 15 (83.3) 3 (16.7)Myalgia Present 54 (75.0) 18 (25.0) 0.016

Absent 14 (50.0) 14 (50.0)Retro orbital pain Present 47 (75.8) 15 (24.2) 0.016

Absent 21 (55.3) 17 (44.7)Painabdomen Present 40 (81.6) 9 (18.4) 0.069

Absent 28 (54.9) 23 (45.1)Rash Present 31 (86.1) 5 (13.9) 0.004

Absent 37 (57.8) 27 (42.2)Arthralgia Present 25 (80.7) 6 (19.4) 0.004

Absent 43 (62.3) 26 (37.7)Bleeding manifestation

Present 22 (100.0) 0 (0.0) 60 /min 44 (63.8) 25 (36.2)Hypotension Present 4 (100.0) 0 (0.0)

0.161Absent 64 (66.7) 32 (33.3)

Icterus Present 15 (93.8) 1 (6.3)0.016

Absent 53 (63.1) 31 (36.9)Oedema Present 9 (100.0) 0 (0.0)

0.031Absent 59 (64.8) 32 (35.2)

RSNormal 51 (63.0) 30 (37.0)

0.073Air entry B/L bases 13 (86.7) 2 (13.3)

B/L Coarse crepitations 4 (100.0) 0 (0.0)

PA Normal 28 (54.9) 23 (45.1)0.004Tenderness

present 40 (81.6) 9 (18.4)

CNS Normal 63 (66.3) 32 (33.7)0.116

Altered sensorium 5 (100.0) 0 (0.0)

51 to 60 years (12%) and 41 to 50 years (10%). The mean age of the study population was 32.48 13.87 years. In the present study all the patients presented with fever 100% followed by headache in 82%, myalgia in 72%, retro-orbital pain in 62% and pain abdomen in 49% of patients. On clinicalexamination, icterus and oedema was observed in 16% and 9% of patients that were statistically significant in NS1 positive patients (p < 0.016 and p

41Journal of The Association of Physicians of India Vol. 63 May 2015

bradycardia in 31% of patients.The NS1 antigen test for the

diagnosis of acute dengue infection had demonstrated considerable variation in sensitivity (49.8%-98.7%) but the specificities reported were more consistent with all being > 90%.9 In this study NS1 test was positive for dengue infection in 68% of patients and 70% of patients were positive for dengue infection on IgM.Ofthese,65werepositivefordengue infection on NS1 while five were negative. The sensitivity of NS1 in predicting dengue infection compared to IgM was 92.86% and specificity was 90% with 95.59% posi t ive predict ive value and 84.38% negative predictive value (p < 0.001). The strength of agreement was considered to be very good based on Kappa statistics (Kappa 0.813; SE of kappa = 0.063; 95% confidenceinterval:From0.689to0.937).

Overa l l t h e p r e s en t s tudyshowed the usefulness of NS1 antigen test which is an excellent tool in addressing potential ly fa ta l , epidemic-prone dengue infection based on its easy and fas t appl i ca t ion compared to immunochromatography based dengue serology tests.

S imi lar ly , c l in ica l features , l i k e r e t r o - o r b i t a l p a i n , m y a l g i a , a r t h r a l g i a , r a s h , bleeding manifestat ions along

Table 5: Complete blood count

Variables Findings Distribution (n=100)PCV (%)

> 50 11< 50 89Total 100Mean SD 42.4 8.3

Plateletcount (/mm3)

TCP 87Normal 13Total 100Mean SD 37121 32305

TLC (/mm3)

Normal 78Leucopenia 16Leucocytosis 6Total 100Mean SD 5500 2490

PCV:Packedcellvolume;TLC:Totalleucocytecount;TCP:Thrombocytopenia

Table 6: Liver function test

Variables Findings Distribution (n=100)Total bilirubin (mg/dl)

Normal 75Raised 25Total 100Mean SD 1.04 1.17

SGOT (IU/L)

Normal 24Raised 76Total 100Mean SD 122 172.3

SGPT (IU/L)

Normal 49Raised 51Total 100Mean SD 91.8 123.6

Alkaline phosphatase

Normal 57Raised 43Total 100Mean SD 144.4 92

wi th laboratory f indings l ike thrombocytopenia, SGOT > SGPTstrongly support the diagnosis of dengue fever.

The limitations of this study were, smaller sample size, and NS1 antigen test was not evaluated consideringPCR,whichhashighersensitivity than NS1 antigen tests though which was beyond the scopeofthisstudy.Furtherstudiescomprising of large sample size with polymerase chain reaction (PCR)would further explore therole of NS1 antigen testing in the diagnosis of dengue infection.

Conclusion

Early identification of dengue infection in acute phase sera using NS1 antigen rapid detection test is valuable in terms of disease progress ion and mortal i ty . In o u r s t u d y , N S 1 R D T s h o we d promising results with sensitivity and specificity of 92.86% and 90% respectively when compared with IgM-ELISA done later in the course of i l lness. However, in highly suspected cases of dengue infection clinical management should not rely on negative serological results.

C o n s i d e r i n g o n l y c l i n i c a l features, in our study we found that presence of retro-orbital pain, myalgia, bleeding manifestations, r a s h e s a n d a r t h r a l g i a w e r e

significantly (p value < 0.05) more in patients who were NS1 positive.

Patientswith dengue fever aremore prone to have liver enzyme derangement as found in our study and preferent ial ly high SGOTmay serve as an early indicator of dengue infection.

Similarly thrombocytopenia was more significant (p value 0.002) in NS1 positive patients and it is a very important indicator of prognosis in dengue fever.

Thus we conclude that dengue infection, which possesses serious public health problem, can be diagnosed early with the help of c l inical features l ike retro-orbital pain, myalgia, bleeding manifestations, thrombocytopenia, SGOT greater than SGPT that issupported by detection of NS1 antigen.

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