Study site and population
The study was conducted at the Singapore General Hospital (SGH). SGH is the country's oldest and largest tertiary hospital and national referral center with 1521 acute-care beds. Admission criteria for suspected dengue is presence of one or more of the following: significant bleeding, severe vomiting or diarrhea that requires intravenous infusion, blood pressure < 90/60 mm Hg and/or pulse rate > 100 beats/minute, severe abdominal pain, dehydration with electrolyte abnormalities and/or postural hypotension, are elderly with medical co-morbidities and are unwell, hematocrit > 50% or platelet count < 80,000 cells/mm3[7]. Adult patients are admitted and managed according to WHO guidelines [8]. The following working day, the patients are transferred to the Department of Family Medicine and Continuing Care, where they were prospectively enrolled into the study. Exclusion criteria were, negative confirmatory test for dengue, failure to follow study protocol and preexisting renal disease.
Clinical and laboratory procedures
Dengue infection was confirmed by the detection of IgM antibodies using the Dengue Fever Virus IgM capture ELISA kit (Focus Technologies™, CA, USA) [9]. In addition, we used real time one step reverse transcriptase PCR (RT-PCR) following standard procedures to confirm suspected cases of dengue fever [10]. False positive cases due to cross-reactivity using the IgM blots and RT-PCR are minimal because Japanese Encephalitis incidence is very low in Singapore, and Yellow Fever has never been detected in this region [11]
Patients' vital signs were measured every one to two hours. Postural blood pressure was taken three times daily. Patients with a postural drop in blood pressure of at least 20 mmHg in systolic and 10 mmHg in diastolic pressure readings from the lying to the standing positions were aggressively hydrated based on intravenous administration of sodium chloride 0.9% given initially at a maximum of 10-20 ml/kg/hour. If clinical improvement is observed, successful reduction of intravenous fluid 10 to 6 and 6 to 3 ml/kg/hour is made till clinical improvement is sustained i.e., hematocrit falls, pulse rate and blood pressure stable, and urine output rises. Patients with postural hypotension were examined twice daily especially for evidence of petechiae, purpura, ecchymoses, bleeding from mucosa, gastrointestinal tract or other sites.
Daily platelet counts and hematocrit were measured. Chest x-ray and/or ultrasound of the abdomen were conducted to verify pleural effusion, ascites or other signs of plasma leakage as clinically indicated. Electrolyte levels, liver function and renal function tests were carried out as clinically indicated.
Urine protein creatinine ratio
Starting at enrollment until discharge, each patient's daily spot urine protein creatinine ratio (UPCR) was measured. As the normal physiologic amount of protein excreted is less than 0.20 gm/mg per day, we consider insignificant proteinuria as 0.06 to 0.19 gm/mg and significant proteinuria as 0.20 gm/mg or greater [12]. Patients who continued to have proteinuria at discharge were followed-up to confirm normalization of his or her UPCR.
Definitions and data analysis
We used WHO clinical criteria to distinguish DHF from DF by the presence of hemorrhagic manifestations, thrombocytopenia and hemo-concentration [8]. Hemo-concentration is defined as an increase of the hematocrit of 20% or more; or evidence of plasma leakage such as pleural effusion, edema and ascites. Thrombocytopenia is defined as platelet count less than 100 × 109/L. DHF cases with circulatory failure (evidenced by a rapid and weak pulse, narrowing of pulse pressure to < 20 mm Hg or hypotension) or profound shock with undetectable blood pressure and pulse were classified as DSS.
The onset of the illness was defined as the first day of fever. The day of defervescence (DDf) was defined as the number of days from the onset till the remission of fever. For the analysis, onset of proteinuria, the day of peak UPCR, and day of lowest platelet count, was rescaled from the DDf, considering DDf as day 0.
Statistical analysis was performed using SPSS software version 17 (IBM Corporation, Somers, NY, USA). Median, together with range, was used to describe continuous variables (UPCR, platelet count, day of peak UPCR, and day of onset of proteinuria, and hematocrit on the day of peak UPCR. The data were compared between the groups with DF and DHF (including DSS) using Fisher t-test and the Mann-Whitney's U-test, respectively. Categorical data were presented as frequency, with percentages shown in parentheses and were analyzed using Chi-square test. Statistical significance was taken as p-value < 0.05.
Ethics
Ethical clearance was obtained from the SingHealth Centralized Institutional Review Board. (CIRB reference 2009/051/E). Waiver of consent was approved by the IRB as the collection of urine samples is non-invasive and did not constitute harm to the patient. Data were anonymized at entry and analysis. The patients were informed that the urinalysis was used to quantify the amount of protein during their illness.