Diagnostic accuracy of peripheral venous lactate and the 2009 WHO warning signs for identifying severe dengue in Thai adults: a prospective observational study

The study’s design was approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University (MUTM 2013-008-02) and the Ethics Committee of Phramongkutklao Hospital, Bangkok, Thailand (IRBRTA 1571/2013). The Standards for Reporting of Diagnostic Accuracy (STARD) were followed in this study [23]. Prior to participating in the study, written informed consent was obtained from all patients, (and/or the patient's guardians if the patient was 15–18 years old).

This prospective observational study was performed among patients who were admitted to the Hospital for Tropical Diseases (Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand) between May 2013 and January 2015. Individuals ≥15 years old were eligible for inclusion if they presented with clinical indicators of dengue, which were defined as acute fever and ≥2 of the following symptoms: 1) headache, 2) retro-orbital pain or ocular pain, 3) myalgia, 4) arthralgia, 5) rash, 6) a positive tourniquet test (≥20 petechiae per 1 square inch), or 7) leukopenia (defined as a white blood cell count <5.0 × 10³ cells/μL). In addition, to be eligible for inclusion, participants had to have the presence of dengue viral infection confirmed via reverse-transcriptase polymerase chain reaction (RT-PCR) from an admission serum sample or dengue-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) using enzyme-linked immunosorbent assays (ELISA) of paired serum samples (taken upon admission and 2 weeks after admission). Patients with a history of underlying medical illness, mixed infection, or who were currently pregnant were excluded from this study.

Laboratory tests at admission included a complete blood count, blood chemistry analysis, and assessment of PVL levels. Blood samples for PVL were subsequently collected every 24 h until the patient exhibited a body temperature of <37.8 °C for at least 24 h. To exclude the presence of other infections, hospitalized patients provided two blood cultures for microbiology analysis and underwent a urinalysis and chest radiography at admission. Other diagnostic tests for infectious diseases were also performed when clinical findings were suspect. The treating physicians were blinded to the results of the PVL tests. All patients with dengue received standard treatment according to the 2009 WHO guidelines for dengue [24]. All patient data, including baseline characteristics, clinical parameters, and laboratory findings, were recorded on a pre-defined case report form.

Using the 2009 WHO dengue case definitions [24], we classified patients as having either non-severe or severe dengue on the basis of their clinical and laboratory data. Patients with non-severe dengue were sub-categorized according to the presence or absence of WSs. Non-severe dengue without WSs was defined as an acute fever with ≥2 of the following symptoms: 1) nausea, 2) vomiting, 3) rash, 4) myalgia, 5) arthralgia, 6) a positive tourniquet test, or 7) leukopenia. The WSs included 1) abdominal pain; 2) persistent vomiting (defined as vomiting with signs of dehydration during physical examination); 3) clinical fluid accumulation that manifested as pleural effusion, ascites, or a serum albumin level <3.5 g/dL; 4) lethargy; 5) a liver span of >15 cm; 6) bleeding from a mucosal area, including the nose, gums, gastrointestinal tract, or vagina; 7) an increase in hematocrit of 2 % above the sex-specific reference range for a healthy Thai adult; and 8) a platelet count of ≤100 × 10³/μL. Patients were diagnosed with severe dengue if they met all of the criteria for non-severe dengue, as well as at least one of the following: 1) severe plasma leakage that caused shock or respiratory distress, 2) severe clinical bleeding (i.e., bleeding in the vital organs or spontaneous bleeding from a mucosal area that required a blood transfusion), or 3) severe organ involvement (evidenced by an aspartate aminotransferase [AST] level >1,000 U/L, an alanine aminotransferase [ALT] level >1,000 U/L, a serum creatinine level ≥3 times above baseline, myocarditis, and/or encephalitis). Plasma leakage was defined as a ≥20 % increase in hematocrit (above the reference range) or clinical evidence of fluid accumulation. Shock was defined as a systolic blood pressure of <90 mmHg with a <0.5 mL/kg/h decrease in urine output, impaired consciousness, AST level >1,000 IU/L, or ALT level >1,000 IU/L. Respiratory distress was defined as a respiratory rate of ≥24 breaths/min with <95 % oxygen saturation on room air and/or the need for oxygen therapy.

Dengue viral RNA was extracted from each patient’s serum using a 2-step PCR method, as described by Lanciotti et al. [25] and modified using the methods outlined by Reynes et al. [26]. The RNA was extracted from the acute serum samples using the PureLink® Viral RNA/DNA Mini Kit (Invitrogen™, USA), according to the manufacturer’s instructions.

All sera were tested using 4 separate captures in ELISA assays for IgM and IgG against the dengue virus and the Japanese encephalitis virus, as described by Innis et al. [27]. To differentiate between dengue and other flavivirus infections, we determined the ratio of dengue IgM to Japanese encephalitis virus IgM, with a ratio of ≥1.0 indicating dengue virus infection and a ratio of <1.0 indicating other flavivirus infection. The ratio of dengue IgM to dengue IgG was also calculated, with a ratio of ≥1.8 indicating a primary dengue infection and a ratio of <1.8 indicating a secondary dengue infection.

Blood samples were collected from a vein in an upper extremity without the use of a tourniquet. The 2 mL blood sample was placed in a vacutainer that contained sodium fluoride, and the sample was immediately placed on ice, sent to the laboratory center, and analyzed for lactate within 10 min of being drawn. A colorimetric assay with an enzymatic reaction that converted lactate to pyruvate was performed using an auto-analyzer (Roche/Hitachi Cobas C Systems, USA), according to the manufacturers' protocols. In this assay, l-lactate is oxidized to pyruvate and hydrogen peroxide by lactate oxidase, and the hydrogen peroxide reacts with peroxidase to generate a colored dye. The intensity of the color is directly proportional to the l-lactate concentration, as measured by the increase in absorbance. Lactate levels are expressed as mmol/L. The laboratory personnel were blinded to the sample sources, and the coefficient of variation for this assay in our laboratory was 1.1 %.

A previous retrospective study at the Hospital for Tropical Diseases indicated that the incidence of severe dengue was 27.9 % among adults who were hospitalized with dengue according to the 2009 WHO definitions [6]. However, it has also been estimated that the incidence of severe dengue would be approximately 20 % among dengue patients if they received close observation throughout treatment and care during their hospitalization [22]. Based on this information, we calculated that a sample size of 120 patients was needed for this study, using a specificity of 90 % with a confidence interval of ±6 %.

All data were analyzed using SPSS software (version 18.0; SPSS Inc., Chicago, IL). Numerical variables were tested for normality using the Kolmogorov-Smirnov test. Variables with non-normal distribution were summarized as a median and inter-quartile range (IQR) and were compared using the Mann–Whitney U test for 2-group comparison. Categorical variables are expressed as frequencies and percentages, and were analyzed using the Chi-square test or Fisher’s exact test, as appropriate. The optimal lactate cutoff value for identifying severe dengue was determined using receiver operating characteristic (ROC) curves. Diagnostic parameters were evaluated using 2 × 2 tables and 95 % confidence intervals (CI) to determine their sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), positive likelihood ratio (LR+), and negative likelihood ratio (LR–). All tests of significance were 2-sided, with a p-value <0.05 indicating statistical significance.