A prospective observational pilot study was designed to validate the diagnostic accuracy of urine LRG in an independent cohort of pediatric and adolescent patients (age 3–16 years) presenting to the outpatient department (OPD) and emergency department (ED) with abdominal pain mimicking acute appendicitis at our tertiary teaching hospital. Data were collected from December 2017 to April 2019. As the incidence of appendicitis and mesenteric lymphadenitis is not known in India, a formal sample size calculation was not done.
All consecutive patients presenting with abdominal pain — central, right lower quadrant, shifting of pain or generalized pain, and associated symptoms such as nausea, vomiting, and fever with a duration of symptoms less than or equal to 72 h (from onset) were included in the study. Written informed consent was obtained from the parent or guardian. Children having urinary tract infection, pyelonephritis, pneumonia, treatment with immune-modulating therapy, prior abdominal surgery, and chronic illness (e.g., diabetes, chronic pancreatitis, inflammatory bowel disease, cystic fibrosis, sickle cell anemia) were excluded from the study. The study was approved by the local institutional ethics committee (T/IM-F/17-18/36). The study was also enrolled in the Clinical Trials Registry of Government of India with registration number CTRI/2018/01/01118.
Study protocol
All patients presenting to the OPD and ED with abdominal pain mimicking acute appendicitis were enrolled. History and physical examination data were recorded in a structured case report form specifically designed for this study. The treating physicians complete the form before sending them for any radiological study (US or CT). Preoperative diagnosis of acute appendicitis was based on history, clinical examination, blood test (Complete blood count [CBC], especially increased Leukocyte count, absolute neutrophil count [ANC]), and by ultrasound examination. Acute appendicitis is considered when the appendix diameter is > 6 mm, non-compressible, increase vascularity, presence of target sign, and appendicolith [8]. Mesenteric lymphadenitis is considered when there is the presence of 3 or more hypervascular mesenteric lymph node of size > 5 mm in short axis and absence of other parameters of appendicitis [1, 8]. A pediatric appendicitis score (PAS) was recorded for each patient. The patients’ attendants have explained the procedure of the collection of urinary samples. Urine samples were collected in a sterile urine container, then aliquoted into two sterile labeled microcentrifuge tubes before storing at − 20 °C within 1 h of collection. The urine samples were analyzed after inclusion in the study. The decision to operate or manage conservatively was always based on the clinical judgment of the surgeon and the radiological findings. In general, all uncomplicated appendicitis cases were managed conservatively and those who did not improve, and those with features of complicated appendicitis were managed with surgery.
Patients with mesenteric lymphadenopathy and other patients with a diagnosis of uncomplicated acute appendicitis (by clinico-radiologic examinations) but managed conservatively were followed clinically until discharge and after 1 month of discharge.
Urine analysis for LRG
Before analyses, urine samples were thawed to room temperature and centrifuged (3500 RPM for 10 min). One of the tubes was used for estimation of urinary creatinine on Beckman Coulter AU5800 fully automated analyzer, by kinetic modification of Jaffe’s procedure, using reagents from Beckman Coulter. The second tube was used to estimate urinary LRG in batches by sandwich ELISA kits from ASSAYPRO, USA, using automated BIOTEK washer and reader by the investigator from the Department of Biochemistry who was blinded to patient information.
Outcome
Clinical outcome was defined as mesenteric lymphadenitis or acute appendicitis and appendicitis treated by conservative management or surgery. The laboratory outcome was defined as the concentration of urine LRG at which it will differentiate between mesenteric lymphadenitis and acute appendicitis and concentration of urine LRG (range) in different types of acute appendicitis (phlegmonous, gangrenous, perforated).
Statistical analysis
Patient data were recorded in an excel database. WBC and absolute neutrophil count (ANC) were converted to dichotomous variable, i.e., yes or no, for leucocytosis and neutrophilia. Urine LRG concentrations were divided by urine creatinine concentration, to adjust for different grades of dehydration among patients. Assessment of association between urine LRG and the presence or absence of appendicitis was done using Fisher’s exact test for the dichotomous variables and by two-tailed Mann-Whitney U test for continuous variables, as urine LRG levels are expected to be asymmetrically distributed. Receiver operator characteristic (ROC) curves with an AUC and 95% confidence interval (CI) was performed to explore the performance of urine LRG, PAS to predict appendicitis (complicated and uncomplicated). From the ROC curve data, the best cut-off values was selected by choosing a value that maximized both sensitivity and specificity (Youden’s index). From this cut-off value, the positive predictive value (PPV) and negative predictive value (NPV) were calculated. Statistical significance was set at p < 0.05.