Study design and setting
This was a descriptive cross-sectional study conducted in paediatrics ward of Mbarara Regional Referral Hospital (MRRH) and Holy Innocent Children’s Hospital (HICH), Mbarara in southwestern Uganda. MRRH receives referrals from health facilities in the neighboring districts in the region and also receives patients from neighboring countries like Tanzania, Democratic republic of Congo and Rwanda. HICH is exclusively a children’s hospital, the only one in the region. The population in the study site visits MRRH/HICH whenever they experience an illness or when referred from peripheral health units. The majority of the people in the study area live in rural areas and they are subsistence crop farmers and practice livestock farming.
Study population
The study included neonates who; presented with suspected clinical sepsis at the study sites, were from exposed contaminated environment and whose parent or legal guardian consent to participate in the study. In the current study, a neonate was defined as a newborn less than one month (30 days) old. Neonates who presented with fever/hypothermia and at least any one of the following; lethargy, poor feeding, full fontanel, vomiting, seizures, diarrhea, respiratory dysfunction and jaundice were enrolled in the study. Exposure status was defined by residence near a stream of water and or its use as source of water, presence of livestock or rodents at home and parents/guardian or caretaker’s occupation that predisposes to contact with contaminated water and or animals. Participants with known underlying etiology such as malaria or any other cause of febrile illness at inclusion or done at bedside as determined by the clinician were excluded because of the possibility of cross reaction in the IgM ELISA analysis.
Sampling and data collection
A total of 103 participants were recruited through convenient sampling method from August, 2018 to November, 2018. An experienced and trained research nurse from each site with the guidance of a clinician noted the clinical presentation and exposure history of neonates admitted at MRRH/HICH and sought consent from parents/caretakers of neonates who fell in the inclusion criteria. Clinical and demographic data was recorded using the predesigned questionnaire from those who consented to participate in the study. The data collected included but not limited to; sex and date of birth (age of the baby, days), duration of fever, source of water for domestic use, presence of animals at home, and signs and symptoms of the infection. From the neonates whose parents/caretakers gave informed consent, about 0.5 mL of blood was drawn aseptically by venipuncture from the arm into EDTA microtube. The blood sample was later used to obtain plasma. Plasma was chosen because of the slightly higher sensitivity for PCR assays compared to whole blood and serum [22] and would later be used for IgM ELISA.
Laboratory procedures
The samples were analyzed at Epicentre research laboratory at Mbarara University of science and technology (MUST). The EDTA whole blood was centrifuged at 3000 rpm for 5 minutes to obtain about 0.2ml (200µl) of plasma. IgM ELISA was done on the plasma (10µl) to detect anti-leptospiral antibodies and the remaining plasma was used for DNA extraction and subsequent detection. ELISA was done using the automated set of analyzer Washer 470 and Reader 270 (BioMérieux) and the Leptospira IgM ELISA kit, EIA-4715 (DRG International, INC). Nine (9) plasma samples were excluded from ELISA analysis either because of anticipated interference due to sample properties (cloudy lipemic samples) or too little volume to proceed with DNA extraction. The procedure was performed and interpreted according to manufacturer’s instructions. For IgM ELISA, positive and negative controls were included for quality control (QC) and the test was considered valid when the QC passed. All the samples were run in duplicates, and checked whether the replicates gave the same cutoff result. Besides the reader, the wells were checked visually with reference to positive and negative control against a white background and the intensity of color formed graded according to the kit insert.
DNA was extracted from the plasma using the QIAamp® DNA Mini, 250 (Qiagen, German), following manufactures protocol. Internal positive and negative control samples were included in each batch of DNA extraction procedure and PCR. A real-time PCR assay using the probe-specific ‘BactoReal Leptospira spp, LipL32’ kit (Ingenetix, Austria) was run for the detection of the LipL32 gene found in pathogenic leptospires with the Rotor-Gene Q real-time PCR instrument according to kit instructions. The sensitivity of the initial PCR protocol was 10 target copies/PCR reactions and Ct of 24, 3. A repeat PCR was done with in-house optimized reaction. The mastermix 5x HOT FIREPol EvaGreen qPCR Supermix (Solis Biodyne, Estonia), Positive DNA controls from Institut Pasteur, and Primers; LipL32F, 5’-AAGCATTACCGCTTGTGGTG-3’ & LipL32R, 5’-GAACTCCCATTTCAGCGATT -3’ (Inqaba biotec, South Africa) with sensitivity of 1 target copies/PCR reaction were used in the optimization of the quantitative PCR. The primers used were designed and described by Picardeau (Institut Pasteur) and colleagues[22]. The final reaction considered had 4µl of mastermix, 0.4µl each of forward and reverse primers (10pmol/µl), 10.2µl molecular grade water and 5µl of template. The temperature profile consisted of initial denaturation at 95°C for 12 minutes and 40 cycles of; 95°C for 15 seconds, 65°C for 30 seconds and 72°C for 30 seconds (acquisition at Green channel). Analysis was done using quantitative, endpoint and melt curve analysis at the Green channel.
Data management and analysis
Data was entered using Epidata and imported to and analyzed using STATA version 12. It was presented by use of pie charts and tables. Continuous variables were presented as mean ± standard deviation. Prevalence was determined as the proportion of positive samples.