Study design and setting
This was a hospital based cross sectional study involving children under 5 years of age in Moshi Municipality presenting with diarrhoea from January 2016 to May 2016.
Four health facilities were purposively selected to cover multiple tiers of health care and these included: St Joseph Hospital, Kilimanjaro Christian Medical Centre (KCMC) Hospital, Majengo and Pasua Health Centre. KCMC is one of the zonal referral hospitals in Tanzania; it serves as a referral, research and teaching hospital, located in the Northeastern part of Tanzania. It serves five regions in the Northern part of Tanzania, namely, Kilimanjaro, Tanga, Arusha, Manyara and Singida with an estimated population over 15,000,000 people. It has a bed capacity of 450 beds. St Joseph is a designated District Hospital which has several departments and admits approximately 1787 pediatric patients annually. Majengo Health Centre and Pasua Health Centre are secondary level health centres. Both serve an average of 4920 patients per year, (out-patient department), 564 in patients annually at Pasua Health Centre and 5491 outpatients annually at Majengo Health Centre.
Study population
All children aged 2–59 months presenting with passage of 3 or more motions of loose or watery stools per 24 h receiving care in the four study centres during the study period were included. Sample size was estimated per hospital attendances. At least 17 patients were expected from each of the 4 study sites each month. Data was collected for 5 months. The expected sample size was 340.
Inclusion and exclusion criteria
We included all children presenting with diarrhoea aged 2–59 months and excluded those children whose parents/guardian did not agree nor sign the consent form.
Data collection and study variables
A questionnaire was used to collect socio-demographic and clinical data from participants with the aid of research assistants. Our outcome variable was rotavirus and predictor variables were age, sex, duration of diarrhoea, frequency of diarrhoea, completion of rotavirus vaccine and degree of dehydration.
The fecal specimens were collected by research assistants and transported to the department of clinical laboratory, molecular biology unit and stored at − 20 °C until used for the detection of group A rotavirus.
Rotavirus Antigen Detection
The specimens were tested by a solid-phase sandwich type enzyme immunoassay method according to manufacturer instructions Rotavirus Antigen ELISA, Epitope diagnostic Inc (Carroll Road San Diego, USA), Catalogue No: KT 841. The optic density (OD) values above the cut off point 1.1 x (mean absorbance of negative control + 0.08) were considered positive for rotavirus antigen.
Molecular typing of rotavirus
Viral RNA extraction
1 ml of 10% (w/v) fecal suspension in phosphate-buffered saline (PBS) was prepared by thorough mixed using vortex and clarified by centrifugation for 5 min at room temperature and 10,000 rpm. The clarified supernatant was collected for viral genome extraction. Double-stranded RNA was extracted according to the manufacturer’s instructions from 140 μLs of collected supernatant using the QIAamp Viral RNA Mini Kit (Qiagen/Westburg, Leusden, Netherlands), Catalogue Number: 52906.
Reverse Transcription and G-typing of Rotaviruses
About 40 μl of extracted nucleic acid were transferred to a PCR tube. The dsRNA was denatured at 97 °C for 5 min, and then chilled on ice for 2 min. The 10X buffer II (Invitrogen) 7.0 μl, 50 mM MgCl2 7.0 μl, Random primers 1.0 μl, dNTPs (10 mM) 2.0 μl, M-MLV (200 U/μl) Invitrogen 2.0 μl, RNase-free H2O 11.0 μl, to make total volume of 30.0 μl. Then 30 μl of RT mix was added to each tube containing the extracted RNA. Incubated at 42 °C for 50 min, and then was incubate again at 95 °C for 5 min followed by chilling on ice for 2 min. The total volume was 70 μl of cDNA was obtained and stored at − 20 °C ready for PCR.
G-typing consensus PCR (VP7)
The first-round PCR mix was prepared with 10X buffer II (Invitrogen) 4.5 μl, 50 mM MgCl2 2.0 μl, dNTPs (10 mM) 1.0 μl, Taq polymerase (5 U/μl) (Invitrogen) 0.2 μl, Primer VP7-F (20 pmol/μl) 1.0 μl, Primer VP7-R (20 pmol/μl) 1.0 μl, RNase-free H2O 35.3 μl, to make total volume 45.0 μl. 45 μl of PCR mix was added to each PCR tube, and 5 μl of cDNA (from the RT reaction) was added, and tubes transferred to thermal cycler and cycle conditions as provided. The samples were then separated in 2% agarose gel electrophoresis to see positive samples.
The second-round PCR mix was then prepared with 10X buffer II 4.8 μl, 50 mM MgCl2 2.5 ul, dNTPs (10 mM) 1.0 ul, Taq polymerase (5 U/μl) 0.2 ul, Primer VP7-R (20 pmol/μl) 1.0 μl, and 1.0 μl (20 pmol/μl) of each strain specific primers G1 to G12, RNase-free H2O 30.5 µl to make total volume of 48.0 μl. 48 μl of second-round mix was then added to each PCR tube and then 2 μl of first round product was added, and PCR was run under the following temperature cycles conditions.
Data analysis
Data was analysed using SPSS version 22. Descriptive summaries were prepared. Categorical and Numerical data were summarised using their respective measures of central tendency and measures of spread and presented using tables.