Respiratory viruses in children hospitalized for acute lower respiratory tract infection in Ghana

This was a hospital based cross-sectional study of children less or equal to five years and hospitalized for acute lower respiratory tract illness.

The study was performed at the children's ward of the Komfo Anokye Teaching Hospital (KATH), Ghana from January to December 2008. KATH is approximately a thousand bed tertiary medical facility located in Ashanti Region, Kumasi, the confluence of the transportation network in the central part of Ghana. Its position makes it the most accessible tertiary and referral medical facility in Ghana attending to a population of over 4.4 million in the Ashanti region and beyond [13].

To have a year round incidence of the various viral agents, recruitment was done throughout the year 2008. Screening and recruitment were started at the beginning of every week till two or three patients were recruited. After a maximum of three patients have been obtained it was suspended till the beginning of the next week. This cycle was repeated till the maximum of 11 patients were recruited for each month.

At the recruitment station all patients arriving at the unit were screened but only those less than five completed years were recruited. Patients recruited into the study should have features of severe pneumonia or very severe pneumonia as defined by the WHO-IMCI (World Health Organisation Integrated Management of Childhood Illness) protocol [14, 15]. Severe pneumonia was said to be present if the child has a history of cough and/or difficult breathing of less than 3 weeks duration, with lower chest wall recession. Severe pneumonia in addition to cyanosis and/or inability to feed or drink was classified as very severe pneumonia. A child was recruited into the study only after the guardian or parents had consented after the objectives of the study had been explained in English or the local dialect. A standardized case record form was then used to record the history of the illness as well as the presenting clinical features of pneumonia.

Five (5 ml) of blood sample was then taken into 25 ml of Brain Heart Infusion Broth (BHIB) and quickly sent to the bacteriological laboratory for culture. Nasopharyngeal specimens were also taken using the nasopharyngeal flocked swab (Copan, Italy). The swab was gently inserted up the nostril towards the pharynx until resistance was felt and then rotated 3 times to obtain epithelial cells. It was then withdrawn and put into 2.5 ml phosphate buffered saline (X1). The samples were transported on ice to the laboratory within few hours of collection. They were then vortexed, transferred into 1.5 ml eppendorf tubes (Eppendorf, Germany) and kept at -80°C for Polymerase Chain Reaction (PCR). Both samples were taken by experienced prior trained nurses of the children ward. Children were excluded if both samples could not be obtained for any reason.

Viral nucleic acids were extracted from samples using QIAamp viral mini kit (Qiagen, Germany) according to the manufacturers' instruction [16]. RT-PCR amplification was performed for the detection of Adv, RSV, Influenza A (Flu A), Influenza B (Flu B), Parainfluenza 1(PIV 1), Parainfluenza 2 (PIV 2) and Parainfluenza 3 (PIV 3) using the light cycler version 1.5 (Roche, Germany). Primers highly specific to each of the viruses were used (Table 1). The reaction mixture for RNA viruses was made up of 5 μl of RNA extract, 1 μl deoxynucleotide (dNTP) triphosphate (10 mM) (Qiagen), 1 μl Bovine Serum Albumin (BSA)(1 mg/ml)(Qiagen), 12.5 μl Qiagen One Step RT-PCR Buffer x5 (containing 12.5 mM MgCl₂) (Qiagen), 1 μl Qiagen One Step Enzyme Mix, 1 μl each of sense and antisense primer(10 μM each), 0.5 μl Probe and 2.0 μl of RNase free water. DNA virus reaction mixture was made up of 5 μl PCR buffer (10X), 1 μl of dNTP mix (10 mM each), 1 μl of MgCl₂ (50 mM), 0.5 μl of Hot Star Taq DNA Polymerase, 0.5 μl of probe, 1 μl of BSA (1 mg/ml) and 1 μl each of sense and antisense primers.

The PCR conditions for PIV, RSV and Influenza viruses consisted of an initial reverse transcription at 20 minutes for 50°C, Taq DNA polymerase activation at 95°C followed by forty five (45) cycles of denaturation at 15 seconds for 95°C and annealing at 15 seconds for 58°C. The initial reverse transcription step was omitted in the case of Adv.

For each batch of tests that were run, RNase free water (Qiagen, Germany) was used as negative control and in vitro transcribed gene of the various viruses was used to determine the detection limit of the assay as positive control. The in vitro transcripts were prepared in our collaborative institution (Bonn Institute of Virology, Bonn, Germany) and transported to Ghana via cooling chain. The in vitro transcripts were prepared by amplifying the genes of interest of the viruses with specific set of primers. The products were ligated to the PCR2.1 vector using the TOPO TA cloning kit (Invitogen Corp, Carlsbad, CA) and transformed into competent E.coli cells. After overnight incubation, desirable colonies were selected and purified using Qiamp Spin-Miniprep Kit (Qiagen, Germany). PCR was performed on the purified product and RNA transcripts were then synthesized from the lowest dilution (with clear band) using Ambion Megascripts T3 kit (Invitogen corp, Calsbad, CA). The final products were purified using Rneasy Mini Kit (Qiagen, Germany) and the concentrations of the transcripts were determined using a spectrophotometer (Themoscientific, U.S.A). The copy numbers of the RNA transcripts were determined following the method of Fronhoffs [21]. Ten-fold dilutions of the RNA transcripts were prepared in Carrier RNA-RNase free water (310 μg of Carrier RNA in 310 ml of RNase free water) and tested. The detection limit was determined as the last dilution after which all other replicates gave negative results. The detection limits were Respiratory Syncytial Virus (RSV): 20 copies/μl, PIV 1: 1 - 78 copies/μl, PIV 2: 400 copies/μl, PIV 3: 30 copies/μl, Influenza A: 120 copies/μl and Influenza B: 480 copies/μl.

To determine the specificities, our assays were tested on other different viruses and they turn out negative.

Bacterial isolates were identified using conventional biochemical methods including urease and indole production, citrate utilization, hydrogen sulphide, gas production and fermentation of sugars. The biochemical media used included Simon's Citrate medium, Urea and Triple Sugar Iron agar (TSI). Coagulase tests were performed for all staphylococci organisms.

Data obtained was double entered into a spreadsheet database prepared with Microsoft^® Excel. It was then compared and cleaned for abnormal wrongful entries. Statistical analysis was done using STATA SE statistical software version 11.2(Texas, USA) after the data had been imported. Categorical variables such as age groups and their association with respiratory agents were analyzed using the Fischer's exact test. Continuous variables were expressed as medians with their inter-quartile ranges. A non-parametric K-sample test on the equality of medians was used to evaluate the differences in the medians of the various subgroups of the continuous variables. For all analysis done, a p-value of less than 0.05 was considered statistically significant.

The study protocol was approved by the Committee for Human Research, Publications and Ethics (CHRPE) of KATH and School of Medical Sciences, KNUST, Kumasi, Ashanti region, Ghana.