RNA viruses in community-acquired childhood pneumonia in semi-urban Nepal; a cross-sectional study

Study participants were recruited from the district of Bhaktapur in the Kathmandu Valley, Nepal. A total of 1,913 (86.2%) of the 2,219 cases were recruited from within Bhaktapur municipality, a semi-urban agricultural based town with a population of approximately 80,000, of which we at any time had approximately 4,500 children 2 to 35 months of age under surveillance for respiratory illness. Low income, low dietary intakes and low consumption of dairy and animal products are widespread, as in most parts of Nepal. Malnutrition, mainly manifested as stunting and anemia, are common among children less than 5 years of age [17].

The Kathmandu Valley is situated at an altitude of 1,300 to 1,350 meters above sea level and has a sub-tropical, temperate climate. There are four distinct seasons; pre-monsoon/spring (March to May), monsoon/summer (June to September), post-monsoon/autumn (October to November) and winter (December to February) [18]. Temperatures may rise to 35°C in summer, while minimum temperatures can fall to 0°C in winter.

We recruited cases from an open cohort of children less than 3 years of age, who were under monthly active and passive surveillance for respiratory illness. Trained fieldworkers referred children with respiratory complaints to the study clinic at the outpatient department (OPD) at Siddhi Memorial Hospital in Bhaktapur, and families could bring their children for free treatment at our clinic for common childhood illnesses. Children residing in Bhaktapur district, but outside the municipality, were only under such passive surveillance. Children aged 2 to 35 months presenting at our study clinic were screened for fast breathing or lower chest wall indrawing (LCI) and classified according to the standard World Health Organization (WHO) algorithm for acute respiratory infection (ARI) [19] (Figure 1). Pneumonia was defined as cough or difficult breathing combined with fast breathing, that is ≥ 50 breaths/min for children 2 to 11 months old, and ≥ 40 breaths/min for children ≥ 12 months old. Severe pneumonia was defined as cough or difficult breathing combined with LCI. Children with audible or auscultatory wheeze were given 2 doses of 2.5 mg nebulized salbutamol administered 15 min apart followed by reassessment after 30 min, which is in accordance with the revised WHO guidelines [20]. A child was included only if he or she had fast breathing or LCI at reassessment.

During a 3-year period from 29 June 2004 to 30 June 2007, we collected 2,230 nasopharyngeal aspirate (NPA) specimens from equally many cases of pneumonia in 1,909 children (some children were included more than once). These children were, after obtaining informed parental consent, included in a study on zinc as adjuvant therapy for CAP (to be presented elsewhere). All included children were randomized to receive either 10 to 20 mg elemental zinc dispersed in water or placebo tablets daily for 14 days. Cases with very severe pneumonia/disease, that is, cough or difficult breathing with stridor when calm or any general danger signs (inability to drink/breastfeed, persistent vomiting, convulsions, lethargy, or unconsciousness) were not included in the study, but instead referred to a tertiary level hospital after initial treatment. Other exclusion criteria are listed in the study profile (Figure 1). Children could not participate in the study again until after 6 months due to the 6-month follow-up scheme of the clinical trial.

The child's respiratory rate (RR) was assessed according to WHO guidelines [19], counting twice for 1 min using a UNICEF timer. The lower of the two counts was used in the analyses. Children were weighed using a UNICEF electronic scale (SECA, Hamburg, Germany) accurate to 100 g with a mother/child-function, so the weight of the child could be determined while held by his or her mother. The child's length/height was measured to the nearest 0.1 cm using a wooden measuring board (as recumbent length in children <2 years of age and as height in children ≥ 2 years of age). Oxygen saturation (SpO₂) was measured either on a finger or a toe with a pulse oxymeter (Siemens MicO2, Siemens Medical Systems Inc, Danvers, MA, USA) using a pediatric sensor (Nellcor, Pleasanton, CA, USA). It was recorded twice 1 min apart after stabilization of the sensor for 1 min. The higher of the two measurements was used in the analyses. The concentration of C-reactive protein (CRP) was determined from a capillary or venous blood specimen using a semi-quantitative rapid test (QuikRead^® CRP, Orion Diagnostica, Espoo, Finland) and a portable photometer (QuikRead^® 101, Orion Diagnostica) according to the manufacturer's instruction. The test had a measurement range of 8 to 160 mg/L and values outside the measurement range were indicated as <8 or >160 mg/L. NPA specimens were obtained using a sterile, disposable suction catheter (Pennine Healthcare Ltd, Derbyshire, UK) with a suction trap (trachea suction set, Unomedical a/s, Birkerød, Denmark) connected to a foot pump (Ambu^® Uni-Suction Pump, Ambu A/S, Ballerup, Denmark). The catheter was inserted through the nostril to a distance equivalent to that between the patient's earlobe and nostril [21]. Suction was applied for a minimum of 10 sec with maximum negative pressure of 200 mmHg. Secretion remaining in the catheter after suction was recovered by rinsing 2 to 3 ml virus transport medium (DiagnoStick^®, Department of Microbiology, University Hospital of North Norway, Tromsø, Norway or UTM, Copan Diagnostics Inc, Corona, CA, USA) through the catheter into the suction trap. The trap was then disconnected and sealed.

The specimens were refrigerated at 2 to 8°C following collection at the field clinic and transported on ice every working day to the main laboratory in Kathmandu, where they were vortexed and divided into three equal aliquots in sterile vials (CryoTubes™, Nunc AS, Roskilde, Denmark). The aliquots analyzed in Nepal were either frozen at -70°C or kept at 2 to 8°C in the refrigerator before analysis. A separate comparative study verified that there were no substantial differences in proportion detected between the two alternative storage temperatures for up to three months (unpublished data). One aliquot was immediately frozen at -70°C and transported to Norway on dry ice and again stored at -70°C in case there should be a need for reanalysis.

One aliquot of each specimen was tested at our research laboratory in Nepal at the Institute of Medicine, Tribhuvan University, for RSV, InfA and InfB, PIV types 1, 2 and 3, and hMPV using a commercially available multiplex reverse transcriptase PCR assay (Hexaplex Plus^®, Prodesse Inc, Waukeshaw, WI, USA) with minor modifications of the manufacturer's instructions [22] and according to previous descriptions [15]. In brief, nucleic acids were extracted from 360 μl of NPA (or plasmid RNA from positive control transcripts) using a nucleic acids extraction kit (Roche High Pure Viral Nucleic Acid Kit, F. Hoffman-La Roche Ltd, Basel, Switzerland) according to the manufacturer's instructions. Each run of the assay included a positive RNA control and a negative control (virus transport medium), starting at nucleic acid isolation. Specimens and negative controls were individually spiked with 40 μl of internal control during nucleic acid isolation to identify any inhibitors. cDNA was produced by reverse transcription using random hexamers, murine leukemia virus reverse transcriptase (ABI, Applied Biosystems, Foster City, CA, USA), RNase inhibitor (ABI) and 3 μl of extracted viral RNA. Amplification reactions were performed using GeneAmp^® PCR System 2700 (ABI). Ten μl of newly synthesized cDNA was added to a mix consisting of 2.5 U of AmpliTaq^® Gold DNA polymerase (ABI) and a Super-Mix containing seven pairs of forward and backward primers flanking unique sequences of the seven viruses (the hemagglutinin neuraminidase gene of PIV types 1, 2 and 3, the matrix protein gene of InfA, the NS1 and NS2 genes of InfB, the NS1 and NS2 genes of RSV and the nucleocapsid gene of hMPV). After initially holding the PCR mixture at 95°C for 10 min, amplification was performed as follows: two cycles at 95°C for 1 min, 55°C for 30 sec and 72°C for 45 sec, and then 38 cycles at 94°C for 1 min, 60°C for 30 sec, 72°C for 30 sec, followed by an additional 7 min at 72°C and immediate cooling to 4°C. After amplification, the PCR products were purified using Qiagen QIAquick PCR Purification Kit (QIAGEN Inc, Valencia, CA, USA) and analyzed by enzyme hybridization assay [15], measuring the optical density at 450 nm (OD ₄₅₀) using a micro-plate reader (Stat Fax^® 2100, Awareness Technology Inc, Palm City, FL, USA).

Three hundred and twenty-four NPA aliquots were stored beyond 3 months at 2 to 8°C before analysis in Nepal. Of these, we re-analyzed the 133 that yielded a negative result, now using the aliquot that had been frozen at -70°C and transported on dry ice to Norway. This was done at the Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway, using the Hexaplex Plus assay and an automated extraction platform (NucliSens^® easyMAG, bioMérieux, Durham, NC, USA). Nucleic acids were extracted from 400 μl of sample, negative and positive processing controls and amplification control using the extraction principle with magnetic particles of this platform.

The criteria for a positive test were OD ₄₅₀ ≥ 0.400 and at least four times greater than the OD ₄₅₀ of the negative control [22]. An OD ₄₅₀ < 0.300 with an OD ₄₅₀ of the internal control >2.00 indicated a negative test. A reading from 0.300 to 0.399 was interpreted as indeterminate and the sample examined again. If the same result was obtained on repeated testing, the NPA was deemed negative. If the OD ₄₅₀ of the internal control for a given NPA was <2.00 and sample absorbance was < 0.400 for all tested agents, the NPA was tested again. If the same result was obtained on repeated testing, the interpretation was indeterminate due to potential inhibition, and the case not included in the analyses.

The data were double entered and compared on a daily basis using Microsoft Visual FoxPro version 6.0 (Microsoft Corporation, Redmond, WA, USA). Statistical analyses were performed using Stata/MP 10.0 for Macintosh (Stata Corporation, College Station, TX, USA). The 95% confidence intervals (CI) for proportions were calculated with binominal exact confidence interval using the 'ci' command. Of the children included in this analysis, 274 were enrolled twice and 18 thrice. We used the 'cluster' option in Stata to adjust the confidence intervals of the proportions for repeated enrollments and thus allowed for possible dependence of observations in a child that was included more than once. Anthropometric measures were expressed as Z-scores, which were generated using the WHO Child Growth Standards 2005 [23]. Meteorological data for the Kathmandu airport weather station (located approximately 10 km from Bhaktapur) were obtained from Department of Hydrology and Meteorology, Ministry of Environment, Science and Technology, Kathmandu, Nepal. Mean daily values for relative humidity and temperature were calculated as the average of two daily measurements (relative humidity at 8.45 AM and 5.45 PM, and maximum and minimum temperature). We estimated the Spearman rank order correlation coefficient to describe the association between the monthly number of infections with each virus and meteorological factors.

The study had ethical clearance from the Research Ethics Committee of the Institute of Medicine at Tribhuvan University in Kathmandu and the Regional Committee for Medical and Health Research Ethics of Western Norway. The implementation of the project was in agreement with the international ethical principles for medical research involving human subjects as stated in the latest version of the Helsinki Declaration.