Acute respiratory infections (ARI) can be very severe in young children, and ARI account for one-fifth of all deaths in children less than five years of age. Of those mortalities, 70% occur in Africa and Southeast Asia [1]. Approximately one-third of children will develop lower respiratory tract infections (LRTI) within the first year of life [2]. Premature infants and/or those with compromised immune systems are particularly susceptible to developing respiratory syncytial virus (RSV) related LRTI [3].

Pediatric patients with either upper or lower respiratory tract infections are typically treated symptomatically as outpatients. Diagnostic specimens are generally only obtained and tested in hospital settings, and even then treatment is usually initiated without etiologic determination. In the US, about 1-2% of infants presenting with LRTI require hospitalization [4].

In studies conducted in Europe, a pathogen was identified in 60-85% of LRTI cases [5–7]. Viral etiology accounted for 39-62% of the cases while S. pneumoniae accounted for 37% of the cases. The viruses identified included RSV in 24-29% of the cases, rhinoviruses in 24%, human parainfluenza viruses (HPIV) in 10%, and human adenoviruses (HAdV) in 7%, influenzaviruses A and B (FLUAV and FLUBV) in 4-5% of the cases [5–8].

The etiology of viral pneumonia in Egypt was described in a study of patients under five years of age published in 1967–68. HAdV, RSV, human parainfluenza viruses 1 through 3 (HPIV-1, HPIV-2, and HPIV-3), and FLUAV infections were diagnosed by seroconversion [9]. In an effort to improve the treatment of patients with respiratory illness, Egypt embarked on formalized physician training in case management of ARI in the 1980s and 1990s [10, 11]. Recently, the burden of atypical pathogens as Chlamydia and Mycoplasma spp. causing LRTI in children was studied in Egypt [12]. HAdV was also detected using serological methods in 30% of the patients [13]. The clinical presentation of RSV and non-RSV infected children were recently compared in Egypt [14]. Moreover, the burden of a number of respiratory viruses in Middle East countries has been described in several studies [15–21]. Nevertheless, there is a dearth of literature and information regarding the viral etiology of respiratory tract infections in pediatric patients in Egypt and Middle East countries.

Respiratory viral diagnostics rely principally on four techniques: virus isolation in cell cultures, antibody detection (serology), antigen detection, and nucleic acid-based molecular methods [22, 23]. For rapid results in a clinical setting, virus isolation is not effective because results can take up to 14 days, which is not timely for treatment decisions. Antigen detection assays such as the direct fluorescence assays (DFA) are more commonly used in clinical settings, because same-day results can be obtained. Nucleic acid-based detection methods such as polymerase chain reaction (PCR) are the gold standard in research laboratories because they are very sensitive; however, these are cost prohibitive in many clinical settings, particularly in newly industrialized and developing nations.

In this study, we sought to determine the predominant viral etiologies of LRTI in pediatric patients and to examine the performance of different laboratory diagnostic methods for the detection of these viruses.

Determining the etiology of LRTIs in children has long been of interest to the research and clinical community. Viruses have been shown to be the causative agent in 36-85% of LRTIs among children [5–7, 21, 24]. Different sampling techniques, detection methodologies, and geographical areas can greatly influence the observed burden from each virus. In our study, we tested for eight of the most common respiratory viruses using three popularly used methodologies and identified at least one virus in 59.9% of the cases. Consistent with results of studies conducted in other countries, among the 324 viruses detected, RSV was the most common viral agent among children under five years of age, followed by HAdV; FLUBV was the least common virus detected [2, 21, 25–27].

DFA has been widely used in the clinical settings because of the high specificity and rapid results. However, DFA is not as sensitive as nucleic acid-based molecular methods. In our study, 22% of samples had a detectable virus by DFA, which is slightly lower than the 32-63% reported by other studies using an indirect immunofluorescence assay [28, 29]. Our DFA results indicate that RSV accounted for 17.6% of all the LRTI, followed by HPIV-3 (1.8%) and HAdV (1.6%); these percentages are on the low end of the ranges reported by other studies using similar methodologies [21, 28, 29]. FLUAV contributed to 0.5% of viruses detected using DFA, while other studies reported rates of 2-15% [28, 29]. This difference may represent a true difference in FLUAV burden between the two studies, particularly because the Tang et al. and Zhang et al. studies tested children up to 16 years of age. Other possibilities exist, such as the different sensitivities of the slightly different methods used or year-to-year variations in FLUAV prevalence. Our findings for HPIV-1 (0.9%), HPIV-2 (0%) and FLUBV (0%) using DFA were similar to another published study that reported rates of HPIV-1 (0.6%), HPIV-2 (0.1%) and FLUBV (0.2%) [28]. In contrast, other studies have reported higher detection levels of these three viruses, which could be due to differences in study populations, sensitivity of the assays, or sample quality. Finally, although the detection of co-infections using the immunofluorescence assay was reported [28, 29], we did not identify any co-infections with DFA, even though we detected co-infections using other methods. The SVC system is a recently developed method using R-Mix™ cells, and decreases viral detection times from 12–14 days for conventional methods to 24–72 hours [30]. Moreover, some studies demonstrated that the R-Mix™ SVC method is more sensitive for respiratory viruses detection than conventional cell culture, and does not significantly increase laboratory virus isolation costs [31, 32]. Our study identified a virus in 26.7% of the patients using the R-Mix™ SVC, which is comparable to a Malaysian study reporting 22% positive by conventional isolation in MDCK, Vero, and Hep-2 cell lines [33]. Using the SVC method, RSV was the most common virus isolated, followed by HPIV-1 and HPIV-3, which is consistent with results using traditional virus isolation methods [33]. LaSala et al. reported that the R-Mix™ system had a low sensitivity for HAdV detection [30] which our results also confirm (Additional files 2). The higher FLUAV prevalence reported by the Malaysian study could be due to the different age group enrolled in the Malaysian study (0–24 months). In both studies, FLUBV was the least common virus among young children with LRTI. Using the SVC system, RSV was successfully isolated. However, better results might have been obtained if the samples were directly inoculated into vials without freezing and thawing, because this is particularly detrimental to the RSV infectivity.

The rt-RT-PCR method detected a viral agent in 59.3% of the participants, which is similar to other studies (35-66%) that used nucleic acid-based techniques [26, 34, 35]. RSV is the most predominant virus among LRTI patients using rt-RT-PCR, DFA, and SVC, in agreement with studies worldwide [25, 26, 36]. Our study demonstrated that HAdV, detected in 18.5%, is the second most common causative viral agent for LRTI. Similar observations were reported by other studies [26].

Although for many years virus isolation was the gold standard method to diagnose respiratory virus infections, molecular methods have demonstrated superior viral detection sensitivity. Virus isolation remains an important aspect of virus detection because it is the only means of obtaining a viable infectious virus for further characterization. Isolation alone greatly underestimates the prevalence of respiratory viruses, based on results from nucleic acid detection methods used in this study. This is particularly true for viruses that do not grow well in culture or are highly susceptible to freeze/thaw cycles. For instance, in this study, the rate of prevalence for HAdV is greatly underestimated by DFA (1.6%) and SVC (3.6%) compared to rt-RT-PCR (18.6%). Studies that used a different laboratory technique for each virus had high detectable rates for viruses identified by PCR compared to viruses identified using DFA or virus isolation [21]. This difference should be considered when designing surveillance studies to estimate the burden of viral etiologies of respiratory diseases.

The greater the number of days between symptom onset and sample collection, the more difficult it is to detect a causative agent. Most respiratory viruses are present in high titers in the respiratory tract in the first three days after symptom onset, whereas viral nucleic acid may remain for longer periods. Therefore, isolation-based methods such as SVC loose sensitivity after the first three days post-onset of symptoms, and DFA is similarly affected when viral titers decrease over the course of infection. In contrast to SVC and DFA, rt-RT-PCR remains a sensitive method for virus detection even after two weeks after symptom onset. The high sensitivity of rt-RT-PCR means it can detect a very low titer virus, or viral nucleic acid long after the virus has disappeared, making it difficult to determine if the detected virus is the primary contributor to disease. Thus, nucleic acid detection results must be interpreted with caution, particularly if the sample was taken late after symptom onset [37].

With the development of the PCR scientists were able to detect co-infections at a level not previously possible [26, 34, 38]. One caveat of this approach is that it is unclear which virus(es) are contributing to disease. By virtue of the nucleic acid-based assay, there is no competition for detection of the various etiologies (unlike SVC) and the amplification step enables detection at lower quantities (unlike DFA). Consequently, PCR is the most useful method to detect co-infections representing near-past and current infections, because of its ability to detect very low viral titers and/or lingering nucleic acid still present later in the infection course. Several common or newly identified respiratory viruses were not assessed in this study, such as picornaviruses, coronaviruses, bocaviruses and newly discovered polyomaviruses, so their contribution to respiratory disease etiology and rates of co-infection in Egypt remain unknown.

We identified a viral etiology in 59.9% of cases of LRTI in children aged five years and under in Egypt. RSV and HAdV were the most commonly detected viruses in this study.