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- Materials and methods
- Supporting Information
Acute respiratory infections are a leading cause of morbidity and mortality worldwide. They represent around 2 million deaths per year, especially in infants. The burden of these infections is particularly important in developing countries. During the last decade, South East Asia received much attention from the international scientific community due to the emergence of respiratory viruses with pandemic potential (SARS-CoV, avian influenza A/H5N1 virus).
Respiratory infections can be caused by numerous viruses, including influenza viruses, parainfluenza viruses, human respiratory syncytial virus (HRSV), human metapneumo-virus (HMPV), human coronaviruses (HCoV), adenoviruses, human bocavirus, and human enteroviruses. Molecular techniques have become more and more popular to detect these viruses. Multiplex reverse transcription–polymerase chain reaction (RT-PCR) has been shown to be a sensitive tool and allows identification of a majority of respiratory viruses, as well as coinfections.[5-7]
In Lao PDR, the etiology of respiratory infections is still poorly documented. To improve the clinical management of the patients, limit unnecessary antibiotic use, and prevent opportunistic secondary infections, it appears important to develop surveillance and tools to assess the etiology of acute respiratory infections in this country.[8, 9]
The purpose of this study was to describe during a limited period of time the viral etiology of acute lower respiratory infections (ALRI) in patients hospitalized in two Lao hospitals by using a set of five multiplex RT-PCR/PCR targeting 18 common respiratory viruses.
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- Materials and methods
- Supporting Information
In this study, we report for the first time in Lao PDR the viral etiologies in patients hospitalized for ALRIs. We identified 186 respiratory viruses in 162 (55%) patients of all ages using 5 multiplex PCR/RT-PCR. Rhinovirus and HRSV were the viruses the most frequently detected, representing 35% and 26% of the total number of viruses observed, respectively. These results are consistent with other studies conducted in the region previously.[6, 15, 16] The majority of the patients included in the study were aged <5 years (64%), and 48% were <2 years old.
Human respiratory syncytial virus is frequently defined as the predominant virus associated with hospitalizations for ALRI in children aged ≤5 years.[17-19] However, in our study, we detected more rhinoviruses (HRhVs) and enteroviruses (35%) than HRSV (26%) among the 292 patients included. Even if HRhVs are typically associated with the common cold, recent studies suggest that these viruses may also be associated with more severe illness, including lower respiratory disease and asthma exacerbations.[21, 22] In this study, HRhV was detected in respiratory specimens from 33% of patients with bronchitis or asthma, in 25% of patients with bronchiolitis, and in 16% of those presenting with pneumonia. Rhinoviruses/enteroviruses were often implicated in coinfections (73% of all the coinfections detected). However, the clinical significance of the detection of a HRhV by a highly sensitive RT-PCR method has been questioned as these viruses can also be detected in asymptomatic children.[23, 24] Rhinoviruses and enteroviruses seem to circulate all year round, without clear seasonality.
Human respiratory syncytial virus was the second most common virus detected in this study with a total of 49 cases (26% of patients with a positive RT-PCR result). This virus is recognized as the leading cause of hospitalizations in children aged ≤5 years for respiratory illness in industrialized countries.[25-27] Similarly to other countries, we demonstrated a substantial burden of HRSV-associated ALRI in Lao PDR. The infants and children aged <5 years were significantly more frequently infected, and then the incidence of HRSV infections decreased with age, probably because of the development of anti-HRSV immunity which is boosted during each subsequent reinfection.[28-30] During the study period, the peak of HRSV activity occurred from June to October, which corresponds to the rainy season (Figure 4). Similar observations were also reported in neighboring countries.[17, 25, 31]
The overall incidence of influenza viruses infection was relatively low (12%), and the majority of the cases detected (69·6%) were among patients older than 5 years with a median age of 12·3 years. These results are in line with those of a preliminary study on influenza-like illness in Lao PDR in which the incidence of influenza was 10·4%. The influenza A virus strains detected during the study period were exclusively 2009 pandemic H1N1 viruses.
As expected and as reported in other studies, the serotypes 1 and 3 were the most frequent PIVs detected in Laos.[12, 17, 33] However, PIV-4 was also identified only in four cases and accounted for 24·5% of all the PIVs detected. PIV-4 is usually uncommon,[17, 34, 35] but has been identified in severe respiratory illnesses,[5, 36] and its role is probably more important than originally thought.[37, 38]
The overall prevalence of HMPV was 4%, which is comparable to the results reported in Greece, the USA, or Thailand. The detection rate of the human bocavirus, another recently discovered respiratory virus, was 3%. This prevalence appears to vary largely between countries: 0·4% in Cambodia where a similar study was conducted, 3·9% in Thailand, 16% in Vietnam, and 24·5% in China. Bocavirus is often implicated in coinfections.[12, 19, 43] In this study, 66% of the bocavirus strains detected were observed during multiple infections.
Human coronaviruses were detected in 4% of the ALRI patients in Lao PDR, which is comparable to other countries (China: 5%; Vietnam: 8%, Cambodia: 8%).[6, 16, 17] HCoV-OC43, HCoV-NL63, and HCoV-229E were identified only in patients aged <4 years while the only case of HCoV-HKU1 infection was observed in a 75 years old patient hospitalized for pneumonia.
Of the 162 patients infected by a respiratory virus, we detected 13·6% of coinfections. The majority of these multiple infections were identified in patients <5 years of age (90%) and associated a rhinovirus (15/22). As rhinoviruses were detected all year round, coinfection cases were also observed regularly each month. Respiratory virus coinfections being frequent,[5, 19, 44] it demonstrates the usefulness of the multiplex RT-PCR approach, which allows the detection of the most important viruses in only few reactions while multiple infections are often undetected in viral culture or by direct immunofluorescence. Nevertheless, the difficult question of the clinical significance of these multiple infections remains unanswered.
In our study, we did not see any association between coinfection and severity of the disease, which is in line with other reports,[5, 19, 24, 45] but this has been subjected to much controversy.[46-48]
We also did not find any significant association between any virus and disease severity.
In this study, bronchitis and pneumonia were the most frequent clinical presentations observed among all the age-groups of patients hospitalized for ALRI. Bronchiolitis was observed almost exclusively in patients <2 years of age (40/43 cases). The viruses that were most frequently detected in patients <2 years of age with bronchiolitis were HRSV (15) and HRhVs (14), which is consistent with previous observations.[49-51] Fifty-seven percent of pneumonia occurred in children below 5 years of age. A better understanding of the roles of the different viruses is of great importance as pneumonia is responsible for approximately 19% of all deaths in children aged <5 years, of which more than 70% take place in sub-Saharan Africa and South-East Asia. The two main viruses observed in pneumonia in Lao PDR were HRhVs and influenza A viruses.
Even if Lao PDR is significantly less populated than neighboring Thailand, Vietnam, and China, the viral etiologies observed in Laotian patients hospitalized with ALRI demonstrate some similarities to those of other South-East Asian countries. However, this study has several limitations. Indeed, it was conducted in only two sites (Vientiane Capital and Luang Prabang), and the second site was included only during the last 6 months of the study. Moreover, our sample size is limited, especially when we stratify by age and viral etiology. Finally, it was difficult to collect sputum, particularly in young children. Thus, identification of bacteria was not possible.
This study aimed at determining the main viral etiologies of patients hospitalized in Lao PDR with ALRI. Rhinoviruses, HRSV, and influenza virus were the more common viruses detected in the patients. Bronchitis and pneumonia accounted for the majority of the hospitalizations for ALRI. These data are consistent with those of the literature. This study demonstrated also the usefulness of multiplex PCR/RT-PCR to detect viral infections and to expand our knowledge of respiratory infections in such country where the data are still sparse. Although the low numbers of some viruses do not allow drawing clear conclusions and considering that bacterial infections cannot be dismissed, this study provides some important preliminary data that can be used for other more focused surveys in a larger population, for instance to better describe the seasonality of the respiratory viruses. The frequency of viral infection should be taken into account by pediatricians to avoid unnecessary use of antibiotics.