For a long time, the number of respiratory infections with clinical symptoms caused by respiratory viruses had no detectable causative agent. With the discovery of human metapneumovirus in 2001 by Bernadette van den Hoogen and her colleagues, virus discovery methods have become a focus of virologists, and there has been a marked increase in the number of newly detected viral pathogens. One of those pathogens is human bocavirus (HBoV), which was initially identified by Tobias Allander in 2005 in 17 respiratory samples from children suffering from a respiratory tract disease of suspected viral origin.

Since the discovery of HBoV, numerous studies related to the epidemiology, pathogenesis, and replication of this newly detected virus have been performed, and they have provided novel insights into viral infection of the respiratory tract and gastrointestinal diseases.

Human bocavirus (HBoV) was discovered in 2005 by Allander et al. in respiratory samples from children with suspected acute respiratory tract infection (ARTI) using a novel technique. This molecular virus screening is based on a random PCR-cloning-sequencing approach and was employed on two chronologically distinct pools of nasopharyngal aspirates (NPAs). It revealed a parvovirus-like sequence, with close relation to the members of the bocavirus genus.

A retrospective study revealed 17 (3.1 %) out of 540 NPAs positive for HBoV, with 14 specimens tested negative for other viruses, giving the suggestion that HBoV is a causative agent of respiratory tract infections.

Bocavirus is a single-stranded DNA virus belonging to the family Parvoviridae, subfamily Parvovirinae, genus Bocavirus. Bocaviruses are unique among parvoviruses because they contain a third ORF between the non-structural and structural coding regions–[2]. The genus bocavirus includes viruses that infect bovine, canine, feline, porcine and some simian species as well as sea lions–[8]. Human bocavirus (HBoV) was first found in children with acute respiratory tract infections in 2005. It was then detected in children with respiratory tract infections in addition to gastroenteritis worldwide–[12]. The virus exists in four different serotypes HBoV1-4–[2],–[14]. Although HBoV 1 and 2 were reported in respiratory samples, all the 4 genotypes of HBoV have been identified in children with acute gastroenteritis.

HBoV has been reported in various countries, indicating its worldwide endemic nature. The virus has been identified in Europe–[17], America–[19], Asia,, Australia–[22], Africa, and the Middle East. The prevalence of HBoV ranges between 1.5 to 19.3%,. Primary infection with HBoV seems to occur early in life and children between the ages of 6–24 months seem to be mostly affected–[10], but older children can also be infected. Newborn children may become protected by maternally derived antibodies. HBoV infections are rarely found in adults–[27]. Lindner et al. detected anti-HBoV antibodies in 94% of healthy blood donors >19 years of age.

HBoV detection has been mostly performed on nasopharyngeal aspirates and swabs and relies mostly on classical,,,,, and real-time PCR,,. Real-time PCR possesses many advantages over conventional PCR, as it offers greater sensitivity, specificity, and reduced expenditure of time.

The current study aims to screen the epidemiological status and molecular phylogeny of HBoV isolates prevailing in pediatric patients with respiratory infection in Saudi Arabia.

Samples were not collected from patients with chronic respiratory ailments; non-consenting caregivers, with history of hospitalization in the preceding 14 days, not admitted in hospital and children aged > 5 years.

Children with SARI, admitted in J. K. Lone Hospital, a pediatric hospital attached to Sawai Man Singh (SMS) Medical College Jaipur were enrolled in the study and tested for respiratory viruses with prior consent of the parent/guardian. Duration of the study was 27 months i.e. between September, 2012 to December, 2014. Children enrolled were ≤ 5 years of age, presenting with fever, cough, sore throat, nasal catarrh, shortness of breath, bronchiolitis, pneumonia, and wheezing.

In 2005, Allander et al., reported the discovery of a previously undescribed human parvovirus in respiratory secretions from children with respiratory tract disease in Sweden. Phylogenetic analysis showed that this virus belonged to the genus Bocavirus (subfamily, Parvovirinae; family, Parvoviridae) and was most closely related to bovine parvovirus (BP) and minute virus of canines (MVC). The virus was thus named "human bocavirus" (HBoV).

Human bocaviruses (HBoV) contain 3 open reading frames (ORFs) encoding a nonstructural protein (NS1, NP1) and two capsid proteins VP1 and VP2, respectively. The genomic organization of HBoV closely resembles that of bovine parvovirus type 1.

HBoV has been reported in respiratory samples from children with acute respiratory tract disease in various parts of the world (including Australia, North America, Europe, Asia, and Africa), suggesting that the virus is circulating worldwide. Pneumonia, acute bronchitis, bronchiolitis, are the main manifestations of HBoV infection.

HBoV seems to be a new member of the community-acquired respiratory viruses such as respiratory syncytial virus, adenovirus, influenza virus, parainfluenza virus, and rhinovirus, which cause common respiratory tract infections in the community. Because of its very high copy numbers in respiratory tract secretions, aerosol and contact transmission are likely effective, as they are for other respiratory viruses.

HBoV has been detected also in nasopharyngeal, serum, fecal and urine samples obtained mainly from young children around the age of 2 years predominantly during the winter season. HBoV was detected in two pediatric patients after organ transplantation, in human immunodeficiency virus-infected pediatric patients, and immunosuppressed adult patients.

Diagnosis of HBoV infection is based on the PCR amplification of viral genome fragments present in human respiratory, serum, stool and urine samples. A great number of different PCR techniques employing varying sets of primers specific for the viral genes NP1[10], NS1, and VP1, VP2 have been described. In addition to the detection of viral genomes by PCR, recent reports describe the detection of HBoV-specific IgG and IgM-antibodies against HBoV VP2 in serum samples using western blot or immunofluorescence assays. Furthermore, a standardized ELISA for the quantitative determination of HBoV-specific antibodies has been established by. The aim of the work was determination of HBoV in respiratory specimens (NPA) of infants by qualitative PCR and determination of acute HBoV infection by estimation of IgM antibodies in serum by ELISA.

We undertook a PCR-based study to identify and characterize respiratory viruses associated with ALRI in young PNG children.

Cultures of nasopharyngeal aspirates from 100 patients were positive in 67% of patients. Cultures yielded growth of one or more pathogen among upper respiratory tract normal flora. Gram positive cocci were isolated from 57/90 (63.3%) of specimens including: Streptococcus pneumoniae 25/90 (27.7%), Staphylococcus aureus 32/90 (35.5%). Gram negative bacilli were detected in 33/90(36.6%) of specimens including, Klebsiella pneumoniae 15/90 (16.6%), Escherichia coli 10/90 (11.1%), Pseudomonas aeruginosa 8/90 (8.8%), while Cultures of the control group showed growth of upper respiratory tract normal flora (table 1).

HBoV is a major pathogen detected in respiratory and gastrointestinal infections. To date, four subtypes have been identified, and they are found worldwide, without any regional, geographic, or border restrictions. Following its initial discovery in Sweden, HBoV has been detected all over Europe, North and South America, Africa, Asia, and Australia.

The four distinct subtypes have been named HBoV1–4. Subtype 1 is mainly associated with respiratory diseases, but can also be found in stool samples from patients suffering from diarrhea. The prevalence in symptomatic patients is approximately 1.5–16% for HBoV-1 21–26% for HBoV-2, approximately 1% for HBoV-3, and 0.6% for HBoV4. It appears that HBoVs have a high frequency of recombination among each other, as some subtypes are derived from recombinations of two others, and novel variants seem to occur more frequently than initially assumed.

The seroprevalence of HBoV is strongly dependent on the age of the investigated patient cohort and ranges from ~40% in children between 18 and 23 months of age up to virtually 100% in children older than two years, with an average of 76.6% in children and 96% in adults. The seroprevalence is lowest for HBoV-4 (0.8–5%), followed by HBoV-3 (10–38.7%), HBoV-2 (34–49.3%), and HBoV-1 (66.9–96%).

Recent clinical studies on respiratory infections that used novel multiplexing assays have shown that more severe infections (i.e., infections that are clinically relevant, require hospitalization, and receive a proper laboratory diagnosis) frequently represent co-infections with up to six different pathogens in a single patient. The range of co-infections or, more precisely, the rate of co-detection of pathogens that occur simultaneously with HBoV (or vice versa), ranges from 60 to 90%. This high rate can be explained by the fact that HBoV can be shed by asymptomatic patients and is able to persist, but it could also be the result of a better study design. During the last several years, it has become impossible to publish a study on respiratory viruses without screening for all viruses known at the time of the study. This requirement is one reason why the aforementioned studies used multiplexing technology and revealed marked frequencies of double or multiple infections (up to 44%) independent of the pathogens investigated. Therefore, HBoV is not exclusively a bystander, but rather, the study cohorts investigated in the past (mainly hospitalized patients) suffered from multiple infections more frequently than previously assumed.

Acute lower respiratory infections (ALRI) are the leading cause of death in children, accounting for approximately two million deaths worldwide annually.1 Currently there is limited information on the role of many of the respiratory viruses in lower respiratory tract infections in children. This is particularly true in developing countries where this knowledge is critically important in improving the management and prevention of childhood respiratory infections. Children in the highlands of Papua New Guinea (PNG) suffer an average of 4.3 ALRIs by the age of 18 months.2 Previous studies of respiratory viral infection in young children in PNG relied on the detection of viral antigens by immunofluorescence and traditional cell culture techniques.3,4 Compared with nucleic acid detection tests, such techniques lack sensitivity for the detection of many respiratory viruses, particularly the recently described human coronaviruses, respiratory polyomaviruses, bocavirus and rhinovirus C.5–10 Furthermore, since a number of these viruses have been detected commonly in the respiratory tract of asymptomatic children in developed countries, their pathogenicity is uncertain.11–15

HBoV is a putative member of the family Parvoviridae, subfamily Parvovirinae, genus Bocavirus. Before identification of HBoV, parvovirus B19 of the genus Erythrovirus was the only known human pathogen in the family of parvoviruses. Parvovirus B19 is widespread and manifestations of infection vary with the immunologic and hematologic status of the host. In immunocompetent children, parvovirus B19 is the cause for erythema infectiosum. In adults it has been associated with spontaneous abortion, non-immune hydrops fetalis, acute symmetric polyarthropathy, as well as several auto-immune diseases.

Based on its genomic structure and amino acid sequence similarity shared with the namesake members of the genus, bovine parvovirus (BPV) and canine minute virus (MVC), HBoV was classified as a bocavirus and therefore provisionally named human bocavirus.

Other subfamily Parvovirinae members known to infect humans are the apathogenic adeno-associated viruses of the genus Dependovirus and parvovirus 4. Parvovirus 4 has not yet been assigned to a genus, but it was proposed to allocate it to the genus Hokovirus as it shares more similarities to the novel porcine and bovine hokoviruses than with other parvoviruses. Recently a second human bocavirus has been identified, HBoV2, with 75.6 % nucleotide similarity to HBoV. HBoV2 was found in stool samples from Pakistani children as well as in samples from Edinburgh (1 of the 3 positive samples was derived from a patient >65 years old), indicating that it is not restricted to one region or to young children.

Saffold virus (SAFV) is an emerging pathogen identified in the United States in 2007. The virus´ name traces back to the name of the first author of the original description. The virus is classified in the species Cardiovirus B (formerly named Theilovirus) within the genus Cardiovirus (genus supergroup 1) of the family Picornaviridae [1–4].

SAFV is closely related to the other members of the Cardiovirus B species, i.e., Theiler's murine encephalomyelitis virus (TMEV), Theiler-like rat virus (Thera virus, TRV), and Vilyuisk human encephalomyelitis virus (VHEV). SAFV is a small non-enveloped virus with a single-stranded RNA of about 8,050 nucleotides. Up to now, eleven different SAFV genotypes have been identified (http://www.picornaviridae.com/).

SAFV-1 has initially been isolated from a fecal specimen of an 8-month-old girl with fever of unknown origin. Following this observation, SAFV was detected frequently in nasal specimens of children with respiratory infections (0.2%-24%,) and stool samples from children suffering from acute gastroenteritis (0.2%-3%,) [1, 4–13]. Due to these findings, SAFV was supposed to be a relevant new human pathogen, especially in children. According to antibody seroprevalence studies, SAFV infection is highly common and occurs early in life, with approximately 80% of seropositive children at the age of 2 years [14–16]. SAFV is spread worldwide [1, 3–4, 6, 9–10, 12–17].

The association of SAFV with various diseases is currently under research. Besides respiratory and gastrointestinal illnesses, the disease spectrum may involve type 1 diabetes and neurological disorders [7, 17]. Furthermore, the virus was detected in nasopharyngeal swabs from children with exudative tonsillitis (9/37,) and in autopsy samples from myocardium, lung and blood from a child with myocarditis. Additionally, the virus was found in feces from Asian children with non-polio acute flaccid paralysis and in cerebrospinal fluid specimens of patients with aseptic meningitis [12, 20–21]. This is of particular importance, because other members of the Cardiovirus B species are neurotropic. E. g., TMEV is known to cause a multiple sclerosis (MS)-like syndrome in mice. However, there are also reports i) describing SAFV detection with similar percentages in healthy and diseased individuals, e.g., in stool samples [9, 22–23] and, ii) reports failing to detect SAFV in samples from diseased patients, e.g., in cerebrospinal fluid from individuals with aseptic meningitis, encephalitis, and MS [24–25], and, finally, iii) reports co-detecting SAFV with common gastroenteritis pathogens in stool samples in case of diarrhea. The true clinical significance and pathophysiology of SAFV thus has to be elucidated.

In order to extend our knowledge on SAFV presence in healthy children, we analyzed adenoid tissue and throat swab samples from children who did not display symptoms of a respiratory tract infection for SAFV RNA. Samples were collected in the course of elective adenoidectomy.

Viral infections of the respiratory tract are responsible for significant mortality and morbidity worldwide. Despite extensive studies in the past decades that have identified a number of etiologic agents, including rhinoviruses, coronaviruses, influenzaviruses, parainfluenzaviruses, respiratory syncytial virus, and adenoviruses, approximately 30% of all cases cannot be attributed to these agents, suggesting that additional respiratory pathogens are likely to exist. In fact, since 2001, six previously undescribed viruses have been identified by analysis of clinical specimens from the human respiratory tract: human metapneumovirus, SARS coronavirus, coronavirus NL63, coronavirus HKU1, human bocavirus, and the recently described KI virus. In some instances, new molecular methods such as VIDISCA, pan-viral DNA microarrays, and high throughput sequencing have played key roles in the identification of these agents. The advent of these new technologies has greatly stimulated efforts to identify novel viruses in the respiratory tract and in other human disease states.

Viruses in the family Polyomaviridae possess double-stranded DNA genomes and infect a variety of avian, rodent, and primate species. To date, two polyomaviruses, BK virus and JC virus, have been unambiguously described as human pathogens. BK and JC viruses are ubiquitous worldwide, and in adult populations, seroprevalence rates approaching 75% and 100%, respectively, have been reported. Although human polyomaviruses have been suggested to utilize a respiratory route of transmission, detection of BK and JC polyomavirus nucleic acids in the respiratory tract has rarely been reported. Infection with these two viruses is predominantly asymptomatic, although in the context of immunosuppression a number of syndromes have been clearly linked to these viruses. JC virus causes primary multifocal leukoencephalopathy, while BK virus has been associated with a variety of renal and urinary tract disorders, most importantly tubular nephritis, which can lead to allograft failure in renal transplant recipients and hemorrhagic cystitis in hematopoietic stem cell transplant recipients. These viruses are believed to persist in a latent phase primarily in the kidney and can periodically undergo reactivation. Excretion of BK and JC viruses in urine has been reported in up to 20% of the general population. Besides JC and BK virus, a very recent report has described a novel polyomavirus, KI, detected in human respiratory secretions and stool. However, the pathogenicity and prevalence of this virus has not yet been established. In addition, in the late 1950s, ∼100 million people in the United States, and many more worldwide, may have been exposed to SV40, a polyomavirus that naturally infects rhesus monkeys via contaminated polio vaccines, leading to widespread debate about whether or not SV40 is capable of sustained infection and replication cycles in humans.

Much of the interest in polyomaviruses and SV40 in particular derives from the transforming properties carried by the early transcriptional region of the viral genome that encodes for the small T antigen (STAg) and and large T antigen (LTAg). T antigen is capable of binding both p53 and Rb proteins and interfering with their tumor suppressor functions. The early region alone is sufficient to transform established primary rodent cell lines and in concert with telomerase and ras transforms primary human cells. This has lead to controversy over whether any human tumors are associated with SV40 infection.

We describe the identification and characterization of a novel polyomavirus initially detected by high throughput sequencing of respiratory secretions from a patient suffering acute respiratory disease of unknown etiology. The virus was detected in the respiratory secretions from an additional 43 patients in two continents, and the complete genomes of multiple isolates were sequenced.

The primary study subjects were 5,298 patients, who underwent multiplex real-time polymerase chain reaction (PCR) for simultaneous detection of respiratory viruses, among who visited the emergency department or outpatient clinic with respiratory symptoms at Ulsan University Hospital from April 2013 to March 2016 (Figure 1). The medical records of all these patients were retrospectively reviewed in detail.

Acute respiratory infection is one of the main causes of hospitalization and death worldwide, although identification of the aetiological agent is not achieved in a majority of cases. Instead, the infections are treated empirically and often successfully with antimicrobial therapy. Nonetheless, the roles of viruses in the aetiology of these infections are becoming clear, especially after the 2009 pandemic of the new Influenza A subtype H1N1. The presence of a virus does not imply either a more benign clinical course or that systemic inflammatory responses or complications will be absent.

Due to its implications for Public Health, the efforts in reinforcing and improving the epidemiological surveillance of respiratory infections have increased. Under this initiative, countries have developed surveillance systems by following cases of influenza-like illness and severe acute respiratory infections (SARIs), which are clinically diagnosed among patients with fever, coughing or sore throat, difficulty breathing and the need for hospitalization. The main aims of surveillance have been to provide information on circulating viruses and the susceptibility of Influenza to available antivirals and also to promote and define vaccination needs in different populations.

The true impact of viral infections in the aetiology of acute respiratory disease requiring hospitalization is unknown. The aims of this study were to identify viral aetiologies in hospitalized adult patients with SARI in Bogotá in 2012 and to describe the characteristics and clinical outcomes among these patients.

Respiratory viral infection is a significant etiology for community-acquired pneumonia123. With the development of detection techniques, respiratory viruses have been detected in 10%–30% patients with community-acquired pneumonia45. Respiratory virus infections are also a frequent cause of bacterial pneumonia. In a systematic review of previous studies, the proportion of bacterial pneumonia in patients with influenza was found to range between 11% and 35%6.

In a study of pandemic and seasonal influenza virus infections, the most common bacterial pathogens found in patients with post-influenza pneumonia were Staphylococcus aureus and Streptococcus pneumoniae78. Elucidation of the pneumonia-causing pathogens in patients with respiratory viral infection is important, because respiratory viral infection complicating bacterial pneumonia is associated with a worse prognosis and high mortality rate compared with respiratory viral infection only 9, although the prognosis can be improved with early and appropriate empirical antibiotic treatment7910. However, the predominant bacterial species causing pneumonia secondary to respiratory viral infections other than influenza remain unknown. Accordingly, the aim of the present study was to know whether the pathogens causing post-viral bacterial pneumonia vary according to the type of preceding respiratory virus.

Acute bronchiolitis (AB), which is the most common acute lower respiratory system disease in infants, is often caused by a viral infection. It is especially the leading cause of hospitalization in infants under 6 months of age.1,2 Epidemic peaks of AB are frequently seen during the winter season. Respiratory syncytial virus (RSV) is usually the cause of 50% to 80% of the cases, but other viruses including adenovirus, influenza virus, and parainfluenza virus have also been reported to cause AB as the sole pathogen or as coinfection with or without RSV.3,4 With various polymerase chain reaction (PCR) techniques, possible new agents like rhinovirus, human metapnomovirus, human bocavirus, Bordatella pertussis, and atypical pathogens were also described as the leading causes of AB.5-7 Having a cardiovascular disease, chronic pulmonary disease, immunodeficiency, and premature birth increase the risk of AB-associated respiratory failure, or even death.8 The World Health Organization has reported that RSV is the causative pathogen for over 30 million new acute lower respiratory infection episodes in children under 5 years of age and it gives rise to more than 3.4 million hospital admissions and 160 000 deaths every year.9,10

The diagnosis of AB is made based on typical history with wheezing and characteristic clinical features such as tachypnea, nasal flaring, chest retractions, and wheezing and/or rales followed by a viral upper respiratory infection in infants. The American Academy of Pediatrics (AAP) 2006 Clinical Practice Guidelines for the Diagnosis and Management of Bronchiolitis described AB as the first episode of wheezing in children under 24 months of age who have respiratory findings during the viral infection episode.11 Chest radiographs and laboratory studies may be thought of on clinical suspicion after evaluating the differential diagnosis for secondary or comorbid bacterial infection, complications, or other conditions. Viral diagnosis methods including antigen detection or immunofluorescence of nasal secretion wash or nasal aspiration, rapid antigen tests, and PCR are only suggested for identifying specific viral agents in children with bronchiolitis if the results will determine discontinuation of palivizumab prophylaxis, initiation or continuation/discontinuation of antibiotic therapy.12-15

Majority of studies have recently researched the burden of respiratory viral tract infection agents in AB with larger groups. In these studies, epidemiological, clinical, and risk factors of AB have also been defined. So, it can be said that AB is frequent in infancy and that there is an increase in the number of admissions to hospitals and bronchiolitis-related morbidity.

The purpose of this study was to evaluate the frequency of pathogens and to determine the differences in clinical and microbiological features among patients under 24 months of age, who were hospitalized with AB in Ege University Children’s Hospital.

The respiratory tract is the most common focus of infection in septic patients. In adults with community-acquired pneumonia (CAP), a bacterial etiology can be established in about 25–50% of cases depending on definitions and methods used [1, 2]. S. pneumoniae is the most often found bacteria, followed by H. influenzae and Mycoplasma pneumoniae. More rarely found agents are Legionella pneumophila, Chlamydophila pneumoniae, and Coxiella burnetti [1, 3]. Several respiratory viruses may also cause severe respiratory disease, including CAP, mainly in children, but also in adults. This is well known for influenza A and B viruses, respiratory syncytial virus, coronavirus, human metapneumovirus, parainfluenza viruses types 1–3, adenovirus, enteroviruses, rhinovirus, and human bocavirus [3–5]. Indeed, viral infections are estimated to cause around 100 million annual cases of CAP worldwide. Many of these viruses have seasonal variation patterns, causing epidemics, often with peaks during winter and early spring. Viral respiratory infections may predispose for bacterial infections by damaging the respiratory epithelium as well as by viral-bacterial interactions. For example, influenza A virus can enhance the pathogenicity of S. pneumoniae, Staphylococcus aureus or H. influenzae. On the other hand it can inhibit the pathogenicity of others, such as M. pneumoniae and C. pneumoniae [8–11]. Using molecular techniques it has become evident that viral infection is present in around 25% of CAP, regardless of severity [1, 12, 13]. Viral co-infections in CAP has been shown to increase both disease severity and length of stay in hospital. In patients with pneumonia requiring intensive care, mixed viral-bacterial infections have demonstrated the highest mortality rate in at least one study.

Commercial multiplex tests are continuously being developed, allowing for rapid etiological diagnosis of a wide range of respiratory viruses and bacteria [4, 15–18]. Diagnosing viral respiratory infections may help in reducing admissions, length of stay, use of antibiotic treatment and, in some cases, target antiviral treatment. Testing is optimal during the first days of infection, when the viral load is high. The clinical significance of a viral finding cannot always be determined. Some respiratory viruses, like rhinovirus, can persist in young children up to 6 weeks after a clinical infection, though persistence and long-term carriage seems to be less frequent in adults [1, 21].

ARIs are one of the major causes of mortality and morbidity in children especially in developing countries. The World Health Organization (WHO) estimated that 1.9 million children die every year due to respiratory tract infections (RTIs), mainly pneumonia in African and Southeast Asian countries. The incidence of influenza-like illness (ILI) is almost similar between developed and developing countries like Nepal; the mortality rates are higher in developing countries. The frequency of mixed infections of noninfluenza ARI viruses with influenza virus varies from 10 to 30%, and studies have suggested an association between mixed viral infections could increase the disease progression or clinical severity.

The most common etiology of ARI worldwide includes influenza virus (InfV), respiratory syncytial virus (RSV), rhinovirus (RV), metapneumovirus (MPV), bocavirus (BV), adenovirus (AV), enterovirus (EV), Mycoplasma pneumoniae, parainfluenza virus (PIV), coronavirus (CoV) OC43, NL63, 229E, and HKU1. Influenza virus types A and B are the leading cause of ARI and serious outbreaks worldwide during the winter season. Annual epidemics of influenza virus alone can cause 5–15% ARI globally. According to WHO, 3–5 million severe cases and 250,000–500,000 deaths occur globally due to annual influenza.

In tropical and subtropical countries in the South East Asia such as Thailand, Singapore, Malaysia, and China, the etiologic agents associated with ILI have been well characterized. However, epidemiology and etiology for ILI is poorly understood in Nepal. A laboratory-based ILI surveillance system is well established in Nepal and has greatly contributed to outbreak investigation, surveillance, and timely response since the emergence of pandemic influenza in 2009. The influenza viruses circulating in Nepal have many similarities with our neighboring countries and the regions. We reported year-round transmission of influenza with a peak activity during the rainy and winter seasons is similar to Thailand, Northern Vietnam, and Lao PDR. An efficient detection method is of great importance for laboratory diagnosis. Rapid antigen detection, viral cultures, and molecular methods, such as PCR assays, are currently used as the viral detection method. In clinical settings, rapid and reliable PCR assays are particularly helpful in making early decisions regarding management and treatment of the patients. In this study, we attempt to characterize the influenza and other ARI pathogens by the multiplex PCR assay in children with influenza-like illness cases.

The definition and criteria for sepsis have changed over the years. Currently, sepsis is defined as a “life-threatening organ dysfunction caused by a dysregulated host response to infection” and said to be present if a patient has an infection and +2 points or more in the SOFA-score. From a clinical viewpoint, a patient is suspected to be septic if there has been a sudden onset of chills and fever accompanied by abnormalities in vital signs, such as an increased respiratory rate, lowered oxygen saturation, tachycardia, hypotension, altered mentation, general malaise, and if laboratory findings such as an increase in leucocytes, C-reactive protein, lactate or procalcitonin support that suspicion. If so, broad-spectrum antibiotic treatment is started on clinical suspicion of bacterial sepsis and according to the preliminary diagnosis. Treatment is later modified according to the results of cultures, other microbiological detection methods, infection biomarkers, and imaging. In most cases, the origin and etiology of the infection cannot be established in the emergency department. Clinical symptoms and signs are helpful but not fully reliable. The inflammatory response in sepsis may itself cause symptoms and signs that can be misleading. For example, a bacteremic urinary tract infection may present with predominating respiratory symptoms and signs. This warrants broad testing early during care.

In this study conducted during the winter season, we pragmatically investigated the clinical relevance of nasopharyngeal viral and bacterial findings from clinically septic patients with suspected respiratory focus or sepsis of unknown origin.

All respiratory viruses may cause symptoms such as nasal congestion, runny nose, wheezing, and cough. We found no significant association between the viruses and a specific symptom.

Acute lower respiratory tract infections (LRTIs) are responsible for significant morbidity and mortality in infants and young children worldwide. Childhood pneumonia accounts for 16% of all deaths of children under 5 years of age and 7–13% of all community cases are life-threatening and require hospitalization. The most common causes of acute LRTIs with divergent presentation are viruses. Respiratory syncytial virus (RSV) is the most common viral cause of pneumonia in young children [1, 2].

Human bocavirus 1 (HBoV1), the second human pathogenic virus in the family Parvoviridae, was discovered in 2005 in nasopharyngeal aspirates (NPAs) of children with acute respiratory tract infection (ARTI). Since then, HBoV1-associated ARTIs have been reported worldwide.

On the one hand, there are studies showing evidence of HBoV1 as a pathogen in association with LRTIs, mainly in children up to 3 years of age [5–10]. On the other hand, HBoV1 has also been found in asymptomatic children [11–14]. These states of affairs at first may seem contradictory; however, HBoV1 has been shown to remain in the nasopharynx for several weeks and even months, thereby causing clinically false polymerase chain reaction (PCR) diagnoses. Moreover, life-threatening and even fatal HBoV1 infections have been reported [15–19]. The definition and diagnosis of acute HBoV1 respiratory tract infection is challenging. Detection of HBoV1 deoxyribonucleic acid (DNA) in blood, messenger ribonucleic acid (mRNA), and viral load assessment in respiratory samples and serology have been recommended as the markers to diagnose active HBoV1 infection [6, 7, 20, 21]. According to the best of our knowledge, there are no studies up to now showing detection of HBoV1 mRNA in the peripheral blood mononuclear cells (PBMCs) as a diagnostic marker for acute HBoV1 infection.

In this study, we report an acute HBoV1 infection in an otherwise healthy child with life-threatening acute bilateral bronchiolitis and right-side pneumonia with detected HBoV1-specific immunoglobulin (Ig) M and DNA in cell-free blood plasma as well as HBoV1 mRNA in PBMC.

The median age of patients was 57.2 years (range, 23–88) and the male-to-female ratio was 1 to 2.27; 219 patients (92.02%) were farmers and 19 (7.98%) were workers or students. Among patients, 52 (21.85%) reported a tick bite within 2 weeks (5–14 days) before the onset of clinical manifestations; the remaining patients did not recall receiving a tick bite.

The main clinical features in confirmed patients included sudden onset of fever (>37.5°C −40°C) lasting up to 10 days, fatigue, anorexia, headache, myalgia, arthralgia, dizziness, enlarged lymph nodes, muscle aches, vomiting and diarrhea, upper abdominal pain, and relative bradycardia (Table 1). A small number of cases suffered more severe complications, including hypotension, mental status alterations, ecchymosis, gastrointestinal hemorrhage, pulmonary hemorrhage, respiratory failure, disseminated intravascular coagulation, multiple organ failure, and/or death. Most patients had a good outcome, but elderly patients and those with underlying diseases, neurological manifestations, coagulopathy, or hyponatremia tended to have a poorer outcome.

Laboratory tests showed that confirmed patients characteristically developed thrombocytopenia, leukopenia, proteinuria, and elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (Table 2). Biochemical tests revealed generally higher levels of lactate dehydrogenase, creatine kinase, AST and ALT enzymes, especially AST.

Children with viral infection in the upper respiratory tract showed symptoms such as runny nose, cough, and hoarseness. Some of them also present lower respiratory tract symptoms such as wheezing, severe cough, breathlessness, and respiratory distress, which may be due to bronchiolitis or pneumonia.

We divided the patients according to three clinical manifestations: pneumonia, bronchiolitis, and bronchitis, and investigated whether the detected viruses were associated with a specific clinical manifestation. Our analysis showed that in most cases, RSV infections induced bronchiolitis (n = 45), followed by pneumonia (n = 22, p=0.004) and bronchitis (n = 6, p=0.0015). EV/Rhi infections more often induced pneumonia or bronchiolitis (n = 19 for both) instead of bronchitis (n = 6, p=0.03) (Figure 2). Other viruses showed a similar prevalence of each clinical manifestation.

In recent years, improved nucleic acid amplification and detection technology has facilitated the identification of pathogens that had previously proved difficult or impossible to detect using traditional culture or immunofluorescent techniques. The increased use of molecular methods has also resulted in the discovery of novel respiratory pathogens including the human coronaviruses NL63, HKU1[2] and the SARS coronavirus, human metapneumovirus, human bocavirus and two human polyomaviruses, KI and WU. Despite these advances, there remain a significant proportion of respiratory disease episodes for which a pathogenic agent can not be identified [8, 9].

The aim of this study was to modify real-time PCR assays to facilitate the rapid screening of respiratory samples for a comprehensive range of viral and bacterial pathogens. Standard PCR assays require extraction of RNA and DNA with simultaneous removal of inhibitors of the PCR process from the sample. Multiple extracts from each sample have been required to set up a comprehensive range of PCR assays for respiratory pathogens, and the sample volume available may be insufficient for all of the required tests. Also, previous attempts at multiplex PCR have been limited by competition between different amplification reactions and by the limited number of fluorescent probes able to be monitored in the same tube. A multiplex tandem PCR assay which utilized a two step PCR system consisting of a limited multiplex PCR followed by a single target PCR has been reported. Our study further modified that procedure to include real-time PCR with specific probes in the second step of the assay. It comprised two first round enrichment PCR assays (A and B) containing 27 and 7 primer pairs respectively, and the second step real-time PCR assays containing 1 – 3 primer pairs and specific Taqman probes. The assay detects several adenovirus genotypes, Bordetella species, human bocavirus, Chlamydophila pneumoniae and psittaci, coronaviruses OC43, 229E, HKU1 and NL63, Haemophilus influenzae, influenza viruses A, B and C, Legionella longbeachae and pneumophila, Moraxella catarrhalis, Mycoplasma pneumoniae, parainfluenzaviruses 1 – 4, Pneumocystis jirovecii, KI and WU polyomaviruses, respiratory syncytial virus types A and B, Streptococcus pyogenes and pneumoniae. Supplementary semi-nested human metapneumovirus and picornavirus PCR assays are required to complete the acute respiratory pathogen profile.

This technique facilitates the investigation of a comprehensive range of respiratory pathogens from a single nucleic acid extract and a single reverse transcription reaction. In addition, the ability to amplify multiple targets efficiently aids in the detection of mixed infections.