Rujipat Samransamruajkit

High prevalence of human rhinovirus C infection in Thai children with acute lower respiratory tract disease

Summary Objective To determine the prevalence of human rhinoviruses (HRV) infections in children with lower respiratory disease in Thailand and monitor the association between species of HRV and clinical presentation in hospitalized paediatric patients. Method Two hundred and eighty-nine nasopharyngeal (NP) suction specimens were collected from hospitalized paediatric patients admitted to King Chulalongkorn Memorial Hospital, Thailand during February 2006–2007. Nucleic acids were extracted from each sample with subsequent amplification of VP4/2 by semi-nested RT-PCR for HRV detection. Other viral respiratory pathogens were also detected by PCR, RT-PCR or real time PCR. Nucleotide sequences of the VP4 region were used for genotyping and phylogenetic tree construction. Result In total, 87 of 289 specimens were positive for HRV indicating an annual prevalence of 30%. Wheezing or asthma exacerbation was the most common clinical presentation observed in infected patients. Sequence analysis and phylogenetic tree showed that 29 (33%) and 8 (9%) specimens belonged to HRV-A and HRV-B, respectively. Most of the HRV positive samples were HRV-C (58%). Moreover, species C was predominantly found in the paediatric population of Thailand in raining season (p <0.05). The frequency of co-infection of HRV-C with other respiratory viral pathogens was approximately 40%. Conclusion HRV-C represents the predominant species and is one of the etiologic agents in acute lower respiratory tract infection, causes of wheezing and asthma exacerbation in infants and young children in Thailand.

Typing (A/B) and subtyping (H1/H3/H5) of influenza A viruses by multiplex real-time RT-PCR assays.

In this study, a specific and sensitive one-step multiplex real-time RT-PCR was developed in two assays by using primers and a number of specific locked nucleic acid (LNA)-mediated TaqMan probes which increase the thermal stability of oligonucleotides. The first assay consisted of primers and probes specific to the matrix (M1) gene of influenza A virus, matrix (M1) gene of influenza B virus and GAPDH gene of host cells for typing of influenza virus and verification by an internal control, respectively. The other assay employed primers and probes specific to the hemagglutinin gene of H1, H3 and H5 subtypes in order to identify the three most prominent subtypes of influenza A capable of infecting humans. The specificity results did not produce any cross reactivity with other respiratory viruses or other subtypes of influenza A viruses (H2, H4 and H6-H15), indicating the high specificity of the primers and probes used. The sensitivity of the assays which depend on the type or subtype being detected was approximately 10 to 10(3)copies/microl that depended on the types or subtypes being detected. Furthermore, the assays demonstrated 100% concordance with 35 specimens infected with influenza A viruses and 34 specimens infected with other respiratory viruses, which were identified by direct nucleotide sequencing. In conclusion, the multiplex real-time RT-PCR assays have proven advantageous in terms of rapidity, specificity and sensitivity for human specimens and thus present a feasible and attractive method for large-scale detection aimed at controlling influenza outbreaks.

Human bocavirus (HBoV) in Thailand: Clinical manifestations in a hospitalized pediatric patient and molecular virus characterization

Summary Objective Human bocavirus (HBoV), a novel virus, which based on molecular analysis has been associated with respiratory tract diseases in infants and children have recently been studied worldwide. To determine prevalence, clinical features and perform phylogenetic analysis in HBoV infected Thai pediatric patients. Methods HBoV was detected from 302 nasopharyngeal (NP) suctions of pediatric patients with acute lower respiratory tract illness and sequenced applying molecular techniques. Results The incidence of HBoV infection in pediatric patients amounted to 6.62% with 40% co-infected with other respiratory viruses. There were no clinical specific manifestations for HBoV; however, fever and productive cough were commonly found. Generalized rales and wheezing were detected in most of the patients as well as perihilar infiltrates. The alignment and phylogenetic analysis of partial VP1 genes showed minor variations. Conclusion Our results indicated that HBoV can be detected in nasopharyngeal aspirate specimens from infants and children with acute lower respiratory tract illness.

Prevalence and molecular characterization of WU/KI polyomaviruses isolated from pediatric patients with respiratory disease in Thailand

Abstract WU and KI polyomaviruses represent novel viruses discovered in respiratory secretions from human patients with acute respiratory tract infection. However, the association between WU/KI polyomaviruses and human disease has remained unclear. In this study, the prevalence of these two novel viruses and occurrence of co-infection with other respiratory viruses were determined in Thai pediatric patients with respiratory disease. Previously described PCR assays were applied to detect WU/KI polyomaviruses as well as other respiratory viruses in 302 nasopharyngeal suction specimens collected from February 2006 through February 2007. The results revealed the anneal prevalence of WU and KI polyomaviruses in the Thai population was 6.29% and 1.99%, respectively. The frequency of co-detection of WU and KI polyomaviruses with other respiratory viral pathogens was 42.11% and 33.33%, respectively. Moreover, each of the two complete genome sequences of WU (CU_295 and CU_302) and KI (CU_255 and CU_258) polyomaviruses were genetically and phylogenetically characterized. Sequence analysis showed that they contained features common to those found in previous studies. However, there were several nucleotide variations within the non-coding regulatory regions and various non-synonymous mutations within the coding regions which may influence virulence and pathogenesis of these viruses. Nevertheless, it is still possible that these viruses are not the causative agents of clinical respiratory disease. Therefore, judging the association of WU/KI polyomavirus infections with a particular disease will be challenging and require more comprehensive case control investigations.

Human metapneumovirus infection in Thai children.

Human metapneumovirus (hMPV) associated with clinical respiratory tract infection (RTI) in children was first isolated in the Netherlands. Of 120 Thai paediatric patients with RTI examined, 5 cases (4.2%) showed detectable hMPV based on N-genespecific RT-PCR. All of them were negative for hRSV infection. Aligning the sequences with a reference strain revealed some nucleotide differences, which necessitates future investigation to evaluate clinical significance and genotype variation.

Effect of frequency of ventilator circuit changes (3 vs 7 days) on the rate of ventilator-associated pneumonia in PICU

PURPOSE Ventilator-associated pneumonia (VAP) is associated with significant morbidity and mortality in pediatric intensive care unit (PICU). Our purpose was to evaluate the effects of ventilator circuit change on the rate of VAP in the PICU. METHODS A prospective randomized controlled trial was conducted at a university hospital PICU. Children (younger than 18 years) who received mechanical ventilation from December 2006 to November 2007 were randomly assigned to receive ventilator circuit changes every 3 or 7 days. RESULTS Of 176 patients, 88 were assigned to receive ventilator circuit every 3 days and 88 patients had a change weekly. The rate of VAP was 13.9/1000 ventilator days for the 3-day circuit change (n = 12) vs 11.5/1000 ventilator days (n = 10) for the 7-day circuit change (odds ratio, 0.8; confidence interval, 0.3-1.9; P = .6). There was a trend toward decreased PICU stay and mortality rate in 7-day change group compared to 3-day change group but did not reach statistical significance. Furthermore, switching from a 3-day to a 7-day change policy could save costs up to US

Human coronavirus infection among children with acute lower respiratory tract infection in Thailand.

22,000/y. CONCLUSIONS The 7-day ventilator circuit change did not contribute to increased rates of VAP in our PICU. Thus, it may be used as a guide to save workload and supply costs.

Levels of protein C activity and clinical factors in early phase of pediatric septic shock may be associated with the risk of death.

Objective: This study was performed to further identify the previously uncharacterized human coronavirus 229E (hCoV-229E) and human coronavirus OC43 (hCoV-OC43) in Thailand by using the RT-PCR technique. In addition, we performed this study in order to delineate the prevalence, the potential clinical impacts and evaluation of the genetic characterization of this pathogen in young children who presented with acute lower respiratory tract infections (ALRI). Methods: We obtained nasopharyngeal secretions (NPs) from 226 children <5 years of age who were either attending the outpatient department or hospitalized with ALRI from March 2002 to July 2003. All clinical, laboratory, RT-PCR, direct sequencing and phylogenetic analysis data were collected and analyzed. Results: Of the 226 NPs samples from infants and young children presented with ALRI, 8 (3.54%) were positive for hCoV-229E, 2 (0.88%) were positive for hCoV-OC43, and 1 (0.44%) had co-infection. The following clinical presentations were noted: fever (100%), rhinitis (44%), acute bronchiolitis (44%), viral pneumonia (33%), viral pneumonia triggering asthma exacerbation (11%) as well as viral pneumonia causing BPD exacerbation (11%). All positive samples were subjected to direct sequencing. The amino acid sequences had 82–99% similarity to previous sequences stored in the GenBank database. Conclusion: The molecular technique we applied to detect human coronavirus appears justified as a valuable diagnostic approach to elucidate the prevalence, cause and clinical implications of ALRI among infants and young children.

Disseminated cytomegalovirus infection associated with Pneumocystis carinii pneumonia in a previously normal infant.

Severe sepsis and septic shock are major causes of morbidity and mortality among children in pediatric intensive care units (PICUs) worldwide. Activated protein C (PC) is a critical endogenous regulator of coagulation and inflammation in patients with sepsis. However, the role of PC in pediatric sepsis is still obscure. We prospectively recruited infants and children aged between 1 month and 15 years old who were admitted to PICU with a clinical diagnosis of systemic inflammatory response syndrome, sepsis, or septic shock. Clinical data were recorded and blood samples kept for further analysis. We then measured the levels of PC activity. Of the approximately 1,100 pediatric patients admitted to PICU from January 1, 2004 to December 31, 2005, 75 were diagnosed with septic shock (6.8%), and 67 samples were available for analysis. Out of these, 41 (61%) were survivors, and 26 (39%) were nonsurvivors. The average plasma PC activity (%) was at 37.8 ± 4.4. Plasma PC activity (%) was significantly lower in the nonsurvivors compared with the survivors at 23.6 ± 4.3 and 46.8 ± 6.3 (P = 0.002), respectively. D-Dimer levels were not significantly different between the survivors (1,461 ± 266 ng/mL) and the nonsurvivors (1,989 ± 489 ng/mL) (P = 0.68). Also, there was no correlation between plasma PC activity and D-dimer levels (r = −0.07; P = 0.6). Importantly, the odds of dying were significantly higher in patients whose level of PC activity was less than 25% (odds ratio = 5.6; P = 0.02). Pediatric patients with septic shock demonstrate very low levels of PC activity, and this may be associated with an increased risk of death.Abbreviations-PICU-pediatric intensive care unit; rhAPC-Recombinant human activated protein C; APC-Activated protein C; PC-Protein C; PRISM-score Pediatric risk of mortality score; SIRS-Systematic inflammatory response syndrome; PCR-Polymerase chain reaction; PT-Prothrombin time; PTT-Partial thromboplastin time; iCa-ionized calcium

Detection and discrimination of WU/KI polyomaviruses by real-time PCR with melting curve analysis

We reported a rare case of dual opportunistic infections of Pneumocystis carinii pneumonia and disseminated cytomegalovirus (CMV) infection in a previously normal female infant. Transient T-cell dysfunction was demonstrated and returned to normal after treatment of CMV infection.