Investigating the aetiology of respiratory tract infections in children admitted to Tygerberg Children’s Hospital using molecular methods and viral culture

Maree, Leana (2012-12)

Thesis (MMed)--Stellenbosch University, 2012.

Includes bibliography

Thesis

ENGLISH ABSTRACT: Introduction Acute respiratory tract infections cause significant morbidity and mortality worldwide, and are the main reason for the utilisation of health care services. Identifying the aetiological cause of lower respiratory tract infections (LRTIs) is difficult at the best of times, and more than 20 viruses and bacteria have been associated with LRTIs, which cannot be distinguished with clinical examination alone. Viruses can be detected in respiratory samples by a variety of methods, and without exception molecular methods have proven to be more sensitive than non-molecular-based tests. The increased sensitivity of molecular methods may assist in expanding our knowledge of the pathogenesis of severe respiratory tract infections, and could have a positive influence on patient management, infection control, vaccination strategies and public health. Aims and objectives 1. Determine the viral causes of lower respiratory tract infections requiring admission in using shell vial culture with immunofluorescent staining and two multiplex PCR assays, the Seeplex® RV15 ACE Detection system (Seeplex® RV15 ACE) and the Respiratory Multiplex Real-Time RT-PCR LightMix® Customised Kit (Resp Multiplex RT-PCR). 2. Compare the Seeplex® RV15 ACE and the Resp Multiplex RT-PCR with shell vial culture for the detection of respiratory viruses in routine diagnostic respiratory samples. 3. Examine the demographic and clinical characteristics associated with each respiratory viral pathogen. Materials and Methods One hundred and thirty-eight paediatric patients, admitted to Tygerberg Children’s Hospital from May 2010 to August 2010 with a presumptive diagnosis of an acute respiratory tract infection were included in the study. Nasopharyngeal or tracheal aspirates were collected, and all samples were tested by all three diagnostic methods. Clinical, demographic and laboratory data were collected through a systematic review of medical and laboratory records and subsequently anonymised Results Thirty-seven viruses were detected in 36 samples (26.1%) by shell vial culture with immunofluorescent staining; 169 viruses in 102 samples (73.9%) with the Seeplex® RV15 ACE; and 90 viruses in 73 samples (52.9%) with the Resp Multiplex RT-PCR. Shell vial culture had excellent specificity, but low sensitivity for all of the respiratory viruses. Conversely, the Seeplex® RV15 ACE had excellent sensitivity for all viruses, but slightly lower specificity. This was due to the detection of additional viruses, which may have been true positives due to the increased sensitivity of this assay. The Resp Multiplex RTPCR had excellent sensitivity and specificity. At least one respiratory pathogen could be identified in 80% of the patients. At least one virus was detected in 57% of patients, bacterial micro-organisms in 6%, and both viral and bacterial pathogens in 17%. Viral-bacterial co-infections were associated with increased severity compared to other infections, as these children were more likely to receive steroids and a blood transfusion (p = 0.002), and more likely to require mechanical ventilation (p < 0.001) and admission to the intensive care unit (p = 0.04). Conclusions We confirmed that molecular techniques are significantly more sensitive than shell vial culture for the detection of respiratory viruses in children. Due to their highly specific nature and the genetic variability observed in viruses, an excellent, continuous quality control programme is essential to ensure the continued superiority of these assays. Viral-bacterial co-infection is associated with increased severity of LRTIs in children. Further research is needed to elucidate the precise pathogenic and immunologic mechanism of this interaction.

AFRIKAANSE OPSOMMING: Inleiding Akute lugweg infeksies is verantwoordelik vir beduidende morbiditeit en mortaliteit wêreldwyd en is die hoofrede vir die benutting van gesondheidsdienste. Identifisering van die oorsaak van laer lugweg infeksies is baie moeilik en meer as 20 virusse en bakterieë word hiermee geassosieer. Ongelukkig kan kliniese ondersoek alleen nie onderskei tussen die verskillende organismes nie. Respiratoriese virusse kan deur ‘n wye verskeidenheid van toets metodes aangetoon word. Molekulêre metodes is sonder uitsondering meer sensitief as nie-molekulêre metodes. Hul verhoogde sensitiwiteit mag help om ons kennis oor die patogenese van erge lugweg infeksies te verbreed en kan ’n positiewe invloed op pasiëntbehandeling, infeksiebeheer, immunisasie strategieë en publieke gesondheidsorg hê. Doel van die Ondersoek 1. Bevestig die virale oorsake van laer lugweg infeksies deur gebruik te maak van “shell vial” kultuur met immunofluoressensie en twee veelvoudige molekulêre toetse, die Seeplex® RV15 ACE en die Resp Multiplex RT-PCR. 2. Vergelyk die Seeplex® RV15 ACE en die Resp Multiplex RT-PCR met “shell vial” kultuur vir die aantoning van respiratoriese virusse in roetine diagnostiese monsters. 3. Ondersoek die demografiese en kliniese eienskappe wat met elke respiratoriese patogeen geassosieer word. Metodiek en Materiaal Een honderd agt-en-dertig kinders wat toegelaat is tot Tygerberg Kinderhopitaal vanaf Mei 2010 tot Augustus 2010 met ’n voorlopige diagnose van ’n akute lugweg infeksie is in die studie ingesluit. Nasofaringeale of trageale aspirate is van elke pasiënt gekollekteer en met al drie diagnostiese metodes ondersoek. Kliniese, demografiese en laboratorium data is gekollekteer deur ’n sistematiese ondersoek van mediese en laboratorium rekords en daarna anoniem gemaak. Resultate Sewe-en-dertig virusse is in 36 monsters (26.1%) aangetoon deur “shell vial” kultuur met immunofluoressensie; 169 virusse in 102 monsters (73.9%) deur die Seeplex® RV15 ACE; en 90 virusse in 73 monsters (52.9%) deur die Resp Multiplex RT-PCR. “Shell vial” kultuur het uitstekende spesifisiteit gehad, maar sensitiwiteit was laag vir al die virusse. Teenoorgesteld hiermee het die Seeplex® RV15 ACE hoë sensitiwiteit vir al die viruses gehad, maar effe laer spesifisiteit. Dit was as gevolg van die aantoning van addisionele virusse, wat moontlik ware positiewe resultate kon wees as gevolg van die verhoogde sensitiwiteit van hierdie toets metode. Die Resp Multiplex RT-PCR het uitstekende sensitiwiteit en spesifisiteit gehad. Ten minste een respiratoriese patogeen is in 80% van die pasiënte geidentifiseer. Een of meer virusse was in 57% van die pasiënte aangetoon, bakterieë in 6% en beide virale en bateriële patogene in 17%. Virale-bakteriële ko-infeksies, in vergelyking met ander infeksies, was geassosieer met meer ernstige lugweg infeksies aangesien hierdie kinders meer geneig was om steroïede en ’n bloedtransfusie te ontvang (p = 0.002). Hulle het ook meer waarskynlik meganiese ventilasie (p < 0.001) en toegang tot die intensiewe sorg eenheid benodig (p = 0.04). Gevolgtrekkings Ons het bevesitg dat molekulêre tegnieke aansienlik meer sensitief is as “shell vial” kultuur vir die aantoning van respiratoriese virusse in kinders. As gevolg van hul hoogs spesifieke aard en die genetiese variasie waargeneem in virusse, is ’n uitstekende deurlopende kwaliteitsbeheer program noodsaaklik vir die voortgesette uitneemendheid van hierdie metodes. Virale-bakteriële ko-infeksies word geassosieer met meer ernstige laer lugweg infeksies in kinders. Verdere navorsing is nodig om die presiese patogenetiese en immunologiese meganisme van hierdie interaksie toe te lig.

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