Thesis (MMed)--Stellenbosch University, 2012. / Includes bibliography / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/71902 |
| Date | 12 1900 |
| Creators | Maree, Leana |
| Contributors | Van Zyl, G. U., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology . Medical Virology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 102 p. : col. ill. |
| Rights | Stellenbosch University |
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