Evaluation of sampling technique and transport media for the diagnostics of adenoviral eye infections. Adenovirus sampling and transport.
BACKGROUND
Human adenoviruses (HAdV) may cause pharyngoconjunctival fever, follicular conjunctivitis or epidemic keratoconjunctivitis (EKC). Especially, outbreaks of the latter may lead to severe economic losses when preventive measures are implemented too late. Thus, a safe sampling method, proper specimen transport conditions and a fast and sensitive diagnostic technique is mandatory.


METHODS
Two commercially available virus transport systems (VTS) were compared with two NaCl-moisturised sampling devices, one of which comprises Dacron-tipped plastic-shafted swabs and the other a cotton-tipped wood-shafted swab, available in most ophthalmologists' offices. Downstream methods for specific detection of HAdV included direct immunofluorescence assay (IFA) of conjunctival swabs, virus isolation by cell culture and quantitative real-time polymerase chain reaction (qPCR). Furthermore, the influence of application of local anaesthetics prior to swabbing on subsequent detection of HAdV was investigated.


RESULTS
Application of local anaesthetics had a positive influence on the amount of swabbed cells, thus increasing the chance of obtaining positive results by IFA. Neither isolation of HAdV by cell culture nor by qPCR was negatively influenced by this pretreatment. Surprisingly, both commercially available VTS performed significantly worse than the NaCl-moisturised swabs. This was shown with regard to virus recovery rates in cell culture as well as viral genome copy numbers in the qPCR.


CONCLUSIONS
Based on our results, the following recommendations are provided to improve sampling, transport and diagnostic techniques regarding conjunctival swabs for diagnosis of human adenovirus infection: (1) application of local anaesthetics, (2) NaCl-moisturised VTS for shipment of specimens, and (3) detection of HAdV by qPCR. The latter method proved to be superior to virus isolation by cell culture, including subsequent identification by IFA, because it is faster, more sensitive and allows simultaneous handling of a number of samples. Hence, countermeasures to prevent further virus spread in an outbreak situation can be implemented earlier, thus reducing the number of subsequent adenoviral infections.
Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, 80937 Munich, Germany. romanwoelfel@bundeswehr.org
Roman
R
Wölfel
Martin
M
Pfeffer
Sandra
S
Essbauer
Sylke
S
Nerkelun
Gerhard
G
Dobler
D000258:Adenovirus Infections, Human / Q000175:diagnosis* / Q000821:virology; D000260:Adenoviruses, Human / Q000302:isolation & purification*; D000772:Anesthesia, Local / Q000379:methods; D000779:Anesthetics, Local / Q000008:administration & dosage; D003228:Conjunctiva / Q000821:virology*; D003236:Conjunctivitis, Viral / Q000175:diagnosis* / Q000821:virology; D003470:Culture Media; D004279:DNA, Viral / Q000032:analysis; D019085:Fluorescent Antibody Technique, Direct; D006801:Humans; D011343:Procaine / Q000008:administration & dosage / Q000031:analogs & derivatives; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods*; D014186:Transportation; D014773:Virology / Q000379:methods*; D014776:Virus Cultivation
D000779:Anesthetics, Local; D003470:Culture Media; D004279:DNA, Viral; D011343:Procaine; C005298:benoxinate