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Detection of Virus in aqueous humour samples from Hospital de la Princesa (MADRID)
J. Martiáñez, E. Lomas, E. Gallego, P. Sánchez, A. Guiu, A. Correa y L. Cardeñoso.
Hospital Universitario de la Princesa
OBJECTIVES
METHODS
Herpesviruses are involved in the pathogenesis of many
ocular diseases: acute anterior uveitis, keratitis, acute retinal
necrosis.
An early diagnosis and an accurate treatment are important in
order to avoid in patients preventable loss of vision and side
effects of an unnecessary medication.
Were included aqueous humour samples processed between
January 2009 and November 2013. All these samples were
analysed for detection of virus.
From January 2009 to May 2012 samples were extracted
(NucliSENS easyMAG-Biomerieux-) and analysed for the
presence of herpes simplex virus 1 (HSV-1) and herpes simplex
virus 2 (HSV-2) by qualitative real time PCR (Affigene® HSV
tracer), varicella zoster virus (Affigene® VZV tracer) by qualitative
real time PCR, Epstein-Barr virus (Affigene® EBV trender) by
quantitative real time PCR and cytomegalovirus (CMV) by
quantitative real time PCR (Affigene® CMV trender)
Since May 2012 samples have been analysed by PCR multiplexhybridation (CLART® Entherpex-Genómica®), after DNA extraction
(NucliSENS easyMAG-Biomerieux-). Fig. 1.
This method allows a qualitative and simultaneous detection of:
HSV-1, HSV-2, EBV, CMV, VZV, human herpesvirus 6 (HHV-6),
human herpes virus 7 (HHV-7), human herpes virus 8 (HHV-8)
and enterovirus (coxsackievirus, poliovirus y echovirus).
Quantitative analysis of rtPCR CMV and EBV viral load was
performed in positive results after hybridation.
Figure 1: Reagents and Instruments.
NucliSENS easyMAG-Biomerieux
CLART® Entherpex-Genómica
Stratagene Real-Time PCR Platform
Affigene® Trender/Tracer
RESULTS
52 aqueous humour samples were studied in this period,
belonging to 46 different patients.
Between 2009 and May 2012, were analysed 29 samples with
7 (24%) positive results. Since May 2012, 23 samples have
been analysed by PCR-Hybridation with 9 (39%) positive
results. In the whole period that we have studied, we have
detected virus in 16 aqueous humour samples (31%),
belonging to 16 different patients (35%). Fig. 2-5.
The different viruses isolated are shown in Table 1:
Most prevalent viruses are: 1. CMV, isolated in 7 samples, 3 of
wich with detectable viral load belonging to patients with
diagnosis of uveitis. 2. HSV-1, isolated in 6 samples, belonging
to 3 patients with a diagnosis of uveitis, 2 with acute retinal
necrosis and 1 with keratitis. 3. VZV, detected in two patients
with diagnosis of acute retinal necrosis. Fig. 6 and Table 2.
Figure 6 y Table 2: Ocular diseases related with viruses studied.
Table 2
CMV
Figures 4-5: Patients studied and results obtained.
VZV
76%
61%
Uveítis (43%)
Acute retinal necrosis
(100%)
CONCLUSION
39%
24%
Figures 2-3: Patients studied and results obtained.
Table 1: Viruses isolated in aqueous humour samples
Herpes Simplex Virus
65%
69%
HSV-1
HSV-2
Human Herpes Virus
HHV-6
HHV-7
VZV
HHV-8
35%
31%
6
-
CMV
6
1
-
EBV
-
-
Enterovirus
-
2
CMV+HHV-6
1
The study of virus in ocular specimens has been generally
limited by the small quantity of available sample.
The development of PCR-hybridation technology allows the
simultaneous detection of multiple viruses using a minimal
quantity of sample with a high sensibility, and has increased the
Laboratory capacity of virus detection: 39 %, opposite to 24%
with individual real time PCR.