Detection of Virus in aqueous humour samples from Hospital de la Princesa (MADRID)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. CardeñosoJ. Martiáñez, E. Lomas, E. Gallego, P. Sánchez, A. Guiu, A. Correa y L. Cardeñoso..

Hospital Universitario de la PrincesaHospital Universitario de la Princesa

OBJECTIVES

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.

METHODS

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 multiplex-hybridation (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.

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 1: Reagents and Instruments.

NucliSENS easyMAG-Biomerieux CLART® Entherpex-Genómica

Stratagene Real-Time PCR Platform Affigene® Trender/Tracer

Figures 4-5: Patients studied and results obtained.

69%

31%

65%

35%

76%

24%

61%

39%

Table 1: Viruses isolated in aqueous humour samples

CMV

Herpes Simplex Virus Human Herpes VirusVZV

EBV Enterovirus CMV+HHV-6

6 -

6 - - 1

HSV-1 HSV-2 HHV-6 HHV-7 HHV-8

- -1 2

Figures 2-3: Patients studied and results obtained.

Figure 6 y Table 2: Ocular diseases related with viruses studied.

CMV

VZV

Uveítis (43%)

Acute retinal necrosis (100%)

Table 2

CONCLUSION

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.