1. EPIDEMIOLOGICAL SURVEILLANCE OF HERPESVIRUS ENCEPHALITIS IN CORDOBA, COLOMBIA SALIM MATTAR, GERMAN ARRIETA, VANEZA TIQUE, FRANCISCO CAMARGO, LUIZ TADEU. UNIVERSITY OF CÓRDOBA, BIOLOGICAL RESEARCH INSTITUTE OF THE TROPICS

2. INTRODUCTION Taxonomy Herpesviridae Alphaherpesvirina e Simplexvirus HHV 1 Oral herpes HHV 2 Genital herpes varicelovirus HHV 3 (VZV) Betaherpesvirinae Citomegalovirus HHV 5 (CMV) Roseolovirus HHV 6A y B HHV7 Gammaherpesvirinae Lynphocryptovirus HHV 4 (EBV) HHV 8

3. INTRODUCTION Commun diseases Murray et al.,2009; Goering et al., 2008 oral and genital Herpes HSV1-2 Varicella and herpes zoster VZV Rash maculopapular in roseola infantum VHH6

4. Viral infections are the main causes of CNS infection in the world. Encephalitis is an inflammatory process in the brain in association with clinical evidence of neurologic dysfunction. Herpes simplex encephalitis is recognized worldwide as the most common infectious encephalitis. INTRODUCTION The brain T2 fluid-attenuated inversion recovery with typical findings for herpes simplex virus encephalitis, which shows a hypertense signal in left temporal lobe (arrow).

5. To establish an epidemiological surveillance of herpes encephalitis in major hospitals of Monteria, Cordoba. OBJETIVE

6. Type of study: A longitudinal descriptive study on meningoencephalitis cases between 2009 to 2011 of Monteria city was performed. Study area: Study was conducted at the San Jerónimo Monteria Hospital METHODOLOGY

7. Target population: Aprox 1,5 million people of the department of Córdoba and 500.000 inhabitants in the capital, Monteria.

8. METHODOLOGY Ethics aspects The study was approved by the ethics committee of the Institute for Tropical Biological Research of the University of Cordoba, according of procedures, Ministry of Health of Colombia.

9. METHODOLOGY Patients and CSF samples (n=265) Inclusion: fever, headache, seizures and signs of brain damage such as altered consciousness and/or personality and/or focal neurological signs were included in the study. Most patients had an increased cell count and/or an increased protein level in CSF. Exclusion: Patients with microorganisms (bacteria and/or fungi) detected by direct examination, KOH, China ink, Gram stain, Ziehl Neelsen and positive culture of CSF.

10. DNA extraction Transport and storage of CSF Collection of clinical and epidemiological data CSF collection METHODOLOGY

11. METHODOLOGY PCR Sequencing PCR products Agarose gel electrophoresis Viral Detection (1-2 round) Multiplex Nested- PCR

12. Pool the primers (1+) 10 µl the primer (HSV1+) 10 µl the primer (VZV1+) 10 µl the primer (CMV1+) 10 µl thee primer (EBV1+) 60 µl ultra pure water free of RNase and Dnase). Mix PCR. 5.0 µl Buffer 10X 4.0 µl MgCl2 (50 mM) 1.5 µl mix dNTPs (10mM) 2.5 µl Pool the primers (1+) 2.5 µl Pool the primers (1-) 0.3 µl Taq ADN Polimerase recombinante (Invitrogen Cat 10342053) (5U/ µl) 29.2 µl ultra pure water Thermocycler conditions : 94ºC x 2 min (35 ciclos) 94°C x 1 min 55 °C x 1 min 72°C x 1 min Final extensión 72°C x 4 min 1° round 5 µl ADN samples Pool the primers (1-) 10 µl the primer (HSV1-) 10 µl the primer (VZV1-) 10 µl the primer (CMV1-) 10 µl the primer (EBV1-) 60 µl ultra pure water free of RNase and Dnase).

13. Pool the primers (2+) 10 µl the primer (HSV2+) 10 µl the primer (VZV2+) 10 µl the primer (CMV2+) 10 µl the primer (EBV2+) 60 µl ultra pure water free of RNase and Dnase). Mix PCR. 5.0 µl Buffer 10X 4.0 µl MgCl2 (50 mM) 1.5 µl mix dNTPs (10mM) 2.5 µl Pool the primers (2+) 2.5 µl Pool the primers (2-) 0.3 µl Taq DNA Polimerase recombinante (Invitrogen Cat 10342053) (5U/ µl) 33.2 µl ultra pure water Thermocycler conditions: 94ºC x 2 min (35 ciclos) 94°C x 1 min 49°C x 1 min 72°C x 1 min Final extensión 72°C x 4 min 2° round 1 µl PCR product 1er round Pool the primers (2-) 10 µl the primer (HSV2-) 10 µl the primer (VZV2-) 10 µl the primer (CMV2-) 10 µl the primer (EBV2-) 60 µl ultra pure water free of RNase and Dnase).

14. Quality control 100, 50 y 25 pb DNA Ladder (Promega. Madinson, WI, USA); OptiQual High Positive Control ACROMETRIX (CMV, EBV, HSV-1, HSV-2); VZV (DNA extracted from lesions in a patient with chickenpox) Agarose gel 3.0 % (NuSieve TM 3:1) in buffered TBE, run at 100 volts x 60 min. Photography AGAROSE GEL ELECTROPHORESIS

15. SEQUENCING PCR PRODUCTS MEGA versión 5.22 comparing known sequencies in GenBank

16. RESULTS AND DISCUSSION Distribution of patients by gender and age ADULT PATIENTS 44.5% >19 years (n=118) PEDIATRIC PATIENTS 55.5% 0-7 years (n=121); 8-14 years (n=14) 15-18 years (n=12) POPULATION (n=265) Male 60% (n=158)Female 40% (n=107)

17. RESULTS AND DISCUSSION Imaging studies n% Computed Axial tomography (CAT) 13651.32 Magnetic resonance (MRN) 5219.62 Treatment Antibiotics 17766.79 Corticoids (dexametazone) 4416.60 Antivirals (aciclovir) 124.53 Underlying disease Non-disease. 16060.38 Congenital or acquired hydrocephalus peritoneal shunt valve or craniotomy. 3914.72 VIH-AIDS. 3212 Preterm neonate (n=14);Syphilis (n=7) diabetes (n=4);hypertension (n=9) 72.64 Evolution Positive outcome22886.04 Mortality3212.08 Sequele Yes 2911 no 23689 Imaging studies n% Computed Axial tomography (CAT) 3256 Magnetic resonance (MRN)1221 Treatment Antibiotics 4172 Corticoids (dexametazone) 1119 Antivirals (aciclovir) 59 Underlying disease Non-disease. 3154 Congenital or acquired hydrocephalus peritoneal shunt valve or craniotomy. 712 VIH-AIDS. 1221 Preterm neonate (n=4);Syphilis (n=1) diabetes (n=1);hypertension (n=1) 710 Evolution Positive outcome4986 Mortality610 Sequele Yes 713 No5087 Patients with herpesvirusTotal patients

18. RESULTS AND DISCUSSION clinical manifestations in patients with viral detection Clinical manifestations Positive Population (%)

19. RESULTAS AND DISCUSSION CSF’S characteristic in studied patients Characteristics CFSNegative patients (n=208)Patients with viral detection (n=57) Glucose (mg/dL)52.7+34.1(0-274)*46.9+27.2(4-140) Proteins (mg/dL)71.7+95.7(0-829)118.5+155.1(13.1 - 851.4) Leukocytes (mm3)51.5+166.5(0-1750)50.4+124.5(0-680) Polymorphonuclear (%)53.8+124.1(0-860)74.0+156.2(0-670) Mononuclear (%)9.48+23.9(0-100)16.77+32(0-99) Erythrocytes (mm3)54.4+46.9(0-100)44.6+46(0-100) Crenate (%)16+30.7(0-100)13.4+29.3(0-100) Intact (%)57.4+45.2(0-100)48+47.1(0-100) *Media + standard deviation (range).

20. RESULTS AND DISCUSSION Detection of viral DNA herpesvirus in CFS Virus N° negative (%)N° positive (%) CI (%) HSV 1-2218 (82,2)47 (17.6)13 – 22 CMV258 (97.3)7 (2.6)0.69 - 4.5 VZV261 (98.4)4 (1.5)0.031 - 2.9 EBV263 (99.2)2 (0.75)0.0 - 1.8 208 (78.4)57(21.5)16.5 - 26.4 CI: Confidence interval (95%).

21. ADULTS POPULATION BRAZIL n=200(12%) 6% CM, 5% VHS1 0.5% VEB Y VVZ 11.3% EV EV/CM-HSV1/CMV BRAZIL n=17 (76.4%) 17.6% VHS1 5.8% CM, 5.8% EV 47% dengue VHS1/VHS2 TAIWAN n=131(25 cases 33%VHS1 50% VVZ 17% CMV 8% EV*, 4%VHS2*, 64%VEB* TAIWAN n=25 8% VHS1, 8%VHS2 M.pneumoniae (2) ROMA n=155(33.5%) 30.9% VVZ, 27.9% VHS1 13.9% VEB Y 9.3% VHS2 y VHH6 VVZ/VHS1 VHS1/VHS2 –VHS2/VEB NEW ZELAND n=411(9%) 27% VVZ, 11% VHS 3% EV 65% No- identified 14% Mortality UK n=787(12%) VHS1, CMV,VEB, VHS1/VEB, VHS2/EV,VEB/VHH6, VEB/CMV

22. PEDIATRIC POPULATION CHILE Izquierdo et al., 2012 case EBV in neonate. FINLAND Martelius et al., 2011 n= 322, 9.9% EBV 47% other clinical suspects. FRANCIA N=253 EBV 2.3%(1case confirmed and 2 probablies)

23. M 1 2 3 4 5 6 7 8 9 10 11 12 13 M 120 Pb 98 Pb 78 Pb 54 Pb Lanes 1-4: positivos controls EBV (54pb), CMV (78pb), VZV (98pb) HSV1-2 (120pb). Lanes 5 and 9 negatives controls. lanes 6-8,10-12 positives CSF EBV (54pb), CMV (78pb), VVZ (98pb); HSV1-2 (120pb). Lane M, 50-100 pbLadder. RESULTS AND DISCUSSION Agarose gel showing amplicons obtained by multiplex-PCR for herpesvirus

24. 120 Pb 98 Pb 78 Pb M 1 2 3 4 5 6 M Lanes 1- 3: positive controls VHS1-2(120pb), VVZ (98pb) CMV (78pb). lane 4: infected patient CMV (78pb + HSV1-2 (120pb) lane 5: negative control lane 6: infected patient VZV (98pb) + VHS1-2 (120pb). Lane M : DNA ladder RESULTS AND DISCUSSION Co-infections

25. PATIENT VHS 2 dentity: 68/71 (96%) acces number JX905318.1 Genbank). RESULTS AND DISCUSSION Sequency results PATIENT EBV Identity: 140/140 (100%) acces number KC617875.1 Genbank)

26. RESULTS AND DISCUSSION Sequency results PATIENT CMV Identity: 206/206(100%) acces number JN048117.1 Genbank). PATIENT VZV Identity: 183/186(98%) acces number KF558391.1Genbank).

27. RESULTS AND DISCUSSION HIV patients 12 Patients (age 3-69 years) Lethal cases (3) Coinfection HSV1,2 + CMV (n=2) HSV 1,2 (n=6) CMV (n=3) VZV (n=1)

28. The incidence of 22% established in this study between 2009-2011 for herpesvirus encephalitis are the first result of surveillance cases in adult and pediatric patients in the department of Córdoba and in the country. The principal member of the herpesvirus family associated with encephalitis in the department of Córdoba was herpes simplex virus 1 and 2. CONCLUSIONS

29. Mortality of encephalitis was of 10.5% (6/57) and neurological sequelae were: 12.2% (7/57). The data suggest the need for active surveillance and follow the cases. It is important to find out the incidence of other virus, like arbovirus. CONCLUSIONS (cont)

30. ACKNOWLEDGEMENTS