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

INTRODUCTION Taxonomy

INTRODUCTION Commun diseases

Murray et al.,2009; Goering et al., 2008

oral and genital Herpes HSV1-2Varicella and herpes zoster

VZV

Rash maculopapular in roseola infantum VHH6

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).

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

OBJETIVE

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

METHODOLOGY

Target population:

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

METHODOLOGY

Ethics aspectsThe 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.

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.

METHODOLOGY

METHODOLOGYPCR

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).

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).

Quality control100, 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

SEQUENCING PCR PRODUCTS

MEGA versión 5.22 comparing known sequencies in GenBank

RESULTS AND DISCUSSION

Distribution of patients by gender and age

RESULTS AND DISCUSSION

Imaging studies n %

Computed Axial tomography (CAT)

136 51.32

Magnetic resonance (MRN) 52 19.62

Treatment

Antibiotics 177 66.79

Corticoids (dexametazone) 44 16.60

Antivirals (aciclovir) 12 4.53

Underlying disease

Non-disease. 160 60.38

Congenital or acquired hydrocephalus peritoneal shunt valve or craniotomy.

39 14.72

VIH-AIDS. 32 12

Preterm neonate (n=14);Syphilis (n=7)diabetes (n=4);hypertension (n=9)

7 2.64

Evolution

Positive outcome 228 86.04

Mortality 32 12.08

Sequele

Yes 29 11

no 236 89

Imaging studies n %

Computed Axial tomography (CAT)

32 56

Magnetic resonance (MRN) 12 21

Treatment

Antibiotics 41 72

Corticoids (dexametazone) 11 19

Antivirals (aciclovir) 5 9

Underlying disease

Non-disease. 31 54

Congenital or acquired hydrocephalus peritoneal shunt valve or craniotomy.

7 12

VIH-AIDS. 12 21

Preterm neonate (n=4);Syphilis (n=1)diabetes (n=1);hypertension (n=1)

7 10

Evolution

Positive outcome 49 86

Mortality 6 10

Sequele

Yes 7 13

No 50 87

Patients with herpesvirusTotal patients

RESULTS AND DISCUSSIONclinical manifestations in patients with viral

detection

Clin

ical m

anif

est

ati

on

s

Positive Population (%)

RESULTAS AND DISCUSSION CSF’S characteristic in studied patients

Characteristics

CFS

Negative 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)

16.7

7 +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).

RESULTS AND DISCUSSIONDetection of viral DNA herpesvirus in CFS

Virus N° negative

(%)

N° positive (%) CI (%)

HSV 1-2 218 (82,2) 47 (17.6) 13 – 22

CMV 258 (97.3) 7 (2.6) 0.69 - 4.5

VZV 261 (98.4) 4 (1.5) 0.031 - 2.9

EBV 263 (99.2) 2 (0.75) 0.0 - 1.8

208 (78.4) 57(21.5) 16.5 - 26.4

CI: Confidence interval (95%). CI: Confidence interval (95%).

ADULTS POPULATION

BRAZILn=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% VVZ17% 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

VHH6VVZ/VHS1

VHS1/VHS2 –VHS2/VEB

NEW ZELAND n=411(9%)

27% VVZ , 11% VHS

3% EV65% No- identified

14% Mortality

UKn=787(12%)

VHS1, CMV,VEB, VHS1/VEB,

VHS2/EV,VEB/VHH6, VEB/CMV

PEDIATRIC POPULATION

CHILE Izquierdo et al., 2012 case EBV

in neonate.

FINLANDMartelius et al.,

2011 n= 322, 9.9% EBV47% other clinical

suspects.

FRANCIAN=253

EBV 2.3%(1case confirmed and 2

probablies)

M 1 2 3 4 5 6 7 8 9 10 11 12 13 M

120 Pb98 Pb78 Pb54 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 DISCUSSIONAgarose gel showing amplicons obtained by multiplex-PCR

for herpesvirus

120 Pb98 Pb78 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 DISCUSSIONCo-infections

VHS2 6 TTGTGCCGCGGTCTCACGGCCGCGGGCCTGGTGGCCATGGGCGACAAGA-GGCGAGCCAC 64

||||||||||| ||||||||||||||||||||||||||||||||||||| ||||||||||

Sbjct 2701 TTGTGCCGCGGCCTCACGGCCGCGGGCCTGGTGGCCATGGGCGACAAGATGGCGAGCCAC 2760

VHS2 65 AT-TCGCGCGC 74

|| ||||||||

Sbjct 2761 ATCTCGCGCGC 2771

PATIENT VHS 2dentity: 68/71 (96%)

acces number JX905318.1 Genbank).

RESULTS AND DISCUSSIONSequency results

EBV 1 CACACGTGTGACATTGCTTGCCTTTTTGCCACATGTTTTCTGGACACAGGACTAACCATG 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 795 CACACGTGTGACATTGCTTGCCTTTTTGCCACATGTTTTCTGGACACAGGACTAACCATG 854

EBV 61 CCATCTCTGATTATAGCTCTGGCACTGCTAGCGTCACTGATTTTGGGCACACTTAACTTG 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 855 CCATCTCTGATTATAGCTCTGGCACTGCTAGCGTCACTGATTTTGGGCACACTTAACTTG 914

EBV 121 ACTACAATGTTCCTTCTCAT 140

||||||||||||||||||||

Sbjct 915 ACTACAATGTTCCTTCTCAT 934

PATIENT EBV Identity: 140/140 (100%)

acces number  KC617875.1 Genbank)

CMV 1 GCAACGTTATGATATCTACAGCAGATACATGCGTCGTATGCCGCCACTTTGCATCATTAC 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1265 GCAACGTTATGATATCTACAGCAGATACATGCGTCGTATGCCGCCACTTTGCATCATTAC 1324

CMV 61 AGACGCCTATAAAGAAACCACGCGTCAGGGTGGCGCAACTTTCACGTGCACGCGCCAAAA 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1325 AGACGCCTATAAAGAAACCACGCGTCAGGGTGGCGCAACTTTCACGTGCACGCGCCAAAA 1384

CMV 121 TCTCACGCTGTACAATCTTACGGTTAAAGATACGGGAGTCTACCTTCTACAGGATCAGTA 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1385 TCTCACGCTGTACAATCTTACGGTTAAAGATACGGGAGTCTACCTTCTACAGGATCAGTA 1444

CMV 181 TACCGGCGATGTCGAGGCTTTCTACC 206

||||||||||||||||||||||||||

Sbjct1445 TACCGGCGATGTCGAGGCTTTCTACC 1470

RESULTS AND DISCUSSION

Sequency results

PATIENT CMVIdentity: 206/206(100%) acces number JN048117.1

Genbank).

VVZ 18 AAAAATATACCCGATTATACTCGGATGTTGTAAGTCCCAGTCTCTTATAATCGGTAAGAC 77

||||||||| || ||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 9833 AAAAATATAAGCGTTTATACTCGGATGTTGTAAGTCCCAGTCTCTTATAATCGGTAAGAC 9892

VVZ 78 AATTTTTATAAATTCATTCCTTTTTAAATATAGGTTATATGGTACACAAATATCATATCC 137

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 9893 AATTTTTATAAATTCATTCCTTTTTAAATATAGGTTATATGGTACACAAATATCATATCC 9952

VVZ 138 CGCGTCTTCTTGGCGTTTTGGATTGATGATATGTTTGTAGGTTAAGGGAACATCGATATG 197

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 9953 CGCGTCTTCTTGGCGTTTTGGATTGATGATATGTTTGTAGGTTAAGGGAACATCGATATG 10012

VVZ 198 GTATTC 203

||||||

Sbjct 10013 GTATTC 10018

PATIENT VZVIdentity: 183/186(98%)

acces number KF558391.1Genbank).

RESULTS AND DISCUSSION HIV patients

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

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)

ACKNOWLEDGEMENTS