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Laboratory exposure to Brucella spp.: challenges for
biosafety.
Veronique Yvette MIENDJE DEYI Microbiology LHUB-ULB
17 May 2018
2
Hôpital Erasme
Institut J. Bordet
CHU Brugmann
CHU St-Pierre
HUDERF
The LHUB-ULB Laboratoire Hospitalier Universtaire de Bruxelles
Universitair Laboratory van Brussels
Introduction Brucella spp. – Brucellosis Biosafety
Clinical case Laboratory exposure & management Microbiological analysis LHUB-ULB NRC Brucella (Sciensano-former CODA-CERVA)
Risk analysis Preventive and correctives measures
Lab Acquired Infections (LAI) worldwide
Laboratory exposure to Brucella spp.: challenges for biosafety.
Gram-negative, nonmotile, coccobacillus bacteria Can easily be confuse with spherical or cocci shape
It causes the foodborne illness Brucellosis
Transmission Contact with secretions of infected animals Consumption of animal products Person to person (breastfeeding, sexual) Lab exposure cases
Brucella spp. - Brucellosis
0
100
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600
1982
1983
1984
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1987
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1989
1990
1991
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1996
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2003
End of year prevalence
Council Directive 77/391/EEC (BE= brucellose-free country)
• Brucella is present in wildlife (wildboar, marine mammals) • +/- 2 human cases per year Courtesy of NRL/NRC Brucella (Sciensano)
Number of outbreaks
Epidemiological animal context BELGIUM
Saegerman C. et al.
Resistance to the immune system Invade blood stream and organs
Clinic Asymptomatic Flu-like illness fatigue, loss of appetite, weakness, and fever.
Undulant fever: characteristic of Brucella infection. Chronic infections develop lasting months or longer. Brain, lungs, heart and bones. 2% of death Often related to B. melitensis cases.
Brucellosis
Diagnosis Undulant fever Serology Culture Lab exposure
Treatment Doxycycline + Rifampicin - 6 to 8 weeks
Prevention Pasteurisation of dairy products: milk, cheese Avoiding consumption of undercooked meat Wearing protective equipment: goggles, gloves, masks… Avoiding Lab exposure Biosafety purpose
Brucellosis
Biosafety Legal context in Belgium
European legislation Incorporated into Belgian law
Precautionary principle
2009/41/CE: Environment Waste Permitting/authorization
Federal level Regional level
2000/54/CE Security at work
Radioactivity
2001/18/CE: GMOs release
Law on the well-being at work
Hygiene at work changing room, toilette, management of dangerous chemicals…
Security at work work under safety conditions, avoiding accident Instructions and formations Health at work protect Lab workers against LAI All the specimens are potentially infectious
Syrian refugee woman, 42 years old Long migration: Turkey, Lebanon, Algeria, Morocco and Spain
Arrival in Belgium for 3 days, from Spain Admitted for abdominal pain, feverless History of metastatic cancer Cervical cancer diagnosed in Spain +/- 1 year Stage IVa infiltrating epidermoid carcinoma, metastatic Bilateral nephrostomy tubes since February 2017
Lab analysis 2 pairs of blood culture Urines: 478 WBC, culture in process
Hospitalization with antalgic & treatment with Temocillin for UTI
Clinical case Emergency unit CHU Saint-Pierre 23/10/2017
2017 23/10 24/10 25/10 26/10 27/10 28/10 29/10 30/10 31/10
Admission Blood culture Urine culture
Temperature 38°6
Temocillin 2g 3x Ceftazidime 2g 3x/24h
Urine culture
Blood culture
P. aeruginosa
P. aeruginosa
A. faecalis
Blood cult + Brucella spp
Doxycycline 100 mg bid Rifadine 600 mg 1x
CT Thorax
Clinical case Evolution (1)
Courtesy of N. Dauby
Transthoracic cardiac echography: normal Brucella mellitensis septicaemia without evidence of endocarditis: Chronic? Relapse?
Cervical echography: left basal mass with invasion of the common jugular vein and left subclavian vein
Progressive deterioration of the respiratory level Supportive care Death on 9/11/2017 due to multi-metastatic cervical uterine neoplasia with
carcinomatous lymphangitis, neoplastic pericarditis, jugular invasion
Clinical case Evolution (2)
Blood cultures Incubation
2 pairs - Emergency unit - 23/10/2017 Negative
2 pairs - 25/10
Saturday 28/10 at 10 pm: Negative Inoculation of the media (PVX et COL) Blood culture re-incubated into the Bactec
Sunday 29/10 at 3 am (night) Gram negative cocci ? Coccobacillus? « Don’t worry . Patient well treated for UTI infection »
Blood culture examination Direct examination
Sunday 29/10 Weak growth (<10h incubation) inoculated media re-incubated
Monday 30/10 ID MALDI biotyper IVD Many times: KO
Vitek-2 ID
Blood culture analysis Culture & Identification MALDI-TOF
Blood culture analysis Thursday 31/10 - Vitek-2 ID result
Blood culture analysis ID confirmed using MALDI-TOF
dB Security Relevant
Serology Rose Bengale (RBT): Negative
Serology - LHUB-ULB
Specimens sent to CODA-CERVA Blood culture bottles Culture Blood sample
Brucella NRC (Sciensano-former CODA-CERVA)
BV Co2 H2S Thionine Fuschine A M Rough
B. melitensis 1 - - + + - + -
2 - - + + + - -
3 - - + + + + -
1 + + - + + - -
2 + + - - + - -
3 + + + + + - -
B.abortus 4 + + - + - + -
5 - - + + - + -
6 - - + + + - -
9 +/- + + + - + -
1 - + + - + - -
2 - - + - + - -
B. suis 3 - - + + + - -
4 - - + - + + -
5 - - + - - + -
B.ovis - - - + - - - +
B.canis - - - + - - - +
ID confirmation: biochemical typing Brucella NRC (Sciensano)
Serology Positive
RBT +++ ELISA IgG POS Wright’s agglutination POS
Serology – Brucella NRC
Americas Western Mediterranean Eastern Mediterranean Africa Unknown “Afganistan” Belgian human isolates
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Italian strains
Morocco strain
Lebanon strains
Peruvian strain
Turkish strains
Molecular typing Brucella NRC (Sciensano)
Method: Multiple Locus Variable Number of Tandem Repeat Analysis
imported human Brucellosis in Belgium Hanot-Mambres D. et al. Imported human brucellosis in Belgium: Bio and molecular typing of bacterial isolates, 1996-2015. 2017, PLoS One.
Clinical case
Courtesy of NRC Brucella (Sciensano)
REMEMBER - Slide of Microbiology lesson at IPL Brucella spp.: Test à effectuer aux Travaux Pratiques?
à la manipulation de Brucella spp. Fièvre de Malte, Fièvre ondulante, complications Manipuler en laboratoire de sécurité biologique de
niveau L3 (aérosols contaminants; haut degré de contagiosité!)
Pas de test au TP Maladie à Déclaration obligatoire!!!
They can’t manipulate this bacteria because of the high risk of infection.
Confirmed Brucella ID Management
• Risk analysis
• Information - Communications • Staff support
• Preventive measures • Corrective measures
List of workers Handing blood culture or strains Present in the Lab during the 3 days
Choose of appropriate guidelines Identification and labeling of the patient's
samples safety handling Plan visits Emergency units Infection units
Information - communications
Immediate correctives actions
Risk analysis – CDC guidelines
Risk analysis – CDC guidelines
Risk analysis – CDC guidelines Saturday 28/10 Tuesday 31/10
26 workers
6 High risks < 5 feet radius
1 particular case
Pregnant W.
12 Low risks > 5 feet radius
7 No risk
L3 Mycobacteria, Encoding, Bacteriology, Parasitology, Mycology, NRC
Far away
MATRA declaration Lab personal Oral communication and distribution of informative flyer
- Support and follow-up (according to risk class)
Occupational medicine & Hospitals management CHU Saint-Pierre Institut J. Bordet CHU-Brugmann Hôpital Erasme
Work accident report (Emergency units)
Information - Communication
Post-Exposure Prophylaxis (PEP) treatment. 3 weeks Bi-therapy: Rifampicin – Doxycycline 4 capsules per day
Pregnant woman: TMP-SMZ (Bactrim)
Liver enzymes control
Infectiology consultations High risks (n= 7)
Week Serology Comment
0 = Baseline Negative 3 Negative 1 positive PCR 6 Negative 12 Negative 18 Negative 24 Negative
Serological monitoring High and Low risks (n = 19)
Not recommended by Guidelines Interest ? PEP treatment Local presentation
1 High risk positive on week 3 Serum (Whole Blood: Neg) Further controls: ALL negative Blood culture – Serology - PCR
Meaning ???? = Case to be investigated by the NRC (Sciensano)
PCR monitoring High and Low risks (n = 19)
Re-organisation of the Blood culture workstation Discussion with Bruker Purpose: Integration Brucella into the MALDI-TOF IVD dB Proceeding in progress
Serology (Rose Bengale) Staff training at the Brucella NRC (Sciensano)
Reminding of Good Laboratory Practices Training on Biosafety in the laboratory Discussion with clinicians Rapid contact with the lab for all suspected cases Brucellosis - Highly contagious disease – Prions..
Corrective & preventive actions
Grand hôpital de Charleroi Hôpital Marie curie UZ Brussels … We are not alone !!! ... but we must always be careful in the aim of preventing LAI
Recent cases of exposure to Brucella spp Clinical Biology Labs - Belgium
Primary Laboratory Acquired Infection (LAI) Summary of 488 publications (1980-2015)
Symptomatic Asymptomatic Total
Bacteria 1417 411 1828 Viruses 764 439 1203 Parasites 170 4 174 Fungi 25 0 25 Total 2376 854 3230
Byers and Harding. 2017. LAI in Biological safety, Principle and Practices, 5th edition. ASM press.
Modifiez les styles du texte du masque Deuxième niveau
Rank Agent N of LAI
1 Brucella spp 426
2 Coxiella burnetti 280
3 Hepatitis B virus 268
4 Salmonella spp 258
5 Francisella tularensis 225
6 M. tuberculosis 194
7 Blastomyces dermatidis 162
8 Venezuelan Equine Encephalitis virus
146
9 Chlamydia psyttaci 116
10 Coccidioides immitis 93
Total 2,168
Rank Agent N of LAI
1 Brucella spp 378
2 M. tuberculosis 255
3 Arboviruses 222
4 Salmonella spp 212
5 Coxiella burnetii 205
6 Hantavirus 189
7 Hepatitis B virus 113
8 Shigella spp
88
9 HIV 48
10 N. meningitidis 43
Total 1,753
From 1930 to 1979 From 1980 to 2015
TOP 10 LAIs reported in Literature
Byers and Harding. 2017. LAI in Biological safety, Principle and Practices, 5th edition. ASM press.
Case of Lab exposure to Brucella melitensis Imported case from a Syrian refugee
Risk analysis 19 workers at risk 7 PEP treatment
Risk management Corrective and preventive measures
Multidisciplinary collaboration
Conclusion
