case report professional acquisition of m. bovis in calabria...

Case ReportProfessional Acquisition of M bovis inCalabria Region (Southern Italy) A Challenging Case ofOsteomyelitis in a Migrant Patient from Bulgaria

Angela Quirino1 Carlo Torti1 Alessio Strazzulla1 Salvatore Nisticograve2

Luisa Galati1 Giorgio Settimo Barreca1 Angelo Giuseppe Lamberti1

Giuseppina Berardelli2 Maria Pacciarini3 Giorgio Gasparini1 Vincenzo Pisani1

Antonio Gambardella1 Maria Carla Liberto1 and Alfredo Focagrave1

1Magna Graeligcia University 88100 Catanzaro Italy2Giovanni Paolo II Hospital 88046 Lamezia Terme Italy3Istituto Zooprofilattico Sperimentale della Lombardia e dellrsquoEmilia Romagna (IZSLER) 25123 Brescia Italy

Correspondence should be addressed to Angela Quirino quirinouniczit

Received 6 April 2015 Accepted 7 July 2015

Academic Editor Lawrence Yamuah

Copyright copy 2015 Angela Quirino et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We report herein the first case of a coinfection with Brucella sppM bovis and Enterobacter cloacae in a butcher who moved fromBulgaria to Italy Molecular typing suggested professional acquisition ofM bovis in Italy So surveillance and preventive measuresneed to be implemented

1 Introduction

Both tuberculosis due to Mycobacterium bovis (M bovis)and brucellosis are zoonotic infections in humans M bovisan acid-fast microorganism belonging to theMycobacteriumtuberculosis complex may infect humans through inhalationingestion contact withmucousmembranes and broken skinButchers are at risk of pulmonary infections due to inhalationof aerosol particles during the handling of infected meatand carcasses Nonpulmonary diseases can also be acquiredby drinking unpasteurized milk from infected cattle [1] Asurvey carried out in the Calabria region during the period2011-2012 on 988 of cattle herds revealed that only lt1 ofanimalswere infected byMbovis [2] Routes of infections canbe heterogeneous and unexpected making it important tocharacterize the infecting strains and possible risk factors toimplement control strategies Indeed in restricted epidemicsstrains ofM boviswere found to be genotypically identical ordifferent only for a single locus [3ndash5]

Brucella melitensis (B melitensis) B abortus B suis andB canis are Gram-negative coccobacilli that infect humans

Occupational brucellosis may be acquired in slaughterhousesand butcher shops during the handling of meat productsand in milk and dairy product industries [6] In the Calabriaregion 82 cases of human brucellosis have been reportedbetween 2007 and 2009 at an average of 273 cases per yearaccounting for 158 of cases reported in Italy [7]

Herein we report the first case of a triple coinfection withBrucella spp and M bovis together with E cloacae in a 45-year-old Bulgarian migrant M bovis was acquired in Italyduring his work as a butcher as suggested by spoligotypingTherefore this case underlines the importance of usingmolecular methods to track the infection ofM bovis shouldthe suspicion of an epidemiological chain arise to guide pre-ventative strategies Moreover the present case strengthensthe importance of continuing surveillance and control mea-sures

2 Case Report

A 45-year-old Bulgarian patient came to Italy in the year2000 In his hometown he worked as a mechanic but once

Hindawi Publishing CorporationCase Reports in Infectious DiseasesVolume 2015 Article ID 794715 4 pageshttpdxdoiorg1011552015794715

2 Case Reports in Infectious Diseases

in Italy he had been working as a butcher for 10 years InJune 2011 after an accidental trauma with an open woundduring work swelling appeared in his second finger of the lefthand progressively extending to the wrist So he had beentreated with several antibiotics not recalled by the patient (apart from levofloxacin at unspecified dosage for ten days)without significant response Then a surgical treatment forcarpal tunnel syndrome was performed in November 2011 InAugust 2012 a subcutaneous abscess was drained while thepatient was in Bulgaria

In February 2013 fever started and an appropriate work-up at the Infectious Diseases Unit of ldquoJohn Paul IIrdquo Hospitalin Lamezia Terme led to a diagnosis of brucellosis basedon clinical manifestations and a positive direct agglutinationreaction (Wright test) for Brucella spp (either B abortus or Bmelitensis) So he was treated with doxycycline (100mg twicedaily) plus rifampicin (600mg once daily) for 2 months Atthe end of treatment a significant improvement was recordedwith absence of fever and meningeal signs but Wright testfor Brucella spp remained positive (1 160 titration) Aftera few days the patient was admitted for meningitis withheadache vomiting and neck stiffness Magnetic resonanceimaging (MRI) of the brain revealed ldquodiffuse hyperintensitiesin T2 sequences at whitematter with small enhanced nodularareasrdquo Also clear cerebrospinal fluid with increased proteinand reduced glucose concentrations was demonstrated So adiagnosis of neurobrucellosis was made For this reason hereceived ceftriaxone (2 g twice daily) for onemonth with vol-untary discharge after improvement (May 2013) During hos-pitalization Wright test became negative (April 16 2013) butleft wrist and hand still remained swollen

In August and September 2013 two skin swabs were posi-tive for Staphylococcus aureus and tobramycin linezolid andlevofloxacin at unknown dosages were prescribed for about30 days without any improvements

The patient presented for consultation at the InfectiousDiseases Unit of the University Hospital of Catanzaro inNovember 2013 On examination he had a swollen lesion inhis left wrist with a spontaneous drainage from a cutaneousfistula Also left knee and the second finger of the right handappeared swollen A MRI of the left hand was prescribedshowing ldquobone lesions attributable to arthriticseptic lesionsrdquo(Figure 1)

The patient was then admitted to our day-hospitalwhere further exams were performed complete blood count(4900000 red blood cells120583L 11700 white cells120583L with689 neutrophils and 295000 platelets120583L) was normaland also renal and liver function tests appeared to be withinthe range of normality Tuberculin skin test (Mantoux test)was positivewith an infiltrate of a diameter of 5 cmMoreoverQuantiferon-TB Gold in-tube (QFT-IT) test was positiveBoth Brucella spp DNA from blood (homemade real-timePCR assay as described below) and Brucella spp antibodies(1 160 titration) were positive However Brucella specifictreatment was postponed waiting for confirmation andresults of bone biopsy that was planned

In January 2014 the patient underwent the bone biopsyof the left wrist at the Orthopedics Unit of the UniversityHospital in Catanzaro leading to isolation of Enterobacter

Figure 1 Magnetic resonance imaging of the left wrist beforestarting antibiotic therapy for Brucella spp and Mycobacteriumbovis The picture shows increased signal intensity of trabecularbone of all carpal bones due to marked medullar edema with manyerosive areas most evident in scaphoid and capitate bones

cloacae (E cloacae) spp cloacae Also PCR for DNA of Mtuberculosis complex (MTBC) and Brucella spp DNA werepositive Afterwards cultures resulted positive for alcohol-acid resistant bacilli subsequently characterized as M bovisssp bovis Drug susceptibility tests (both genotypic and phe-notypic) were performed for rifampicin isoniazid ethamb-utol streptomycin pyrazinamide amikacin ofloxacin andlinezolid A molecular line specific probe assay (GenotypeMTBRD plus Hain Lifesciences GmbH Germany) was usedfollowing manufacturerrsquos instructions Only resistance topyrazinamide was detected

Regarding methods the samples were processed accord-ing to national and international guidelines using an N-acetyl-l-cysteine-NaOH decontamination procedure inocu-lated into BACTEC MGIT 960 tubes (Becton Dickinson andCo Cockeysville MD USA) and onto solid slant medium(Lowenstein-Jensen) and incubated at 37∘C for up to 4 and 6weeks respectively

MTBC-DNA was amplified with Strand DisplacementAmplification (SDA) technology (ProbeTec ET SystemBecton-Dickinson and Co Cockeysville MD USA) Ampli-fied MTBC-DNA was revealed only in the soft tissue ofthe left wrist biopsy Identification of species was per-formed using GenoType MTBC assay (Hain LifesciencesNehren Germany) Genotype MTBC assay allowed us toidentify the isolate as M bovis ssp bovis For Brucella DNAextraction either in blood or in bone samples a Qiagenkit was used following a modified procedure suggested bythe manufacturer (Qiagen Hilden Germany) For genus-specific real-time PCR assay the forward primer B4 51015840-TGG-CTCGGTTGCCAATATCAA-31015840 and reverse primer B5 51015840-CGCGCTTGCCTTTCAGGTCTG-31015840 were used to amplify a223 bp portion of the BCSP31 gene [8] The PCR conditionswere denaturation at 95∘C for 10min followed by 45 cycles(95∘C for 10 s 60∘C for 10 s and 72∘C for 9 s) After amplifi-cation melting curve analysis was carried out by evaluating amelting temperature of 8816 plusmn 005∘C

Case Reports in Infectious Diseases 3

In February 2014 the patient was admitted to theNeurology Unit of University Hospital of Catanzaro wherehe underwent brain MRI showing ldquodisseminated foci withsignal alteration most evident in supratentorial areas andmedulla oblongata (bulbus)rdquo However the foci appeared tobe reduced in number and size with respect to evaluationperformed in April 2013 A lumbar puncture was performedand subsequent cerebrospinal fluid examination showedclear aspect pH = 8 glucose concentration erythrocytesleukocytes and proteins within the ranges of normality whilecultures andDNAdetection forMTBCandBrucella spp werenegative

After considering the above findings from January 30to February 7 2014 the patient was treated for E cloacaessp cloacae infection with ertapenem 1 gday intravenouslywith a partial recovery of pain and swelling Then oral anti-TB therapy was started on February 10 2014 with isoniazid300mgday plus rifampicin 600mgday (with the objectiveof treating also brucellosis) plus pyrazinamide 2 gday (inter-rupted once molecular identification was available showingM bovis naturally resistant to this drug) plus ethambutol16 gday (interrupted after two months for therapy simplifi-cation) plus moxifloxacin 400mgday (with the objective oftreating also brucellosis) Lastly on February 24 2014 intra-venous amikacin 1 grday plus oral doxycycline 100mg twicedaily was prescribed for treating brucellosis Amikacin wascontinued intravenously for 20 days After 3months of antibi-otic therapy conditions improved with significant reductionof edema and resolution of functional impairment both inleft wrist and in knee Then a MRI of left wrist confirmedreduction of the bone lesions (Figure 2)

Interestingly genomic diversity ofM bovis isolated fromour patient was assessed and compared to M bovis bovinestrains isolated from a cattle herd in the same area aroundLamezia Terme It has to be noticed that this cattle herdprovided the animals to the slaughterhouse and butcherrsquosshop where the patient worked so a transmission chain isfurther supported Indeed by typing of a 24-locus-basedmycobacterial interspersed repetitive unit-variable numbertandem repeat (MIRU-VNTR) [9] we revealed that just alocus (locus 577) was deleted in the human strain suggestingepidemiological relatedness Furthermore those strains wereanalyzed by spoligotyping [10] and multilocus variable-number tandem-repeat analysis (MLVA) using 12 markersof VNTRMIRU ETRA ETRB ETRC ETRD and ETRE[11] and VNTR2163a VNTR2163b VNTR3155 VNTR4052VNTR1895 VNTR3232 and MIRU26 [12] The loci analyzedcomprised the 6 loci recommended by the EuropeanNetworkVENoMYC [13] These molecular typing methods showedthat the strains studied were correlated

3 Discussion

We investigated a unique coinfection withM bovis Brucellaspp and E cloacae Brucella spp infection involved threedifferent sites (blood brain and bone) as demonstrated bymolecular methods So these methods were important for anetiological diagnosis in different body compartments

Figure 2 Magnetic resonance imaging of the left wrist after threemonths of specific antibiotic therapy versus Brucella spp andMycobacterium bovis The picture shows slight decrease of signalintensity of trabecular bone of radium and carpal bone and mod-erate reduction of edema extension in scaphoid and capitate bones

For M bovis molecular analysis strongly suggested thatthe isolate from our patient belonged to the same chain oftransmission of animal strains in the same area and the sameperiod of time In fact cattle breeding slaughterhouse andbutcherrsquos shop were part of a unique facility In previousstudies mutations frequency was demonstrated to be verylow so genotypes could be strictly correlated with the sametransmission chain in animals even after 4-5 years especiallyin a geographic restricted area [3ndash5]

Since no M bovis infections were notified in the slaugh-terhousewhere the patientwasworking but the isolated strainwas related to strains circulating in animals in the same areathe present case indicates that surveillance systems should beimplemented especially in regions where these infections arestill endemic

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr Emanuele Borroni (S Raffaele Hos-pital Milan Italy) and Dr Maria Beatrice Boniotti (IstitutoZooprofilattico Sperimentale della Lombardia e dellrsquoEmiliaRomagna (IZSLER) Brescia Italy) for helping with molec-ular typing of M bovis strains The authors also thank DrChiara Costa (Infectious Diseases Unit ldquoMater DominirdquoTeachingHospital Catanzaro Italy) and all the dedicated staffwho followed the patient and processed the samples

References

[1] S Bilal M Iqbal P Murphy and J Power ldquoHuman bovinetuberculosismdashremains in the differentialrdquo Journal of MedicalMicrobiology vol 59 part 11 pp 1379ndash1382 2010


[2] Italian Ministry of Health httpwwwsalutegovit[3] C Allix K Walravens C Saegerman J Godfroid P Supply

and M Fauville-Dufaux ldquoEvaluation of the epidemiologicalrelevance of variable-number tandem-repeat genotyping ofMycobacteriumbovis and comparison of themethodwith IS6110restriction fragment length polymorphism analysis and spolig-otypingrdquo Journal of Clinical Microbiology vol 44 no 6 pp1951ndash1962 2006

[4] R Brosch S V Gordon M Marmiesse et al ldquoA new evolu-tionary scenario for the Mycobacterium tuberculosis complexrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 99 no 6 pp 3684ndash3689 2002

[5] A L Gibson G Hewinson T Goodchild et al ldquoMolecularepidemiology of disease due toMycobacterium bovis in humansin the United Kingdomrdquo Journal of Clinical Microbiology vol42 no 1 pp 431ndash434 2004

[6] A L C Rodrigues S K L D Silva B L A Pinto J B D Silvaand U Tupinambas ldquoOutbreak of laboratory-acquired Brucellaabortus in Brazil a case reportrdquo Revista da Sociedade Brasileirade Medicina Tropical vol 46 no 6 pp 791ndash794 2013

[7] Italian Ministry of Health ldquoEpidemiological Bullettinrdquo httpwwwsalutegovitportaletemidatidefconsMalattiejsp

[8] M I Queipo-Ortuno J D Colmenero J M Reguera et alldquoRapid diagnosis of human brucellosis by SYBR green I-basedreal-time PCR assay and melting curve analysis in serumsamplesrdquo Clinical Microbiology and Infection vol 11 no 9 pp713ndash718 2005

[9] E Mazars S Lesjean A-L Banuls et al ldquoHigh-resolutionminisatellite-based typing as a portable approach to global anal-ysis of Mycobacterium tuberculosis molecular epidemiologyrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 4 pp 1901ndash1906 2001

[10] J Kamerbeek L Schouls AKolk et al ldquoSimultaneous detectionand strain differentiation of Mycobacterium tuberculosis fordiagnosis and epidemiologyrdquo Journal of Clinical Microbiologyvol 35 no 4 pp 907ndash914 1997

[11] R Frothingham and W A Meeker-OrsquoConnell ldquoGenetic diver-sity in the Mycobacterium tuberculosis complex based on vari-able numbers of tandem DNA repeatsrdquo Microbiology vol 144no 5 pp 1189ndash1196 1998

[12] M B Boniotti M Goria D Loda et al ldquoMolecular typing ofMycobacterium bovis strains isolated in Italy from 2000 to 2006and evaluation of variable-number tandem repeats for geo-graphically optimized genotypingrdquo Journal of Clinical Microbi-ology vol 47 no 3 pp 636ndash644 2009

[13] P Supply ldquoProtocol and guidelines for multilocus variablenumber tandem repeat genotyping of M bovis VENoMYC(veterinary network of laboratories researching into improveddiagnosis and epidemiology of mycobacterial diseases) WP7rdquoin Proceedings of the WP7Workshop VENoMYC CoordinationAction EU SSPE-CT-2004-501903 pp 15ndash16 Toledo SpainOctober 2006

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in Italy he had been working as a butcher for 10 years InJune 2011 after an accidental trauma with an open woundduring work swelling appeared in his second finger of the lefthand progressively extending to the wrist So he had beentreated with several antibiotics not recalled by the patient (apart from levofloxacin at unspecified dosage for ten days)without significant response Then a surgical treatment forcarpal tunnel syndrome was performed in November 2011 InAugust 2012 a subcutaneous abscess was drained while thepatient was in Bulgaria

In February 2013 fever started and an appropriate work-up at the Infectious Diseases Unit of ldquoJohn Paul IIrdquo Hospitalin Lamezia Terme led to a diagnosis of brucellosis basedon clinical manifestations and a positive direct agglutinationreaction (Wright test) for Brucella spp (either B abortus or Bmelitensis) So he was treated with doxycycline (100mg twicedaily) plus rifampicin (600mg once daily) for 2 months Atthe end of treatment a significant improvement was recordedwith absence of fever and meningeal signs but Wright testfor Brucella spp remained positive (1 160 titration) Aftera few days the patient was admitted for meningitis withheadache vomiting and neck stiffness Magnetic resonanceimaging (MRI) of the brain revealed ldquodiffuse hyperintensitiesin T2 sequences at whitematter with small enhanced nodularareasrdquo Also clear cerebrospinal fluid with increased proteinand reduced glucose concentrations was demonstrated So adiagnosis of neurobrucellosis was made For this reason hereceived ceftriaxone (2 g twice daily) for onemonth with vol-untary discharge after improvement (May 2013) During hos-pitalization Wright test became negative (April 16 2013) butleft wrist and hand still remained swollen

In August and September 2013 two skin swabs were posi-tive for Staphylococcus aureus and tobramycin linezolid andlevofloxacin at unknown dosages were prescribed for about30 days without any improvements

The patient presented for consultation at the InfectiousDiseases Unit of the University Hospital of Catanzaro inNovember 2013 On examination he had a swollen lesion inhis left wrist with a spontaneous drainage from a cutaneousfistula Also left knee and the second finger of the right handappeared swollen A MRI of the left hand was prescribedshowing ldquobone lesions attributable to arthriticseptic lesionsrdquo(Figure 1)

The patient was then admitted to our day-hospitalwhere further exams were performed complete blood count(4900000 red blood cells120583L 11700 white cells120583L with689 neutrophils and 295000 platelets120583L) was normaland also renal and liver function tests appeared to be withinthe range of normality Tuberculin skin test (Mantoux test)was positivewith an infiltrate of a diameter of 5 cmMoreoverQuantiferon-TB Gold in-tube (QFT-IT) test was positiveBoth Brucella spp DNA from blood (homemade real-timePCR assay as described below) and Brucella spp antibodies(1 160 titration) were positive However Brucella specifictreatment was postponed waiting for confirmation andresults of bone biopsy that was planned

In January 2014 the patient underwent the bone biopsyof the left wrist at the Orthopedics Unit of the UniversityHospital in Catanzaro leading to isolation of Enterobacter

Figure 1 Magnetic resonance imaging of the left wrist beforestarting antibiotic therapy for Brucella spp and Mycobacteriumbovis The picture shows increased signal intensity of trabecularbone of all carpal bones due to marked medullar edema with manyerosive areas most evident in scaphoid and capitate bones

cloacae (E cloacae) spp cloacae Also PCR for DNA of Mtuberculosis complex (MTBC) and Brucella spp DNA werepositive Afterwards cultures resulted positive for alcohol-acid resistant bacilli subsequently characterized as M bovisssp bovis Drug susceptibility tests (both genotypic and phe-notypic) were performed for rifampicin isoniazid ethamb-utol streptomycin pyrazinamide amikacin ofloxacin andlinezolid A molecular line specific probe assay (GenotypeMTBRD plus Hain Lifesciences GmbH Germany) was usedfollowing manufacturerrsquos instructions Only resistance topyrazinamide was detected

Regarding methods the samples were processed accord-ing to national and international guidelines using an N-acetyl-l-cysteine-NaOH decontamination procedure inocu-lated into BACTEC MGIT 960 tubes (Becton Dickinson andCo Cockeysville MD USA) and onto solid slant medium(Lowenstein-Jensen) and incubated at 37∘C for up to 4 and 6weeks respectively

MTBC-DNA was amplified with Strand DisplacementAmplification (SDA) technology (ProbeTec ET SystemBecton-Dickinson and Co Cockeysville MD USA) Ampli-fied MTBC-DNA was revealed only in the soft tissue ofthe left wrist biopsy Identification of species was per-formed using GenoType MTBC assay (Hain LifesciencesNehren Germany) Genotype MTBC assay allowed us toidentify the isolate as M bovis ssp bovis For Brucella DNAextraction either in blood or in bone samples a Qiagenkit was used following a modified procedure suggested bythe manufacturer (Qiagen Hilden Germany) For genus-specific real-time PCR assay the forward primer B4 51015840-TGG-CTCGGTTGCCAATATCAA-31015840 and reverse primer B5 51015840-CGCGCTTGCCTTTCAGGTCTG-31015840 were used to amplify a223 bp portion of the BCSP31 gene [8] The PCR conditionswere denaturation at 95∘C for 10min followed by 45 cycles(95∘C for 10 s 60∘C for 10 s and 72∘C for 9 s) After amplifi-cation melting curve analysis was carried out by evaluating amelting temperature of 8816 plusmn 005∘C





3 Discussion







Acknowledgments


References




















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Discussion







Acknowledgments


References




















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















case report professional acquisition of m. bovis in calabria...

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