Educational Workshop EW13: Infections due to non-tuberculous mycobacteria

Arranged with the ESCMID Study Group for Mycobacterial Infections

Convenors: J.S. Friedland (London, GB) J. Gonzalez (Barcelona, ES)

Faculty: E. Tortoli (Milan, IT) E. Cambau (Paris, FR) G. Bloemberg (Zurich, CH) M. Viveiros (Lisbon, PT)

1

2 Tortoli - The world of nontuberculous mycobacteria

The world of nontuberculous mycobacteria

Enrico Tortoli

23rd ECCMID, Berlin April 27, 2013

Emerging Bacterial Pathogens Unit

The origin of mycobacteria

Merhej et al. Biology Direct 2009

Emerging Bacterial Pathogens Unit

The

KINGDOM TYPE Actinobacteria CLASS Actinobacteria ORDER Corynebacteriales SUB-ORDER Corynebacterineae FAMILY Mycobacteriaceae

Gao and Gupta. Microbiol.Mol.Biol.Rev. 2012

Emerging Bacterial Pathogens Unit

3 Tortoli - The world of nontuberculous mycobacteria

M. ulcerans coeval of dinosaurs?

Hayman. Lancet. 1984

Emerging Bacterial Pathogens Unit

The NonTuberculous Mycobacteria

Environmental and opportunistic pathogens of humans and animals

Emerging Bacterial Pathogens Unit

The history

• 1889. M. smegmatis “agent of syphilis” • 1953. Buhler and Pollak: Two cases of pulmonary disease caused by “yellow bacillus” • 1956. Prissick et Masson: Cervical lymphadenitis in children caused by chromogenic mycobacteria • 1959. Runyon’s classification • 1979. Wolinsky: Nontuberculous mycobacteria and associated diseases • 1982. First reports of disseminated MAC infections in AIDS

Emerging Bacterial Pathogens Unit

4 Tortoli - The world of nontuberculous mycobacteria

Habitat

• Waters – natural waters • acidic swamps – drinking water distribution systems • biofilms • aerosols •Soil – forests – peats – potting

Emerging Bacterial Pathogens Unit

The cell wall

Lipid-rich hydrophobic impermeable

Emerging Bacterial Pathogens Unit

The hydrophobicity

• Promotes the attachment to surfaces • Provides protection from a wide range of antimicrobial agents • Is responsible of biofilm formation • Causes the concentration at air-water interfaces such facilitating the aerosol formation • Allows the utilization of hydrocarbons

Emerging Bacterial Pathogens Unit

5 Tortoli - The world of nontuberculous mycobacteria

The slow growth

The growth rate is limited by: • the costly synthesis of mycolic acids • the impermeability to hydrophilic nutrients • the low number of copies of RNA operons which affects the rate of protein synthesis

Emerging Bacterial Pathogens Unit

The slow growth

• Disadvantages – poor competitors in nutrient-rich habitats • Advantages – survival and proliferation in marginal environments – adequate time to adapt to changed conditions • dormant state to survive anaerobiosis or starvation • increased resistance in biofilms

Emerging Bacterial Pathogens Unit

Other factors influencing environmental distribution

• Growth at low pH – acidic swamps and peats • Temperature resistance – survival in hot springs and hot water pipes • Oligotrophism – survival in environments with low level of nutrients, in particular when competitors have been eliminated by disinfectants

Emerging Bacterial Pathogens Unit

6 Tortoli - The world of nontuberculous mycobacteria

Intracellular organisms

Intracellular growth, in either amoebae or macrophages, results in increased virulence

Emerging Bacterial Pathogens Unit

Slow- and rapid-growers

Clear phylogenetic separation of slow- and rapid-growers with the latter being the more ancestral

Emerging Bacterial Pathogens Unit

The transmission of human infections

• Aerosol droplets (shower, hot tub, SPAs) – Pulmonary infections • Waterborne transmission – Cervical lymphadenitis in children – Cutis and soft tissues infections • Soil and dust – Post-traumatic infections (cutis and soft tissues, bone and joints)

Emerging Bacterial Pathogens Unit

7 Tortoli - The world of nontuberculous mycobacteria

Differences in pathogenicity

M. gordonae M AC M. malmoense M. terrae M. xenopi M. kansasii M. triviale M. celatum M. szulgai ...... M. shimoidei

Emerging Bacterial Pathogens Unit

. . . and in the genomic size

SPECIES ORFs M. smegmatis 6,940 M. rhodesiae 6,340 M. fortuitum 6,300 M. chubuense 6,070 M. kansasii 5,960 M. vanbaalenii 6,050 M. kansasii 5,960 M. marinum 5,500 M. avium 5,400 M. intracellulare 5,280 M. abscessus 4,990 M. ulcerans 4,310 M. tuberculosis 4,060 M. leprae 2,750 http://www.genomesonline.org

Emerging Bacterial Pathogens Unit

NTMs in the European Union

Croatia

Denmark M. avium Estonia M. gordonae Finland M. intracellulare Germany M. fortuitum

Greece M. xenopi M. malmoense Italy

Luxembourg

Netherlands

Slovenia

0 10203040506070 ECDC. Eurosurveillance. In press

Emerging Bacterial Pathogens Unit

8 Tortoli - The world of nontuberculous mycobacteria

NTMs in the European Union

M. avium M. gordonae M. intracellulare M. fortuitum M. xenopi M. chelonae M. abscessus M. kansasii M. malmoense Others

ECDC. Eurosurveillance. In press

Emerging Bacterial Pathogens Unit

NTMs, “green” bacteria?

• Evidence of the presence of rapid-growers in polluted soils • Ability to metabolize a variety of hydrocarbon pollutants

Emerging Bacterial Pathogens Unit

9 Cambau - Procedures for drug susceptibility testing in rapid and slow growing NTM

10 Cambau - Procedures for drug susceptibility testing in rapid and slow growing NTM

11 Cambau - Procedures for drug susceptibility testing in rapid and slow growing NTM

12 Cambau - Procedures for drug susceptibility testing in rapid and slow growing NTM

13 Cambau - Procedures for drug susceptibility testing in rapid and slow growing NTM

14 Cambau - Procedures for drug susceptibility testing in rapid and slow growing NTM

15 Cambau - Procedures for drug susceptibility testing in rapid and slow growing NTM

16 Cambau - Procedures for drug susceptibility testing in rapid and slow growing NTM

17 Cambau - Procedures for drug susceptibility testing in rapid and slow growing NTM

18 Bloemberg - Molecular mechanisms of drug resistance in NTM

Institut für Medizinische Mikrobiologie

Molecular mechanisms of drug resistance in NTM

Dr. Guido V. Bloemberg Institute of Medical Microbiology University of Zürich Zürich, Switzerland

12:00-12:30 Saturday 27th April ©CNRT

Institut für Medizinische Mikrobiologie Medical relevance of nontuberculous mycobacteria (NTM)

Examples:

M. leprae Leprosy M. abscessus, M. kansasii Lung disease M. avium Lymphadenitis M. ulcerans Cutaneous ulcers M. haemophilum Joint infections M. genavense Disseminated infections

Tortoli. Clinical manifestations of nontuberculous mycobacteria infections. Clin Microbiol Infect. 2009 Oct;15(10):906-10.

Institut für Medizinische Mikrobiologie Non-tuberculous mycobacteria - Slow growers; approx.60 species - M. avium - M. intracellulare - M. kansasii - M. genavense - M. leprae - M. xenopi

- Fast growers; approx. 70 species - M. fortuitum group - M. chelonae/M.abscessus group - M. smegmatis group - M. margeritense / M. wolinskyi group

19 Bloemberg - Molecular mechanisms of drug resistance in NTM

Institut für Medizinische Mikrobiologie What do we detect in the diagnostic laboratory?

Sequence based identification of NTM directly in clinical specimens during a 6-months period:

16S rRNA gene sequence identification n M. chelonae/abscessus complex 36 M. kansasii/gastri 10 M. avium 9 M. fortuitum 7 M. xenopi 3 M. mucogenicum 2 M. asiaticum 1 M. intracellulare 1 M. malmoense 1 Non-pathogenic mycobacteria 52 (mostly rapid growers)

Institut für Medizinische Mikrobiologie Most common used antibiotics for treatment of NTM infections

- Macrolides - Aminoglycosides - Rifampicin - Ethambutol - Fluoroquinolones - Tetracyclines

Institut für Medizinische Mikrobiologie

Resistance against antibiotics

- Intrinsic resistance - Modifying enzymes

- Acquired resistance - Chromosomal mutations

20 Bloemberg - Molecular mechanisms of drug resistance in NTM

Institut für Medizinische Mikrobiologie Reviews

Institut für Medizinische Mikrobiologie

Resistance against macrolides (protein synthesis)

Resistance against clarithromycin / erythromycin

- Intrinsic resistance - erm genes - Acquired resistance - 23S rRNA gene

Institut für Medizinische Mikrobiologie Resistance against aminoglycosides (protein synthesis)

- Intrinsic resistance: - Aminoglycoside modifying enzymes - Acetyltransferases - Phosphotransferases - Acquired resistance: - Mutations in 16S rRNA gene - Aminoglycoside phosphotransferase

21 Bloemberg - Molecular mechanisms of drug resistance in NTM

Institut für Medizinische Mikrobiologie Resistance against rifampin (RNA synthesis)

Fast growing non-tuberculous mycobacteria: - naturally resistant - possibly ADP ribosylases

Slow growing non-tuberculous mycobacteria: acquired resistance: mutations in rpoB

Institut für Medizinische Mikrobiologie

Resistance against ethambutol (arabinogalactan – arabinomannan synthesis)

- Acquired resistance - Mutations in embB, embR and other genes in the emb operon

- Intrinsic resistance - Polymorphisms in embB, lfrA, efflux pump

Institut für Medizinische Mikrobiologie

Clarithromycin resistance in abscessus

22 Bloemberg - Molecular mechanisms of drug resistance in NTM

Institut für Medizinische Mikrobiologie M. abscessus

General information - Pulmonary infections; chronic infections - Risk groups: underlying pulmonary diseases, e.g. bronchiectasis and cystic fibrosis - a major complication following lung transplantation in CF patients - Treatment - surgical resection of focal bronchiectasis and cavitie - combination therapy including the macrolide larithromycin - only drug of demonstrated efficacy that can be administered orally

Institut für Medizinische Mikrobiologie Clarithromycin

- Macrolide - Binds to the 23 rRNA - Inhibits translation - Acid-stable, orally taken - High concentration in phagocytes

Institut für Medizinische Mikrobiologie Clarithromycin resistance in M. abscessus Two main resistance mechanisms: - Mutations in the drug-binding pocket, in particular at nucleotide positions 2058 and 2059, of the bacterial 23S rRNA gene confering high level Cla resistance (MIC = 256 mg/L) - Inducible erm gene - erm gene encodes: ribosomal methylase Erm (41) - mono- or di-methylate the adenine at position 2058 of the 23S rRNA - Erm genes have been described in many species of rapidly growing mycobacteria - Functionality dependent on the nucleotide at position 28 - WT 28T inducible resistance; mutant 28C loss of functionality

23 Bloemberg - Molecular mechanisms of drug resistance in NTM

Institut für Medizinische Mikrobiologie Does M. abscessus acquire resistance mutations in the rrl gene in addition to the presence of an inducible Erm(41) methylase ?

- Monitoring 5 patients over 2-4 years - 29 M. abscessus clinical isolates - Genetic and phenotypic characterisation - In three out of five patients acquisition of resistance mutations in the rrl gene in addition to the presence of an inducible Erm methylase

Institut für Medizinische Mikrobiologie

Phenotypic and genotypic monitoring

Institut für Medizinische Mikrobiologie Genotyping of M. abscessus isolates using RAPD-PCR.

Maurer F P et al. J. Antimicrob. Chemother. 2012;67:2606- 2611 © The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]

24 Bloemberg - Molecular mechanisms of drug resistance in NTM

Institut für Medizinische Mikrobiologie Conclusion

Clarithromycin resistance mutations in the 23S rRNA

peptidyltransferase region provide an additional

selective advantage independent of a functional

erm(41) gene.

Institut für Medizinische Mikrobiologie Species dependent clarithromycin resistance

Interspecies differences in erm genes:

The Erm methylase in M. abscessus subsp. massiliense is dysfunctional due to: - 2 bp deletion of nucleotides 64–65 - 274 bp deletion of nucleotides 159–432

Institut für Medizinische Mikrobiologie Acknowledgements

Institute of Medical Microbiology

Florian Maurer Vera Rüegger Claudia Ritter Erik Böttger

25 Viveiros - Epidemiology of infections due to NTM

Educational Workshop / TAE Trainees Day Speaker

EW13: Infections due to non-tuberculous mycobacteria (NTM)

Epidemiology of infections due to NTM

Miguel Viveiros 27 April 2013

Instituto de Higiene e Medicina Tropical da Universidade Nova de Lisboa. Lisbon, Portugal

https://www.escmid.org/research_projects/study_groups/mycobacterial_infection/

Genus Mycobacterium: Mycobacterium tuberculosis 1882: Robert Koch 1896: Lehmann & Neumann Family: Mycobacteriaceae Order: Actinomycetales Class: Actinomycetes 1935: Pinner “Atypical acid-fast microorganisms” 1938: da Costa Cruz 1948: MacCallum et al. 1949: Cuttino & McCabe “Nocardia intracellulare” (M. intracellulare) 1951: Norden & Linell “M. balnei” (= M. marinum, 1926) 1955: Buhler & Pollak M. kansasii 1956: Masson & Prissick M. scrofulaceum 1959: Runyon: classification of “atypical” mycobacteria 1967: International Working Group on Mycobacterial Taxonomy (IWGMT)

Genus Mycobacterium

> 150 species

Mycobacterium tuberculosis complex M. tuberculosis, M. bovis, M. africanum, M. microti, M. canettii (M. caprae, M. pinnipedii, M. mungi)

v.s.

NTM – Non tuberculous mycobacteria (MNT) or MOTT - Mycobacteria other than tuberculosis or Atypical mycobacteria

26 Viveiros - Epidemiology of infections due to NTM

Genus Mycobacterium

> 150 species

Mycobacterium tuberculosis complex M. tuberculosis, M. bovis, M. africanum, M. microti, M. canettii (M. caprae, M. pinnipedii, M. mungi)

v.s.

NTM – Non tuberculous mycobacteria (MNT) or MOTT - Mycobacteria other than tuberculosis or Atypical mycobacteria – Nowdays became typical mycobacteria!

Genera Mycobacterium

> 150 species to date

Classification of nontuberculous mycobacterial diseases:

i) Risk of infection

ii) Clinical criteria

iii) The laboratorial diagnosis of NTM disease is complex and requires good communication between clinicians, radiologists, and microbiologists!

Swamps and bogs Ecology of the NTMs

• Ubiquitous, mostly environmental species • High adaptability to different ecological niches but the the still waters/ ponds/swamps/bogs are the most important source of NTMs • High temperatures, low [O2], low pH, high concentrations of [Zn] and [Fe], high concentrations of humic acids. • Hydrophobic nature >>> biofilms • Higher resistance to water treatments • Higher antibiotic resistance • Usually considered non-pathogenic • Pathogenicity depends on the immune status of the individual • Differentiation based on time of growth (slow growers vs. fast growers) and presence or absence of carotenoid pigmentation

27 Viveiros - Epidemiology of infections due to NTM

Laboratory classification of NTMs

Runyon criteria: growth rate; pigmentation

- Slow growers (> 7 days, solid media) (Runyon I, II, III) Runyon I (photochromogens: carotenoids accumulation after 1h of light exposure) e.g M. kansasii, M. marinum

Runyon II (scotochromogens: constitutive carotenoid accumulation) e.g M. gordonae, M. scrofulaceum

Runyon III (non chromogens: no pigmentation) e.g M. avium complex (M. avium, M. intracellulare) *Variable pigmentation

- Fast growers (< 7 dias) (Runyon IV) Runyon IV (non chromogens: no pigmentation) e.g M. fortuitum, M. chelonae, M. abcessus

NTM clinical syndromes in humans PULMONARY DISEASE LYMPHADENITIS Mainly in adults, rare in children, specially Commonest form of infection by in older persons with or without underlying NTM in children chronic lung disease and patients with cystic Initial injury in the gum or fibrosis; pharyngeal mucosa with subsequent Major NTM pathogens: M. avium complex lymphatic drainage to satellite and M. kansasii lymph nodes M. avium complex up to 80% in Symptoms similar to the pre-existing lung Southern Europe and in northern disease and TB Europe, M. malmoense and M. Fast growers: M. abscessus , M. fortuitum, haemophilum. M. xenopi, M. malmoense, M. szulgai, and M. simiae

NTM clinical syndromes in humans SKIN AND SOFT TISSUE DISSEMINATED DISEASE INFECTION Usually as a consequence of direct Mainly in severely immunocompromised inoculation, caused primarily by M. patients marinum and M. ulcerans and the Most commonly caused by MAC and less rapidly growing mycobacteria spp. commonly by the rapidly growing (RGM) infections in this category mycobacteria, M. abscessus, M. fortuitum, may be nosocomial, including and M. chelonae. surgical and esthetical site infections.

M. ulcerans M. avium complex M. marinum

28 Viveiros - Epidemiology of infections due to NTM

"CLASSIC“ CUTANEOUS INFECTIONS BY NTM

Cutaneous infections by and Mycobacterium ulcerans – difficult to isolate cultures in the laboratory, incubation period may vary from one week to two years.

M. marinum M. fortuitum, M. chelonae, M. abscessus, M. ulcerans

It inhabits swamps and bogs in West Africa, Agent of “tuberculosis" in fish from fresh or low Australia, China, Mexico and Latin America, salinity water that causes erythematous nodules and causing Buruli ulcer (Uganda, 1948) or granulomatous, single or multiple, and can progress to Baimsdale (Australia, 1960), which features necrosis and ulceration of the skin in aquarist slow and progressive evolution with significant (aquarium hobbyists) and users of pools, thermal destruction of skin and underlying tissues. baths or jacuzzis (granuloma of the pools).

Geographical distribution for Buruli´s ulcer

(Boleira et al. An. Bras Dermatol. 2010;85:281)

“MODERN” NON-TUBERCULOUS MYCOBACTERIA CUTANEOUS INFECTIONS

http://www.ncbi.nlm.nih.gov/sites/entrez

29 Viveiros - Epidemiology of infections due to NTM

“MODERN” CUTANEOUS INFECTION BY NTM

• M. abscessus •M. chelonae Apparatus for liposuction •M. fortuitum Mesotherapy solutions •M. xenopi Solutions and appliances for tattoos •M. scrofulaceum •M. gordonae •M. avium •M. furunculosis

 Post-treatment subcutaneous abscesses in non-hospital environment  Rapid dissemination in immunosuppression

“MODERN” CUTANEOUS INFECTION BY NTM

Recreational waters • M. abscessus •M. chelonae Pools 18-25ºC •M. xenopi Saunas 35-40ºC •M. fortuitum •M. gordonae (10x more mycobacteria) •M. avium Spa tubs  aerosols •M. furunculosis • M. marinum

 Patients with HIV-AIDS - dental clinics and hemodialysis Lifeguards and pool regulars - long exposure to aerosols Hospital hydrotherapy pools - children with cystic fibrosis - M. chelonae Skin infections in Spas and Beauty Salons Wound infection and transplantation. Risk of transmission during immunosuppression. http://www.stoptb.org; http://www.google.pt

"CLASSIC“ MAJOR RESPIRATORY INFECTIONS BY NTM

Respiratory infections by Mycobacterium avium and Mycobacterium kansasii - difficult to distinguish from M. tuberculosis in time for the implementation of appropriate therapy, resistant to 1st line tuberculostatic drugs

M. avium M. intracellulare , M. abscessus , M. kansasii

It exists in all soils and damp Agent of “bird tuberculosis“; causes lung environments causing lung infection very infection in immunocompetents, easily similar to tuberculosis rarely disseminated in immunosuppressed patients disseminated, even in (AIDS, leukemias, neoplasies, immunosuppressed patients. Frequent immunosuppressive therapy). It exists in soils, cause of cervical lymphadenitis in moist environments and water pipes. children.

30 Viveiros - Epidemiology of infections due to NTM

“MODERN” NON-TUBERCULOUS MYCOBACTERIA RESPIRATORY INFECTIONS CLEAR ASSOCIATION WITH CYSTIC FIBROSIS AND/OR COPD

THE MOST DIFFICULT CASES AT ANY AGE GROUP

http://www.ncbi.nlm.nih.gov/sites/entrez

CLINICAL SIGNIFICANCE

 Isolation of M. kansasii and M. malmoense (in northwestern Europe) from pulmonary specimens usually indicates disease, whereas Mycobacterium gordonae and, to a lesser extent, M. simiae or M. chelonae are typically contaminants rather than causative agents of true disease.  M. avium complex (MAC), M. xenopi, and M. abscessus form an intermediate category between these two extremes. (Jakko van Ingen, Diagnosis of Nontuberculous Mycobacterial Infections, Semin Respir Crit Care Med 2013; 34(01): 103-109

ROUTES OF TRANSMISSION OF NTMs

 Immunocompromised patients - leukemia, transplant recipients, AIDS M. avium complex Aerosols

 Patients with pre-existing lung diseases (CF & COPD) M. chelonae, M. fortuitum, Aerosols Tap-pipe-drinking M. abscessus /bolletii/massiliensis and M. xenopi and non sterile Water !  Hemophiliacs Dialysis machines M. chelonae, M. fortuitum, M. abscessus

 Surgery patients - Medical supplies - needles, catheters, pacemaker contaminated solutions, bronchoscopes Contaminated material M. chelonae, M. fortuitum, M. abscessus /bolletii/massiliensis and M. xenopi http://www.stoptb.org/http://www.ncbi.nlm.nih.gov/sites/entrez

31 Viveiros - Epidemiology of infections due to NTM

NTMs geographical & epidemiological distribution Significant increase in NTM isolates over time in Canada, United States, United , Netherlands, France, Spain and Portugal. Significant predominance of M. avium complex in respiratory infections.

Estimated prevalence of average >10 per 100,000 in Europe » Marras, T. K et al. Thorax 2007 » Van Ingen J, et al. Thorax 2009 » Couto I, et al. CMI 2010 » Prevots DR, et al. AJRCCM 2009 and 2010 » Amorim A et al. Scand J Infect Dis. 2010 » McCallum AD et al. J R Coll Physicians Edinb. 2011 » Couderc C, AD et al. Med Mal Infect. 2011 » García-Martos P & García-Agudo L. Enferm Infecc Microbiol Clin. 2012.

Estimated prevalence of 14 to 35 per 100,000 in Canada and the U.S. » Marras, T. K et al. AJRCCM 2008 » Billinger ME et al. Emerg Infect Dis. 2009 » Winthrop K, et al. AJRCCM 2010 » Prevots DR, et al. AJRCCM 2010

» Kendall BA &Winthrop KL.Semin Respir Crit Care Med. 2013 http://www.ncbi.nlm.nih.gov/sites/entrez

NTMs geographical & epidemiological distribution

Significant increase in NTM isolates over time in Asia with a significant predominance of M. avium complex

THE EXAMPLE OF PORTUGAL

Mycobacteria Lab. IHMT/UNL : January 2005 to December 2007

32 Viveiros - Epidemiology of infections due to NTM

Opportunistic infections of the immunosupressed

M. avium complex: Mycobacterium Mycobacterium avium avium avium complex subspecies MAC Mycobacterium avium subspecies hominissuis Mycobacterium avium subspecies paratuberculosis Mycobacterium intracellulare

The most frequent of all NTMs causing infection in humans globally also found in Portugal

Which M. avium subspecies is most prevalent?

www.healthcaremagic.com

80% Mycobacterium avium subspecies hominissuis

Submitted to IJMM, 2013

28 strains isolated from 69 strains isolated from human samples - porcine samples (lymph hospitals from the nodes) - regions across Lisbon Health Region mainland Portugal between between 2005 and 2011 2004 and 2006

Submitted to IJMM, 2013

Typed by Mycobacterium avium tandem repeats (MATR)-VNTR

The MAH isolates were found to be genetically diverse and genotypes are randomly distributed across the country.

None of the human strains shared identical profiles with porcine isolates, albeit a few profiles differed by only a single tandem repeat copy.

Both humans and pigs seem to be infected by the pool of environment-inhabiting opportunistic MAH strains rather than by specific and/or potentially high virulent and prevalent clones.

Comparative analysis using Portuguese MAH environmental strains, including from soil or water sources, are envisaged to ascertain if environmental strains are the main source of infections.

33 Viveiros - Epidemiology of infections due to NTM

Opportunistic infections of the immunosupressed RECOMMENDED READING

NTMs ?

Look in the environment !

www.healthcaremagic.com

Acknowledgments THE IHMT TEAM

INSTITUTE OF HYGIENE AND TROPICAL MEDICINE

THE INIAV & FCT/UNL TEAM Célia Leão Ilda Santos Sanches João Inácio Ana Botelho

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