dionisio, e et al 2013., rev inst med trop sp.pdf

8/11/2019 Dionisio, E et al 2013., REv Inst Med Trop SP.pdf

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ISSN 0036-4665ISSN 1678-9946 on line

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The purpose of the Revista do Instituto de Medicina Tropical de So Paulo (Journal of theSo Paulo Institute of Tropical Medicine) is to publish the results of researches which contri-bute significantly to knowledge of all transmissible diseases.

REVISTA DO INSTITUTO DE MEDICINA TROPICAL DE SO PAULO(JOURNAL OF THE S. PAULO INSTITUTE OF TROPICAL MEDICINE).

So Paulo, SP-Brasil, 1959 -v. ilust. 28 cm

1959-2013, 1-551973-2002 (supl. 1-12)2003 (supl. 13 - on-line only)2005-2012 (supl. 14-18)

ISSN 0036-4665ISSN 1678-9946 on line


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Rev. Inst. Med. Trop. Sao Paulo Vol. 55 No. 6 P. 371-440 November-December, 2013

ISSN 0036-4665ISSN 1678-9946 on line

ADDRESSINSTITUTO DE MEDICINA TROPICAL DE SO PAULO

Av. Dr. Enas de Carvalho Aguiar, 47005403-000 So Paulo, SP - BrazilPhone/Fax: 55.11.3062.2174; 55.11.3061-7005

e-mail: [email protected]

SUBSCRIPTIONSFOREIGN COUNTRIES

One year (six issues) ........ U$ 200.00Single issue ...................... U$ 50.00

CONTENTS

MYCOLOGYFirst report on Cryptococcus neoformans in pigeon excreta from public and residential locations in the metropolitan area of Cuiab,State of Mato Grosso, Brazil - D.T. TAKAHARA, M.S. LAZRA, B. WANKE, L. TRILLES, V. DUTRA, D.A.J. PAULA, L. NAKAZATO,M.C. ANZAI, D.P. LEITE JNIOR, C.R. PAULA & R.C. HAHN ...............................................................................................................................371

Distribution of dermatophytes from soils of urban and rural areas of cities of Paraiba State, Brazil - Z.B.V.S. PONTES, A.C. OLIVEIRA,F.Q.S. GUERRA, L.R.A. PONTES & J.P. SANTOS .....................................................................................................................................................377

Molecular typing of Candida albicansisolates from hospitalized patients - P.S. BONFIM-MENDONA, A. FIORINI,C.S. SHINOBU-MESQUITA, L.C. BAEZA, M.A. FERNANDEZ & T.I.E. SVIDZINSKI .................................. ....................................... .................385

LEISHMANIASISApplicability of kDNA-PCR for routine diagnosis of American tegumentary leishmaniasis in a tertiary reference hospital -M.M. SATOW, E.H. YAMASHIRO-KANASHIRO, M.C. ROCHA, L.K. OYAFUSO, R.C. SOLER, P.C. COTRIM & J.A.L. LINDOSO ................393

PCRComparison of six commercially-available DNA polymerases for direct PCR - M. MIURA, C. TANIGAWA, Y. FUJII & S. KANEKO ...................401

PHLEBOTOMINESPhlebotomine sandflies in rural locations in the state of Parana, Southern Brazil - S.C.C.S. MELO, W. CELLA, R. MASSAFERA,N.M.M.G. SILVA, R. MARQUI, M.D.B. CARVALHO & U. TEODORO .................................. ....................................... ........................................ ...407

PARASITOLOGYPotentially pathogenic free-living amoebae in some flood-affected areas during 2011 Chiang Mai flood - A. WANNASAN,P. UPARANUKRAW, A. SONGSANGCHUN & N. MORAKOTE ............................... ....................................... ....................................... .................411

MICROBIOLOGYSmqnrvariants in clinical isolates of Stenotrophomonas maltophiliain Brazil - J.I. GRACIA-PAEZ, J.R. FERRAZ,I.A. FRANA E SILVA, F. ROSSI, A.S. LEVIN & S.F. COSTA ................................... ....................................... ....................................... .................417

BRIEF COMMUNICATIONOvicidal effect of Piperaceae species onBiomphalaria glabrata,Schistosoma mansoni host - L.N. RAPADO,

P.O.M. LOPES,

L.F. YAMAGUCHI& E. NAKANO ...............................................................................................................................................................................421

CASE REPORTCase study of a patient with HIV-AIDS and visceral leishmaniasis co-infection in multiple episodes - E.D. SILVA, L.D. ANDRADE,P.S.R. ARAJO, V.M. SILVEIRA, C.E. PADILHA, M.A.L. SILVA & Z.M. MEDEIROS ................................... ....................................... .................425

Usefulness of kDNA PCR in the diagnosis of visceral leishmaniasis reactivation in co-infected patients - A.C. NICODEMO, V.S. AMATO,F.F. TUON, R.M. SOUZA, T.S. OKAY & L.M.A. BRAZ .................................................... ........................................ ....................................... ..........429

LETTERS TO THE EDITORDifferential diagnosis of respiratory viruses by using real time RT-PCR methodology - R.S. PAULINO, M.A. BENEGA, K.C.O. SANTOS,D.B.G. SILVA, J.C. PEREIRA, N.A. SASAKI, P.E. SILVA, S.P. CURTI, M.I. OLIVEIRA, T.R.M.P. CARVALHANAS, T. PERET,

D. ERDMAN & T.M. PAIVA..........................................................................................................................................................................................432

High prevalence of hepatitis A antibodies among recyclable waste pickers, Central Brazil - H.O. SOARES, C.L.R. LOPES, N.R. FREITAS,.M. COSTA E SILVA, L.R. MOURA & R.M.B. MARTINS ....................................... ........................................ ....................................... .................433

Analogies in medicine: violin strings adhesions - J.S. ANDRADE-FILHO ..................................................................................................................435

AUTHOR INDEX ......................................................................................................................................................................................................437

SUBJECT INDEX .....................................................................................................................................................................................................439

Impact Factor: 0.959


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ENDEREOINSTITUTO DE MEDICINA TROPICAL DE SO PAULO

Av. Dr. Enas de Carvalho Aguiar, 47005403-000 So Paulo, SP - BrasilFone/Fax: 55.11.3062.2174; 55.11.3061-7005

e-mail: [email protected]

Rev. Inst. Med. Trop. Sao Paulo Vol. 55 No. 6 P. 371-440 Novembro-Dezembro, 2013

CONTEDO

ISSN 0036-4665ISSN 1678-9946 on line

MICOLOGIAPrimeiro registro de Cryptococcus neoformans em excretas de pombos provenientes de locais pblicos e residenciais de rea metropolitana deCuiab, Estado do Mato Grosso, Brasil - D.T. TAKAHARA, M.S. LAZRA, B. WANKE, L. TRILLES, V. DUTRA, D.A.J. PAULA,L. NAKAZATO, M.C. ANZAI, D.P. LEITE JNIOR, C.R. PAULA & R.C. HAHN .................................................... ........................................ .......371

Distribuio de dermatfitos isolados de solos de cidades do Estado da Paraba, Brasil - Z.B.V.S. PONTES, A.C. OLIVEIRA,F.Q.S. GUERRA, L.R.A. PONTES & J.P. SANTOS .....................................................................................................................................................377

Tipagem molecular de Candida albicansisoladas de pacientes hospitalizados - P.S. BONFIM-MENDONA, A. FIORINI,C.S. SHINOBU-MESQUITA, L.C. BAEZA, M.A. FERNANDEZ & T.I.E. SVIDZINSKI ..................................... ....................................... ..............385

LEISHMANIOSEAplicao do kDNA-PCR para diagnstico de rotina de leishmaniose tegumentar americana em um hospital de referncia - M.M. SATOW,E.H. YAMASHIRO-KANASHIRO, M.C. ROCHA, L.K. OYAFUSO, R.C. SOLER, P.C. COTRIM & J.A.L. LINDOSO ..........................................393

PCRComparao de seis polimerases de DNA disponveis comercialmente para o PCR direto - M. MIURA, C. TANIGAWA, Y. FUJII &S. KANEKO ....................................................................................................................................................................................................................401

FLEBOTOMNEOSFlebotomneos em localidades rurais do Estado do Paran, Sul do Brasil - S.C.C.S. MELO, W. CELLA, R. MASSAFERA, N.M.M.G. SILVA,R. MARQUI, M.D.B. CARVALHO & U. TEODORO .................................. ........................................ ....................................... .................................. 407

PARASITOLOGIAAmebas potencialmente patognicas de vida livre em algumas reas afetadas durante a inundao de 2011 em Chiang Mai - A. WANNASAN,P. UPARANUKRAW, A. SONGSANGCHUN & N. MORAKOTE ................................. ........................................ ....................................... ..............411

MICROBIOLOGIAVariantes de Smqnrde isolados clnicos de Stenotrophomonas maltophiliano Brasil - J.I. GRACIA-PAEZ, J.R. FERRAZ,I.A. FRANA E SILVA, F. ROSSI, A.S. LEVIN & S.F. COSTA ......................................................... ....................................... .................................. 417

COMUNICAO BREVEEfeito ovicida de espcies de Piperaceae emBiomphalaria glabrata, hospedeiro do Schistosoma mansoni - L.N. RAPADO,

P.O.M. LOPES,

L.F. YAMAGUCHI& E. NAKANO ...............................................................................................................................................................................421

RELATO DE CASOEstudo de caso de paciente com mltiplos episdios da coinfeco HIV-AIDS e leishmaniose visceral - E.D. SILVA, L.D. ANDRADE,P.S.R. ARAJO, V.M. SILVEIRA, C.E. PADILHA, M.A.L. SILVA & Z.M. MEDEIROS ...................................... ....................................... ..............425

Utilidade da kDNA PCR no diagnstico de reativao de leishmaniose visceral em pacientes co-infetados sintomticos - A.C. NICODEMO,V.S. AMATO, F.F. TUON, R.M. SOUZA, T.S. OKAY & L.M.A. BRAZ ...................................... ........................................ ....................................... .429

CARTAS AO EDITORDifferential diagnosis of respiratory viruses by using real time RT-PCR methodology - R.S. PAULINO, M.A. BENEGA, K.C.O. SANTOS,

D.B.G. SILVA, J.C. PEREIRA, N.A. SASAKI, P.E. SILVA, S.P. CURTI, M.I. OLIVEIRA, T.R.M.P. CARVALHANAS, T. PERET,D. ERDMAN & T.M. PAIVA..........................................................................................................................................................................................432

High prevalence of hepatitis A antibodies among recyclable waste pickers, Central Brazil - H.O. SOARES, C.L.R. LOPES, N.R. FREITAS,.M. COSTA E SILVA, L.R. MOURA & R.M.B. MARTINS.......................................................................................................................................433

Analogies in medicine: violin strings adhesions - J.S. ANDRADE-FILHO ...................................................... ....................................... .....................435

NDICE DE AUTORES ...........................................................................................................................................................................................437

NDICE DE ASSUNTOS .........................................................................................................................................................................................439

Impact Factor: 0.959


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Rev. Inst. Med. Trop. Sao Paulo

55(6):371-376, November-December, 2013

doi: 10.1590/S0036-46652013000600001

(1) Laboratrio de Micologia, Faculdade de Medicina, Universidade Federal do Mato Grosso, Cuiab, MT, Brazil.(2) Laboratrio de Micologia, Instituto de Pesquisas Clnicas Evandro Chagas, Fundao Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.(3) Laboratrio de Biologia Molecular Veterinria, Faculdade de Agronomia e Medicina Veterinria, Universidade Federal do Mato Grosso, Cuiab, MT, Brazil.

(4) Laboratrio de Leveduras Patognicas, Instituto de Cincias Biolgicas, Universidade de So Paulo, So Paulo, SP, Brazil.Correspondence to: Prof Rosane Hahn. Laboratrio de Micologia/Investigao/FM/UFMT. Av. Fernando Corra da Costa 2369, Bairro Boa Esperana, 78060-900 Cuiab, MT, Brasil.

Phone: 55 65 3615-8809. E-mail: [email protected]

FIRST REPORT ON Cryptococcus neoformansIN PIGEON EXCRETA FROM PUBLICAND RESIDENTIAL LOCATIONS IN THE METROPOLITAN AREA OF CUIAB,

STATE OF MATO GROSSO, BRAZIL

Doracilde Terumi TAKAHARA(1), Mrcia dos Santos LAZRA(2), Bodo WANKE(2), Luciana TRILLES(2), Valria DUTRA(3), Daphine Ariadne Jesus de PAULA(3),Luciano NAKAZATO(3), Mariana Caselli ANZAI(1), Diniz Pereira LEITE JNIOR(1), Claudete Rodrigues PAULA(4) & Rosane Christine HAHN(1)

SUMMARY

Cryptococcosis is a severe systemic mycosis caused by two species of Cryptococcusthat affect humans and animals: C. neoformansand C. gattii. Cosmopolitan and emergent, the mycosis results from the interaction between a susceptible host and the environment. Theoccurrence of C. neoformanswas evaluated in 122 samples of dried pigeon excreta collected in 49 locations in the City of Cuiab, Stateof Mato Grosso, Brazil, including public squares (n = 5), churches (n = 4), educational institutions (n = 3), health units (n = 8), openareas covered with asbestos (n = 4), residences (n = 23), factory (n = 1) and a prison (n = 1). Samples collected from July to Decemberof 2010 were seeded on Niger seed agar (NSA). Dark brown colonies were identified by urease test, carbon source assimilation testsand canavanine-glycine-bromothymol blue medium. Polymerase chain reaction primer pairs specific for C. neoformanswere alsoused for identification. Cryptococcus neoformansassociated to pigeon excreta was isolated from eight (6.6%) samples correspondingto six (12.2%) locations. Cryptococcus neoformanswas isolated from urban areas, predominantly in residences, constituting a riskof acquiring the disease by immunocompromised and immunocompetent individuals.

KEYWORDS:Cryptococcus neoformans;Pigeon excreta; Urban environment; State of Mato Grosso.

INTRODUCTION

Although cryptococcosis has been studied since 1894, over thepast 40 years many important advances have been achieved regardingtaxonomy, epidemiology, capsular structure, virulence factors, serotypesand specific genotypes25. In Brazil, reports have been registered in moststates11,16,23,29,34,39, but in the State of Mato Grosso little research has beenconducted in relation to clinical and environmental isolates of the agentsof cryptococcosis. The first description of these microorganisms in HIV-positive patients in Mato Grosso was reported by FAVALESSA et al., whodetected 26 Cryptococcusneoformansand 10 Cryptococcus gattiiisolatesin distinct clinical materials from seropositive and seronegative patients10.

The genus Cryptococcuscomprises more than 38 species, two ofwhich are considered potentially pathogenic: Cryptococcus neoformansand C. gattii17,18,20. Although both are found worldwide, their mainecological niches present some differences:Cryptococcus neoformansismost commonly isolated from pigeon droppings, while C. gattiiis morefrequently isolated from decaying wood and soil14,24,25.

The presence of Cryptococus neoformans in soil and old driedpigeon excreta has been widely studied in several countries7,8. Poultry

manure is considered a natural substrate for C. neoformans. Pigeonscan even carry it on their beaks, feathers and legs, as well as presentingcolonization by this agent on the crop. They act as dispersers in theenvironment, generating a source of infection for humans. Regardingthe primary habitat of C. neoformans, species of plants and aged woodscan be considered locations where the yeasts may naturally developtheir sexual state32.

Considering the complete lack of data reported in the literature todate, the present study aimed to evaluate the possible environmentaldistribution of C. neoformans in public places (churches, squares,educational institutions, prisons, factories and health facilities) andresidences within the City of Cuiab and the neighbor metropolitan area.

MATERIAL AND METHODS

Study periods and locations: The samples were collected betweenJuly and December of 2010 in the metropolitan area of Cuiab.

Mato Grosso is located in the Midwest region of Brazil. The stateoccupies an area of 903,357km, being the third largest from Braziland it is the only one to have three characteristic biomes, Pantanal


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TAKAHARA, D.T.; LAZRA, M.S.; WANKE, B.; TRILLES, L .; DUTRA, V.; PAULA, D.A.J.; NAKAZATO, L.; ANZAI, M.C.; LEITE-JNIOR, D.P.; PAULA, C.R. & HAHN, R.C.- Firstreport on Cryptococcus neoformans in pigeon excreta from public and residential locations in the metropolitan area of Cuiab, State of Mato Grosso, Brazil.Rev. Inst. Med. Trop. SaoPaulo, 55(6): 371-6, 2013.

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(marshland), Cerrado (Brazilian savanna) and Amazon. Its capital is theCity of Cuiab, which has about 551,000 inhabitants. In its territory issituated the geodesic center of South America, at 153556 South and

560605 West (Fig. 1).

The climate is characterized by a mean annual rainfall of 1,469.4mm and average annual temperature of 24 to 26 C. Despite unequallydistributed, the region is well supplied with rain and seasonality istypically tropical, with maximal temperatures in summer and minimalin winter. Over 70% of the total rainfall accumulated during the periodof November to March. The winters are excessively dry, due to veryscarce rainfall35. The average temperature during the collecting periodwas 27 C (July to December 2010), with maximal that reached 40 Cfor several times in August, September and October15.

The sites selected were characterized as follows: five public squares,four churches, three educational institutions, eight public health units,

four open areas covered with asbestos, 23 residences, one factory andone prison.

Inclusion criteria: At all the sites selected, aspects related to theexcreta collected were evaluated according to the following parameters:excreta presenting a dried aspect; deposited on the surfaces of public orresidential environments; the presence of pigeons close to the excreta;the presence of chicks or nests; and sufficient quantity for posteriorweighing (> one gram) and analysis.

Sample processing: Following homogenization, 1 g of each samplewas suspended in 50 mL of sterile physiological saline with 0.4 g/Lchloramphenicol, shaken for five min and allowed to settle for 30 min.

The supernatant was aspirated, inoculated onto Niger seed agar (NSA)medium (0.1 mL of supernatant per plate, 10 plates per sample), incubatedat room temperature (25 C to 27 C) and observed for five to seven days.

Yeast colonies on NSA were selected by observing the shiny, smooth,and dark brown colonies (due to melanin production). The brown colonieswere sub-cultivated onto Sabouraud (Merck) medium for urease test andother biochemical tests as well microscopic analysis with India ink tovisualize the capsule21,22

.For the biochemical tests, auxanogram technique

was used, in which the assimilation of eleven carbon sources (dextrose,lactose, maltose, sucrose, inositol, galactose, cellobiose, melezitose,melibiose, rhamnose and erythritol)and two nitrogen sources (peptoneand potassium nitrate)18,22were used to identify the cryptococcal isolates.

The dark brown colonies were also sub-cultivated onto NSA mediumwhich is recommended to confirm phenoloxidase activity11. After passagethrough NSA medium, dark brown colonies were seeded on CGB medium

(L-canavanine glycine bromothymol blue) for species identification19.No alteration in the yellow-green original color of the CGB mediumconfirms C. neoformans.

For molecular identification of the cryptococcal isolates, the protocoldescribed by POETA et al.33was used, with modifications, for DNAextraction. Yeast cells were suspended in 0.5 mL TENTS [10 mM, TrispH 8.0, 5% sodium dodecyl sulfate (SDS)]. Then, 0.5g of 0.5-mm glassbeads were added and boiled at 100 C for 10 min. It was then added 0.5mL of phenol: chloroform and samples were vortexed for two min. Aftercentrifugation for 10 min in a microfuge at 14,500 x g, the aqueous phasewas transferred to a tube with one volume of isopropanol and 0.3 M ofsodium acetate was added, and samples were placed at -20 C overnight.

Fig. 1 - Location of the City of Cuiab, State of Mato Grosso, Brazil.


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TAKAHARA, D.T.; LAZRA, M.S.; WANKE, B.; TRILLES, L.; DUTRA, V.; PAULA, D.A.J.; NAKAZATO, L.; ANZAI, M.C.; LEITE-JNIOR, D.P.; PAULA, C.R. & HAHN, R.C.- Firstreport on Cryptococcus neoformans in pigeon excreta from public and residential locations in the metropolitan area of Cuiab, State of Mato Grosso, Brazil.Rev. Inst. Med. Trop. SaoPaulo, 55(6): 371-6, 2013.

373

DNA collected was precipitated, washed with 70% ethanol, re-suspendedin 50 L of ultrapure water and stored at -20 C.

To confirm the species of the isolates, pairs of primersCNA70A (5-ATTGCGTCCATGTTACGTGGC-3) and CNA70S(5-ATTGCGTCCACCAAGGAGCTC-3 ) specific for C. neoformanswere used, resulting in amplification products of 695 bp2,13.

RESULTS

All the brown colonies isolated on NSA medium were encapsulatedyeast forms, and have been observed in microscopy with India Ink.All were thermotolerant to 37 C, urease-producing and inhibited bycycloheximide. In Canavanine-glycine-bromothimol blue medium (CGB)didnt have color change and this confirmed specie C. neoformans, as wellas the assimilation of carbohydrates (glucose, maltose, sucrose, galactose,cellobiose, inositol, xylose, raffinose, trehalose, dulcitol) and no nitrate

assimilation. All colonies isolated was confirmed by PCR (PolymeraseChain Reaction) from the use of specific primers. Further analysis shouldbe performed to investigate the molecular types of these isolates.

One hundred and twenty-two dry pigeon excreta samples were chosenat random from different locations (Table 1).

The presence of excreta was detected in the eight groups evaluated.However, considering the squares, the presence of excreta was onlyobserved in four of the eleven surveyed. Similarly, in four of the tenchurches and three of the five schools the same fact was observed,concomitant presence of pigeons and excreta. According to the isolation ofC. neoformans, it was possible to determine that these yeasts were mostlydetected in the pigeon excreta collected from the residences assessed.

Regarding the different groups evaluated, C. neoformanswas detectedin one of the four churches, specifically in the tower, where the presenceof both pigeons and excreta were observed. C. neoformanswas isolated

in one of the three educational institutions inhabited by pigeons whereexcreta were also observed. Isolates of C. neoformanswere similarlyidentified in samples from four of the 23 residences evaluated.

The presence of pigeon excreta was observed in 49 (78%) of the 63sites visited. The presence of these substrata according to the differentsites is presented in Table 1.

Isolation of C. neoformanswas obtained from six (12.2%) of the 49sites analyzed, where eight (6.6%) out of 122 samples of dried pigeonexcreta collected were positive.

Two samples collected from the church were positive for C.neoformans, eight colonies were detected. In the educational institution, C.neoformanswas detected in only one of the 13 samples analyzed and in thissample, four colonies were detected. Regarding the residences, five samplespositive for C. neoformanswere obtained and 60 colonies were detected.

The identification of C. neoformansisolates was confirmed by PCRusing specific primers (Fig. 2).

DISCUSSION

The deposition of pigeon excreta (Columba livia) in public placescan serve as a source of infectious agents of importance for public health,such as C. neoformans. In this study, certain facts observed during samplecollection deserve attention: the amount of excreta obtained was variable,in that frequent cleaning was observed in several of the public spacesevaluated. Thus, despite the presence of pigeons, the presence of excretawas not verified at all the sites selected.

Furthermore, in the majority of the sites visited, there were nomechanical barriers to prevent access by pigeons, a resource currentlyused to hinder the approach of pigeons to windows, air conditioning unitsand other physical barriers.

Table 1Types and number of sites investigated (Groups) and positivity (%) associated with the presence of Cryptococcus neoformans in pigeon excreta of environments in

the Cuiab City, State of Mato Grosso, Brazil

Groups/type of location NumberPresence of

excretaSample (n)

Samplepositive

Isolation

absolute relative

n %

Group I: squares 11 5 12 0 0 0.0Group II: churches 10 4 13 2 1/4 25.0

Group III: educational institutions 5 3 13 1 1/3 33.0

Group IV: health units 8 8 20 0 0 0.0

Group V: open areas* 4 4 11 0 0 0.0

Group VI: residences 23 23 44 5 4/23 17.0

Group VII: factories 1 1 3 0 0 0.0

Group VIII: prisons 1 1 6 0 0 0.0

Total 63 49 (78%) 122 8 (6.6%) 6/49 12.2

*with asbestos covering.


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The isolation of virulent strains of the fungus from soil sampleswas first reported by SILVA & CAPUANO37 in Brazil as early as 1960.

MACHADO et al.27

also reported recovering this fungus from the soil inan attempt to correlate the clinical-epidemiological history of patientssuffering from cryptococcosis in the Santa Casa of Porto Alegre, in theState of Rio Grande do Sul (RS), Brazil.

In this study, only samples of pigeon excreta were collected, soilsamples were not included. However, when considering studies thatexamined samples of pigeon excreta, positivity rates for the isolation ofthe fungus in Brazil ranged from 4.3 to 31.3%3,6,9,13,16,23,27,28,29,31,34,37,39. Thefindings of this study show a positivity rate of 12% for C. neoformans,values that are compatible with the rates of isolation in Brazil previouslyreported in the literature. Most of the total samples analyzed (44/122)were from residences, sites which presented expressive positivity (17%).This finding may represent a risk for the acquisition of cryptococcosis,

since in several of the evaluated residences the habit of feeding pigeons byresidents was frequently observed, luring them and indirectly encouragingthem to reproduce. Food scraps were also found in these places, reflectingpoor hygiene care in the common areas of residential estates.

Ten churches were visited and the presence of excreta was investigatedin four, though positivity for C. neoformanswas demonstrated only inone. BARONI et al.3 also evaluated the presence of C. neoformansin ten churches in the City of Rio de Janeiro and C. neoformans wasfound in every church selected and was present in 37.8% of 219 pigeondropping samples. Samples of excreta were obtained, in addition to airsamples in church towers and from the surrounding areas. It is knownthat high summer temperatures can inhibit the growth of C. neoformans,

possibly due to inactivation of the yeast36,40. Cuiab is known for its hightemperatures, a factor that should be considered in relation to the low ratesof detection of C. neoformansin pigeon excreta at the sites evaluated.

According to BULMER4, the problem is the long viability of C.neoformansin dried excreta, about two years. Based on this information,old buildings and towers of old churches can be considered potentialsources for C. neoformansand should be periodically evaluated bypublic health authorities. In Cuiab, most churches are fairly old (over50 years-old) and are considered historical monuments of the city, whichcompleted 292 years in 2011.

Uninfected pigeon excreta can become infected when exposedto air containing aerosolized cells of C. neoformans5. Consideringall the locations where pigeon excreta might be deposited within theurban areas of Cuiab, the aerial dispersion of cryptococcal propagulesfrom the positive sites to the surroundings is probably occurring. The

positivity (12%) rate for the isolation of C. neoformans from pigeonexcreta detected in this study is in agreement with the values obtained byLOPEZ-MARTINEZ et al.26, who analyzed 711 samples from numerousenvironmental sources in Mexico City, including bird droppings, fruitsand vegetables. They reported the presence of C. neoformansin 9.5%of excreta samples, 9.5% in fruits and 4.2% vegetables. In contrast, inanother study in Bogota (Colombia), 480 samples of debris from treesand 89 excreta samples were investigated. Among the plant samples,99% were characterized as C. gattiiand 1% as C. neoformans, while inthe excreta samples, only C. neoformans was isolated12.

Considering the public squares in the present study, the findings inCuiab contrast with those obtained in Porto Alegre, Rio Grande do SulState, by REOLON et al.34They affirmed that in all five squares in which

the investigation of yeasts of the genus Cryptococcuswas conducted, atotal of 88 samples, positivity was obtained in all 88 (100%) samples. Inour study, 11 squares were evaluated, but it was not possible to isolateyeasts of the genus Cryptococcus, despite the presence of excreta in fiveof the squares. The authors who conducted the study in Porto Alegre didnot mention the period or season in which the materials were collected,making it virtually impossible to compare the factors that could interferewith the isolation of yeasts in cities with very different bioclimaticconditions, such as Cuiab and Porto Alegre.

In the City of Pelotas, Rio Grande do Sul State, FARIA et al.9evaluated 70 environments, including squares (n = 1), historic buildings(n = 8), church towers (n = 1), rice mills and warehouses (n = 7) andoutdoor locations (n = 9). Considering all these sites, the isolation of

C. neoformanswas verified in 26.9% (n = 7/26). Among the 14 squaresevaluated in Pelotas, only one had a mean quantity excreta from which C.neoformans was isolated. The City of Pelotas has no extreme temperaturesand relative humidity is high. This contrasts with the bioclimaticconditions of Cuiab, where temperatures in August, September andOctober, rise considerably and the relative humidity remains extremelylow, reaching critical levels. Sun light exposure associated with theclimate of Cuiaba may be critical for the survival of C. neoformansinopen areas of the city. Moreover the agent was mainly isolated fromprotected places in Cuiab, such as an educational institution, a churchand four residences. These findings reveal the risk of exposure forimmunosuppressed and even immunocompetent individuals in dailyactivities or living in these microenvironments. Measures are required to

Fig. 2 - PCR amplification of Cryptococcus neoformans: M, 100 pb DNA ladder, 1 negativecontrol (NC), 1 positive control (PC) and sample isolates A1 (church), A2 (educational

institution), A3 (residence 1) and A4 (residence 2).


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TAKAHARA, D.T.; LAZRA, M.S.; WANKE, B.; TRILLES, L.; DUTRA, V.; PAULA, D.A.J.; NAKAZATO, L.; ANZAI, M.C.; LEITE-JNIOR, D.P.; PAULA, C.R. & HAHN, R.C.- Firstreport on Cryptococcus neoformans in pigeon excreta from public and residential locations in the metropolitan area of Cuiab, State of Mato Grosso, Brazil.Rev. Inst. Med. Trop. SaoPaulo, 55(6): 371-6, 2013.

375

reduce the number of birds through the maintenance of adequate hygiene,aeration, lighting and ventilation1,11. Simply performing adequate cleaningof such environments could be effective, as well as not offering food topigeons, particularly in residential areas.

RESUMO

Primeiro registro de Cryptococcus neoformans em excretas depombos provenientes de locais pblicos e residenciais de rea

metropolitana de Cuiab, Estado do Mato Grosso, Brasil

A criptococose micose sistmica potencialmente grave causada porduas espcies do gnero Cryptococcusque acometem tanto homens comoanimais: Cryptococcus neoformanse C. gattii. So infeces cosmopolitase emergentes, resultantes da interao do hospedeiro - humano e animalversusmeio ambiente.A proposta deste trabalho foi avaliar a ocorrnciade C. neoformansem 122 amostras de excretas secas de pombos coletadas

em 49 locais na cidade de Cuiab, Estado do Mato Grosso, Brasil,incluindo: praas pblicas (n = 5), igrejas (n = 4), instituies de ensino(n = 3), unidades de sade (n = 8), reas abertas exibindo cobertura deamianto (n = 4), conjuntos residenciais domiciliares (n = 23), uma fbrica(n = 1) e um presdio (n = 1). Semeadura de suspenso de amostras emmeio gar niger (NSA), identificao fenotpica por provas bioqumicase teste em meio de canavanina-glicina-azul de bromotimol, das colniasisoladas com pigmentao marrom escura. Foi tambm utilizada a tcnicada reao em cadeia da polimerase com pares de iniciadores especficospara identificao de C. neoformans.As amostras foram coletadas dejulho a dezembro de 2010.Cryptococcus neoformans foi isolado emoito (6,6%) de 122 amostras correspondendo a seis (12,2%) dos 49 stiosanalisados. Cryptococcus neoformansassociado a excretas de pombosocorre em reas de Cuiab, predominando em residncias nas amostras

analisadas, constituindo fator de risco potencial para aquisio da doenatanto para indivduos imunocomprometidos como imunocompetentes.

ACKNOWLEDGMENTS

Financial support for this study was provided by FAPEMAT -Fundao de Amparo Pesquisa no Estado de Mato Grosso [State ofMato Grosso Foundation for the Support of Science].

REFERENCES

1. Abegg MA, Cella FL, Faganello J, Valente P, Schrank A, Vainstein MH. Cryptococcusneoformans and Cryptococcusgattiiisolated from the excreta of psittaciformes in asouthern Brazilian zoological garden. Mycopathologia. 2006;161:83-91.

2. Aoki FH, Imai T, Tanaka R, Mikami Y, Taguchi H, Nishimura NF, et al.New PCRprimer pairs specific for Cryptococcus neoformans serotype A or B prepared on thebasis of random amplified polymorphic DNA fingerprint pattern analyses. J ClinMicrobiol. 1999;37:315-20.

3. Baroni FA, Paula CR, Silva EG, Viani FC, Rivera ING, Oliveira MTB, et al.Cryptococcus neoformansstrains isolated from church towers in Rio de Janeiro City,RJ, Brazil. Rev Inst Med Trop Sao Paulo. 2006;48:71-5.

4. Bulmer GS. Twenty-five years with Cryptococcus neoformans. Mycopathologia.1990;109:111-22.

5. Casadevall A, Perfect JR. Cryptococcus neoformans. Washington: American Societyfor Microbiology Press; 1998.

6. Casali AK, Goulart L, Rosa e Silva LK, Ribeiro AM, Almeida AA, et al. Moleculartyping of clinical and environmental Cryptococcus neoformans isolates in theBrazilian State Rio Grande do Sul. FEMS Yeast Res. 2003;3:405-15.

7. Currie BP, Freundlich LF, Casadevall A. Restriction fragment length polymorphismanalysis of Cryptococcus neoformansisolates from environmental (pigeon excreta)and clinical sources in New York City. J Clin Microbiol. 1994;32:1188-92.

8. Emmons CW. Saprophytic sources of Cryptococcus neoformansassociated with thepigeon (Columba livia). Amer J Hyg. 1955;62:227-32.

9. Faria RO, Nascente PS, Meinerz ARM, Cleff MB, Antunes TA, Silveira ES, et al.Ocorrncia de Cryptococcus neoformans em excretas de pombos na cidade de Pelotas,Estado do Rio Grande do Sul. Rev Soc Bras Med Trop. 2010;43:198-200.

10. Favalessa OC, Ribeiro LC, Tadano T, Fontes CJF, Dias FB, Coelho BP,et al.Primeiradescrio da caracterizao fenotpica e susceptibilidade in vitroa drogas de levedurasdo gnero Cryptococcusspp isoladas de pacientes HIV positivos e negativos, Estadode Mato Grosso. Rev Soc Bras Med Trop. 2009;42:661-5.

11. Fili WF, Wanke B, Agena SM, Vilela VO, Macedo RC, Lazra MS. Cativeiro de

aves como fonte de Cryptococcus neoformansna cidade de Campo Grande, MatoGrosso do Sul, Brasil. Rev Soc Bras Med Trop. 2002;35:591-5.

12. Granados DP, Castaeda E. Isolation and characterization of Cryptococcus neoformansvarieties recovered from natural sources in Bogot, Colombia, and study of ecologicalconditions in the area. Microb Ecol. 2005;49:282-90.

13. Horta JA, Staats CC, C asali AK, Ribeiro AM , Schrank IS, Schrank A, et al.Epidemiological aspects of clinical and environmental Cryptococcus neoformansisolates in the Brazilian state Rio Grande do Sul. Med Mycol. 2002;40:565-71.

14. Idnurm A, Bahn Y, Nielsen K, Lin X, Fraser JA, Heitman J. Deciphering the modelpathogenic fungus Cryptococcus neoformans. Nat Rev Microbiol. 2005;3:753-64.

15. Instituto Nacional de Meteorologia (INMET) [Internet]. Brasilia, DF, Brasil: INMET;[Updated 2011 January 2001; Cited 2011 October 29] Available from: http://www.inmet.gov.br/html/observacoes.php?/

16. Kobayashi CC, Souza LK, Fernandes OF, Brito SC, Silva AC, Sousa ED, et al.Characterization of Cryptococcus neoformans isolated from urban environmentalsources in Goinia, Gois, Brazil. Rev Inst Med Trop Sao Paulo. 2005;47:203-7.

17. Kon AS, Grumach AS, Colombo AL, Penalva ACO, Wanke B, Telles FQ, et al.Consenso em criptococose - 2008. Rev Soc Bras Med Trop. 2008;41:524-44.

18. Kwon-Chung KJ, Bennet JE. Cryptococcosis.In:Kwon-Chung KJ, Bennet JE, editors.Medical Mycology. Philadelphia: Lea & Febiger; 1992. p. 426-36.

19. Kwon-Chung KJ, B ennet JE. Culture media and reagents. In: Kwon-Chung KJ,Bennet JE, editors. Medical Mycology. Appendix B. Philadelphia: Lea & Febiger;1992. p. 816-26.

20. Kwon-Chung KJ, Boekhout T, Fell T J, Diaz M. Proposal to conserve the name

Cryptococcus gattiiagainst C. hondurianusand C. bacillisporus(Basidiomycota,Hymenomycetes, Tremellomycetidae). Taxon. 2002;51:804-6.

21. Kwon-Chung KJ, Polacheck I, Bennett JE. Improved diagnostic medium for separationof Cryptococcus neoformansvar. neoformans (serotypes A and D) and Cryptococcusneoformansvar. gattii(serotypes B and C). J Clin Microbiol. 1982;15:535-7.

22. Lacaz CS, Porto E, Martins JEC, Heins-Vaccari EM, Mello NT. Criptococcose.In:Lacaz CS, Porto E, Martins JEC, Heins-Vaccari EM, Mello NT, editors. Tratado deMicologia Mdica. 9th. ed. So Paulo: Sarvier; 2002. p. 416-40.

23. Lazra MS, Wanke B, Nishikawa MM. Isolation of both varieties of Cryptococcusneoformans var. neoformansfrom saprophytic sources in the city of Rio de Janeiro,Brazil. J Med Vet Mycol. 1993;31:449-54.


10/80


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24. Levitz SM. The ecology of Cryptococcus neoformans and the epidemiology ofcryptococcosis. Rev Infect Dis. 1991;13:1163-9.

25. Lin X, Heitman J. The biology of the Cryptococcus neoformans species complex.Ann Rev Microbiol. 2006;8:69-105.

26. Lpez-Martnez R, Castan-Olivares LR. Isolation of Cryptococcus neoformansvar. neoformans from bird droppings, fruits and vegetables in Mexico City.Mycopathologia. 1995;129:25-8.

27. Machado CC, Amaral AA, Severo LC. Cryptococcus neoformans var. neoformansisolado do solo. Rev Inst Med Trop Sao Paulo. 1993;35:77-9.

28. Montenegro H, Paula CR. Environmental isolation of Cryptococcus neoformansvar. gattii and C. neoformans var. neoformans in the city of So Paulo, Brazil. MedMycol. 2000;38:385-90.

29. Melo NT, Nigro RC, Pereira AD, Huggins D, Lacaz CS. Isolamento de Cryptococcusneoformans de fezes de pombos, do solo e ninhos de pombos. Rev Bras Med.1987;44:6-9.

30. Pappalardo MC, Melhem MS. Cryptococcosis: a review of the Brazilian experiencefor the disease. Rev Inst Med Trop Sao Paulo. 2003;45:299-305.

31. Passoni LF, Wanke B, Nis hikawa MM , Laz ra MS. Cryptococcus neoformansisolated from human dwellings in Rio de Janeiro, Brazil: an analysis of the domesticenvironmental of AIDS patients with and without cryptococcosis. Med Mycol.1998;36:305-11.

32. Passoni LF. Wood, animals and human beings as reservoirs for human Cryptococcusneoformansinfection. Rev Iberoam Micol. 1999;16:77-81.

33. Poeta MD, Toffaletti DL, Rude TH, Dykstra CC, Heitman J, Perfect JR. TopoisomeraseI is essential in Cryptococcus neoformans: role in pathobiology and as an antifungaltarget. Genetics. 1999;152:167-78.

34. Reolon A, Perez LRR, Mezzari A. Prevalncia de Cryptococcus neoformans nospombos urbanos da cidade de Porto Alegre, Rio Grande do Sul, Brasil. J Bras PatolMed Lab. 2004;40:293-8.

35. Rolim GS, Camargo MBP, Lania DG, Moraes JFL.Classificao climtica de Kppene Thornthwaite e sua aplicabilidade na determinao de zonas agroclimticas para oEstado de So Paulo. Bragantia. 2007;66:711-20.

36. Rosario I, Hermoso-de-Mendonza M, Dniz S, Soro G, lamo I, Acosta B. Isolationof Cryptococcus species including C. neformansfrom cloaca of pigeons. Mycoses.2005;48:421-4.

37. Silva JO, Capuano DM. Ocorrncia de Cryptococcus spp e de parasitas de interesseem sade pblica, no excreta de pombos na cidade de Ribeiro Preto, SP, Brasil. RevInst Adolfo Lutz. 2008;67:137-41.

38. Silva ME, Paula LA. Isolamento de Cryptococcus neoformansde excrementos e

ninhos de pombos (Columba livia) em Salvador, Bahia. Rev Inst Med Trop SaoPaulo. 1963;5:9-11.

39. Silva ME. Ocorrncia de Cryptoccocus neoformans e Microsporum gypseum emsolos da Bahia, Brasil. Bol Fund Gonalo Moniz. 1960;17:1-14.

40. Sorrell TC, Ellis DH. Ecology of Cryptococcus neoformans. Rev Iberoam Micol.1997;14:42-3.

Received: 10 December 2012Accepted: 4 April 2013


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Rev. Inst. Med. Trop. Sao Paulo

55(6):377-383, November-December, 2013

doi: 10.1590/S0036-46652013000600002

(1) Laboratory of Mycology, Department of Pharmaceutical Sciences, Federal University of Paraba, Joo Pessoa, PB, Brazil.(2) Laboratory of Ceramic, Department of Mechanical Engineering, Federal University of Paraba,Joo Pessoa, PB, Brazil.(3) Department of Statistic, Federal University of Paraba,Joo Pessoa, PB, Brazil.

Correspondence to:Felipe Queiroga Sarmento Guerra, Tel.: 55.83.9602-1666. E-mail: [email protected]

DISTRIBUTION OF DERMATOPHYTES FROM SOILS OF URBAN AND RURAL AREASOF CITIES OF PARAIBA STATE, BRAZIL

Zlia Braz Vieira da Silva PONTES(1), Aurylene Carlos de OLIVEIRA(1), Felipe Queiroga Sarmento GUERRA(1),Luiz Renato de Arajo PONTES(2) & Jozemar Pereira dos SANTOS(3)

SUMMARY

The dermatophytes, keratinophilic fungi, represent important microorganisms of the soil microbiota, where there are cosmopolitanspecies and others with restricted geographic distribution. The aim of this study was to broaden the knowledge about the presence ofdermatophytes in soils of urban (empty lots, schools, slums, squares, beaches and homes) and rural areas and about the evolution of

their prevalence in soils of varying pH in cities of the four mesoregions of Paraiba State, Brazil. Soil samples were collected from 31cities of Paraiba State. Of 212 samples, 62% showed fungal growth, particularly those from the Mata Paraibana mesoregion (43.5%),which has a tropical climate, hot and humid. Soil pH varied from 4.65 to 9.06, with 71% of the growth of dermatophytes occurringat alkaline pH (7.02 - 9.06) (= 0.000). Of 131 strains isolated, 57.3% were geophilic species, particularly Trichophyton terrestre(31.3%) andMycrosporum gypseum(21.4%).M. nanumand T. ajelloiwere isolated for the first time in Paraiba State. The zoophilicspecies identified were T. mentagrophytesvar. mentagrophytes (31.3 %) and T. verrucosum(7.6 %), and T. tonsuranswas isolated asan anthropophilic species. The soils of urban areas including empty lots, schools, slums and squares of cities in the mesoregions ofParaiba State were found to be the most suitable reservoirs for almost all dermatophytes; their growth may have been influenced byenvironmental factors, soils with residues of human and/or animal keratin and alkaline pH.

KEYWORDS:Dermatophytes; Keratinophilic fungi; Soil; pH conditions; Brazil.

INTRODUCTION

The dermatophytes (Trichophyton, MicrosporumandEpidermophyton),keratinophilic fungi, represent important microorganisms of the soilmicrobiota, where there are cosmopolitan species and others withrestricted geographic distribution1,2,6,10,17,21. There have been reports ofthe isolation of T. ajelloi, T. rubrum, T. mentagrophytes, T. verrucosum,T. terrestre, T. tonsurans, T. simii, T. schoenleinii, M.gypseum,M. canis,M. audouinii,M. nanum,M. cookeiand/orE. floccosum, from the soilsof various Brazilian states and locals around the world8,20,24,25,30,32,34.

The occurrence of fungi in the soil can also be influenced by

non-biological factors such as soil temperature, humidity, rainfall,environmental light, climate, chemical composition, quantity of organicmatter in the soil and pH. Some have a wide range of tolerance foracidic to alkaline soils2,7,14,16. However, studies of soil pH in relation tooccurrence of dermatophytes are uncommon in Brazil.

The study of the diversity of dermatophytes in the soil is importantbecause changes in the distribution of species of dermatophytes due toecological factors, socio-economic, therapeutic, and migration processes

of livestock populations, reflect the epidemiology of dermatophytosis,which are one of the source infections of the soil2,3,16,18,31. Thus, the aimof this study was to broaden the study into the presence of dermatophytesfrom soils of urban and rural areas of cities of four mesoregions of ParaibaState and the influence of pH on fungi growth.

MATERIALS AND METHODS

The state of Paraiba is situated in the eastern portion of NortheastBrazil, with coordinates between 6 and 8 S and between 34 and 38 W;therefore, it is included in the tropical zone. It comprises an area of 56,372km2 and is divided into four mesoregions (Mata Paraibana, Borborema,

Agreste Paraibano and Serto Paraibano) and into 23 geographicmicroregions, including a total of 223 cities. In the Mata Paraibana, thepredominant climate is warm, humid tropical (As) with an average annualrainfall of 1,800 mm, temperature of 26 C and relative humidity of 80%.The soils are sandy and muddy, which are influenced by sea water andhave especially coastal vegetation of mangrove swamp, rainforest andcerrado. In Borborema, the predominant climate is semi-arid (Bsh), warmand dry with average annual rainfall of 500 mm, temperature of 26 C andrelative humidity of 75%. The soils are shallow stony soil with caatinga


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PONTES, Z.B.V.S.; OLIVEIRA, A.C.; GUERRA, F.Q.S.; PONTES, L.R.A. & SANTOS, J.P. - Distribution of dermatophytes from soils of urban and rural areas of cities of Paraiba State,

Brazil.Rev. Inst. Med. Trop. Sao Paulo, 55(6): 377-83, 2013.

378

vegetation. The climate Bsh, together with As are observed in AgresteParaibano. However, in Serto Paraibano, the predominant climate is semi-humid (Aw) with an average annual rainfall of 800 mm, temperature of27 C and relative humidity of 70%. In the two last mesoregions, a slowdevelopment of soils with caatinga vegetation (Fig. 1)28.

An ecological study was performed with a total of 212 soil samples.The sampling was non-probabilistic, as it was done by convenienceand accessibility to the members of the team, taking into considerationconglomerates of cities in Paraiba mesoregions. Each mesoregion wasrepresented by a city of great geographical and population density: JooPessoa for Mata Paraibana, Monteiro for Borborema, Campina Grandefor Agreste Paraibano and Patos for Serto Paraibano. The other citieswere randomly included.

Soil samples were selected from urban (empty lots, schools, slums,squares, homes and beaches) and rural areas of cities. The samplingsites were selected on the basis of the likely presence of soil with keratinresidues from humans and animals.

The collection, processing and pH of soil solutions were accordingto the techniques described by VANBREUSEGHEM33. Approximately100g of soil at a depth of three to five centimeters was collected, placedin polyethylene bags and brought to be processed at the Laboratory ofMycology in the Department of Pharmaceutic Sciences and Laboratoryof Ceramic, Department of Mechanical Engineering at the FederalUniversity of Paraiba.

Using a pHmetrer, the pH of each soil sample (20 g) was measured

Fig. 1 - Location of 31 cities, according to four mesoregions, soils type, vegetation and climate of the state of Paraba, Brazil. Adapted from RODRIGUEZ28.


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379

after dilution in distilled sterile water (20 mL) with 20 minutes of agitationand decantation. Each sample was distributed in sterile Petri plates,moistened with sterile water (20 mL) and some sterile human hair stripswere placed over each surface. The plates were identified and incubated(27-30 C) and from the 5thto the 70thday the hair strips were regularlyobserved with magnifying glasses for signs of fungal growth. Hair stripswith a development of prominent fungal growth around them, wereplaced between slide and cover slid, colored in lactophenol blue cottonand examined in a microscope (10X and 40X). They were cultivated inSabouraud dextrose agarmedium with chloramphenicol (0.05 mg mL-1)and in Mycobiotic agar and incubated at room temperature for anotherminimum period of two weeks.

The identification of the species was based on macromorphology andmicromorphology features (slide-culturing) and physiological tests (ureahydrolysis, in vitrohair perforation, vitamin requirement and sensitivemedia). The classification was based on BARNETT & HUNTER5,

REBELL & TAPLIN

27

and HOOG et al.12

.The data were subjected to statistical analysis, which consisted of

the Binomial test. The process was carried out by computing SPSS 1322,allowing to verify if the dermatophytes growth soil acidic pH is equalto alkaline pH.

RESULTS

In 31 cities of four mesoregions of the state of Paraiba (Fig. 1), 62%of the growth of dermatophytes occurred in soil with different pH. Incities from Mata Paraibana, isolations were observed in 43.5% of samples,where this rate was 84% in the capital, Joo Pessoa. In cities from SertoParaibano, the isolation rate was 20.6%, whereas 23.7% in cities from

Agreste Paraibano and 12.2% in cities from Borborema (Table 1).

A total of 131 strains of dermatophytes were isolated, where 57.3%of the geophilic species were identified. T. terrestre(31.3%) was themost common species, followed by M. gypseum (21.4%), M. nanum(3%), T. ajelloi(0.8%) andAnthroderma gypsea(0.8%), a teleomorphform ofM. gypseum, observed in sample soil.M. nanumand T. ajelloiwere isolated for the first time in Paraiba State. The zoophilic speciesidentified included T. mentagrophytesvar. mentagrophytes(31.3%) andT. verrucosum(7.6%). T. tonsurans(3.8%) was the only anthropophilicspecies isolated. The growth of more than one fungal species wasobserved in 13 samples (Table 1).

The soils that showed the highest rates of dermatophytes were those of

urban areas (95%), especially in soils of empty lots (25.2% of isolations),around schools (22.9%), in slums (21.4%) and squares (19.8%), comparedto around homes (3.8%) and on beaches (2.3%) (Table 2).

Dermatophytes developed in a wide pH range: acid to alkaline(4.65 - 9.06), with 71% in alkaline pH (7.02 - 9.06). T. terrestredevelops within the pH range of 5.76 - 8.90. T. mentagrophytesvar.mentagrophytesandM. gypseumdevelop within the pH range 4.65 -9.06 and 5.77 - 8.31, respectively and T. verrucosumwas reported fromurban areas at pH 6.65 - 8.05. In acid pH soil, an inhibition of growthM. nanum, A. gypseaand T. ajelloiwas observed. The dermatophytesgrowth in soil of alkaline pH was significantly different from the acidicpH (= 0.000) (Table 3).

DISCUSSION

Studies worldwide have examined various variables, such as soiltype, pH, climate, temperature, moisture and organic matter content, andhave revealed the presence of dermatophytes and other keratinophilicfungi in soil1,3,6,9,14,21,31. In Brazil, there are few reports on the isolationof dermatophytes in soil, specifically in the Northeast region16,26,32. Inthe mesoregion of Mata Paraibana, with an As climate and sandy andmuddy soils28, dermatophytes were isolated in 43.5% of samples. Aprevious study reported that 55.7% of 68 soil samples from the city ofJoo Pessoa-Paraiba State (PB), showed the growth of dermatophytes26. InBorborema, the isolation rate was 12.2%. This area has a Bsh climate andshallow rocky soil. In other mesoregions, the lack of water for prolongedperiods accounts for the slow development of soil. The distribution ofclimates is related to the geographic localization, that is, the closer tothe coast the more humid and the farther from the coast the drier. Thefour mesoregions of Paraiba have predominantly caatinga vegetation,

except Mata Paraibana

28

. Although the roles of fungi in ecosystems havebeen well documented, knowledge about their population dynamicsand community structure and of the diversity of soil fungi is still poor.Further studies of Paraiba soils are necessary to analyze the changes andinfluence of variables such as types of climate, soil and vegetation on thedevelopment of dermatophytes.

The pH range of 7.2 - 8.0 is favorable for the production of proteolyticenzymes (keratinases) by keratinophilic fungi, which are necessary fortheir growth, along with other soil conditions15. However, the results ofthis study indicate the growth of dermatophytes in acid and alkaline pH,where 71% of isolations were observed in the alkaline pH range between7.02 and 9.06 (= 0.000). These results, obtained with different soilsamples, confirm the importance of pH in the habitat to the occurrence and

distribution of dermatophytes. In acidic soils, there is growth inhibitionof dermatophytes and other keratinophilic fungi, but soils that are weaklyacidic to neutral or alkaline are optimal for their growth 14,16,21,23. In thisinvestigation, in acid pH soils, the growth ofA. gypseum, M. nanum andT. ajelloiwas inhibited. Some authors6observed that the frequency ofT. ajelloi(33%) increased with a decrease in pH, reaching a maximumin strongly acidic soil.

Eight species of dermatophytes were identified in the soils of citiesin Paraiba. Of the geophilic species (57.3%), T. terrestre(31.3%) wasespecially found in soils from squares, empty lots, schools, slums andbeaches. This variable distribution rate can be related to the samplingsites, where the presence of people and animals are frequent, providingresidues of organic matter, which are essential for the growth of these

fungi. The results obtained are close to those for other cities in Brazilsuch as: Belo Horizonte and So Paulo29and in soils of countries such asGermany and Argentina7,21. However, the frequency of this species waslow in Italy25and India31. T. terrestrehas been found to be a pathogenparticularly in pets and humans including the elderly who exhibitcomplications related to immunological factors25.

Other geophilic species that were isolated included M. gypseum(21.4%), M. nanum (3%), T. ajelloi (0.8%) and A. gypsea (0.8%) atalkaline pH, exceptM. gypseum,which also showed growth at acid pH.Similar results were obtained in soils from the Brazilian states of Rio deJaneiro (31%)10, So Paulo (30%)29and Bahia (28.8%)32. However, inRecife, Pernambuco State, 5.6% isolation was observed for this species


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squares of cities of mesoregions of Paraiba State were found to be themost suitable reservoirs for almost all dermatophytes. Its growth mayhave been influenced by environmental factors such as residues of humanand/or animal keratin and alkaline pH.

RESUMO

Distribuio de dermatfitos isolados de solos de cidades doEstado da Paraba, Brasil

Os dermatfitos, fungos queratinoflicos, representam importantesmicrorganismos da microbiota do solo, onde existem espciescosmopolitas e outras de distribuio geogrfica restrita. Este estudo tevecomo objetivo ampliar o conhecimento da distribuio de dermatfitosdo solo proveniente de reas urbanas (terrenos baldios, escolas, favelas,praas, praias e residncias) e rurais de quatro mesorregies paraibanase da influncia do pH na adaptao desse grupo de fungos. Amostras

de solos urbanos e rurais foram coletadas de 31 cidades do estado daParaba, Brasil. De 212 amostras 62% apresentaram crescimento fngico,destacando-se a Mesorregio da Mata Paraibana (43.5%), a qual apresentaclima tropical, quente e mido. O pH das amostras de solo variou de4.65 a 9.06, com crescimento de 71% dos dermatfitos em pH alcalino(7.02 - 9.06) (= 0.000). Das 131 cepas isoladas 57.3% eram espciesgeoflicas, destacando-se Trichophyton terrestre(31.3%) eMicrosporumgypseum(21.4%).M. nanume T. ajelloiforam isolados pela primeiravez no estado da Paraba. Entre as espcies zooflicas foram identificadasT. mentagrophytesvar. mentagrophytes (31.3%) e T. verrucosum(7.6%)e como espcie antropoflica foi isolada T. tonsurans. Os solos deterrenos baldios, escolas, favelas e praas de cidades paraibanas so osreservatrios mais adequados dos dermatfitos, cujo crescimento pode tersido influenciado por fatores ambientais, solos com resduos de queratina

humana e ou animal e pH alcalino.

ACKNOWLEDGEMENTS

The authors would like to thank to the Laboratory of Ceramics forcollecting and measuring the pH of soils samples.

REFERENCES

1. Al-Musallam AA, Al-Zarban SS, Al-San NA, Ahmed TM. A report on thepredominant occurrence of a dermatophytes species in cultivated soil from Kuwait.Mycopathologia. 1995;130:159-61.

2. Alvarez DP, Luque AG, Marini P. Influencia del sustrato queratinoso de suelos dePradera sobre la colonizacin por dermatofitos geoflicos. Bol Micol. 1986;3:25-9.

3. Amaral CDP, Pereira DIB, Meireles MCA. Caracterizao da microbiota por fungosfilamentosos no tratamento hpico de bovinos de corte. Ci Rural. 2011;41:2137-42.

4. Anbu P, Hilda A, Gopinath SC. Keratinophilic fungi of poultry farm and featherdumping soil in Tamil Nadu, India. Mycopathologia. 2004;158:303-9.

5. Barnett HL, Hunter BB. Illustrated genera of imperfect fungi. 4th ed. New York:Burgess; 1986.

6. Bohacz J, Kowalska TK. Species diversity of keratinophilic fungi in various soiltypes. Cent Eur JBiol. 2012;7:259-66.

7. Bhme H, Ziegler W. The distribution of geophilic dermatophytes and otherkeratinophilic fungi in relation to the pH of the soil. Mycopathol Mycol Appl.1969;38:247-55.

8. Fischman O, Ramos CD. Geophilic dermatophytes recovered from Rio Grande doSul soil. Mycopathol Mycol Appl. 1967;33:157-60.

9. Ganaie MA, Sood S, Rizvi G, Khan TA. Isolation and identification of keratinophilicfungi from different soil samples in Jhansi city (India). Plant Pathol J. 2010;9:194-7.

10. Goulart EG, Lima SMF, Carvalho MA, Oliveira JA, Jesus MM, Campos RE, et al.Isolamento de fungos patognicos do solo no municpio do Rio de Janeiro, RJ, Brasil.Folha Md. 1986;93:15-20.

11. Hayashi N, Toshitani S. Human infections with Microsporum gypseum in Japan.Mykosen. 1983;26:337-45.

12. Hoog GS, Guarro J, Figueras MJ. Atlas of clinical fungi. 2nd ed. Utrecht:Centraalbureau voor Schimmelcultures; 2000. 1126p.

13. Iovannitti CA, Malliarchuk O, Casanova A, Dawson M. Estudio micolgico enmuestras de tierra de la ciudad de la Plata. Rev Argent Micol. 1985;8:9-11.

14. Jain N, Sharma M. Distribution of dermatophytes and other related fungi in Jaipurcity, with particular reference to soil pH. Mycoses. 2011;54:52-8.

15. Kaul S, Sumbali G. Impact of some ecological factors on the occurrence of poultrysoil-inhabiting keratinophiles. Mycopathologia. 1998;143:155-9.

16. Leal AFG, Macdo DPC, Laranjeira D, Souza-Motta CM, Fernandes MJS, MagalhesOMC, et al.Correlao epidemiolgica entre fungos queratinoflicos isolados do soloe agentes de dermatomicoses. Rev Soc Bras Med Trop. 2009;42:471-3.

17. Lee MJ, Park JS, Chung H, Jun JB, Bang YJ. Distribuition of soil keratinophilicfungi isolated in summer beaches of the east sea in Korea. Korea J Med Mycol.2011;16:44-50.

18. Lima OE, Pontes ZBVS, Oliveira NMC, Carvalho MFFP, Guerra MFL, Santos JP.Freqncia de dermatofitoses em Joo Pessoa, Paraba, Brasil. An Bras Dermat.1999;74:127-32.

19. Lpez Martnez R. Investigacin de algunas fuentes de infeccin en las dermatofitoses:estudio de suelos, animales y hombre. Gac Md Mx. 1986;122:167-72.

20. Mahmoudabadi AZ, Zarrin M. Isolation of dermatophytes and related keratinophilicfungi from the two public parks in Ahvaz. Jundishapur J Microbiol. 2008;1:20-3.

21. Mangiaterra ML, Alonso JM. Keratinophilic fungi in soils of Corrientes city(Argentina). Bol Micol. 1989;4:129-33.

22. Norusis MJ. SPSS for Windows-Base System Users Guide, Release 13.0. Chicago:SPSS.

23. Ogbonna CI, Pugh GJ. Keratinophilic fungi from Nigerian soil. Mycopathologia.1987;99:115-8.

24. Oyeka CA, Okoli I. Isolation of dermatophytes and non-dermatophytic fungi fromsoil in Nigeria. Mycoses. 2003;46:336-8.

25. Papini R, Mancianti F, Grassotti G, Cardini G. Survey of keratinophilic fungi isolatedfrom city park soils of Pisa, Italy. Mycopathologia. 1998;143:17-23.

26. Pontes ZBVS, Oliveira AC. Dermatophytes from urbain soils in Joo Pessoa, Paraba,Brazil. Rev Arg Microbiol. 2008;40:161-3.

27. Rebell G, Taplin D. Dermatophytes: their recognition and identification. Coral Gables;University of Miami; 1974.

28. Rodriguez JL. Atlas escolar da Paraba. 3. ed. Joo Pessoa: Grafiset; 2002.

29. Rogers AL, Beneke EJ. Human pathogenic fungi recovered from Brazilian soil.Mycopathol Mycol Appl. 1964;22:15-20.


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30. Shadzi S, Chadeganipour M, Alimoradi M. Isolation of keratinophilic fungi fromelementary schools and public parks in Isfahan, Iran. Mycoses. 2002;45:496-9.

31. Sharma M. Incidence of dermatophytes and other keratinophilic fungi in the schoolsand college playground soils of Jaipur, India. Afr J Microbiol Res. 2010;4:2647-54.

32. Silva ME. Ocorrncia de Cryptococcus neoformans eMicrosporum gypseum emsolos da Bahia, Brasil. Bol Fund Gonalo Moniz. 1960;17:1-14.

33. Vanbreuseghem R. Technique biologique pour lisolement des dermatophytes de sol.Ann Soc Belge Md Trop. 1952;32:173-8.

34. Vilela EM, Moraes MAP. Isolamento de Microsporum gypseum e Trichophytonmentagrophytesno solo da cidade de Manaus, Amazonas (Brasil). Rev Inst MedTrop Sao Paulo. 1962;4:299-301.

35. Zampronha VCC, Oliveira IP, Monteiro MSR, Souza H, Santos KJG, Arajo AA.Isolamento e identificao de dermatfitos presentes no contnuo do solo de cerradodo campus II da Universidade Catlica de Gois. Rev Eletrn Fac Montes Belos.2005;1:37-46.

Received: 16 January 2012Accepted: 28 March 2013


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BONFIM-MENDONA, P.S.; FIORINI, A.; SHINOBU-MESQUITA, C.S.; BAEZA, L.C.; FERNANDEZ, M.A. & SVIDZINSKI, T.I.E. - Molecular typing ofCandida albicansisolates from

hospitalized patients.Rev. Inst. Med. Trop. Sao Paulo, 55(6): 385-91, 2013.

386

of C. albicans from hospitalized patients by using the RAPD andmicrosatellites assays.

MATERIAL AND METHODS

Microorganisms: A total of 39 strains of C. albicans isolatedfrom different sources of patients hospitalized at University Hospitalof Maringa in 2009, were used in this study. The yeasts were screenedin chromogenic media CHROMagar Candida and identified byconventional phenotypic methods (germ tube, microculture in cornmealagar supplemented with 1% Tween 80, auxanogram and zymogram test)39.

Yeasts were taken from the following sources: urine 51.5% (N = 20),blood 20.5% (N = 8), catheter tip 15.5% (N = 6), orotracheal discharge10% (N = 4) and peritoneal fluid 2.5% (N = 1). Regarding hospital sectors,49% (N = 19) were isolated from adult Intensive Care Unit (ICU), 18%(N = 7) medical clinic, 13% (N = 5) pediatrics, 7.5% (N = 3) Neonatal

Intensive Care Unit (NICU), 5% (N = 2) surgical clinic and 7.5% (N =3) pediatric ICU.

DNA Extraction: The yeast strains were grown overnight at 25 Cusing Sabouraud Dextrose Broth (SDB, Difco, USA) and genomic DNAextracted as described by CHONG et al.12. The concentration (260 nm)and purity (260/280) of the genomic DNA obtained were determined byoptical density in a spectrophotometer, the visualization was made outin agarose gel at 0.7% with 1X TBE buffer (Tris-base 90 mmol l-1, Boricacid 90 mmol l-1, EDTA 2 mmol l-1pH 8.0).

Nested-PCR: Identification of yeasts was confirmed by Nested-PCR which comprised two amplification stages, according to LUO &MITCHELL23. Briefly, primers that amplified Internal Transcribed Spacer

(ITS) fragments of DNAr and identified the genus Candida were usedin the first reaction. Amplification of species specific primers was usedin the second reaction.

RAPD (Random Amplified Polymorphic DNA): RAPD wasperformed using the kitReady-To-GoRAPDAnalysis Beads(AmershamBiosciences Corporation, Piscataway, NJ, USA) as described by themanufacturer. The RAPD reactions were performed by adding 30 ng ofgenomic DNA, one mol l-1oligonucleotide and water for a final volume of25 L to each tube containingReady-To-Go beads. The oligonucleotidesused were M2 (5-CTTGATTGCC-3)25and P4 (5-AAGAGCCCGT-3- Analysis Kit Ready-To-Go/RAPD Beads). The reaction was conductedin a in a Eppendorf Mastercycler Gradient Thermocycleras follows: 95C for five min, followed by 45 cycles consisting of 95 C for one min,

36 C for one min and 72 C for five min. Control tubes without templateDNA were included in each run and reproducibility of the method waschecked by repeating the amplification using different DNA extractionsfrom two isolates and at least three different days.

The PCR products were electrophoresed in 2% agarose gel (w/v) in1X TBE buffer at 150 volts for three hours. Amplicons in the gel werestained with ethidium bromide (0.5 mg mL -1) and visualized underUV transillumination (UVP Bioimaging Systems, Upland, CA). TheRAPD profiles were analyzed using Bionumericssoftware version 4.6(Applied Maths).

The similarity was verified by the coefficient (SAB

) between eachpair of standards for A and B isolates and calculated with the formulaS

AB= 2E / (2E + a + b),whereEis the number of common bands in

the patterns A and B, ais the number of bands with an a pattern and noB correlated patterns, and bis the number of bands withB pattern and

no correlation in pattern A. From the similarity matrix, the units weregrouped by UPGMA (Unweighted Pair-Group Method with ArithmeticalAverage). An S

ABvalue of 1.00 indicates that the pattern of bands for line

A is identical to B; values between 0.80 to 0.99 represent very similarclinical isolates but not identical, and may suggest microevolution ofa single strain; S

ABvalues less than 0.80 represent independent lines12.

Microsatellites: Samples were genotyped using two microsatellitemarkers, CDC3 and HIS3, whose primer sequences were shown in Table1, and all technical procedure was performed as described by BOTTERELet al.7. The amplification products were analyzed by electrophoresis inpolyacrylamide gel at 8% (w/v) in 1X TBE buffer for five hours at 140volts. For the determination of the sizes of the fragments we used themolecular size marker 25 bp (Invitrogen). After the run, the gel was

stained with ethidium bromide (0.5 mg mL-1

) and photodocumentedunder UV transillumination (UVP Bioimaging Systems, Upland, CA).The size of the amplified fragments was determined by image analysissoftware LabImage 1D (Loccus Biotech).

The results were expressed according to the tested locus name andsize of the two alleles observed in base pairs. The reproducibility of thisstep was ensured by the inclusion of analysis of a strain ofC. albicansATCC 38696 which provided repeatable and consistent results with thoseobtained by BOTTEREL et al.7.

RESULTS

Analysis by Nested-PCR: Amplifications with primers ITS1/ITS4

Table 1Primers used for genotyping of C. albicans isolates by Microsatellite and RAPD

Locus(GenBank access number), chromosome Primer Nucleotide sequence (forward and reverse)

Microsatellite

CDC3 (Z25869), chromosome 1 CDC35-CAGATGATTTTTTGTATGAGAAGAA-35-CAGTCACAAGATTAAAATGTTCAAG-3

HIS3(AF006605), chromosome 2 HIS35-TGGCAAAAATGATATTCCAA-35-TACACTATGCCCCAAACACA-3

RAPD- P4 5-AAGAGCCCGT-3

- M2 5-CTTGATTGCC-3


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DISCUSSION

RAPD and microsatellite analysis were able to show similarityamong C. albicans isolates recovered from a hospital. Microsatelliteanalysis supplied a good DP with markers used, it allowed formation ofvarious genotypes grouping samples, confirming the similarity betweenthem, which reinforces the interpretation of the data found in RAPD.Additionally, it was possible to prove the high similarity (100%) of

the same yeast which was from colonization (urinary catheter, trachealsecretions) and later detected in blood cultures from the same patient.

The RAPD results showed SAB

values of 0.84 0.126 for the primerM2 and S

AB0.88 0.08 for P4 (Fig. 1 and 2). It is important to highlight

that the strains that are considered identical by a primer are not alwaysnecessarily also considered identical or belong to the same cluster whenanalyzed by another primer. This should be referred as a limitation of thetechnique, nevertheless according to CHONG et al.12the values found,in RAPD, indicate high similarity between the isolates.

In microsatellite analysis we were able to verify the presence of sixdifferent alleles with the primer CDC3 and seven alleles with primerHIS3, of which 113bp, 117bp, 125bp, 150bp, 154bp, 158bp and 162bp

have already been recognized by other authors1,6,7. These primers amplifymicrosatellite regions highly polymorphic for C. albicans.Moreover,these regions are stable over generations and were chosen because theyare located on different chromosomes, which increase the chances offinding polymorphisms7. The discriminatory power (DP) found usingmarkers CDC3andHIS3, was 0.85 and 0.90 respectively. These resultsand especially the combined value of DP (0.91) are considered by severalauthors as reliable studies of molecular typing7,22. The data presented inTable 2 show the prevalence of genotypes (D, E, F, M), however, thereis no relation with sites of isolation of yeasts. This type of observationhas already been described in another study using the same genotypingtechnique2. Finally, by putting together epidemiological data (Fig. 1 and2, Table 2), it is possible to observe the formation of groups with high

similarity (90-100%). These are mostly from patients hospitalized in ICUwhere the evidence of common origin is of great importance. Accordingto AL-KARAAWI et al.2, the clinical isolates of C. albicanstend to begenetically similar to each other if they were isolates from patients with asimilar profile, as those interned in ICU. CHAVES et al.10recently showedthat candidemic patients had highly related microsatellites genotype incolonizing and bloodstream isolates. However, it should be noted thatthe detection of yeasts highly similar in our study was not associated

with hospital unit. The same profile was found in various hospital areassuch as pediatric and internal medicine. These data reinforce that mostC. albicans infections are from endogenous sources. They should alsosuggest that these strains may be circulating in the various units, but notcharacterizing the occurrence of outbreaks.

Although the infection of different patients from different sectors withyeasts of the same genetic profile insinuates cross-transmission17,19, highsimilarity among samples suggests an adaptation to the environmentalconditions, thus characterizing microevolutions28. Five (14.70%) of allpatients enrolled in this study are particularly interesting since C. albicanswere isolated from different sites. In all cases the analysis confirmed thatthe clinical isolates were identical to each other (Table 3) indicating themigration of yeasts from colonization (urine catheters, tracheal secretion)

into the blood, suggesting the source of systemic infection. This resultindicates that each isolated pair has genotypic identity, suggestingclonal origin. This fact has been demonstrated by molecular typing, inseveral studies10,24,29,37and helps confirm that previous colonization is animportant predisposing factor for systemic infection.

Despite the small number of samples analyzed, this study contributeswith the understanding on epidemiology of fungal infections in hospitals.The analyzed data allow us to conclude that both techniques generatedreproducible profiles showing similarity among the isolates. Thesetechniques are suitable for epidemiological molecular studies of C.albicansand can be applied in larger populations. The good performanceof these techniques allows its use for genotyping of outbreaks of hospital

Table 3Similarity by Random Amplified Polymorphic DNA and genotype by Microsatellite of Candida albicansisolated in two sources from a same patient

Patient Source Genotype RAPDCDC3(bp)

HIS3(bp)

SAB

Primer M2 SAB

Primer P4

11B Catheter tip 137:121 158:162 1.00 1.00

1A Blood 137:121 158:162 1.00 1.00

22B TOT 129:125 150:194 1.00 1.00

2A Blood 129:125 150:194 1.00 1.00

33B Catheter tip 129:125 150:162 1.00 1.00

3A Blood 129:125 150:162 1.00 1.00

44B Urine 129:121 158:158 1.00 1.00

4A Blood 129:121 158:158 1.00 1.00

55A TOT 129:121 158:158 1.00 1.00

5B Catheter tip 129:121 158:158 1.00 1.00

TOT: endotracheal aspirate.


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origin or not, and characterization of isolates from different sites,including recurrent infections such as vulvovaginal candidiasis andinvestigations before and after treatments. Knowledge of the relationshipof clinical isolates involved in infections is extremely important for thedevelopment and application of the correct therapeutic strategy and tobetter understand the epidemiology of these infections.

RESUMO

Tipagem molecular de Candida albicansisoladas de pacienteshospitalizados

Introduo: A maioria das infeces fngicas hospitalares socausadas por Candidaspp. e C. albicans a espcie mais comumenteidentificada. Mtodos moleculares so ferramentas importantes para aavaliao da origem das leveduras isoladas em hospitais. Mtodos:Este um estudo sobre o perfil gentico de 39 isolados clnicos nosocomiais

de C. albicansatravs das tcnicas de RAPD e microssatlite, foramusados dois diferentes iniciadores para cada tcnica. Resultados:RAPD forneceu 10 e 11 diferentes perfis com valores de SAB 0,84 0,126 e 0,88 0,08 para osprimersM2 e P4, respectivamente. A anlisede microssatlites, usando os marcadores CDC3 e HIS3permitiu aobservao de seis e sete diferentes alelos respectivamente, com poderdiscriminatrio combinado de 0,91. Concluses:Embora seja clara avariabilidade gentica, foi possvel identificar alta similaridade, sugerindoorigem comum para pelo menos parte deles. importante enfatizar quefoi comprovada origem comum de leveduras isoladas de colonizao(urina, cateter ou secreo orotraqueal) e hemocultura do mesmopaciente, indicando que a candidemia deve ter iniciado a partir de umstio de colonizao. A combinao das tcnicas RAPD e microssatlitesfornece uma anlise rpida e eficiente para investigao de similaridade

entre isolados nosocomiais de C. albicans.

REFERENCES

1. Adachi H, Shimizu K, Hattori H, Tanaka R, Chibana H, Takagi Y, et al. Genotyping ofCandida albicans by fragment analysis of microsatellites combined with 25S rDNAand RPS-based strategies. Nihon Ishinkin Gakkai Zasshi. 2009;50:167-74.

2. Al-Karaawi ZM, Manfredi M, Waugh ACW, McCullough MJ, Jorge J, Scully C, et al.Molecular characterization of Candidaspp. isolated from the oral cavities of patientsfrom diverse clinical settings. Oral Microbiol Immunol. 2002;17:44-9.

3. Arajo SM, Fontes CJ, Leite Jnior DP, Hahn RC. Fungal agents in different anatomicalsites in public health services in Cuiab, state of Mato Grosso, Brazil. Rev Inst MedTrop Sao Paulo. 2012;54:5-10.

4. Asmundsdottir LR, Erlendsdottir H, Haraldsson G, Guo H, Xu J, Gottfredsson M.Molecular epidemiology of candidemia: evidence of clusters of smolderingnosocomial infections. Clin Infect Dis. 2008;47:17-24.

5. Ben Abdeljelil J, Saghrouni F, Emira N, Valentin-Gomez E, Chatti N, Boukadida J,et al.Molecular typing of Candida albicansisolates from patients and health care workersin a neonatal intensive care unit. J Appl Microbiol. 2011;111:1235-49.

6. Beretta S, Fulgencio JP, Enache-Angoulvant A, Bernard C, El Metaoua S, Ancelle T,etal. Application of microsatellite typing for the investigation of a cluster of cases ofCandida albicanscandidaemia. Clin Microbiol Infect. 2006;12:674-6.

7. Botterel F, Desterke C, Costa C, Bretagne S. Analysis of microsatellite markers ofCandidaalbicansused for rapid typing. J Clin Microbiol. 2001;39:4076-81.

8. Bougnoux ME, Kac G, Aegerter P, dEnfert C, Fagon JY, CandiRea Study Group.Candidemia and candiduria in critically ill patients admitted to intensive care unitsin France: incidence, molecular diversity, management and outcome. Intensive CareMed. 2008;34:292-9.

9. Chang MR, Correia FP, Costa LC, Xavier PCN, Palhares DB, Taira DL,et al. Candidabloodstream infection: data from a teaching hospital in Mato Grosso do Sul, Brazil.Rev Inst Med Trop Sao Paulo. 2008;50:265-8.

10. Chaves GM, Santos FP, Colombo AL. The pers istence of mul tifocal colon isationby a single ABC genotype of Candida albicansmay predict the transition fromcommensalism to infection. Mem Inst Oswaldo Cruz. 2012;107:198-204.

11. Chong PP, Abdul Hadi SR, Lee YL, Phan CL, Tan BC, Ng KP, et al. Genotyping anddrug resistance profile of Candidaspp. in recurrent and one-off vaginitis, and highassociation of non-albicans species with non-pregnant status. Infect Genet Evol.2007;7:449-56.

12. Chong PP, Lee YL, Tan BC, Ng KP. Genetic relatedness ofCandidastrains isolated fromwomen with vaginal candidiasis in Malaysia. J Med Microbiol. 2003;52:657-66.

13. Chvez-Galarza J, Pais C, Sampaio P. Microsatellite typing identifies the major cladesof the human pathogen Candida albicans. Infect Genet Evol. 2010;10:697-702.

14. Colombo AL, Nucci M, Park BJ, Nour SA, Arthington-Skaggs B, Da Matta DA, etal.Epidemiology of candidemia in Brazil: a nationwide sentinel surveillance ofcandidemia in eleven medical centers. J C lin Microbiol. 2006;44:2816-23.

15. Dalle F, Dumont L, Franco N, Mesmacque D, Caillot D, Bonnin P, et al. Genotyping ofCandida albicansoral strains from healthy individuals by polymorphic microsatellitelocus analysis. J Clin Microbiol. 2003;41:2203-5.

16. Dassanayake RS, Samaranayake LP. Amplification-based nucleic acid scanning techniquesto assess genetic polymorphism in Candida. Crit Rev Microbiol. 2003;29:1-24.

17. De Pinho Resende JC, Franco GR, Rosa CA, Hahn RC, Hamdan JS. Phenotypic andgenotypic identification of Candidaspp. isolated from hospitalized patients. RevIberoam Micol. 2004;21:24-8.

18. DiNubile MJ, Lupinacci RJ, Strohmaier KM, Sable CA, Kartsonis NA. Invasivecandidiasis treated in the intensive care unit: observations

dionisio, e et al 2013., rev inst med trop sp.pdf

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