Molecular characterization of Mycobacterium massiliense and Mycobacterium bolletii in isolates collected from outbreaks of infections after laparoscopic surgeries and cosmetic procedures

An outbreak of infections affecting 311 patients who had undergone different invasive procedures occurred in 2004 and 2005 in the city of Belem, in the northern region of Brazil. Sixty-seven isolates were studied; 58 were from patients who had undergone laparoscopic surgeries, 1 was from a patient with a postinjection abscess, and 8 were from patients who had undergone mesotherapy. All isolates were rapidly growing nonpigmented mycobacteria and presented a pattern by PCR-restriction enzyme analysis of the hsp65 gene with BstEII of bands of 235 and 210 bp and with HaeIII of bands of 200, 70, 60, and 50 bp, which is common to Mycobacterium abscessus type 2, Mycobacterium bolletii, and Mycobacterium massiliense. hsp65 and. rpoB gene sequencing of a subset of 20 isolates was used to discriminate between these three species. hsp65 and rpoB sequences chosen at random from 11 of the 58 isolates from surgical patients and the postinjection abscess isolate presented the highest degrees of similarity with the corresponding sequences of M. massiliense. In the same way, the eight mesotherapy isolates were identified as M. bolletii. Molecular typing by pulsed-field gel electrophoresis (PFGE) grouped all 58 surgical isolates, while the mesotherapy isolates presented three different PFGE patterns and the postinjection abscess isolate showed a unique PFGE pattern. In conclusion, molecular techniques for identification and typing were essential for the discrimination of two concomitant outbreaks and one case, the postinjection abscess, not related to either outbreak all of which were originally attributed to a single strain of M. abscessus.

Candidemia neonatal, em hospital público do Mato Grosso do Sul

O objetivo de nosso estudo foi realizar tipagem molecular de 25 amostras clínicas de Candida spp, isoladas de crianças com candidemia, internadas na unidade de terapia intensiva neonatal de um Hospital Universitário entre 1998 a 2006. Dados demográficos e clínicos foram obtidos de prontuários para conhecimento dos aspectos clínicos e epidemiológicos. Identificação das leveduras foi feita por método convencional e a susceptibilidade antifúngica por método de microdiluição. O perfil genético foi determinado pela técnica de RAPD-PCR. Candida albicans (11; 44%) e Candida parapsilosis (10; 40%) foram as mais isoladas. Dezessete (68%) dos recém-nascidos tinham peso inferior a 1.500g. Prematuridade (92%), uso de cateter venoso central (100%), foram as condições de risco mais associados. Dezenove (76%) pacientes foram a óbito. Apenas uma cepa de Candida parapsilosis, mostrou ser sensível dose dependente ao fluconazol. Na análise molecular, foram observados 11 padrões genéticos distintos. Somente em dois casos foi observada relação epidemiológica, sugerindo mesma fonte de infecção.

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE) x S(SM)(EK) x S(SM)(SSRs)). Clustering analyses showed a mean of 9 +/- 12.4 isolates per cluster (3.8 +/- 8 isolates/taxon) for MLEE, 6.2 +/- 4.9 isolates per cluster (4 +/- 4.5 isolates/taxon) for SSRs, and 4.1 +/- 2.3 isolates per cluster (2.6 +/- 2.3 isolates/taxon) for EK. A total of 45 (13%), 39(11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (Si) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented with similarity and grouping analysis. Therefore, the use of genotyping systems that give results which offer minimum disparity, or the combination of the results of these systems, can provide greater security and consistency in the determination of strains and their genetic relationships. (C) 2010 Elsevier B.V. All rights reserved.

Candida colonisation as a source for candidaemia

Candida spp. are important healthcare-associated pathogens. Identifying the source of infection is important for prevention and control strategies. The objective of this study was to evaluate candida colonisation sites as potential sources for candidaemia. Sixty-three consecutive patients with a positive blood culture for candida were included. Surveillance cultures were collected from urine, rectum, oropharynx, skin, intravascular catheter tip and skin around catheter. Molecular typing was performed when the same species of candida was isolated from blood and surveillance sites of a patient. C. albicans was associated with 42% of candidaemias, C. parapsilosis 33%, C. tropicalis 16% and C. guilliermondii, C. krusei, C. glabrata, C. holmii and C. metapsilosis were all 2% each. Six of 10 C. parapsilosis catheter tip isolates were indistinguishable from corresponding blood isolates (all in neonates). C. albicans isolates from blood were indistinguishable from corresponding gastrointestinal, tract isolates in 13 of 26 patients and from catheter tip isolates in two patients. In conclusion, the results suggest that gastrointestinal colonisation is the probable source of C. albicans candidaemia and C. parapsilosis is exogenous. (C) 2009 The Hospital, Infection Society. Published by Elsevier Ltd. All rights reserved.

Differences Between ""Classical"" Risk Factors for Infections Caused by Methicillin-Resistant Staphylococcus aureus (MRSA) and Risk Factors for Nosocomial Bloodstream Infections Caused by Multiple Clones of the Staphylococcal Cassette Chromosome mec Type IV MRSA Strain

OBJECTIVE. To identify risk factors associated with nosocomial bloodstream infections caused by multiple clones of the staphylococcal cassette chromosome mec (SCCmec) type IV strain of methicillin-resistant Staphylococcus aureus (MRSA). DESIGN. An unmatched case-control study (at a ratio of 1 : 2) performed during the period from October 2002 through September 2003. SETTING. A 2,000-bed tertiary care teaching hospital affiliated with the University of Sao Paulo in Sao Paulo, Brazil. METHODS. Case patients (n = 30) were defined either as patients who had a bloodstream infection due to SCCmec type IV strains of MRSA diagnosed at least 48 hours after hospital admission or as neonates with the infection who were born in the hospital. Control patients (n = 60) were defined as patients with SCCmec type III MRSA infection diagnosed at least 48 hours after hospital admission. Genes n = 60 encoding virulence factors were studied in the isolates recovered from case patients, and molecular typing of the SCCmec type IV MRSA isolates was also done by pulsed-field gel electrophoresis and multilocus sequence typing. RESULTS. In multivariate analysis, the following 3 variables were significantly associated with having a nosocomial bloodstream infection caused by SCCmec type IV strains of MRSA: an age of less than 1 year, less frequent use of a central venous catheter (odds ratio [OR], 0.07 [95% confidence interval {CI}, 0.02-0.28]; P = .001), and female sex. A second analysis was performed that excluded the case and Pp. 001 control patients from the neonatal unit, and, in multivariate analysis, the following variables were significantly associated with having a nosocomial bloodstream infection caused by SCCmec type IV strains of MRSA: less frequent use of a central venous catheter (OR, 0.12 [95% CI, 0.03-0.55]; P = .007), lower Acute Physiology and Chronic Health Evaluation II score on admission (OR, 0.14 [95% CI, 0.03-0.61];), less frequent surgery (OR, 0.21 [95% CI, 0.06-0.83];), and female sex (OR, 5.70 [95% CI, 1.32-24.66]; P =.020). P = .009 Pp. 025 Pp). Of the 29 SCCmec type IV MRSA isolates recovered from case patients, none contained the Panton-Valentine leukocidin, gamma-hemolysin, enterotoxin B or C, or toxic shock syndrome toxin-1. All of the isolates contained genes for the LukE-LukD leukocidin and alpha-hemolysin. Genes for enterotoxin A were present in 1 isolate, and genes for beta-hemolysin were present in 3 isolates. CONCLUSIONS. ""Classical"" risk factors do not apply to patients infected with the SCCmec type IV strain of MRSA, which is an important cause of nosocomial bacteremia. This strain infects a patient population that is less ill and has had less frequent invasive procedures than a patient population infected with the multidrug-resistant strain of SCCmec type III MRSA. We found that virulence factors were rare and that Panton-Valentine leukocidin was absent. There were multiple clones of the SCCmec type IV strain in our hospital. Children under 1 year of age were at a higher risk. There was a predominant clone ( sequence type 5) in this patient population.

Salvage of long-term central venous catheters during an outbreak of Pseudomonas putida and Stenotrophomonas maltophilia infections associated with contaminated heparin catheter-lock solution

objective. To describe the management of patients with long-term central venous catheters (CVCs) during an outbreak of infection due to Pseudomonas putida and Stenotrophomonas maltophilia associated with contaminated heparin catheter-lock solution. design. Descriptive study. setting. Private, 250-bed tertiary-care hospital. methods. In March 2003, we identified 2 febrile cancer patients with P. putida bacteremia. Over 2 days, 7 cases of bacteremia were identified; lots of syringes prefilled with heparin catheter-lock solution, supplied by a compounding pharmacy, were recalled and samples were cultured. More cases of bacteremia appeared during the following days, and any patient who had had a catheter lock infused with the suspect solution was asked to provide blood samples for culture, even if the patient was asymptomatic. Isolates that were recovered from culture were typed by pulsed-field gel electrophoresis. Antimicrobial salvage treatment of long-term CVCs was attempted. results. A total of 154 patients had had their catheter lock infused with solution from the lots that were suspected of being contaminated. Only 48 of these patients had CVCs. By day 7 of the outbreak, 18 of these patients had become symptomatic. Twenty-six of the remaining 30 asymptomatic patients then also provided blood samples for culture, 10 of whom developed fever shortly after samples were collected. Thirty-two patients were identified who had P. putida bacteremia; 9 also had infection due to S. maltophilia. Samples from 1 of the 3 lots of prefilled syringes in use at the time of the outbreak also grew P. putida on culture. Molecular typing identified 3 different clones of P. putida from patients and heparin catheter-lock solution, and 1 clone of S. maltophilia. A total of 27 patients received antimicrobial therapy regimens, some of which included decontamination of the catheter lock with anti- infective lock solution. Of 27 patients, 19 (70%) retained their long-term CVC during the 6-month follow-up period. conclusions. To our knowledge, this is one of the largest prospective experiences in the management of bloodstream infection associated with long-term CVCs. The infections were caused by gram-negative bacilli and were managed without catheter removal, with a high response rate. We emphasize the risks of using intravenous formulations of medications supplied by compounding pharmacies that produce large quantities of drugs.

Frequency of human metapneumovirus infection in hematopoietic SCT recipients during 3 consecutive years

Respiratory viruses can cause significant morbidity in immunocompromised hosts. Human metapneumovirus (hMPV) has been increasingly associated with lower respiratory tract infection in hematopoietic SCT (HSCT) recipients, with mortality rates up to 50%. No data on the occurrence of hMPV infection in HSCT recipients have been reported in the southern hemisphere. We conducted a retrospective study including 228 nasal wash samples from 153 HSCT recipients with respiratory symptoms during 2001, 2002 and 2003. hMPV was detected by real-time PCR with primers complementary to the nucleocapsid region of hMPV genome. Eleven of the 153 patients (7.2%) acquired hMPV infection during the study period (6.4% in 2001, 4.7% in 2002 and 11.1% in 2003). Among the 11 HSCT recipients with hMPV infection, 1 died 8 days after the diagnosis, but the role of hMPV in the patient`s death could not be established. In 2001 and 2003, hMPV group A prevailed over group B. In 2002, both groups were detected equally. hMPV infections were diagnosed in late winter and spring. The frequency of hMPV infection in HSCT recipients living in Brazil was similar to those observed in the northern hemisphere. Sensitive techniques to detect hMPV should be included in the diagnostic assessment of HSCT recipients with respiratory symptoms.

Species diversity of Leishmania (Viannia) parasites circulating in an endemic area for cutaneous leishmaniasis located in the Atlantic rainforest region of northeastern Brazil

OBJECTIVES To identify the aetiological agents of cutaneous leishmaniasis and to investigate the genetic polymorphism of Leishmania (Viannia) parasites circulating in an area with endemic cutaneous leishmaniasis (CL) in the Atlantic rainforest region of northeastern Brazil. METHODS Leishmania spp. isolates came from three sources: (i) patients diagnosed clinically and parasitologically with CL based on primary lesions, secondary lesions, clinical recidiva, mucocutaneous leishmaniasis and scars; (ii) sentinel hamsters, sylvatic or synanthropic small rodents; and (iii) the sand fly species Lutzomyia whitmani. Isolates were characterised using monoclonal antibodies, multilocus enzyme electrophoresis (MLEE) and polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region rDNA locus. RESULTS Seventy-seven isolates were obtained and characterised. All isolates were identified as Leishmania (Viannia) braziliensis serodeme 1 based on reactivity to monoclonal antibodies. MLEE identified 10 zymodemes circulating in the study region. Most isolates were classified as zymodemes closely related to L. (V.) braziliensis, but five isolates were classified as Leishmania (Viannia) shawi. All but three of the identified zymodemes have so far been observed only in the study region. Enzootic transmission and multiclonal infection were observed. CONCLUSIONS Our results confirm that transmission cycle complexity and the co-existence of two or more species in the same area can affect the level of genetic polymorphism in a natural Leishmania population. Although it is not possible to make inferences as to the modes of genetic exchange, one can speculate that some of the zymodemes specific to the region are hybrids of L. (V.) braziliensis and L. (V.) shawi.

Clonal relationships determined by multilocus sequence typing among enteropathogenic Escherichia coli isolated in Brazil

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infantile diarrhea in developing nations. Multilocus sequence typing (MLST) characterizes bacterial strains based on the sequences of internal fragments in housekeeping genes. Little is known about strains of EPEC analyzed by MLST from Brazil. In this study, a diverse collection of 29 EPEC strains isolated from patients with diarrhea, admitted to the University Hospital of Ribeirao Preto, was characterized by MLST. Strain analysis demonstrated 22 different sequence types (STs), of which almost half (48%) were new, indicating a high genotype diversity. The 22 STs were divided by eBURST into 12 clonal complexes. It was not possible to correlate typical and atypical EPEC with other strains in the MLST database. This is the first study that analyzed EPEC strains from South America that are included in the E. coli MLST database. Nine (31%) out of 29 strains are part of the CC10 clonal complex, the major clonal complex in the database, which comprises 174 strains and 86 different STs, suggesting that these strains might be the most important intestinal pathogenic E. coli worldwide. Genetic relationships between typical and atypical EPEC, enterohemorrhagic E. coli, and enteroaggregative E. coli strains were not established by MLST.

Molecular Characterization of Strains of Respiratory Syncytial Virus Identified in a Hematopoietic Stem Cell Transplant Outpatient Unit Over 2 Years: Community or Nosocomial Infection?

Respiratory syncytial virus (RSV) is recognized as the leading cause of nosocomial respiratory infection among hematopoietic stem cell transplant (HSCT) recipients, causing considerable morbidity and mortality. RSV is easily transmitted by contact with contaminated surfaces, and in HSCT units, more than 50% of RSV infections have been characterized as of nosocomial origin. From April 2001 to October 2002, RSV was identified by direct immunofluorescent assay in 42 symptomatic HSCT recipients. Seven RSV strains from 2001 and 12 RSV strains from 2002 were sequenced. RNA extraction, cDNA synthesis, and seminested polymerase chain reaction (PCR) with primers complementary to RSV genes G and F were pet-formed. PCR products were analyzed by nucleotide sequencing of the C-terminal region of gene G for typing (in group A or B). Of the 7 strains analyzed in 2001, only 2 belonged to group B; the other 5 belonged to group A. Of these 7 strains, 3 were identical and were from recipients receiving outpatient care. In 2002, of the 12 strains analyzed, 3 belonged to group A and the other 9 belonged to group B. Of these 9 strains, 7 were genetically identical and were also from recipients receiving outpatient care. Therefore, multiple strains of RSV cocirculated in the hematopoietic stem cell transplant units (ward and outpatient units) between 2001 and 2002. Nosocomial transmission was more likely to occur at the HSCT outpatient unit than in the HSCT ward. Infection control practices should also be implemented in the outpatient setting.

Molecular characteristics of extended-spectrum beta-lactamase-producing Escherichia coli from Rio de Janeiro, Brazil

Twenty-five extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clinical isolates from Rio de Janeiro, Brazil were characterized by isoelectric focusing, PCR and sequencing of bla(ESBL) genes, plasmid-mediated quinolone resistance determinants, phylogenetic groups, replicon typing, pulsed-field electrophoresis, and multilocus sequencing typing. Twenty-three (92%) ESBL-producing E. coli isolates were positive for bla(CTX-M) genes, aac(6`)-lb-cr, and qnrB. Genetic relatedness of ESBL producers clustered seven (28%) CTX-M-15-producing isolates as sequence type (ST) 410, clonal complex (CC) 23, and two (8%) as clone O25-ST131. Our results illustrate the predominance of phylo-group A (52%), ST410 (CC 23) and CTX-M-15 among ESBL-producing E. coli isolates from hospitals in Rio de Janeiro.

Molecular systematics of Thorea (Rhodophyta, Thoreales) species in Brazil

This study aimed to evaluate species level taxonomy and phylogenetic relationship among Thorea species in Brazil and other regions of the world using two molecular markers - RUBISCO large subunit plastid gene (rbcL) and nuclear small-subunit ribosomal DNA (SSU rDNA). Three samples of Thorea from Brazil (states of Mato Grosso do Sul and São Paulo) and one sample from Dominican Republic (DR) were sequenced. Analyses based on partial sequences of rbcL (1,282 bp) and complete sequences of SSU (1,752 bp) were essentially congruent and revealed that Thoreales formed a distinct monophyletic clade, which had two major branches with high support, representing the genera Thorea and Nemalionopsis. Thorea clade had four main branches with high support for all analyses, each one representing the species: 1) T. gaudichaudii C. Agardh from Asia (Japan and Philippines) - this clade occurred only in the rbcL analyses; 2) T. violacea Bory from Asia (Japan) and North America (U.S.A. and DR); 3) T. hispida (Thore) Desvaux from Europe (England) and Asia (Japan); 4) a distinct group with the three Brazilian samples (sequence identity: rbcL 97.2%, 1,246 bp; SSU 96.0-98.1%, 1,699-1,720 bp). The Brazilian samples clearly formed a monophyletic clade based on both molecular markers and was interpreted as a separate species, for which we resurrected the name T. bachmannii Pujals. Morphological and molecular evidences indicate that the Thoreales is well-resolved at ordinal and generic levels. In contrast, Thorea species recognized by molecular data require additional characters (e.g. reproductive and chromosome numbers) to allow consistent and reliable taxonomic circumscription aiming at a world revision based on molecular and morphological evidences.

Thaptomys Thomas 1915 (Rodentia, Sigmodontinae, Akodontini) with karyotypes 2n = 50, FN = 48, and 2n = 52, FN = 52: two monophyletic lineages recovered by molecular phylogeny

A novel karyotype with 2n = 50, FN = 48, was described for specimens of Thaptomys collected at Una, State of Bahia, Brazil, which are morphologically indistinguishable from Thaptomys nigrita, 2n = 52, FN = 52, found in other localities. It was hence proposed that the 2n = 50 karyotype could belong to a distinct species, cryptic of Thaptomys nigrita, once chromosomal rearrangements observed, along with the geographic distance, might represent a reproductive barrier between both forms. Phylogenetic analyses using maximum parsimony and maximum likelihood based on partial cytochrome b sequences with 1077 bp were performed, attempting to establish the relationships among the individuals with distinct karyotypes along the geographic distribution of the genus; the sample comprised 18 karyotyped specimens of Thaptomys, encompassing 15 haplotypes, from eight different localities of the Atlantic Rainforest. The intra-generic relationships corroborated the distinct diploid numbers, once both phylogenetic reconstructions recovered two monophyletic lineages, a northeastern clade grouping the 2n = 50 and a southeastern clade with three subclades, grouping the 2n = 52 karyotype. The sequence divergence observed between their individuals ranged from 1.9% to 3.5%.

Use of chromosome microdissection in fish molecular cytogenetics

Chromosome microdissection is a technique in which whole chromosomes or chromosomal segments are dissected under an inverted microscope yielding chromosome-specific sequences. Several protocol modifications introduced during the past 15 years reduced the number of chromosomes required for most applications. This is of particular interest to fish molecular cytogenetics, since most species present highly uniform karyotypes which make impossible the collection of multiple copies of the same chromosome. Probes developed in this manner can be used to investigate chromosome homologies in closely related species. Here we describe a protocol recently used in the gymnotiform species group Eigenmannia and review the major steps involved in the generation of these markers focusing on protocol modifications aiming to reduce the number of required chromosomes.