Evaluation of methodology for serotyping invasive and nasopharyngeal isolates of Haemophilus influenzae in the ongoing surveillance in Brazil

To assess the magnitude of discrepant results obtained by routine Haemophilus influenzae serotyping, 258 isolates, collected by the epidemiological surveillance system in Brazil from individuals with invasive diseases or carriage, were evaluated by two slide agglutination (SlAg) methods: SlAg method 1, by which strains were initially screened with a serotype b-specific antiserum, and SlAg method 2, by which strains were tested against all serotype-specific antisera in parallel. Investigators comparing results of the two SlAg methods with those obtained by capsule type-specific PCR were blinded to the method used. The serotype prevalence rates found by the three methods were significantly different, involving discrepancies mainly between serotype b and noncapsulated (NC) isolates. For invasive isolates (n = 131), the overall agreement rate between SlAg method 1 or 2 and PCR was 68.0 or 88.3%, respectively, whereas for colonizing isolates (n = 127) the corresponding rate was 46.5 or 94.2%, respectively. SlAg method 2 improved the ascertainment of serotypes over that obtained with SlAg method 1, demonstrating good correlation with PCR. Use of the polyvalent antiserum as a screening reagent for SlAg for invasive and colonizing isolates showed poor discriminatory power, with a sensitivity of 65.8% and a specificity of 91.7%. We stress the importance of using a well-standardized SlAg methodology and suggest that reference laboratories should utilize PCR routinely to confirm SlAg results and to check all nonspecific SlAg reactions and apparent NC isolates by SlAg in order to provide reliable data on the prevalence of H. influenzae serotypes in the H. influenzae type b vaccine era.     

Genetic diversity among clinical isolates of Acremonium strictum determined during an investigation of a fatal mycosis

Primarily saprophytic in nature, fungi of the genus Acremonium are a well-documented cause of mycetoma and other focal diseases. More recently, a number of Acremonium spp. have been implicated in invasive infections in the setting of severe immunosuppression. During the course of routine microbiological studies involving a case of fatal mycosis in a nonmyeloablative hematopoietic stem cell transplant patient, we identified a greater-than-expected variation among strains previously identified as Acremonium strictum by clinical microbiologists. Using DNA sequence analysis of the ribosomal DNA intergenic transcribed spacer (ITS) regions and the D1-D2 variable domain of the 28S ribosomal DNA gene (28S), the case isolate and four other clinical isolates phenotypically identified as A. strictum were found to have <99% homology to the A. strictum type strain, CBS 346.70, at the ITS and 28S loci, while a sixth isolate phenotypically identified only as Acremonium sp. had >99% homology to the type strain at both loci. These results suggest that five out of the six clinical isolates belong to species other than A. strictum or that the A. strictum taxon is genetically diverse. Based upon these sequence data, the clinical isolates were placed into three genogroups.     

Genetic diversity of respiratory syncytial virus isolated during an epidemic period from children of northeastern Brazil

Outbreaks of human respiratory syncytial virus (HRSV) are the leading cause of serious acute lower respiratory viral disease in many countries in different continents. Data on clinical and epidemiological aspects of HRSV infections in this country have been reported, but there is lack of data regarding the molecular epidemiology of this virus in Salvador. The genetic variability of HRSV isolated during an outbreak in Salvador, Brazil (1999) has been analysed. Partial sequences of the G protein gene of 13 isolates from antigenic group A and 4 isolates from antigenic group B of HRSV were determined. Nucleotide sequences of C-terminal G gene were compared to sequences of HRSV isolates from countries of South America and from the rest of the world available at the GenBank. Brazilian group A and B isolates were clustered into previously characterised genotypes: GA5, GA2, GA7, and GB3, SAB3, respectively. This is the first study of GA7 and SAB3 genotypes circulation in South American countries. It is interesting to point out that viruses isolated in Salvador appear to be closer related with those from Montevideo-Uruguay and Buenos Aires, Argentina strains, suggesting circulation of similar strains among different South American countries in different seasons. Moreover, viruses closely related genetically circulated in the same year in Salvador and distant places such as Mozambique, supporting the previous suggestion on the complexity of HRSV strain circulation patterns, and the high capability of HRSV spreading world-wide.     

Clonal diversity among streptogramin A-resistant Staphylococcus aureus isolates collected in French hospitals

We analyzed 62 clinical isolates of streptogramin A-resistant (SGA(r)) Staphylococcus aureus collected between 1981 and 2001 in 14 hospitals located in seven French cities. These isolates, including five with decreased susceptibility to glycopeptides, were distributed into 45 antibiotypes and 38 SmaI genotypes. Each of these genotypes included between 1 and 11 isolates, the SmaI patterns of which differed by no more than three bands. Although numerous clones were identified, we observed the spread of monoclonal isolates either within the same hospital or within hospitals in distinct cities and at large time intervals. Hybridization with probes directed against 10 SGA(r) genes (vatA, vatB, vatC, vatD, vatE, vgaA, vgaB, vgaAv, vgbA, and vgbB) revealed six patterns: vgaAv (21 isolates), vatA-vgbA (24 isolates), vgaAv-vatB-vgaB (14 isolates), vgaAv-vatA-vgbA (1 isolate), vgaAv-vatA-vgbA-vatB-vgaB (1 isolate), and vgaA (1 isolate). We detected at least one SGA(r) determinant in all of the tested isolates. vgaAv, which is part of the recently characterized transposon Tn5406, was found in 59.7% of the tested isolates. Of the 16 streptogramin B-susceptible isolates, 14 carried vgaAv alone and were susceptible to the mixtures of streptogramins, whereas the 2 isolates carrying vgaAv-vatB-vgaB were resistant to these mixtures. vatA-vgbA was found on plasmids of the same apparent size in 26 (42%) of the tested clinical isolates from 18 unrelated SmaI genotypes. The possible dissemination of some of the multiple clones characterized in the present study with an expected increased selective pressure of streptogramins following the recent licensing of Synercid (quinupristin-dalfopristin) must be carefully monitored.     

Genetic diversity of isoniazid-resistant Mycobacterium tuberculosis isolates collected in Poland and assessed by spoligotyping

The genetic compositions of 71 isoniazid-resistant Mycobacterium tuberculosis strains from Poland were determined by spoligotyping. Nearly 80% of the isolates belonged to either the T or the Haarlem family. The genotypic diversity was largely due to variation within those families. The scarcity of imported genotypes suggested that the M. tuberculosis population studied has an endemic nature.     

Genetic diversity among clinical Coccidioides spp. isolates in Arizona

Increasing coccidioidomycosis rates in Arizona may indicate the development of a hypervirulent strain. One hundred and twenty-one clinical Coccidioides spp. isolates were collected over 16 months from Maricopa, Graham, Yuma, and Pima counties in Arizona. The patient age distribution ranged from 9 to 91 years, with a median age of 58 years; 36% were female, and 64% male. All isolates were analyzed by measuring length polymorphisms in nine distinct microsatellite regions. The three microsatellites found to have the greatest discriminatory power for Coccidioides posadasii were: K03 (0.87), GA37 (0.83), and K01 (0.78). The majority of isolates (n=119) were C. posadasii. Duplicate isolates (n=28) from 13 patients showed single strain infections. Phylogenetic analysis of the microsatellite data showed no dominant microsatellite pattern. We conclude that the increase in reported cases of coccidioidomycosis in Arizona is not linked to a dominant, hypervirulent strain of Coccidioides posadasii.     

Genetic diversity among clinical and environmental isolates of Aspergillus fumigatus.

To determine if cases of invasive aspergillosis (IA) were caused by strains of Aspergillus fumigatus with unique characteristics, strains from immunosuppressed patients with IA were compared to strains obtained from sputa of patients with cystic fibrosis and to strains from the environment. An extremely high genomic diversity was observed among the 879 strains typed by Southern blotting with a retrotransposon-like element from A. fumigatus (C. Neuvéglise, J. Sarfati, J. P. Latgé, and S. Paris, Nucleic Acids Res. 24:1428-1434, 1996). Analysis of Southern blot hybridization patterns showed the absence of clustering between environmental isolates and clinical isolates from patients with IA or cystic fibrosis. In addition, strains could not be clustered depending on their geographical location. This study implies that practically any strain of A. fumigatus is potentially pathogenic and can provoke a case of IA when it encounters a favorable environment in an immunosuppressed host.     

Pilot study of the genetic diversity of the pneumococcal nasopharyngeal flora among children attending day care centers

A pilot study was conducted to determine the genetic diversity of multiple colonies of pneumococci recovered from 37 nasopharyngeal (NP) samples of children. A total of 239 pneumococcal isolates (typically, six to eight colonies per sample) were typed by pulsed-field gel electrophoresis (PFGE). In most NP samples (89%) the multiple colonies shared common PFGE types and serotypes. However, four samples were heterogeneous (samples A through D): each contained two strains with different PFGE types, antibiotypes, and serotypes. Samples A and B each contained one strain of a vaccine capsular type and another expressing a non-vaccine type (according to the currently licensed seven-valent conjugate vaccine). In samples B and C the penicillin MIC for one strain was elevated and the other strain was susceptible. In each of the heterogeneous samples, one of the strains was a representative of an internationally disseminated clone. Samples A, C, and D contained strains which carried prophages that were inducible by mitomycin C and that could be visualized by electron microscopy. The comC gene allele (which encodes the competence-stimulating peptide) was the same in both strains found in each of samples A, B, and D. Carriage of multiple pneumococci with distinct properties should favor genetic exchange and provide a dynamic population structure for pneumococci in their ecological reservoir. Quantitative resolution of majority and minority components of the pneumococcal NP flora will be of importance for evaluation of the impact of intervention strategies such as vaccination or introduction of new antimicrobial agents.     

Genetic diversity of the DBLalpha region in Plasmodium falciparum var genes among Asia-Pacific isolates

In Plasmodium falciparum a highly polymorphic multi-copy gene family, var, encodes the variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1), which has an important role in cytoadherence and immune evasion. Using previously described universal PCR primers for the first Duffy binding-like domain (DBLalpha) of var we analysed the DBLalpha repertoires of Dd2 (originally from Thailand) and eight isolates from the Solomon Islands (n=4), Philippines (n=2), Papua New Guinea (n=1) and Africa (n=1). We found 15-32 unique DBLalpha sequence types among these isolates and estimated detectable DBLalpha repertoire sizes ranging from 33-38 to 52-57 copies per genome. Our data suggest that var gene repertoires generally consist of 40-50 copies per genome. Eighteen DBLalpha sequences appeared in more than one Asia-Pacific isolate with the number of sequences shared between any two isolates ranging from 0 to 6 (mean=2.0 +/-1.6). At the amino acid level DBLalpha sequence similarity within isolates ranged from 45.2 +/- 7.1 to 50.2 +/- 6.9%, and was not significantly different from the DBLalpha amino acid sequence similarity among isolates (P>0.1). Comparisons with published sequences also revealed little overlap among DBLalpha sequences from different regions. High DBLalpha sequence diversity and minimal overlap among these isolates suggest that the global var gene repertoire is immense, and may potentially be selected for by the host's protective immune response to the var gene products, PfEMP1.     

Genetic diversity of bat rabies viruses in Brazil

Summary Thirty-three Brazilian bat rabies viruses (RVs) were studied by sequence analysis and were compared against sequences of bat-related RVs from other regions of the Americas. Phylogenetic analysis revealed that bat-related RVs formed several monophyletic lineages and that these were associated with bat species. Brazilian bat RVs were found to include nine major lineages, one of which grouped with RVs isolated from Lasiurus spp. from different regions of the Americas. These results suggest that there is considerable diversity among Brazilian bat RV variants and that some of these RV variants may be associated with bats from other countries.     

Genetic diversity of Burkholderia pseudomallei isolates in Australia

Melioidosis is caused by the gram-negative saprophytic bacterium Burkholderia pseudomallei, which is endemic to southeast Asia and northern Australia. We have previously found evidence of geographic localization of strains based on multilocus sequence typing (MLST). In this study, we examined the diversity of 277 isolates from northern Australia, which were resolved into 159 different sequence types. No sequence types were common to both Queensland and the Northern Territory, and there was significant differentiation between the alleles present in the two regions. The considerable diversity in sequence types contrasts with the limited diversity of alleles at MLST loci, supporting previous work suggesting a high rate of recombination relative to mutation in B. pseudomallei, where new sequence types are primarily generated by reassortment of existing alleles.     

Genetic diversity among Mycobacterium bovis isolates: a preliminary study of strains from animal and human sources.

Mycobacterium bovis has the broadest host range of species in the Mycobacterium tuberculosis complex and is responsible for disease in humans and diverse animal species. We report on genotypic differences at multiple loci among 13 isolates derived from a range of human and animal infections. All isolates were classified as M. bovis by phenotypic analysis but could be subdivided into five distinct genotypes based on polymorphisms at the pncA and oxyR loci, the status of the RD5 deletion region, and the spoligotype pattern. These findings suggest the existence of a spectrum of strains with genotypic characteristics between those of M. tuberculosis and M. bovis.     

Genetic diversity of the M RNA segment among Crimean-Congo hemorrhagic fever virus isolates in China

The complete nucleotide sequences of the medium (M) segment of seven Chinese isolates of Crimean-Congo hemorrhagic fever (CCHF) virus were determined. The M-segment RNA of CCHF virus comprises 5356-5377 nucleotides depending on the isolate and encodes a protein comprising 1689-1697 amino acids in a viral complementary sense. Phylogenetic analysis of the M segment showed that the Chinese CCHF virus isolates were clustered into three groups, one of which was more closely related to a Nigerian isolate. Pairwise comparison of a precursor protein showed that amino-terminal regions comprising 250 amino acids were extraordinary heterogeneous, with a 22.4% identity in amino acids being observed between the most distantly related isolates. Since all the viruses were isolated from 1966 to 1988 within a restricted area in the Xinjiang Autonomous Region in western China, the results indicate that a multisource virus population is endemic in this region.     

Genetic diversity among Borrelia burgdorferi isolates from wood rats and kangaroo rats in California.

Twenty-nine Borrelia burgdorferi isolates, obtained from dusky-footed wood rats (Neotoma fuscipes) and California kangaroo rats (Dipodomys californicus) in California, were analyzed genetically. Chromosomal DNA was examined by restriction endonuclease analysis (REA) and gene probe restriction fragment length polymorphism. Pulsed-field gel electrophoresis was used to analyze the plasmid profiles of the isolates. REA, the method with the greatest discrimination, disclosed 24 distinct restriction patterns among the 29 isolates. These restriction patterns were sorted into four restriction fragment length polymorphism groups on the basis of their gene hybridization patterns. Results of the REA and plasmid profile analysis supported this grouping. The degree of genetic diversity among Californian isolates demonstrated by our findings is greater than that previously reported among other groups of North American isolates and is similar or greater than the diversity reported among European isolates.     

Genetic diversity of the internal transcribed spacers (ITS) and 5.8S rRNA genes among the clinical isolates of Candida parapsilosis in Brazil and Japan.

The internal transcribed spacer (ITS) region including 5.8S rDNA sequences of 58 isolates of Candida parapsilosis in Brazil and Japan was analyzed. Although most of the C. parapsilosis strains tested were confirmed to belong to three already reported genetically distinct groups (I, II and III) based on their ITS region sequences, 5 strains of the Brazilian isolates showed different sequences from those heretofore reported and suggested a presence of new genotype. For these strains of C. parapsilosis, we proposed a new genetic group (IV). The sequence similarities of this new group of IV to I, II and III were 87.4%, 94.7% and 87.3% in the ITS1 region, respectively. Genetic diversity in ITS regions of the remaining C. parapsilosis strains in Brazil and Japan was also discussed.     

Genetic diversity within isolates of mutans streptococci recognized by an rRNA gene probe.

A total of 79 clinical isolates of mutans streptococci and five laboratory strains representing serotypes c, d, e, f, and g were genotyped by a nonradioactive hybridization method with the rrnB rRNA operon of the Escherichia coli chromosome as a probe. The hybridization patterns of chromosomal DNA fragments obtained by digestion with restriction endonucleases HindIII, SmaI, and BamHI revealed genotypic heterogeneity among the serotypes and among isolates of the same serotype recovered from unrelated subjects. Diversity also existed among isolates obtained from a single subject. For 5 of 13 subjects studied, two or three genotypes within serotypes were found, while eight subjects harbored the same number of genotypes as serotypes. The data show that the method utilizing the rRNA gene probe is of value in determining the molecular epidemiology of isolates of mutans streptococci.     

Genetic diversity of carbapenem-resistant isolates of Acinetobacter baumannii in Europe.

In total, 96 carbapenem-resistant isolates of Acinetobacter baumannii were obtained from 25 hospitals in 17 European countries. Imipenem MICs ranged from <4 to 128 mg/L on retesting by Etest, with MICs > or =16 mg/L being associated with the carriage of genes encoding at least one other class D carbapenemase in addition to the intrinsic OXA-51-like enzyme. Molecular typing results obtained by random amplified polymorphic DNA analysis, followed by pulsed-field gel electrophoresis (PFGE) of ApaI-digested chromosomal DNA, were highly congruent, with 17 different PFGE types being delineated at a cut-off similarity level of 85%. With few exceptions, multiple isolates from a single hospital belonged to the same PFGE type. Seven sequence groups were identified among the 96 A. baumannii isolates, with the majority of isolates (n = 81) belonging to the previously defined sequence groups 1 and 2, which each included eight PFGE types. These two multinational lineages included the previously defined European clones II and I, respectively, but the problem of resistant A. baumannii in Europe appeared not to be confined solely to these two European clones. Rather, two broader lineages of carbapenem-resistant A. baumannii now seem to be spreading throughout Europe.     

Molecular characteristics of extended-spectrum beta-lactamases among gram-negative isolates collected in Cairo University Hospital

Phenotyping is commonly used for detection of extended-spectrum beta-lactamase (ESBL) production in gram-negative isolates. ESBLs are mainly coded for by four important genes, namely bla (TEM), bla (SHV), bla (CTX-M), and bla (OXA). Our aim in this study is to assess use of a multiplex PCR as a rapid method to identify four common genes responsible for ESBL production in different gram-negative isolates. All 793 clinical isolates are subjected to both screen and confirmatory testing for ESBL production using double disc synergy testing (DDST). Two hundred isolates with the ESBL phenotype are subjected to multiplex PCR for detection of the four genes bla (TEM, SHV, CTX-M, and OXA). The isolates were obtained from various clinical specimens: 68 (34 %) were isolated from urine cultures, 43 (21.5 %) from sputum, 26 (13 %) from wounds, 34 (17 %) from blood culture, 20 (10 %) from stool of healthy carrier and nine (4.5 %) from bronchoalveolar lavages. In this study, 83 isolates (41.5 %) were from outpatients (urine and stool specimens only), and the remaining 117 isolates (58.5) were from inpatients. By PCR technique, 181 isolates were found to be ESBL producers. blaTEM was the commonest genotype (39.2 %), followed by blaSHV (32.5 %) and blaCTX-M (30.9 %), either alone or in combination. Acinetobacter baumannii isolate had none of the ESBL genes. Eighteen (9.9 %) out of 181 isolates carried more than one type of beta-lactamase genes. Our study demonstrated rapid detection of bla (TEM, SHV, CTX-M, and OXA) in isolates belonging to Enterobacteriaceae and other nonfermenting clinical isolates using multiplex PCR. This genotypic method provided a rapid and efficient differentiation of ESBLs in the laboratory.     

Extended spectrum cephalosporin resistance among clinical isolates of Enterobacteriaceae in West Norway during 2006–2013; a prospective surveillance study

Routine surveillance of resistance to broad‐spectrum cephalosporins in Enterobacteriaceae and phenotypic identification of underlying mechanisms using a simple strategy was commenced in 2006 at our laboratory, serving West Norway. This report focuses on the results until 2013. The classical plasmid‐mediated extended spectrum beta‐lactamase (ESBL_A) among clinically relevant Escherichia coli isolates showed an increase from 0.6% to 4.3% during the surveillance period, while prevalence for other mechanisms remained stable, below 0.7%. ESBL_A in Klebsiella pneumoniae had similar prevalence in 2006 (0.6%) and 2013 (4.4%), but in between it peaked to 3.9% in 2008 and to 9.3% in 2011. Within the other species, the numbers of clinically relevant isolates and isolates‐producing ESBL_A were much lower. An increasing resistance due to hyperproduction of AmpC enzymes was seen in Enterobacter and Citrobacter, with prevalence increasing from 18% and 12.2% in 2006 to 27.5% and 26.1% in 2013, respectively. Hyperproduction of KOXY enzyme in Klebsiella oxytoca remained below 9.5% and did not show an increasing trend. The overall increase in the proportions of isolates‐producing ESBL_A in E. coli/K. pneumoniae and hyperproduction of AmpC in Enterobacter/Citrobacter necessitates measures to hinder the spread of resistant bacteria and vigilant antibiotic stewardship.