Molecular epidemiology of clinical Cryptococcus neoformans strains from India

Little is known about the molecular epidemiology of the human pathogenic fungus Cryptococcus neoformans in India, a country now in the midst of an epidemic of AIDS-related cryptococcosis. We studied 57 clinical isolates from several regions in India, of which 51 were C. neoformans var. grubii, 1 was C. neoformans var. neoformans, and 5 were C. neoformans var. gattii. This strain set included 18 additional sequential isolates from 14 patients. Strains were characterized phenotypically by measuring the polysaccharide capsule and by determining the MICs of standard antifungals. Molecular typing was performed by a PCR-based method using the minisatellite-specific core sequence (M13), by electrophoretic karyotyping, by restriction fragment length polymorphisms with the C. neoformans transposon 1 (TCN-1), and by URA5 DNA sequence analysis. Overall, Indian isolates were less heterogeneous than isolates from other regions and included a subset that clustered into one group based on URA5 DNA sequence analysis. In summary, our results demonstrate (i) differences in genetic diversity of C. neoformans isolates from India compared to isolates from other regions in the world; (ii) that DNA typing with the TCN-1 probe can adequately distinguish C. neoformans var. grubii strains; (iii) that TCN-1 sequences are absent in many C. neoformans var. gattii strains, supporting previous studies indicating that these strains have a limited geographical dispersal; and (iv) that human cryptococcal infection can be associated with microevolution of the infecting strain and by simultaneous coinfection with two distinct C. neoformans strains.     

Gene disruption in Cryptococcus neoformans and Cryptococcus gattii by in vitro transposition

Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous fungi that infect immunocompromised and immunocompetent people. We developed an insertional mutagenesis strategy for these species based on in vitro transposition and we tested the method by disrupting the URA5 gene in a strain of C. neoformans and the CAP10 gene in three strains of C. gattii. We targeted plasmid DNA containing the URA5 gene or plasmid DNA containing the CAP10 gene from genomic libraries from the shotgun sequencing project for the C. gatti strain WM276. In the latter case, the availability of the end sequences of the clones from the assembled genomic sequence allows rapid selection of target genes for disruption. Modified transposons containing the nourseothricin (NAT) or neomycin (Neo) resistance cassettes were randomly inserted into the target DNA by in vitro transposition. The disrupted genes were used for biolistic transformation and homologous integration was subsequently confirmed by PCR and Southern blot analysis. These results demonstrate that the emerging genomic resources, combined with in vitro transposition into plasmid DNAs from shotgun sequencing libraries or cloned PCR products, will facilitate high-throughput genetic analysis in Cryptococcus species.     

Differentiation of Cryptococcus neoformans varieties and Cryptococcus gattii using CAP59-based loop-mediated isothermal DNA amplification

Members of the Cryptococcus species complex (C. neoformans and C. gattii) are opportunistic pathogens responsible for frequently fatal cases of meningoencephalitis. These yeasts have been classified into five serotypes. Serotypes A andDare assigned to C. neoformans var. grubii and C. neoformans var. neoformans, respectively, Serotype AD strains are hybrids and serotype B and C strains are considered to belong to the related but distinct species C. gattii. Previous studies have identified ‘serotype-associated' alleles of several genes in the Cryptococcus species complex. We developed a loop-mediated isothermal DNA amplification method using CAP59 allele-specific primers to identify the serotypes A, D and B/C of the Cryptococcus species complex.     

Environmental prevalence of Cryptococcus neoformans and Cryptococcus gattii in India: an update

An overview of work done to-date in India on environmental prevalence, population structure, seasonal variations and antifungal susceptibility of Cryptococcus neoformans and Cryptococcus gattii is presented. The primary ecologic niche of both pathogens is decayed wood in trunk hollows of a wide spectrum of host trees, representing 18 species. Overall, C. neoformans showed a higher environmental prevalence than that of C. gattii which was not found in the avian habitats. Apart from their arboreal habitat, both species were demonstrated in soil and air in close vicinity of their tree hosts. In addition, C. neoformans showed a strong association with desiccated avian excreta. An overwhelming number of C. neoformans strains belonged to genotype AFLP1/VNI, var. grubii (serotype A), whereas C. gattii strains were genotype AFLP4/VGI, serotype B. All of the environmental strains of C. neoformans and C. gattii were mating type α (MATα). Contrary to the Australian experience, Eucalyptus trees were among the epidemiologically least important and, therefore, the hypothesis of global spread of C. gattii through Australian export of infected Eucalyptus seeds is rebutted. Reference is made to long-term colonization of an abandoned, old timber beam of sal wood (Shorea robusta) by a melanin positive (Mel(+)) variant of Cryptococcus laurentii that was pathogenic to laboratory mice.     

Molecular typing of selected Enterococcus faecalis isolates: pilot study using multilocus sequence typing and pulsed-field gel electrophoresis

The present study compared the recently developed multilocus sequence typing (MLST) approach with a well-established molecular typing technique, pulsed-field gel electrophoresis (PFGE), for subspecies differentiation of Enterococcus faecalis isolates. We sequenced intragenic regions of three E. faecalis antigen-encoding genes (ace, encoding a collagen and laminin adhesin; efaA, encoding an endocarditis antigen; and salA, encoding a cell wall associated antigen) and one housekeeping gene (pyrC) of 22 E. faecalis isolates chosen largely for their temporal and geographical diversity, but also including some outbreak isolates. MLST analysis of polymorphic regions of these four genes identified 13 distinct sequence types (STs) with different allelic profiles; the composite sequences generated from the four sequenced gene fragments of individual isolates showed 98.3 to 100% identity among the 22 isolates. We also found that the allelic profiles from two sequences, ace and salA, were sufficient to distinguish all 13 STs of this study. The 13 STs corresponded to 12 different PFGE types, with one previously designated PFGE clone (a widespread U.S. clone of beta-lactamase-producing isolates) being classified into two highly related STs which differed at 2 of 2,894 bases, both in the same allele. MLST also confirmed the clonal relationships among the isolates of two other PFGE clonal groups, including vancomycin resistant isolates. Thus, this pilot study with representative E. faecalis isolates suggests that, similar to PFGE, the sequence-based typing method may be useful for differentiating isolates of E. faecalis to the subspecies level in addition to identifying outbreak isolates.     

Molecular typing and in vitro resistance of Cryptococcus neoformans clinical isolates obtained in Germany between 2011 and 2017

Cryptococcosis is a fungal infection of the central nervous system predominantly caused by Cryptococcus neoformans in immunocompromised patients. In several countries worldwide, up to 50% of isolates show in vitro resistance to clinically used antifungals including fluconazole. No prospective data on susceptibility to antifungal drugs are available for Germany. In this study, we characterised all C. neoformans isolates collected from individual patients’ samples at the German reference laboratory for cryptococcosis 2011 and 2017 (n = 133) by multi-locus sequence typing and phenotypic drug susceptibility testing. We identified serotype A/genotype VNI isolates belonging to clonal complexes previously described from Europe, Africa, Asia and South America as the most prevalent agents of cryptococcosis in Germany. Overall, we observed minimal inhibitory concentrations (MICs) above the epidemiological cut-offs (ECVs) in 1.6% of isolates regarding fluconazole and 2.3% of isolates regarding 5-flucytosine. Here, two C. neoformans var. grubii isolates displayed decreased drug susceptibility to fluconazole, one of them additionally to 5-flucytosine. We also found 5-flucytosine MICs above the ECV for two C. neoformans var. neoformans isolates. We identified a novel mutation in the ERG11 gene which might be associated with the elevated fluconazole MIC in one of the isolates. The clinical importance of the detected in vitro resistance is documented by patient histories showing relapsed infection or primary fatal disease. Of note, sertraline demonstrated antifungal activity comparable to previous reports. Systematic collection of susceptibility data in combination with molecular typing of C. neoformans is important to comprehensively assess the spread of isolates and to understand their drug resistance patterns.     

Identification of genotypically diverse Cryptococcus neoformans and Cryptococcus gattii isolates by Luminex xMAP technology

A Luminex suspension array, which had been developed for identification of Cryptococcus neoformans and Cryptococcus gattii isolates, was tested by genotyping a set of 58 mostly clinical isolates. All genotypes of C. neoformans and C. gattii were included. In addition, cerebrospinal fluid (CSF) obtained from patients with cryptococcal meningitis was used to investigate the feasibility of the technique for identification of the infecting strain. The suspension array correctly identified haploid isolates in all cases. Furthermore, hybrid isolates possessing two alleles of the Luminex probe region could be identified as hybrids. In CSF specimens, the genotype of the cryptococcal strains responsible for infection could be identified after optimization of the PCR conditions. However, further optimization of the DNA extraction protocol is needed to enhance the usability of the method in clinical practice.     

Molecular typing of clinical Cryptococcus neoformans isolates collected in Germany from 2004 to 2010

Cryptococcosis is a fungal infection mostly caused by Cryptococcus neoformans. We identified agents of cryptococcosis diagnosed in Germany from 2004 to 2010. We used multi-locus sequence typing (MLST) to understand the molecular epidemiology of cryptococcosis. Sero- and mating types of individual patient isolates were determined by PCR. MLST was performed using the seven-locus scheme. Allele and nucleotide diversity was calculated for each locus of C. neoformans var. grubii and C. neoformans var. neoformans. Phylogenetic relations were assessed by dendrograms. Clinical data were compared between infections caused by the two variants. We studied 101 isolates. Eight were identified as hybrids (8 %). All non-hybrids were of the α mating type. Among 78 C. neoformans var. grubii (77 %), 16 sequence types (STs) were identified including three novel STs. They clustered in four groups, previously isolated in Asia, Europe or worldwide. Among 15 C. neoformans var. neoformans (15 %), 10 STs were identified, without clustering. These isolates showed higher allele, and nucleotide diversity compared with C. neoformans var. grubii. C. neoformans var. neoformans was more likely to cause soft-tissue infections (3/9, 33 vs. 1/63, 2 %, p = 0.005) and to affect non-AIDS patients (7/14, 50 vs. 15/76, 20 %, p = 0.036). C. neoformans var. grubii is the predominant agent of cryptococcosis in Germany. MLST suggests that a part of these cases are acquired abroad by immigrants or tourists. C. neoformans var. neoformans isolates represent a greater genetic diversity and are associated with more variable clinical presentations.     

Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based method for discrimination between molecular types of Cryptococcus neoformans and Cryptococcus gattii

We evaluated the usefulness of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for Cryptococcus identification at the species and subspecies levels by using an in-house database of 25 reference cryptococcal spectra. Eighty-one out of the 82 Cryptococcus isolates (72 Cryptococcus neoformans and 10 Cryptococcus gattii) tested were correctly identified with respect to their molecular type designations. We showed that MALDI-TOF MS is a practicable alternative to conventional mycology or DNA-based methods.     

Development of a Singleplex PCR Assay for Rapid Identification and Differentiation of Cryptococcus neoformans var. grubii,Cryptococcus neoformans var. neoformans,Cryptococcus gattii,and Hybrids

A singleplex PCR assay using a single primer pair targeting the putative sugar transporter gene was developed here to distinguish Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii according to the distinct size of the amplicon. The interspecies and intravarietal hybrids were also characterized on the basis of distinct combined profiles of amplicons. This PCR assay is a rapid, simple, and reliable approach suitable for laboratory diagnoses and large-scale epidemiologic studies.     

Elucidating global epidemiology of Burkholderia multivorans in cases of cystic fibrosis by multilocus sequence typing

Burkholderia multivorans is a prominent B. cepacia complex (BCC) species causing infection in people with cystic fibrosis. Despite infection control measures being introduced to reduce the spread of BCC there is a continued emergence of infections by B. multivorans. Our objective was to analyze a global collection of B. multivorans isolates, comparing those from environmental and clinical sources with those from reported outbreaks. Multilocus sequence typing (MLST) was performed on 107 B. multivorans isolates to provide a detailed analysis of the global population biology of this species. MLST resolved 64 B. multivorans sequence types. Twelve of these were globally distributed and associated with human infection; two of these (ST-21 and ST-375) were also composed of environmental isolates. These global lineages included strains previously linked to large outbreaks (e.g., French epidemic clone ST-16). Though few environmental isolates of B. multivorans were available for analysis, of six strains identified, three were identical to strains recovered from cystic fibrosis (CF) infection. Although the ability of B. multivorans to cause CF outbreaks is known, our report here concerning the existence of globally distributed B. multivorans CF strains is a new observation for this emerging B. cepacia complex pathogen and suggests that certain strain types may be better adapted to human infection than others. Common transmission-associated risk factors were not obviously linked to the globally distributed strains; however, the overlap in strains recovered from water environments, industrial products, and human infection suggests that environmental sources may be an important reservoir for infection with B. multivorans.     

Irish strains of Neisseria meningitidis: characterisation using multilocus sequence typing

A total of 56 Neisseria meningitidis strains are analysed using multilocus sequence typing (MLST). Twenty-nine distinct sequence types (STs) were identified, eight of which were new. Four known hypervirulent clones--ST-11 (electrophoretic type [ET]-37) complex, ST-44 complex (lineage 3), ST-32 (ET-5) complex and ST-8 complex (cluster A4)--were identified by MLST in 35 disease-associated and four carrier strains. Two other clones (ST-22 complex and ST-269 complex) were identified in nine disease-associated and one carrier strain. The remaining strains were heterogeneous. Additional sequencing within the FumC gene further distinguished the ET-15 clone within the ST-11 (ET-37) clonal complex. This resolution of isolates into genetic clones by MLST enhances the more traditional techniques of serotyping and serosubtyping. The data obtained established that hyperendemic meningococcal disease in Ireland could be attributed to strains belonging to four major hypervirulent clones, all of which account for elevated levels of disease worldwide. The extra information provided by MLST will be used to study the population structure and epidemiology of N. meningitidis and will allow a comparison of Irish strains with those circulating globally.     

Isolation of Cryptococcus neoformans var. gattii from Eucalyptus camaldulensis in India.

Cryptococcus neoformans var. gattii has an ecological association with five Eucalyptus species: E. blakelyi, E. camaldulensis, E. gomphocephala, E. rudis, and E. tereticornis. After human infections due to C. neoformans var. gattii were diagnosed in the states of Punjab, Himachal Pradesh, and Karnataka, India, a study was undertaken to investigate the association of C. neoformans var. gattii with Indian eucalypts, especially in the state of Punjab. A total of 696 specimens collected from E. camaldulensis, E. citriodora and E. tereticornis (hybrid) trees were examined for the presence of C. neoformans var. gattii. Flowers from two trees of E. camaldulensis in the Chak Sarkar forest and one from the village of Periana near the Ferozepur area yielded five isolates of C. neoformans var. gattii. The origin of the trees could be traced to Australia, thus providing evidence that the distribution of E. camaldulensis correlated with the distribution of human cryptococcosis cases caused by C. neoformans var. gattii in northern India.     

Molecular epidemiology of clinical Acinetobacter baumannii and Acinetobacter genomic species 13TU isolates using a multilocus sequencing typing scheme

To further expand the limited multilocus sequence typing (MLST) database for Acinetobacter baumannii , 53 clinical isolates from various outbreaks in Europe and the USA, collected between 1991 and 2004, plus the A. baumannii reference strain ATCC 19606^T and 20 clinical Acinetobacter genomic species 13TU isolates from the same period, were analyzed using a new MLST scheme based on fragments of the gltA , gyrB , gdhB , recA , cpn60 , gpi and rpoD genes. Data were compared with typing results generated using pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD)-PCR. In total, 50 sequence types (STs) were distinguished among the A. baumannii isolates investigated, and the MLST data were in high concordance with the PFGE and RAPD-PCR results. Only five clonal complexes were identified by eBURST analysis, including the 21 STs listed in a previous study, suggesting high diversity among the A. baumannii isolates. With one exception, there was no relatedness among isolates from outbreaks in different countries (Europe) or regions (USA). No intercontinental spread was revealed. Acinetobacter genomic species 13TU isolates could also be analyzed using the A. baumannii MLST scheme (18 different STs) and could be distinguished from A. baumannii isolates according to characteristic sequences. It was concluded that the MLST scheme provides a high level of resolution and is a promising tool for studying the epidemiology of A. baumannii and Acinetobacter genomic species 13TU.     

Molecular epidemiology of meningococci: Application of DNA sequence typing

Neisseria meningitidis is an invasive pathogen contributing significantly to childhood mortality worldwide. The organism is adapted to the human host and transmitted by close contact or droplet aerosols. In comparison to healthy carriage, invasive disease is a rare event. Nevertheless, due to a high case-fatality rate and the fact that meningococcal infection is a communicable disease, molecular typing of meningococci has been driven forward considerably in the past decades. Multilocus and antigen sequence typing data are assembled in large databases accessible via the internet. For epidemiological purposes, representative case ascertainment strategies are necessary if data are to be exploited for trend analysis, geographic visualization, detection of abnormalities such as outbreaks, and prediction of vaccine coverage. In Europe, a consensus for molecular typing has been achieved.     

Rapid identification of Cryptococcus neoformans and Cryptococcus gattii by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Compared to DNA sequence analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) correctly identified 100% of Cryptococcus species, distinguishing the notable pathogens Cryptococcus neoformans and C. gattii. Identification was greatly enhanced by supplementing a commercial spectral library with additional entries to account for subspecies variability.     

Experimental systemic infection with Cryptococcus neoformans var. grubii and Cryptococcus gattii in normal and immunodeficient mice.

Cryptococcus neoformans (Cn) var. grubii or Cryptococcus neoformans var. neoformans infection is usually associated with immunocompromised hosts, whereas Cryptococcusgattii more frequently causes disease in immunocompetent hosts. We examined the effects of immunodeficiency and glucocorticoid-induced immunosuppression on systemic murine infection induced by i.v. inoculation with these pathogens. SCID and immunocompetent BALB/c and C57BL/6 mice were infected with 相似文献   

Distribution of Cryptococcus gattii and Cryptococcus neoformans in decayed trunk wood of Syzygium cumini trees in north-western India.

The aim of this study is to report the regional distribution of Cryptococcus. gattii and Cryptococcus. neoformans in decayed wood inside trunk hollows of Syzygium cumini trees (Java plum, Indian black berry) investigated in Amritsar (Panjab), Meerut Cantt. and Bulandshahr (Uttar Pradesh) and Delhi, in north-western India. Two hundred and seventeen wood samples collected from 74 S. cumini trees were investigated. This includes 7 known positive S. cumini trees in Delhi subjected to a mycological surveillance for perennial colonization by C. gattii and C. neoformans. Cryptococcus gattii showed the highest prevalence (89%) in S. cumini trees in Delhi, followed by 27%, 12.5% and 9% prevalence in Bulandshahr, Amritsar City and Meerut Cantt., respectively. In contrast, C. neoformans had the highest prevalence (54%) in Amritsar, followed by 44% in Delhi, 9% in Bulandshahr and 0% in Meerut Cantt. Furthermore, 44% of the S. cumini trees in Delhi, 9% in Bulandshahr and 8% in Amritsar were concomitantly colonized by both C. gattii and C. neoformans. A mycological surveillance over 4.8-5.2 years of 7 selected S. cumini trees in Delhi revealed perennial colonization by both the Cryptococcus species. In addition, air samples taken close to the decayed trunk hollows of 4 of the perennially colonized S. cumini trees contained strains of the C. neoformans species complex. Of a random sample of 48 isolates serotyped, 26 (54%) were C. neoformans, serotype A, and 22 (46%) C. gattii, serotype B. Determination of mating type alleles was done in 44 of the isolates, comprising 31 of C. neoformans, serotype A and 13 of C.gattii, serotype B. All of them proved to be mating type alpha (MATalpha). The data on high prevalence, fungal population density, perennial colonization and aerial isolations indicate that decayed wood in trunk hollows of S. cumini trees is to-date the main well documented primary environmental niche of C. gattii and C. neoformans in north-western India. Attention is drawn to the likely health hazard posed by the environmental reservoirs of C. gattii and C. neoformans occurring in tree trunk hollows in proximity to human and animal habitations.     

Pathogenicity of Cryptococcus neoformans var. gattii in an immunocompetent mouse model.

The pathogenicity of two different genomic profiles of Cryptococcus neoformans var. gattii serotype B isolated from goats that died from cryptococcal pneumonia was assessed in an experimental model of immunocompetent mice. One strain of each randomly amplified polymorphic DNA (RAPD) profile (GR52 and GR56) and three reference C. neoformans isolates representing serotypes B, D and C were used. BALB/c male mice were inoculated by the intraperitoneal route with each strain. After 4 weeks of follow-up, the animals were sacrificed and autopsy specimens of testes, liver, spleen, kidney, lungs and brain were cultured and stained for histopathology. Although spontaneous mortality was only 2% (one animal), all mice except for those inoculated with serotype C showed positive cultures in almost one organ. The strain GR52 isolated from goat showed the highest rate of positive cultures (80%) followed by serotype D (77%). Serotype B reference strain and second goat strain GR56 were both isolated from 70% of samples. Serotype C was recovered in only 33% of organs, and never from brain or lung specimens. GR52 grew abundantly from all lung cultures, and yeast cells with large capsules were seen in histopathology inside the alveoli, peribronchial vessels and interalveolar spaces. They appeared to elicit no inflammatory response. We conclude that intraperitoneally inoculated C. neoformans var. gattii shows high virulence in this immunocompetent mouse model. Strain GR52 was highest in pathogenicity and had marked lung tropism. In contrast, the serotype C reference strain showed the lowest pathogenicity and seemed not to spread outside the abdominal viscera.     

Isolation of Cryptococcus gattii and Cryptococcus neoformans var. grubii from the flowers and bark of Eucalyptus trees in India.

The association of Cryptococcus gattii with Eucalyptus trees has been well established. Here we report the isolation of both C. gattii and Cryptococcus neoformans var. grubii from the flowers and bark of Eucalyptus trees in India. We investigated a total of 233 samples of Eucalyptus trees: 120 flowers, 81 fragments of bark, and 32 leaves. C. gattii was isolated from two samples of flowers of Eucalyptus terreticornis. C. neoformans var. grubii was recovered twice from the bark of Eucalyptus camaldulensis, initially from one of three samples, and again 2 months later, from one of four samples collected beneath the canopy of the tree. The primary isolation medium was Nigerseed agar, and brown colonies were presumptively identified as C. gattii or C. neoformans. The species identification was confirmed by morphological and biochemical characteristics. Using the Crypto-Check kit (Iatron, Tokyo, Japan), the first two isolates were identified as serotype B (C. gattii) and the other two were serotype A (C. neoformans var. grubii). PCR analysis of the isolates of C. neoformans var. grubii revealed that they possessed the MATalpha mating type allele. Molecular typing by amplified fragment length polymorphism markers indicated that both isolates of C. neoformans var. grubii possessed the same genotype. This study demonstrates that C. neoformans var. grubii, as well as C. gattii, may be associated with Eucalyptus trees.