Prevalence of respiratory viruses in community-acquired pneumonia in children: a systematic review and meta-analysis.

BACKGROUND: Respiratory viruses are increasingly detected in children with community-acquired pneumonia but prevalence estimates vary substantially. We aimed to systematically review and pool estimates for 22 viruses commonly associated with community-acquired pneumonia. METHODS: We conducted a systematic review and meta-analysis to determine the prevalence of each of the common respiratory viruses detected by any diagnostic method in children aged up to 18 years with community-acquired pneumonia. We searched MEDLINE, PubMed, Embase, Web of Science, and Scopus databases with no language restrictions for relevant published articles and reports published between Jan 1, 1995, and Dec 31, 2019, restricting the review to pre-COVID-19 pandemic years. Three independent reviewers screened articles and extracted data using a predefined protocol. We calculated the pooled prevalence for each virus in childhood pneumonia using DerSimonian-Laird random-effects models. We assessed bias using the Newcastle-Ottawa Scale. The review protocol was registered in PROSPERO (CRD42016034047). FINDINGS: We identified 186 eligible articles that represented 152 209 children up to age 18 years with community-acquired pneumonia. One or more respiratory viruses were detected in 55·0% (95% CI 50·4-59·7) of paediatric patients with a diagnosis of community-acquired pneumonia; heterogeneity was high (I2=99·4%). Respiratory syncytial virus (22·7%, 20·9-24·5) and rhinovirus (22·1%, 19·5-24·7) were the most commonly detected causes of paediatric pneumonia globally, with other viruses detected in 1-9% of cases. There was non-significant variation in prevalence by the country's national income, under-5 mortality rate, or WHO region. INTERPRETATION: Respiratory viruses are frequently detected in community-acquired pneumonia among children of all ages and geographical regions, with non-significant variation by country's national income or region. Further strategies to limit antibiotic use in children with viral pneumonia and develop treatment and prevention approaches targeting common respiratory viruses are expected to have a substantial effect on the residual burden of childhood pneumonia. FUNDING: None.

Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood.

Species of Candida frequently cause life-threatening infections in neonates, transplant and intensive care unit (ICU) patients, and others with compromised host defenses. The successful management of systemic candidiasis depends upon early, rapid diagnosis. Blood cultures are the standard diagnostic method, but identification requires days and less than half of the patients are positive. These limitations may be eliminated by using real-time polymerase chain reaction (PCR) to detect Candida DNA in the blood specimens of patients at risk. Here, we optimized a PCR protocol to detect 5-10 yeasts in low volumes of simulated and clinical specimens. We also used a mouse model of systemic candidiasis and determined that candidemia is optimally detectable during the first few days after infection. However, PCR tests are often costly, labor-intensive, and inconvenient for routine use. To address these obstacles, we evaluated the innovative microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.), which has the potential for full automation and rapid turnaround. Eleven and nine of 16 specimens from individual patients with culture-proven candidemia tested positive for C. albicans DNA by conventional and microfluidic real-time PCR, respectively, for a combined sensitivity of 94%. The microfluidic platform offers a significant technical advance in the detection of microbial DNA in clinical specimens.

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.

Dynamic and heterogeneous mutations to fluconazole resistance in Cryptococcus neoformans.

Infections with the human pathogenic basidiomycetous yeast Cryptococcus neoformans are often treated with fluconazole. Resistance to this antifungal agent has been reported. This study investigated the patterns of mutation to fluconazole resistance in C. neoformans in vitro. The MIC of fluconazole was measured for 21 strains of C. neoformans. The MICs for these 21 strains differed (0.25 to 4.0 microg/ml), but the strains were selected for this study because they exhibited no growth on plates of yeast morphology agar (YMA) containing 8 microg of fluconazole per ml. To determine their mutation rates, six independent cultures from a single original colony were established for each of the 21 strains. Each culture was then spread densely on a YMA plate with 8 microg of fluconazole per ml. A random set of putative mutants was subcultured, and the MIC of fluconazole was determined for each mutant. The 21 strains evinced significant heterogeneity in their mutation rates. The MICs of the putative mutants ranged widely, from their original MIC to 64 microg of fluconazole per ml. However, for this set of 21 strains, there was no significant correlation between the original MIC for a strain and the mutation rate of that strain; the MIC for the mutant could not be predicted from the original MIC. These results suggest that dynamic and heterogeneous mutational processes are involved in generating fluconazole resistance in C. neoformans.

Multiple gene genealogies reveal recent dispersion and hybridization in the human pathogenic fungus Cryptococcus neoformans.

Cryptococcus neoformans (= Filobasidiella neoformans) is a significant emerging fungal pathogen of humans. To understand the evolution of this pathogen, 34 strains were obtained from various locations around the world and fragments of four genes were sequenced from each. These strains represented all three varieties and five serotypes. The four sequenced genes are: (i) the mitochondrial large ribosomal subunit RNA; (ii) the internal transcribed spacer region of the nuclear rRNA, including ITS1, 5.8S rRNA subunit and ITS2; (iii) orotidine monophosphate pyrophosphorylase; and (iv) diphenol oxidase. Phylogenetic analyses indicated considerable divergence among lineages, which corresponded to the current classification of C. neoformans into three varieties. However, there is no apparent phylogeographic pattern. Significant incongruences were observed among gene genealogies. The analyses indicated that the major lineages in C. neoformans diverged tens of millions of years ago but have undergone recent dispersion and hybridization.

Assessment of genetic relatedness of vaginal isolates of Candida albicans from different geographical origins.

PCR fingerprinting with single non-specific primers was used to type vaginal isolates of C. albicans from Portugal, Angola, Madagascar, and two regions of Germany (Berlin and Munich). In addition to analysing isolates that exhibited the normal biotype of C. albicans, the study included atypical strains that failed to assimilate glucosamine and N-acetylglucosamine, which were isolated from women in Angola and Madagascar. A total of 212 strains of C. albicans were studied, representing 87 different multi-locus genotypes. The genotypes of strains from each geographical population were highly similar but not identical. There was one exception: a strain from Portugal grouped with the typical strains from Angola. The typical and especially the atypical populations from Africa displayed less genotype variation than the populations from Europe. The Portuguese samples exhibited the greatest genotypic heterogeneity. Distance analysis (UPGMA) revealed a statistically weak correlation between genotype and geographical origin of the C. albicans isolates.

Overview of basic medical mycology.

To introduce the general mycologic aspects of fungal rhinosinusitis, this article reviews, in brief, the biology of fungi and the principles of fungal pathogenesis. A glossary of frequently used mycologic terms is provided. The basis of fungal classification and strategies for the diagnosis of mycotic infections are summarized. The morphologic criteria for the identification of the common etiologic agents of rhinosinusitis are presented.

Clonal and spontaneous origins of fluconazole resistance in Candida albicans.

The genotypes and susceptibilities to fluconazole of 78 strains of the human pathogenic yeast Candida albicans were compared. The strains comprised two sets of samples from Durham, N.C.: one from patients infected with the human immunodeficiency virus (HIV) and the other from healthy volunteers. For each strain, the MIC of fluconazole was determined by the standard National Committee for Clinical Laboratory Standards protocol. Genotypes were determined by PCR fingerprinting with five separate primers. The analysis revealed little evidence for genotypic clustering according to HIV status or body site. However, a small group of fluconazole-resistant strains isolated from patients infected with HIV formed a distinct cluster. In addition, two fluconazole-resistant strains were isolated from individuals who never took fluconazole, one from a patient infected with HIV and the other from a healthy person. The results suggest both clonal and spontaneous origins of fluconazole resistance in C. albicans.

Uniparental mitochondrial transmission in sexual crosses in Cryptococcus neoformans.

Restriction fragment length polymorphism (RFLP) in the large ribosomal RNA region of the mitochondrial DNA (mtDNA) was developed as a genetic marker for investigating mitochondrial transmission in sexual crosses of the human pathogenic basidiomycetous yeast Cryptococcus neoformans. Strain JEC20 of C. neoformans var. neoformans (mat a) was mated with six strains of C. neoformans var. grubii (mat alpha). Successful mating was indicated by the formation of hyphae and basidiospores. These basidiospores were examined for mtDNA RFLP genotypes. All 570 basidiospores examined from the six crosses showed the mtDNA genotype of strain JEC20. The failure to recover the C. neoformans var. grubii mtDNA in any cross indicates that the C. neoformans var. grubii mtDNA is either selectively eliminated in the newly formed dikaryon or selectively excluded in the immediate dikaryotic hyphae of the newly formed dikaryon.

Molecular typing of pathogenic fungi.

In this Round Table, the application of several methods of molecular typing were discussed in reference to four important pathogenic fungi: Coccidioides immitis, Histoplasma capsulatum, Candida albicans and Paracoccidioides brasiliensis. Among the different methods the following were discussed: restriction fragment length polymorphisms (RFLP), single nucleotide polymorphisms, random amplified polymorphic DNA (RAPD), polymerase chain reaction (PCR)-RFLP and microsatellites. By means of these methods, several important biological questions related to speciation, mode of reproduction and population genetics could be approached. The basic information obtained from this approach has implications in the understanding of these pathogenic fungi in relation to their behavior and the development of pathogenic features, such as resistance to antimicrobials and virulence factors used for colonization of mammalian hosts. The knowledge obtained from these studies could also be used for the development of innovative diagnostic methods, as well as for novel therapeutic approaches and production of vaccines.

Development and characterization of a genetic linkage map of Cryptococcus neoformans var. neoformans using amplified fragment length polymorphisms and other markers.

A segregating population of single basidiospore isolates from a sexual cross was used to generate the first moderately dense genetic linkage map of Cryptococcus neoformans var. neoformans (Serotype D). Polymorphic DNA markers were developed using amplified fragment length polymorphisms, random amplified polymorphic DNA, and gene-encoding sequences. These markers were used to analyze 100 meiotic progeny. All markers were tested for distorted segregation with a goodness of fit test. Of the total of 181 markers, 148 showed balanced (1:1) segregation ratios. Segregation distortion was observed for 33 markers. Based on all the markers, a linkage map was generated that consists of 14 major linkage groups with 127 markers, several small linkage groups, and 2 linkage groups that consist only of highly skewed markers. The genetic distance of the linkage map is 1356.3 cM. The estimated total haploid genome size for C. neoformans var. neoformans was calculated using Hulberts method and yielded a map size of 1917 cM. The number of major linkage groups correlates well with the proposed number of 13 chromosomes for C. neoformans var. neoformans. Several genes, including CAP64, CnLAC, and the mating-type locus, were mapped, and their associations were consistent with published data. To date, 6 linkage groups have been assigned to their corresponding chromosomes. This linkage map should provide a framework for the ongoing genome sequencing project and will be a useful tool for studying the genetics and pathogenicity of this important medical yeast.

[Genetic structure of geographically different populations of candida albicans]. / Untersuchungen zur genetischen Struktur von Candida-albicans-Populationen unterschiedlicher geographischer Herkunft.

Codominant single-locus markers were developed by amplifying genomic DNA of C. albicans with pairs of random primers. Monomorphic PCR products were screened for polymorphisms by the SSCP technique. Sequencing confirmed that SSCP's were mostly due to single nucleotide substitutions in the polymorphic fragments. A total of 85 polymorphic loci were observed within 13 PCR fragments. Populations from Africa displayed less genotype variation than the populations from Europe and USA. Two genetically similar African C. albicans populations exhibiting an atypical biotype were strictly clonal and perhaps represent a geographically distributed clone. Analyses of "typical" C. albicans populations of different geographical origin provided however evidence for both clonality and recombination. Evidence for clonality was supported by the absence of segregation genotypes, and by deviation of genotypic frequencies from Hardy-Weinberg expectations. Tests for nonrandom association of alleles across loci revealed less evidence for linkage disequilibrium than expected for strictly clonal populations. Although all C. albicans populations tested were primarily clonal, evidence for recombination suggests that sexual reproduction or some other form of genetic exchange occurs in this species.

Genetic structure of typical and atypical populations of Candida albicans from Africa.

Atypical isolates of the pathogenic yeast Candida albicans have been reported with increasing frequency. To investigate the origin of a set of atypical isolates and their relationship to typical isolates, we employed a combination of molecular phylogenetic and population genetic analyses using rDNA sequencing, PCR fingerprinting, and analysis of co-dominant DNA nucleotide polymorphisms to characterize the population structure of one typical and two atypical populations of C. albicans from Angola and Madagascar. The extent of clonality and recombination was assessed in each population. The analyses revealed that the structure of all three populations of C. albicans was predominantly clonal but, as in previous studies, there was also evidence for recombination. Allele frequencies differed significantly between the typical and the atypical populations, suggesting very low levels of gene flow between them. However, allele frequencies were quite similar in the two atypical C. albicans populations, suggesting that they are closely related. Phylogenetic analysis of partial sequences encoding the nuclear 26S rDNA demonstrated that all three populations belong to a single monophyletic group, which includes the type strain of C. albicans.

Species and genotypic diversities and similarities of pathogenic yeasts colonizing women.

We examined the patterns of strain relatedness among pathogenic yeasts from within and among groups of women to determine whether there were significant associations between genotype and host condition or body site. A total of 80 yeast strains were isolated, identified, and genotyped from 49 female volunteers, who were placed in three groups: (i) 19 women with AIDS, (ii) 11 pregnant women without human immunodeficiency virus (HIV) infection, and (iii) 19 women who were neither pregnant nor infected with HIV. Seven yeast species were recovered, including 59 isolates of Candida albicans, 9 isolates of Candida parapsilosis, 5 isolates of Candida krusei, 3 isolates of Candida glabrata, 2 isolates of Saccharomyces cerevisiae, and 1 isolate each of Candida tropicalis and Candida lusitaniae. Seventy unique genotypes were identified by PCR fingerprinting with the M13 core sequence and by random amplified polymorphic DNA analysis. Of the nine shared genotypes, isolates from three different hosts were of one genotype and pairs of strains from different body sites of the same host shared each of the other eight genotypes. Genetic similarities among groups of strains were calculated and compared. We found no significant difference in the patterns of relatedness of strains from the three body sites (oral cavity, vagina, and rectum), regardless of host conditions. The yeast microflora of all three host groups had similar species and genotypic diversities. Furthermore, a single host can be colonized with multiple species or multiple genotypes of the same species at the same or different body sites, indicating dynamic processes of yeast colonization on women.

Lack of genetic differentiation between two geographically diverse samples of Candida albicans isolated from patients infected with human immunodeficiency virus.

The patterns of genetic variation of samples of Candida albicans isolated from patients infected with human immunodeficiency virus in Durham, N.C., and Vitória, Brazil, were compared. Genotypes for 126 strains were obtained at 16 polymorphic restriction sites distributed on nine PCR fragments. The results indicated evidence of clonality both within and between these two geographically diverse samples. The samples are genetically very similar, with little evidence of genetic differentiation.

PCR-restriction fragment length polymorphism (RFLP) analyses reveal both extensive clonality and local genetic differences in Candida albicans.

Using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to obtain genotypes for the diploid pathogenic yeast, Candida albicans, we analysed 204 C. albicans isolates from three populations of the Duke University community: two from clinical sources [one from patients infected with human immunodeficiency virus (HIV) and the other from patients without HIV infection], and the third from healthy student volunteers. The results indicated: (i) extensive evidence for clonality within and between populations of C. albicans; and (ii) greater genotypic and gene diversities in the nonclinical population than those derived from clinical specimens, regardless of HIV status. The two clinical populations were genetically more similar to each other than either was to the population consisting of isolates from healthy people. Within each population sample there was a general lack of heterozygotes, and random associations of alleles within and between loci were found in less than 50% of the loci or pairs of loci. These findings were consistent between the two sets of samples analysed: those including all isolates and those including only clone-corrected isolates. Possible mechanisms are presented to explain the observed patterns of genetic variation within and between C. albicans populations.

Pathogenesis of Cryptococcus neoformans is associated with quantitative differences in multiple virulence factors.

Two isolates of Cryptococcus neoformans were previously described as being highly divergent in their level of capsule synthesis in vivo and in their virulence for mice. The highly virulent isolate (NU-2) produced more capsule than a weakly virulent isolate (184A) in vitro under tissue culture conditions and in vivo. This investigation was done to determine if there were differences between the two isolates in other factors that might also contribute to virulence. Growth rate was not a factor as NU-2 grew more slowly than 184A. Based on PCR fingerprinting the two isolates were genetically different providing an opportunity to examine differences in multiple virulence traits. Quantitative analysis revealed that NU-2 expressed significantly more melanin and mannitol than did 184A. Although the isolates expressed the same capsular chemotype, NU-2 produced an additional structure reporter group (SRG) under tissue culture conditions that was not present when grown in glucose salts/urea/basal medium (GSU). Capsular polysaccharide SRGs of 184A were unaffected by shifting the growth conditions from GSU to tissue culture conditions. Our results suggest that pathogenesis of a C. neoformans strain is dictated by the quantitative expression of the strain's combined virulence traits. Regulators of the expression of these genes may be playing key roles in virulence.

Colony size can be used to determine the MIC of fluconazole for pathogenic yeasts.

This report describes a new statistical method for estimating the MIC of fluconazole for yeasts pathogenic to humans. This method is based on comparison of the colony sizes on solid media containing different concentrations of fluconazole. By this method, the MICs of fluconazole for 10 yeast strains were comparable to results obtained by the standard method recommended by the National Committee for Clinical Laboratory Standards. This method is simple to perform and easy to interpret. The turnaround time is faster than other methods. The method should be applicable to the determination MICs of other antifungal drugs for yeasts.

Comparison of use of phenotypic and genotypic characteristics for identification of species of the anamorph genus Candida and related teleomorph yeast species.

A total of 49 type and neotype isolates and 32 clinical isolates of the anamorph genus Candida and related teleomorph genera were obtained from different culture collections and clinical laboratories. Isolates were subjected to two phenotypic methods of identification, Vitek yeast biochemical card (YBC) and API ID 32C, both based on carbohydrate assimilation, and one genotypic method, PCR fingerprinting, based on the detection of DNA polymorphisms between minisatellite-specific sequences with the primer M13 (5' GAGGGTGGCGGTTCT 3'). The correct identification of a strain at the Centraalbureau voor Schimmelcultures was used as the gold standard for the identification of an isolate. When the study was restricted to species included in the respective biochemical databases, the Vitek YBC and API ID 32C systems performed adequately with positive identification rates of 87.3 and 76.8%, respectively. When uncommon species were added to the study, several of which are not included in the databases, the identification efficiencies were 76.5 and 77.5%, respectively. By comparison, all isolates were correctly identified by PCR fingerprinting, with 63 reference species profiles in the databank. Sufficient polymorphisms among the total set of banding patterns were observed, with adequate similarity in the major patterns obtained from a given species, to allow each isolate to be assigned unambiguously to a particular species. In addition, variations in minor bands allowed for differentiation to the strain level. PCR fingerprinting was found to be rapid, reproducible, and more cost-effective than either biochemical approach. Our results provide reference laboratories with an improved identification method for yeasts based on genotypic rather than phenotypic markers.

Induction of interleukin-6 mRNA in rat alveolar macrophages by in vitro exposure to both Cryptococcus neoformans and anti-C. neoformans antiserum.

Lewis rat alveolar macrophages (AM) were harvested and exposed in vitro to Cryptococcus neoformans to investigate the induction of inflammatory cytokines. AM in tissue culture wells were incubated with viable yeast cells of C. neoformans or the capsular polysaccharide, glucuronoxylomannan (GXM), with or without rabbit anti-C. neoformans antiserum. At 3, 6, 12 and 24 h, AM were washed, lyzed and total RNA was isolated. Using reverse transcription-PCR, the transcripts of cytokine genes were semi-quantified by comparison with constitutive transcripts. Incubation of AM with lipopolysaccharide, as positive control, induced elevated levels of the three transcripts measured: interleukin (IL)-1 alpha, IL-6 and tumour necrosis factor (TNF)-alpha. Under the same conditions, no obvious changes were observed in the levels of transcription of these cytokines by AM after exposure to several strains of C. neoformans. However, AM that were incubated with both the yeast cells and rabbit polyclonal antisera to C. neoformans manifested significantly increased levels of mRNA for IL-6, but not IL-1 alpha or TNF-alpha. This increased level of IL-6 mRNA was detectable after incubation for 6 or 12 h. Levels of transcription in AM were unaffected by exposure to normal rabbit serum, specific antiserum alone. GXM at concentrations of 10, 100 or 500 micrograms ml-1, or GXM and antiserum. Adsorption of the antiserum with heat-killed yeast cells of C. neoformans diminished its ability to induce IL-6 mRNA in combination with fresh, viable yeast cells. The induction of IL-6 mRNA by yeast cells and antiserum does not require intact complement. In the absence of complement, the rabbit antiserum served as a potent opsonin and markedly increased phagocytosis of C. neoformans by AM. These results indicate that antibody-opsonized C. neoformans are readily phagocytosed by rat AM, and that antibody-mediated phagocytosis may differ from complement-mediated phagocytosis in the subsequent stimulation of IL-6.