Genomic epidemiology of Cryptococcus yeasts identifies adaptation to environmental niches underpinning infection across an African HIV/AIDS cohort

Emerging infections caused by fungi have become a widely recognized global phenomenon and are causing an increasing burden of disease. Genomic techniques are providing new insights into the structure of fungal populations, revealing hitherto undescribed fine-scale adaptations to environments and hosts that govern their emergence as infections. Cryptococcal meningitis is a neglected tropical disease that is responsible for a large proportion of AIDS-related deaths across Africa; however, the ecological determinants that underlie a patient's risk of infection remain largely unexplored. Here, we use genome sequencing and ecological genomics to decipher the evolutionary ecology of the aetiological agents of cryptococcal meningitis, Cryptococcus neoformans and Cryptococcus gattii, across the central African country of Zambia. We show that the occurrence of these two pathogens is differentially associated with biotic (macroecological) and abiotic (physical) factors across two key African ecoregions, Central Miombo woodlands and Zambezi Mopane woodlands. We show that speciation of Cryptococcus has resulted in adaptation to occupy different ecological niches, with C. neoformans found to occupy Zambezi Mopane woodlands and C. gattii primarily recovered from Central Miombo woodlands. Genome sequencing shows that C. neoformans causes 95% of human infections in this region, of which over three-quarters belonged to the globalized lineage VNI. We show that VNI infections are largely associated with urbanized populations in Zambia. Conversely, the majority of C. neoformans isolates recovered in the environment belong to the genetically diverse African-endemic lineage VNB, and we show hitherto unmapped levels of genomic diversity within this lineage. Our results reveal the complex evolutionary ecology that underpins the reservoirs of infection for this, and likely other, deadly pathogenic fungi

The Diagnostic Utility of Induced Sputum Microscopy and Culture in Childhood Pneumonia.

BACKGROUND.: Sputum microscopy and culture are commonly used for diagnosing the cause of pneumonia in adults but are rarely performed in children due to difficulties in obtaining specimens. Induced sputum is occasionally used to investigate lower respiratory infections in children but has not been widely used in pneumonia etiology studies. METHODS.: We evaluated the diagnostic utility of induced sputum microscopy and culture in patients enrolled in the Pneumonia Etiology Research for Child Health (PERCH) study, a large study of community-acquired pneumonia in children aged 1-59 months. Comparisons were made between induced sputum samples from hospitalized children with radiographically confirmed pneumonia and children categorized as nonpneumonia (due to the absence of prespecified clinical and laboratory signs and absence of infiltrate on chest radiograph). RESULTS.: One induced sputum sample was available for analysis from 3772 (89.1%) of 4232 suspected pneumonia cases enrolled in PERCH. Of these, sputum from 2608 (69.1%) met the quality criterion of <10 squamous epithelial cells per low-power field, and 1162 (44.6%) had radiographic pneumonia. Induced sputum microscopy and culture results were not associated with radiographic pneumonia, regardless of prior antibiotic use, stratification by specific bacteria, or interpretative criteria used. CONCLUSIONS.: The findings of this study do not support the culture of induced sputum specimens as a diagnostic tool for pneumonia in young children as part of routine clinical practice

Standardization of Laboratory Methods for the PERCH Study.

The Pneumonia Etiology Research for Child Health study was conducted across 7 diverse research sites and relied on standardized clinical and laboratory methods for the accurate and meaningful interpretation of pneumonia etiology data. Blood, respiratory specimens, and urine were collected from children aged 1-59 months hospitalized with severe or very severe pneumonia and community controls of the same age without severe pneumonia and were tested with an extensive array of laboratory diagnostic tests. A standardized testing algorithm and standard operating procedures were applied across all study sites. Site laboratories received uniform training, equipment, and reagents for core testing methods. Standardization was further assured by routine teleconferences, in-person meetings, site monitoring visits, and internal and external quality assurance testing. Targeted confirmatory testing and testing by specialized assays were done at a central reference laboratory

Microscopic Analysis and Quality Assessment of Induced Sputum From Children With Pneumonia in the PERCH Study.

BACKGROUND.: It is standard practice for laboratories to assess the cellular quality of expectorated sputum specimens to check that they originated from the lower respiratory tract. The presence of low numbers of squamous epithelial cells (SECs) and high numbers of polymorphonuclear (PMN) cells are regarded as indicative of a lower respiratory tract specimen. However, these quality ratings have never been evaluated for induced sputum specimens from children with suspected pneumonia. METHODS.: We evaluated induced sputum Gram stain smears and cultures from hospitalized children aged 1-59 months enrolled in a large study of community-acquired pneumonia. We hypothesized that a specimen representative of the lower respiratory tract will contain smaller quantities of oropharyngeal flora and be more likely to have a predominance of potential pathogens compared to a specimen containing mainly saliva. The prevalence of potential pathogens cultured from induced sputum specimens and quantity of oropharyngeal flora were compared for different quantities of SECs and PMNs. RESULTS.: Of 3772 induced sputum specimens, 2608 (69%) had 25 PMNs per LPF, measures traditionally associated with specimens from the lower respiratory tract in adults. Using isolation of low quantities of oropharyngeal flora and higher prevalence of potential pathogens as markers of higher quality, 25 PMNs per LPF) was the microscopic variable most associated with high quality of induced sputum. CONCLUSIONS.: Quantity of SECs may be a useful quality measure of induced sputum from young children with pneumonia

Detection of Pneumococcal DNA in Blood by Polymerase Chain Reaction for Diagnosing Pneumococcal Pneumonia in Young Children From Low- and Middle-Income Countries.

BACKGROUND.: We investigated the performance of polymerase chain reaction (PCR) on blood in the diagnosis of pneumococcal pneumonia among children from 7 low- and middle-income countries. METHODS.: We tested blood by PCR for the pneumococcal autolysin gene in children aged 1-59 months in the Pneumonia Etiology Research for Child Health (PERCH) study. Children had World Health Organization-defined severe or very severe pneumonia or were age-frequency-matched community controls. Additionally, we tested blood from general pediatric admissions in Kilifi, Kenya, a PERCH site. The proportion PCR-positive was compared among cases with microbiologically confirmed pneumococcal pneumonia (MCPP), cases without a confirmed bacterial infection (nonconfirmed), cases confirmed for nonpneumococcal bacteria, and controls. RESULTS.: In PERCH, 7.3% (n = 291/3995) of cases and 5.5% (n = 273/4987) of controls were blood pneumococcal PCR-positive (P < .001), compared with 64.3% (n = 36/56) of MCPP cases and 6.3% (n = 243/3832) of nonconfirmed cases (P < .001). Blood pneumococcal PCR positivity was higher in children from the 5 African countries (5.5%-11.5% among cases and 5.3%-10.2% among controls) than from the 2 Asian countries (1.3% and 1.0% among cases and 0.8% and 0.8% among controls). Among Kilifi general pediatric admissions, 3.9% (n = 274/6968) were PCR-positive, including 61.7% (n = 37/60) of those with positive blood cultures for pneumococcus. DISCUSSION.: The utility of pneumococcal PCR on blood for diagnosing childhood pneumococcal pneumonia in the 7 low- and middle-income countries studied is limited by poor specificity and by poor sensitivity among MCPP cases

Genome-wide identification of potato long intergenic noncoding RNAs responsive to <i>Pectobacterium carotovorum</i> subspecies <i>brasiliense</i> infection

BACKGROUND: Long noncoding RNAs (lncRNAs) represent a class of RNA molecules that are implicated in regulation of gene expression in both mammals and plants. While much progress has been made in determining the biological functions of lncRNAs in mammals, the functional roles of lncRNAs in plants are still poorly understood. Specifically, the roles of long intergenic nocoding RNAs (lincRNAs) in plant defence responses are yet to be fully explored. RESULTS: In this study, we used strand-specific RNA sequencing to identify 1113 lincRNAs in potato (Solanum tuberosum) from stem tissues. The lincRNAs are expressed from all 12 potato chromosomes and generally smaller in size compared to protein-coding genes. Like in other plants, most potato lincRNAs possess single exons. A time-course RNA-seq analysis between a tolerant and a susceptible potato cultivar showed that 559 lincRNAs are responsive to Pectobacterium carotovorum subsp. brasiliense challenge compared to mock-inoculated controls. Moreover, coexpression analysis revealed that 17 of these lincRNAs are highly associated with 12 potato defence-related genes. CONCLUSIONS: Together, these results suggest that lincRNAs have potential functional roles in potato defence responses. Furthermore, this work provides the first library of potato lincRNAs and a set of novel lincRNAs implicated in potato defences against P. carotovorum subsp. brasiliense, a member of the soft rot Enterobacteriaceae phytopathogens. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2967-9) contains supplementary material, which is available to authorized users

Discovery and profiling of small RNAs responsive to stress conditions in the plant pathogen <i>Pectobacterium atrosepticum</i>

BACKGROUND: Small RNAs (sRNAs) have emerged as important regulatory molecules and have been studied in several bacteria. However, to date, there have been no whole-transcriptome studies on sRNAs in any of the Soft Rot Enterobacteriaceae (SRE) group of pathogens. Although the main ecological niches for these pathogens are plants, a significant part of their life cycle is undertaken outside their host within adverse soil environment. However, the mechanisms of SRE adaptation to this harsh nutrient-deficient environment are poorly understood. RESULTS: In the study reported herein, by using strand-specific RNA-seq analysis and in silico sRNA predictions, we describe the sRNA pool of Pectobacterium atrosepticum and reveal numerous sRNA candidates, including those that are induced during starvation-activated stress responses. Consequently, strand-specific RNA-seq enabled detection of 137 sRNAs and sRNA candidates under starvation conditions; 25 of these sRNAs were predicted for this bacterium in silico. Functional annotations were computationally assigned to 68 sRNAs. The expression of sRNAs in P. atrosepticum was compared under growth-promoting and starvation conditions: 68 sRNAs were differentially expressed with 47 sRNAs up-regulated under nutrient-deficient conditions. Conservation analysis using BLAST showed that most of the identified sRNAs are conserved within the SRE. Subsequently, we identified 9 novel sRNAs within the P. atrosepticum genome. CONCLUSIONS: Since many of the identified sRNAs are starvation-induced, the results of our study suggests that sRNAs play key roles in bacterial adaptive response. Finally, this work provides a basis for future experimental characterization and validation of sRNAs in plant pathogens. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2376-0) contains supplementary material, which is available to authorized users

A mini-review on household solid waste management systems in low-income developing countries: A case study of urban Harare City, Zimbabwe

Among other African cities, in terms of solid waste management (SWM), Harare has been ranked as one of the poorest. The municipality struggles to efficiently provide SWM services to its residents. Considering that SWM systems are complex, the individual behaviour of its components (waste generation, collection, and disposal) determines the overall performance of the system. This mini-review thus aims to understand the management system of the urban city’s biggest source of solid waste which is household solid waste (HSW). The systematic literature review methodology using NVIVO was used to analyse the literature. Out of a selected sample size of 500 journal articles, 26 were selected based on their relevance to the study. The uncertainty of the data provided by the Harare City Council (HCC) and of HSW generation data harvested in the literature was calculated. According to the HCC data records, Harare has an average daily waste generation per capita of on average 0.38±0.1 kg producing about 207, 635, 294±56, 027, 040 kg of HSW generated annually. With the waste collection efficiency in Harare at about 72.4%±7.5%, the city collects approximately 170, 385, 600±33 384 209 kg of HSW annually. All the collected HSW is disposed of in the city’s major MSW dumpsite called Pomona and only about 23, 498, 400±3, 988, 817 kg MSW is recovered annually with the average recovery efficiency at around 9.5%±2.8%, where most of the waste recovery is done by the waste pickers

Identifying the Leverage Points in the Household Solid Waste Management System for Harare, Zimbabwe, Using Network Analysis Techniques

Managing household solid waste (HSW) has gone beyond what the Harare local government can handle. Inadequate knowledge of the interactions existing between issues that affect the efficient running of waste management systems is one of the major hindrances in waste management planning in developing countries like Zimbabwe. The complexity of the waste management system for a given municipal area needs to be identified and understood to generate appropriate and efficient waste management strategies. Network analysis (NA) is a methodology extensively used in research to help reveal a comprehensive picture of the relationships and factors related to a particular phenomenon. The methodology reduces the intricacy of large systems such as waste management to smaller and more understandable structures. In this study, NA, which was done mainly using the R software environment, showed a result of 1.5% for network density, thus signifying that for Harare, waste management strategies need to be ‘seeded’ in various parts of the system. The Pareto principle and the 3Rs (Reduce, Reuse, Recycle) concept were applied to suggest the issues to prioritize and generate strategies that could potentially affect significant change to the city’s waste management system. The key issues identified, in their order of importance, are an increase in uncollected waste, low waste collection efficiency, increase in illegal waste dumping, the deteriorating country’s economy, reduced municipal financial capacity, reduced municipal workforce capacity, inadequate or unreliable waste data, increase in waste volume, increase in the number of street vendors, no waste planning and monitoring unit, no engineered landfills in the city, increase in waste collection pressure, low waste collection frequency, increase in the unemployment rate, reduced municipal technical capacity, few waste collection vehicles, limited vehicles maintenance, distinct socio-economic classes, high vehicles breakdown, and increase in population

Global gene expression analysis of cross-protected phenotype of pectobacterium atrosepticum

© 2017 Gorshkov et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The ability to adapt to adverse conditions permits many bacterial species to be virtually ubiquitous and survive in a variety of ecological niches. This ability is of particular importance for many plant pathogenic bacteria that should be able to exist, except for their host plants, in different environments e.g. soil, water, insect-vectors etc. Under some of these conditions, bacteria encounter absence of nutrients and persist, acquiring new properties related to resistance to a variety of stress factors (cross-protection). Although many studies describe the phenomenon of cross-protection and several regulatory components that induce the formation of resistant cells were elucidated, the global comparison of the physiology of cross-protected phenotype and growing cells has not been performed. In our study, we took advantage of RNA-Seq technology to gain better insights into the physiology of cross-protected cells on the example of a harmful phytopathogen, Pectobacterium atrosepticum (Pba) that causes crop losses all over the world. The success of this bacterium in plant colonization is related to both its virulence potential and ability to persist effectively under various stress conditions (including nutrient deprivation) retaining the ability to infect plants afterwards. In our previous studies, we showed Pba to be advanced in applying different adaptive strategies that led to manifestation of cell resistance to multiple stress factors. In the present study, we determined the period necessary for the formation of cross-protected Pba phenotype under starvation conditions, and compare the transcriptome profiles of non-adapted growing cells and of adapted cells after the cross-protective effect has reached the maximal level. The obtained data were verified using qRT-PCR. Genes that were expressed differentially (DEGs) in two cell types were classified into functional groups and categories using different approaches. As a result, we portrayed physiological features that distinguish cross-protected phenotype from the growing cells