Genomic characterisation of invasive non-typhoidal Salmonella enterica Subspecies enterica Serovar Bovismorbificans isolates from Malawi.

BACKGROUND: Invasive Non-typhoidal Salmonella (iNTS) are an important cause of bacteraemia in children and HIV-infected adults in sub-Saharan Africa. Previous research has shown that iNTS strains exhibit a pattern of gene loss that resembles that of host adapted serovars such as Salmonella Typhi and Paratyphi A. Salmonella enterica serovar Bovismorbificans was a common serovar in Malawi between 1997 and 2004. METHODOLOGY: We sequenced the genomes of 14 Malawian bacteraemia and four veterinary isolates from the UK, to identify genomic variations and signs of host adaptation in the Malawian strains. PRINCIPAL FINDINGS: Whole genome phylogeny of invasive and veterinary S. Bovismorbificans isolates showed that the isolates are highly related, belonging to the most common international S. Bovismorbificans Sequence Type, ST142, in contrast to the findings for S. Typhimurium, where a distinct Sequence Type, ST313, is associated with invasive disease in sub-Saharan Africa. Although genome degradation through pseudogene formation was observed in ST142 isolates, there were no clear overlaps with the patterns of gene loss seen in iNTS ST313 isolates previously described from Malawi, and no clear distinction between S. Bovismorbificans isolates from Malawi and the UK. The only defining differences between S. Bovismorbificans bacteraemia and veterinary isolates were prophage-related regions and the carriage of a S. Bovismorbificans virulence plasmid (pVIRBov). CONCLUSIONS: iNTS S. Bovismorbificans isolates, unlike iNTS S. Typhiumrium isolates, are only distinguished from those circulating elsewhere by differences in the mobile genome. It is likely that these strains have entered a susceptible population and are able to take advantage of this niche. There are tentative signs of convergent evolution to a more human adapted iNTS variant. Considering its importance in causing disease in this region, S. Bovismorbificans may be at the beginning of this process, providing a reference against which to compare changes that may become fixed in future lineages in sub-Saharan Africa.

Widespread pyocyanin over-production among isolates of a cystic fibrosis epidemic strain.

BACKGROUND: Some isolates of the Liverpool cystic fibrosis epidemic strain of Pseudomonas aeruginosa exhibit an unusual virulence-related phenotype, characterized by over-production of quorum sensing-regulated exoproducts such as pyocyanin and LasA protease. Our aim was to determine the prevalence of this unusual phenotype amongst isolates of the epidemic strain, and to study other intraclonal phenotypic and genotypic variations. RESULTS: The unusual phenotype was detected in at least one epidemic strain isolate from the majority of cystic fibrosis patients tested, and can be retained for up to seven years during chronic infection. Multiple sequential isolates of the epidemic strain taken from six patients over a period of up to nine years exhibited a wide range of phenotypes, including different antimicrobial susceptibilities. Our data suggest that each sputum sample contains a mixture of phenotypes and genotypes within the epidemic strain population, including within colony morphotypes. Many isolates exhibit premature (during early rather than late exponential growth) and over-production of pyocyanin, which has a number of toxic effects directly relevant to cystic fibrosis. CONCLUSION: The widespread occurrence of this unusual phenotype suggests that it may play an important role in the success of the epidemic strain.

Predominance of rotavirus P[4]G2 in a vaccinated population, Brazil.

We identified 21 rotaviruses in 129 patients with diarrhea in a Brazilian city with high rotavirus vaccine coverage. All rotaviruses were genotype P[4]G2 with 1 mixed infection with P[NT]G9. Although virus predominance could have occurred randomly, the vaccine may be less protective against P[4]G2. Prospective surveillance is urgently needed.

Characterisation of community acquired non-typhoidal Salmonella from bacteraemia and diarrhoeal infections in children admitted to hospital in Nairobi, Kenya.

BACKGROUND: In sub-Saharan Africa community-acquired non-typhoidal Salmonella (NTS) is a major cause of high morbidity and death among children under 5 years of age especially from resource poor settings. The emergence of multidrug resistance is a major challenge in treatment of life threatening invasive NTS infections in these settings. RESULTS: Overall 170 (51.2%) of children presented with bacteraemia alone, 28 (8.4%) with gastroenteritis and bacteraemia and 134 (40.4%) with gastroenteritis alone. NTS serotypes obtained from all the cases included S. Typhimurium (196; 59%), S. Enteritidis (94; 28.3%) and other serotypes in smaller numbers (42; 12.7%); distribution of these serotypes among cases with bacteremia or gastroenteritis was not significantly different. A significantly higher proportion of younger children (< 3 years of age) and those from the slums presented with invasive NTS compared to older children and those from upper socio-economic groups (p < 0.001). One hundred and forty-seven (44.3%) NTS were resistant to 3 or more antibiotics, and out of these 59% were resistant to ampicillin, chloramphenicol and tetracycline. There was no significant difference in antibiotic resistance between the two serotypes, S. Typhimurium and S. Enteritidis. Ceftriaxone and ciprofloxacin were the only antibiotics tested to which all the NTS were fully susceptible. Using Pulsed Field Gel Electrophoresis (PFGE) there were 3 main patterns of S. Typhimurium and 2 main patterns of S. Enteritidis among cases of bacteraemia and gastroenteritis. CONCLUSION: Serotype distribution, antibiotic susceptibility and PFGE patterns of NTS causing bacteraemia and gastroenteritis did not differ significantly. The high prevalence of NTS strains resistant to most of the commonly used antimicrobials is of major public health concern.

Human metapneumovirus and respiratory syncytial virus disease in children, Yemen.

Factors increasing the severity of respiratory infections in developing countries are poorly described. We report factors associated with severe acute respiratory illness in Yemeni children (266 infected with respiratory syncytial virus and 66 with human metapneumovirus). Age, indoor air pollution, and incomplete vaccinations were risk factors and differed from those in industrialized countries.

Cryptosporidiosis: prevalence, genotype analysis, and symptoms associated with infections in children in Kenya.

Cryptosporidium parasites are leading causes of enteric disease, especially in children. A prospective survey on the prevalence of cryptosporidiosis in children less than five years of age was undertaken at six microbiology laboratories in Kenya on fecal samples submitted for routine parasite and ova investigations. Analysis of 4,899 samples over a two-year study period showed an overall prevalence of cryptosporidiosis of 4% that was highest between November to February. Investigations on the nature of enteric diseases prompting ova and cyst examination requests showed 66.4% had acute diarrhea, 9% had persistent diarrhea, and 21% had recurrent diarrhea. The main symptoms were abdominal pain (51.1%), vomiting (51.6%), and abdominal swelling (11%). The prevalence of cryptosporidiosis was highest among children 13-24 months of age (5.2%) and least among those 48-60 months of age (2%). No significant differences were observed by sex but vomiting was slightly higher in males than in females (65% males and 52% females; P = 0.07). Cryptosporidiosis was significantly associated with persistent diarrhea (P = 0.0001, odds ratio [OR] = 2.193, 95% confidence interval [CI] = 1.463-3.29), vomiting (P = 0.0273, OR = 1.401, 95% CI = 1.04-1.893), and abdominal swelling (P = 0.0311, OR = 1.56, 95% CI = 1.04-2.34). Genotype analysis based on polymerase chain reaction-restriction fragment length polymorphism of the 18S rRNA gene fragment showed that 87% (153 of 175) of the Cryptosporidium isolates were C. hominis, 9% (15 of 175) were C. parvum, and remaining 4% were C. canis, C. felis, C. meleagridis, and C. muris. The most common protozoa in coinfected patients were Entamoeba histolytica/E. dispar, E. coli, and Giardia intestinalis (6%, 5%, and 2%, respectively). Our results show that Cryptosporidium is among the most common protozoan parasites in children with enteric diseases and that anthroponotic species are the leading cause of human cryptosporidiosis in Kenya, which suggests that human-to-human transmission is the main mode of spread.

Evidence for persistence of adenovirus in the tear film a decade following conjunctivitis.

Chronic papillary conjunctivitis has been described following adenoviral conjunctivitis. It is unknown however, how long adenovirus is able to persist in the tear film and conjunctiva. To determine if adenovirus persists in the ocular surface following adenoviral conjunctivitis, 304 patients with a history of adenovirus conjunctivitis from whom an adenovirus had been isolated 10 years previously were sent a questionnaire regarding persistent or recurrent symptoms and were invited to attend. Patients were examined and samples of tears and conjunctival cells were collected from both eyes using tear film washes, filter paper, and swabs, the latter for virus isolation. Extracted DNA from the ocular samples was amplified using primers for herpes simplex virus (thymidine kinase) and adenovirus (hexon) genes. Adenovirus amplicons were sequenced and compared to original serotype. Thirty patients attended, 19 of whom had persistent papillary conjunctivitis. Evidence of adenovirus DNA was detected in 17 of 30 patients, 15 of whom also had evidence of a chronic papillary conjunctivitis. Adenovirus DNA was significantly associated with papillary conjunctivitis (P = 0.03). Adenovirus amplicons were successfully sequenced from six patients. Four patients harbored type 3 adenovirus, the same serotype with which they were infected originally 10 years previously. Two patients were infected originally with adenovirus serotype 3 but the current serotype was type 4. Infection of the ocular surface with adenovirus may predispose to the development of a persistent or recurrent conjunctivitis, the presence of which, appears to be associated with evidence of long term persistence of adenovirus DNA.

Suppression of thrombospondin 1 and 2 production by herpes simplex virus 1 infection in cultured keratocytes.

PURPOSE: Stromal vascularization is a frequent occurrence in herpes simplex keratitis (HSK) and carries a poor prognosis for penetrating keratoplasty. The pathogenesis may involve disruption of the normal equilibrium between angiogenic and anti-angiogenic factors in and around the cornea. Thrombospondin (TSP) 1 and 2 are multifunctional matricellular glycoproteins with potent anti-angiogenic properties and are expressed by human keratocytes in a stromal wound repair model. We hypothesize that the synthesis of these anti-angiogenic proteins by keratocytes is inhibited by HSV1 and that such a mechanism may contribute to stromal vascularization in HSK. METHODS: Nonconfluent monolayers of human keratocytes were infected with HSV1 at a multiplicity of infection of 5 virus particles/keratocyte. Expression of TSP1 and TSP2 was determined by immunohistochemistry and SDS-polyacrylamide gel electrophoresis at 0, 2, 4, 6, 8, 24, 48, and 72 h after infection (ai). Expression of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) served as a control. Expression of immediate early and late viral proteins was also determined. Protein expression was quantified by densitometric analysis of the immunoblot bands. RESULTS: Human keratocytes supported the growth of HSV1 at all times ai. TSP1 and TSP2 were downregulated as early as 4 h ai to a 50% reduction by 8 h (p<0.002), and were absent from 24 h ai (p<0.001). There was no change in the level of expression of GAPDH throughout the duration of the experiment. Immediate early viral proteins (HSV1:ICP27) could be detected from 6 h ai reaching maximum intensity 24 h ai and late proteins (HSV:1gD) were expressed from 24 h. CONCLUSIONS: The synthesis of TSP1 and TSP2 is selectively downregulated by HSV1 infection in human keratocytes. Addition of these proteins or their angio-active peptides in early stage HSK therapy may be an important adjuvant in controlling HSV1 induced corneal vascularization.

A randomized double-blinded placebo-controlled crossover trial of nebulized taurolidine in adult cystic fibrosis patients infected with Burkholderia cepacia.

Burkholderia cepacia is an aggressive pathogen that colonizes cystic fibrosis (CF) patients, causing greatly increased morbidity and mortality. It is resistant to most antibiotics, but sensitive in vitro to a novel agent, taurolidine. This has not previously been used against B. cepacia, nor given in nebulized form. We assessed the effect of nebulized taurolidine on United Kingdom epidemic (ET12) B. cepacia infection in 20 adult CF patients attending our regional adult cystic fibrosis outpatient clinic using a prospective, randomized, double-blinded placebo-controlled crossover trial. Nebulized taurolidine (4 mL 2% solution) or saline (4 mL 0.9% solution) was given twice daily. Each arm lasted 4 weeks, with a 2-week intervening washout period. Sputum B. cepacia colony counts (primary outcome measure), spirometry, and symptoms (secondary outcome measures) were assessed. Eighteen patients completed the study. There was no change in B. cepacia colony counts or spirometry, nor symptom scores. We conclude that, although taurolidine is well tolerated in nebulized form, in this study it had no in vivo anti-B. cepacia activity.