Use of Bulk Segregant Analysis for Determining the Genetic Basis of Azole Resistance in the Opportunistic Pathogen Aspergillus fumigatus.

A sexual cycle was described in 2009 for the opportunistic fungal pathogen Aspergillus fumigatus, opening up for the first time the possibility of using techniques reliant on sexual crossing for genetic analysis. The present study was undertaken to evaluate whether the technique 'bulk segregant analysis' (BSA), which involves detection of differences between pools of progeny varying in a particular trait, could be applied in conjunction with next-generation sequencing to investigate the underlying basis of monogenic traits in A. fumigatus. Resistance to the azole antifungal itraconazole was chosen as a model, with a dedicated bioinformatic pipeline developed to allow identification of SNPs that differed between the resistant progeny pool and resistant parent compared to the sensitive progeny pool and parent. A clinical isolate exhibiting monogenic resistance to itraconazole of unknown basis was crossed to a sensitive parent and F1 progeny used in BSA. In addition, the use of backcrossing and increasing the number in progeny pools was evaluated as ways to enhance the efficiency of BSA. Use of F1 pools of 40 progeny led to the identification of 123 candidate genes with SNPs distributed over several contigs when aligned to an A1163 reference genome. Successive rounds of backcrossing enhanced the ability to identify specific genes and a genomic region, with BSA of progeny (using 40 per pool) from a third backcross identifying 46 genes with SNPs, and BSA of progeny from a sixth backcross identifying 20 genes with SNPs in a single 292 kb region of the genome. The use of an increased number of 80 progeny per pool also increased the resolution of BSA, with 29 genes demonstrating SNPs between the different sensitive and resistant groupings detected using progeny from just the second backcross with the majority of variants located on the same 292 kb region. Further bioinformatic analysis of the 292 kb region identified the presence of a cyp51A gene variant resulting in a methionine to lysine (M220K) change in the CYP51A protein, which was concluded to be the causal basis of the observed resistance to itraconazole. The future use of BSA in genetic analysis of A. fumigatus is discussed.

Profound diarrhoea and weight loss in an immunocompromised patient.

A 75-year-old man was admitted with a 3-month history of worsening diarrhoea and weight loss. He was on long-term immunosuppression following cardiac transplantation. Investigations revealed herpes simplex oesophagitis and stool samples were positive for norovirus. Treatment with acyclovir and nitazoxanide resulted in a complete resolution of symptoms. Norovirus is a common cause of infectious gastroenteritis, but immunosuppressed patients may present with chronic diarrhoea rather than an acute illness. This case highlights the importance of a low clinical threshold for testing for norovirus infection in immunocompromised patients.

Culturing and Mating of Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic human fungal pathogen, capable of causing invasive aspergillosis in patients with compromised immune systems. The fungus was long considered a purely asexual organism. However, a sexual cycle was reported in 2009, with methods described to induce mating under laboratory conditions. The presence of a sexual cycle now offers a valuable tool for classical genetic analysis of the fungus, such as allowing determination of whether traits of interest are mono- or poly-genic in nature. For example, the sexual cycle is currently being exploited to determine the genetic basis of traits of medical importance such as resistance to azole antifungals and virulence, and to characterize the genes involved. The sexual cycle can also be used to assess the possibility of gene flow between isolates. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. This unit describes protocols for culturing of A. fumigatus and for inducing sexual reproduction between compatible MAT1-1 and MAT1-2 isolates of the species. The unit also provides working methods for harvesting sexual structures, isolating single-spore progeny and confirming whether sexual recombination has occurred. © The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Repeat induction of labour for post-term pregnancy.

BACKGROUND: A previous post-term pregnancy is thought to influence the gestation of a subsequent pregnancy. Adverse maternal and perinatal outcomes are associated with post-term pregnancy and routine induction of labour by 42+0 weeks is advised to reduce these complications. OBJECTIVE: To determine the recurrence rate of a post-term pregnancy and the need for repeat induction of labour. METHODS: This observational cohort study featured women with a first and second singleton pregnancy, based on data recorded in the Aberdeen Maternity and Neonatal Databank (1986-2012). Term and post-term pregnancies were defined as ≥37+0 - 40+6 and ≥41+0 weeks, respectively. The exposure was a post-term pregnancy and the control a term pregnancy. Logistic regression was used to assess post-term recurrence and repeat induction of labour. RESULTS: The study population consisted of 25,669 women with 33% of the women delivering post-term in their first pregnancy. In these women, the rate of a subsequent post-term pregnancy was 35.7% compared to 18.6% for women with an initial term pregnancy (adjusted odds ratio (aOR) 2.27, 95% confidence interval (CI) 2.11-2.44). Compared to women who had spontaneous term deliveries in both pregnancies, women who were induced post-term in the second pregnancy had increased odds of having been induced post-term in their first pregnancy. The adjusted odds ratio was found to be 6.08 (95% CI 5.30-6.98). CONCLUSIONS: Women with a first post-term pregnancy are less likely to labour spontaneously and more likely to have a second post-term pregnancy with a higher risk of repeat induction of labour, than women who have previously delivered at term. These findings could be useful in the counselling of women with a previous post-term pregnancy. Given that these women are less likely to labour spontaneously, the offer of an elective induction could also be considered to potentially improve maternal and perinatal outcomes.