Pneumocystis jirovecii genetic diversity in a Spanish tertiary hospital.

Pneumocystis jirovecii is associated with non-noxious colonization or severe pneumonia in immunocompromised hosts. Epidemiological investigations have been hampered by the lack of a standardized typing scheme. Thus, only partial molecular data on Spanish P. jirovecii cases are available. Recently, a new ISHAM consensus multilocus sequence typing scheme (MLST) targeting ß-TUB, mt26S, CYB, and SOD with a publicly accessible database has been launched to overcome this problem. The molecular epidemiology of P. jirovecii from immunocompromised patients either colonized (n = 50) or having pneumonia (n = 36) seen between 2014 and 2018 at a single center in Barcelona, Spain, was studied. The new ISHAM consensus MSLT scheme was used to investigate the local epidemiology and identify possible unnoticed outbreaks. Mutations in the DHPS gene, not included in the scheme but giving information about potential sulfa treatment failure, were also studied. The study assigned 32 sequence types (ST) to 72.2% pneumonia and 56% colonization cases. The most frequent STs were ST21 (18.5%), ST22 (14.8%), and ST37(14.8%). For non-unique STs, ST3, ST30 and ST31 were found only in pneumonia cases, whereas ST27 was associated exclusively to colonizations. Despite 38 patients sharing similar STs, only two were involved in a potential cross transmission event. No DHPS mutations were identified. The new consensus typing scheme was useful to ascertain the molecular epidemiology of P. jirovecii in our center revealing a high genetic diversity and the potential association of specific STs to colonization and pneumonia cases. LAY SUMMARY: A newly described MLST scheme aims at providing a standardized tool to study and compare Pneumocystis jirovecii epidemiology. A high diversity among P. jirovecii isolates from patients in Barcelona, Spain, and a potential association between specific STs and infection/colonization were identified.

Prevalence of cryptococcal antigenemia and nasal colonization in a free-ranging koala population.

Cryptococcosis, caused by environmental fungi in the Cryptococcus neoformans and Cryptococcus gattii species complexes, affects a variety of hosts, including koalas (Phascolarctos cinereus). Cryptococcal antigenemia and nasal colonization are well characterized in captive koalas, but free-ranging populations have not been studied systematically. Free-ranging koalas (181) from the Liverpool Plains region of New South Wales, Australia, were tested for cryptococcal antigenemia (lateral flow immunoassay) and nasal colonization (bird seed agar culture). Results were related to environmental and individual koala characteristics. Eucalypt trees (14) were also randomly tested for the presence of Cryptococcus spp. by bird seed agar culture. In sum, 5.5% (10/181) and 6.6% (12/181) of koalas were positive for antigenemia and nasal colonization, respectively, on at least one occasion. And 64.3% (9/14) of eucalypts were culture-positive for Cryptococcus spp. URA5 restriction fragment length polymorphism analysis identified most isolates as C. gattii VGI, while C. neoformans VNI was only found in one koala and one tree. Colonized koalas were significantly more likely to test positive for antigenemia. No associations between antigenemia or colonization, and external environmental characteristics (the relative abundance of Eucalyptus camaldulensis and season), or individual koala characteristics (body condition, sex, and age), could be established, suggesting that antigenemia and colonization are random outcomes of host-pathogen-environment interactions. The relationship between positive antigenemia status and a relatively high abundance of E. camaldulensis requires further investigation. This study characterizes cryptococcosis in a free-ranging koala population, expands the ecological niche of the C. gattii/C. neoformans species complexes and highlights free-ranging koalas as important sentinels for this disease.

A Possible Link between the Environment and Cryptococcus gattii Nasal Colonisation in Koalas (Phascolarctos cinereus) in the Liverpool Plains, New South Wales.

Cryptococcosis caused by yeasts of the Cryptococcus gattii species complex is an increasingly important mycological disease in humans and other mammals. In Australia, cases of C. gattii-related cryptococcosis are more prevalent in the koala (Phascolarctos cinereus) compared to humans and other animals, likely due to the close association that both C. gattii and koalas have with Eucalyptus species. This provides a cogent opportunity to investigate the epidemiology of spontaneous C. gattii infections in a free-living mammalian host, thereby offering insights into similar infections in humans. This study aimed to establish a link between nasal colonisation by C. gattii in free-ranging koalas and the tree hollows of Eucalyptus species, the key environmental source of the pathogen. We (i) detected and genotyped C. gattii from nine out of 169 free-ranging koalas and representative tree hollows within their home range in the Liverpool Plains, New South Wales, and (ii) examined potential environmental predictors of nasal colonisation in koalas and the presence of C. gattii in tree hollows. Phylogenetic analyses based on multi-locus sequence typing (MLST) revealed that the koalas were most likely colonised by the most abundant C. gattii genotypes found in the Eucalyptus species, or closely related genotypes. Importantly, the likelihood of the presence of C. gattii in tree hollows was correlated with increasing hollow size.

Metal Complexes as Antifungals? From a Crowd-Sourced Compound Library to the First In Vivo Experiments.

There are currently fewer than 10 antifungal drugs in clinical development, but new fungal strains that are resistant to most current antifungals are spreading rapidly across the world. To prevent a second resistance crisis, new classes of antifungal drugs are urgently needed. Metal complexes have proven to be promising candidates for novel antibiotics, but so far, few compounds have been explored for their potential application as antifungal agents. In this work, we report the evaluation of 1039 metal-containing compounds that were screened by the Community for Open Antimicrobial Drug Discovery (CO-ADD). We show that 20.9% of all metal compounds tested have antimicrobial activity against two representative Candida and Cryptococcus strains compared with only 1.1% of the >300,000 purely organic molecules tested through CO-ADD. We identified 90 metal compounds (8.7%) that show antifungal activity while not displaying any cytotoxicity against mammalian cell lines or hemolytic properties at similar concentrations. The structures of 21 metal complexes that display high antifungal activity (MIC ≤1.25 µM) are discussed and evaluated further against a broad panel of yeasts. Most of these have not been previously tested for antifungal activity. Eleven of these metal complexes were tested for toxicity in the Galleria mellonella moth larva model, revealing that only one compound showed signs of toxicity at the highest injected concentration. Lastly, we demonstrated that the organo-Pt(II) cyclooctadiene complex Pt1 significantly reduces fungal load in an in vivo G. mellonella infection model. These findings showcase that the structural and chemical diversity of metal-based compounds can be an invaluable tool in the development of new drugs against infectious diseases.

Multilocus Sequence Typing Reveals Extensive Genetic Diversity of the Emerging Fungal Pathogen Scedosporium aurantiacum.

Scedosporium spp. are the second most prevalent filamentous fungi after Aspergillus spp. recovered from cystic fibrosis (CF) patients in various regions of the world. Although invasive infection is uncommon prior to lung transplantation, fungal colonization may be a risk factor for invasive disease with attendant high mortality post-transplantation. Abundant in the environment, Scedosporium aurantiacum has emerged as an important fungal pathogen in a range of clinical settings. To investigate the population genetic structure of S. aurantiacum, a MultiLocus Sequence Typing (MLST) scheme was developed, screening 24 genetic loci for polymorphisms on a tester strain set. The six most polymorphic loci were selected to form the S. aurantiacum MLST scheme: actin (ACT), calmodulin (CAL), elongation factor-1α (EF1α), RNA polymerase subunit II (RPB2), manganese superoxide dismutase (SOD2), and ß-tubulin (TUB). Among 188 global clinical, veterinary, and environmental strains, 5 to 18 variable sites per locus were revealed, resulting in 8 to 23 alleles per locus. MLST analysis observed a markedly high genetic diversity, reflected by 159 unique sequence types. Network analysis revealed a separation between Australian and non-Australian strains. Phylogenetic analysis showed two major clusters, indicating correlation with geographic origin. Linkage disequilibrium analysis revealed evidence of recombination. There was no clustering according to the source of the strains: clinical, veterinary, or environmental. The high diversity, especially amongst the Australian strains, suggests that S. aurantiacum may have originated within the Australian continent and was subsequently dispersed to other regions, as shown by the close phylogenetic relationships between some of the Australian sequence types and those found in other parts of the world. The MLST data are accessible at http://mlst.mycologylab.org. This is a joined publication of the ISHAM/ECMM working groups on "Scedosporium/Pseudallescheria Infections" and "Fungal Respiratory Infections in Cystic Fibrosis".

Cryptococcus gattii Species Complex as an Opportunistic Pathogen: Underlying Medical Conditions Associated with the Infection.

The Cryptococcus gattii species complex has often been referred to as a primary pathogen due to its high infection frequency among apparently immunocompetent patients. In order to scrutinize the immune status of patients and the lineages of etiologic agents, we analyzed patient histories and the molecular types of etiologic agents from 135 global C. gattii cases. Eighty-six of 135 patients had been diagnosed as immunocompetent, although some of them had underlying medical issues, and 49 were diagnosed as immunocompromised with risk factors similar to those seen in Cryptococcus neoformans infection. We focused on the 86 apparently immunocompetent patients and were able to obtain plasma from 32 (37%) to analyze for the presence of autoantibodies against the granulocyte-macrophage colony-stimulating factor (GM-CSF) since these antibodies have been reported as a hidden risk factor for C. gattii infection. Among the 32 patients, 25 were free from any known other health issues, and 7 had various medical conditions at the time of diagnosis for cryptococcosis. Importantly, plasma from 19 (76%) of 25 patients with no recognized underlying medical condition showed the presence of GM-CSF autoantibodies, supporting this antibody as a major hidden risk factor for C. gattii infection. These data indicate that seemingly immunocompetent people with C. gattii infection warrant detailed evaluation for unrecognized immunologic risks. There was no relationship between molecular type and underlying conditions of patients. Frequency of each molecular type was related to its geographic origin exemplified by the overrepresentation of VGIV in HIV-positive (HIV+) patients due to its prevalence in Africa. IMPORTANCE The C. neoformans and C. gattii species complex causes cryptococcosis. The C. neoformans species complex is known as an opportunistic pathogen since it primarily infects immunocompromised patients. C. gattii species complex has been referred to as a primary pathogen due to its high infection frequency in apparently immunocompetent people. We analyzed 135 global cases of C. gattii infection with documented patient history. Eighty-six of 135 patients were originally diagnosed as immunocompetent and 49 as immunosuppressed with similar underlying conditions reported for C. neoformans infection. A significant number of C. gattii patients without known underlying conditions possessed autoantibodies against granulocytes-macrophage colony-stimulating factor (GM-CSF) in their plasma, supporting the presence of GM-CSF antibodies as a hidden risk factor for C. gattii infection. No relationship was found between C. gattii lineages and the underlying conditions except for overrepresentation of the molecular type VGIV among HIV+ patients due to the prevalence of VGIV in Africa.

Consensus Multilocus Sequence Typing Scheme for Pneumocystis jirovecii.

Pneumocystis jirovecii is an opportunistic human pathogenic fungus causing severe pneumonia mainly in immunocompromised hosts. Multilocus sequence typing (MLST) remains the gold standard for genotyping of this unculturable fungus. However, the lack of a consensus scheme impedes a global comparison, large scale population studies and the development of a global MLST database. To overcome this problem this study compared all genetic regions (19 loci) currently used in 31 different published Pneumocystis MLST schemes. The most diverse/commonly used eight loci, ß-TUB, CYB, DHPS, ITS1, ITS1/2, mt26S and SOD, were further assess for their ability to be successfully amplified and sequenced, and for their discriminatory power. The most successful loci were tested to identify genetically related and unrelated cases. A new consensus MLST scheme consisting of four genetically independent loci: ß-TUB, CYB, mt26S and SOD, is herein proposed for standardised P. jirovecii typing, successfully amplifying low and high fungal burden specimens, showing adequate discriminatory power, and correctly identifying suspected related and unrelated isolates. The new consensus MLST scheme, if accepted, will for the first time provide a powerful tool to investigate outbreak settings and undertake global epidemiological studies shedding light on the spread of this important human fungal pathogen.

Identification of the environmental source of infection for a domestic ferret with cryptococcosis.

Cryptococcosis, caused by the Cryptococcus gattii and C. neoformans species complexes, is an environmentally acquired mycosis affecting a broad range of host species. Among 9 communally housed ferrets, a 5-y-old castrated male ferret domiciled in an outdoor enclosure in Sydney, Australia was diagnosed with sinonasal cryptococcosis. Clinical signs resolved during 18 mo of itraconazole therapy, but the ferret was eventually euthanized because of splenic hemangiosarcoma. At postmortem, microscopic foci of persistent cryptococcosis were detected. The diagnosis raised concerns that the owners and other ferrets were exposed to a common environmental source of infection, thus prompting an investigation. Soil samples, swabs of a hollow eucalypt log (used for behavioral enrichment), and nasal swabs from 8 asymptomatic ferrets were collected. Nasal exudate (obtained at diagnosis) and tissues (collected at postmortem) were available from the clinical case. Bird seed agar culture resulted in a heavy growth of Cryptococcus spp. from one environmental site (the log), one nasal swab, and nasal exudate and tissues from the clinical case. All other samples were culture-negative. Sub-cultured isolates from the log were a mixture of C. gattii molecular type VGI and C. neoformans molecular type VNI. Ferret isolates were a similar mixture of C. gattii VGI (all disease isolates) and C. neoformans VNI (nasal-colonizing isolate). Multilocus sequence typing further revealed the ferret isolates as identical to environmental isolates collected from the log, confirming the log as the source of clinical disease and nasal colonization. The log was removed to prevent further exposure to a high environmental load of Cryptococcus spp.

Rhabdomyolysis in pontocerebellar hypoplasia type 2 (PCH-2).

Pontocerebellar hypoplasia type 2, an autosomal recessive neurodegeneration with prenatal onset, is characterised by progressive microcephaly and chorea/dystonia and has not previously been associated with muscular involvement. The gene associated with PCH-2 is unknown. An episode of rhabdomyolysis is reported in two non-related children with PCH-2, fatal in one, precipitated by intercurrent disease. Muscle biopsies in two other PCH-2 patients, and in one rhabdomyolysis patient whose biopsy antedated this complication showed areas of myofibrillar disruption or necrosis. Postmortem muscle sampled in another case without neuromuscular symptoms revealed focal necrosis, regenerating small fibres and upregulation of HLA-ABC. Random serum creatine kinase values in six other PCH-2 patients without clinical signs of neuromuscular involvement were increased in four. Collected data provide preliminary evidence of a subclinical myopathy associated with PCH-2.

The effect of adjunct caudal block on postoperative analgesia in robot-assisted laparoscopic radical prostatectomy: A prospective randomized controlled, single blinded pilot study in a tertiary centre.

OBJECTIVE: Caudal block provides satisfactory postoperative pain relief in lower abdominal operations. This pilot study explores its safety and effect on postoperative pain control in patients who underwent robot-assisted laparoscopic radical prostatectomy (RARP). METHODS: From 2013 to 2014, 40 consecutive patients were randomized into two groups - one received caudal block using ropivacaine immediately after operation, the other received standard analgesia. Primary outcome measure was pain score based on 11-point Likert scale (0-10) recorded at recovery room, and at 6, 12, 24, 48, and 72 h after operation. All analgesic requirements, opioid-related adverse events and time to passage of flatus were examined. RESULTS: Mean age of the two groups was similar (60.4 vs. 62.3 years, p = 0.33), as was American Society of Anaesthesiologists (ASA) class, body mass index (BMI) and operation times. No significant difference in median pain scores was reported in recovery room (2 vs. 3, p = 0.34), and at 6 h (2 vs. 2, p = 0.94), 12 h (0 vs. 0, p = 0.62), 24 h (1 vs. 0, p = 0.58), 48 h (1 vs. 0, p = 0.36) and 72 h (0 vs. 0, p = 0.78) postoperatively between control and caudal block groups, respectively. There was a higher mean opioid usage in the caudal block group which was not statistically significant. Although this was statistically insignificant while no significant difference in mean paracetamol usage was observed postoperatively. Median time to passage of flatus was similar (2.0 vs. 2.0 days, p = 0.97). There was one case of superficial wound infection and no opioid-related adverse events observed. Hospital stay was similar in both groups (2.5 vs. 2.5 days, p = 0.96). CONCLUSION: Although a safe modality, caudal block in post RARP patients does not seem to improve pain control nor reduce analgesia requirements.

Dermatofibrosarcoma protuberans in children.

Dermatofibrosarcoma protuberans (DFSP) is a relatively rare neoplasm affecting the skin. It is an infiltrative tumour of intermediate malignancy, with a limited potential for metastasis but a high rate of recurrence. The incidence in children is even less frequent, although a proportion of those identified in adulthood may reflect a delay in diagnosis of childhood DFSP. We report the experience of DFSP seen at The Children's Hospital at Westmead (Sydney, Australia). Three children aged 5, 10 and 11 years of age underwent surgical excision of their lesions. Recurrence was evident in one child whose initial histopathology was not definitive for DFSP, and whose initial surgery had not involved wide local excision. All three children were male, and all had lesions affecting their trunk. One child whose lesion was thought to have been evident since birth may have represented congenital DFSP.

A case of chronic intestinal pseudo-obstruction secondary to primary visceral myopathy.

BACKGROUND: A 13-year-old girl presented to the emergency room at her local hospital with an acute onset of vomiting, severe abdominal pain and distension. There was evidence of small-bowel obstruction on plain abdominal x-ray. Throughout the girl's adolescent years she was admitted to hospital numerous times for recurrent abdominal symptoms and underwent multiple sequential laparotomies. She had marked weight loss and a poor quality of life. The patient's symptoms were initially managed with intravenous fluids, bowel rest, and nasogastric decompression of the upper gut. INVESTIGATIONS: Peripheral blood tests with biochemistry and measurement of serum folate, vitamin B(12), albumin, 25-hydroxyvitamin D, inflammatory markers, autoantibodies and thyroid function; gastrointestinal imaging (plain abdominal x-ray, small-bowel series, colonic transit study, and abdominal CT with oral contrast); MRI of the brain and lumbar puncture; upper endoscopy; and laparotomy with sero-muscular biopsy of the small bowel. DIAGNOSIS: Chronic intestinal pseudo-obstruction secondary to primary visceral myopathy. MANAGEMENT: Prokinetic agents including oral cisapride and tegaserod, a venting gastrostomy, and total parenteral feeding.

Pigmented hypertrichotic dermatosis and insulin dependent diabetes: manifestations of a unique genetic disorder?

A novel pigmented dermatosis was observed in four unrelated boys, three of whom had insulin-dependent diabetes. Three patients were the offspring of consanguineous parents. All four boys had pigmented hypertrichotic patches or induration on the upper inner thighs, with variable involvement of the genitalia, trunk, and limbs. Two boys had episcleritis and orbital proptosis with similar facies and musculoskeletal abnormalities including clinodactyly, flat feet, and short stature. One child had paraaortic and inguinal lymphadenopathy and three patients had an enlarged liver and spleen. A large, swollen pancreas was observed on ultrasound imaging in one patient with insulin dependent diabetes who also had echocardiographic evidence of pericardial inflammation. Three boys had elevated laboratory markers of inflammation. Biopsy specimens from the skin and orbit showed a chronic inflammatory cell infiltrate composed of polyclonal lymphocytes, histiocytes, and plasma cells; fibrosis was observed in two patients, one of whom had previously received radiation therapy to the orbit. Two boys responded to treatment with subcutaneous interferon-alpha, combined with a short course of oral prednisone in the child without diabetes. We believe these inflammatory pigmented skin lesions represent a unique dermatosis associated with diabetes mellitus and systemic disease. The pathogenesis is unknown. The presence of consanguinity in three of four families, and similar dysmorphic features in two boys, suggest a genetic disorder, possibly with autosomal recessive inheritance.

A novel mutation of WT1 exon 9 in a patient with Denys-Drash syndrome and pyloric stenosis.

We report a novel mutation in WT1 exon 9 (1214 A>G) resulting in an amino acid change from H to R at codon 405 in a 46 XY female patient who had congenital hypertrophic pyloric stenosis, pseudohermaphroditism masculinus, renal failure, and Wilms tumor, and died at the age of 22 months. The patient demonstrated the difficulty in diagnosing a patient with intersex before conclusive genetic characterization.