EV71:TLLcho virus murine model of enterovirus A71 neurological disease does not exhibit neurogenic pulmonary oedema.

Small animal models play an important role in investigating and revealing the molecular determinants and mechanisms underlying neuro-virulence of enterovirus A71 (EV-A71). In our previous study, we successfully developed two mouse cell-line replication competent EV-A71 strains (EV71:TLLm and EV71:TLLmv) which were capable of inducing neuro-invasion in BALB/c mice. The more virulent EV71:TLLmv exhibited ability to induce acute encephalomyelitis accompanied by neurogenic pulmonary oedema. EV71:TLLcho virus strain was generated from EV71:TLLm by a series of passages in CHO-K1 cells. EV71:TLLcho demonstrated a broader range of infectivity across various mammalian cell lines and exhibited complete cytopathic effects (CPE) within 48 hours post-inoculation in comparison to EV71:TLLm or EV71:TLLmv. EV71:TLLcho consistently yielded higher levels of viral replication at all time points examined. In comparison to EV71:TLLm, EV71:TLLcho consistently induced more severe disease and increased mortality in one-week old BALB/c mice. However, unlike mice challenged with EV71:TLLmv, none of the mice challenged with EV71:TLLcho progressed to severe acute encephalomyelitis and developed neurogenic pulmonary oedema.

Effect of molnupiravir on SARS-CoV-2 evolution in immunocompromised patients: a retrospective observational study.

INTRODUCTION: Continued SARS-CoV-2 infection among immunocompromised individuals is likely to play a role in generating genomic diversity and the emergence of novel variants. Antiviral treatments such as molnupiravir are used to mitigate severe COVID-19 outcomes, but the extended effects of these drugs on viral evolution in patients with chronic infections remain uncertain. This study investigates how molnupiravir affects SARS-CoV-2 evolution in immunocompromised patients with prolonged infections. METHODS: The study included five immunocompromised patients treated with molnupiravir and four patients not treated with molnupiravir (two immunocompromised and two non-immunocompromised). We selected patients who had been infected by similar SARS-CoV-2 variants and with high-quality genomes across timepoints to allow comparison between groups. Throat and nasopharyngeal samples were collected in patients up to 44 days post treatment and were sequenced using tiled amplicon sequencing followed by variant calling. The UShER pipeline and University of California Santa Cruz genome viewer provided insights into the global context of variants. Treated and untreated patients were compared, and mutation profiles were visualised to understand the impact of molnupiravir on viral evolution. FINDINGS: Patients treated with molnupiravir showed a large increase in low-to-mid-frequency variants in as little as 10 days after treatment, whereas no such change was observed in untreated patients. Some of these variants became fixed in the viral population, including non-synonymous mutations in the spike protein. The variants were distributed across the genome and included unique mutations not commonly found in global omicron genomes. Notably, G-to-A and C-to-T mutations dominated the mutational profile of treated patients, persisting up to 44 days post treatment. INTERPRETATION: Molnupiravir treatment in immunocompromised patients led to the accumulation of a distinctive pattern of mutations beyond the recommended 5 days of treatment. Treated patients maintained persistent PCR positivity for the duration of monitoring, indicating clear potential for transmission and subsequent emergence of novel variants. FUNDING: Australian Research Council.

Bronchiectasis is associated with delayed diagnosis and adverse outcomes in the New Zealand Common Variable Immunodeficiency Disorders cohort study.

Common variable immunodeficiency disorders (CVID) are multi-system disorders where target organ damage is mediated by infective, autoimmune and inflammatory processes. Bronchiectasis is probably the most common disabling complication of CVID. The risk factors for bronchiectasis in CVID patients are incompletely understood. The New Zealand CVID study (NZCS) is a nationwide longitudinal observational study of adults, which commenced in 2006. In this analysis, the prevalence and risk factors for bronchiectasis were examined in the NZCS. After informed consent, clinical and demographic data were obtained with an interviewer-assisted questionnaire. Linked electronic clinical records and laboratory results were also reviewed. Statistical methods were applied to determine if variables such as early-onset disease, delay in diagnosis and increased numbers of infections were associated with greater risk of bronchiectasis. One hundred and seven adult patients with a diagnosis of CVID are currently enrolled in the NZCS, comprising approximately 70% of patients known to have CVID in New Zealand. Fifty patients (46·7%) had radiologically proven bronchiectasis. This study has shown that patients with compared to those without bronchiectasis have an increased mortality at a younger age. CVID patients with bronchiectasis had a greater number of severe infections consequent to early-onset disease and delayed diagnosis. Indigenous Maori have a high prevalence of CVID and a much greater burden of bronchiectasis compared to New Zealand Europeans. Diagnostic latency has not improved during the study period. Exposure to large numbers of infections because of early-onset disease and delayed diagnosis was associated with an increased risk of bronchiectasis. Earlier diagnosis and treatment of CVID may reduce the risk of bronchiectasis and premature death in some patients.

Phylogenetic and Timescale Analysis of Barmah Forest Virus as Inferred from Genome Sequence Analysis.

Barmah Forest virus (BFV) is a medically important mosquito-borne alphavirus endemic to Australia. Symptomatic disease can be a major cause of morbidity, associated with fever, rash, and debilitating arthralgia. BFV disease is similar to that caused by Ross River virus (RRV), the other major Australian alphavirus. Currently, just four BFV whole-genome sequences are available with no genome-scale phylogeny in existence to robustly characterise genetic diversity. Thirty novel genome sequences were derived for this study, for a final 34-taxon dataset sampled over a 44 year period. Three distinct BFV genotypes were characterised (G1-3) that have circulated in Australia and Papua New Guinea (PNG). Evidence of spatio-temporal co-circulation of G2 and G3 within regions of Australia was noted, including in the South West region of Western Australia (WA) during the first reported disease outbreaks in the state's history. Compared with RRV, the BFV population appeared more stable with less frequent emergence of novel lineages. Preliminary in vitro assessment of RRV and BFV replication kinetics found that RRV replicates at a significantly faster rate and to a higher, more persistent titre compared with BFV, perhaps indicating mosquitoes may be infectious with RRV for longer than with BFV. This investigation resolved a greater diversity of BFV, and a greater understanding of the evolutionary dynamics and history was attained.

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis from pooled rectal, pharyngeal and urine specimens in men who have sex with men.

OBJECTIVES: Screening of men who have sex with men (MSM) for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) requires sampling from anorectal and pharyngeal sites in addition to urogenital sampling. Due to the cost of testing multiple anatomical sites individually testing of pooled specimens has potential merit. The Cepheid GeneXpert CT/NG assay (GeneXpert), which also has potential for point-of-care nucleic acid testing in the sexual health clinic, has not been assessed for pooled specimen testing. METHODS: We prospectively compared GeneXpert testing of pooled pharyngeal and rectal swabs with urine samples to standard of care testing of individual specimens from 107 participants using the Roche cobas 4800 CT/NG assay (cobas) for CT and NG in high-risk MSM attending an inner city sexual health clinic. RESULTS: We found testing of pooled pharyngeal, rectal and urine samples by the GeneXpert to have 100% agreement for NG and 94% overall agreement for CT when compared with individual specimen testing by cobas. For CT testing, 14 cases were detected for both tests, 4for cobas only, 2 for GeneXpert only and 89 participants were negative for both tests. CONCLUSIONS: Pooled specimen CT and NG testing by the GeneXpert was accurate when compared with single specimen testing and has potential for screening MSM for CT and NG. The role of pooled specimen testing with the GeneXpert as a point-of-care nucleic acid test in MSM requires further investigation.

Improved blood culture identification by FilmArray in cultures from regional hospitals compared with teaching hospital cultures.

Rapid identification of bacteria isolated from blood cultures by direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now in wide spread use in major centres but is not yet feasible in smaller hospital laboratories. A FilmArray multiplex PCR panel for blood culture isolate identification (BCID) provides an alternative approach to near point-of-care microbial identification in regional hospitals. We assessed the accuracy and time to identification of the BCID FilmArray in a consecutive series of 149 blood cultures from 143 patients in a teaching hospital and smaller regional hospitals, currently identified by direct MALDI-TOF and proprietary molecular methods. The BCID FilmArray contained 18 of 34 species and 20 of 23 species isolated from teaching and regional hospital, respectively. Overall, 85 % of the teaching hospital and 100 % of the regional hospital monomicrobial blood cultures were identified, compared with 60 and 68 %, respectively, for direct MALDI-TOF on the same cultures. There were no incorrect results from blood cultures containing Staphylococcus aureus, streptococci, Pseudomonas aeruginosa or Enterobacteriaceae. The three discrepant results were all in mixed cultures. The mean reduction in time to identification of blood culture isolates was 53 h, which did not include the time required to transport cultures from regional centres to a central laboratory. The overall performance of the BCID FilmArray is stronger in blood cultures from smaller regional hospitals that encounter a narrower range of bacterial species dominated by the commonest species. This approach is more suited to smaller clinical laboratories than the MALDI-TOF direct method.

Anti-tumour necrosis factor-alpha therapy for severe enteropathy in patients with common variable immunodeficiency (CVID).

We present three common variable immunodeficiency (CVID) patients with severe inflammatory bowel disease of unknown aetiology, resistant to steroid treatment, treated with infliximab. After exclusion of any infection, infliximab was given at a dose of 5 mg/kg every 4 weeks for a 3 month induction followed by every 4-8 weeks depending on clinical response. Two of these patients had predominantly small bowel disease; they both showed clinical response to infliximab with weight gain and improvement of quality of life scores. The third patient had large bowel involvement with profuse watery diarrhea; this patient improved dramatically within 48 hours of having infliximab treatment. All three patients have been maintained on infliximab treatment for between 5 and 53 months (mean 37 months) with no evidence of increased susceptibility to infections in the patients with small bowel disease, although the third patient developed two urinary tract infections and a herpes zoster infection following therapy. This is the first small case series to show that infliximab is a useful addition to current therapy in this rare group of patients with potentially life threatening enteritis.

[Laparoscopic hysterectomy: technique, indications]. / Hystérectomie coelioscopique: technique, indications.

Today, hysterectomy is, after caesarean section, the most frequent surgical intervention performed in fertile women. Introduced in 1989, laparoscopic hysterectomy remains poorly diffused: today, less than 5% of all hysterectomies remain done by laparoscopy. Nevertheless after a correct learning curve, laparoscopic hysterectomy finds perfect indications in benign and even some malignant indications. In these conditions, the complication rate is similar to those of the other surgical routes. Currently the limitations of this technique are the very bulky uterus, contraindicated uterine morcellation, the lengthening of the operative time potentially generated by this technique, and the lack of experienced instructors.

Cutaneous IgA-associated vasculitis induced by alcohol.

A patient with elevated levels of serum IgA developed purpuric lesions histologically resembling Henoch-Schönlein purpura brought on by consuming alcohol. Alcohol challenge with 5 units of alcohol reproduced the lesions, with a rapid rise of circulating CD4+ and CD8+ T cells followed by a fall of serum IgA and C3 concentration. The skin lesions and serum abnormalities resolved spontaneously within 6 weeks of the alcohol challenge.

Effect of chemical fogging on immature Aedes mosquitoes in natural field conditions.

INTRODUCTION: Dengue and dengue haemorrhagic fever are common and serious arboviral diseases endemic in a number of countries situated in both the tropical and subtropical belts. METHODS: A prospective study was carried out to examine the environmental factors influencing the ovipositing behaviour of gravid female Aedes mosquitoes in a typical urbanised residential environment in Malaysia. This study reports the effect of the usual ultra-low volume fogging of insecticides carried out by public health officers on the collection of immature Aedes mosquitoes using ovitraps. RESULTS: Throughout the study, no dead immature Aedes mosquitoes was noted in any of the ovitraps set up in all of the fogging and immediate post-fogging periods. The mean number of days of ovitrapping for immediate pre-fogging, fogging and post-fogging periods were 10.3, 10.1 and 10.4 days, respectively. There was no statistically significant difference in the mean duration of ovitrapping cycle among the immediate pre-fogging, fogging and immediate post-fogging periods. The total number of immature Aedes mosquitoes collected in the immediate post-fogging periods was more than the immediate pre-fogging periods, and both were more than the fogging periods. However, there was no statistically significant difference in the total number of immature Aedes mosquitoes collected at various periods. It was not unusual to find dead insects, spiders and even small animals collected in ovitraps or environment in the fogged locality within 48 hours of chemical fogging. CONCLUSION: In this study, the usual chemical fogging in natural environment was ineffective in breaking the reproductive lifecycle by eliminating gravid female Aedes mosquitoes.

Distinct phenotype associated with a cryptic subtelomeric deletion of 19p13.3-pter.

Telomeres are gene rich regions with a high recombination rate. Cryptic subtelomeric rearrangements are estimated to account for 5% of mental retardation/malformation syndromes. Here we present the first patient with a deletion of 19p13.3, identified by subtelomeric FISH analysis. His features included a distinctive facial appearance, cleft palate, hearing impairment, congenital heart malformation, keloid scarring, immune dysregulation, and mild learning difficulties. Subtelomeric FISH analysis identified a deletion of 19p13.3-pter. The deletion size was determined to be 1.2 Mb by FISH analysis. It extended from within the chromosomal region covered by BAC RP11-50C6 to 19pter. The deleted area encompassed approximately 60 genes. Fifteen possible candidate genes were considered with respect to the phenotype, including follistatin-related precursor 3 (FSTL3) and serine-threonine kinase 11 (STK-11).

A modified mycological medium for isolation and culture of Malassezia furfur.

A mycological medium was developed for primary isolation and culture of lipophilic yeasts. It was initially based on published information of nutrients and trace components that would promote the growth of these yeasts. It was subsequently modified and adjusted to specifically promote the growth of lipophilic yeasts and simultaneously avoid the luxurious growth of other fungi and bacteria. With this medium, the conventional bacteriological procedures such as microbial streaking for pure culture and anti-microbial sensitivity testing could be carried out for these lipophilic yeasts.

Differential environmental preferences of gravid female Aedes mosquitoes in ovipositing their eggs.

A prospective field study was carried out to investigate any preferential differences of gravid female Aedes mosquitoes in ovipositing their eggs in man-made containers placed in different environmental conditions. The findings of this study show that gravid female Aedes mosquitoes preferred to breed in containers found in the outdoor garden than those placed on the patio and or inside the house. The findings also show that if the breeding habitats in the garden were removed, they would favorably use the breeding habitats found on the patio or inside the house as alternatives. An incidental interesting finding in this study shows that ultra-low volume fogging of insecticides using the vehicle-mounted equipment carried out outside the house may promote the gravid female Aedes mosquitoes to enter the house to breed.

Differential preferences of oviposition by Aedes mosquitos in man-made containers under field conditions.

A study was made of the oviposit behavior of gravid female Aedes mosquitos in man-made habitats under field conditions. The study showed that the gravid female Aedes mosquitos preferred containers with relatively easy access but not too open to external environmental influence. The dark surface of the containers served as the initial and long-range attractant to the breeding sites. Volatile chemicals generated by the decaying vegetation in the container may serve as a close-range attractant. Finally, the water quality and the quantity of 'food' derived from decaying vegetative matter in the water determined the amount of eggs deposited in each container. The study confirmed previous findings that each gravid female Aedes mosquito had the tendency to lay her eggs in more than one container. However, the results of the study suggests that under favorable conditions, each gravid female Aedes mosquito could be encouraged to lay all her eggs in a single breeding site.

A new cell culture tube in diagnostic virology for virus isolation.

Virus isolation and accurate characterization plays a crucial role in the rapid identification of the causative agents of infectious disease outbreaks especially if the causative viruses are novel where no pre-existing diagnostic reagents would be available. A new cell culture tube, named Jui Meng (JM) Cell Culture Tube, was developed to reduce the cost and improve the efficiency and biosafety of work pertaining to virus isolation. The design of the tube is based heavily on the principle of practicability, functionality, biosafety and long-term cost saving for diagnostic laboratory work in virus isolation. It is designed to culture an initial inoculum of one milliliter of culture medium containing 1 x 10(4) to 1 x 10(5) cells/ml.

Scaling laws for wall shear stress through stenoses under steady and pulsatile flow conditions.

Most patients with atherosclerosis exhibit isolated stenoses of one or more epicardial coronary arteries. The wall shear stresses produced in high-grade stenosis are important in the understanding of atheromatous plaque rupture and thrombosis. This study is designed to establish a method which can be used to scale the different wall shear stresses obtained under different flow conditions to be normalized and subsequently collapsed on to a single general curve. The simulations include both steady and pulsatile flow. The reduced area percentages of the stenoses studied are 50, 75 and 90 per cent. Scaling laws for steady and pulsatile flow conditions are proposed and presented. It can be found from the results that the scaling analysis for pulsatile flow conditions is more complicated than for steady flow conditions and is restricted to, and only valid at, certain time intervals.

A clinical review of granulosa cell tumours of the ovary cases in KKH.

INTRODUCTION: Granulosa cell tumour (GCT) represents the largest group of sex-cord stromal tumours and comprises 1.5-3% of ovarian malignancy. The aim is to determine the incidence of the disease, study the profile of local patients, and assess the use of imaging studies in the diagnosis of the tumour. MATERIAL AND METHODS: Clinical records of 19 patients diagnosed with GCT between October 1988 and July 1997 in Kandang Kerbau Hospital (KKH) were reviewed. RESULTS: GCT accounts for 3.5% of ovarian malignancy (54 out of 1552) in Singapore, of which 94.7% are adult GCT. In our study, patients are mainly peri/postmenopausal women (63.2%) in their 50s who experience post-menopausal bleeding. There is a high incidence of association with endometrial hyperplasia (40%). Ultrasound scans are able to predict the size and involvement of the tumour rather accurately. In our study sample, 13 patients (68.4%) presented with Stage 1 of the disease, none with Stage 2, 1 with Stage 3 (5.3%) and none with Stage 4. The other 5 patients (26.3%) were unstaged. Only one patient required adjuvant chemotherapy. CONCLUSION: The local data with regards to GCT is congruent with those found in foreign literature. However, in our study, there were no patients with recurrence whereas GCTs are known to be late recurring in up to 20% of patients 10-20 years after diagnosis. This is probably attributed to the relatively short period of follow-up in this study. Thus, despite the fact that there is no evidence of recurrence of disease in our current study, we still recommend a vigilant follow-up protocol on all patients as literature has proven that with early detection of recurrences, it is possible to achieve complete cure.