Somatic activating BRAF variants cause isolated lymphatic malformations.

Somatic activating variants in PIK3CA, the gene that encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), have been previously detected in ∼80% of lymphatic malformations (LMs).1 , 2 We report the presence of somatic activating variants in BRAF in individuals with LMs that do not possess pathogenic PIK3CA variants. The BRAF substitution p.Val600Glu (c.1799T>A), one of the most common driver mutations in cancer, was detected in multiple individuals with LMs. Histology revealed abnormal lymphatic channels with immunopositivity for BRAFV600E in endothelial cells that was otherwise indistinguishable from PIK3CA-positive LM. The finding that BRAF variants contribute to low-flow LMs increases the complexity of prior models associating low-flow vascular malformations (LM and venous malformations) with mutations in the PI3K-AKT-MTOR and high-flow vascular malformations (arteriovenous malformations) with mutations in the RAS-mitogen-activated protein kinase (MAPK) pathway.3 In addition, this work highlights the importance of genetic diagnosis prior to initiating medical therapy as more studies examine therapeutics for individuals with vascular malformations.

Respiratory virus infection among hospitalized adult patients with or without clinically apparent respiratory infection: a prospective cohort study.

OBJECTIVES: To determine the viral epidemiology and clinical characteristics of patients with and without clinically apparent respiratory tract infection. METHODS: This prospective cohort study was conducted during the 2018 winter influenza season. Adult patients with fever/respiratory symptoms (fever/RS group) were age- and sex-matched with patients without fever/RS (non-fever/RS group) in a 1:1 ratio. Respiratory viruses were tested using NxTAG™ Respiratory Pathogen Panel IVD, a commercially-available multiplex PCR panel. RESULTS: A total of 214 acutely hospitalized patients were included in the final analysis, consisting of 107 with fever/RS (fever/RS group), and 107 age- and sex-matched patients without fever/RS (non-fever/RS group). Respiratory viruses were detected in 34.1% (73/214) of patients, and co-infection occurred in 7.9% (17/214) of patients. The incidence of respiratory virus was higher in the fever/RS group than in the non-fever/RS group (44.9% (48/107) versus 23.4% (25/107), p 0.001). Influenza B virus, enterovirus/rhinovirus and coronaviruses were detected more frequently in the fever/RS group, whereas parainfluenza virus 4B and adenovirus were detected more frequently in the non-fever/RS group. Among the non-fever/RS group, chest discomfort was more common among patients tested positive for respiratory viruses than those without respiratory virus detected (44% (11/25) versus 22% (18/82), p 0.04). CONCLUSIONS: Respiratory viruses can be frequently detected among hospitalized patients without typical features of respiratory tract infection. These patients may be a source of nosocomial outbreaks.

Use of the human colorectal adenocarcinoma (Caco-2) cell line for isolating respiratory viruses from nasopharyngeal aspirates.

The human colorectal adenocarcinoma-derived Caco-2 cell line was evaluated as a means isolating common respiratory viruses from nasopharyngeal aspirates for the diagnosis of respiratory diseases. One hundred eighty-nine direct immunofluorescence positive nasopharyngeal aspirates obtained from patients with various viral respiratory diseases were cultured in the presence of Caco-2 cells or the following conventional cell lines: LLC-MK2, MDCK, HEp-2, and A549. Caco-2 cell cultures effectively propagated the majority (84%) of the viruses present in nasopharyngeal aspirate samples compared with any positive cultures obtained using the panel cells (78%) or individual cell line MDCK (38%), HEp-2 (21%), LLC-MK2 (27%), or A549 (37%) cell lines. The differences against individual cell line were statistically significant (P = < 0.000001). Culture in Caco-2 cells resulted in the isolation of 85% (36/42) of viruses which were not cultivated in conventional cell lines. By contrast, 80% (24/30) of viruses not cultivated in Caco-2 cells were isolated using the conventional panel. The findings indicated that Caco-2 cells were sensitive to a wide range of viruses and can be used to culture a broad range of respiratory viruses.

Studying the transmission dynamics of meticillin-resistant Staphylococcus aureus in Hong Kong using spa typing.

This study investigated the transmission dynamics of meticillin-resistant Staphylococcus aureus (MRSA) in a tertiary referral surgical unit with 300 beds. All adult patients were actively screened for MRSA by culture at hospital admission and twice weekly thereafter during hospitalisation from 1 October to 31 December 2008. The colonisation pressure per 1000 patient-days and the incidence density of nosocomial MRSA transmission per 1000 colonisation-days were calculated for the different spa types of MRSA. In total, 6619 nasal swabs were obtained from 2289 patients. One-hundred and forty-eight (7%) patients had MRSA in nasal swabs at admission screening, of which 68/148 (46%) were residents of elderly care homes. Fifty-two of 2141 (2%) patients had conversion of nasal MRSA carriage status from negative to positive during hospitalisation. Among the 200 patients with MRSA, spa types t1081 and t037 were found in 99 (50%) and 30 (15%) patients, respectively. The colonisation pressure per 1000 patient-days was 40.9 for t0181, 22.2 for t037 and 26.3 for the less common spa types. The incidence densities of nosocomial MRSA transmission per 1000 colonisation-days were significantly higher for t1081 (28.5 vs 4.0, P<0.01) and t037 (21.5 vs 4.0, P=0.03) compared with the less common spa types. Proactive screening of MRSA in patients from elderly care homes and targeted isolation of these patients, especially those carrying spa types with high transmissibility, are important for the control of MRSA in hospitals.

Differential susceptibility of different cell lines to swine-origin influenza A H1N1, seasonal human influenza A H1N1, and avian influenza A H5N1 viruses.

BACKGROUND: The novel swine-origin influenza A H1N1 virus (S-OIV) causes the current pandemic. Its tissue tropism and replication in different cell lines are not well understood. OBJECTIVE: Compare the growth characteristics of cell lines infected by S-OIV, seasonal influenza A H1N1 (sH1N1) and avian influenza A H5N1 (H5N1) viruses and the effect of temperature on viral replication. STUDY DESIGN: Cytopathic effect (CPE), antigen expression by immunofluorescence (IF) and viral load profile by quantitative RT-PCR in 17 cell lines infected by S-OIV, sH1N1 and H5N1 were examined. Comparison of their replication efficiency in chick embryo was performed. The effect of temperature on viral replication in Madin-Darby canine kidney (MDCK) cells was determined by TCID(50) at 33 degrees C, 37 degrees C and 39 degrees C for 5 consecutive days. RESULTS: S-OIV replicated in cell lines derived from different tissues or organs and host species with comparable viral load to sH1N1. Among 13 human cell lines tested, Caco-2 has the highest viral load for S-OIV. S-OIV showed a low viral load with no CPE or antigen expression in pig kidney cell PK-15, H5N1 demonstrated the most diverse cell tropism by CPE and antigen expression, and the highest viral replication efficiency in both cell lines and allantoic fluid. All three viruses demonstrated best growth at 37 degrees C in MDCK cells. CONCLUSION: Cell line growth characteristics of S-OIV, sH1N1 and H5N1 appear to correlate clinically and pathologically with involved anatomical sites and severity. Low replication of S-OIV in PK-15 suggests that this virus is more adapted to human than swine.

Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer.

BACKGROUND: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis. METHODS: RAMP expression was examined by real-time quantitative RT-PCR, immunohistochemistry and western blotting. Inhibition of RAMP expression was performed by siRNA-mediated knockdown. The functional effects of RAMP on cell kinetics were measured by cell viability assay, colony formation assay and flow cytometry. Cell lines stably expressing RAMP were established to investigate the oncogenic effects of RAMP in vitro. RESULTS: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001). In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia. Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G(2)/M arrest. In additional, knockdown RAMP induced cell apoptosis is dependent on functional accumulation of p53 and p21 and induction of cleaved caspases-9, caspases-3 and PARP. Strikingly, overexpression of RAMP promoted anchorage-independent cell growth in soft agar. CONCLUSION: Our findings demonstrate that RAMP plays an oncogenic role in gastric carcinogenesis. Inhibition of RAMP may be a promising approach for gastric cancer therapy.

Comparative host gene transcription by microarray analysis early after infection of the Huh7 cell line by SARS coronavirus and human coronavirus 229E.

During the early stages of infection, SARS-CoV produces more severe perturbation of host cell gene expression in a human epithelial cell line of liver origin than the HCoV-229E.

High frequency of polyoma BK virus shedding in the gastrointestinal tract after hematopoietic stem cell transplantation: a prospective and quantitative analysis.

The polyoma BK virus (BKV) remains latent after primary infection and may reactivate during immunosuppression. The uroepithelium is the main latency site defined. This study addressed whether the gastrointestinal tract might be another latency site. To test this hypothesis, we prospectively quantified fecal BKV by quantitative PCR reaction in 40 patients undergoing hematopoietic SCT (HSCT). Urinary BKV was similarly quantified. Fecal BKV excretion was positive in 16/40 patients, of whom 10 were transient (<3 consecutively positive samples), six were persistent (> or =3 consecutively positive samples) and three were persistent with peaking (> or =10(3)-fold increase in viral load over baseline, reaching 5.11 x 10(6), 4.68 x 10(7) and 2.75 x 10(8) copies/sample at 14, 14 and 21 days post-HSCT, respectively). Urinary BKV excretion was positive in 25/40 patients. Fecal BKV excretion was significantly correlated with that of the urine (P=0.036) and was significantly associated with allogeneic HSCT (P=0.037) and persistent and peaking of urinary BKV excretion (P<0.001). Binary logistic regression showed that BKV viruria was the only significant risk factor for fecal BKV excretion (P=0.021). Fecal BKV excretion occurred in 40% patients undergoing HSCT, implicating the gastrointestinal tract as a BKV latency site.

Safety of vaccinating sibling donors with live-attenuated varicella zoster vaccine before hematopoietic stem cell transplantation.

Reactivation of varicella zoster virus (VZV), clinically manifested as herpes zoster (HZ) is a common complication after hematopoietic stem cell transplantation (HSCT). The optimum prophylaxis for this disease has not been defined. In this study, we examined the effects of vaccinating donors with a live-attenuated vaccine with particular reference to their immune responses and the outcome of HSCT patients. Forty prospective HLA-matched sibling donors were vaccinated before HSCT. There were humoral immune responses in both sero-positive (P<0.01) and sero-negative (P=0.058) donors. Cellular immune response was assayed in 26 donors. Significant correlation was observed between cellular immune responses as enumerated by thymidine incorporation and interferon gamma secretion (P<0.001) and the latter was used in subsequent analyses. Significant response was observed in sero-negative (6/26) and a group of sero-positive (13/26) donors while 7/26 sero-positive donors showed no response. Thirty-four HSCT were performed. These patients have a lower, albeit insignificant, risk of HZ compared with historical controls and only 3/34 patients developed single dermatomal HZ at 6, 9 and 28 months after HSCT. No patients developed VZV-related mortality. Vaccinating donors with live-attenuated VZV vaccine was safe, but whether it confers a significant protection to the patients would require further study.

Foam sclerotherapy of venous malformations.

Venous malformations may occur either as localized or segmental lesions. Radiologic imaging defines the extent of involvement but magnetic resonance imaging is the best modality: it gives a bright hypersignal on T2-weighted spin-echo sequences. During a 30-month period, 1427 patients were investigated for venous disorders and 1% were found to have venous angiomata (9 women and 5 men). The age range was 15 to 76 years (mean 30.8+/-18.6 years). Foam was produced by the Tessari technique using 1% or 2% concentration of polidocanol. The duplex Doppler was used for ultrasound guidance to insure intravenous flow of foam and to monitor effects of treatment. A goal of pain-free healing of ulcers or cosmetic improvement was set for each patient. The mean number of treatments was 3.6+/-2.8 (range 1-10). Pain-free healing was achieved in patients with non-healing ulcerations and cosmetically, all of the patients were improved. Sclerosant foam is useful in treating low-flow venous malformations.

Rapidly progressive necrotising fasciitis following a stonefish sting: a report of two cases.

Two patients developed rapidly progressive necrotising fasciitis after being stung by a stonefish. Both were given a hot-water bath for pain relief. The hot water may have accelerated bacterial growth and the consequent development of necrotising fasciitis. Vibrio vulnificus was cultured from one patient. It is recommended that patients should receive high dose of oral and intravenous antibiotic prophylaxis for vibrio prevention. Antibiotics should be given before or during, not after, a hot-water bath, and the patient's condition should be monitored closely.

Molecular diagnostics in tuberculosis.

Molecular diagnostics in tuberculosis has enabled rapid detection of Mycobacterium tuberculosis complex in clinical specimens, identification of mycobacterial species, detection of drug resistance, and typing for epidemiological investigation. In the laboratory diagnosis of tuberculosis, the nucleic acid amplification (NAA) test is rapid and specific but not as sensitive as culture of mycobacteria. The primary determinant of successful NAA testing for tuberculosis depends on the shedding of mycobacterial DNA in secretions from caseating granulomas and its dissemination into sterile body fluids or tissue biopsies. In multibacillary diseases with a high mycobacterial load, a positive Ziehl-Neelsen smear with a positive NAA test is diagnostic of active tuberculosis, whereas a positive Ziehl-Neelsen smear with a negative NAA test in the absence of inhibitors would indicate nontuberculous mycobacterial disease. The role of the NAA test is more important in paucibacillary diseases with low mycobacterial loads. The presence of polymerase chain reaction (PCR) inhibitors, however, especially in extrapulmonary specimens, may produce false-negative results. Although this problem can be overcome to some extent by extra extraction steps, the additional processing invariably leads to the loss of mycobacterial DNA. To circumvent this problem, a brief culture augmentation step is carried out before the NAA test is performed, which can enhance the mycobacterial load while concomitantly diluting inhibitors, thereby maintaining the sensitivity of the test without excessively increasing turnaround time.

Toxoplasma gondii serology and stem cell transplantation in Chinese.

We report the screening and monitoring results of toxoplasma activity in 602 cases of hematopoietic stem cell transplantation (HSCT) in Chinese. A total of 13 recipients and 12 donors (including one donor-recipient pair) were serologically positive for toxoplasma, giving an incidence of 2.1% and 2.3%, respectively. All patients except those with glucose-6-phosphate dehydrogenase (G6PD) deficiency received sulfamethoxazole-trimethoprim (septrin) prophylaxis post-HSCT. None of the 292 deaths, including eight seropositive (either donor or recipient) cases, were attributable to toxoplasma. Only two of 16 seropositive survivors remained seropositive, while none of 60 seronegative cases (including six with G6PD deficiency) seroconverted. We concluded that in low incidence areas, toxoplasmosis is not a significant complication of HSCT with septrin prophylaxis. Most patients are also expected to remain seronegative after HSCT.

Medical treatment of viral pneumonia including SARS in immunocompetent adult.

Since no randomized controlled trials have been conducted on the treatment of viral pneumonia by antivirals or immunomodulators in immunocompetent adults, a review of such anecdotal experience are needed for the more rational use of such agents. Case reports (single or case series) with details on their treatment and outcome in the English literature can be reviewed for pneumonia caused by human or avian influenza A virus (50 patients), varicella zoster virus (120), adenovirus (29), hantavirus (100) and SARS coronavirus (SARS-CoV) (841). Even with steroid therapy alone, the mortality rate appeared to be lower when compared with conservative treatment for pneumonia caused by human influenza virus (12.5% vs. 42.1%) and hantavirus (13.3% vs. 63.4%). Combination of an effective antiviral, acyclovir, with steroid in the treatment of varicella zoster virus may be associated with a lower mortality than acyclovir alone (0% vs. 10.3%). Combination of interferon alfacon-1 plus steroid, or lopinavir/ritonavir, ribavirin plus steroid were associated with a better outcome than ribavirin plus steroid (0% vs. 2.3% vs. 7.7%, respectively). Combination of lopinavir/ritonavir plus ribavirin significantly reduced the virus load of SARS-CoV in nasopharyngeal, serum, stool and urine specimens taken between day 10 and 15 after symptom onset when compared with the historical control group treated with ribavirin. It appears that the combination of an effective antiviral and steroid was associated with a better outcome. Randomized therapeutic trial should be conducted to ascertain the relative usefulness of antiviral alone or in combination with steroid.