Multiplex sequencing of SARS-Cov-2 genome directly from clinical samples using the Ion Personal Genome Machine (PGM).

Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64- Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.

Multiplex sequencing of SARS-Cov-2 genome directly from clinical samples using the Ion Personal Genome Machine (PGM)

@#Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.

Psychological and work-related factors predicting work engagement in Malaysian employees.

BACKGROUND: Past research on work engagement has focused almost exclusively on either psychological or work-related factors in almost wholly separate literature. There is therefore a need to examine how these factors collectively influence work engagement. AIMS: To determine levels of work engagement and to identify psychological and work-related characteristics predicting work engagement in employees in Malaysia. METHODS: We recruited 5235 employees from 47 public and private organizations in Malaysia who responded to an online health survey. We assessed work engagement with the 9-item Utrecht Work Engagement Scale (UWES-9) and psychological distress using the 6-item Kessler scale. We performed multiple linear regression to determine predictors of work engagement. RESULTS: Employee mean age was 33.8 years (standard deviation [SD] ± 8.8). The mean work engagement score on the UWES-9 was 3.53 (SD ± 0.94). Eleven of 18 variables on multiple regression predicted work engagement, F(18, 4925) = 69.02, P < 0.001, R2 = 0.201. Factors that predicted higher work engagement were age, marital status, education level, job type, job permanency, longer sleep duration, lower psychological distress and no history of workplace bullying. CONCLUSIONS: Key factors associated with poorer work engagement in Malaysian employees include inadequate sleep, psychological distress and a history of workplace bullying. These are modifiable factors that individuals and employers can target to improve work engagement, ideally tailored according to occupational type.

Detailed assessment of nutritional status and eating patterns in children with gastrointestinal diseases attending an outpatients clinic and contemporary healthy controls.

BACKGROUND/OBJECTIVES: In the era of modern multidisciplinary clinical management, very little is known about the prevalence and presentation of malnutrition in children with gastrointestinal disorders (GastroD) particularly employing composite, global measures of nutritional status. SUBJECTS/METHODS: Anthropometry, body composition, dietary intake, eating habits and grip strength were assessed with bedside methods in 168 patients from outpatient gastroenterology clinics (n, median (IQR) years; Crohn's disease (CD): n=53, 14.2 (11.6:15.4); ulcerative colitis (UC): n=27, 12.2 (10.7:14.2); coeliac disease: n=31, 9.3 (7.5:13.6); other GastroD: n=57, 9.8 (7.2:13.8)) and compared with 62 contemporary healthy controls (n, median (IQR): 9.8 (6.9:13.8)) and the results of the recent UK, National Diet and Nutritional Survey (NDNS). RESULTS: Children with CD had lower BMI z-scores than controls (median (IQR): -0.3 (-0.9:0.4) vs 0.3 (-0.6:1.4); P=0.02) but only 2% were classified as thin (BMI z-score <-2 s.d.). The prevalence of obesity in children with UC was 19%, 6% in CD, 11% in children with other GastroD and 15% in controls. No difference was found in grip strength measurement between groups. Except for CD children, the proportion of patients with suboptimal micronutrient intake was similar to that of controls and the cohort of children from the latest NDNS. A higher proportion of children with CD had suboptimal intake for riboflavin, vitamin B6 and calcium and consumed significantly more meat products, juices (including carbonated drinks), spreads/jams and crisps and savoury snacks and significantly fewer portions of dairy, fish, fruits and vegetables compared with healthy controls. CONCLUSIONS: GastroD affect children's body composition, growth, strength, dietary intake and eating habits, particularly CD, but to a lesser extent than expected.

Magnetic nanoparticles encapsulated within a thermoresponsive polymer.

This study describes a facile two-step approach to modify the surface of nanoparticles, thereby imparting a core-shell structure to the system. The core consists of magnetic nanoparticles and the shell is composed of thermoresponsive hydroxypropyl cellulose, using a coupling agent to covalently bind the core to the shell. Hydroxypropyl cellulose is known for its biocompatibility and biodegradability, and its thermoresponsive properties make it an excellent candidate for fabricating biocompatible stimuli-responsive magnetic nanoparticles. We report the synthesis of magnetic nanoparticles and the successful binding of the polymer to them. X-ray diffraction studies show that the surface modification of the magnetic nanoparticles does not result in any phase change and the size of the magnetic core thus calculated (7 nm) reveals that such hybrid core-shell system is superparamagnetic in nature, as further confirmed by magnetization measurements. The size obtained by X-ray diffraction is in good agreement with that obtained by transmission electron microscope. Evidence of binding is given by Fourier transform infrared spectroscopy and a quantitative analysis of the polymeric content obtained by thermogravimetry analysis. Dynamic light scattering as a function of temperature reveals the thermoresponsive behavior of the particles with a lower critical solution temperature around 41 degrees C, which is also the temperature at which cellulose undergoes a coil-to-globule transition.

Identification of novel BRCA large genomic rearrangements in Singapore Asian breast and ovarian patients with cancer.

Large genomic rearrangements have been reported to account for about 10-15% of BRCA1 gene mutations. Approximately, 90 BRCA rearrangements have been described to date, all of which but one have been reported in Caucasian populations of predominantly Western European descent. Knowledge of BRCA genomic rearrangements in Asian populations is still largely unknown. In this study, we have investigated for the presence of BRCA rearrangements among Asian patients with early onset or familial history of breast or ovarian cancer. Using multiplex ligation-dependent probe amplification (MLPA), we have analyzed 100 Singapore patients who previously tested negative for deleterious BRCA mutations by the conventional polymerase chain reaction-based mutation detection methods. Three novel BRCA rearrangements were detected, two of which were characterized. The patients with the rearrangements, a BRCA1 exon 13 duplication, a BRCA1 exon 13-15 deletion and a BRCA2 exon 4-11 duplication, comprise 3% of those previously tested negative for BRCA mutations. Of the BRCA1 and BRCA2 pathogenic mutations identified in our studies on Asian high-risk breast and ovarian patients with cancer to date, these rearrangements constitute 2/19 and 1/2 of the BRCA1 and BRCA2 pathogenic mutations, respectively. Given the increasing number of rearrangements reported in recent years and their contribution to the BRCA mutation spectrum, the presence of BRCA large exon rearrangements in Asian populations should be investigated where clinical, diagnostic service is recommended.

The future of medical education: the second 100 years.

This is a proud year for the medical profession in Singapore, as we celebrate 100 years of medical education. A centennial should force us to ponder whether we are producing a doctor that meets Singapore's future needs. A few of the issues that we face include how much time staff allocate to quality teaching; the continual loss of many of the best senior clinical staff in all the teaching hospitals; whether the medical profession should reconsider what, and how, it teaches given the pace of new knowledge; how we incorporate advances into standard practice; how we incorporate issues such as patient safety and effective communication into a curriculum already overcrowded with traditional topics; and how we subsidise the cost of medical education. We have undertaken a major revision of how we choose applicants, the content of the curriculum, how it is taught, the way it is assessed, and the means to recruit and retain role models in academic medicine.

Therapeutic strategies in gastric cancer.

Gastric cancer continues to be a major public health problem and is the second most common cause of cancer-related deaths in the world. These statistics led the American Society of Clinical Oncology (ASCO) International Affairs Committee to choose gastric cancer as the topic for the International Symposium held at the 2003 ASCO Annual Meeting. Dr Yoshiaki Ito will discuss the role of RUNX3 in the genesis and progression of human gastric cancer. Dr Pelayo Correa will present a compelling argument on the use of Helicobacter pylori therapy and antioxidants in selected high-risk population as chemoprevention strategies for gastric cancer. The controversy regarding the role of extended lymph node dissection for gastric cancer will be discussed by Dr Cornelis J.H. Van De Velde and Dr Mitsuru Sasako. Dr Van De Velde will present the European surgical approach to gastric cancer, and Dr Sasako will review the Japanese experience. The issues of whether certain patients benefit from more aggressive surgical dissection and the potential risks compared with benefits will also be discussed. Dr John Macdonald will discuss the role of adjuvant chemotherapy and adjuvant chemoradiotherapy in resected gastric cancer, as well as the role of chemotherapy in metastatic gastric cancer.

Anticancer drug discovery and development throughout the world.

This year's American Society of Clinical Oncology International Symposium devoted 2 hours to a lively discussion of various aspects of anticancer drug discovery and development throughout the world. The scientific program started with an overview of efforts directed toward promoting international collaboration in natural product-derived anticancer drug discovery. This was followed by a discussion on the importance of interethnic differences and pharmacogenetics in anticancer drug development. Thereafter, this part of the program was completed by a description of the activities of the newly created Singapore-Hong Kong-Australia Drug Development Consortium and an overview of the contribution of Japan to anticancer drug development. The logistics and regulatory aspects of clinical trials with new anticancer agents in different parts of the world were then presented, with an emphasis on Europe, North America, and Japan. The program was completed with a panel discussion of the efforts to harmonize the exchange of clinical data originating from one region of the globe with other territories, with input from official representatives of the United States Food and Drug Administration and the Medical Devices Evaluation Center of Japan.

Eosinophilia and fleeting pulmonary shadows in a middle aged man.

We present an unusual case of a malignancy associated with severe eosinophilia and fleeting pulmonary shadows. Initially limited to the lung, the illness progressed to involve multiple organs with marked increase in eosinophilia.

Clinical characteristics and treatment outcome of 218 patients with non-Hodgkin's lymphoma in a Singaporean institution.

A retrospective analysis of 218 patients with non-Hodgkin's lymphoma (NHL) seen at a single institution in Singapore over a ten-year period was conducted. Twenty percent, 56% and 24% of patients had low-, intermediate- and high-grade disease respectively using the Working Formulation, and 25% of patients immunophenotyped had T-cell NHL. Forty-nine percent had primary extranodal disease, with the commonest sites of involvement being the gastrointestinal tract, nasal cavity, and tonsils. 86% and 73% respectively of patients with intermediate and high grade disease received combination chemotherapy as first line treatment, with CHOP being the most commonly used regime. Seventy-four percent of patients with low grade lymphoma received first line chemotherapy, 5% each was treated with radiotherapy or surgery alone, and 21% was treated symptomatically. Patients with low grade B-cell lymphoma had a 5-year survival of 80% and 10-year survival of 42%. One-year survival for intermediate and high grade B-cell lymphoma was 76% and 42%, while 2-year survival was 67% and 42% respectively. 1-, 2-, and 3-year survival for patients with T-cell lymphoma was 67%, 46% and 37% respectively. The difference in survival between low-, intermediate- and high-grade B-cell lymphoma was statistically significant (p = 0.0018 using the log rank test), but that between B- and T-cell lymphoma was not. Using the Cox regression model, International prognostic index, grade and extranodal disease were found to be statistically significant predictors of survival (p = 0.0001, p = 0.0157, p = 0.0343) respectively.

Phase II trial of docetaxel in Asian patients with inoperable stage III non-small cell lung cancer.

Docetaxel has a response rate of greater than 30% in first-line treatment of Western patients with advanced non-small cell lung cancer (NSCLC). The goal of this open-label. phase II study was to evaluate the activity and safety profile of docetaxel in Asian patients with inoperable untreated stage III NSCLC. Docetaxel was given at 100 mg/m2 as a 1-h infusion every 3 weeks. Prophylactic dexamethasone was given to reduce hypersensitivity reactions and edema. Thirty-five patients were enrolled in the study. The response rate was 34% (95% CI, 19%-50%) according to intent-to-treat analysis. No complete response was observed. Twenty-four patients (69%) had grade 3 or 4 neutropenia in cycle 1, and febrile neutropenia was seen in 12 patients. Six patients (17%) experienced mild fluid retention. Docetaxel is an active agent in first-line treatment of Asian patients with locally advanced NSCLC, with the main toxicity being neutropenia. Fluid retention was a minor problem in this study.

A prospective randomized study of Chop versus Chop plus alpha-2B interferon in patients with intermediate and high grade non-Hodgkin's lymphoma: the International Oncology Study Group NHL1 Study .

UNLABELLED: The addition of a brief alpha interferon regimen to each CHOP induction cycle, plus one year of alpha interferon thrice weekly maintenance therapy, has no early effect on response rates or survival in patients with Intermediate or High grade cell NHL. BACKGROUND: The CHOP (Cyclophosphamide, Adriamycin. Vincristine, Prednisone) regimen is the most widely used first-line therapy for patients with Intermediate or High Grade (IG/HG) non-Hodgkin's lymphoma (NHL). Alpha 2b interferon (INF) enhances response rates and improves survival in low-grade NHL. The International Oncology Study Group (IOSG) conducted a prospective randomized study comparing CHOP alone or combined with INF in patients with IG/HG-NHL. The primary study aim was to compare the objective response rates in these patient cohorts. PATIENTS AND METHODS: Patients with a confirmed diagnosis of measurable NHL of International Working Formulation (IWF) groups D to H histology were randomized to receive CHOP alone or CHOP with 5Mu INF s.c. for 5 days on days 22 to 26 of each 28 day cycle with INF 5 million units (Mu) given three times per week subcutaneously for 52 weeks in those patients who responded to CHOP plus INF. RESULTS: The overall response rates were equivalent in both groups: CHOP alone (214 patients) 81% (complete 55%, partial 26%); CHOP plus INF (221 patients) 80% (complete 54%, partial 26%). At 36 months, the actuarial survival rate was equivalent in both groups. CONCLUSIONS: There is no apparent early advantage in terms of response or survival conferred by adding the study INF regimen to CHOP therapy for patients with IG/HG-NHL.

Screening for colorectal cancer in Singapore.

The incidence of colorectal cancer in Singapore has risen relentlessly since 1955. Today, the large bowel is the most frequent gastrointestinal cancer site among both men and women. Survival with this cancer has shown little improvement during this period. The prospects for further reduction in mortality through more radical surgery, adjuvant radiotherapy, chemo- or immunotherapy remain limited. Theoretically, detection of colorectal cancer at an earlier stage or detection of its precursors will reduce mortality. Screening for colorectal cancer is advocated to achieve this end. Success with screening programmes will depend on the diffusion of current knowledge about this disease to both health professionals and the general public.

Hypoglycaemia misdiagnosed by capillary glucose measurement.

The diagnosis of hypoglycaemia has become much easier with the introduction of bedside capillary glucose measurements which require a single drop of blood from a finger prick. However, such a diagnosis may be wrongly made in patients with poor peripheral circulation. This case report illustrates the common circumstances where misdiagnosis occurs A drop of venous blood should be used for bedside glucose measurement if the diagnosis is in doubt.