Iron burden and endocrine complications in transfusion-dependent thalassemia patients In Sarawak, Malaysia: a retrospective study.

INTRODUCTION: Thalassaemia is one of the major health problems in Malaysia. With safe blood transfusion regime, the lifespan of patients with transfusion-dependent thalassaemia (TDT) has improved but at the cost of a higher risk of developing endocrine disorders. It is crucial for us to monitor the iron overload to prevent end organ damage. This study aims to evaluate the iron burden and prevalence of endocrinopathies in patients with TDT in Sarawak. MATERIALS AND METHODS: This retrospective cohort study was conducted between January 2020 to June 2020 in six government hospitals in Sarawak. A total of 89 patients with TDT, aged 10 years and above, were recruited. RESULTS: Out of the 89 patients, there were 54 males (60.7%) and 35 females (39.3%) with a median age of 21 years (range 10.0-65.0). Sixty-seven (75.3%) patients had betathalassaemia major and 15 (16.9%) patients had haemoglobin E beta-thalassaemia (HbE beta-thalassaemia), remaining seven patients had other genotypes. Thirty-one (34.8%) patients had mean serum ferritin 2500ng/ml and above, and 44 (66.6%) had liver iron concentration (LIC) ≥7mg/g. The prevalence of endocrine disorders in our cohort was 69.7%. The most common endocrinopathies were short stature (n=46, 51.7%), followed by hypogonadism (n=24, 26.9%), delayed puberty (n=23, 25.8%), hypothyroidism (n=10, 11.2%), diabetes mellitus (n=9, 10.1%), impaired glucose tolerance (n=6, 6.7%) and hypoparathyroidism (n=3, 3.3%). Endocrinopathies were significantly associated with age (p=0.01), age at initiating regular blood transfusion (p<0.01) and duration of regular blood transfusion (p<0.01). CONCLUSION: Our data shows that the development of endocrinopathies in TDT can be time dependent. Early detection of endocrine-related complications and prompt treatment with iron chelation therapy are important to improve morbidity and mortality. A multidisciplinary approach with good patient-doctor collaboration is the key to improving patient care in our settings.

Demographics and outcome of patients with congenital haemophilia in Sarawak, Malaysia.

INTRODUCTION: Sarawak has a population that is geographically and characteristically widely varied. This study aimed to determine the demographic profile of patients in Sarawak, Malaysia. Materials and Methods - A cross-sectional study was conducted in 2019 at four major haemophilia treatment centres in Kuching, Sibu, Bintulu and Miri Hospitals, Sarawak. Demographic and clinical data were collected with consents from patients. RESULTS AND DISCUSSION: Ninety-six haemophilia patients were identified - 79(82.3%) haemophilia A(HA) and 17(17.7%) haemophilia B(HB). Severe haemophilia patients were noted in 45.6% (36/79) of HA and 64.7% (11/17) of HB. In all 44.3% of the HA and 52.9% of the HB population had no identifiable family history of haemophilia. Two-thirds of the patients with severe HA were on prophylaxis [24/36 (66.7%)] and only onethird [4/11 (36.4%)] in severe HB. Inhibitors developed in 9/79 (11.4%) of the HA population [3/79 (3.8%) high responders]. The median inhibitor titre was not significantly different between the different treatment groups - on demand versus prophylaxis (1.0BU versus 2.0BU; z statistic -1.043, p-value 0.297, Mann-Whitney test). None of the patients developed inhibitory alloantibodies to factor IX. Four HA patients (5.1%) underwent immune tolerance induction where one case had a successful outcome. Three severe HA patients received emicizumab prophylaxis and showed remarkable reduction in bleeding events with no thromboembolic events being reported. One female moderate HA patient received PEGylated recombinant anti-haemophilic factor. Eleven patients underwent radiosynovectomy. One mild HB patient succumbed to traumatic intracranial bleeding. Our data reported a prevalence (per 100,000 males) of 5.40 cases for all severities of HA, 2.46 cases for severe HA; 1.16 cases for all severities of HB, and 0.75 cases for severe HB. The overall incidence of HA and HB was 1 in 11,500 and 1 in 46,000, respectively. CONCLUSION: This study outlines the Sarawakian haemophilia landscape and offers objective standards for forward planning. Shared responsibilities among all parties are of utmost importance to improve the care of our haemophilia population.

Paediatric middle aortic syndrome with endovascular treatment in East Malaysia.

Middle-aortic syndrome is a surgically curable cause of childhood hypertension. Open surgery is traditionally offered but with the advance of medical technology, endovascular approached is available in many country. Failure to control BP in open surgery is as low as 4.1% compares to 13% in endovascular approaches. However, mortality is 4% in open surgery almost 2 times higher than 2.3% in endovascular approach. This article presents a case of 10 years old child treated successfully without complication with endovascular balloon dilatation, as a first case of such disease in East Malaysia.

Interventions for preventing cardiomyopathy due to anthracyclines: a Bayesian network meta-analysis.

Background: The relative efficacy of interventions for primary prevention of anthracycline-associated cardiotoxicity is unknown. Methods: We conducted a systematic review of randomized controlled trials for primary prevention of anthracycline-associated cardiotoxicity in adult cancer patients. We used hierarchal outcome definitions in the following order of priority: (1) composite of heart failure or decline in left ventricular ejection fraction, (2) decline in ejection fraction, or (3) heart failure. Data were analyzed using a Bayesian network meta-analysis with random effects. Results: A total of 16 trials reported cardiotoxicity as a dichotomous outcome among 1918 patients, evaluating dexrazoxane, angiotensin antagonists, beta-blockers, combination angiotensin antagonists and beta-blockers, statins, Co-enzyme Q-10, prenylamine, and N-acetylcysteine. Compared with control, dexrazoxane reduced cardiotoxicity with a pooled odds ratio (OR) of 0.26 (95% credible interval [CrI] 0.11-0.74) and had the highest probability (33%) of being most effective. No other agent was demonstrably better than placebo. Angiotensin antagonists had an 84% probability of being most effective in a sensitivity analysis excluding one outlying study (OR 0.06 [95% CrI 0.01- 0.24]). When the outcome was restricted to heart failure, dexrazoxane was associated with an OR of 0.12 (95% CrI 0.06-0.23) relative to control and had 58% probability of being most effective, while angiotensin antagonists had an OR of 0.18 (95% CrI 0.05-0.55). Available data suggested that dexrazoxane and angiotensin antagonists did not affect malignancy response rate or risk of death. Conclusion: Moderate quality data suggest that dexrazoxane, and low quality data suggest angiotensin antagonists, are likely to be effective for cardiotoxicity prevention.

Clinical spectrum of children receiving palliative care in Malaysian Hospitals.

INTRODUCTION: Awareness for paediatric palliative care has resulted in the impetus for paediatrician-led palliative care services across Malaysia. However, there is paucity of local data on patients receiving hospital-based paediatric palliative care. We aim to review the clinical spectrum of patients referred to these services. METHODS: An observational study of children aged between 0-18 years receiving palliative care at 13 hospitals between 1st January and 31st December 2014 was carried out. RESULTS: There were 315 patients analysed, 90 (28.6%) and 46 (14.6%) were neonates and adolescents respectively. The main ICD-10 diagnostic categories for all patients were identified to be 'Congenital malformations, deformations and chromosomal abnormalities' 117 (37.1%), 'Diseases of nervous system' 76 (24.1%) and 'Neoplasms' 60 (19.0%). At referral 156 (50%) patients had holistic needs assessments. Patients with 'Diseases of nervous system' were assessed to have significantly more physical needs than the other two diagnostic categories. Majority of patients who knew of their diagnosis and prognosis were those with malignancy. Over a fifth of referrals were at their terminal admission. Of 144 who died, 111 (77.1%) had advanced care plans. There was bereavement follow-up in 98 (68.1%) patients. CONCLUSION: Patients referred for palliative care have varied diagnoses and needs. To ensure all paediatricians are competent to deliver quality care to all children, further education and training initiatives is imperative.

Frequent occurrence of gastric cancer in Asian kindreds with Li-Fraumeni syndrome.

Type of cancer and age of onset in individuals with inherited aberrations in the tumour suppressor gene TP53 are variable, possibly influenced by genetic modifiers and different environmental exposure. Since 2009, the modified Chompret criteria (MCC) have been used to identify individuals for TP53 mutation screening. Using the TP53 mutation database maintained by the International Agency for Research on Cancer (IARC), we investigated if the MCC, mainly developed for a Caucasian population, was also applicable in Asia. We identified several differences in Asian families compared with similar Caucasian cohorts, suggesting that identification and management of Li-Fraumeni syndrome in Asia do not completely mirror that of North America and Western Europe. Early gastric cancer (<40 years) may be considered a new addition to the MCC especially for Asian families.

Prevention of early-onset Group B Streptococcal disease - the Northern Ireland experience.

OBJECTIVES: To ascertain guideline adherence for prevention of Group B Streptococcal (GBS) neonatal infection and establish prevalence and outcomes in Northern Ireland (NI). DESIGN: Retrospective observational study. SETTING: Northern Ireland maternity units. POPULATION: Using NI Health Information Systems the following were identified: (1) a cohort of women with one or more risk factors for GBS disease in 2009-2010, (2) all culture-positive cases of GBS in babies aged 0-89 days (2008-2010), (3) stillbirths due to GBS (2009-2010). METHODS: Information was analysed for a 15% randomised sample of the available cases. Maternal and infant case notes were reviewed for confirmed cases of neonatal early onset GBS (EOGBS) during 2008-2010. MAIN OUTCOME MEASURES: Adherence to the 2003 RCOG guideline on prevention of GBS disease (2009-2010). Number of neonatal GBS infections: antenatal risk factors, management and neonatal outcomes (2008-2010). The number of stillbirths related to GBS (2009-2010). RESULTS: Five hundred and seventy-four women had one or more identifiable risk factors for GBS disease; intrapartum antibiotic prophylaxis (IAP) was administered in 42% of cases. Improved administration of IAP was noted in the presence of escalating risk factors. At best, guideline adherence was 50-70%. Forty-three neonates had proven early-onset Group B Streptococcal disease; 55.8% had maternal risk factors. Of the total identified cases, 25.5% received IAP. The total mortality rate was 11.46%. The incidence of EOGBS disease in NI was 0.57/1000 live births. CONCLUSIONS: Prevalence of EOGBS is higher in NI than the UK as a whole. Risk factors are present in 55.8% of mothers; IAP does not prevent all cases of EOGBS.

An investigation of arsenic contamination in Peninsular Malaysia based on Centella asiatica and soil samples.

The first objective of this study was to provide data of arsenic (As) levels in Peninsular Malaysia based on soil samples and accumulation of As in Centella asiatica collected from 12 sampling sites in Peninsular Malaysia. The second objective was to assess the accumulation of As in transplanted C. asiatica between control and semi-polluted or polluted sites. Four sites were selected which were UPM (clean site), Balakong (semi-polluted site), Seri Kembangan (semi-polluted site) and Juru (polluted site). The As concentrations of plant and soil samples were determined by Instrumental Neutron Activation Analysis. The As levels ranged from 9.38 to 57.05 µg/g dw in soils, 0.21 to 4.33 µg/g dw in leaves, 0.18 to 1.83 µg/g dw in stems and 1.32-20.76 µg/g dw in roots. All sampling sites had As levels exceeding the CCME guideline (12 µg/g dw) except for Kelantan, P. Pauh, and Senawang with P. Klang having the highest As in soil (57.05 µg/g dw). In C. asiatica, As accumulation was highest in roots followed by leaves and stems. When the As level in soils were higher, the uptake of As in plants would also be increased. After the transplantation of plants to semi-polluted and polluted sites for 3 weeks, all concentration factors were greater than 50 % of the initial As level. The elimination factor was around 39 % when the plants were transplanted back to the clean sites for 3 weeks. The findings of the present study indicated that the leaves, stems and roots of C. asiatica are ideal biomonitors of As contamination. The present data results the most comprehensive data obtained on As levels in Malaysia.

Factors affecting team leadership skills and their relationship with quality of cardiopulmonary resuscitation.

OBJECTIVE: This study aims to explore the relationship between team-leadership skills and quality of cardiopulmonary resuscitation in an adult cardiac-arrest simulation. Factors affecting team-leadership skills were also assessed. DESIGN: Forty advanced life-support providers leading a cardiac arrest team in a standardized cardiac-arrest simulation were videotaped. Background data were collected, including age (in yrs), sex, whether they had received any leadership training in the past, whether they were part of a professional group, the most recent advanced life-support course (in months) they had undergone, advanced life-support instructor/provider status, and whether they had led in any cardiac arrest situation in the preceding 6 months. MEASUREMENTS AND MAIN RESULTS: Participants were scored using the Cardiac Arrest Simulation test score and Leadership Behavior Description Questionnaire for leadership skills. Process-focused quality of cardiopulmonary resuscitation data were collected directly from manikin and video recordings. Primary outcomes were complex technical skills (measured as Cardiac Arrest Simulation test score, preshock pause, and hands-off ratio). Secondary outcomes were simple technical skills (chest-compression rate, depth, and ventilation rate). Univariate linear regressions were performed to examine how leadership skills affect quality of cardiopulmonary resuscitation and bivariate correlations elicited factors affecting team-leadership skills.Teams led by leaders with the best leadership skills performed higher quality cardiopulmonary resuscitation with better technical performance (R = 0.75, p < .001), shorter preshock pauses (R = 0.18, p < .001), with lower total hands-off ratio (R = 0.24, p = .01), and shorter time to first shock (R = 0.14, p = .02). Leadership skills were not significantly associated with more simple technical skills such as chest-compression rate, depth, and ventilation rate. Prior training in team leader skills was independently associated with better leadership behavior. CONCLUSIONS: There is an association between team leadership skills and cardiac arrest simulation test score, preshock pause, and hands off ratio. Developing leadership skills should be considered an integral part of resuscitation training.

Molecular characterisation of hemagglutinin and neuraminidase genes of the first highly pathogenic Avian Influenza H5N1 2.3.2.1c virus isolated from Sabah, Malaysia.

Highly Pathogenic Avian Influenza (HPAI) is a highly contagious disease in poultry. The outbreaks can lead to flock mortality up to 100% in two to three days. In July 2018, high mortality in a commercial layer farm in Kauluan village, Sabah was reported. Samples were sent to Veterinary Research Institute Ipoh for diagnosis. Virus isolation and molecular detection is carried out simultaneously. The causative agent was then identified as AI H5N1 virus by real time reverse transcription-polymerase chain reaction (RT-PCR). The virus was then subjected for further nucleotide sequencing of full length hemagglutinin (HA) and neuraminidase (NA) gene. The PQRERRRKR/GLF motif at the HA cleavage site indicated that the isolate was of HPAI virus. Phylogenetic analysis of the HA gene showed that the isolate was belonged to the clade 2.3.2.1c virus. In the HA gene, besides the S133A substitution, the virus possesses conserved amino acid at most of the avian receptor binding sites including the glutamine (Q) and glycine (G) at position 222 and 224 respectively, indicating that the virus retains the avian-type receptor binding preference. As such, the zoonotic potential of the virus was relatively low. On the other hand, though the N154D and T156A substitution were detected in the same gene, the pandemic potential of this Sabah 2.3.2.1c virus is low in the absence of the Q222L, G224S, H103Y, N220K and T315I. A typical 20 amino acid deletion with loss of four corresponding glycosylation sites in the NA stalk region was visible. Though three NA resistance markers were detected, the virus was predicted to be sensitive to NA inhibitor. This is the first HPAI H5N1 outbreak in Sabah. The introduction of this virus into East Malaysia for the first time raised an alert alarm of the future epidemic potential. Strict farm biosecurity, continuous surveillance programme in poultry, wild birds, migratory birds; molecular epidemiology as well as risk assessment for the virus with pandemic potential are needed in dealing with emergence of new influenza virus in the country.

Safety and outcomes of short-term use of peripheral vasoactive infusions in critically ill paediatric population in the emergency department.

Early restoration of oxygen delivery to end organs in paediatric patients experiencing shock states is critical to optimizing outcomes. However, obtaining central access in paediatric patients may be challenging in non-intensive care settings. There is limited literature on the use of peripheral vasoactive infusions in the initial resuscitation of paediatric patients in the emergency department. The aims of this study were to report the associated complications of peripheral vasoactive infusions and describe our local experience on its use. This was a single-centre, retrospective study on all paediatric patients who received peripheral vasoactive infusions at our paediatric emergency department from 2009 to 2016. 65 patients were included in this study. No patients had any local or regional complications. The mean patient age was 8.29 years old (± 5.99). The most frequent diagnosis was septic shock (45, 69.2%). Dopamine was the most used peripheral vasoactive agent (71.2%). The median time to central agents was 2 h (IQR 1-4). 16(24.2%) received multiple peripheral infusions. We reported no complications of peripheral vasoactive infusions. Its use could serve as a bridge till central access is obtained. Considerations on the use of multiple peripheral vasoactive infusions in the emergency department setting needs further research.

Cross-clade immunogenicity and safety of an AS03A-adjuvanted prepandemic H5N1 influenza vaccine in Hong Kong.

OBJECTIVE: To present Hong Kong'Äìspecific data from a large Asian population (also involving Thailand, Singapore, and Taiwan) on safety and manufacturing consistency across four AS03(A)-adjuvanted H5N1 vaccine formulations in terms of immune response against the A/Vietnam/1194/2004 strain. Immunogenicity against the heterologous A/Indonesia/05/2005 strain was also assessed. NCT Number: 00449670. DESIGN: Prospective, observer-blind study. SETTING: Out-patient clinic of a tertiary hospital in Hong Kong. PARTICIPANTS: A total of 360 subjects aged 18 to 60 years were randomised into six groups to receive two doses (21 days apart) of the study vaccine. INTERVENTIONS: One of the four adjuvanted formulations (3.75 microgram H5N1 haemagglutinin [HA]+AS03(A)) of the vaccine (H5N1-AS03(A)) or one of the two non-adjuvanted (3.75 microgram H5N1 [HA]) formulations of the vaccine (H5N1-DIL). MAIN OUTCOME MEASURES: Blood samples collected before vaccination and 21 days after each vaccine dose were analysed using haemagglutination-inhibition and neutralisation assays. Solicited, unsolicited, and serious adverse events were recorded. RESULTS: Manufacturing consistency across all four vaccine formulations was demonstrated. After two doses, the AS03(A)-adjuvanted prepandemic influenza vaccine demonstrated high seroprotection rates against the A/Vietnam/1194/2004 strain (95.8%) and good immunogenicity against the heterologous A/Indonesia/05/2005 strain (45.7%), as compared to the non-adjuvanted vaccine (4.6% and 1.5%, respectively). The seroconversion rates induced by the adjuvanted formulations in terms of viral neutralising antibodies against the two strains were much higher than those induced by the non-adjuvanted formulations. There were no safety concerns for any of the adjuvanted vaccine formulations. CONCLUSIONS: The AS03(A)-adjuvanted H5N1 prepandemic influenza vaccine demonstrated good immunogenicity and an acceptable safety profile in Hong Kong.

Serum uric acid does not predict cardiovascular or all-cause mortality in type 2 diabetes: the Fremantle Diabetes Study.

AIMS/HYPOTHESIS: To determine whether serum uric acid: (1) is associated with cardiovascular disease (CVD) death and/or all-cause mortality in type 2 diabetes; and (2) consistent with published data, predicts these outcomes in older patients and those of southern European ethnicity. METHODS: We studied those 1,268 (98%) of 1,294 type 2 participants in the observational Fremantle Diabetes Study who had a fasting serum uric acid measured at baseline. Mortality data were collected over a mean (+/-SD) 10.3 +/- 3.9 years. Cox proportional hazards modelling was used to determine independent baseline predictors of CVD and all-cause death including fasting serum uric acid as a continuous variable and quartiles. RESULTS: During follow up, 525 deaths occurred (41.4% of the cohort) of which 271 (51.6%) were attributed to CVD. In univariate analyses, patients in the highest uric acid quartile had the greatest CVD and all-cause mortality (p = 0.007 and p = 0.001). After adjustment for significant variables in the most parsimonious model, baseline serum uric acid was not an independent associate of CVD or all-cause mortality whether entered as a continuous variable (HR 1.11 [95% CI 0.96-1.27] and 1.10 [95% CI 0.98-1.22] for a 0.1 mmol/l increase, respectively) or as quartiles (p > 0.10). Analyses of 638 patients >65 years of age and 231 of southern European ethnicity produced similar results. CONCLUSIONS/INTERPRETATION: Serum uric acid was not an independent predictor of CVD or all-cause mortality in our community-based type 2 patients. Fasting serum uric acid concentrations do not appear to be prognostically useful in type 2 diabetes.

Activation of the c-myb locus by viral insertional mutagenesis in plasmacytoid lymphosarcomas.

Rearrangement in the c-myb locus of each of four independently derived BALB/c plasmacytoid lymphosarcoma (ABPL's) is due to the insertion of a defective Moloney murine leukemia virus (M-MuLV) into a 1.5-kilobase-pair stretch of cellular DNA at the 5' end of the v-myb-related sequences. This retroviral insertion is associated with abnormal transcription of myb sequences and probably represents a step in the neoplastic transformation of ABPL cells.

Mild autosomal recessive osteopetrosis: successful treatment with bone marrow transplant.

We describe a 5 1/2 year old boy who was diagnosed with mild autosomal recessive osteopetrosis based on the presence of bony sclerosis, extramedullary haematopoeisis, leukoerythroblastosis and visual impairment who had an allogeneic bone marrow transplant from a matched sibling donor. Conditioning regime was busulphan 16 mg/kg and cyclophosphamide 200 mg/kg. Apart from transient hypercalcaemia, there were no major post transplant complications. Four years post transplant, the extramedullary haematopoeisis has resolved completely with normal blood counts. Apart from a fracture after a trivial fall two months after transplant, he has not suffered any fracture related limb deformities.

Evaluation of the VITEK 2 AST N-054 test card for the detection of extended-spectrum beta-lactamase production in Escherichia coli with CTX-M phenotypes.

OBJECTIVES: A new VITEK 2 antibiotic susceptibility testing (AST) card, AST N-054, was introduced for aerobic gram-negative bacilli in 2007 and has been widely adopted for routine use in the UK. We evaluated its performance for detecting extended-spectrum beta-lactamase (ESBL) production in Escherichia coli. METHODS: ESBL-producing faecal isolates of E. coli (n = 137) from residents in nursing homes were tested using the AST N-054 card on VITEK 2 and with MASTDISCS ID ESBL detection disc diffusion tests (Mast Diagnostics, Bootle, UK). The susceptibility result recommended by the VITEK 2 software was also recorded. RESULTS: The AST N-054 card detected ESBL production in 93 of the 137 isolates tested [test sensitivity 67.9% (95% CI, 59.7-75.1)]. E. coli strain A, a widespread lineage in the UK with a low-level CTX-M enzyme production, accounted for most of the detection failures, with 35/73 strain A isolates incorrectly reported versus 9/64 non-strain A isolates (P < 0.0001). The MASTDISCS correctly detected ESBL in 135/137 isolates [test sensitivity 98.5% (95% CI, 94.5-99.9)]. Of the 44 isolates found to be negative for ESBL production by VITEK 2, the Advanced Expert System misreported 29 as susceptible to cefotaxime and all as susceptible to ceftazidime and aztreonam. CONCLUSIONS: These data suggest that the AST N-054 card for the VITEK 2 system is less reliable than other previously reported cards for the detection of CTX-M beta-lactamase-producing E. coli circulating in the UK, particularly strain A isolates.

Molecular Characterization of Avian Infectious Bronchitis Virus Isolated in Malaysia during 2014-2016.

Avian Infectious Bronchitis (IB) is a highly contagious disease which can cause huge economic losses to the poultry industry. Forty five IB viruses (IBV) were isolated from poultry in Malaysia during 2014-2016. Phylogenetic analysis of the spike glycoprotein 1 (S1) gene revealed that all isolates were clustered into five distinct groups. The predominant type of IBV isolated was QX strains (47%), second was 4/91 type (27%), followed by Malaysian strain MH5365/95 (13%), Massachusetts type (11%) and finally Taiwanese strains (2%). Four types of S1 protein cleavage recognition motifs were found among the isolates which includes HRRRR, RRSRR, RRFRR and RRVRR. To our knowledge, this is the first report describing the motif RRVRR and are unique to Malaysian strains. Six IBVs were grouped in Malaysian MH5365/95 strains. Among these, one isolate was different from others where it only shared 82% identity with MH5365/95 and to others. It formed its own branch in the Malaysian cluster suggesting it may be a variant unique to Malaysia. Alignment analysis of the S1 amino acid sequences indicated that point mutations, insertions and deletions contribute to the divergence of IB variants. This study indicated at least five groups of IBV are circulating in Malaysia with most of the isolates belonged to QX strains. As new IBV variants continue to emerge, further study need to be carried out to determine whether the current available vaccine is able to give protection against the circulating virus.

Rapid differentiation between fluconazole-sensitive and -resistant species of Candida directly from positive blood-culture bottles by real-time PCR.

In view of both the delay in obtaining identification by conventional methods following blood-culture positivity in patients with candidaemia and the close relationship between species and fluconazole (FLC) susceptibility, early speciation of positive blood cultures has the potential to influence therapeutic decisions. The aim was to develop a rapid test to differentiate FLC-resistant from FLC-sensitive Candida species. Three TaqMan-based real-time PCR assays were developed to identify up to six Candida species directly from BacT/Alert blood-culture bottles that showed yeast cells on Gram staining at the time of initial positivity. Target sequences in the rRNA gene complex were amplified, using a consensus two-step PCR protocol, to identify Candida albicans, Candida parapsilosis, Candida tropicalis, Candida dubliniensis, Candida glabrata and Candida krusei; these are the most commonly encountered Candida species in blood cultures. The first four of these (the characteristically FLC-sensitive group) were identified in a single reaction tube using one fluorescent TaqMan probe targeting 18S rRNA sequences conserved in the four species. The FLC-resistant species C. krusei and C. glabrata were detected in two further reactions, each with species-specific probes. This method was validated with clinical specimens (blood cultures) positive for yeast (n=33 sets) and the results were 100 % concordant with those of phenotypic identification carried out concomitantly. The reported assay significantly reduces the time required to identify the presence of C. glabrata and C. krusei in comparison with a conventional phenotypic method, from approximately 72 to <3 h, and consequently allows optimization of the antifungal regimen at an earlier stage.

A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5'-disrupted myb loci.

The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein. Here we report the identification of an 85-kilodalton (p85c-myb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c-myb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-myb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z ore-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.

Two modes of c-myb activation in virus-induced mouse myeloid tumors.

Two modes of disruption of the protooncogene c-myb by viral insertional mutagenesis in mouse myeloid tumor cells are described. The first mode was found in six tumors in which a Moloney murine leukemia virus component had inserted in the same transcriptional orientation upstream of the 5'-most exon with v-myb homology (vE1). cDNA sequence data indicate the presence of a truncated c-myb mRNA that is initiated in the upstream 5' long terminal repeat of the integrated provirus and processed via a cryptic splice donor sequence in the gag region to the splice acceptor site in vE1 of the c-myb gene, thus removing the remaining downstream viral and myb intronic sequences. Unlike most gag-onc transcripts, the gag and myb sequences in the hybrid transcript were not in the same reading frame. It is presumed that the gag sequence provides a cryptic translation initiation site for the novel amino-truncated c-myb protein. The second mode of disruption was by downstream virus insertion at the 3' side of the c-myb, which results in the synthesis of a small (approximately 2 kilobase) myb transcript. The 5' long terminal repeat of the inserted provirus provides a TGA termination codon that results in the elimination of 240 normal c-myb amino acid residues from the carboxyl terminus of the tumor-specific myb protein. These results suggest that truncated myb proteins play a role in neoplastic transformation of myeloid cells.