Clinical utility of tumour marker velocity of cancer antigen 15-3 (CA 15-3) and carcinoembryonic antigen (CEA) in breast cancer surveillance.

BACKGROUND: Serum tumour markers, cancer antigen 15-3 (CA 15-3) and carcinoembryonic antigen (CEA) are not routinely recommended for detecting breast cancer recurrence and monitoring treatment. In this study, we aim to evaluate the diagnostic accuracy of absolute CA 15-3 and CEA levels and report on the clinical utility of tumour marker velocity in breast cancer surveillance. METHODS: 67 consecutive patients over a 15-year period (1998-2012) with available serial serum CA 15-3 and CEA measurements at recurrence were matched to a control group of patients. Tumour marker velocity was derived from the average change in consecutive tumour marker values over time, expressed in unit/year. Logistic regression analysis was performed to investigate the association between tumour characteristics, tumour marker velocity and disease recurrence. RESULTS: Using the Youden index values, the optimal cut-off values for absolute CA 15-3 and CEA corresponded to the normal assay reference range while tumour marker velocity values were derived to be 2.5U/mL/year and 1.2ng/mL/year respectively. CA 15-3 velocity > 2.5U/mL/year had the highest AUROC value of 0.85 than CEA velocity alone. When either tumour marker velocity exceeded threshold values, the sensitivity, specificity, negative predictive value and positive predictive value were 94.0%, 73.1%, 92.5%, and 77.8% respectively. In the multivariate logistic regression analysis, having both CA 15-3 and CEA velocity exceeding the cut-off values was shown to be a significant predictor for disease recurrence (p = 0.01). CONCLUSION: These findings highlighted the clinical utility of serial tumour markers measurements and its velocity in breast cancer surveillance.

Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance, a Novel Antimicrobial Resistance Multilocus Typing Scheme for Tracking Global Dissemination of N. gonorrhoeae Strains.

A curated Web-based user-friendly sequence typing tool based on antimicrobial resistance determinants in Neisseria gonorrhoeae was developed and is publicly accessible (https://ngstar.canada.ca). The N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) molecular typing scheme uses the DNA sequences of 7 genes (penA, mtrR, porB, ponA, gyrA, parC, and 23S rRNA) associated with resistance to ß-lactam antimicrobials, macrolides, or fluoroquinolones. NG-STAR uses the entire penA sequence, combining the historical nomenclature for penA types I to XXXVIII with novel nucleotide sequence designations; the full mtrR sequence and a portion of its promoter region; portions of ponA, porB, gyrA, and parC; and 23S rRNA sequences. NG-STAR grouped 768 isolates into 139 sequence types (STs) (n = 660) consisting of 29 clonal complexes (CCs) having a maximum of a single-locus variation, and 76 NG-STAR STs (n = 109) were identified as unrelated singletons. NG-STAR had a high Simpson's diversity index value of 96.5% (95% confidence interval [CI] = 0.959 to 0.969). The most common STs were NG-STAR ST-90 (n = 100; 13.0%), ST-42 and ST-91 (n = 45; 5.9%), ST-64 (n = 44; 5.72%), and ST-139 (n = 42; 5.5%). Decreased susceptibility to azithromycin was associated with NG-STAR ST-58, ST-61, ST-64, ST-79, ST-91, and ST-139 (n = 156; 92.3%); decreased susceptibility to cephalosporins was associated with NG-STAR ST-90, ST-91, and ST-97 (n = 162; 94.2%); and ciprofloxacin resistance was associated with NG-STAR ST-26, ST-90, ST-91, ST-97, ST-150, and ST-158 (n = 196; 98.0%). All isolates of NG-STAR ST-42, ST-43, ST-63, ST-81, and ST-160 (n = 106) were susceptible to all four antimicrobials. The standardization of nomenclature associated with antimicrobial resistance determinants through an internationally available database will facilitate the monitoring of the global dissemination of antimicrobial-resistant N. gonorrhoeae strains.

Antipsychotics affect multiple calcium calmodulin dependent proteins.

Calcineurin is a calmodulin (CaM) dependent protein phosphatase recently found to be altered in the brains of patients suffering from schizophrenia and by repeated antipsychotic treatment in rats. Some data suggest, however, that antipsychotics and schizophrenia may have a more widespread effect on the CaM signaling axis than calcineurin alone. In the current study, the effects of selected psychoactive drugs were investigated using Western blotting, in situ hybridization and immunocytochemistry to determine if they target CaM, calmodulin-dependent protein kinases (CaMK) or calcineurin. Results indicated that repeated treatment with haloperidol, clozapine or risperidone increased CaM protein and CaMII mRNA levels but decreased calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) IV (CaMKIV), kinase alpha (CaMKKalpha), kinase beta (CaMKKbeta) and calcineurin protein levels in the striatum of Sprague-Dawley rats (Rattus Norvegicus). Closer examination of CaMKIV, CaMKKalpha and CaMKKbeta revealed that the observed decreases in protein levels were short-lived following antipsychotic treatment and reversed (i.e. upregulated) 24 h post-treatment similar to what was previously reported for calcineurin. The D(2)/D(3)dopamine receptor antagonist raclopride mimicked the decreases in CaMKIV, CaMKKalpha, CaMKKbeta and calcineurin observed following antipsychotic treatment whereas increases in these proteins were observed in an amphetamine model of the positive symptoms of schizophrenia. Mood stabilizers such as lithium and valproic acid or the antidepressant fluoxetine had no effect on CaMKIV, CaMKKalpha, CaMKKbeta and calcineurin with the exception of an increase in CaMKKbeta following lithium treatment. The results collectively suggest that antipsychotic specifically target several proteins associated with CaM signaling.

Use of Bordetella pertussis BP3385 to establish a cutoff value for an IS481-targeted real-time PCR assay.

This study utilized the Bordetella pertussis single-copy PCR target BP3385 as a means of confirming IS481 PCR-positive reactions with cycle threshold (C(T)) values of >35. IS481 PCRs with C(T) values of >35 cycles may represent PCR conditions where there is <1 CFU of B. pertussis per PCR.

Knowledge and perceptions regarding colorectal cancer screening among Chinese--a community-based survey in Singapore.

OBJECTIVE: Despite the increase in colorectal cancer (CRC) incidence among Chinese in Asia, there are no data on predictors of CRC screening uptake in this population. This study investigated how knowledge and perceptions about CRC correlated with screening behavior in Singaporean-Chinese. METHODS: A community-based cross-sectional study was carried out on Singaporean-Chinese at least 50 years old in Queenstown Estate, Singapore between 1/1/2006 and 1/2/2006. A questionnaire administered via face-to-face interviews elicited knowledge, perceptions and screening behavior of subjects. RESULTS: The response rate was 72.4%, with 514 completed responses. Expense was the commonest perceived barrier to screening (56.6% agreed), unlike for other populations. Social influence is important, with 67.5% agreeing to the statement "I would go for CRC screening if my family wanted me to". After excluding confounders, Chinese who had been for fecal occult blood test (FOBT) screening had higher knowledge score (p<0.001), lower perceived severity (p<0.01), were more likely to have been influenced by their family/friends to go for screening (p=0.04) and to have attended screening tests for other diseases (p<0.001). CONCLUSION: FOBT screening uptake is associated with specific areas of knowledge and perception among Singaporean-Chinese. To increase screening uptake within Chinese populations, clinicians should consider these factors in their approach to patients.

The use of human serum in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells.

AIM: To evaluate the use of human serum (HS) in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells, and compare it with fetal bovine serum (FBS) and bovine pituitary extract (BPE). METHODS: Conjunctival epithelial cells were cultivated in media supplemented with HS (5%, 10%), FBS (5%, 10%), and BPE (70 microg/ml, 140 microg/ml). The colony forming efficiency (CFE), bromodeoxyuridine (BrdU) ELISA proliferation assay, and cell generations were analysed. Cells were evaluated for keratin (K4, K19, and K3) and MUC5AC expression by immunostaining and RT-PCR. Conjunctival equivalents constructed on amniotic membranes were transplanted onto severe combined immune deficient (SCID) mice for 10 days and analysed histologically. RESULTS: The proliferation assays of HS supplemented cultures (CFE, 6.7% (SD 1.8%); BrdU absorbance, 0.86 (0.16)) were comparable to FBS supplemented (CFE, 9.3% (1.8%); BrdU absorbance, 1.11 (0.18)) and BPE supplemented cultures (CFE, 5.9 (1.5); BrdU absorbance, 0.65 (0.12)). Goblet cell densities for HS, FBS, and BPE supplemented media were 52 cells/cm(2), 60 cells/cm(2), and 50 cells/cm(2), respectively. HS supplemented cultures formed stratified epithelial sheets in vivo following transplantation. CONCLUSIONS: The proliferative capacity of conjunctival epithelial cells cultivated in HS supplemented cultures was comparable to FBS and BPE supplemented cultures. The elimination of animal material from the culture system is advantageous when cultivating cells for clinical transplantation.

Constitutive shedding of the amyloid precursor protein ectodomain is up-regulated by tumour necrosis factor-alpha converting enzyme.

The amyloid precursor protein (APP) of Alzheimer's disease is a transmembrane protein that is cleaved within its extracellular domain, liberating a soluble N-terminal fragment (sAPP alpha). Putative mediators of this process include three members of the ADAM (a disintegrin and metalloprotease) family, ADAM9, ADAM10 and ADAM17/TACE (tumour necrosis factor-alpha converting enzyme). Tumour necrosis factor-alpha protease inhibitor (TAPI-1), an inhibitor of ADAMs, reduced constitutive and muscarinic receptor-stimulated sAPP alpha release in HEK-293 cells stably expressing M3 muscarinic receptors. However, the former was less sensitive to TAPI-1 (IC(50)=8.09 microM) than the latter (IC(50)=3.61 microM), suggesting that these processes may be mediated by different metalloproteases. Constitutive sAPP alpha release was increased several-fold in cells transiently transfected with TACE, and this increase was proportional to TACE expression. In contrast, muscarinic-receptor-activated sAPP alpha release was not altered in TACE transfectants. TACE-dependent constitutive release of co-transfected APP(695) was inhibited by TAPI-1 with an IC(50) of 0.92 microm, a value significantly lower than the IC(50)s for inhibition of either constitutive or receptor-regulated sAPP alpha shedding mediated by endogenous secretases. The results indicate that TACE is capable of catalysing constitutive alpha-secretory cleavage of APP, but it is likely that additional members of the ADAM family mediate endogenous constitutive and receptor-coupled release of sAPP alpha in HEK-293 cells.

Quantitation of peripheral blood cytomegalovirus DNA for monitoring recurrent cytomegalovirus retinitis in pediatric solid organ transplant recipients.

Cytomegalovirus (CMV) infection is a major concern following solid organ transplantation, especially in the pediatric population who remain at high risk of primary infection. CMV disease leads not only to increased patient and graft morbidity, but also to increased health care costs. This study describes the usefulness of a quantitative CMV polymerase chain reaction (PCR) technique for monitoring peripheral blood CMV DNA in pediatric recipients of kidney and liver allografts who had recurrent CMV retinitis. The incidence of CMV disease in 28 pediatric transplant recipients was 28.6%, one-half of whom developed retinitis. Two of these patients had recurrent retinitis on cessation of anti-viral treatment. A peripheral blood CMV DNA copy number of > or =500/microg of DNA was associated with recrudescence of the retinitis in these patients. We conclude that the measurement of peripheral blood CMV DNA by PCR is a useful tool for the surveillance of disease resolution and recurrence. This is particularly important in patients with CMV retinitis, who may remain asymptomatic for a period of time, despite recurrences.

Müllerian-inhibiting substance type II receptor expression and function in purified rat Leydig cells.

Müllerian-inhibiting substance (MIS), a gonadal hormone in the transforming growth factor-beta superfamily, induces Müllerian duct involution during male sexual differentiation. Mice with null mutations of the MIS ligand or receptor develop Leydig cell hyperplasia and neoplasia in addition to retained Müllerian ducts, whereas MIS-overexpressing transgenic mice have decreased testosterone concentrations and Leydig cell numbers. We hypothesized that MIS directly modulates Leydig cell proliferation and differentiated function in the maturing testis. Therefore, highly purified rat Leydig and Sertoli cells were isolated to examine cell-specific expression, binding, and function of the MIS type II receptor. These studies revealed that this receptor is expressed abundantly in progenitor (21-day) and immature (35-day) Leydig cells as well as in Sertoli cells. Prepubertal progenitor Leydig cells exhibit high affinity (Kd = 15 nM), saturable binding of MIS. No binding, however, is detected with either peripubertal immature Leydig cells or Sertoli cells at either age. Moreover, progenitor, but not immature Leydig cells, respond to MIS by decreasing DNA synthesis. These data demonstrate that functional MIS type II receptors are expressed in progenitor Leydig cells and support the hypothesis that MIS has a direct role in the regulation of postnatal testicular development.

Th1 and Th2 cytokine mRNA profiles in childhood nephrotic syndrome: evidence for increased IL-13 mRNA expression in relapse.

Idiopathic nephrotic syndrome of childhood is thought to be associated with T lymphocyte dysfunction often triggered by viral infections, with the production of circulating factor(s) resulting in proteinuria. In view of the conflicting evidence of T cell activation and Th1 or Th2 pattern of cytokine synthesis in this disease, this study examined the mRNA expression of interleukin-2 (IL-2), interferon-gamma, IL-4, and IL-13 from CD4+ and CD8+ T cells in steroid-responsive nephrotic patients in relapse and remission. Fifty-five children with steroid-responsive nephrotic syndrome were included in this study, together with 34 normal controls and 24 patient controls with viral infections. RNA was isolated from purified CD4+ or CD8+ cells from peripheral blood and subjected to reverse transcription-PCR. Cytokine mRNA expression was measured semiquantitatively, and a cytokine index was derived from densitometric readings, with cyclophilin as the housekeeping gene. Both cross-sectional and paired data showed an increased CD4+ and CD8+ IL-13 mRNA expression in patients with nephrotic relapse as compared to remission, normal, and patient controls (P < 0.008). This was also associated with increased cytoplasmic IL-13 expression in phorbol myristate acetate/ionomycin-activated CD3+ cells (6.66+/-3.39%) from patients with nephrotic relapse compared to remission (2.59+/-1.35%) (P < 0.0001). However, there was no significant difference in CD4+ or CD8+ IL-2, interferon-gamma and IL-4 mRNA expression. IL-13 is an important T cell cytokine with anti-inflammatory and immunomodulatory functions on B cells and monocytes. It is conceivable that IL-13 may act on monocytes to produce vascular permeability factor(s) involved in the pathogenesis of proteinuria in patients with relapse nephrotic syndrome.

Soft tissue cover in compound and complicated tibial fractures using microvascular flaps.

This is a retrospective review of the use of microvascular flaps in the salvage of severely injured lower limb. From January 1992 to December 1994, we treated 10 patients using 10 microvascular flaps; 8 patients with Type III compound tibial fractures and 2 patients with infected implants following internal fixation of comminuted tibial fractures. The size of soft tissue defect ranged from 6 x 3 cm to 20 x 10 cm. One patient had a 6 cm bony defect. Soft tissue cover was achieved in 9 patients using microvascular muscle flaps with meshed split skin grafts and 1 patient using a scapula osteocutaneous flap. They were performed within 5 to 27 days following injury or removal of implants (mean 18 days). All the microvascular flaps were successful (100%). One re-exploration was performed. The mean period of hospitalization following microsurgical flap cover was 16.8 days. Eight out of 10 patients were followed up for a mean period of 48 months. All the 8 lower limbs were successfully salvaged. Three out of 8 patients (37.5%) had chronic osteomyelitis which required sequestrectomy and bone grafting. No amputation was necessary. Two patients required adjunctive cancellous bone grafting to accelerate bony union. Six out of 8 patients (75%) achieved bony union and full weight bearing capacity. The use of microvascular flap in achieving soft tissue cover plays an important role in the salvage of severely traumatized lower limb. Microvascular muscle flap is preferred when only soft tissue defect is present. Successful soft tissue cover facilitates secondary bone grafting procedures. Although the timing of surgery per se does not adversely affect the success rate of microvascular flaps, delayed soft tissue cover is associated with a high chronic infection rate (37.5%). An adequate surgical debridement is crucial in reducing the complication of deep infection.

Verrucous haemangioma--a case report.

Verrucous haemangioma is an uncommon skin lesion which increases in size and also evolves in appearance with time. Early diagnosis is important so that surgical treatment can be instituted early for better cosmetic results. Its similarities and differences to angiokeratoma circumscriptum are also highlighted, and the importance of distinguishing the two in terms of management is discussed.

Use of patient-controlled analgesia with alfentanil for burns dressing procedures: a preliminary report of five patients.

The use of patient-controlled analgesia with alfentanil (PCA-alfentanil) as a form of pain relief for dressing procedures in patients during the acute phase of their burn injuries was investigated. Five ASA 1 and 2 patients with 10-30 per cent total body surface area (TBSA) thermal burns, had PCA-alfentanil for their dressing procedures after standard fluid resuscitation. One patient who did not receive a loading dose and a background infusion of alfentanil had unsatisfactory pain relief. Four patients had good pain relief after a loading dose of IV alfentanil 1 mg followed by a continuous background infusion of 200-800 micrograms/h. Demand dose ranged from 200 to 400 micrograms and lockout time ranged from 1 to 3 min. The total dose of alfentanil delivered ranged from 0.8 to 4.48 mg and duration of the dressings ranged from 30 to 60 min. All patients were mildly sedated, calm, communicative and cooperative during dressing procedures. None of them experienced hypotension or respiratory depression. One patient experienced nausea but no vomiting, no other adverse effects of alfentanil were noted. From the pilot study, PCA-alfentanil may be an effective form of pain relief for dressing procedures in patients during their acute phase of burn injuries. The optimal PCA-alfentanil setting has yet to be determined.

Vancomycin-resistant Enterococcus in the Singapore National Burns Centre: a case report.

Vancomycin-resistant Enterococcus (VRE) is becoming an important cause of nosocomial infections. An outbreak of VRE in a burns unit, if it ever occurs, will be a catastrophe as vancomycin-resistance can potentially be transferred to other organisms like methicillin-resistant Staphylococcus aureus. We report a case of VRE in our burns centre in which it was detected and the patient isolated from other patients early. Measures to control the occurrence of VRE include the restriction of the use of vancomycin and the practice of other established infection-control measures.

Age- and sex-related changes in lymphocyte subpopulations of healthy Asian subjects: from birth to adulthood.

Flow cytometric analysis of lymphocyte subsets were evaluated in 391 healthy Asian subjects ranging in age from birth to 40 years. Lymphocyte subsets were analysed using specific monoclonal antibodies: CD20 (B cells), CD3 and CD2 (T cells), CD16 and CD56+ (NK cells), CD4/CD3+ (helper-inducer T cells), CD8/ CD3+ (suppressor/cytotoxic T cells), HLA-DR expression on CD3 and CD25 (Tac) on CD3. The total white cell count, absolute lymphocyte counts, and B cell percentages peaked in infancy and declined steadily with age. Absolute counts of each subset, which were derived from absolute lymphocyte counts, also followed this trend. Increases with age were seen in the NK, T cell (CD2, CD3), and CD8 percentages. Males tended to have higher NK and CD8 percentages than females, and, conversely, females had higher CD3 and CD4 percentages than males. Comparison of our results with studies involving Caucasian subjects indicated higher NK percentages in our Asian population and lower CD4 absolute counts in the males of our population. These results indicate the presence of age, sex, and probable racial differences in lymphocyte subset expression. Our results may serve as reference standards for the Asian population.

Immunoglobulin (Ig) and IgG subclasses in Asian children with bronchial asthma.

Selective immunoglobulin deficiencies have been shown to be associated with atopic disease. In this study, serum immunoglobulin (Ig) G, A, M, E and IgG subclasses of 92 Asian asthmatic children were studied and compared with those of age-matched controls. The children, aged between 0.7 and 17.4 years (mean age 7.5 years), were recruited from the National University Hospital, Singapore. The serum Ig levels were measured by enzyme-linked immunosorbent assay, except for IgE which was measured by the fluorescent allergosorbent test. As expected, serum total IgE levels were markedly higher in the asthmatic children than in the controls (geometric mean = 513 units/ml and 164 units/ml, respectively; p < 0.0001). Serum IgM levels were also slightly higher in the asthmatic patients than in the controls (geometric mean = 1.74 and 1.51 milligrams, respectively; p < 0.04). Mean serum IgG and A and IgG subclasses (1-4) levels in the asthmatics did not differ significantly from those in the controls. However, four asthmatic children were found to have selective IgA deficiency (serum IgA < 0.08 milligrams). None of the patients was found to be IgG subclass-deficient.

Semi-nested PCR using NS3 primers for the detection and typing of dengue viruses in clinical serum specimens.

BACKGROUND: More rapid, specific and sensitive tests for the laboratory diagnosis of dengue virus infections are needed. OBJECTIVE: To develop a semi-nested polymerase chain reaction (PCR) assay based on primers within the NS3 gene for the simultaneous detection and typing of dengue viruses in human sera. STUDY DESIGN: A first round of single-step reverse transcription-polymerase chain reaction (RT-PCR) was carried out with a pair of consensus primers, followed by a second round of semi-nested amplification using the upstream consensus primer and four type-specific down-stream primers. The sensitivity and specificity of the semi-nested PCR assay were determined using plaque- or TCID(50)-titrated virus-infected tissue culture fluid, and total RNA extracted from C6/36 cells infected with dengue and other flaviviruses, respectively. A retrospective study was performed on acute sera from thirteen patients with dengue (confirmed by virus isolation) employing semi-nested PCR in parallel with virus re-isolation and a single-step RT-PCR method for the typing of dengue viruses in human sera. RESULTS: The semi-nested PCR assay could detect up to 1 pfu of dengue virus, but not other flaviviruses. The semi-nested PCR and single-step RT-PCR assays correctly typed dengue viruses in twelve and five sera, respectively, whereas none of the sera was positive by virus re-isolation. CONCLUSIONS: This semi-nested PCR assay is a sensitive and specific tool for the detection and typing of dengue viruses from viremic human sera.

Rapid, single-step RT-PCR typing of dengue viruses using five NS3 gene primers.

In order to detect and type dengue viruses in serum specimens, four type-specific downstream primers were designed for use with a consensus upstream primer in a reverse transcription and polymerase chain reaction (RT-PCR) assay. RT-PCR using these five primers amplified NS3 gene fragments of diagnostic sizes of 169, 362, 265 and 426 base pairs for dengue virus types 1, 2, 3 and 4, respectively, but not for Japanese encephalitis, Kunjin and yellow fever viruses. The conventional two-step RT-PCR procedure was simplified by combining RT and PCR in a single-step format with a "hot start". This RT-PCR protocol was applied successfully to dengue virus-spiked serum and dengue patient serum samples, and could detect as few as one PFU of dengue virus. This assay offers a rapid, specific and sensitive molecular technique for the simultaneous detection and typing of dengue viruses.

Inflammatory response in bacterial meningitis: cytokine levels in the cerebrospinal fluid.

Inflammatory response plays an important role in the pathogenesis of cerebral injury in bacterial meningitis. In this study, we evaluated the cytokine levels of interleukin 1-beta (IL1 beta), tumour necrosis factor alpha (TNF alpha) and interleukin 6 (IL6) in the cerebrospinal fluid (CSF), and determined their correlation with acute clinical complications and with changes in CSF biochemistry. Interleukin 6, TNF alpha and IL1 beta were present in 9/9, 3/9 and 4/9 patients, respectively. The CSFs with detectable TNF alpha or IL1 beta had higher levels of IL6 (p < 0.02), protein (NS) and lower glucose levels (p < 0.02), compared with those in which TNF alpha and IL1 beta were absent. Tumour necrosis factor alpha and IL1 beta levels also correlated with the presence of prolonged fever, fits, spasticity and death (logTNF alpha: r = 0.70, p < 0.05; logIL1 beta: r = 0.62, p = 0.08). The cytokine levels reflect the degree of inflammatory response and are positively correlated with the severity of acute clinical complications. Modulation of this inflammatory response in bacterial meningitis may improve its morbidity and mortality.