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.

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.

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.

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.

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.

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.

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.

Fibula osteoseptocutaneous flap for reconstruction of composite mandibular defects.

The fibula osteoseptocutaneous flap is a versatile method for reconstruction of composite-tissue defects of the mandible. The vascularized fibula can be osteotomized to permit contouring of any mandibular defect. The skin flap is reliable and can be used to resurface intraoral, extraoral, or both intraoral and extraoral defects. Twenty-seven fibula osteoseptocutaneous flaps were used for composite mandibular reconstructions in 25 patients. All the defects were reconstructed primarily following resection of oral cancers (23), excision of radiation-induced osteonecrotic lesions (2), excision of a chronic osteomyelitic lesion (1), or postinfective mandibular hypoplasia (1). The mandibular defects were between 6 and 14 cm in length. The number of fibular osteotomy sites ranged from one to three. All patients had associated soft-tissue losses. Six of the reconstructions had only oral lining defects, and 1 had only an external facial defect, while 18 had both lining and skin defects. Five patients used the skin portion of the fibula osteoseptocutaneous flaps for both oral lining and external facial reconstruction, while 13 patients required a second simultaneous free skin or musculocutaneous flap because of the size of the defects. Four of these flaps used the distal runoff of the peroneal pedicles as the recipient vessels. There was one total flap failure (96.3 percent success). There were no instances of isolated partial or complete skin necrosis. All osteotomy sites healed primarily. The contour of the mandibles was good to excellent.

Skin graft and skin equivalent in burns.

The problem in extensive burns is the relative lack of available donor sites for skin grafting. Before the burnt areas can be covered, infection often sets in and the patients may then succumb. Skin grafts can be expanded in a limited way. Biological dressings or synthetic dressings can be used to buy time till donor sites become available. Cultured epithelium grown in the tissue culture laboratory permits massive expansion of skin and has been used with success as epithelial autografts in patients. Composite skin equivalents or skin substitutes have been developed and show promise.

Medical progress in Singapore--a review of the last three decades, 1951-1980.

Graduating in 1951 and up-to-date, I have been engaged in hospital practice. It is appropriate at this juncture, the year of the Silver Jubilee of the Academy of Medicine, to recollect and record the changes and progress of medicine in Singapore, during the last three decades, 1951 to 1980.

Hypoglycemia misdiagnosed as cerebrovascular accident.

Hypoglycemia can give rise to a variety of neurological signs and symptoms. This may occur in the absence of the well known signs of acute hypoglycemia taught to diabetics on insulin. In the past year, 6 patients with hypoglycemia were admitted with a diagnosis of cerebrovascular accident. They were all being treated with oral hypoglycemic drugs. The neurological signs all reversed immediately with the restoration of a normal blood glucose. Their circumstances of admission illustrate some of the difficulties in making the correct diagnosis in this group of patients.

Human keratinocyte cell culture for the burns patients--a preliminary report.

One of the major problems in extensive burns is the relative lack of available donor sites for skin grafting. Keratinocyte cell culture in the laboratory was carried out successfully in Singapore General Hospital and shows promise as an alternative source for skin replacement. Our experience further proved that a fibroblast base is necessary for keratinocyte cell culture. It is observed that heterogenous cell source can grow concurrently and become confluent. The colony forming efficiency from trypsinized skin is about 1-4% in primary cultures and 35-40% in secondary cultures. The time taken to reach confluency are 20-21 days and 10-12 days respectively. The thickness of cultured skin is estimated as 0.5 mm (5-6 layers) under light microscope. The size of the harvested cultured skin is approximately one third of the cultured area due to contraction. The expansion ratio before shrinkage is estimated to be approximately 6000-fold based on our data.

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.

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.

Haemodynamic profile of acebutolol in acute myocardial infarction.

This paper evaluates the haemodynamics of intravenous Acebutolol (SECTRAL) (0.5 mgm/Kgm) in the acute phase of myocardial infarction uncomplicated by hypertension, cardiac failure or conduction abnormalities. Nineteen observations were made on 15 consecutive patients. Haemodynamic parameters were recorded just before, and at 15 and 30 min after injections, using Swan-Ganz Catheter-Thermister and Edslab Cardiac Output Computer (9520) in the Intensive Care Unit. All patients survived; none had extension of infarction. The Heart Rate dropped by 9 +/- 1% (+/- SEM) (from 90.2 +/- 4.0 to 81.6 +/- 3.1 per min, P less than 0.001) but systolic and mean Blood Pressures were not significantly altered. Pulmonary Capillary Pressure was elevated by 2.5 +/- 6% (from 11.6 +/- 0.8 to 14.4 +/- 0.9 mmHg P less than 0.001) but cardiac failure hardly ever developed clinically. The mean Pulmonary Arterial Pressure rose by 10 +/- 2% (P less than 0.005) while the Right Atrial mean increased from 6.0 +/- 1.0 to 8.3 +/- 1.3 mm Hg (P less than 0.005). Although the Cardiac Index was depressed by 11 +/- 2% (from 3.0 +/- 0.1 to 2.7 +/- 0.1 L/min/M2; P less than 0.001), the Stroke Index remained virtually unaffected. Myocardial oxygen consumption per min as reflected by Heart Rate x BP product declined by 12 +/- 2% (P less than 0.001), while the Stroke Work Index was lowered by 9 +/- 3% (P less than 0.005). The haemodynamic profile indicates that intravenous Acebutolol in uncomplicated infarcts is well tolerated, and that it could be employed with advantage to manipulate determinants of myocardial oxygen consumption through reduction of Heart Rate Pressure product and Stroke Work Index.

Crohn's disease--a diagnostic rarity in Singapore.

Nine cases of Crohn's disease from the Department of Medicine, Singapore General Hospital were collected over a duration of 9 years (1978-1986). Male (5): Female (4) ratio was nearly equal. Predominantly young people (mean age 30.5 year, range 12-59 year) from all races in Singapore were affected. Presentation could be acute (1), subacute (2), or chronic (6). The commonest symptoms were abdominal pain (8) diarrhoea (6) and weight loss (6). Three patients had a palpable right iliac fossa mass, 3 had definite malabsorption from ileal disease and 1 had perianal involvement leading to an anal stricture. The only extraintestinal manifestations of disease were clubbing and sacroiliatis. Haematological (haemoglobin, total white count, erythrocyte sedimentation rate) and biochemical (albumin) parameters generally reflected the degree of activity and chronicity of disease prior to presentation. The diagnosis and assessment of disease sites were based on a combination of radiological, endoscopic, operative and histological criteria. Ileal disease (4) per se was commonest followed by ileocolic disease (3) and colonic disease (2). Medical treatment consisted of sulphasalazine +/- steroids in all patients. Azathioprine and metronidazole were used for steroid sparing and perianal disease respectively. Laparotomy was performed in 2 patients. Six patients were well with infrequent (less than or equal to 2 times/year) or no relapses during follow up. Of the remaining three, 2 had either chronically active disease or frequent relapses (greater than 2 times/year) and one severe recurrent disease despite repeated gut resection.

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.

Liver disease in asymptomatic hepatitis B surface antigen carriers.

Fifty-five Chinese healthy asymptomatic Hepatitis Bs Antigen carriers, 49 males and 6 females, were studied over a six year period, 1978 to 1984. Seventeen patients (30.9%) had normal transaminases, and 38 patients (69.1%) had fluctuating levels of transaminases throughout the period of study. 83.6% of patients were below 40 years, and mean age was 29.5 years (range 18 to 49 years). Twenty three patients (41.8%) had liver biopsy, and a spectrum of chronic liver diseases was seen, with Chronic Lobular Hepatitis (CLH) and Chronic Persistent Hepatitis (CPH) being the commonest. Six patients (26.1%) had normal histology, seven patients (30.4%) had CLH, seven patients (30.4%) had CPH, two patients (8.7%) had Chronic Active Hepatitis (CAH), and one patient (4.4%) with Active Cirrhosis (AC). An attempt to correlate the severity of histological changes with the levels of transaminases at the time of biopsy failed to show any definite correlation. However, three patients with transaminases of more than four times normal had severe liver diseases (2 CAH and 1 AC).

Adriamycin in the treatment of resectible and irresectible primary hepatocellular carcinoma.

The treatment of 205 patients with primary hepatocellular carcinoma in Singapore from 1975 to 1980 is reviewed. Adriamycin given at a dose of 40 mg/m(2) intravenously every 4 weeks for irresectible tumours achieved a response rate of 32% alone and has significantly prolonged the median survival time to 4 1/2 months (135 days) in these individuals. Despite this response only 1/34 (3%) patient showed complete remission and is alive at 2 1/2 years. Use of other combinations with Adriamycin e.g. 5 Fluorouracil and Vincristine produced a significant response in the first 16 weeks (p < 0.005) but not thereafter, when completed with the historical group. To improve the response and to consolidate this response, split doses of total hepatic irradiation (to a maximum of 3,150 rads) and alternate day administration of intravenous Adriamycin 10 mg (to a maximum of 90 mg) were given. 12/23 patients (50%) responded. This was followed by a four weekly Adriamycin, 5 F.U. and Vincristine. Median survival time was 5 1/2 months (165 days). Relapse occurred in all patients. Similarly in 5 patients who had resections, early recurrences occurred in 3/5 (60%) within 3 months, inspite of postoperative chemotherapy with Adriamycin, 5 F.U., Vincristine and Cyclophosphamide given every four weeks. In view of the high recurrence rate following effective treatment, further studies on the biological behaviour of these abnormal livers is required.

Ultrasonography in the diagnosis of cholestatic jaundice.

Fifty consecutive patients (32 males and 18 females) with cholestatic jaundice were examined by grey-scale ultrasound from June 1981 to June 1983. All patients were studied without access to case notes. All patients had a diagnosis established by subsequent liver biopsy, clinical course, surgery or autopsy. Using the presence or absence of a dilated biliary system as the criterion, intrahepatic or extrahepatic cholestasis was correctly differentiated in 48 of the 50 patients, giving an overall accuracy of 96%. All 16 patients with intrahepatic cholestasis were correctly identified. Two of the 34 patients with extrahepatic cholestasis, each of whom had biliary stones, were misdiagnosed as intrahepatic cholestasis. In the 34 patients with extra-hepatic cholestasis, site of obstruction was defined in 55.9%, and specific aetiology diagnosed in 44.1%. Ultrasound proves to be an accurate method for the evaluation of cholestatic jaundice, and would have a definite value as a screening test before proceeding to invasive studies.