Harmine promotes periodontal ligament cell-induced tissue regeneration.

OBJECTIVE: to investigate whether harmine has a promotive effect on human periodontal ligament cells (hPDLCs)-induced tissue regeneration. MATERIALS AND METHODS: Various concentrations of harmine on hPDLCs proliferation were tested. Osteogenic and cementogenic characteristics were examined in hPDLC/rhBMP-2 and hPDLC/harmine by alizarin red S staining, real-time PCR, and Western blotting assay. The activity of harmine was investigated in an ectopic transplantation nude mouse model. RESULTS: We determined that 10 µM of harmine was the threshold concentration. hPDLC/harmine showed similar mineralized nodule formation in alizarin S staining compared to hPDLC/rhBMP-2. In real-time PCR, the highest gene expression level was observed for Runx2 in hPDLC/harmine at all time points. The level of CEMP-1 in hPDLC/harmine was higher at 7 days than hPDLCs alone. Thicker band of Runx2 in hPDLC/harmine was observed than in hPDLC/rhBMP-2 at 7 days by Western blotting. The band for CEMP-1 in hPDLC/harmine was thicker than hPDLCs alone at both 7 and 14 days. In ectopic transplantation, hPDLCs with harmine showed a comparable amount of mineralized tissue formation compared to rhBMP-2. hPDLCs with harmine or rhBMP-2 formed both bone and cementum-like tissue with Sharpey's fiber-like collagen insertion. CONCLUSION: Harmine can be a potential candidate for promoting hPDLCs-induced tissue regeneration.

Role of PIN1 on in vivo periodontal tissue and in vitro cells.

BACKGROUND: Although expression of peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1) was reported in bone tissue, the precise role of PIN1 in periodontal tissue and cells remain unclear. MATERIAL & METHODS: To elucidate the roles of PIN1 in periodontal tissue, its expression in periodontal tissue and cells, and effects on in vitro 4 osteoblast differentiation and the underlying signaling mechanisms were evaluated. RESULTS: PIN1 was expressed in mouse periodontal tissues including periodontal ligament cells (PDLCs), cementoblasts and osteoblasts at the developing root formation stage (postnatal, PN14) and functional stage of tooth (PN28). Treatment of PIN1 inhibitor juglone, and gene silencing by RNA interference promoted osteoblast differentiation in PDLCs and cementoblasts, whereas the overexpression of PIN1 inhibited. Moreover, osteogenic medium-induced activation of AMPK, mTOR, Akt, ERK, p38 and NF-jB pathways were enhanced by PIN1 siRNA, but attenuated by PIN1 overexpression. Runx2 expressions were induced by PIN1 siRNA, but downregulated by PIN1 overexpression. CONCLUSION: In summary, this study is the first to demonstrate that PIN1 is expressed in developing periodontal tissue, and in vitro PDLCs and cementoblasts. PIN1 inhibition stimulates osteoblast differentiation, and thus may play an important role in periodontal regeneration.

Case report: A rare case of extravesical, extraperitoneal metastasis after transuretheral resection of urothelial carcinoma.

Bladder perforation secondary to transurethral resection of bladder tumour (TURBT) increases the risk of tumour cell seeding and eventual extravesical metastasis. Here we presented a case where a patient with localised bladder tumour was initially managed with repeated TURBTs for tumour recurrence. Subsequently he was found to have extravesical pelvic metastasis. This was likely secondary to microperforation of bladder and tumour cell seeding. Microscopic bladder perforation is difficult to diagnose. However patients with confirmed bladder perforation during TURBT would justify systemic radiological cancer surveillance in view of higher risk of metastatic disease.

Effects of irradiation on cytokine production in a mouse model of Behçet's disease.

OBJECTIVE: Low-dose whole-body irradiation is known to have anti-inflammatory effects. The objectives of this study were to verify that cytokine augmentation is induced by irradiation in vivo, and to assess the effectiveness of radiation in treating Behçet's disease (BD). METHODS: Whole-body and half-body irradiation with single doses of 10cGy, 2Gy and 10Gy were delivered to normal mice, and cytokine and chemokine levels were analyzed in PBMC and sera. BD-like mice were treated with low-dose, half-body 10cGy irradiation. RESULTS: In normal mice, PBMC cytokine mRNA levels peaked four days after irradiation. Of the cytokines and chemokines examined, the levels of IL-4, IL-6, IL-12p40, TNF-alpha, TGF-b, MIP-1alpha and IL-18 were all influenced by radiation treatment. Of these, IL-4, an ameliorating factor for BD, was the most elevated following low-dose irradiation (10cGy group). FACS analysis showed intracellular IL-4-staining of 7.24+/-0.92% of PBMC from irradiated mice compared to 1.3+/-0.1% from non-irradiated, normal mice (p<0.005). Serum IL-4 levels were also significantly increased (6.08+/-1.7 pg/ml) relative to control (1.83+/-0.8; p<0.005). CONCLUSION: Augmentation of cytokine production may contribute to the anti-inflammatory effects of low dose irradiation and amelioration of BD symptoms in this mouse model.

Pathotypes and genetic relationship of worldwide collections of Elsinoë spp. causing scab diseases of citrus.

Two scab diseases are recognized currently on citrus: citrus scab, caused by Elsinoë fawcettii, and sweet orange scab, caused by E. australis. Because the two species cannot be reliably distinguished by morphological or cultural characteristics, host range and molecular methods must be used to identify isolates. Four pathotypes of E. fawcettii and two of E. australis have been described to date based on host range. The host specificity and genetic relationships among 76 isolates from Argentina, Australia, Brazil, Korea, New Zealand, and the United States were investigated. Based on pathogenicity tests on eight differential hosts, 61 isolates were identified as E. fawcettii and 15 as E. australis. Of 61 isolates of E. fawcettii, 24 isolates were identified as the Florida broad host range (FBHR) pathotype, 7 as the Florida narrow host range (FNHR) pathotype, 10 as the Tryon's pathotype, and 3 as the "Lemon" pathotype. Two new pathotypes, the "Jingeul" and the satsuma, rough lemon, grape-fruit, clementine (SRGC), are described, and four isolates did not fit into any of the known pathotypes of E. fawcettii. Of the 15 isolates of E. australis from Argentina and Brazil, 9 belonged to the sweet orange pathotype and 6 from Korea to the natsudaidai pathotype. E. fawcettii and E. australis were clearly distinguishable among groups by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) assays and the E. fawcettii group was divided into three subgroups, A-1, A-2, and A-3. The A-1 group was composed of the FBHR, FNHR, and SRGC pathotypes; some Lemon pathotypes; and the uncertain isolates. The A-2 subgroup included all of the Tryon's pathotype isolates and one of the three Lemon pathotype isolates and the A-3 group contained the Jingeul pathotype isolates. E. australis was differentiated into two groups: B-1, the natsudaidai pathotype isolates, and B-2, the sweet orange pathotype isolates. Isolates of E. fawcettii and E. australis were clearly distinguishable by sequence analysis of the internal transcribed spacer (ITS) region and the translation elongation factor 1 alpha (TEF) gene. There were also fixed nucleotide differences in the ITS and TEF genes that distinguished subgroups separated by RAPD-PCR within species. We confirmed two species of Elsinoë, two pathotypes of E. australis, and at least six pathotypes of E. fawcettii and described their distribution in the countries included in this study.

Development of PCR Assays for the Identification of Species and Pathotypes of Elsinoë Causing Scab on Citrus.

Two scab pathogens of citrus, Elsinoë fawcettii and E. australis, cause citrus scab and sweet orange scab, respectively, and pathotypes of each species have been described. The two species cannot be readily distinguished by morphological or cultural characteristics and can be distinguished only by host range and the sequence of the internal transcribed spacer (ITS) region. In this study, random amplified polymorphic DNA (RAPD) assays clearly distinguished E. fawcettii and E. australis, and the sweet orange and natsudaidai pathotypes within E. australis also could be differentiated. We developed specific primer sets, Efaw-1 for E. fawcettii; Eaut-1, Eaut-2, Eaut-3, and Eaut-4 for E. australis; and EaNat-1 and EaNat-2 for the natsudaidai pathotype within E. australis using RAPD products unique to each species or pathotype. Other primer sets, Efaw-2 and Eaut-5, which were specific for E. fawcettii and E. australis, respectively, were designed from previously determined ITS sequences. The Efaw-1 and Efaw-2 primer sets successfully identified E. fawcettii isolates from Korea, Australia, and the United States (Florida) and the Eaut-1 to Eaut-5 primer sets identified both the sweet orange pathotype isolates of E. australis from Argentina and the natsudaidai pathotype isolates from Korea. The EaNat-1 and EaNat-2 primer sets were specific for isolates of the natsudaidai pathotype. The Efaw-1 and Efaw-2 primer sets successfully detected E. fawcettii from lesions on diseased leaves and fruit from Korea and primer pairs Eaut-1, Eaut-2, Eaut-3, Eaut-4, and Eaut-5 detected E. australis from lesions on sweet orange fruit from Brazil.

Characterization of alpha-adrenoceptors in the nucleus reticularis gigantocellularis involved in the cardiovascular depressant effects of guanabenz in the rat.

The participation of alpha-adrenoceptors in the nucleus reticularis gigantocellularis in the hypotensive, negative inotropic and chronotropic effects induced by guanabenz, was examined in rats anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Pretreatment with alpha-adrenoceptor antagonists yohimbine (10 micrograms), phentolamine (2.5 micrograms) and phenoxybenzamine (20 micrograms), which were injected bilaterally into the nucleus reticularis gigantocellularis, significantly antagonized the cardiovascular suppressant effects normally produced by systemic administration of guanabenz (10 micrograms/kg, i.v.). Pretreatment with prazosin (0.25 microgram) did not affect the vasodepressive, but significantly attenuated the bradycardic actions of guanabenz. The general trend of "antagonization potency" shown by the alpha-adrenergic blockers, against the cardiovascular effects of guanabenz, was in the order: yohimbine greater than phentolamine greater than phenoxybenzamine greater than prazosin. It is concluded that while the alpha 2-adrenoceptors in the nucleus reticularis gigantocellularis are more critically involved in the antihypertensive actions of guanabenz, the possibility exists that alpha 1-adrenoceptors may also participate, in part.

Participation of nucleus reticularis gigantocellularis in guanabenz-promoted hypotension, decrease in cardiac contractility and bradycardia in rats.

The possible participation of the nucleus reticularis gigantocellularis in the hypotension, decrease in cardiac contractility and bradycardia induced by guanabenz was examined in rats that were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Guanabenz (10 micrograms/kg, i.v.) elicited an initial, transient hypertension, accompanied by an increase in cardiac contractility, followed by a significant and sustained hypotension, as well as decrease in the force and rate of cardiac contraction. In rats receiving bilateral focal electrolytic lesions of the nucleus reticularis gigantocellularis, the same injection produced only the initial transient responses, without the subsequent depressant effects. Microinjection of guanabenz directly into the ventro-medial portion of the nucleus reticularis gigantocellularis, at an ineffective systemic concentration (500 ng), produced significant and prolonged reduction in arterial pressure, cardiac contractility and heart rhythm. On the other hand, local application of the same concentration (500 ng) of guanabenz into the lateral portion of the same nucleus produced only minor hypotension and bradycardia, with no decrease in cardiac contractility. It is concluded that the nucleus reticularis gigantocellularis is at least one of the central sites through which guanabenz may produce its cardiovascular suppressant effects.

Tyrosinase production in recombinant E. coli containing trp promoter and ubiquitin sequence.

We have successfully expressed the active tyrosinase of Streptomyces antibioticus in Escherichia coli under the control of the trp promoter by fusing the sequence to the ORF438 gene. Because our attempt to connect the polycistronic gene of ORF438 and tyrosinase directly to the trp promoter of E. coli resulted in the expression of functionally inactive tyrosinase, we decided to fuse the COOH-terminus of ubiquitin sequence to the NH2-terminus of ORF438. Ubiquitin fusion has been shown to augment the yield of cloned gene products in E. coli by increasing the stability or translational efficiency of the fusion proteins. As a result, E. coli transformants harboring a plasmid pTRUBF that contains the ubiquitin-fused ORF438 and the tyrosinase gene produced the strong black pigment of melanin. About 300 units of tyrosinase per liter of batch culture were detected when cultivated in M9 medium containing casamino acids, L-tyrosine, and copper supplements. The black pigment, however, was not seen when grown in LB medium, suggesting that the trp promoter is well regulated. When recombinant E. coli cells grown in LB medium were transferred to a tryptophan-deficient minimal medium with phenol, we observed that phenol was removed from the solution, and the color of the medium turned black. This is due to the fact that the tyrosinase has polyphenol oxidase properties. We expect to use this recombinant E. coli for the waste treatment of phenolic compounds.

The roles of alpha 2-adrenoceptors in the nucleus reticularis gigantocellularis and vagal mechanism in the cardiovascular suppressive effects of guanabenz in the rat.

In pentobarbital-anesthetized rats, pretreatment with yohimbine (10 micrograms), which was microinjected into the bilateral nucleus reticularis gigantocellularis (NRGC), significantly antagonized the reduction in arterial pressure, and the force and rate of heart contraction normally promoted by systemic administration of guanabenz (10 micrograms/kg, i.v.). At the same time, the vasodepressive as well as negative inotropic and chronotropic effects of direct application of guanabenz (500 ng) into the NRGC were attenuated by bilateral cervical vagotomy or atropine sulfate (1 mg/kg, i.v.). We conclude that guanabenz may promote antihypertension by activating the alpha 2-adrenoceptors in the NRGC, which in turn elicits cardiovascular suppression by at least facilitating the vagal outflows to the heart.

Pulmonary edema as a cause for fulminant death following microinjection of kainic acid into the rat medulla oblongata.

The present study confirmed histopathologically the suggestion that pulmonary edema may underlie the fulminant death following microinjection of kainic acid into many medullary sites in rats anesthetized with pentobarbital sodium. We also observed a sustained elevation in systemic arterial pressure, cardiac contractility and heart rate before the animal succumbed to kainate neurotoxicity. It is suggested that the increased systemic hydrostatic pressure may retrogradely promote pulmonary hypertension that precipitates pulmonary edema.

Bioreactor studies on temperature induction of the Q- mutant of bacteriophage lambda in Escherichia coli.

The parasite-host interactions between bacteriophage lambda (denoted as lambda) and Escherichia coli bacteria were studied in different bioreactor systems. Although the replicated lambda-DNA of Q- mutant remains naked for a longer time to reach a high gene expression, the epidemic of lambda-infection and the coevolutionary host-phage relations limit the temperature induction efficiency. The temperature induction is strongly dependent upon the susceptible population density at which lambda-infection is activated. Maximum beta-galactosidase expression occurs at the threshold of the infection system. According to this concept, the lethal level of parasitic lambda to hosts is approx. 5 x 10(6) pfu/ml. Since a higher phage lambda burden is exerted upon host cells at a low ODsh, the system moves towards virulence reduction for total survival. Prey-predator isocline analysis is used to consider the stability of the outcome of infection. The host growth has a destabilizing effect at lower population densities and a stabilization effect at higher population. Based upon the predictions, a substrate enrichment enhances bacterial growth and reporter protein production. However, the operations still need to follow the trajectory of threshold tie line to guarantee maximal productivity. Since the washout of infected cells reduces induction performance in continuous cultures, a batch mode of operation is better than continuous stirred tank reactor (CSTR) mode to achieve high gene expression. The threshold cell density regulates induction performances and therefore produces the optimal gene expression by maintaining maximal viable cells that provide sufficient resources for lambda expression.

Temperature induction of bacteriophage lambda mutants in Escherichia coli.

The paper presents temperature induction in Escherichia coli cells with phage lambda on the target-protein production and cell growth. Replicated lambda-DNA particles in the Q- and S- mutants remain naked for a longer time by preventing DNA packaging and cell lysis, and therefore the expression of the foreign genes is high. However, the parasitic infection of phage-lambda causes on significant losses of host cell viability in the induction phase. The temperature effects on cell growth and targeted-gene product formation were investigated. Gene amplification was found to be growth phase dependent for both Qam73 (Q mutation) and Sam100 (S mutation) mutants. Maximum induction occurs in the early exponential phase and under the optimal cell density. The total beta-galactosidase activity at this optimal induction condition increases roughly 8-10-fold with respect to that without thermal induction. To maximize the induction efficiency for the gene-product beta-galactosidase activity, several operating parameters were investigated. In this study, temperature induction is strongly dependent upon the population density of 'susceptible' cells at which time the temperature is shifted to 38-42 degrees C. This may be due to the 'threshold' population density to regulate the infection of lambda to hosts and control the productivity of target gene expression.

Out of hospital cardiac arrests--the experience of one hospital in Singapore.

In Singapore, all public emergency ambulances are equipped with semi-automatic external defibrillators and the crew is trained in their use. This is the first paper from Singapore reporting the survival rate in patients presenting to an urban public hospital with acute coronary syndrome (ACS) who developed out-of-hospital cardiac arrest (OHCA). All consecutive patients who presented to the ED of a public hospital with OHCA or ACS were surveyed from 1 April 1999 to 30 September 1999. There were 392 patients among whom 115 (28.5%) had OHCA. There was no significant difference in age and gender distribution between the OHCA and non-OHCA patients. More than 2/3 of the OHCA patients had no report of chest pain or breathlessness before they collapsed. Forty five (39.1%) of the 115 OHCA patients were noted to have initial rhythms of ventricular tachycardia (VT) or ventricular fibrillation (VF) and received pre-hospital defibrillation. The mean time from collapse to first DC shock was 12.07+/-7.2 min. Twenty (17.4%) of the OHCA patients had return of spontaneous circulation after resuscitation in the ED. Four patients (3.5%), all with an initial rhythm of VF were discharged alive from the hospital. Much remains to be done to reduce the time interval to first DC shock for the OHCA group.

Theoretical analysis of the effect of cell recycling on recombinant cell fermentation processes.

A cell recycle system is studied for two-stage continuous fermentation. Cell recycle around the second stage provides higher cell concentrations than processes without recycle and a longer residence time of the cell, which is necessary for inducible products, especially in recombinant cell fermentation. Residence time distribution of the cell in the fermentor is important for the optimization of inducible products. The residence time distributions are studied for the cases with and without significant cell growth in the second stage. With cell growth in the second stage, three cases are considered. These are the cases of (1) zero residence time for two daughter cells after the cell division, (2) zero residence time of one daughter cell after the cell division and inherited residence time for the other daughter cell from the mother cell after the cell division, and (3) two daughter cells having the residence time of the mother cell after the cell division.

Nitrazepam-induced cricopharyngeal dysphagia, abnormal esophageal peristalsis and associated bronchospasm: probable cause of nitrazepam-related sudden death.

Nitrazepam was used in the treatment of resistant myoclonic epilepsy in 38 children. After the occurrence of nitrazepam-associated swallowing incoordination, high-peaked esophageal peristalsis and related bronchospasm in one patient, we initiated a prospective study of esophageal manometry using a station pull-through technique with a pediatric 4-channel continuous perfusing system. Three more patients were found to have delayed cricopharyngeal relaxation and high-peaked esophageal peristaltic waves. The initial patient developed severe respiratory distress and bronchospasm necessitating ventilatory support while on nitrazepam and improved dramatically with subsequent normal manometric study following nitrazepam discontinuation. Nitrazepam was reintroduced for its anticonvulsant and cognitive benefits and was tolerated at a reduced dosage. We postulate a central nervous system effect of nitrazepam promoting parasympathetic overactivity or vagotonia which can cause potentially fatal respiratory distress. Care must be exercised in nitrazepam use and esophageal manometry may be helpful in defining patients at greater risk for sudden death.

Hyperekplexia and sudden neonatal death.

Fifteen patients with hyperekplexia were identified in 3 families; diagnostic clinical characteristics were defined which allowed for early recognition and treatment. During the first 24 hours of life, spontaneous apnea and sluggish feeding effort were observed. After the first 24 hours, surviving infants exhibited the hyperekplexic startle response to nose tapping. This startle response is characterized by sudden muscular rigidity, feeding-induced oropharyngeal incoordination, and poor air exchange often with apnea, persisting with repetitive nose tapping. Untreated infants experienced recurring apnea until 1 year of age. Three of 15 patients died unexpectedly during the neonatal period. Patients treated with clonazepam (0.1-0.2 mg/kg/day) had no serious apneic episodes and startle reflexes were diminished. The pathophysiologic mechanism for hyperekplexia remains obscure. Electroencephalographic studies were consistently normal. The response to and tolerance of benzodiazepines are striking in newborns and infants and suggest an aberrant central nervous system reflex as the etiology; therefore, hyperekplexia should be considered in the evaluation of neonates and infants with apnea, aspiration pneumonia, episodic muscular rigidity, hyperexcitability, and near-miss sudden infant death syndrome. The need for immediate monitoring of at-risk infants, observation for signs of hyperekplexia, and initiation of clonazepam in these patients are emphasized. Hyperekplexic startle response to nose tapping should be included in the routine examination of all newborns.

Meniscal flounce on MR: correlation with arthroscopic or surgical findings.

The purpose of this study was to investigate the frequency of the meniscal flounce on MR imaging in patients who underwent arthroscopy or open surgery due to symptoms related to internal derangement of the knee, and to investigate associated findings in patients with meniscal flounce by comparing and analyzing the findings from MR imaging and surgery. MR images obtained from 116 knees before surgery were reviewed retrospectively. Seven medial menisci showed buckled, wavy flounce on sagittal MR images. None of the lateral menisci showed flounce. We reviewed the surgical records of all seven patients and the videotapes of six of the patients undergoing arthroscopy or open surgery. The frequency of flounce on sagittal MR images was 6.0% in the medial meniscus and was completely absent in the lateral meniscus. On coronal MR images, the truncated appearance of the affected meniscus was demonstrated in five patients, and a valgus deformity was seen in three patients. Five patients showed a moderate to large amount of joint effusion. On MR imaging and in surgery, ligament injuries were found in six patients (six medial collateral ligament injuries, five anterior cruciate ligament injuries, and two posterior cruciate ligament injuries). Non-specific synovitis was found in the one remaining patient. In the surgery of all seven patients, no tears were found at the meniscus itself showing flounce. In conclusion, the meniscal flounce seen on sagittal MR imaging can be a rare appearance of a transient distortion of a normal meniscus due to a valgus deformity caused by a MCL tear and/or due to an external rotation induced by cruciate ligament injury or positioning of knee joint within the magnet. The meniscal flounce should be interpreted carefully because it frequently appears truncated on the coronal scan and can simulate a meniscal tear.

Computerised prostate boundary estimation of ultrasound images using radial bas-relief method.

A new method is presented for automatic prostate boundary detection in ultrasound images taken transurethrally or transrectally. This is one of the stages in the implementation of a robotic procedure for prostate surgery performed by a robot known as the robot for urology (UROBOT). Unlike most edge detection methods, which detect object edges by means of either a spatial filter (such as Sobel, Laplacian or something of that nature) or a texture descriptor (local signal-to-noise ratio, joint probability density function etc.), this new approach employs a technique called radial bas-relief (RBR) to outline the prostate boundary area automatically. The results show that the RBR method works well in the detection of the prostate boundary in ultrasound images. It can also be useful for boundary detection problems in medical images where the object boundary is hard to detect using traditional edge detection algorithms, such as ultrasound of the uterus and kidney.

Vision screening of 4-year-old children in Singapore.

AIM OF THE STUDY: To evaluate the feasibility of an improved visual acuity screening program for Singapore 4-year-old preschool children and to draw up an appropriate referral criteria as well as evaluating the rates and outcomes of these referrals. METHOD: A total of 450 children aged 4 to 4 1/2 years, who attended 3 polyclinics of the Family Health Service (FHS) for their 4-year-old Developmental Health Screening during the study period from 1/4/1997 to 30/6/1997 were recruited for the study. Children who were tested with Snellen (or Sloan) visual acuity chart resulting in visual acuity of 6/9 or worse, or failed to pass the 3 mm medium plate at 30 cm distance (300 seconds of arc) in the Frisby Stereotest, or were found to have strabismus, or were untestable in either visual acuity test or stereotest were offered referral to ophthalmologists in the hospitals for specialist assessment. RESULT: 82.7% of the 450 children were successfully screened with Snellen (or Sloan) chart while 91.6% were successfully screened with Frisby Stereotest. In all, 180 children were evaluated by ophthalmologists. Majority of the children were referred because of their abnormal visual acuity test while only 2 children were referred for failing stereotest alone. Among the 180 children referred, 63 (35.0%) were found to have refractive errors for which spectacles were prescribed. Eight children had amblyopia and 2 children had strabismus which were not detected at the polyclinic screening. The untestable children evaluated had significantly higher abnormality rate (37.5%) than that of children who had 6/9 vision (8.8%) therefore they should be offered referral for further evaluation. There was high "refused referral" rate of 39.0%. Parents of children who were untestable or had 6/9 vision were found to be more likely to refuse offer of referral. If these two groups of children were excluded, the "refused referral" rate dropped to 13.3%. When the referral criteria for visual acuity was reset at 6/12 instead of 6/9, the referral rate dropped from 39.6% to a more manageable 26.7% and the positive predictive value improved from 35.4% to 48.3% and none of the children with amblyopia were missed being screened-out. CONCLUSION: The study confirmed the feasibility of doing visual acuity screening at 4 to 41/2 year-old. The referral criteria for abnormal visual acuity should be set at 6/12. The efficacy of adding Frisby stereotest needs further evaluation.