An investigation on the knowledge, attitudes and practice towards vaginal candidiasis amongst women of reproductive age attending the University of the West Indies, St. Augustine Campus

From 20th century, our views, understanding and treatment of pathogenic infections have drastically changed. Pathogenic organisms were discovered, classified and treatments were subsequently implemented. Candidiasis spp. was discovered and linked to the condition; Vulvovaginal Candidiasis (VVC), commonly known as yeast infection, which affects the female lower genital tract, vulva and vagina. Symtoms of such an infection include itching, burning, soreness and a creamy vaginal discharge. Given the nature, location and symptoms of such an infection, individuals are often self-conscious and hesitant to discus it or seek medical attention until symptoms become unbearable. VVC is one of the most common infections in reproductive age females with 75% of women experiencing infection at least once in their lives. Due to the qualitative gap in literature toward women affected by VVC in our country, this study aimed to highlight the knowledge, attitudes and practices towards VVC among reproductive age in Trinidad and Tobago.

Poster - Thur Eve - 67: Clinical results of deep inspiration breath hold radiation treatment for the left breast patients.

Adjuvant radiotherapy for left breast cancers increases local tumor control, but also increases the risk of radiation-induced cardiac disease. Deep Inspiration Breath Hold (DIBH) can minimize dose to the heart for left breast patients where the heart is within the tangential field. In this study, we evaluated the dosimetric benefit of DIBH technique comparing to free breathing (FB) radiotherapy for left breast cancer patients. Five patients with left breast cancer treated with DIBH technique were selected randomly. The CT scans of breath hold (BH) and FB were taken for every DIBH patient. Standard clinical DIBH intensity-modulated radiotherapy (IMRT) plans were generated with BH scan dataset using the Varian Eclipse TP system. The prescription dose is 4250 cGy in 16 fractions. The BH plan was copied to the FB scan dataset and shifted accordingly to have the same coverage for the breast tissue, and the dose was re-calculated. Dose-volume histograms (DVH) of the heart and lung; mean dose and maximum dose of the heart were calculated and compared from the BH and FB plans for every patient. The lung volume is increased during BH and hence the heart is moved out of the field, resulting in the lower heart maximum dose. The mean dose is almost less than 1 Gy for all BH plans. The average mean heart dose is 0.8 Gy for BH plan compared to 1.6 Gy for FB plan. Patients benefit significantly from DIBH technique due to the very low heart dose.

Long-term outcome of high-dose melphalan and autologous stem cell transplantation for AL amyloidosis.

Light chain (AL) amyloidosis is the result of a clonal plasma cell expansion, in which amyloidogenic monoclonal light chains deposit in various tissues resulting in organ dysfunction and organ failure. The median survival of patients with AL amyloidosis without therapy is 10-14 months. Several phase II studies report haematological and clinical remission in up to 50% of patients after high-dose melphalan and autologous stem cell transplantation. We analysed retrospectively the long-term outcome of 19 patients treated in this way between August/1996 and December/2001. We observed a relatively high treatment-related mortality of 26%, but 12 patients (63%) were high-risk candidates. Eight patients (42%) surviving longer than 100 days achieved haematological remission and long-term survival, whereas 6 (32%) obtained no clear benefit from high-dose therapy. However, 62% of patients survived beyond 2 years and the median survival from transplant was 48 months (range 0-104 months).

Outcome of local excision of rectal carcinoma.

PURPOSE: This study was designed to determine the results of patients with rectal adenocarcinoma treated with local excision. METHODS: A retrospective, chart review was conducted for all patients treated with local excision for rectal adenocarcinoma from 1984 to 1998. RESULTS: Sixty-four patients were retained for analysis. The median follow-up was 37 (range, 9-125) months. There were 15 local failures with a median time to local failure of 12 months. Seven patients were salvaged with further operation (4 by repeat local excision, 4 by abdominoperineal resection, and 1 by low anterior resection). The incidence of local recurrence increased with advancing stage of the carcinoma (T1, 13 percent; T2, 24 percent; T3, 71 percent), histologic grade of differentiation, (well, 12 percent; moderately, 24 percent; poorly, 44 percent), and margin status (negative, 16 percent; close (within 2 mm), 33 percent; positive, 50 percent). Sixteen percent of carcinomas < or = 3 cm failed compared with 47 percent for carcinomas > 3 cm. Nine percent (1/11) of T2 patients treated with adjuvant radiation therapy recurred locally compared with 36 percent (5/14) without radiation therapy. Three of four T3 patients who received radiation therapy failed locally compared with two of three who did not. Using the Kaplan-Meier method, the overall survival at five years was 71 percent, and disease-free survival was 83 percent. Actuarial local failure was 27 percent and freedom from distant metastasis was 86 percent. The sphincter preservation rate was 90 percent at five years. CONCLUSIONS: Local excision alone is an acceptable option for well-differentiated, T1 carcinomas, < or = 3 cm. Adjuvant radiation is recommended for T2 lesions. The high local recurrence rate in patients after local excision of T3 lesions with or without adjuvant radiotherapy would mandate a radical resection.

Geometry of the DNA Substrates in Cre-loxP Site-Specific Recombination.

Abstract Cre recombinase is a member of a large family of site-specific recombination enzymes that performs a cut-and-paste operation between two specific DNA sequences. Our goal has been to understand the mechanism of this complex reaction by trapping and characterizing the three-dimensional structures of each of the reaction intermediates. This work has led to high resolution crystallographic models of (i) the initial synaptic complex, (ii) the covalent Cre- DNA intermediate, and (iii) the Holliday junction intermediate. The Cre-loxP system appears to function by creating at the outset a protein-DNA architecture that resembles that of the Holliday junction intermediate that is eventually formed. The "arms" of the loxP sites are initially bent by about 75° in the synaptic complex, forming a nearly planar arrangement that is held fixed, while cleavage and strand exchange occur in the central region between the arms. The simplest view of the recombination pathway is that it contains two symmetrical halves, each of which uses this Holliday junction-like architectural framework to mediate the cleavage and ligation steps. The two halves are linked by a subtle isomerization of the Holliday intermediate that switches the roles of the recombinase subunits and allows exchange of the second pair of DNA strands. In this paper, we summarize recent structural results from our laboratory, with an emphasis on the geometry of the DNA substrates.

Determination of domperidone in human serum and human breast milk by high-performance liquid chromatography-electrospray mass spectrometry.

A sensitive and selective method for the determination of domperidone in human breast milk and serum has been developed. The same method may be successfully applied to both matrices to a lower limit of quantitation of 0.5 ng/ml. Samples are processed by a liquid-liquid extraction, and analyzed by LC-ESI-MS in positive ion mode. There was no interference, on the domperidone quantitation, from over 30 drugs. Samples from patients, at various times post-dose, were analyzed and a large number showed significant levels of domperidone in the breast milk as well as in the serum.

Asymmetric DNA bending in the Cre-loxP site-specific recombination synapse.

Cre recombinase catalyzes site-specific recombination between two 34-bp loxP sites in a variety of DNA substrates. At the start of the recombination pathway, the loxP sites are each bound by two recombinase molecules, and synapsis of the sites is mediated by Cre-Cre interactions. We describe the structures of synaptic complexes formed between a symmetrized loxP site and two Cre mutants that are defective in strand cleavage. The DNA in these complexes is bent sharply at a single base pair step at one end of the crossover region in a manner that is atypical of protein-induced DNA bends. A large negative roll (-49 degrees) and a positive tilt (16 degrees) open the major groove toward the center of the synapse and compress the minor groove toward the protein-DNA interface. The bend direction of the site appears to determine which of the two DNA substrate strands will be cleaved and exchanged in the initial stages of the recombination pathway. These results provide a structural basis for the observation that exchange of DNA strands proceeds in a defined order in some tyrosine recombinase systems. The Cre-loxS synaptic complex structure supports a model in which synapsis of the loxP sites results in formation of a Holliday junction-like DNA architecture that is maintained through the initial cleavage and strand exchange steps in the site-specific recombination pathway.

Structure and mechanism in site-specific recombination.

Three-dimensional structural information on the integrase family of site-specific recombinases has only recently become available, with the crystal structures of catalytic domains, full-length proteins and protein-DNA complexes of this family reported over the past two years. These results have led to a model for the overall architecture and active site stereochemistry of the recombination pathway that addresses a number of interesting mechanistic issues.

Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination.

We have determined the X-ray crystal structures of two DNA Holliday junctions (HJs) bound by Cre recombinase. The HJ is a four-way branched structure that occurs as an intermediate in genetic recombination pathways, including site-specific recombination by the lambda-integrase family. Cre recombinase is an integrase family member that recombines 34 bp loxP sites in the absence of accessory proteins or auxiliary DNA sequences. The 2.7 A structure of Cre recombinase bound to an immobile HJ and the 2.5 A structure of Cre recombinase bound to a symmetric, nicked HJ reveal a nearly planar, twofold-symmetric DNA intermediate that shares features with both the stacked-X and the square conformations of the HJ that exist in the unbound state. The structures support a protein-mediated crossover isomerization of the junction that acts as the switch responsible for activation and deactivation of recombinase active sites. In this model, a subtle isomerization of the Cre recombinase-HJ quaternary structure dictates which strands are cleaved during resolution of the junction via a mechanism that involves neither branch migration nor helical restacking.

Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse.

During site-specific DNA recombination, which brings about genetic rearrangement in processes such as viral integration and excision and chromosomal segregation, recombinase enzymes recognize specific DNA sequences and catalyse the reciprocal exchange of DNA strands between these sites. The bacteriophage recombinase Cre catalyses site-specific recombination between two 34-base-pair loxP sites. The crystal structure at 2.4 A resolution of Cre bound to a loxP substrate reveals an intermediate in the recombination reaction, in which a Cre molecule has cleaved the substrate to form a covalent 3'-phosphotyrosine linkage with the DNA. Four recombinases and two loxP sites form a synapsed structure in which the DNA resembles models of four-way Holliday-Junction intermediates. The Cre-loxP complex challenges models of site-specific recombination that require large changes in quaternary structure. Subtle allosteric changes at the carboxy termini of the Cre subunits may instead coordinate the cleavage and strand-exchange reactions.

Urine culture screening in lithotripsy compared with traditional methods.

The authors compare biochemical screening tests with microscopy, cultures, and patient symptoms in 257 patients who visited the Urology-Lithotripsy Unit. They conclude that strip screening and microscopy were poor methods to screen for UTI in patients with stents and raise the question whether all stent patients or just symptomatic stent patients should have urine cultures. The authors also suggest that UTI can be eliminated as a diagnosis in 45% of non-stent patients by using strip screening and not doing further testing. These recommendations have quality improvement and cost effectiveness implications.

Inosine-uridine nucleoside hydrolase from Crithidia fasciculata. Genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue.

Protozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidia fasciculata is the inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase). The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to the amino acid sequences of tryptic fragments and to the miniexon of C. fasciculata. The full-length cDNA was expressed in Escherichia coli and the protein purified to > 99% homogeneity. The open reading frame encodes a protein of 315 amino acids. Enzyme purified from C. fasciculata was missing the N-terminal Met and gave a major mass peak of 34 194 amu by mass spectrometry. Predicted mass from the DNA sequence for the Met-processed enzyme was 34 196. A pET3d-IUNH construct expressed in E. coli introduced MetAla instead of MetPro at the N-terminus. Enzyme purified from this construct also had a processed N-terminus and gave predicted and observed masses of 34 168 and 34 170 amu, respectively. The amino acid sequence for IU-nucleoside hydrolase has no close relatives among the known proteins. A cDNA clone of unknown function from Leishmania major shows near identity in the N-terminal deduced amino acid sequence. Open reading frames near 1 and 47 min on the E. coli chromosome and from two yeast genomes encode for proteins of similar size with substantial amino acid identity. Mutation of His241Ala caused a 2100-fold loss in k(cat) for inosine but a 2.8-fold increase in k(cat) with p-nitrophenyl beta-D-ribofuranoside, establishing the location of the catalytic site and implicating His241 as a proton donor for leaving group activation. IU-nucleoside hydrolase from C. fasciculata and the protein expressed in E. coli were crystallized and diffract to 2.5 and 2.1 A resolution, respectively. Both belong to the P2(1)2(1)2 orthorhombic space group with unit cell parameters a = 63.5 A, b = 131.9 A, c = 90.1 A, and alpha = beta = gamma = 90 degrees. Two subunits of the tetrameric enzyme are present in the asymmetric unit. The following paper reports the X-ray crystal structure for this enzyme.

Three-dimensional structure of the inosine-uridine nucleoside N-ribohydrolase from Crithidia fasciculata.

Protozoan parasites rely on the host for purines since they lack a de novo synthetic pathway. Crithidia fasciculata salvages exogenous inosine primarily through hydrolysis of the N-ribosidic bond using several nucleoside hydrolases. The most abundant nucleoside hydrolase is relatively nonspecific but prefers inosine and uridine as substrates. Here we report the three-dimensional structure of the inosine-uridine nucleoside hydrolase (IU-NH) from C. fasciculata determined by X-ray crystallography at a nominal resolution of 2.5 A. The enzyme has an open (alpha, beta) structure which differs from the classical dinucleotide binding fold. IU-nucleoside hydrolase is composed of a mixed eight-stranded beta sheet surrounded by six alpha helices and a small C-terminal lobe composed of four alpha helices. Two short antiparallel beta strands are involved in intermolecular contacts. The catalytic pocket is located at the C-terminal end of beta strands beta 1 and beta 4. Four aspartate residues are located at the bottom of the cavity in a geometry which suggests interaction with the ribose moiety of the nucleoside. These groups could provide the catalytically important interactions to the ribosyl hydroxyls and the stabilizing anion for the oxycarbonium-like transition state. Histidine 241, located on the side of the active site cavity, is the proposed proton donor which facilitates purine base departure [Gopaul, D. N., Meyer, S. L., Degano, M., Sacchettini, J. C., & Schramm, V. L. (1996) Biochemistry 35, 5963-5970]. The substrate binding site is unlike that from purine nucleoside phosphorylase, phosphoribosyltransferases, or uracil DNA glycosylase and thus represents a novel architecture for general acid-base catalysis. This detailed knowledge of the architecture of the active site, together with the previous transition state analysis [Horenstein, B. A., Parkin, D. W., Estupiñán, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795], allows analysis of the interactions leading to catalysis and an explanation for the tight-binding inhibitors of the enzyme [Schramm, V. L., Horenstein, B. A., & Kline, P. C. (1994) J. Biol. Chem. 269, 18259-18262].

Improving quality and resource utilization through the clinical technologist.

This paper reports on the significant benefits realized during the implementation of a Clinical Technologist position in two wards of a major teaching hospital. In addition to efficient and effective handling of specimens and test results, the program results in significant savings and in enhanced patient care.

Refinement of the structure of Escherichia coli-derived rat intestinal fatty acid binding protein with bound oleate to 1.75-A resolution. Correlation with the structures of the apoprotein and the protein with bound palmitate.

The structure of rat intestinal fatty acid binding protein (I-FABP) with bound oleate (C18:1) has been refined with x-ray diffraction data to a resolution of 1.75 A. The protein contains 10 anti-parallel beta strands composed of 99 residues and 2 short helices of 14 residues. Oleate is located in the interior of the protein in a bent conformation with C1-C12 more ordered than C13-C18. Two of the eight ordered waters in I-FABP:oleate are part of a hydrogen bond network that includes the carboxylate of oleate, the guanidinium group of Arg106, the nitrogen of the indole group of Trp82, and the side chain of Gln115. Most of the methylenes of bound oleate reside in a crevice formed by hydrophobic and aromatic side chains. Tyr70 and Tyr117 envelop the acyl chain from C3 to C8 forming contacts with both the convex and concave faces of its van der Waals surface. The hydroxyls of each phenolic side chain hydrogen bond to ordered water molecules. Two ordered waters make van der Waals contact with the concave face of the bound fatty acid. The omega-terminal methyl of oleate is oriented so that it points toward the center of the benzene of Phe55 allowing it to form van der Waals interactions with its component methylenes. Comparison of the structure of I-FABP:oleate with a recently refined 1.19-A model of apoI-FABP and an earlier 2.0-A model of I-FABP:palmitate revealed a remarkable degree of similarity in the positions of their main chain and side chain atoms and in the conformations of the bound oleate and palmitate. The principal differences were confined to a few discrete regions of the protein. The helical domain, the type I turn between beta strands C and D, and the ring of Phe55 together form a solvent-accessible portal to the interior of the protein. They are repositioned in I-FABP:oleate (and I-FABP:palmitate) so that the binding cavity is even more accessible to solvent and its volume is increased. The side chain of Phe55 which shows discrete disorder in the apoprotein functions as an omega-terminal "sensing device": moving progressively outward toward the surface as the chain length of the bound fatty acid increases by 2 methylenes. Tyr70 and Tyr117 which also show discrete disorder in the apoprotein structure due to rotation around their C alpha-C beta bonds, are stabilized in a single, well ordered position in the holoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of blood cultures with corresponding venipuncture site cultures of specimens from hospitalized premature neonates.

We compared the presence and identities of isolates from blood culture samples obtained by percutaneous venipuncture with those of commensal skin organisms cultured from respective venipuncture sites after skin cleansing; 677 blood and skin site culture pairs from 488 infants were compared. Organisms grew in 58 blood cultures; nine of these cultures had corresponding venipuncture site cultures that also grew organisms. Forty-two blood culture isolates were coagulase-negative staphylococci; five of these were associated with similar venipuncture site cultures. According to restriction-endonuclease fingerprinting of chromosomal DNA and plasmid analysis, three pairs of blood and venipuncture site cultures were identical and two pairs were different. Thus only 7% (3/42) of coagulase-negative staphylococcal blood isolates were associated with identical contamination at the venipuncture site. We conclude that, if the venipuncture site has been carefully cleansed, the growth of coagulase-negative staphylococci in blood cultures of specimens from premature neonates indicates bacteremia rather than skin contamination in the vast majority of cases.

Isolation of Agrobacterium radiobacter from a central venous catheter.

A case of septicemia caused by Agrobacterium radiobacter is reported in a patient undergoing chemotherapy treatment who had recently been neutropenic. Agrobacterium radiobacter was isolated from the Hickman line blood culture. The patient responded favorably to removal of the Hickman catheter and treatment with amikacin and piperacillin. The molecular and biochemical characteristics of the isolate are presented.

Comparison of two rapid methods used in the identification of Haemophilus and Moraxella species.

Both Haemophilus influenzae and Moraxella catarrhalis cause pneumonia in children and adults. The timely isolation and identification of these two organisms is important for the initiation of antibiotic therapy. This paper compares two commercial systems with traditional biochemical methods with respect to accuracy, cost and turn-around-time.