Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand.

Background & objectives: El Tor Vibrio cholerae O1 carrying ctxBC trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand. Methods: A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpAC, tcpAE, hlyAC and hlyAE were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles. Results: All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid. Interpretation & conclusions: In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxBC).

Validation of salmonellosis and shigellosis diagnostic test kits at a provincial hospital in Thailand.

Rapid Diagnosis of salmonellosis and shigellosis was performed using six different diagnostic test kits which recently have been made available commercially. They were Salmo-Dot, Typhi-Dot, Shigel Dot A, B, C, and D test kits for detection of Salmonella spp., group D salmonellae, and groups A, B, C, and D Shigella spp., respectively. The principle of all test kits is a membrane (dot) ELISA using specific monoclonal antibodies to the respective pathogens as the detection reagents. The present study was designed to validate the accuracy of the test kits, at a laboratory in a provincial hospital in Thailand, in comparison with the conventional bacterial culture method alone or with the combined results of the culture and the Western blot analysis (WB) for detecting the respective bacterial lipopolysacchharides (LPS) in specimens. Five hundred rectal swab samples of patients with diarrhea who seeked treatment at the hospital, were evaluated. The diagnostic accuracy of the Salmo-Dot was 91.0% when compared with the conventional bacterial culture method alone but was 100.0% in comparison with the combined results of the culture and the WB. The Typhi-Dot and the Shigel-Dot A, B, C, and D showed 100%, 99.2%, 95.0%, 94.0% and 96.4%, respectively when compared with the culture alone and all were 100% in comparison with the combination of the results of the bacterial culture and the WB. The Shigel-Dot A revealed antigen of type 1 Shigella dysenteriae in several specimens in which the bacteria could not be recovered by the culture method. This difference is important as type 1 Shigella dysenteriae have high epidemic potential and often cause severe morbidity. Unawareness of their presence by the conventional culture may have great impact on disease surveillance for public health. The pathogen detection using the six diagnostic test kits is sensitive, specific, rapid, and relatively simple and less expensive. Several specimens can be tested at the same time without much increase in turn around time. Moreover, these kits produce no contaminated waste as compared with the bacterial culture method. The test kits should be used for rapid screening of specimens of patients with diarrhea especially in areas where culture facilities are inadequate.

Trichinella spiralis-specific monoclonal antibodies and affinity-purified antigen-based diagnosis.

Hybridomas secreting monoclonal antibodies (MAbs) to Trichinella spiralis were produced. Myeloma cells were fused with splenocytes of a mouse immunized with excretory-secretory (E-S) antigen of infective larvae. A large percentage of growing hybrids secreted antibodies cross-reactive to many of 23 heterologous parasites tested. Only 6 monoclones (designated 3F2, 5D1, 10F6, 11E4, 13D6 and 14D11) secreted MAbs specific to the E-S antigen and/or a crude extract (CE) of T. spiralis infective larvae. The 6 monoclones secreted IgM, IgG3, IgM, IgG3, IgG3 and IgG3, respectively. Clone 5D1 was selected to mass produce MAbs which were then coupled to CNBr-activated Sepharose CL-4B to prepare an affinity-purified antigen. Dot-blot ELISA with either purified antigen or CE was evaluated. There were 17 patients with acute trichinellosis and 76 individuals convalescing from T. spiralis infection (group 1). Controls were 170 patients with parasitic infections other than trichinellosis (group 2) and 35 healthy parasite-free controls (group 3). CE-ELISA was positive in all group 1 patients. However, sera from many group 2 patients also were reactive (opisthorchiasis-44.2%, schistosomiasis-44%, gnathostomiasis-30%, paragonimiasis-28.6%, taeniasis-27.3%, strongyloidiasis-23.1% and hookworm infections-20%). Affinity-purified antigen was 100% specific, all sera from group 2 and group 3 individuals tested negative. Although 74 of 76 patients (97.4%) with convalescing trichinellosis tested positive, sera from only 3 of 17 patients (17.6%) with acute T. spiralis were reactive. Thus, CE antigen is appropriate when sensitivity is needed, while purified antigen should be used when specificity is required. Dot-blot ELISA is easier to perform, more rapid and less expensive than indirect ELISA. Many samples can be assayed simultaneously, special equipment is not required, and results can be preserved for retrospective analysis. Dot-blot ELISA is therefore the method of choice for the rapid diagnosis of trichinellosis, particularly when more complex laboratory tests are unavailable.

Epidemiological analysis of methicillin resistant Staphylococcus aureus in Thailand.

The geographical distribution of 65 clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) recovered from 7 hospitals in Thailand was investigated. The presence of mecA gene in MRSA was determined by specific PCR with the use of primers 5'-GTAGTTGTCGGGTTTGGT-3' and 5'-GGTATCATCTTGTACCCA-3'. Chromosomal DNA restriction analysis with SmaI was resolved by pulsed-field gel electrophoresis (PFGE) compared with antibiotype analysis and phage type analysis. All 65 strains carried mecA gene. They all were resistant to penicillin, tetracycline, erythromycin, amoxicillin/clavulanic acid and variably resistant to gentamicin, ofloxacin, trimethoprim-sulfamethoxazole, chloramphenicol, fosfomycin and clindamycin; and all isolates were susceptible to vancomycin. A total of 19 PFGE patterns designated as type A, A1, A2, A3, A4, B, B1, C, D, E, E1, E2, F, F1, F2, G, H, I and J was identified. Type A4 and E were commonly found in every studied areas. Phage typing showed even greater variability that 52 (80%) isolates belonged to 25 different phage types; 13 (20%) isolates were non-typable. The clarity and polymorphism of the PFGE patterns enable us to discriminate between isolates which could not be differentiated by antibiogram or phage type analysis. The findings demonstrate the existence of a common epidemic MRSA clone in Thailand.

Detection of Salmonella contamination in food samples by dot-ELISA, DNA amplification and bacterial culture.

A dot-blot enzyme-linked immunosorbent assay (dot-ELISA) employing a genus Salmonella specific monoclonal antibody (MAb) was used for detection of the bacteria in food samples in comparison with the conventional culture method and the DNA amplification. Among the 200 chicken and pork samples (100 each) tested, 9% and 33%, 7% and 20% and 7 and 23% were positive for salmonellae by the dot-ELISA, the culture method and the DNA amplification, respectively. Statistical analyses revealed that the sensitivity, specificity, efficacy, and positive and negative predictive values of the detection of Salmonella in the food samples by dot-ELISA compared with the culture method were 93.33%, 91.76%, 92%, 66.66% and 98.73%, respectively. Comparison of the DNA amplification and the culture method revealed the sensitivity, specificity, efficacy, and positive and negative predictive values of 100%, 91.58%, 92%, 65.21% and 100%, respectively. The dot-ELISA and the DNA amplification results were in a better agreement when the two assays were compared. The sensitivity, specificity, efficacy, positive and negative predictive values of the dot-ELISA compared to the DNA amplification were 91.3%, 100%, 98%, 100% and 97.5%, respectively. From this study, the dot-ELISA is rapid, simple, sensitive, specific at low cost with limited amount of infectious waste to be disposed and offers another advantage in that it detects only the smooth LPS of Salmonella which implies the possible presence of the virulent organisms.

Determinação do tempo da dilatação máxima do canalículo da célula parietal após injeção de pentagastrina em espaços de tempo fracionados / Timing of canalicular maximal dilation of the parietal cell after pentagastrin injection at fractionized spaces of time

Doze ratos da linhagem Wistar de 90 dias de idade, pesando aproximadamente 300g, foram mantidos em jejum, só com água, durante 48h, em regime circadiano. Três animais do grupo controle foram sacrificados após o período de jejum. Outros 9 ratos, pertencentes aos 3 grupos experimentais, receberam intraperitonealmente 250microgram/kg de pentagastrina e foram sacrificados respectivamente 3, 20 e 90 minutos depois da injeção. As observações histológicas das células parietais baseadas na histomorfometria comparativa seguida de citometria permitiu-nos confirmar a evidência da dilatação máxima dos canalículos aos 20 minutos, após injeção de pentagastrina

Isolation of haemolytic Escherichia coli from patients with urinary tract infection

One hundred and eighty-five isolates of Escherichia coli isolated from different sources, such as 131 isolates form 58 diarrhoea cases, 33 isolates from 21 control cases and 21 isolates from 9 urinary tract infection cases with different age groups were studied for haemolytic activity. It was observed that one case from diarrhoea cases showed alpha haemolytic activity; none of the control cases showed any haemolytic activity; and 3 cases of urinary tract infection showed beta haemolytic activity. The haemolytic E. coli isolates were also performed for the plasmid encoded haemolysin determinant and demonstrated to possess the haemolytic phenotype Hly and encodes 107,000 kilodalton. E. coli isolated from the urinary tract infections were also testted for antibiotic sensitivity and found that most of them were resistant to ampicillin, streptomycin and tetracycline.

Preparation of enterotoxigenic Escherichia Coli (heat labile) antisera and its use in BIKEN assay (Part 2)

The purified Escherichia Coli (LT) toxin was immunized in rabbits. The specificity of the antisera was demonstrated by Ouchterlony double-gel diffusion tests and by neutralization of the activities of CT and LT to cause morphological changes in Chinese Hamster Ovary cells assays. The optimum titre of the antisera was checked and BIKEN assay was used to check the sensitivity and specificity of the antisera. A total of 965 isolates were used to checked for the identification of LT by BIKEN assay and found that 27 isolates produced LT.

Disinfectants efficacy on bacteria with the contaminants

The study was carried out to determine the effect of contaminants on the efficacy of routinely used disinfectants on bacteria and call on attention of hospital staff and medical care personnel for correct usage of disinfectants. Disinfectant efficacy of Hibitane, Osban, Ethanol, Isodine and Tego 51 on organisms such as P. aeruginosa, E. Cole, S. aureus and B. subtilis contaminated with organic substances such as human blood, egg white and animal oil were investigated. The disinfectant efficacy was also determined on clinical specimens. 70

Ethanol was totally ineffective on B. subtilis. Osban and Ethanol did not effect on P. aeruginosa, and B. subtilis when they were contaminated with the organic substances. Tego 51 was weak in the efficacy on sputum and urine specimens. Hibitane and Isodine were effective on tested organisms even when contaminated with organic substances and also on the clinical specimens.

Purification of enterotoxigenic Escherichia Coli heat labile toxin (Part 1)

Purification of Escherichia Coli heat labile toxin (LT) was done from 9 litres of culture. The cells were suspended in 0.01M Tris (hydroxymethyl) amino-methane (Tris)-hydrochloride buffer (pH 8.6) containing 0.9 percent sodium chloride. The suspension was sonicated. The supernatant was then treated with ammonium sulphate and disolved in TEAN buffer and dialysed. This crude LT was then passed through A5M Biogel and collected by D-galactose and concentrated to about 3 ml. It was then passed through Sephacryl S-200 column and again concentrated by Amicon PM 10 membrane and used as purified LT. Separation of subunits were carried out by using 6M urea solution in 0.1M propionic acid (pH 4.0). Every steps of fractions were checked by Ouchterlony double gel diffusion test using anti cholera sera and by CHO cell assay. Protein assay was done and specificity of the protein was also checked by SDS polyacrylamide slab gel electrophoresis.

Effect of the neonatal steroid hormone treatment of rats on adult hormone levels and the reactivity of seminal vesicles to parasympathomimetic drugs

Seminal vesicle reactivity to cholinergic agents, plasma testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) conmcentrations and seminal vesicle testosterone concentrations were determined in adult male rats treated during the first 6 h of life with 1.0 ml peanut oil (oil-treated), 1.0 mg testosterone propionate (TP-treated) or 1.2 mg 19-nor-testosterone homofarnesate (19-NT-treated). At 90-100 days of age, the neonatally treated animals presented atrophied accessory genital organsand increased (TP-treated, N=10) or unchanged (19-NT-treated, N=11) pD, values for acetylcholine (vehicle; 5.18 ñ 0.06, N=10; TP-treated; 5.26 ñ 0.06, N+10; 19-NT-treated; 5.14 ñ 0.09, , N=11), and acetyyl-beta-methylcholine (vehicle: 5.19 ñ 0.07; TP-treated: 5.43 ñ 0.06; 19-NT-treated 5.25 ñ 0.07). The relative intrinsic activity, alpha, of acetyl-beta-methylcholine increased after both hormonal treatments (vehicle: 0.85 ñ 0.03; TP-treated:0.95 ñ 0.02; 19-NT-treated: 0.92 ñ 0.03). No variation in mean adult plasma testosterone concentration was observed after neonatal treatmentwith either TP or 19-NT (vehicle:752.93 ñ 273.66, N+8; TP-treated; 459.05 ñ 88.32, N=8; 19-NT-treated: 836.86 ñ 113.08, N=7). However, testosterone content of seminal vesicles of adult rats was decreased in the animalstreated with TP (N=5) and 19-NT (N=6) compared to controls. These results indicate a specific effect of neonatal hormone treatment on androgen metabolism which is demonstrable in the adult

Structural and ultrastructural alterations of epithelial cells of vas deferens caused by contrast medium

In a attempt to verify experimentally any possible alterations of epithelial cells of vas deferens caused by radiopaque substance, the right and left vasa deferential of 3 rats were injected respectively with saline solution (0.9 per cent NaCl) and Conray-400 (66.8 per cent sodium iodomethamate). 72 hours later, the animals were killed by cranial trauma and histolofical sections and electronmiucrographs of their vasa deferentia were observed. The histological sections of epithelial cells of left vasa deferentia showed a decrease in height and slight changes in stereocilia as compared with those of right contralateral vasa deferentia. Electronmicrographs displayed a reduction in number and size of stereocilia and alterations of many mitochondria in epithelial cells of left vasa deferentia

Effects of castration and antiandrogens (RU 23908 and cyproterone acetate) on glandulan activity and on the secretory epithelium of the rat submandibular gland

The influence of hormones on the submandibular gland of rodents has attracted more attention since the observation of the sexual dimorphism of these organs. Androgens enhance both the development and secretory activity of the gland. In the present investigation we have studied the differences in wet weight, protein content, glandulain activity and morphometry of granular convoluted tubules of submandibular glands excised from control, castrated and antiandrogen-teated (RU 23908 or cyproterone acetate) male adult albino rats. Castration and antiandrogenic treatment did not affect the wet weight or protein content of the organs. Castration or treatment with RU 23908, but not treatment with cyproterone acetate, decreased glandulain activity and the height of the secretory epithelium of granular ducts. The morphology and glandulain activity of submandibular gland granular ducts do not seem to be related solely to plasma testosterone concentration. Different hormonal treatments are needed to identify other factors implicated in the phenomenon

Estudo morfometrico da mucosa jejunal na esquistossomose mansonica humana. / Morphometric study of the jejunal mucosa in human schistosomiasis mansoni

Os autores estudam morfometricamente a mucosa jejunal de humanos em 4 grupos: controle, esquistossomoticos hepatointestinais e hepatoesplenicos, constituidos por 30 individuos cada um atraves de biopsias perorais e outro grupo de 10 pacientes esquistossomoticos com ma absorcao enterica atraves do estudo de biopsias cirurgicas.Os varios paramentros analisados a luz da estatistica mostraram, no grupo com ma absorcao, uma reducao da altura das vilosidades jejunais, as custas da reducao de celulas absorventes, ocorrendo tambem, aumento do numero de vilosidades achatadas e do indice mitotico. As criptas glandulares nao sofrem modificacoes. As celulas caliciformes e de Paneth nao sofreram alteracoes significativas em seu numero nos diversos grupos. O encontro de ovos de S.mansoni ocorreu apenas nos grupos esquistossomose hepatoesplenica e ma absorcao.Nesses grupos, a densidade de eosinofilos foi superior aos demais

Acao da lidocaina e bupivacaina na rata prenhe: efeito soobre o desenvolvimento ponderal das crias. / Effect of lidocaine and bupivocaine in pregnant rats. Effect on the ponderal development of the litters

Foi estudado o efeito da injecao intraperitoneal de lidocaina e bupivacaina em ratos (Rattus novergicus albinus) prenhes sobre o desenvolvimento ponderal das crias Verificou-se que estes anestesicos locais embora nao influenciem na velocidade de crescimento; os pesos medios das crias, cujas maes, receberam lidocaina durante a prenhez sao menores do que os pesos medios das crias do grupo controle e do grupo que recebeu bupivacaina.periodo de latencia medio observado foi d

Fertility of unilaterally vasectomized rats and ultrastructure of sperm granuloma.

Os testiculos, epididimos e ductos deferentes de ratos albinos, com vasectomia unilateral apresentaram histologia normal. Os granulomas de esperma aumentaram progressivamente de tamanho durante os 280 dias de experiencia e as eletromicrografias de sua parede mostram celulas gigantes de corpo estranho enfileiradas fagocitando fragmentos de espermatozoides.Setenta e cinco por cento dos ratos unilateralmente vasectomizados mantiveram a sua capacidade reprodutora