Development of a ceuE-based multiplex polymerase chain reaction (PCR) assay for direct detection and differentiation of Campylobacter jejuni and Campylobacter coli in Thailand.

A novel ceuE-based multiplex PCR system was developed as an efficient diagnostics test to detect and differentiate C. jejuni and C. coli. There is no cross reactivity between C. jejuni and C. coli. In addition, the assay does not produce a positive signal from other enteric bacteria including Salmonella, Shigella and Escherichia coli strains. Campylobacter detection sensitivity was determined to be equivalent to previously reported PCR for other enteric bacteria. We also noticed that silicon dioxide extraction can improve Campylobacter detection sensitivity from infected stool samples. It was demonstrated that the PCR assay developed in this study had a much better Campylobacter detection rate than the traditional culturing method (77% versus 56%). However, we also identified small numbers of culture positive stools (8%, or 16 out of 202 samples) that did not yield PCR positive results for Campylobacter. These PCR negative/culture positive stools were proven to be inhibitory to PCR amplification.

Prospective study of the incidence of diarrheal disease and Helicobacter pylori infection among children in an orphanage in Thailand.

To evaluate the hypothesis that gastric infection with Helicobacter pylori increases risk for diarrheal disease in children, we conducted a yearlong prospective study among 160 orphanage children < 5 years of age in Nonthaburi, Thailand. Serum samples collected at six-month intervals were examined by ELISA for antibodies to H. pylori, and children were followed daily for the development of diarrhea. Seven percent of children were seropositive on enrollment, 59% were seronegative, and 34% were indeterminate. Among the seronegative children, seroconversion occurred at a rate of 7% per six months. Forty-six percent of children developed 214 total episodes of diarrhea. By age group, children < 18 months, 18-24 months and > 24 months of age experienced 2.6, 1.1, and 0.2 mean diarrhea episodes per six months. The incidence of diarrhea was not significantly different between children by H. pylori serostatus. We conclude that H. pylori infection was not associated with an increased risk of diarrheal disease.

Coli surface antigens associated with enterotoxigenic Escherichia coli strains isolated from persons with traveler's diarrhea in Asia.

Enterotoxigenic Escherichia coli (ETEC) strains were isolated from travelers or military personnel who developed diarrhea after visiting Nepal or who were deployed to Thailand, Indonesia, or the Philippines. ETEC isolates were examined for colonization factor antigen (CFA). CFAs were identified on 59% (40 of 68) of the isolates examined. The lack of a detectable CFA on 41% (28 of 68) of the isolates is of concern for the development of an effective ETEC vaccine.

Distribution of colonization factor antigens among enterotoxigenic Escherichia coli strains isolated from patients with diarrhea in Nepal, Indonesia, Peru, and Thailand.

Samples (1,318) of enterotoxigenic Escherichia coli (ETEC) isolated in 1994-1995 from children with diarrhea from Nepal, Indonesia, Peru, and Thailand were examined for colonization factor antigen (CFA) and coli surface (CS) antigens. Fifty-five percent of 361 heat-labile and heat-stable (LT-ST), 14% of 620 LT-only, and 48% of 337 ST-only ETEC had CFA/CS antigens. LT-ST ETEC strains were predominantly in the CFA II group, and ST only strains were in the CFA IV group. Additional studies are needed to identify ETEC strains that do not have CFA/CS antigens.

A new enzymatic method for the measurement of creatinine involving a novel ATP-dependent enzyme, N-methylhydantoin amidohydrolase.

A new enzymatic method for the measurement of serum and urine creatinine is described. The method is based on a novel microbial creatinine degradation pathway via N-methylhydantoin [Shimizu et al., Clin. Chim. Acta, 185, 241-252 (1989)]. By using two novel enzymes, N-methylhydantoin amidohydrolase and N-carbamoylsarcosine amidohydrolase, as key enzymes, coupled with a colorimetric procedure for hydrogen peroxide detection, the creatinine level can be measured. The results obtained for human serum and urine show good correlation with those obtained by a standard chemical method based on the Jaffe reaction. The new method is simple and specific, and shows excellent sensitivity and reliability.

Purification and characterization of an ATP-dependent amidohydrolase, N-methylhydantoin amidohydrolase, from Pseudomonas putida 77.

N-Methylhydantoin amidohydrolase, an ATP-dependent amidohydrolase involved in microbial degradation of creatinine, was purified 70-fold to homogeneity, with a 62% overall recovery, and was crystallized from Pseudomonas putida 77. The enzyme has a relative molecular mass of 300,000. It is a tetramer of two identical small subunits (M(r) 70,000) and two identical large subunits (M(r) 80,000). The enzyme requires ATP for the amidohydrolysis of N-methylhydantoin and vice versa. Mg2+, Mn2+ or Co2+, and K+, NH4+, Rb+ or Cs+, were absolutely required concomitantly for the enzyme activity as divalent and monovalent cations, respectively. The Km and Vmax values for N-methylhydantoin were 32 microM and 9.0 mumol.min-1.mg protein-1. The hydrolysis of amide compounds and coupled hydrolysis of ATP were observed with hydantoin, DL-5-methylhydantoin, glutarimide and succimide in addition to N-methylhydantoin. 2-Pyrrolidone, 2-oxazolidone, delta-valerolactam, 2,4-thiazolidinedione, 2-imidazolidone, D-5-oxoproline methyl ester, DL-5-oxoproline methyl ester, and naturally occurring pyrimidine compounds, i.e. dihydrouracil, dihydrothymine, uracil, and thymine, effectively stimulated ATP hydrolysis by the enzyme without undergoing detectable self-hydrolysis.

Typing of human group A rotavirus with alkaline phosphatase-labeled oligonucleotide probes or a monoclonal enzyme immunoassay in unfrozen stools of children with diarrhea in Bangkok.

In developed countries, serotypes (or G types) have been identified in > 70% of group A rotavirus using monoclonal enzyme immunoassays (MEIAs); however, these assays have identified < 50% of rotavirus G types from developing countries presumably because the VP7 antigens were damaged by freezing and thawing during transportation of specimens. The VP7 (G) serotypes of rotavirus in unfrozen stool collected from children with acute diarrhea in Bangkok were determined using MEIA and compared to hybridization with alkaline phosphatase-labeled oligonucleotide probes. Reverse transcription of dsRNA coding for VP7 followed by polymerase chain reaction amplification of cDNA was used as an additional step prior to hybridization for 98 specimens that did not hybridize with the oligonucleotide probes. Of 251 rotavirus specimens, 208 (83%; 99% Cl = 76-89%) hybridized with G type specific oligonucleotides compared to 146 (58%; 99% Cl = 50-66%) that were typeable by MEIA. Forty-five (82%) of 55 stools containing G type 1, 80 of 84 (95%) containing G type 2, 0 of 3 containing G type 3, and 2 of 4 (50%) containing G type 4 as identified by MEIA hybridized with G type specific oligonucleotides. Differences in nucleotide sequences coding for VP7, in addition to destruction of the VP7 antigen by freezing and thawing of the specimen, may explain why not all rotavirus hybridized with G type specific probes.

Diagnosis of Helicobacter pylori infection in a developing country: comparison of two ELISAs and a seroprevalence study.

Serology to detect antibodies to Helicobacter pylori is not frequently used as a diagnostic tool in developing countries. When compared to a commercial ELISA, an ELISA constructed and validated in Thailand had a higher sensitivity (98% vs. 85%), specificity (76% vs. 66%), and negative predictive value (97% vs. 76%) for the detection of H. pylori infection among 104 patients with dyspepsia evaluated by endoscopy. The positive predictive value was 88% for both tests. Serum antibody levels fell significantly 5-8 months after eradication of infection in 8 Thai patients (P = .009). By 8 years of age, > 50% of Thai persons living in urban and rural locations were seropositive. The low negative predictive value of the commercial ELISA limits the usefulness of this assay as a diagnostic tool in Thailand and suggests a need to reevaluate H. pylori serologic tests when used in populations living in developing countries.

Diarrhoeal disease in children less than one year of age at a children's hospital in Guangzhou, People's Republic of China.

We performed a case-control study of diarrhoea to determine its causes in children less than 1 year old in Guangzhou, People's Republic of China, in April to September 1989. Stools were cultured for Salmonella, Shigella, Campylobacter and vibrios by standard techniques; rotavirus (RV) was identified by polyacrylamide gel electrophoresis; and specific deoxyribonucleic acid probes were used to identify Escherichia coli containing genes coding for Shiga-like toxin I and II, enteropathogenic E. coli adherence factor, and enteroinvasive E. coli (EIEC). E. coli isolates were tested for heat-labile toxin (LT) and heat-stable toxin (ST) production and mannose-resistant adherence to HeLa cells. Rotavirus was identified in 13 of 174 children with diarrhoea (cases) and in 2% of 174 age-matched children without diarrhoea (controls), P less than 0.001. C. jejuni was identified in 10% of cases and 2% of controls, P = 0.003. Giardia lamblia was identified in 4 cases, LT and ST enterotoxigenic E. coli (ETEC) in 2, and S. flexneri in 1 case; they were not found in controls. ETEC that produced LT only was isolated from 5 cases and 3 controls, P = 0.721; E. coli that adhered to HeLa cells in a diffuse pattern was isolated from 30 cases and 40 controls, P = 0.229; and E. coli that adhered in an aggregative pattern was isolated from 20 cases and 18 controls, P = 0.863. EIEC was not isolated from cases or controls. Nine cases (5%) developed persistent diarrhoea (greater than 14 d duration). C. jejuni and aggregative E. coli were isolated from different children with persistent diarrhoea.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of sarcoplasmic reticulum Ca2+-adenosine triphosphatase from vitamin E-deficient dystrophic rabbit skeletal muscle with iron-ascorbate-treated and untreated enzyme.

Sarcoplasmic reticulum Ca2+-ATPase from rabbit skeletal muscle has an Arrhenius curve of enzyme activity with a discontinuity at about 20 degrees C. Preparations treated with FeSO4 and ascorbic acid and from a vitamin E-deficient dystrophic rabbit have 22% of the normal activity and a linear Arrhenius curve (Promkhatkaew, D., Komaratat, P., & Wilairat, P. (1985) Biochem. Int. 10, 937-943). All three preparations were cross-linked to the same extent by dimethyl suberimidate and copper-phenanthroline reagent at temperatures above and below the temperature of the Arrhenius discontinuity. Both iron-ascorbate-treated Ca2+-ATPase and that from a vitamin E-deficient animal had 50% of the normal sulfhydryl content, but the disulfide and free amino contents were unaltered. These observations suggest that loss of sulfhydryl groups through lipid peroxidation, both in vivo and in vitro, resulted in reduction of Ca2+-ATPase activity and loss of the break in the Arrhenius plot. Changes in Ca2+-ATPase polypeptide aggregational state could not account for the discontinuity in the Arrhenius curve as revealed by the similar extent of cross-linking of the three enzyme preparations at temperatures above and below the temperature of the Arrhenius discontinuity.