Clinical and prognostic categorization of extraintestinal nontyphoidal Salmonella infections in infants and children.

The study included 172 patients, aged 0-15 years, for whom at least 1 nonfecal, nonurinary specimen was culture-positive for nontyphoidal Salmonella. Ninety-five percent had positive blood cultures. Immunocompromising diseases were found in 19% of 74 infants and 77% of 98 children. Associations between the study factors and outcomes, as localized infection or death, were assessed by logistic regression analysis. Thirty-three patients had localized infections. An adjusted risk factor for development of localized infections was an age of <12 months (P=.003). There were 17 deaths. The case-fatality rates were 43% and 10% for immunocompromised and 5% and 0% for nonimmunocompromised infants and children, respectively. Adjusted risk factors for death were age of <12 months (P=.006), inappropriate antimicrobial therapy (P=.014), meningitis or culture-proven pneumonia due to nontyphoidal Salmonella (P=.004), and immunocompromised status (P<.001). The clinical courses and prognoses for infants and children with extraintestinal infection due to nontyphoidal Salmonella can be categorized into 4 groups according to the characteristics of age (infants vs. children) and host status (immunocompromised vs. nonimmunocompromised).

Primers for Salmonella serovar detection by polymerase chain reaction.

Salmonella serovar detection was studied by polymerase chain reaction (PCR). The primers were designed from Salmonella specific clone, A18:2 which was previously constructed and studied for genus specificity through colony hybridization. The primers were subsequently tested for specificity and sensitivity and showed that they amplified DNA fragment of all Salmonellae tested but did not amplify all isolates of non-Salmonellae tested. The amplified fragment was confirmed and increased sensitivity by nested PCR. Salmonella isolates amplified by the primers in the first round PCR were all positive in the second round. The sensitivity in the first and second round were 7 pg and 80 fg, respectively. The result indicated that the primers can be used as molecular tool for future field survey of Salmonella both in food and in clinical specimens.

Construction of specific DNA probe for the detection of Salmonella in food.

The Salmonella specific DNA fragment from genomic DNA of S. typhimurium ATCC 23566 was cloned in E. coli and successfully used as a digoxigenin labeled probe for detecting the presence of Salmonella serotypes in both artificially contaminated food and natural contaminated food samples.

Salmonella enteritidis outbreak in Thailand: study by random amplified polymorphic DNA (RAPD) analysis.

An outbreak of Salmonella enteritidis in Thailand was reported in 1990. The majority of isolates were found in chicken and human throughout the country. The continuation of a high rate of spreading which is presently continuing prompted us to investigate possible clonal involvement in the outbreak. One hundred and twenty five isolates of S. enteritidis which were isolated between 1990-1993 were clonally identified by the technique of Random Amplified Polymorphic DNA (RAPD) analysis. Eight profiles were found indicating the presence of 8 clones, designated no. 1-8. The predominant clone was profile no. 4 which was encountered in 93.6% of tested isolates while the rest of the profile comprised only 0.8-1.6%. The predominant clone was distributed mainly in isolates from chickens and humans which is suggestive that the profile no. 4 is the major clone involved in this outbreak and that chickens were the source of S. enteritidis infection. The information from the Microbiology Laboratory at Ramathibodi Hospital revealed that nearly 40% of S. enteritidis were isolated from blood specimens. This may reflect the invasiveness of S. enteritidis in Thailand. We concluded that the outbreak involved the single clone, RAPD profile no. 4 which may disperse dominantly during the epidemic.

Electron microscopy study of the mode of growth of Pseudomonas pseudomallei in vitro and in vivo.

The mode of growth of Pseudomonas pseudomallei in culture media and in the lung tissue of infected humans and animals was studied using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). In culture media, P. pseudomallei cells were seen to be entrapped in microcolonies within large amounts of intercellular fibrous material. The lung tissue of infected humans and animals showed that bacterial cells growing in lung tissue were surrounded by radially arranged fibres that constitute a very well defined glycocalyx structure. In the infected areas of the animal lung tissue, bacterial cell could be seen to have formed glycocalyx enclosed microcolonies that displaced host cell components, e.g. the nucleus of a phagocyte. The presence of bacteria in unusual locations indicated that effective phagocytosis was not occurring. The demonstration that cells of P. pseudomallei produce exopolysaccharide glycocalyces and form glycocalyx enclosed microcolonies in laboratory media and in lung tissue of infected humans and animals and the presence of bacteria in unusual locations contribute to a new understanding of the mechanism whereby this organism can cause persistent chronic infections.

Resistance of Pseudomonas pseudomallei growing as a biofilm on silastic discs to ceftazidime and co-trimoxazole.

We have examined the resistance of Pseudomonas pseudomallei biofilm cells to ceftazidime and cotrimoxazole. A large number of these biofilm cells remained viable at 12 and at 24 h, except in the biofilm treated with 200 times the MIC of ceftazidime. The inherent resistance of P. pseudomallei biofilms to conventional antibiotics may explain the lack of success in the treatment of the chronic manifestations of this bacterial infection.

Morphological variation in pathogenic strains of Penicillium marneffei.

Penicillium marneffei is a dimorphic fungus known to be pathogenic to animals and man. The natural reservoir of this organism was known to be bamboo rats found in South Vietnam, Thailand and China. The first two human infections were reported in 1959 and 1973 from the United States. Up to 1984, five new cases of human penicillosis were reported from Thailand. Since then several more cases have been reported from different parts of the world mainly from the southern part of China. However, there are very limited mycological descriptions of this fungi. In this report, five Thai strains were studied for colonial morphology in comparison with Reference strain PLM 689. Variation in mycelial pigment was observed ranging from yellowish-green to orange with water soluble red pigment produced in every strain which can be seen early from the reverse side. Ultrastructural study by both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) was compared with that of the reference strain PLM 689. PLM 689 strain had only biverticillate penicilli, but all five strains from Thailand had both monoverticillate and biverticillate penicilli which occasionally appeared on the same branch. The conidia of the Thai isolates were oval in shape and 1.3-2 x 0.7-1.6 microns in size smaller than those of PLM 689 which were 2.5-4 x 2-3 microns. Phialides were also smaller and a little shorter but the number of phialides was similar to those of PLM 689 ranging 4-10 except for one strain which had 3-16 phialides. All Thai strains have stipes smaller and somewhat longer than those of PLM 689.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunodiffusion test for diagnosis and monitoring of human pythiosis insidiosi.

To facilitate the laboratory diagnosis of human cases of pythiosis insidiosi, an immunological test was evaluated. A soluble antigen was prepared from a human isolate of Pythium insidiosum, an aquatic, thermotolerant oomycete that causes infections in cattle, dogs, horses, and humans. Sera from seven proven cases of disseminated human pythiosis insidiosi were tested in an immunodiffusion test along with appropriate control sera from patients with a variety of actinomycotic, bacterial, and mycotic diseases as well as sera from uninfected individuals. Titers ranged from 1:1 to 1:32 in the seven serum samples from the disseminated cases of pythiosis insidiosi of varying severity. The heterologous sera gave negative reactions. The rapidity and specificity of the immunodiffusion test makes it a useful diagnostic tool for the serodiagnosis of P. insidiosum infections.

A community-based study of acute respiratory tract infection in Thai children.

A 2-year longitudinal study was conducted among the population of a socioeconomically depressed urban community in Bangkok, Thailand, from January 1986 through December 1987 to determine the incidence, etiologic agents, and risk factors associated with acute respiratory tract infection (ARI) in children less than 5 years of age. Data were obtained for a total of 674 children, who were visited twice weekly for detection of signs and symptoms of ARI. During the first year of the study, throat-swab specimens were obtained for bacterial culture from both ill and healthy children and a nasal wash was performed on mildly ill children for detection of virus. During both years of the study, nasopharyngeal aspiration for identification of virus was performed for children with more severe infection. The overall incidence of ARI was 11.2 episodes per child-year. The highest (14.9) and lowest (8.8) rates per child-year occurred in age groups 6-11 months and 48-59 months, respectively. Respiratory syncytial virus, parainfluenza virus, adenovirus, Streptococcus pneumoniae, and Haemophilus influenzae were the prevalent pathogenic agents identified. Factors associated with higher risk of ARI were low family income, working mothers, mothers with allergies, chronic malnutrition, and crowding in the home.

Salmonella krefeld in Thailand: I. Epidemiology, infection and drug resistance.

Information from the National Salmonella Shigella Center (NSSC), Thailand indicated that the most frequently isolated Salmonella serotype from humans during 1974-1975 was Salmonella typhi (33.1%), during 1976-1982 was S. krefeld (26.6%) and during 1983-1987 was S. derby (12.6%). Antimicrobial susceptibility study of various Salmonella serotypes indicated that S. krefeld was the serotype with multiple drug resistance persisting for the longest period of time. Human salmonellosis due to S. krefeld is very rare. During 1976-1978, a large outbreak of S. krefeld gastroenteritis occurred in Thailand, mainly in children. The outbreak spread countrywide and is currently endemic. Gastrointestinal symptoms are severe in young infants. Systemic invasion with bacteremia, meningitis and pneumonitis were reported. The antimicrobial susceptibility pattern of isolates varied from sensitive to multiply drug resistant. The common antibiotic resistances were to ampicillin (75-92%), chloramphenicol (33-75%), kanamycin (67-90%) and sulfamethoxazole-trimethoprim (15-52%). Resistance to gentamicin and sulfamethoxazole-trimethoprim declined after the period of the epidemic. Antimicrobial resistance patterns of 150 S. krefeld strains isolated in Thailand during 1978-1987 showed multiple drug resistance with up to seven drugs. The most common patterns were ApCmKmSuTp and ApCmKmSmSuTc.

Salmonella krefeld in Thailand: II. Molecular biology of drug resistance.

Human salmonellosis due to Salmonella krefeld is very rare. During 1976-1978, a large outbreak of S. krefeld gastroenteritis occurred in Thailand, mainly in children. The majority of strains were multiply drug resistant with high minimum inhibitory concentration (MIC). The MIC for these drugs were ampicillin (Ap) 256-4096 mg/l, chloramphenicol (Cm) 256-512 mg/l, kanamycin (Km) 512- greater than 4096 mg/l, streptomycin (Sm) greater than 1024 mg/l, sulfamethoxazole (Su) 4096- greater than 8192 mg/l, tetracycline (Tc) 64-128 mg/l and trimethoprim (Tp) 64-256 mg/l. Resistance to Su and Tp declined after the period of the epidemic. The resistance genes were found to be highly transferable at a rate of 10(-2) to 10(-4). All strains with more than five resistance markers had large molecular weight plasmids of 120-140 megadaltons. The restriction profile analysis of plasmids from isolates collected from various regions of the country showed similarity of DNA fragment pattern. These isolates were resistant to Ap, Cm, Km, Sm, Su and Tc.

Epidemiological study of sulfonamide and trimethoprim resistance genes in Enterobacteriaceae.

Sulfonamide (Su) and trimethoprim (Tp) resistance are known to caused by the production of drug resistant dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR), respectively. Sulfonamide and trimethoprim are often used in combination under the name cotrimoxazole. Cotrimoxazole resistance in various enteric bacteria isolated at Ramathibodi Hospital was studied. The rate of resistance from 1984-1989 of many genera was rather constant at 40%-60% except in Shigella spp in which the rate increased rapidly in 1987 till 1989. Seventy-five percent of Su-Tp resistant (Sur-Tpr) bacteria were also found to be resistant to other drugs such as ampicillin, aminoglycosides, tetracycline and chloramphenicol in addition to cotrimoxazole. Two hundred and forty Su-Tp resistant strains were analysed for the presence of type I and II dihydropteroate synthase as well as type I and V dihydrofolate reductase genes by hybridization with the corresponding gene probes. Type I DHPS gene predominated in Su-Tp resistant bacteria at 60.8% whereas type II DHPS was found in only 25%. Some strains (11.7%) had both genotypes but 2.5% did not have any. In the trimethoprim resistance study, the DHFR type I gene was also found more frequently (30%) whereas type V DHFR was only 19%. The remaining of Tp resistance (51%) was unclassified. The coexistence of Su and Tp resistance genes of each type was investigated among 118 Su and Tp resistant strains. It was found that type I DHPS gene was found together with either type I or V DHFR gene and type II DHPS was found with type I DHFR gene at about the same rate (28.9%, 27.1% and 26.3%, respectively). However, the presence of type II DHPS together with type V DHFR was rather low, only 5.9% of isolates were found to have both types of genes.

Drug resistance of Haemophilus ducreyi.

Chancroid, the disease caused by H. ducreyi is one of the common sexually transmitted diseases (STD) in Thailand and other tropical countries. In Thailand, the diagnosis of chancroid is still based on clinical appearance which may be confused with other STD manifested by genital ulcers. In recent years the increasing resistance strains of H. ducreyi to these antimicrobial agents has been reported so that cultivation and antimicrobial susceptibility tests of this organism have become more important. This study showed that MBV is the best medium for isolation with a success rate of 48%. All strains tested from isolates of this study were resistant to ampicillin, due to production of beta-lactamase. Approximately 99% of the strains were resistant to tetracycline 92% of strains were resistant to sulfamethoxazole and 32% were resistant to trimethoprim. All isolates were susceptible to chloramphenicol, ceftriaxone, erythromycin and the fluorinated quinolones ciprofloxacin, norfloxacin, ofloxacin and pefloxacin. Beta-lactamase enzymes produced by 37 strains of H. ducreyi were determined for their isoelectric point (pI). All had pI of 5.4, indicative of plasmid-mediated beta-lactamase type TEM-1.

Haemophilus influenzae and Streptococcus pneumoniae in children with acute respiratory infection.

The incidence of Haemophilus influenzae and Streptococcus pneumoniae in children with acute respiratory infection (ARI) under 5 years was carried out by throat swab culture, blood culture, body fluid or tissue culture in 688 patients from a community, 744 patients from a teaching hospital in Bangkok, 766 normal children from the community and 303 children from a hospital well baby clinic. H. influenzae was found in the throats of 15-20% of patients and in the throats of 4-6% of normal children (p less than 0.001 for both hospital and community patients). Only 12/332 strains (3.6%) of H. influenzae were type b. The rest of H. influenzae were non type b. The most common biotype of H. influenzae non type b was biotype II. S. pneumoniae was found in hospital patients in highly significant numbers compared to the controls (12% vs 4%). No significant difference was observed in strains from the community patients.

Biotinylated probes for epidemiological studies of drug resistance in Salmonella krefeld.

A gene probe for ampicillin resistance and one for sulphonamide resistance were prepared to study the origin and the relation of multiple drug resistances in Salmonella krefeld. The resistance genes were cloned into the pACYC184 vector of Escherichia coli from a common plasmid of S. krefeld that encoded for resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, sulphonamide and tetracycline resistance. Restriction map analysis and deletion analysis of a recombinant plasmid (pACSS1) showed that the gene determining ampicillin resistance was located on a 1.34 and 1.12 kb PstI fragment, and that the gene for sulphonamide resistance was located on a 0.85 kb PstI fragment. These fragments were used as probes. Their specificity was tested by colony hybridization with various bacterial species, including sensitive and resistance S. krefeld isolates. Further study indicated that the ampicillin resistance gene probe reacted with the gene for TEM-1 beta-lactamase and that the gene probe for sulphonamide resistance reacted with the gene for type II dihydropteroate synthase. The two probes were sufficiently specific to allow study of the epidemiology of resistance in S. krefeld and other enteric bacteria.

Surveillance of antibiotic resistance in South East Asia.

Antibiotic resistance in Gram-negative bacteria, particularly Salmonella and Shigella, requires surveillance worldwide. This study describes results of surveys in Hong Kong, Bangkok and Kuala Lumpur. All strains were isolated in hospitals which have large community catchment areas in addition to specialised hospital units. The prevalence of resistant strains was high in all areas. Gram-negative bacteria such as Enterobacter associated with hospital infections were resistant to penicillins and cephalosporins, with gentamicin resistance ranging from about 20% in Kuala Lumpur and Hong Kong, to 35% in Bangkok. Ninety-seven percent of Shigella isolated in Thailand were resistant to ampicillin. About 10% of Salmonella were resistant to chloramphenicol in all three centres.

Predictors for extraintestinal infection in Salmonella enteritis in Thailand.

To identify the risks and predictors for extraintestinal Salmonella infection (ETI) in infants and children with nontyphoidal Salmonella enteritis, we performed a retrospective review of 326 infants and children with diarrhea and rectal swab cultures positive for nontyphoidal Salmonella enteritis seen at Ramathibodi Hospital between 1981 and 1983. Nineteen patients had bacteremia. The overall rate of bacteremia was 5.8% which was 24.3% of those having blood cultures taken. Salmonella typhimurium was the most common cause of ETI and the most invasive among the common serotypes causing enteritis. The clinical characteristics of the patients with high probabilities of having ETI were: younger than 6 months of age; high body temperature; and immunocompromising conditions. The observed frequency of ETI in these patients was 21.9 to 26.3% compared with 0 to 0.7% in patients without those risk factors.