Gnathostoma spinigerum: growth and development of third-stage larvae in vitro.

Advanced third-stage larvae of Gnathostoma spinigerum were cultured in RPMI-1640, with various supplements at 37 C under 5% CO2 in air for 300 days. The most suitable medium supplement for worm development was 10% fetal calf serum, 1% dog serum, and 0.25% dog hemolysate. After approximately 180 days of cultivation, some larvae molted to the fourth stage as distinguished by 8 transverse rows of cephalic hooklets and well differentiated sex organs. The maximum body length and width of these larvae were 18.6 mm and 1.1 mm, respectively. Six of 50 larvae (12%) developed to the fourth stage, with a 32% survival rate at the end of cultivation. Although the highest survival rate (70%) of the worms was observed in the medium supplemented with 25 mM NaHCO3, only 4% developed into fourth stage larvae. The addition of fetal calf serum, dog serum, and dog hemolysate was essential for growth and development.

Use of culture-filtrated antigen in an ELISA and a dot immunoassay for the diagnosis of melioidosis.

An enzyme-linked immunosorbent assay (ELISA) and a dot immunoassay with culture-filtrated antigen were developed for detection of Burkholderia pseudomallei specific antibodies in melioidosis patients. Sixty-eight sera of bacteriologically confirmed melioidosis patients, 45 sera of other bacterial infected patients and 80 sera of healthy blood donors from endemic area were investigated. The samples were subjected to those assays im comparison with indirect hemagglutination (IHA). The sensitivity, specificity, positive and negative predictive values in this dot immunoassay were 94.1%, 99.2%, 98.5% and 96.9%, respectively, with cut-off dilution at 1:4,000, whereas those in ELISA were 92.6%, 96.8%, 94.0% and 96.0%, respectively, with cut-off value of OD = 0.47 at 490 nm. Meanwhile, those in IHA were 64.7%, 93.6%, 84.6%, 83.0% respectively, with a cut-off value of > or = 1:80. The results in this study demonstrated that the dot immunoassay was more reliable and rapid than ELISA as the serological test for diagnosis of melioidosis.

Construction of a specific DNA probe for diagnosis of melioidosis and use as an epidemiological marker of Pseudomonas pseudomallei.

Pseudomonas pseudomallei is a causative agent of melioidosis. The disease manifestations range from fulminant sepsis to asymptomatic seroconversion. In septicemic cases, a mortality rate of 80-90% is reported. Rapid and specific diagnosis has become important to the clinical microbiology laboratory. We have developed a P. pseudomallei-specific DNA probe. The cloned fragment, herein designated pKKU-S23L, contained 1.5 kb of P. pseudomallei chromosomal DNA. A radioactively labelled pKKU-S23L insert could detect 1.5 ng of its genomic DNA or 40,000 P. pseudomallei cells. The probe was highly specific for P. pseudomallei DNA and did not cross-hybridize with DNAs prepared from other related bacteria. Using pKKU-S23L as a probe in total cellular DNA digestions and Southern blot hybridization, we were able to classify 60 P. pseudomallei clinical isolates obtained from individual melioidosis patients into eight categories. Therefore, this probe has a potential not only for use in development of specific detection of bacterial DNA in clinical specimens but also for application in epidemiological studies of P. pseudomallei.

Characterization of Pseudomonas pseudomallei antigens by SDS-polyacrylamide gel electrophoresis and western blot.

Immunological characterization of various Pseudomonas pseudomallei preparations was carried out by SDS-PAGE and Western blot using sera from infected humans and from patients with other bacterial infections. Somatic (SOM) and partially purified cell extracts (PCE) gave more complex SDS-PAGE patterns: M(r) ranged from 86 to 12.7 and 48 to 10 kDa, respectively. The culture-filtrated antigens (CF) from 3 different kinds of synthetic media consisted of fairly simple profiles with common bands M(r) of 40, 26 and 16 kDa. PCE and CF reacted specifically with infected human sera; SOM did not. The components with M(r) of 40 kDa in CF reacted consistently with all infected sera but failed to react with sera infected with Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, Proteus mirabilis, Salmonella spp., Staphylococcus aureus, Streptococcus spp., Pseudomonas aeruginosa and P. stutzeri. This peptide was demonstrated to be a major component in CF thus suggesting its potential for development of immunodiagnostic methods for melioidosis.

Prevalence and intensity of Opisthorchis viverrini in rural community near the Mekong River on the Thai-Laos border in northeast Thailand.

The prevalence and intensity of Opisthorchis viverrini in fourteen villages in Nakhon-Phanom province, Northeast, Thailand have been investigated. Overall prevalence of O. viverrini infection was 66.4 per cent in a total population of 2,412 individuals. The prevalence was 18.5 per cent in children under 5 years, 38.9 per cent in those aged 5-9 years, and ranged from 64.9 per cent to 82.2 per cent in the age group above 10 years. The intensity of O. viverrini infection increased with age. The mean faecal egg output was highest in the 30-34 year age group and remained relatively constant through older ages. In all age groups the prevalence and intensity of infection in both men and women were similar. The population was divided according to the presence and intensity of infection as follow, 33 per cent were uninfected, 59 per cent had light infections (less than 1,000 eggs per g of faeces; EPG), 7 per cent had moderate infections (1,000-10,000 EPG), and 1 per cent had heavy (greater than 10,000 EPG). Other important intestinal infections found in this community are hookworm, Taenia spp. and Trichuris trichiura with the prevalence of 17.9 per cent, 1.1 per cent and 1.1 per cent respectively.

Value of an ELISA for mycobacterial antigen detection as a routine diagnostic test of pulmonary tuberculosis.

A double antibody sandwich ELISA was carried out with commercially available anti-BCG and peroxidase labeled anti-BCG, for the detection of mycobacterial antigens. By using purified protein derivative of tuberculin (PPD) as the antigen, the lowest detection limit of the assay was found to be 0.05 microgram/ml. At the cut off level of absorbance index (Al) greater than or equal to 5. positive results of ELISA were obtained from 24/25 sputum specimens which were positive for staining of acid fast bacilli (AFB), 5/16 specimens positive for culture of Mycobacterium tuberculosis and 67/69 specimens positive for both tests. The assay was positive in only 11/164 specimens negative for both staining of AFB and culture of M. tuberculosis. 4 of which were known to have tuberculosis. Thus, with sputum specimens, the sensitivity, specificity, efficiency, positive predictive value and negative predictive value of the ELISA were 87.27, 93.29, 90.88, 89.72 and 91.62 percent respectively. Positive results were also obtained in 2/111 sputum specimens which were positive for other bacteria but the presence of AFB in these specimens could not be ruled out. With pleural fluid specimens, positive ELISA with Al greater than 1 was found in 3/26 specimens of patients with tuberculous pleurisy and 0/11 of those with malignancy. Twenty-six sera and urine specimens of tuberculous patients and also all control specimens (138 sera and 86 urine specimens) assayed, gave negative ELISA results (Al less than 1).