Sensitivity and specificity of polymerase chain reaction for the detection of Toxoplasma gondii DNA added to laboratory samples.

We studied the sensitivity and specificity of PCR to detect T. gondii DNA by aliquoting various concentration of tachyzoites into laboratory specimens from 60 positive and 10 negative buffy-coat samples. We were able to detect the specific gene from purified DNA samples containing as few as 0.25 parasites per 100,000 human leukocytes. These results had an impressive initial 100% specificity but later it decreased because of false-negative data.

PCR technique for detecting Toxoplasma gondii in animal amniotic fluid.

The goal of diagnosing congenital toxoplasmosis is early detection of maternofetal transmission, for early treatment to prevent unwanted sequelae. Polymerase chain reaction (PCR) is a method used recently for detecting toxoplasmosis during pregnancy. Amniotic fluid is a the clinical specimen used, since it provides a rapid, simple and safe method to obtain accurate results. The advantages of the PCR technique are high sensitivity, specificity and positive predictive value compared with other laboratory methods. To determine the sensitivity, specificity and lower detection limits in our laboratory, amplification of the B1 gene by nested PCR was performed on Toxoplasma gondii tachyzoites added to animal amniotic fluid samples. From 48 samples, our technique detected T. gondii in 30 out of 41 positive samples, and gave negative results for all the negative samples. The sensitivity for this nested PCR was 73%, the specificity was 100%, and the efficiency of the test was 77.1%. The nested PCR technique is recommended as a diagnostic method for detecting T. gondii in suspected congenital toxoplasmosis animals.

Application of real-time polymerase chain reaction (PCR) analysis for detection and discrimination of malaria parasite species in Thai patients.

A TaqMan real-time PCR system was used to detect and discriminate the 4 species of human malaria parasites in clinical blood samples. A 150-base pair (bp) region of the small subunit ribosomal RNA (SSU rRNA) gene of each malaria parasite, including species-specific sequences to be detected by TaqMan probe, was used as a target for PCR analysis. The PCR method used universal primers and species-specific TaqMan probes for Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. The detection threshold for the method, as determined with serial dilution of cultured P. falciparum-infected erythrocytes, was 5 parasite-infected erythrocytes per reaction. Fifty blood samples of falciparum malaria and a second set of 50 samples of vivax malaria, diagnosed by microscopic examination at the Hospital for Tropical Diseases, Mahidol University, Thailand, were analyzed by real-time PCR. In the 50 samples of microscopically-diagnosed falciparum malaria, 40 were regarded as P. falciparum single infection, 7 were P. falciparum and P. vivax mixed infections, and 3 were P. vivax single infection by real-time PCR. In the second set of 50 samples of microscopically diagnosed vivax malaria, all were considered P. vivax single infection by PCR. Neither P. ovale nor P. malariae infection was identified in the 100 blood samples. Real-time PCR analysis was shown to be more sensitive and accurate than routine diagnostic methods. Application and extension of the PCR method reported here will provide a powerful tool for further studies of malaria.

Toxoplasma gondii antibody in Thai cats and their owners.

Humans are thought to acquire Toxoplasma infection by three major routes: ingesting food and water contaminated with oocysts from cat excreta, consumption of under-cooked infected meat, and transplacental transfer. Congenital clinical toxoplasmosis in the newborn indicating definite transplacental transmission had been reported in Thailand, whilst studies concerning infection due to the other two routes were inconclusive. Since the way domestic cats live and eat and also the eating behavior of Thai people differ from those in the West, we conducted a sero-epidemiological study of T. gondii in cats and their owners in Bangkok metropolitan area. Among 327 humans, the prevalence of Toxoplasma antibody was 6.4% and in 315 cats it was 7.3%. These relatively low prevalence rates may result from the predominantly well-cooked fish and rice diet of stray cats, which congregate in temples where they are fed. Toxoplasma antibody seropositive was associated with living in close proximity to seropositivity cats [OR (95% CI) = 5.43 (1.28-23.04); p=0.01]. Risks were increased in and around temples, particularly if courtyards were of earth or grass, suggesting ground temperature was an important determinant of oocyst survival.