Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction

Abstract

We applied the polymerase chain reaction to detection of the pathogenic protozoan Toxoplasma gondii based on our identification of a 35-fold-repetitive gene (the BI gene) as a target. Using this procedure, we were able to amplify and detect the DNA of a single organism directly from a crude cell lysate. This level of sensitivity also allowed us to detect the BI gene from purified DNA samples containing as few as 10 parasites in the presence of 100,000 human leukocytes. This is representative of the maximal cellular infiltration (105/ml) in 1 mi of cerebrospinal fluid obtained from patients with toxoplasmic encephalitis. The BJ gene is present and conserved in all six T. gondii strains tested to date, including two isolates from patients with acquired immunodeficiency syndrome. No signal was detected by using this assay and DNAs from a variety of other organisms, including several which might be found in the central nervous system of an immunocompromised host. This combination of sensitivity and specificity should make detection of the BJ gene based on polymerase chain reaction amplification a very useful method for diagnosis of toxoplasmosis both in immunocompromised hosts and in congenitally infected fetuses.

Sometimes Sperm Whales (Physeter macrocephalus) Cannot Find Their Way Back to the High Seas: A Multidisciplinary Study on a Mass Stranding

“…DNA extracts were subjected to a nested PCR specific for the B1 gene of Toxoplasma gondii [70]. This gene was selected being a repetitive DNA sequence of T. gondii mostly used for the detection of the protozoan by nested PCR on biological samples for diagnostic purposes [71].…”

Specificity of the histopathological triad for the diagnosis of toxoplasmic lymphadenitis: Polymerase chain reaction study*

“…The target DNA was the pub-lished sequence of the 35-fold repetitive B1 gene of T. gondii RH. 7 The primer TOXO 1 (5¢-GGAACTGCATCCGTTCAT-GAG-3¢; B1 gene nucleotides 694-714) with primer TOXO 2 (5¢-TCTTTAAAGCGTTCGTGGTC-3¢; B1 gene nucleotides 887-868) was used for the first round of amplification. The primer TOXO 2 with primer TOXO 4 (5¢-TGCATAG-GTTGCAGTCACTG-3¢; B1 gene nucleotides 757-776) was used for the second round of amplification.…”

“…Frozen tissues of 12 lymph nodes showing the histopathological triad (florid reactive follicular hyperplasia, clusters of epithelioid histiocytes around and / or within germinal centers, and focal sinusoidal distention of monocytoid B cells) 1 on hematoxylin and eosin (HE)-stained sections ( Fig. 1) and tissues of 27 lymph nodes from patients with various other conditions were studied for the presence of T. gondii DNA by the PCR method of Burg et al 7 The 27 lymph nodes without the triad were from 13 cases of follicular lymphoid hyperplasia (FLH), three cases of dermatopathic lymphadenopathy (DPL), two cases of plasmacytosis, two cases of Castleman's disease of the hyaline-vascular type, two cases of metastatic Pathology International 2001; 51: [619][620][621][622][623] Toxoplasmosis is a common cause of lymphadenopathy, but toxoplasmic cysts are not usually found in histological sections used for establishing diagnosis, except on extremely rare occasions. The histopathological triad of florid reactive follicular hyperplasia, clusters of epithelioid histiocytes, and focal sinusoidal distention by monocytoid B cells has been considered to be diagnostic of toxoplasmic lymphadenitis, but the validity of the histopathological triad is based indirectly on serological correlation only.…”

Real-time PCR targeting a 529-bp repeat element for diagnosis of toxoplasmosis

“…A variety of real-time PCR assays and PCR targets have been developed for the rapid and sensitive detection of T. gondii [4][5][6], including the B1 gene, which occurs in 35 copies in the parasite genome [7][8][9][10][11][12][13]. A further repeat element of unknown function, which occurs in 200-300 copies, has recently been described in T. gondii [14].…”