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Diagnosing schistosomiasis by detection of cell-free parasite DNA in human plasma.

Wichmann D, Panning M, Quack T, Kramme S, Burchard GD, Grevelding C, Drosten C - PLoS Negl Trop Dis (2009)

Bottom Line: Serological tests on the other hand do not distinguish between active and past disease.Patients in whom no viable eggs could be detected and who had been treated for schistomiasis in the past (n = 30) showed lower detection rates (33.3%) and significantly lower CFPD concentrations.Further studies are needed to confirm the clinical usefulness of CFPD quantification in therapy monitoring.

View Article: PubMed Central - PubMed

Affiliation: Sektion Infektiologie und Tropenmedizin, I. Medizinische Klinik, Universitätsklinikum Eppendorf, Hamburg, Germany.

ABSTRACT

Introduction: Schistosomiasis (bilharzia), one of the most relevant parasitoses of humans, is confirmed by microscopic detection of eggs in stool, urine, or organ biopsies. The sensitivity of these procedures is variable due to fluctuation of egg shedding. Serological tests on the other hand do not distinguish between active and past disease. In patients with acute disease (Katayama syndrome), both serology and direct detection may produce false negative results. To overcome these obstacles, we developed a novel diagnostic strategy, following the rationale that Schistosoma DNA may be liberated as a result of parasite turnover and reach the blood. Cell-free parasite DNA (CFPD) was detected in plasma by PCR.

Methodology/principal findings: Real-time PCR with internal control was developed and optimized for detection of CFPD in human plasma. Distribution was studied in a mouse model for Schistosoma replication and elimination, as well as in human patients seen before and after treatment. CFPD was detectable in mouse plasma, and its concentration correlated with the course of anti-Schistosoma treatment. Humans with chronic disease and eggs in stool or urine (n = 14) showed a 100% rate of CFPD detection. CFPD was also detected in all (n = 8) patients with Katayama syndrome. Patients in whom no viable eggs could be detected and who had been treated for schistomiasis in the past (n = 30) showed lower detection rates (33.3%) and significantly lower CFPD concentrations. The duration from treatment to total elimination of CFPD from plasma was projected to exceed one year.

Conclusions/significance: PCR for detection of CFPD in human plasma may provide a new laboratory tool for diagnosing schistosomiasis in all phases of clinical disease, including the capacity to rule out Katayama syndrome and active disease. Further studies are needed to confirm the clinical usefulness of CFPD quantification in therapy monitoring.

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Related in: MedlinePlus

DNA copies per mL of pooled mouse plasma (y-axis, four mice per datum point) in mice infected intraperitoneally with 100 cercariae of S. mansoni.After completion of parasite maturation on day 42, mice were treated orally with praziquantel on day 45 (120 mg per kg). At the indicated times (x-axis), four mice were sacrificed, their blood pooled, and 1 mL of pooled plasma was tested as described in the Materials and Methods section for cell-free Schistosoma DNA. The untreated group is marked with an asterisk (*).
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pntd-0000422-g001: DNA copies per mL of pooled mouse plasma (y-axis, four mice per datum point) in mice infected intraperitoneally with 100 cercariae of S. mansoni.After completion of parasite maturation on day 42, mice were treated orally with praziquantel on day 45 (120 mg per kg). At the indicated times (x-axis), four mice were sacrificed, their blood pooled, and 1 mL of pooled plasma was tested as described in the Materials and Methods section for cell-free Schistosoma DNA. The untreated group is marked with an asterisk (*).

Mentions: To determine systematically under which conditions and at what quantities CFPD was detectable in schistosomiasis, a quantitative real-time PCR assay for a Schistosoma multi-copy gene was established as described in the Materials and Methods section. A well-established mouse model of schistosomiasis was employed. In a first step it was tested whether CFPD circulated in plasma during the phase of chronic schistosomiasis. Four adult BALB/c mice were infected with 100 cercariae of S. mansoni and sacrificed after completion of the replication cycle on day 42 after infection. To enable testing of a large volume of mouse plasma, blood was pooled from four mice and one mL of pooled plasma was extracted. Quantitiative PCR with an absolute quantification standard (refer to Materials and Methods section) yielded a DNA concentration of 128.27 copies of CFPD target gene per mL of plasma (Figure 1, marked datum point).


Diagnosing schistosomiasis by detection of cell-free parasite DNA in human plasma.

Wichmann D, Panning M, Quack T, Kramme S, Burchard GD, Grevelding C, Drosten C - PLoS Negl Trop Dis (2009)

DNA copies per mL of pooled mouse plasma (y-axis, four mice per datum point) in mice infected intraperitoneally with 100 cercariae of S. mansoni.After completion of parasite maturation on day 42, mice were treated orally with praziquantel on day 45 (120 mg per kg). At the indicated times (x-axis), four mice were sacrificed, their blood pooled, and 1 mL of pooled plasma was tested as described in the Materials and Methods section for cell-free Schistosoma DNA. The untreated group is marked with an asterisk (*).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667260&req=5

pntd-0000422-g001: DNA copies per mL of pooled mouse plasma (y-axis, four mice per datum point) in mice infected intraperitoneally with 100 cercariae of S. mansoni.After completion of parasite maturation on day 42, mice were treated orally with praziquantel on day 45 (120 mg per kg). At the indicated times (x-axis), four mice were sacrificed, their blood pooled, and 1 mL of pooled plasma was tested as described in the Materials and Methods section for cell-free Schistosoma DNA. The untreated group is marked with an asterisk (*).
Mentions: To determine systematically under which conditions and at what quantities CFPD was detectable in schistosomiasis, a quantitative real-time PCR assay for a Schistosoma multi-copy gene was established as described in the Materials and Methods section. A well-established mouse model of schistosomiasis was employed. In a first step it was tested whether CFPD circulated in plasma during the phase of chronic schistosomiasis. Four adult BALB/c mice were infected with 100 cercariae of S. mansoni and sacrificed after completion of the replication cycle on day 42 after infection. To enable testing of a large volume of mouse plasma, blood was pooled from four mice and one mL of pooled plasma was extracted. Quantitiative PCR with an absolute quantification standard (refer to Materials and Methods section) yielded a DNA concentration of 128.27 copies of CFPD target gene per mL of plasma (Figure 1, marked datum point).

Bottom Line: Serological tests on the other hand do not distinguish between active and past disease.Patients in whom no viable eggs could be detected and who had been treated for schistomiasis in the past (n = 30) showed lower detection rates (33.3%) and significantly lower CFPD concentrations.Further studies are needed to confirm the clinical usefulness of CFPD quantification in therapy monitoring.

View Article: PubMed Central - PubMed

Affiliation: Sektion Infektiologie und Tropenmedizin, I. Medizinische Klinik, Universitätsklinikum Eppendorf, Hamburg, Germany.

ABSTRACT

Introduction: Schistosomiasis (bilharzia), one of the most relevant parasitoses of humans, is confirmed by microscopic detection of eggs in stool, urine, or organ biopsies. The sensitivity of these procedures is variable due to fluctuation of egg shedding. Serological tests on the other hand do not distinguish between active and past disease. In patients with acute disease (Katayama syndrome), both serology and direct detection may produce false negative results. To overcome these obstacles, we developed a novel diagnostic strategy, following the rationale that Schistosoma DNA may be liberated as a result of parasite turnover and reach the blood. Cell-free parasite DNA (CFPD) was detected in plasma by PCR.

Methodology/principal findings: Real-time PCR with internal control was developed and optimized for detection of CFPD in human plasma. Distribution was studied in a mouse model for Schistosoma replication and elimination, as well as in human patients seen before and after treatment. CFPD was detectable in mouse plasma, and its concentration correlated with the course of anti-Schistosoma treatment. Humans with chronic disease and eggs in stool or urine (n = 14) showed a 100% rate of CFPD detection. CFPD was also detected in all (n = 8) patients with Katayama syndrome. Patients in whom no viable eggs could be detected and who had been treated for schistomiasis in the past (n = 30) showed lower detection rates (33.3%) and significantly lower CFPD concentrations. The duration from treatment to total elimination of CFPD from plasma was projected to exceed one year.

Conclusions/significance: PCR for detection of CFPD in human plasma may provide a new laboratory tool for diagnosing schistosomiasis in all phases of clinical disease, including the capacity to rule out Katayama syndrome and active disease. Further studies are needed to confirm the clinical usefulness of CFPD quantification in therapy monitoring.

Show MeSH
Related in: MedlinePlus