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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

Box plot analysis of cell-free DNA concentrations in patients with Katayama syndrome (first visits only), patients with chronic disease, and all patients who had positive plasma PCR after treatment (pooled from treated Katayama syndrome patients and patients examined retrospectively after treatment).Boxes represent the innermost two quartiles (25%–75% percentiles = interquartile range, IQR) of data. The whiskers represent an extension of the 25th or 75th percentiles by 1.5 times the IQR. The notches represent the median +/−1.57 IQR √n. If the notches of two boxes do not overlap, the medians ( = notch centers) are significantly different (true for the active disease vs. the treated group).
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pntd-0000422-g004: Box plot analysis of cell-free DNA concentrations in patients with Katayama syndrome (first visits only), patients with chronic disease, and all patients who had positive plasma PCR after treatment (pooled from treated Katayama syndrome patients and patients examined retrospectively after treatment).Boxes represent the innermost two quartiles (25%–75% percentiles = interquartile range, IQR) of data. The whiskers represent an extension of the 25th or 75th percentiles by 1.5 times the IQR. The notches represent the median +/−1.57 IQR √n. If the notches of two boxes do not overlap, the medians ( = notch centers) are significantly different (true for the active disease vs. the treated group).

Mentions: A third field of application is the monitoring of therapy. In order to prevent relapse and long term sequelae from insufficient treatment, it is important to achieve a laboratory confirmation of the success of treatment [18], [43]–[45]. Unfortunately, patients after therapy as well as patients after a long course of disease with spontaneous healing (“burnt out bilharzia”) are difficult to judge based on clinical or laboratory findings [16],[18]. Several repetitive, parallel samplings are necessary to increase the statistical chance of detection of eggs by microscope, and thus to increase the clinical sensitivity of laboratory diagnostics [20],[22],[46]. This problem applies not only to microscopy, but also to conventional PCR on stool or urine samples [21],[47]. In the latter tests, there are additional issues such as PCR inhibition in stool samples. We have shown here that the concentration of CFPD in plasma was significantly reduced after therapy. The average CFPD concentration in those patients who still had detectable DNA after treatment (25.1 copies per mL) was significantly lower than in patients with Katayama syndrome (first visits, 537 copies per mL) or active disease (323.6 copies per mL), as determined by ANOVA (F-test, p<0.035; refer to Figure 4 for a Box Plot diagram). The decline of CFPD concentration in patients before and after treatment may thus become an effective parameter for monitoring patients under therapy. On the contrary, we were surprised to see that it took considerably longer in humans than in mice for CFPD PCR to become entirely negative after treatment. Lo et al. have determined that the half-life of fetal DNA in mother's plasma after birth ranges between 4 and 30 minutes [48]. In our study, pooled data from patients followed prospectively and patients re-examined retrospectively after treatment suggest that it may take more than one year until CFPD becomes entirely undetectable. Although we have no experimental evidence, it can be speculated that inactive eggs may release DNA with very slow kinetics. The greater number of eggs in humans with chronic disease as opposed to mice in our experiments may be responsible for a considerably longer duration until CFPD is totally eliminated in humans. Future studies should address the utility of paired CFPD determinations in individual patients before and after treatment, rather than insisting on negative CFPD results for a confirmation of treatment success.


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)

Box plot analysis of cell-free DNA concentrations in patients with Katayama syndrome (first visits only), patients with chronic disease, and all patients who had positive plasma PCR after treatment (pooled from treated Katayama syndrome patients and patients examined retrospectively after treatment).Boxes represent the innermost two quartiles (25%–75% percentiles = interquartile range, IQR) of data. The whiskers represent an extension of the 25th or 75th percentiles by 1.5 times the IQR. The notches represent the median +/−1.57 IQR √n. If the notches of two boxes do not overlap, the medians ( = notch centers) are significantly different (true for the active disease vs. the treated group).
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000422-g004: Box plot analysis of cell-free DNA concentrations in patients with Katayama syndrome (first visits only), patients with chronic disease, and all patients who had positive plasma PCR after treatment (pooled from treated Katayama syndrome patients and patients examined retrospectively after treatment).Boxes represent the innermost two quartiles (25%–75% percentiles = interquartile range, IQR) of data. The whiskers represent an extension of the 25th or 75th percentiles by 1.5 times the IQR. The notches represent the median +/−1.57 IQR √n. If the notches of two boxes do not overlap, the medians ( = notch centers) are significantly different (true for the active disease vs. the treated group).
Mentions: A third field of application is the monitoring of therapy. In order to prevent relapse and long term sequelae from insufficient treatment, it is important to achieve a laboratory confirmation of the success of treatment [18], [43]–[45]. Unfortunately, patients after therapy as well as patients after a long course of disease with spontaneous healing (“burnt out bilharzia”) are difficult to judge based on clinical or laboratory findings [16],[18]. Several repetitive, parallel samplings are necessary to increase the statistical chance of detection of eggs by microscope, and thus to increase the clinical sensitivity of laboratory diagnostics [20],[22],[46]. This problem applies not only to microscopy, but also to conventional PCR on stool or urine samples [21],[47]. In the latter tests, there are additional issues such as PCR inhibition in stool samples. We have shown here that the concentration of CFPD in plasma was significantly reduced after therapy. The average CFPD concentration in those patients who still had detectable DNA after treatment (25.1 copies per mL) was significantly lower than in patients with Katayama syndrome (first visits, 537 copies per mL) or active disease (323.6 copies per mL), as determined by ANOVA (F-test, p<0.035; refer to Figure 4 for a Box Plot diagram). The decline of CFPD concentration in patients before and after treatment may thus become an effective parameter for monitoring patients under therapy. On the contrary, we were surprised to see that it took considerably longer in humans than in mice for CFPD PCR to become entirely negative after treatment. Lo et al. have determined that the half-life of fetal DNA in mother's plasma after birth ranges between 4 and 30 minutes [48]. In our study, pooled data from patients followed prospectively and patients re-examined retrospectively after treatment suggest that it may take more than one year until CFPD becomes entirely undetectable. Although we have no experimental evidence, it can be speculated that inactive eggs may release DNA with very slow kinetics. The greater number of eggs in humans with chronic disease as opposed to mice in our experiments may be responsible for a considerably longer duration until CFPD is totally eliminated in humans. Future studies should address the utility of paired CFPD determinations in individual patients before and after treatment, rather than insisting on negative CFPD results for a confirmation of treatment success.

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