Edited by: Thiago Almeida Pereira, Stanford University, United States
Reviewed by: Jose Mauro Peralta, Federal University of Rio de Janeiro, Brazil; Bonnie Webster, Natural History Museum, United Kingdom
*Correspondence: Edward Oliveira,
This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
The laboratorial diagnosis of the intestinal schistosomiasis is always performed using Kato-Katz technique. However, this technique presents low sensitivity for diagnosis of individuals with low parasite burden, which constitutes the majority in low endemicity Brazilian locations for the disease. The objective of this study was developed and to validate a real-time PCR assay (qPCR) targeting 121 bp sequence to detect
In Brazil, intestinal schistosomiasis is caused by
This situation could be partly attributed to the lack of accurate diagnostic techniques to detect intestinal schistosomiasis in endemic areas. The use of the Kato-Katz technique to detect
Serological assays have been used for schistosomiasis diagnosis by detecting antibodies against schistosomal antigens. However, they are unable to discriminate between active infections and past exposures, especially in individuals living in regions endemic for schistosomiasis (
Alternatively, the detection of schistosome DNA through DNA amplification techniques provides advantages compared to the many parasitological techniques and serological tests, due their high sensitivity, specificity, and accuracy. Furthermore, DNA amplification techniques can detect early pre-patent infections. Although the PCR assay is widely used for laboratory diagnosis of many infectious and parasitic diseases, its application for schistosomiasis was reported for the first time for our research group. We showed that PCR targeting 121 bp, described by Hamburger et al. (
Since then, we have extensively worked with this 121 bp sequence as a target in the PCR assays for diagnosing intestinal schistosomiasis. The 121 bp sequence was used successfully in conventional PCR (
Thus, the main goal of this study was to develop a qPCR assay targeting 121 bp sequence to detect
In this study we tried contaminate negative fecal samples with
To contorn this limitation, genomic DNA was extracted from adult
As negative controls we used DNA extracted from three negative
A forward 5′-CCG ACC AAC CGT TCT ATG A-3′ and reverse 5′-CAC GCT CTC GCA AAT AAT CTA AA-3′ primers and a 5′-6[FAM]/TCG TTG TAT CTC CGA AACCAC TGG ACG/[3BHQ1] probe were designed to amplify and detect a 90 bp fragment of a highly repetitive 121 bp sequence of
Diagram showing anneling positions of the primers and probes in the 121 bp and human β-actin gene sequences. SnapGene software (from Insightful Science; available at
The reaction was performed with a final volume of 25 μl containing 12.5 μl of TaqMan® Universal PCR Master Mix (Life Technologies, Thermo Fisher Scientific Inc., USA),
Extraction and amplification protocols were performed in different rooms to minimize the possibility of contamination. All experiments were performed in a laminar flow chamber, previously irradiated with ultraviolet light, and employing only sterile disposable products, including barrier tips.
The lower LOD of the qPCR was defined by the amplification curve of a positive control containing 38 ng, 3.8 ng, 380 pg, 38 pg, 3.8 pg, 380 fg, 0.38 fg, and 0.038 fg of genomic DNA of adult worms diluted 1:5 in linear acrylamide solution, in triplicate. The mean of Ct from the triplicates was used to define the point in the amplification curve. The amplification efficiency assay was analyzed according to the amplification efficiency (
DNA from
The repeatability test was carried out using six DNA samples extracted from human feces (three negatives and three positives for the presence of
The validation of the qPCR was performed through a cross-sectional-based study carried out in the Tabuas and Estreito de Miralta districts (
STARD flow diagram followed to select the studied groups.
All residents of the mentioned locations with over 1 year of age, of both gender, and who agreed to participate in the study and signed the informed consent form were included. Furthermore, the diagnostic tests were applied to assess the cure after specific treatment of low parasite burden individuals.
The stools samples were provided by the participants at day 0 and examined using the Kato-Katz and Saline Gradient techniques in both locations. The participants who presented
The fecal samples from the residents of Tabuas and Estreito de Miralta were submitted to the Kato-Katz technique using the Helm-Test® produced by BioManguinhos-Fiocruz (Rio de Janeiro, RJ, Brazil). Twenty-four slides of the same stools sample, which correspond to the 1,000 mg of feces, were examined to perform a quantitative comparison between the parasitological tests. Infection intensity was calculated by the number of
The Saline Gradient technique was performed according to the protocol published by Coelho et al. (
The DNA of 1,000 mg fecal samples obtained from residents of Tabuas and Estreito de Miralta was extracted using the QIAamp DNA Stools Mini Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s recommendations and following the protocols of DNA Isolation from Stool for Pathogen Detection and DNA Isolation from Large Amounts of Stool. The DNA concentration was measured by absorbance at 260 nm in a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The A260/A280 absorbance ratio was analyzed to verify the purity of the DNA obtained.
The qPCR assay was performed using fecal samples according to the conditions standardized. The DNA samples that did not present amplification for the human β-actin gene were retested for ensure the efficiency of DNA extraction and PCR-amplification.
The database was built in Microsoft Office Excel 2007 spreadsheets and analyzed using GraphPad Prism version 6.0 (San Diego, CA, USA) or Open Epi software version 3.0 (
The use of human samples was approved following the standards of the Ethical Review Committee of the IRR/FIOCRUZ, Brazil (CEPSH 03/2008) and National Committee of Ethical Research (784/2008, CONEP 14886) in accordance with the Brazilian legislation (RDC 466/2012). The written informed consent was obtained from all the participants/parents or guardians before collecting the samples.
A standard curve was constructed and the analytical sensitivity assay showed that the
The analytical specificity was assessed using DNA from
In Tabuas, 84.5% (148/175) of the population participated of this study. From these, 73 were females and 75 males, aged between 1 and 86 years. Ninety-six individuals were residents in the Tabuas district and 52 in the Ribeirão de Tabuas, an adjacent location. The reasons for non-participation in the study were: 1) refusal; 2) insufficient biological sample for performing all techniques, and 3) health reasons.
The
Positivity rates of intestinal schistosomiasis found by parasitological techniques and qPCR in the population from Tabuas district, Minas Gerais state, Brazil.
Kato–Katz (n = 148) | Saline gradient (n = 148) | “Reference test” (n = 148) | qPCR (n = 148) | ||||||
---|---|---|---|---|---|---|---|---|---|
One |
Two |
Three slides | Six |
Twelve slides | Twenty-four |
1,000 mg |
KK (24 slides) plus SG Results | 1,000 mg |
|
Positivity |
12.2 |
15.5 |
16.9 |
19.6 |
19.6 |
20.6 |
29 |
31 |
30.4 |
*95% Confidence interval.
Positivity of intestinal schistosomiasis in the participants from Tabuas district, diagnosed by Kato-Katz (24 slides), Saline Gradient, and qPCR and distributed by age ranges.
The SG technique detected 43/148 participants with
To create a “reference test,” the results obtained by KK (24 slides) and SG were combined and the positivity increased to 31.0% (46/148), with a statistical difference regarding the previous KK positivity (
Besides
Considering the results of the KK technique with two slides as a definitive diagnostic, the qPCR presented 95.7% sensitivity (95% CI: 79–99.2), 81.6% specificity (95% CI: 73.9–87.4), and 83.8% accuracy (95% CI: 77–88.9). Considering the results of the KK technique with 24 slides, the qPCR presented 96.7% sensitivity (95% CI: 83.8–99.4), 87.2% specificity (95% CI: 79.9–92.0), and 89.2% accuracy (95% CI: 83.2–93.2).
Considering the SG results, the qPCR presented 81.4% sensitivity (95% CI: 67.3–90.2), 90.5% specificity (95% CI: 83.3–94.7), and 87.8% accuracy (95% CI: 81.6–92.2). Based in the results from the “reference test,” the qPCR presented 82.6% (95% CI: 69.3–90.9), 93.1% (95% CI: 86.5–96.6), and 89.8% (95% CI: 83.9–93.7) of sensitivity, specificity, and accuracy rates, respectively (
Performance of qPCR considering the parasitological techniques and “reference test” applied in the population from Tabuas district, Minas Gerais state, Brazil.
Kato-Katz (2 slides) as reference (n = 148) | Kato-Katz (24 slides) as reference (n = 148) | Saline gradient as reference (n = 148) | “Reference test” as reference (n = 148) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Sensitivity |
Specificity |
Accuracy |
Sensitivity |
Specificity |
Accuracy |
Sensitivity |
Specificity |
Accuracy |
Sensitivity |
Specificity |
Accuracy |
|
qPCR | 95.7 |
81.6 |
83.8 |
96.8 |
87.2 |
89.2 |
81.4 |
90.5 |
87.8 |
82.6 |
93.1 |
89.9 |
*95% Confidence interval.
Agreement analysis of the qPCR results in relation to the parasitological techniques and reference test in the population from Tabuas district, Minas Gerais state, Brazil.
Kato–Katz (2 slides) | Kato-Katz (24 slides) | Saline gradient | Reference test | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
P | N | T | P | N | T | P | N | T | P | N | T | ||
qPCR | P | 22 | 23 | 45 | 30 | 15 | 45 | 35 | 10 | 45 | 38 | 7 | 45 |
N | 1 | 102 | 103 | 1 | 102 | 103 | 8 | 95 | 103 | 8 | 95 | 103 | |
T | 23 | 125 | 148 | 31 | 117 | 148 | 43 | 105 | 148 | 46 | 102 | 148 | |
Kappa index | 0.56 (0.41–0.7) | 0.72 (0.56–0.88) | 0.71 (0.55–0.87) | 0.76 (0.6–0.92) |
P, Positive; N, Negative; T, Total; ( ), Confidence interval with 95%.
In the crosstabulation between SG and qPCR, eight participants presented positive results with the SG technique and negative with the qPCR (with positive amplification of human β-actin gene). On the other hand, 10 participants presented negative results with the SG technique but were positive with the qPCR. Thirty-five positive and 95 negative results were consistent between the SG technique and qPCR assay (Kappa index: 0.71). Forty-five individuals were positive by the qPCR, of which seven cases were not detected by the “reference test.” In contrast, eight cases were positive by the “reference test” and were not detected by the qPCR (with positive amplification of human β-actin gene), resulting in a Kappa index of 0.76.
Presumed cure rates measured by parasitological techniques and qPCR 30, 90, and 180 days pos treatment of the
Diagnostic tests | Assessment at the 30 days | Assessment at the 90 days | Assessment at the 180 days |
---|---|---|---|
Kato-Katz |
100% |
94.4% |
78.4% |
Saline gradient | 100% |
94.4.2% |
78.4% |
qPCR | 100% |
83.3% |
62.1% |
*Relation of treated and presumably cured participants in each assessment moment pos treatment.
Kato-Katz (24 slides) and Saline gradient: difference between 30 and 90, p = 0.14; 90 and 180 days post treatment, p = 0.05.
qPCR: difference between 30 and 90, p = 0.008; 90 and 180 days post treatment, p = 0.04.
In Estreito de Miralta, 87.1% (142/163) of the population was included in this study, of which 77 were females and 65 males, aged from 01 to 86 years, 96 residents of the Estreito de Miralta and 46 of Serra Verde, an adjacent location. The reasons for non-participation in the study were the same as those considered for the Tabuas location.
The positivity rates obtained by the KK technique were 9.2% (13/142), 10.5% (15/142), 11.3% (16/142), 12% (17/142), 16.2% (23/142), and 19.7% (28/142) using 1, 2, 3, 6, 12, and 24 slides, respectively (
Positivity rates of intestinal schistosomiasis found by parasitological techniques and qPCR in the population from Estreito de Miralta district, Minas Gerais state, Brazil.
Kato-Katz (n = 142) | Saline gradient (n = 142) | “Reference test” (n = 142) | qPCR (n = 142) | ||||||
---|---|---|---|---|---|---|---|---|---|
One |
Two |
Three slides | Six |
Twelve slides | Twenty-four slides | 1,000 mg |
KK (24 slides) plus SG Results | 1,000 mg |
|
Positivity |
9.2 |
10.5 |
11.3 |
12 |
16.2 |
19.7 |
18.3 |
24.6 |
18.3 |
*95% Confidence interval.
Positivity of intestinal schistosomiasis in the participants from Estreito de Miralta district, diagnosed by Kato–Katz (24 slides), Saline Gradient, and qPCR and distributed by the age ranges.
Likewise, “reference test” was created with the results obtained by the KK (24 slides) and SG techniques. The positivity increased to 24.6% (35/142), with no statistical difference regarding the previous KK (
Other intestinal parasites were also detected by both parasitological techniques. The KK and SG techniques detected 14 (9.9%) individuals positive for hookworms, eight (5.6%) for
Performance of qPCR considering the parasitological techniques and “reference test” applied in the population from Estreito de Miralta district, Minas Gerais state, Brazil.
Kato–Katz (2 slides) as reference (n = 142) | Kato–Katz (24 slides) as reference (n = 142) | Saline gradient as reference (n = 142) | “Reference test” as reference (n = 142) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Sensitivity |
Specificity |
Accuracy |
Sensitivity |
Specificity |
Accuracy |
Sensitivity |
Specificity |
Accuracy |
Sensitivity |
Specificity |
Accuracy |
|
qPCR | 80 |
89 |
88 |
64.3 |
92.9 |
87.3 |
69.2 |
93.1 |
88.7 |
57.1 |
94.4 |
85.2 |
The qPCR results were crosstabulated with the parasitological techniques and the results are presented in the
Agreement analysis of the qPCR results in relation to the parasitological techniques and “reference test” in the population from Estreito de Miralta district, Minas Gerais state, Brazil.
Kato-Katz (2 slides) | Kato-Katz (24 slides) | Saline gradient | Reference test | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
P | N | T | P | N | T | P | N | T | P | N | T | ||
qPCR | P | 12 | 14 | 26 | 18 | 8 | 26 | 18 | 8 | 26 | 20 | 6 | 26 |
N | 3 | 113 | 116 | 10 | 106 | 116 | 8 | 108 | 116 | 15 | 101 | 116 | |
T | 15 | 127 | 142 | 28 | 114 | 142 | 26 | 116 | 142 | 35 | 107 | 142 | |
Kappa index | 0.52 (0.36–0.68) | 0.59 (0.42–0.75) | 0.62 (0.46–0.79) | 0.56 (0.4–0.73) |
P, Positive; N, Negative; T, Total; ( ), Confidence interval with 95%.
The crosstabulation of the SG and qPCR results showed that 126 were consistent and 16 discordants. Among the discordant results, eight were qPCR positive and SG negative and eight were qPCR negative (with positive amplification for the human β-actin gene) and SG positive (Kappa index: 0.62). There were 21/142 qPCR and “reference test” discordant results, of which six presented positive qPCR and negative “reference test” results. In contrast, 15 individuals presented negative qPCR (with positive amplification of human β-actin gene) and positive “reference test” results, resulting in a Kappa index of 0.56 (
In the follow-up for cure assessment 30 days post-treatment, new stool samples were collected from 30/35 positive participants. This evaluation showed two participants diagnosed as positive for
The follow-up at 180 days post-treatment consisted of 29/35 samples for new stool samples for the last cure assessment. This evaluation showed only one participant diagnosed with intestinal schistosomiasis by the KK technique and qPCR, resulting in a presumed cure rate of 96.5% (
Presumed cure rates measured by parasitological techniques and qPCR 30, 90, and 180 days post treatment of the
Diagnostic tests | Assessment at the 30 days | Assessment at the 90 days | Assessment at the 180 days |
---|---|---|---|
Kato-Katz |
93.3% |
97% |
96.5% |
Saline gradient | 93.3% |
97% |
100% |
qPCR | 93.3% |
93.9% |
96.5% |
*Relation of treated and presumably cured participants in each assessment moment post treatment.
Kato-Katz (24 slides): difference between 30 and 90, p = 0.51; 90 and 180 days post treatment, p = 0.93.
Saline gradient: difference between 30 and 90, p = 0.51; 90 and 180 days post treatment, p = 0.34.
qPCR: difference between 30 and 90, p = 0.92; 90 and 180 days post treatment, p = 0.63.
Despite the parasitological technique presenting the best cost-benefit conditions, the assessment of more sensitive techniques is essential for an efficient diagnosis in endemic areas. Current scenarios show that molecular tests are a promising tool for diagnosis and cure assessment of intestinal schistosomiasis in individuals of low parasite burden (
In the conditions defined in this study, the qPCR was extremally sensitive and capable of detecting 0.38 fg of
The qPCR assay was highly specific for detecting
In Tabuas, the positivity rate presented by the KK technique increased until six slides and kept relatively constant from 6 to 24 slides examined of the same fecal sample (
The positivity of qPCR was higher than that of the KK (24 slides,
The sensitivity of the qPCR was high (96.7%) but the specificity was low (87.2%) when the KK (24 slides) results were taken as reference. Only one participant positive with eggs diagnosed by the Kato-Katz technique was not identified by the qPCR assay, which can be explained by the absence of eggs in the sample examined. The qPCR detected 15 positive participants not identified by the Kato-Katz, by examining 24 slides. This discordance was probably due to the limitation of parasitological technique for detecting parasite eggs in stools samples from residents of low endemicity areas, where most of the carriers present low parasite burden (<100 epg). In these cases, the PCR assay detects more cases of infection than the evaluations of many slides by the Kato-Katz technique, suggesting that it can be a useful diagnostic tool. In contrast, the sensitivity rates were lower (81.4 and 82.6%) and the specificity rates were high (90.4 and 93.4%) when the SG or “reference test” were considered as a reference. Thus, the accuracy ranged from 87.8 to 89.8%, with no statistical difference (
In a cross-sectional population-based study, the qPCR targeting 121 bp was compared with POC-CCA®, KK (18 slides), Saline Gradient, and Helmintex techniques. The qPCR assay presented sensitivity of 91.4%, specificity of 86.9%, and Kappa index of 0.71, when the results of the three parasitological techniques were considered as a “reference test.” Moreover, the qPCR assay diagnosed 86.9% of the participants with very low parasite burden (<12 epg) while the POC-CCA® diagnosed 50.8% (
Furthermore, retrotransposon (SjR2), a portion of a mitochondrial gene (nad1) and cell-free parasite DNA (cfDNA) detection by Droplet Digital PCR (ddPCR) has shown to be applicable to the diagnosis of schistosomiasis (
It is difficult to obtain a valid comparison between parasitological techniques and PCR assays since they are methodologies with different principles. These discrepant results may be related to irregular distribution of eggs in the feces when the number of eggs per gram of feces (epg) is small (
The presumed cure rate of 100% post-treatment was expected. However, we observed that 5.6, 21.6 and 16.7, 37.9% of the individuals from Tabuas were positive for
There are insufficient data regarding the clearance of
In Estreito de Miralta, the positivity obtained by the KK technique (24 slides) was high than the SG technique (
In both districts (Tabuas and Estreito de Miralta), the high positivity rates for schistosomiasis were found in participants aged from 10 to 19 years, followed by participants aged between 20 and 29 (
Despite the low parasite load presented by the infected participants in Estreito de Miralta, the qPCR presented a positivity rate of 18.3%, approximately twice as high as that obtained by the Kato-Katz technique (two slides), as is performed in the Brazilian Schistosomiasis Program Control. Thus, approximately 50% of the individuals infected with
Apparently, the sensitivity rates of the qPCR assay (64.3, 69.2, 57.1%) were impaired and the specificity (92.9, 93.1, 94.4%) were favored when considering the KK (24 slides) and SG techniques or “reference test” as true results. These data indicate the influence of individual parasite burden in the performance of the diagnostic techniques used. Some authors reported that false negative results obtained by PCR may be due to a few factors such as inhibition of the amplification reaction by fecal compounds or DNA degradation during transportation of the sample from the field (
In contrast to the presumed cure rate observed in the population from Tabuas, the presumed cure rates in Estreito de Miralta were high and more consistent in the follow up at 30, 90, and 180 days post-treatment, with no statistical difference (
In conclusion, the diagnostic techniques presented different performance in the populations from two districts (Tabuas and Estreito de Miralta). In Tabuas, the positivity rate was higher, with the participants presenting low, moderate, and high parasite burdens and a considerable percentage of participants positive with
The qPCR was an acceptable diagnostic tool, with added value to microscopy in low endemicity areas for the diagnosis of intestinal schistosomiasis in fecal samples, which makes it particularly useful in low transmission areas, and consequently, in post-treatment settings. Moreover, the qPCR can be multiplexed for the diagnosis of other intestinal parasites making the assay more useful. On other hand, because of the relative high costs, the qPCR assay is not cost-effective for the routine diagnosis of schistosomiasis in endemic countries. However, the qPCR assay has become less expensive over time, with increase of the number of research centers with qPCR infrastructure. Thus, the qPCR assay can be used in the routine diagnosis of helminth infections in countries of low economic power.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
LMVS aided with the field work and parasitological techniques, and performed the molecular techniques and analyzed the results. CS assisted with the qPCR assay and analyzed the results. ÁAO aided with the enrollment of the participants, assisted with the parasitological techniques, and assisted with the field work. NFFC assisted with the enrollment of the participants and field work. LG assisted with the qPCR assay and critically reviewed the manuscript for intellectual content. AR supported with the study design and critically reviewed the manuscript for intellectual content. PMZC supported with the study design and critically reviewed the manuscript for intellectual content. EO assisted with the study design, data analysis, drafted the manuscript, and critically reviewed the manuscript for intellectual content. All authors contributed to the article and approved the submitted version.
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001; DECIT/CNPq program 2012 #404405/2012-6; PROEP/Pesquisa Clínica Program MCT-CNPq/FIOCRUZ No 03/2012. EO is supported by CNPq-Brazil (Conselho Nacional de Desenvolvimento Científico e Tecnológico, (Proc. 301159/2016-5).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
LMVS thanks the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the PhD-scholarship.