Traditionally, automated slide scanning involves capturing a rectangular grid of field-of-view (FoV) images which can be stitched together to create whole slide images, while the autofocusing... Show moreTraditionally, automated slide scanning involves capturing a rectangular grid of field-of-view (FoV) images which can be stitched together to create whole slide images, while the autofocusing algorithm captures a focal stack of images to determine the best in-focus image. However, these methods can be time-consuming due to the need for X-, Y- and Z-axis movements of the digital microscope while capturing multiple FoV images. In this paper, we propose a solution to minimise these redundancies by presenting an optimal procedure for automated slide scanning of circular membrane filters on a glass slide. We achieve this by following an optimal path in the sample plane, ensuring that only FoVs overlapping the filter membrane are captured. To capture the best in-focus FoV image, we utilise a hill-climbing approach that tracks the peak of the mean of Gaussian gradient of the captured FoVs images along the Z-axis. We implemented this procedure to optimise the efficiency of the Schistoscope, an automated digital microscope developed to diagnose urogenital schistosomiasis by imaging Schistosoma haematobium eggs on 13 or 25 mm membrane filters. Our improved method reduces the automated slide scanning time by 63.18% and 72.52% for the respective filter sizes. This advancement greatly supports the practicality of the Schistoscope in large-scale schistosomiasis monitoring and evaluation programs in endemic regions. This will save time, resources and also accelerate generation of data that is critical in achieving the targets for schistosomiasis elimination. Show less
Hoogerwerf, M.A.; Janse, J.J.; Kuiper, V.P.; Schuijlenburg, R. van; Kruize, Y.C.M.; Sijtsma, J.C.; ... ; Microbe, L. 2023
Background Vaccine development against hookworm is hampered by the absence of the development of protective immunity in populations repeatedly exposed to hookworm, limiting identification of... Show moreBackground Vaccine development against hookworm is hampered by the absence of the development of protective immunity in populations repeatedly exposed to hookworm, limiting identification of mechanisms of protective immunity and new vaccine targets. Immunisation with attenuated larvae has proven effective in dogs and partial immunity has been achieved using an irradiated larvae model in healthy volunteers. We aimed to investigate the protective efficacy of immunisation with short-term larval infection against hookworm challenge.Methods We did a single-centre, placebo-controlled, randomised, controlled, phase 1 trial at Leiden University Medical Center (Leiden, Netherlands). Healthy volunteers (aged 18-45 years) were recruited using advertisements on social media and in publicly accessible areas. Volunteers were randomly assigned (2:1) to receive three short-term infections with 50 infectious Necator americanus third-stage filariform larvae (50L3) or placebo. Infection was abrogated with a 3-day course of albendazole 400 mg, 2 weeks after each exposure. Subsequently all volunteers were challenged with two doses of 50L3 at a 2-week interval. The primary endpoint was egg load (geometric mean per g faeces) measured weekly between weeks 12 and 16 after first challenge, assessed in the per-protocol population, which included all randomly assigned volunteers with available data on egg counts at week 12-16 after challenge. This study is registered with ClinicalTrials.gov, NCT03702530.Findings Between Nov 8 and Dec 14, 2018, 26 volunteers were screened, of whom 23 enrolled in the trial. The first immunisation was conducted on Dec 18, 2018. 23 volunteers were randomly assigned (15 to the intervention group and eight to the placebo group). Egg load after challenge was lower in the intervention group than the placebo group (geometric mean 571 eggs per g [range 372-992] vs 873 eggs per g [268-1484]); however, this difference was not statistically significant (p=0 center dot 10). Five volunteers in the intervention group developed a severe skin rash, which was associated with 40% reduction in egg counts after challenge (geometric mean 742 eggs per g [range 268-1484] vs 441 eggs per g [range 380-520] after challenge; p=0 center dot 0025) and associated with higher peak IgG1 titres. Interpretation To our knowledge, this is the first study to describe a protective effect of short-term exposure to hookworm larvae and show an association with skin response, eosinophilic response, and IgG1. These findings could inform future hookworm vaccine development. Copyright (c) 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Show less
Koopman, J.P.R.; Houlder, E.L.; Janse, J.J.; Casacuberta-Partal, M.; Lamers, O.A.C.; Sijtsma, J.C.; ... ; Roestenberg, M. 2023
Background A controlled human infection model for schistosomiasis (CHI-S) can speed up vaccine development and provides insight into early immune responses following schistosome exposure. Recently,... Show moreBackground A controlled human infection model for schistosomiasis (CHI-S) can speed up vaccine development and provides insight into early immune responses following schistosome exposure. Recently, we established CHI-S model using single-sex male-only Schistosoma mansoni (Sm) cercariae in Schistosoma-naive individuals. Given important differences in antigenic profile and human immune responses to schistosomes of different sex, we pioneered a single-sex female-only CHI-S model for future use in vaccine development.Methods We exposed 13 healthy, Schistosoma-naive adult participants to 10 (n = 3) or 20 (n = 10) female cercariae and followed for 20 weeks, receiving treatment with praziquantel (PZQ) 60 mg/kg at week 8 and 12 after exposure.Findings The majority (11/13) participants reported rash and/or itch at the site of exposure, 5/13 had transient symptoms of acute schistosomiasis. Exposure to 20 cercariae led to detectable infection, defined as serum circulating anodic antigen levels >1.0 pg/mL, in 6/10 participants. Despite two rounds of PZQ treatment, 4/13 participants showed signs of persistent infection. Additional one-or three-day PZQ treatment (1 x 60 mg/kg and 3 x 60 mg/kg) or artemether did not result in cure, but over time three participants self-cured. Antibody, cellular, and cytokine responses peaked at week 4 post infection, with a mixed Th1, Th2, and regulatory profile. Cellular responses were (most) discriminative for symptoms.Interpretation Female-only infections exhibit similar clinical and immunological profiles as male-only infections but are more resistant to PZQ treatment. This limits future use of this model and may have important implications for disease control programs. Show less
Sarink, M.J.; Koelewijn, R.; Stelma, F.; Kortbeek, T.; Lieshout, L. van; Smit, P.W.; ... ; Hellemond, J.J. van 2023
Purpose:The purpose of this study was to assess the variation in methods and to determine whether an External Quality Assessment Scheme (EQAS) for polymerase chain reaction (PCR) detection of... Show morePurpose:The purpose of this study was to assess the variation in methods and to determine whether an External Quality Assessment Scheme (EQAS) for polymerase chain reaction (PCR) detection of Acanthamoeba keratitis is valuable for the diagnostic process.Methods:A multicenter EQAS was introduced, covering 16 diagnostic laboratories. Using Acanthamoeba castellanii ATCC strain 30010, 3 sets of samples were prepared, containing different amounts of DNA, cysts, or trophozoites. Samples were masked and sent to the participants with instructions for use and a questionnaire concerning the applied methodologies. Special attention in this questionnaire was given to the used pretreatment methods to assess existing variations in these procedures.Results:A large variation in the methodologies and substantial differences in the diagnostic performance were found between participants. In contrast to the DNA samples where all participants had a perfect score, several false negative results were reported for the samples containing cysts or trophozoites. Only 9 participants had an optimal score, whereas one participant reported all samples as negative, one participant reported failures due to inhibition, and the other 5 reported in total 7 false negative results. A clear correlation was noticed between the PCR detection rate and the number of cysts or trophozoites in the sample.Conclusions:The results indicate that a pretreatment procedure can be a risky step in PCR-based detections of Acanthamoeba, but it improves the sensitivity and reliability, especially of samples containing cysts. Therefore, participation in an EQAS is informative for routine diagnostic laboratories and can assist in improving the laboratory procedures used for the diagnosis of Acanthamoeba keratitis. Show less
Oyibo, P.; Meulah, B.; Bengtson, M.; Lieshout, L. van; Oyibo, W.; Diehl, J.C.; ... ; Agbana, T. 2023
Purpose: Automated diagnosis of urogenital schistosomiasis using digital microscopy images of urine slides is an essential step toward the elimination of schistosomiasis as a disease of public... Show morePurpose: Automated diagnosis of urogenital schistosomiasis using digital microscopy images of urine slides is an essential step toward the elimination of schistosomiasis as a disease of public health concern in Sub-Saharan African countries. We create a robust image dataset of urine samples obtained from field settings and develop a two-stage diagnosis framework for urogenital schistosomiasis.Approach: Urine samples obtained from field settings were captured using the Schistoscope device, and S. haematobium eggs present in the images were manually annotated by experts to create the SH dataset. Next, we develop a two-stage diagnosis framework, which consists of semantic segmentation of S. haematobium eggs using the DeepLabv3-MobileNetV3 deep convolutional neural network and a refined segmentation step using ellipse fitting approach to approximate the eggs with an automatically determined number of ellipses. We defined two linear inequality constraints as a function of the overlap coefficient and area of a fitted ellipses. False positive diagnosis resulting from over-segmentation was further minimized using these constraints. We evaluated the performance of our framework on 7605 images from 65 independent urine samples collected from field settings in Nigeria, by deploying our algorithm on an Edge AI system consisting of Raspberry Pi + Coral USB accelerator.Result: The SH dataset contains 12,051 images from 103 independent urine samples and the developed urogenital schistosomiasis diagnosis framework achieved clinical sensitivity, specificity, and precision of 93.8%, 93.9%, and 93.8%, respectively, using results from an experienced microscopist as reference.Conclusion: Our detection framework is a promising tool for the diagnosis of urogenital schistosomiasis as our results meet the World Health Organization target product profile requirements for monitoring and evaluation of schistosomiasis control programs. (c) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. Show less
Korne, C.M.D.; Lieshout, L. van; Leeuwen, F.W.B. van; Roestenberg, M. 2023
Imaging of parasites is central to diagnosis of many parasitic diseases and has thus far played an important role in the development of antiparasitic strategies. The development of novel imaging... Show moreImaging of parasites is central to diagnosis of many parasitic diseases and has thus far played an important role in the development of antiparasitic strategies. The development of novel imaging technologies has revolutionized medicine in fields other than parasitology and has also opened up new avenues for the visu-alization of parasites. Here we review the role imaging technology has played so far in parasitology and how it may spur further advancement. We point out possibilities to improve current microscopy-based diagnostic methods and how to extend them with radiological imaging modalities. We also highlight in vivo tracking of parasites as a readout for efficacy of new antiparasitic strate-gies and as a source of fundamental insights for rational design. Show less
Background Female genital schistosomiasis (FGS) occurs when Schistosoma haematobium eggs are deposited in reproductive tissue. Female genital schistosomiasis in the cervical mucosa is associated... Show moreBackground Female genital schistosomiasis (FGS) occurs when Schistosoma haematobium eggs are deposited in reproductive tissue. Female genital schistosomiasis in the cervical mucosa is associated with increased vascularity. If FGS is associated with the presence of hemoglobin in cervicovaginal lavage (CVL), the use of urinary reagent strips to detect hemoglobin in CVL could supplement FGS diagnosis. Methods Nonmenstruating, nonpregnant, sexually active women aged 18-31 participating in the HPTN 071 (PopART) Population-Cohort were invited in 2 Zambian communities. Genital self-swabs and a urine specimen were collected at a home visit, and CVL and hand-held colposcopy were performed at a midwife led clinic visit. Urinary reagent strips were used to identify hemoglobin in CVL. Eggs and circulating anodic antigen (CAA) were detected from urine. Visual-FGS was defined as the presence of sandy patches, rubbery papules, or abnormal blood vessels. Polymerase chain reaction (PCR)-FGS was defined as Schistosoma deoxyribonucleic acid detected by real-time PCR on CVL or cervical or vaginal swab. Results Of 209 women with home genital swabs and companion CVL specimens, 66% (138 of 209) had detectable CVL hemoglobin, 13.4% (28 of 209) had PCR-defined FGS, and 17.2% (36 of 209) had visual-FGS. Active Schistosoma infection, diagnosed by CAA or urine microscopy, was present in 21.0% (44 of 209) participants. Active Schistosoma infection (P = .4), PCR-FGS (P = 0.7), and visual-FGS (P = 0.3) were not associated with CVL hemoglobin presence. Results did not differ in subgroups with high infection burden (cycle threshold < 35 or 2-3 positive genital PCR). Conclusions Polymerase chain reaction-FGS, visual-FGS, and active Schistosoma infection were not associated with the presence of CVL hemoglobin. Further research is needed to establish accessible community-based FGS diagnostics.Urinary reagent strips (URS) are useful for estimating S haematobium prevalence. Female genital schistosomiasis (FGS) occurs as a complication of S haematobium infection and is associated with cervical mucosal changes. This study evaluates URS in cervicovaginal lavage for FGS diagnosis. Show less
Hoekstra, P.T.; Casacuberta-Parta, M.; Lieshout, L. van; Corstjens, P.L.A.M.; Tsonaka, R.; Assare, R.K.; ... ; Dam, G.J. van 2022
Background: Most studies assessing praziquantel (PZQ) efficacy have used relatively insensitive diagnostic methods, thereby overestimating cure rate (CR) and intensity reduction rate (IRR). To... Show moreBackground: Most studies assessing praziquantel (PZQ) efficacy have used relatively insensitive diagnostic methods, thereby overestimating cure rate (CR) and intensity reduction rate (IRR). To determine accurately PZQ efficacy, we employed more sensitive DNA and circulating antigen detection methods. Methodology: A sub-analysis was performed based on a previously published trial conducted in children from Cote d'Ivoire with a confirmed Schistosoma mansoni infection, who were randomly assigned to a standard (single dose of PZQ) or intense treatment group (4 repeated doses of PZQ at 2-week intervals). CR and IRR were estimated based on PCR detecting DNA in a single stool sample and the up-converting particle lateral flow (UCP-LF) test detecting circulating anodic antigen (CAA) in a single urine sample, and compared with traditional KatoKatz (KK) and point-of-care circulating cathodic antigen (POC-CCA). Principal findings: Individuals positive by all diagnostic methods (i.e., KK, POC-CCA, PCR, and UCP-LF CAA) at baseline were included in the statistical analysis (n = 125). PCR showed a CR of 45% (95% confidence interval (CI) 32-59%) in the standard and 78% (95% CI 66-87%) in the intense treatment group, which is lower compared to the KK results (64%, 95% CI 52-75%) and 88%, 95% CI 78-93%). UCP-LF CAA showed a significantly lower CR in both groups, 16% (95% CI 11-24%) and 18% (95% CI 12-26%), even lower than observed by POCCCA (31%, 95% CI 17-35% and 36%, 95% CI 26-47%). A substantial reduction in DNA and CAA-levels was observed after the first treatment, with no further decrease after additional treatment and no significant difference in IRR between treatment groups. Conclusion/Significance:The efficacy of (repeated) PZQ treatment was overestimated when using egg-based diagnostics (i.e. KK and PCR). Quantitative worm-based diagnostics (i.e. POC-CCA and UCPLF CAA) revealed that active Schistosoma infections are still present despite multiple treatments. These results stress the need for using accurate diagnostic tools to monitor different PZQ treatment strategies, in particular when moving toward elimination of schistosomiasis.Author summaryEfficacy of praziquantel (PZQ) for the treatment of schistosomiasis is usually assessed by classical microscopic detection of parasite eggs in stool or urine. Due to low sensitivity, especially in case of low-intensity infections, the prevalence of infection is underestimated leading to an overestimated cure rate (CR) when using these methods. In a repeated treatment trial, the efficacy of one versus four repeated PZQ treatments, given at 2-week intervals, was investigated in school-aged children from Cote d'Ivoire by applying a range of diagnostic methods, including traditional microscopy as well as more sensitive DNA and circulating antigen detection methods. Our results demonstrate that PZQ efficacy measurements vary based on the diagnostic method used: while egg-based diagnostics (stool microscopy and DNA detection methods) show an improved CR after repeated treatment, the CR determined by worm-based diagnostics (urine circulating antigen detection methods) remained poor over time. Although all four diagnostic methods showed a significant reduction in intensity of infection already after a single treatment, more accurate antigen diagnostics revealed that, in most cases, worms remain present even after multiple treatments. Hence, using accurate diagnostic tools is essential to determine the true infection status and to monitor and evaluate treatment programs. Show less
Meulah, B.; Oyibo, P.; Bengtson, M.; Agbana, T.; Lontchi, R.A.L.; Adegnika, A.A.; ... ; Lieshout, L. van 2022
Conventional microscopy is the standard procedure for the diagnosis of schistosomiasis, despite its limited sensitivity, reliance on skilled personnel, and the fact that it is error prone. Here, we... Show moreConventional microscopy is the standard procedure for the diagnosis of schistosomiasis, despite its limited sensitivity, reliance on skilled personnel, and the fact that it is error prone. Here, we report the performance of the innovative (semi-)automated Schistoscope 5.0 for optical digital detection and quantification of Schistosoma haematobium eggs in urine, using conventional microscopy as the reference standard. At baseline, 487 participants in a rural setting in Nigeria were assessed, of which 166 (34.1%) tested S. haematobium positive by conventional microscopy. Captured images from the Schistoscope 5.0 were analyzed manually (semiautomation) and by an artificial intelligence (AI) algorithm (full automation). Semi- and fully automated digital microscopy showed comparable sensitivities of 80.1% (95% confidence interval [CI]: 73.2-86.0) and 87.3% (95% CI: 81.3-92.0), but a significant difference in specificity of 95.3% (95% CI: 92.4-97.4) and 48.9% (95% CI: 43.3-55.0), respectively. Overall, estimated egg counts of semi- and fully automated digital microscopy correlated significantly with the egg counts of conventional microscopy (r50.90 and r50.80, respectively, P, 0.001), although the fully automated procedure generally underestimated the higher egg counts. In 38 egg positive cases, an additional urine sample was examined 10 days after praziquantel treatment, showing a similar cure rate and egg reduction rate when comparing conventional microscopy with semiautomated digital microscopy. In this first extensive field evaluation, we found the semiautomated Schistoscope 5.0 to be a promising tool for the detection and monitoring of S. haematobium infection, although further improvement of the AI algorithm for full automation is required. Show less
Hoekstra, P.T.; Madinga, J.; Lutumba, P.; Grootveld, R. van; Brienen, E.A.T.; Corstjens, P.L.A.M.; ... ; Lieshout, L. van 2022
Detection of Schistosoma eggs in stool or urine is known for its low sensitivity in diagnosing light infections. Alternative diagnostics with better sensitivity while remaining highly specific,... Show moreDetection of Schistosoma eggs in stool or urine is known for its low sensitivity in diagnosing light infections. Alternative diagnostics with better sensitivity while remaining highly specific, such as real-time PCR and circulating antigen detection, are progressively used as complementary diagnostic procedures but have not yet replaced microscopy. This study evaluates these alternative methods for the detection of Schistosoma infections in the absence of microscopy. Schistosomiasis presence was determined retrospectively in 314 banked stool and urine samples, available from a previous survey on the prevalence of taeniasis in a community in the Democratic Republic of the Congo, using real-time PCR, the point-of-care circulating cathodic antigen (POC-CCA) test, as well as the up-converting particle lateral flow circulating anodic antigen (UCP-LF CAA) test. Schistosoma DNA was present in urine (3%) and stool (28%) samples, while CCA (28%) and CAA (69%) were detected in urine. Further analysis of the generated data indicated stool-based PCR and the POC-CCA test to be suitable diagnostics for screening of S. mansoni infections, even in the absence of microscopy. A substantial proportion (60%) of the 215 CAA-positive cases showed low antigen concentrations, suggesting that even PCR and POC-CCA underestimated the "true" number of schistosome positives. Show less
Grau-Pujol, B.; Gandasegui, J.; Escola, V.; Marti-Soler, H.; Cambra-Pelleja, M.; Demontis, M.; ... ; Munoz, J. 2022
Soil-transmitted helminth (STH) cornerstone control strategy is mass drug administration (MDA) with benzimidazoles. However, MDA might contribute to selection pressure for anthelmintic resistance,... Show moreSoil-transmitted helminth (STH) cornerstone control strategy is mass drug administration (MDA) with benzimidazoles. However, MDA might contribute to selection pressure for anthelmintic resistance, as occurred in livestock. The aim of this study is to evaluate the treatment response to albendazole and the relationship with the presence of putative benzimidazole resistance single-nucleotide polymorphisms (SNPs) in the 3-tubulin gene of STH in Southern Mozambique. After screening 819 participants, we conducted a cohort study with 184 participants infected with STH in Manhiga district, Southern Mozambique. A pretreatment and a posttreatment stool samples were collected and the STH infection was identified by duplicate Kato-Katz and quantitative polymerase chain reaction (qPCR). Cure rate and egg reduction rates were calculated. Putative benzimidazole resistance SNPs (F167Y, F200T, and E198A) in Trichuris trichiura and Necator americanus were assessed by pyrosequencing. Cure rates by duplicate Kato-Katz and by qPCR were 95.8% and 93.6% for Ascaris lumbricoides, 28% and 7.8% for T. trichiura, and 88.9% and 56.7% for N. americanus. Egg reduction rate by duplicate Kato-Katz was 85.4% for A. lumbricoides, 34.9% for T. trichiura, and 40.5% for N. americanus. Putative benzimidazole resistance SNPs in the 3-tubulin gene were detected in T. trichiura (23%) and N. americanus (21%) infected participants at pretreatment. No statistical difference was observed between pretreatment and posttreatment frequencies for none of the SNPs. Although treatment response to albendazole was low, particularly in T. trichiura, the putative benzimidazole resistance SNPs were not higher after treatment in the population studied. New insights are needed for a better understanding and monitoring of human anthelmintic resistance. Show less
Background: Soil-transmitted helminths (STH), Schistosoma spp. and Plasmodium falciparum are parasites of major public health importance and co-endemic in many sub-Saharan African countries.... Show moreBackground: Soil-transmitted helminths (STH), Schistosoma spp. and Plasmodium falciparum are parasites of major public health importance and co-endemic in many sub-Saharan African countries. Management of these infections requires detection and treatment of infected people and evaluation of large-scale measures implemented. Diagnostic tools are available but their low sensitivity, especially for low intensity helminth infections, leaves room for improvement. Antibody serology could be a useful approach thanks to its potential to detect both current infection and past exposure. Methodology: We evaluated total IgE responses and specific-IgG levels to 9 antigens from STH, 2 from Schistosoma spp., and 16 from P. falciparum, as potential markers of current infection in a population of children and adults from Southern Mozambique (N = 715). Antibody responses were measured by quantitative suspension array Luminex technology and their performance was evaluated by ROC curve analysis using microscopic and molecular detection of infections as reference. Principal findings: IgG against the combination of EXP1, AMA1 and MSP2 (P. falciparum) in children and NIE (Strongyloides stercoralis) in adults and children had the highest accuracies (AUC = 0.942 and AUC = 0.872, respectively) as markers of current infection. IgG against the combination of MEA and Sm25 (Schistosoma spp.) were also reliable markers of current infection (AUC = 0.779). In addition, IgG seropositivity against 20 out of the 27 antigens in the panel differentiated the seropositive endemic population from the non-endemic population, suggesting a possible role as markers of exposure although sensitivity could not be assessed. Conclusions: We provided evidence for the utility of antibody serology to detect current infection with parasites causing tropical diseases in endemic populations. In addition, most of the markers have potential good specificity as markers of exposure. We also showed the feasibility of measuring antibody serology with a platform that allows the integration of control and elimination programs for different pathogens. Show less
For many parasitic diseases, the microscopic examination of clinical samples such as urine and stool still serves as the diagnostic reference standard, primarily because microscopes are accessible... Show moreFor many parasitic diseases, the microscopic examination of clinical samples such as urine and stool still serves as the diagnostic reference standard, primarily because microscopes are accessible and cost-effective. However, conventional microscopy is laborious, requires highly skilled personnel, and is highly subjective. Requirements for skilled operators, coupled with the cost and maintenance needs of the microscopes, which is hardly done in endemic countries, presents grossly limited access to the diagnosis of parasitic diseases in resource-limited settings. The urgent requirement for the management of tropical diseases such as schistosomiasis, which is now focused on elimination, has underscored the critical need for the creation of access to easy-to-use diagnosis for case detection, community mapping, and surveillance. In this paper, we present a low-cost automated digital microscope-the Schistoscope-which is capable of automatic focusing and scanning regions of interest in prepared microscope slides, and automatic detection of Schistosoma haematobium eggs in captured images. The device was developed using widely accessible distributed manufacturing methods and off-the-shelf components to enable local manufacturability and ease of maintenance. For proof of principle, we created a Schistosoma haematobium egg dataset of over 5000 images captured from spiked and clinical urine samples from field settings and demonstrated the automatic detection of Schistosoma haematobium eggs using a trained deep neural network model. The experiments and results presented in this paper collectively illustrate the robustness, stability, and optical performance of the device, making it suitable for use in the monitoring and evaluation of schistosomiasis control programs in endemic settings. Show less
Hoekstra, P.T.; Chernet, A.; Dood, C.J. de; Brienen, E.A.T.; Corstjens, P.L.A.M.; Labhardt, N.D.; ... ; Lieshout, L. van 2022
The increasing number of refugees coming from or passing through Schistosoma-endemic areas and arriving in Europe highlights the importance of screening for schistosomiasis on arrival, and focuses... Show moreThe increasing number of refugees coming from or passing through Schistosoma-endemic areas and arriving in Europe highlights the importance of screening for schistosomiasis on arrival, and focuses attention on the choice of diagnostic test. We evaluate the diagnostic performance of circulating anodic antigen (CAA) detection in 92 asymptomatic refugees from Eritrea. Results were compared with already-available stool microscopy, serology, and urine point-of-care circulating cathodic antigen (POC-CCA) data. For a full diagnostic comparison, real-time polymerase chain reaction (PCR) and the POC-CCA were included. All outcomes were compared against a composite reference standard. Urine and serum samples were subjected to the ultra-sensitive and highly specific up-converting particle lateral flow CAA test, Schistosoma spp. real-time PCR was performed on urine and stool, and the POC-CCA was used on urine using the G-score method. CAA was detected in 43% of urine and in 40% of serum samples. Urine PCR was negative in all 92 individuals, whereas 25% showed Schistosoma DNA in stool. POC-CCA was positive in 30% of individuals. The CAA test confirmed all microscopy positives, except for two cases that were also negative by all other diagnostic procedures. Post-treatment, a significant reduction in the number of positives and infection intensity was observed, in particular regarding CAA levels. Our findings confirm that microscopy, serology, and POC-CCA lack the sensitivity to detect all active Schistosoma infections. Accuracy of stool PCR was similar to microscopy, indicating that this method also lacks sensitivity. The CAA test appeared to be the most accurate method for screening active Schistosoma infections and for monitoring treatment efficacy. Show less
Amaruddin, A.I.; Koopman, J.P.R.; Muhammad, M.; Lenaerts, K.; Eijk, H.M.H. van; Brienen, E.A.T.; ... ; Yazdanbakhsh, M. 2022
BackgroundFemale genital schistosomiasis (FGS) is a neglected and disabling gynecological disease that can result from infection with the parasitic trematode Schistosoma haematobium. Accurate... Show moreBackgroundFemale genital schistosomiasis (FGS) is a neglected and disabling gynecological disease that can result from infection with the parasitic trematode Schistosoma haematobium. Accurate diagnosis of FGS is crucial for effective case management, surveillance and control. However, current methods for diagnosis and morbidity assessment can be inaccessible to those at need, labour intensive, costly and unreliable. Molecular techniques such as PCR can be used to reliably diagnose FGS via the detection of Schistosoma DNA using cervicovaginal lavage (CVL) samples as well as lesser-invasive vaginal self-swab (VSS) and cervical self-swab samples. PCR is, however, currently unsuited for use in most endemic settings. As such, in this study, we assessed the use of a rapid and portable S. haematobium recombinase polymerase amplification (Sh-RPA) isothermal molecular diagnostic assay, coupled with simplified sample preparation methodologies, to detect S. haematobium DNA using CVL and VSS samples provided by patients in Zambia.Methodology/Principal findingsVSS and CVL samples were screened for FGS using a previously developed Sh-RPA assay. DNA was isolated from VSS and CVL samples using the QIAamp Mini kit (n = 603 and 527, respectively). DNA was also isolated from CVL samples using two rapid and portable DNA extraction methods: 1) the SpeedXtract Nucleic Acid Kit (n = 223) and 2) the Extracta DNA Tissue Prep Kit (n = 136). Diagnostic performance of the Sh-RPA using VSS DNA extacts (QIAamp Mini kit) as well as CVL DNA extracts (OIAamp Mini kit, SpeedXtract Nucleic Acid Kit and Extracta DNA Tissue Prep Kit) was then compared to a real-time PCR reference test.Results suggest that optimal performance may be achieved when the Sh-RPA is used with PuVSS samples (sensitivity 93.3%; specificity 96.6%), however no comparisons between different DNA extraction methods using VSS samples could be carried out within this study. When using CVL samples, sensitivity of the Sh-RPA ranged between 71.4 and 85.7 across all three DNA extraction methods when compared to real-time PCR using CVL samples prepared using the QIAamp Mini kit. Interestingly, of these three DNA extraction methods, the rapid and portable SpeedXtract method had the greatest sensitivity and specificity (85.7% and 98.1%, respectively). Specificity of the Sh-RPA was >91% across all comparisons.Conclusions/SignificanceThese results supplement previous findings, highlighting that the use of genital self-swab sampling for diagnosing FGS should be explored further whilst also demonstrating that rapid and portable DNA isolation methods can be used to detect S. haematobium DNA within clinical samples using RPA. Although further development and assessment is needed, it was concluded that the Sh-RPA, coupled with simplified sample preparation, shows excellent promise as a rapid and sensitive diagnostic tool capable of diagnosing FGS at the point-of-care in resource-poor schistosomiasis-endemic settings. Show less
Coinfection with Plasmodium falciparum and helminths may impact the immune response to these parasites because they induce different immune profiles. We studied the effects of coinfections on the... Show moreCoinfection with Plasmodium falciparum and helminths may impact the immune response to these parasites because they induce different immune profiles. We studied the effects of coinfections on the antibody profile in a cohort of 715 Mozambican children and adults using the Luminex technology with a panel of 16 antigens from P. falciparum and 11 antigens from helminths (Ascaris lumbricoides, hookworm, Trichuris trichiura, Strongyloides stercoralis, and Schistosoma spp.) and measured antigen-specific IgG and total IgE responses. We compared the antibody profile between groups defined by P. falciparum and helminth previous exposure (based on serology) and/or current infection (determined by microscopy and/or qPCR). In multivariable regression models adjusted by demographic, socioeconomic, water, and sanitation variables, individuals exposed/infected with P. falciparum and helminths had significantly higher total IgE and antigen-specific IgG levels, magnitude (sum of all levels) and breadth of response to both types of parasites compared to individuals exposed/ infected with only one type of parasite (P <= 0.05). There was a positive association between exposure/infection with P. falciparum and exposure/infection with helminths or the number of helminth species, and vice versa (P <= 0.001). In addition, children coexposed/coinfected tended (P = 0.062) to have higher P. falciparum parasitemia than those single exposed/infected. Our results suggest that an increase in the antibody responses in coexposed/coinfected individuals may reflect higher exposure and be due to a more permissive immune environment to infection in the host.IMPORTANCE Coinfection with Plasmodium falciparum and helminths may impact the immune response to these parasites because they induce different immune profiles. We compared the antibody profile between groups of Mozambican individuals defined by P. falciparum and helminth previous exposure and/or current infection. Our results show a significant increase in antibody responses in individuals coexposed/coinfected with P. falciparum and helminths in comparison with individuals exposed/infected with only one of these parasites, and suggest that this increase is due to a more permissive immune environment to infection in the host. Importantly, this study takes previous exposure into account, which is particularly relevant in endemic areas where continuous infections imprint and shape the immune system. Deciphering the implications of coinfections deserves attention because accounting for the real interactions that occur in nature could improve the design of integrated disease control strategies. Show less
Casacuberta-Partal, M.; Lieshout, L. van; Diepen, A. van; Sijtsma, J.C.; Ozir-Fazalalikhan, A.; Koopman, J.P.R.; ... ; Roestenberg, M. 2021
Assays which enable the detection of schistosome gut-associated circulating anodic (CAA) and cathodic (CCA) antigen in serum or urine are increasingly used as a diagnostic tool for schistosome... Show moreAssays which enable the detection of schistosome gut-associated circulating anodic (CAA) and cathodic (CCA) antigen in serum or urine are increasingly used as a diagnostic tool for schistosome infection. However, little is known about the production and clearance of these circulating antigens in relation to the sex and reproductive maturity of the parasite. Here we describe CAA and CCA excretion patterns by exploring a mouse model after exposure to 36 male-only, female-only and mixed (male/female) Schistosoma mansoni cercariae. We found that serum and urine CAA levels, analysed at 3 weeks intervals, peaked at 6 weeks post-infection. Worms recovered after perfusion at 14 weeks were cultured ex vivo. Male parasites excreted more circulating antigens than females, in the mouse model as well as ex vivo. In mixed infections (supporting egg production), serum CAA levels correlated to the number of recovered worms, whereas faecal egg counts or Schistosoma DNA in stool did not. No viable eggs and no inflammation were seen in the livers from mice infected with female worms only. Ex vivo, CAA levels were higher than CCA levels. Our study confirms that CAA levels reflect worm burden and allows detection of low-level single-sex infections. Show less
Background Microscopic examination of thick and thin blood films is the gold standard in current guidelines for the diagnosis of malaria, but guidelines do not uniformly agree on which combination... Show moreBackground Microscopic examination of thick and thin blood films is the gold standard in current guidelines for the diagnosis of malaria, but guidelines do not uniformly agree on which combination of other methods should be used and when. Methods Three questionnaires were sent between March 2018 and September 2019 to laboratories subscribing to the external quality assessment scheme for the diagnosis of blood and intestinal parasites of the Dutch Foundation for Quality Assessment in Medical Laboratories in order to investigate how much variation in the laboratory diagnosis of malaria between different clinical laboratories is present in the Netherlands. Results The questionnaires were partially or fully completed by 67 of 77 (87%) laboratories. Only 9 laboratories reported 10 or more malaria positive patients per year. Most laboratories use a different diagnostic strategy, within office versus outside office hours depending on the screening assay result. Within office hours, 62.5% (35/56) of the responding laboratories perform an immunochromatographic test (ICT) in combination with microscopic examination of thick and thin blood films without additional examinations, such as Quantitative Buffy Coat and/or rtPCR analysis. Outside office hours 85.7% (48/56) of laboratories use an ICT as single screening assay and positive results are immediately confirmed by thick and thin blood films without additional examinations (89.6%, 43/48). In case of a negative ICT result outside office hours, 70.8% (34/48) of the laboratories perform microscopic examination of the thick film the next morning and 22.9% (11/48) confirm the negative ICT result immediately. Furthermore, substantial differences were found in the microscopic examinations of thick and thin blood films; the staining, theoretical sensitivity of the thick film and determination of parasitaemia. Conclusions This study demonstrated a remarkably high variation between laboratories in both their diagnostic strategy as well as their methods for microscopic examination for the diagnosis of malaria in a clinical setting, despite existing national and international guidelines. While the impact of these variations on the accuracy of the diagnosis of malaria is yet unknown, these findings should stimulate clinical laboratories to critically review their own diagnostic strategy. Show less
Grau-Pujol, B.; Marti-Soler, H.; Escola, V.; Demontis, M.; Jamine, J.C.; Gandasegui, J.; ... ; Munoz, J. 2021
World Health Organization goals against soil-transmitted helminthiases (STH) are pointing towards seeking their elimination as a public health problem: reducing to less than 2% the proportion of... Show moreWorld Health Organization goals against soil-transmitted helminthiases (STH) are pointing towards seeking their elimination as a public health problem: reducing to less than 2% the proportion of moderate and heavy infections. Some regions are reaching WHO goals, but transmission could rebound if strategies are discontinued without an epidemiological evaluation. For that, sensitive diagnostic methods to detect low intensity infections and localization of ongoing transmission are crucial. In this work, we estimated and compared the STH infection as obtained by different diagnostic methods in a low intensity setting. We conducted a cross-sectional study enrolling 792 participants from a district in Mozambique. Two stool samples from two consecutive days were collected from each participant. Samples were analysed by Telemann, Kato-Katz and qPCR for STH detection. We evaluated diagnostic sensitivity using a composite reference standard. By geostatistical methods, we estimated neighbourhood prevalence of at least one STH infection for each diagnostic method. We used environmental, demographical and socioeconomical indicators to account for any existing spatial heterogeneity in infection. qPCR was the most sensitive technique compared to composite reference standard: 92% (CI: 83%-97%) for A. lumbricoides, 95% (CI: 88%-98%) for T. trichiura and 95% (CI: 91%-97%) for hookworm. qPCR also estimated the highest neighbourhood prevalences for at least one STH infection in a low intensity setting. While 10% of the neighbourhoods showed a prevalence above 20% when estimating with single Kato-Katz from one stool and Telemann from one stool, 86% of the neighbourhoods had a prevalence above 20% when estimating with qPCR. In low intensity settings, STH estimated prevalence of infection may be underestimated if based on Kato-Katz. qPCR diagnosis outperformed the microscopy methods. Thus, implementation of qPCR based predictive maps at STH control and elimination programmes would disclose hidden transmission and facilitate targeted interventions for transmission interruption. Show less
