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BackgroundWith the World Health Organization's (WHO) publication of the 2021-2030 neglected tropical diseases (NTDs) roadmap, the current gap in global diagnostics became painfully apparent. Improving existing diagnostic standards with state-of-the-art technology and artificial intelligence has the potential to close this gap. Methodology/Principal findingsWe prototyped an artificial intelligence-based digital pathology (AI-DP) device to explore automated scanning and detection of helminth eggs in stool prepared with the Kato-Katz (KK) technique, the current diagnostic standard for diagnosing soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura and hookworms) and Schistosoma mansoni (SCH) infections. First, we embedded a prototype whole slide imaging scanner into field studies in Cambodia, Ethiopia, Kenya and Tanzania. With the scanner, over 300 KK stool thick smears were scanned, resulting in total of 7,780 field-of-view (FOV) images containing 16,990 annotated helminth eggs (Ascaris: 8,600; Trichuris: 4,083; hookworms: 3,623; SCH: 684). Around 90% of the annotated eggs were used to train a deep learning-based object detection model. From an unseen test set of 752 FOV images containing 1,671 manually verified STH and SCH eggs (the remaining 10% of annotated eggs), our trained object detection model extracted and classified helminth eggs from co-infected FOV images in KK stool thick smears, achieving a weighted average precision (+/- standard deviation) of 94.9% +/- 0.8% and a weighted average recall of 96.1% +/- 2.1% across all four helminth egg species. Conclusions/SignificanceWe present a proof-of-concept for an AI-DP device for automated scanning and detection of helminth eggs in KK stool thick smears. We identified obstacles that need to be addressed before the diagnostic performance can be evaluated against the target product profiles for both STH and SCH. Given that these obstacles are primarily associated with the required hardware and scanning methodology, opposed to the feasibility of AI-based results, we are hopeful that this research can support the 2030 NTDs road map and eventually other poverty-related diseases for which microscopy is the diagnostic standard. Author summaryRecently, the World Health Organization (WHO) published its 2021-2030 road map for neglected tropical diseases (NTDs). While diagnostics are clearly pivotal to steer the NTD endemic countries towards the ambitious targets set, the current gap in the global diagnostic armamentarium for NTDs once more becomes painfully apparent. As most NTD programs mainly rely on microscopic examination of slides, automation of slide scanning coupled with artificial intelligence (AI) has the potential to close this diagnostic gap by 2030. Therefore, we developed a device to automate scanning of stool smears and deployed it in four endemic countries to build an image data base for both intestinal and blood-dwelling worms. After the images were annotated, we used 90% to train and 10% to validate an AI model. As the AI model was able to reliably recognise worm eggs, we provided a proof-of-concept for automated scanning and detection of worm eggs in stool smears. We identified important obstacles for both the slide scanning device and the application of AI, but we are hopeful that this research can support the 2030 NTDs roadmap and eventually other NTDs for which microscopic examination is the diagnostic standard.

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated., Author summary Today’s diagnosis of infection with the filarial parasite Onchocerca volvulus mainly depends on the microscopic analysis of skin biopsies and serological testing. The work presented here describes the use of multiple mass spectrometry-based screening methods (metabolomics and lipidomics) to search for biomarkers indicative of infection with Onchocerca volvulus. This resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine as biomarker for O. volvulus. Further evaluation of these biomarkers in a geographically distinct non-endemic population however invalidated the use of urine cis-cinnamoylglycine. These findings are of utmost importance as it not only opens new avenues in the development of non-invasive diagnostic tools for filarial infections, but also emphasizes the need for evaluation and validation of newly discovered biomarkers in different populations from different geographies.

Current diagnostic tools to determine infection with the helminth parasite Onchocerca volvulus have limited performance characteristics. In previous studies, a proteome-wide screen was conducted to identify linear epitopes in this parasite’s proteome, resulting in the discovery of 1110 antigenic peptide fragments. Here, we investigated three of these peptides using peptide ELISA’s and evaluated their sensitivity and specificity. Epitope mapping was performed, and peptides were constructed that contained only the minimal epitope, flanked by a linker. Investigation of the performance of these minimal epitope peptides demonstrated that all three of them have a specificity (as defined by lack of response in non-helminth-infected individuals) of 100%, low cross-reactivity (5.6%, 5.6%, and 9.3%, respectively), but low sensitivity (36.9%, 46.5%, and 41.2%, respectively). Some cross-reactivity was observed in samples from individuals infected with soil-transmitted helminths or Brugia malayi. Combining these three minimal epitopes in a single peptide, called OvNMP-48, resulted in a performance that exceeded the sum of the individual epitopes, with a sensitivity of 76.0%, a specificity of 97.4%, and a cross-reactivity of 11.1%. Cross-reactivity was observed in some STH and Brugia malayi-infected individuals. This work opens the opportunity to start exploring how these novel linear epitope markers might become part of the O. volvulus diagnostic toolbox. Electronic supplementary material The online version of this article (10.1007/s00436-019-06345-3) contains supplementary material, which is available to authorized users.

Infections with intestinal worms, such as Ascaris lumbricoides, affect hundreds of millions of people in all tropical and subtropical regions of the world. Through large-scale deworming programs, World Health Organization aims to reduce moderate-to-heavy intensity infections below 1%. Current diagnosis and monitoring of these control programs are solely based on the detection of worm eggs in stool. Here we describe how metabolome analysis was used to identify the A. lumbricoides-specific urine biomarker 2-methyl pentanoyl carnitine (2-MPC). This biomarker was found to be 85.7% accurate in determining infection and 90.5% accurate in determining a moderate-to-heavy infection. Our results also demonstrate that there is a correlation between 2-MPC levels in urine and A. lumbricoides DNA detected in stool. Furthermore, the levels of 2-MPC in urine were shown to rapidly and strongly decrease upon administration of a standard treatment (single oral dose of 400 mg albendazole). In an Ascaris suum infection model in pigs, it was found that, although 2-MPC levels were much lower compared to humans, there was a significant association between urinary 2-MPC levels and both worm counts (p = 0.023) and the number of eggs per gram (epg) counts (p A. lumbricoides-specific biomarker that can be used to monitor infection intensity.

Intestinal worms, or soil-transmitted helminths (STHs), affect hundreds of millions of people in all tropical and subtropical regions of the world. The most prevalent STH is Ascaris lumbricoides. Through large-scale deworming programs, World Health Organization aims to reduce morbidity, caused by moderate-to-heavy intensity infections, below 2%. In order to monitor these control programs, stool samples are examined microscopically for the presence of worm eggs. This procedure requires well-trained personnel and is known to show variability between different operators interpreting the slides. We have investigated whether ABA-1, one of the excretory-secretory products of A. lumbricoides can be used as a coproantigen marker for infection with this parasite. Polyclonal antibodies were generated and a coproantigen ELISA was developed. Using this ELISA, it was found that ABA-1 in stool detected Ascaris infection with a sensitivity of 91.5% and a specificity of 95.3%. Our results also demonstrate that there is a correlation between ABA-1 levels in stool and A. lumbricoides DNA detected in stool. Using a threshold of 18.2 ng/g stool the ABA-1 ELISA correctly assigned 68.4% of infected individuals to the moderate-to-heavy intensity infection group, with a specificity of 97.1%. Furthermore, the levels of ABA-1 in stool were shown to rapidly and strongly decrease upon administration of a standard anthelminthic treatment (single oral dose of 400 mg albendazole). In an Ascaris suum infection model in pigs, it was found that ABA-1 remained undetectable until day 28 and was detected at day 42 or 56, concurrent with the appearance of worm eggs in the stool. This report demonstrates that ABA-1 can be considered an Ascaris -specific coproantigen marker that can be used to monitor infection intensity. It also opens the path for development of point-of-care immunoassay-based tests to determine A. lumbricoides infection in stool at the sample collection site., Author summary Intestinal worms are one of the most common infections in tropical and subtropical parts of the world. The roundworm Ascaris lumbricoides is the most prevalent and efforts are ongoing to use preventive chemotherapy to reduce both prevalence and intensity of this infection. To monitor these programs, stool-based microscopy is currently used. We have investigated the possibility of using ABA-1, an abundantly secreted protein from the worm, as a biomarker in stool of infected individuals. We have developed an ELISA and using this assay determined that ABA-1 as stool biomarker had a sensitivity of 91.5% and a specificity of 95.3% to detect infection with A. lumbricoides. We also showed that ABA-1 in stool rapidly and strongly decreased upon administration of a standard anthelminthic treatment. The main asset of this novel stool biomarker is its potential to be used in point-of-care immunoassay-based tests to determine A. lumbricoides infection in stool at the sample collection site.

Diagnostic tools for the detection of infection with Onchocerca volvulus are presently limited to microfilaria detection in skin biopsies and serological assessment using the Ov16 immunoglobulin G4 (IgG4) rapid test, both of which have limited sensitivity. We have investigated the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on immunodominant linear epitopes previously discovered. Peptides that were used in these assays were designated O. volvulus motif peptides (OvMP): OvMP-1 (VSV-EPVTTQET-VSV), OvMP-2 (VSV-KDGEDK-VSV), OvMP-3 (VSV-QTSNLD-VSV), and the combination of the latter two, OvMP-23 (VSV-KDGEDK-VSV-QTSNLD-VSV). Sensitivity (O. volvulus infection), specificity (non-helminth infections), and cross-reactivity (helminth infections) were determined using several panels of clinical plasma isolates. OvMP-1 was found to be very sensitive (100%) and specific (98.7%), but showed substantial cross-reactivity with other helminths. Of the other peptides, OvMP-23 was the most promising peptide with a sensitivity of 92.7%, a specificity of 100%, and a cross-reactivity of 6%. It was also demonstrated that these peptides were immunoreactive to IgG but not IgG4, and there is no correlation with the Ov16 IgG4 status, making them promising candidates to complement this already available test. Combination of the Ov16 IgG4 rapid test and OvMP-23 peptide ELISA led to a sensitivity of 97.3% for the detection of O. volvulus infection, without compromising specificity and with minimal impact on cross-reactivity. The available results open the opportunity for a "clinical utility use case" discussion for improved O. volvulus epidemiological mapping.

Most tissue samples available for cancer research are archived as formalin-fixed paraffin-embedded (FFPE) samples. However, the fixation process and the long storage duration lead to DNA fragmentation and hinder epigenome analysis. The use of droplet digital PCR (ddPCR) to detect DNA methylation has recently emerged. In this study, we compare an optimized ddPCR assay with a conventional qPCR assay by targeting a dilution series of control DNA. In addition, we compare the ddPCR technology with results from Infinium arrays targeting two separate CpG sites on a set of colon adenoma FFPE samples. Our data demonstrate that qPCR and ddPCR assess methylation status equally well on dilution controls with a high DNA input. However, the methylation detection on low-input samples is more accurate using ddPCR. The proposed primer design (methylation-independent primers with amplification of solely the converted DNA target) will allow for methylation detection, independent of bisulfite conversion efficiency. Those data show that ddPCR can be used for methylation analysis on FFPE samples with a wide range of DNA input and that the precision of the assay depends largely on the total amount of amplifiable DNA fragments. Due to accessibility of the ddPCR technology and its accuracy on high- as well as low-DNA input samples, we propose the use of this approach for studies involving degraded FFPE samples.

We present a lab-on-a-disk technology for fast identification and quantification of parasite eggs in stool. We introduce a separation and packing method of eggs contained in 1 g of stool, allowing for removal of commonly present solid particles, fat droplets and air bubbles. The separation is based on a combined gravitational and centrifugal flotation, with the eggs guided to a packed monolayer, enabling quantitation and identification of subtypes of the eggs present in a single field of view (FOV). The prototype was tested with stool samples from pigs and humans infected with intestinal parasites (soil-transmitted helminths eggs). The quality of the images created by this platform was appropriate for identification and quantification of egg types present in the sample.

Summary In our previous study, a proteome‐wide screen was conducted to identify linear epitopes in this parasite's proteome, resulting in the discovery of three immunodominant motifs. Here, we investigated whether such antigenic peptides were found in proteins that were already known as vaccine candidates and excretome/secretome proteins for Onchocerca volvulus This approach led to the identification of 71 immunoreactive stretches in 46 proteins. A deep‐dive into the immunoreactivity profiles of eight vaccine candidates that were chosen as most promising candidates for further development (Ov‐CPI‐2, Ov‐ALT‐1, Ov‐RAL‐2, Ov‐ASP‐1, Ov‐103, Ov‐RBP‐1, Ov‐CHI‐1, and Ov‐B20), resulted in the identification of a poly‐glutamine stretch in Ov‐RAL‐2 that has properties for use as a serodiagnostic marker for O. volvulus infection. A peptide ELISA was developed, and the performance of this assay was evaluated. Based on this assessment, it was found that this assay has a sensitivity of 75.0% [95% CI: 64.9%‐83.5%] and a specificity of 98.5% [95% CI: 94.6%‐99.8%]. Furthermore, 8.7% reactivity in Asian parasite‐infected individuals (8 out of 92) was observed. Besides this identification of a linear epitope marker, the information on the presence of linear epitopes in vaccine candidate proteins might be useful in the study of vaccines for river blindness.

Background Ov16 serology is considered a reference method for Onchocerca volvulus epidemiological mapping. Given the suboptimal sensitivity of this test and the fact that seroconversion takes more than a year after infection, additional serological tests might be needed to guide onchocerciasis elimination programmes. Recently, two linear epitopes encoded in OvMP-1 and OvMP-23 peptides were introduced as serological markers, but the observed antibody cross-reactivity in samples originating from Onchocerca volvulus non-endemic areas required further investigation. Methods We evaluated both peptide markers in an O. volvulus hypo-endemic setting in Jimma Town, Ethiopia using peptide ELISA. For all individuals (n = 303), the infection status with soil-transmitted helminths and Schistosoma mansoni was known. Results We found that 11 (3.6%) individuals were positive for anti-Ov16 IgG4 antibodies, while 34 (11.2%) and 15 (5.0%) individuals were positive for OvMP-1 and OvMP-23, respectively. Out of the 34 OvMP-1 positive samples, 33 were negative on the Ov16 IgG4 ELISA. Similarly, out of the 15 OvMP-23 positive samples, 14 scored negative on this reference method. No difference in seroprevalence for all three markers could be observed between uninfected individuals and individuals infected with different soil-transmitted helminths or S. mansoni. Moreover, the intensity of the response to OvMP-1, OvMP-23 or Ov16 was not significantly stronger in individuals carrying patent STH or S. mansoni infections, nor was there any correlation between the intensities of the responses to the three different antigens. Conclusions This study demonstrates that a patent infection with either soil-transmitted helminths or S. mansoni does not lead to increased antibody recognition of both OvMP-1 and OvMP23. Electronic supplementary material The online version of this article (10.1186/s13071-019-3308-z) contains supplementary material, which is available to authorized users.

Three O. volvulus immunogenic peptide sequences recently discovered by peptide microarray were adapted to a lateral flow assay (LFA). The LFA employs gold nanoshells as novel high-contrast reporter nanoparticles and detects a serological response against the 3 peptides, found in OvOC9384, OvOC198, and OvOC5528, respectively. When tested on 118 sera from O. volvulus infected patients and 208 control sera, the LFA was 90%, 63%, and 98% sensitive for each peptide, respectively, and 99-100% specific vs samples from healthy volunteers. Samples of other filarial infections cross-reacted by 7-24%. The sensitivity, specificity, and cross-reactivity values matched those obtained by ELISA with the same sample set. While the exact choice of peptide(s) will require fine-tuning, this work establishes that O. volvulus peptides identified by peptide microarray can be translated into an antibody-based LFA and that gold nanoshells provide the same sensitivity, specificity, and cross-reactivity as the corresponding ELISA assays.

Diagnostic tools for the detection of infection with Onchocerca volvulus are presently limited to microfilaria detection in skin biopsies and serological assessment using the Ov16 immunoglobulin G4 (IgG4) rapid test, both of which have limited sensitivity. We have investigated the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on immunodominant linear epitopes previously discovered. Peptides that were used in these assays were designated O. volvulus motif peptides (OvMP): OvMP-1 (VSV-EPVTTQET-VSV), OvMP-2 (VSV-KDGEDK-VSV), OvMP-3 (VSV-QTSNLD-VSV), and the combination of the latter two, OvMP-23 (VSV-KDGEDK-VSV-QTSNLD-VSV). Sensitivity (O. volvulus infection), specificity (non-helminth infections), and cross-reactivity (helminth infections) were determined using several panels of clinical plasma isolates. OvMP-1 was found to be very sensitive (100%) and specific (98.7%), but showed substantial cross-reactivity with other helminths. Of the other peptides, OvMP-23 was the most promising peptide with a sensitivity of 92.7%, a specificity of 100%, and a cross-reactivity of 6%. It was also demonstrated that these peptides were immunoreactive to IgG but not IgG4, and there is no correlation with the Ov16 IgG4 status, making them promising candidates to complement this already available test. Combination of the Ov16 IgG4 rapid test and OvMP-23 peptide ELISA led to a sensitivity of 97.3% for the detection of O. volvulus infection, without compromising specificity and with minimal impact on cross-reactivity. The available results open the opportunity for a “clinical utility use case” discussion for improved O. volvulus epidemiological mapping.

JC Polyomavirus (JCPyV) is a widespread polyomavirus that usually resides latently in its host. As reactivation of the virus upon immune-modulating conditions holds serious risk, it is of importance to properly determine who is infected with this virus. Assessment of infection with JCPyV currently is based on the detection of antibodies against the major capsid protein VP1. However, specific antibodies against the peptide JCPyV_VP2_167-15mer have been shown to hold potential as a novel serological marker for infection with JCPyV. We have immunized rabbits with this peptide and the resulting hyperimmune serum was further characterized by detailed epitope mapping. The results demonstrated that the rabbit immune response is polyclonal in nature, recognizing two different epitopes in the 15-mer peptide. The strongest epitope consisted of L173PALTSQEI181, while a second moderate epitope consisted of D171DLPALT177. While some of the essential amino acid residues are the same as the ones for human plasma samples (P174, L176), some others are different. L173, T177, and I181 are essential for the rabbit hyperimmune serum, but not for human plasma samples, while E180 was essential for the human plasma samples and not for the rabbit hyperimmune serum. In conclusion, we generated polyclonal rabbit antibodies with strong reactivity against JCPyV_VP2_167-15mer recognizing at least two different epitopes in this peptide.

Background Diagnostic procedures for the diagnosis of infection with the nematode parasite Onchocerca volvulus are currently based on the microscopic detection of microfilariae in skin biopsies. Alternative approaches based on amplification of parasitic DNA in these skin biopsies are currently being explored. Mostly this is based on the detection of the O-150 repeat sequence using PCR based techniques. Methods An isothermal, loop-mediated amplification method has been designed using the mitochondrial O. volvulus cox1 gene as a target. Results Analysis of dilution series of synthetic DNA containing the targeted sequence show a non-linear dose-response curve, as is usually the case for isothermal amplification methods. Evaluation of cross-reactivity with the heterologous sequence from the closely related parasites Wuchereria bancrofti, Loa loa and Brugia malayi demonstrated strong specificity, as none of these sequences was amplified. The assay however amplified both O. volvulus and O. ochengi DNA, but with a different melting point that can be used to discriminate between the species. Evaluation of this assay in a set of skin snip biopsies collected in an endemic area in Ghana showed a high correlation with O-150 qPCR and also demonstrated a similar sensitivity. Compared to qPCR, LAMP had a sensitivity of 88.2% and a specificity of 99.2%. Conclusions We have developed a sensitive and specific loop-mediated amplification method for detection of O. volvulus DNA in skin biopsies that is capable of providing results within 30 min. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1913-7) contains supplementary material, which is available to authorized users.

River blindness, caused by infection with the filarial nematode Onchocerca volvulus, is a neglected tropical disease affecting millions of people. There is a clear need for diagnostic tools capable of identifying infected patients, but that can also be used for monitoring disease progression and treatment efficacy. Plasma-derived parasitic microRNAs have been suggested as potential candidates for such diagnostic tools. We have investigated whether these parasitic microRNAs are present in sufficient quantity in plasma of Onchocerca-infected patients to be used as a diagnostic biomarker for detection of O. volvulus infection or treatment monitoring. Plasma samples were collected from different sources (23 nodule-positive individuals and 20 microfilaridermic individuals), microRNAs (miRNAs) were extracted using Qiagen miRNeasy kit, and a set of 17 parasitic miRNAs was evaluated on these miRNA extracts using miRCURY Locked Nucleic Acid (LNA) Universal RT microRNA PCR system. Of the 17 miRNAs evaluated, only 7 miRNAs were found to show detectable signal in a number of samples: bma-miR-236-1, bma-miR-71, ov-miR71-22nt, ov-miR-71-23nt, ov-miR-100d, ov-bantam-a, and ov-miR-87-3p. Subsequent melting curve analysis, however, indicated that the signals observed for ov-miR-71 variants and ov-miR-87-3p are non-specific. The other miRNAs only showed positive signal in one or few samples with Cq values just below the cutoff. Our data indicate that parasitic miRNAs are not present in circulation at a sufficiently high level to be used as biomarker for O. volvulus infection or treatment monitoring using LNA-based RT-qPCR analysis.

Flu is caused by the influenza virus that, due to mutations, keeps our body vulnerable for infections, making early diagnosis essential. Although immuno-based diagnostic tests are available, they have low sensitivity and reproducibility. In this paper, the prospect of detecting influenza A virus using digital ELISA has been studied. To appropriately select bioreceptors for this bioassay, seven commercial antibodies against influenza A nucleoprotein were methodically tested for their reactivity and binding affinity. The study has been performed on two markedly different platforms, being an enzyme-linked immunosorbent assay and a surface plasmon resonance system. The selected antibodies displayed completely different behavior on the two platforms and in various assay configurations. Surprisingly, the antibodies that showed overall good reactivity on both platforms had the highest dissociation constant among the tested antibodies, suggesting that, although important, binding affinity is not the only parameter to be considered when selecting antibodies. Moreover, only one antibody had the capacity to capture the nucleoprotein directly in lysis buffer used for releasing this viral protein, which might pose a huge advantage when developing assays with a fast time-to-result. This antibody was implemented on an in-house developed digital ELISA platform for ultrasensitive detection of recombinant nucleoprotein, reaching a detection limit of 4 ± 1 fM in buffer and 10 ± 2 fM in 10-fold diluted nasopharyngeal swabs, which is comparable to currently available fast molecular detection techniques. These results point to a great potential for ultrasensitive immuno-based influenza detection. ispartof: Analytical Chemistry vol:88 issue:17 pages:8450-8458 ispartof: location:United States status: published

Background Onchocerciasis, also known as river blindness is one of the neglected tropical diseases affecting millions of people, mainly in sub-Saharan Africa and is caused by the filarial nematode Onchocerca volvulus. Efforts to eliminate this disease are ongoing and are based on mass drug administration programs with the microfilaricide ivermectin. In order to monitor the efficacy of these programs, there is an unmet need for diagnostic tools capable of identifying infected patients. We have investigated the diagnostic potential of urinary N-acetyltyramine-O,β-glucuronide (NATOG), which is a promising O. volvulus specific biomarker previously identified by urine metabolome analysis. Methods A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was used to assess the stability characteristics of NATOG and to evaluate the levels of NATOG in study samples. An LC-fluorescence method was also developed. Results Stability characteristics of NATOG were investigated and shown to be ideally suited for use in tropical settings. Also, an easy and more accessible method based on liquid chromatography coupled to fluorescence detection was developed and shown to have the necessary sensitivity (limit of quantification 1 μM). Furthermore, we have evaluated the levels of NATOG in a population of 98 nodule-positive individuals from Ghana with no or low levels of microfilaria in the skin and compared them with the levels observed in different control groups (endemic controls (n = 50), non-endemic controls (n = 18) and lymphatic filariasis (n = 51). Only a few (5 %) of nodule-positive individuals showed an increased level (> 10 μM) of NATOG and there was no statistical difference between the nodule-positive individuals and the control groups (P > 0.05). Conclusions Results of the present study indicate the limited potential of NATOG as a diagnostic biomarker for O. volvulus infection in amicrofilaridermic individuals. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1582-6) contains supplementary material, which is available to authorized users.

Understanding the immune response upon infection with the filarial nematode Onchocerca volvulus and the mechanisms that evolved in this parasite to evade immune mediated elimination is essential to expand the toolbox available for diagnostics, therapeutics and vaccines development. Using high-density peptide microarrays we scanned the proteome-wide linear epitope repertoire in Cameroonian onchocerciasis patients and healthy controls from Southern Africa which led to the identification of 249 immunodominant antigenic peptides. Motif analysis learned that 3 immunodominant motifs, encompassing 3 linear epitopes, are present in 70, 43, and 31 of these peptides, respectively and appear to be scattered over the entire proteome in seemingly non-related proteins. These linear epitopes are shown to have an atypical isotype profile dominated by IgG1, IgG3, IgE and IgM, in contrast to the commonly observed IgG4 response in chronic active helminth infections. The identification of these linear epitope motifs may lead to novel diagnostic development but further evaluation of cross-reactivity against common co-infecting human nematode infections will be needed., Author summary Infection with the filarial parasite Onchocerca volvulus is the cause of river blindness. We analyzed the immune response against this parasite in infected individuals in order to identify linear epitopes. Using high-density peptide microarrays we discovered three immunodominant motifs in the Onchocerca volvulus proteome that induce a broad IgG response, but the typical IgG4 immune response against parasites was absent. Our study led to the identification of novel potential epitope sequences that can potentially be used for detection of infection with Onchocerca volvulus.

Background JC polyomavirus (JCPyV) is a widespread human polyomavirus that usually resides latently in its host, but can be reactivated under immune-compromised conditions potentially causing Progressive Multifocal Leukoencephalopathy (PML). JCPyV encodes its own microRNA, jcv-miR-J1. Methods We have investigated in 50 healthy subjects whether jcv-miR-J1-5p (and its variant jcv-miR-J1a-5p) can be detected in plasma or urine. Results We found that the overall detection rate of JCPyV miRNA was 74% (37/50) in plasma and 62% (31/50) in urine. Subjects were further categorized based on JCPyV VP1 serology status and viral shedding. In seronegative subjects, JCPyV miRNA was found in 86% (12/14) and 57% (8/14) of plasma and urine samples, respectively. In seropositive subjects, the detection rate was 69% (25/36) and 64% (23/36) for plasma and urine, respectively. Furthermore, in seropositive subjects shedding virus in urine, higher levels of urinary viral miRNAs were observed, compared to non-shedding seropositive subjects (P

Background JC polyomavirus (JCPyV) is a widespread human polyomavirus that usually resides latently in its host, but can be reactivated under immune-compromised conditions potentially causing Progressive Multifocal Leukoencephalopathy (PML). Detection of antibodies against the major capsid protein VP1 currently is the main marker for assessment of infection with JCPyV. Methods Based on a peptide microarray, peptide JCPyV_VP2_167-15mer was selected and a peptide ELISA was developed for detection of antibodies directed against this peptide. Epitope mapping and computational modelling was performed to further characterize this peptide. In a cohort of 204 healthy subjects it was investigated whether antibodies against JCPyV_VP2_167-15mer were correlated with VP1 serology or urinary viral load. Results Epitope mapping of peptide JCPyV_VP2_167-15mer showed that the minimal epitope consisted of L173PALTSQEI181 with amino acids P174, L176 and E180 being essential for antibody recognition. Computational analysis was used to predict that this epitope is located at an exposed domain of the VP2 capsid protein, readily accessible for immune recognition upon infection. No correlation could be observed with JCPyV VP1 antibody levels, or urinary viral load. Conclusion This work indicates that specific antibodies against JCPyV_VP2_167-15mer might be considered as a novel serological marker for infection with JCPyV. Electronic supplementary material The online version of this article (doi:10.1186/1743-422X-11-174) contains supplementary material, which is available to authorized users.

OBJECTIVES: Combination microbicide vaginal rings may be more effective than single microbicide rings at reducing/preventing sexual transmission of HIV. Here we report the pre-clinical development and macaque pharmacokinetics of matrix-type silicone elastomer vaginal rings containing dapivirine and darunavir. METHODS: Macaque rings containing 25 mg dapivirine 100 mg dapivirine 300 mg darunavir or 100 mg dapivirine+300 mg darunavir were manufactured and characterized by differential scanning calorimetry. In vitro release was assessed into isopropanol/water and simulated vaginal fluid. Macaque vaginal fluid and blood serum concentrations for both antiretrovirals were measured during 28 day ring use. Tissue levels were measured on day 28. Ex vivo challenge studies were performed on vaginal fluid samples and IC50 values were calculated. RESULTS: Darunavir caused a concentration-dependent reduction in the dapivirine melting temperature in both solid drug mixes and in the combination ring. In vitro release from rings was dependent on drug loading the number of drugs present and the release medium. In macaques serum concentrations of both microbicides were maintained between 10(1) and 10(2) pg/mL. Vaginal fluid levels ranged between 10(3) and 10(4) ng/g and between 10(4) and 10(5) ng/g for dapivirine and darunavir respectively. Both dapivirine and darunavir showed very similar concentrations in each tissue type; the range of drug tissue concentrations followed the general rank order: vagina (1.8 x 10(3)-3.8 x 10(3) ng/g) > cervix (9.4 x 10(1)-3.9 x 10(2) ng/g) > uterus (0-108 ng/g) > rectum (0-40 ng/g). Measured IC50 values were >2 ng/mL for both compounds. CONCLUSIONS: Based on these results and in light of recent clinical progress of the 25 mg dapivirine ring a combination vaginal ring containing dapivirine and darunavir is a viable second-generation HIV microbicide candidate. (c) The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions please e-mail: journals.permissions@oup.com.

The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae controls a variety of properties that depend on the nutrient composition of the medium. High activity of the pathway occurs in the presence of rapidly fermented sugars like glucose or sucrose, but only as long as growth is maintained. Growth arrest of fermenting cells or growth on a respirative carbon source, like glycerol or ethanol, is associated with low activity of the PKA pathway. We have studied how different nutrients trigger rapid activation of the pathway. Glucose and sucrose activate cAMP synthesis through a G-protein-coupled receptor system, consisting of the GPCR Gpr1, the Gα protein Gpa2 and its RGS protein Rgs2. Glucose is also sensed intracellularly through its phosphorylation. Specific mutations in Gpr1 abolish glucose but not sucrose signalling. Activation of the PKA pathway by addition of a nitrogen source or phosphate to nitrogen- or phosphate-starved cells, respectively, is not mediated by an increase in cAMP. Activation by amino acids is triggered by the general amino acid permease Gap1, which functions as a transporter/receptor. Short truncation of the C-terminus results in constitutively activating alleles. Activation by ammonium uses the ammonium permeases Mep1 and Mep2 as receptor. Specific point mutations in Mep2 uncouple signalling from transport. Activation by phosphate is triggered a.o. by the Pho84 phosphate permease. Several mutations in Pho84 separating transport and signalling or triggering constitutive activation have been obtained.

In eukaryotic cells, G-protein-coupled receptors (GPCRs), non-transporting nutrient carrier homologues and active nutrient carriers have been recently shown to function as sensors that directly monitor the level of nutrients in the extracellular environment. The plasma membrane is not only the cellular boundary at which signalling molecules that govern metabolism and proliferation are detected, but also the boundary across which nutrients that sustain the generation of energy and building blocks are transported. Nutrient sensors combine these functions in various ways. Classical receptor proteins detect the presence of nutrients, carriers combine the functions of nutrient transporters and receptors, and carrier homologues have lost their transport capacity and become pure receptors. The activation of signal transduction pathways by nutrients adds a new layer to the regulatory network that controls metabolism and proliferation. Nutrient sensors highlight the importance of both nutrients as signalling molecules and nutrient carriers as receptors for signalling pathways.

Addition of a nitrogen source to yeast (Saccharomyces cerevisiae) cells starved for nitrogen on a glucose-containing medium triggers activation of protein kinase A (PKA) targets through a pathway that requires for sustained activation both a fermentable carbon source and a complete growth medium (fermentable growth medium induced or FGM pathway). Trehalase is activated, trehalose and glycogen content as well as heat resistance drop rapidly, STRE-controlled genes are repressed, and ribosomal protein genes are induced. We show that the rapid effect of amino acids on these targets specifically requires the general amino acid permease Gap1. In the gap1Delta strain, transport of high concentrations of l-citrulline occurs at a high rate but without activation of trehalase. Metabolism of the amino acids is not required. Point mutants in Gap1 with reduced or deficient transport also showed reduced or deficient signalling. However, two mutations, S391A and S397A, were identified with a differential effect on transport and signalling for l-glutamate and l-citrulline. Specific truncations of the C-terminus of Gap1 (e.g. last 14 or 26 amino acids) did not reduce transport activity but caused the same phenotype as in strains with constitutively high PKA activity also during growth with ammonium as sole nitrogen source. The overactive PKA phenotype was abolished by mutations in the Tpk1 or Tpk2 catalytic subunits. We conclude that Gap1 acts as an amino acid sensor for rapid activation of the FGM signalling pathway which controls the PKA targets, that transport through Gap1 is connected to signalling and that specific truncations of the C-terminus result in permanently activating Gap1 alleles.

The study of signal transduction in microorganisms has become a major research topic in molecular and cellular biology. In this era, thorough knowledge of microbial physiology is no longer the sole and exclusive interest of academic research. It is now being acknowledged as a major importance for food, feed, and nutritional R&D. Detailed investigation of the mechanisms by which cells respond to environmental stimuli is contributing largely to both our fundamental and applied understanding of microorganisms.

BACKGROUND: JC polyomavirus (JCPyV) is a widespread human polyomavirus that usually resides latently in its host. It can be reactivated under immunomodulating conditions and cause Progressive Multifocal Leukoencephalopathy (PML). Circulating microRNAs (miRNAs) are emerging as promising biomarkers for several pathologies. In this study, we have investigated whether circulating miRNAs exist that are differentially expressed between JCPyV seropositive and JCPyV seronegative on the one hand or between JCPyV shedders and JCPyV non-shedders on the other hand. METHODS: Human miRNA expression profiling was performed in a small set of plasma samples obtained from seronegative subjects, seropositive shedders and seropositive non-shedders. A set of 10 miRNAs was selected for further analysis in a larger group of samples. RESULTS: Based on the plasma profiling experiment of 30 samples, 6 miRNAs were selected that were possibly differentially expressed between seropositive and seronegative subjects and 4 miRNAs were selected that were possibly differentially expressed between shedders and non-shedders. Subsequently, expression of these 10 selected miRNAs was assessed in an independent set of 100 plasma samples. Results indicated that none of them were differentially expressed. CONCLUSION: This study could not identify circulating human miRNAs that were differentially expressed between plasma from JCPyV seropositive and JCPyV seronegative subjects or between JCPyV shedders and JCPyV non-shedders. ispartof: Virology Journal vol:11 issue:41 ispartof: location:England status: published

Polyomaviruses are a family of non-enveloped DNA viruses infecting several species, including humans, primates, birds, rodents, bats, horse, cattle, raccoon and sea lion. They typically cause asymptomatic infection and establish latency but can be reactivated under certain conditions causing severe diseases. MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in several cellular processes by binding to and inhibiting the translation of specific mRNA transcripts. In this review, we summarize the current knowledge of microRNAs involved in polyomavirus infection. We review in detail the different viral miRNAs that have been discovered and the role they play in controlling both host and viral protein expression. We also give an overview of the current understanding on how host miRNAs may function in controlling polyomavirus replication, immune evasion and pathogenesis.