HIV Diagnostics and Vaccines: It Takes Two to Tango.

Current serological tests for HIV screening and confirmation of infection present challenges to the adoption of HIV vaccines. The detection of vaccine-induced HIV-1 antibodies in the absence of HIV-1 infection, referred to as vaccine-induced seropositivity/seroreactivity, confounds the interpretation of test results, causing misclassification of HIV-1 status with potential affiliated stigmatization. For HIV vaccines to be widely adopted with high community confidence and uptake, tests that are agnostic to vaccination status (i.e., only positive for true HIV-1 infection) of tested individuals are needed. Successful development and deployment of such tests will require HIV vaccine developers to work in concert with diagnostic developers. Such tests will need to match today's high-performance standards (accuracy, cost-effectiveness, simplicity) for use in both vaccinated and unvaccinated populations, especially in low- and middle-income countries with high HIV burden. Herein, we discuss the challenges and strategies for developing modified serological HIV tests for concurrent deployment with HIV vaccines.

Towards Novel HIV-1 Serodiagnostic Tests without Vaccine-Induced Seroreactivity.

Vaccine-induced seroreactivity/positivity (VISR/P) poses a significant and common challenge to HIV vaccine implementation, as up to 95% of vaccine recipients may be misclassified as having HIV infection by current HIV screening and confirmatory serological assays. We investigated whether internal HIV proteins could be used to overcome VISR and discovered a set of 4 antigens (gp41 endodomain, p31 integrase, p17 matrix protein, and Nef) that are recognized by antibodies produced in individuals with HIV infection but not in vaccinated individuals. When evaluated in a multiplex double-antigen bridging ELISA, this antigen combination had specificities of 98.1% prevaccination and 97.1% postvaccination, demonstrating the assay is minimally impacted by vaccine-induced antibodies. The sensitivity was 98.5%, further increasing to 99.7% when p24 antigen testing was included. Results were similar across HIV-1 clades. Although more technical advancements will be desired, this research provides the groundwork for the development of new fourth-generation HIV tests unaffected by VISR. IMPORTANCE While the detection of HIV infection is accomplished by several methods, the most common are serological tests that detect host antibodies produced in response to viral infection. However, the use of current serological tests may present a significant challenge to the adoption of an HIV vaccine in the future because the antibodies to HIV antigens detected in currently available tests also tend to be included as antigens in the HIV vaccines in development. The use of these serological tests may thus result in the misclassification of vaccinated HIV-negative individuals, which can have potential for significant harms for individuals and could prevent the widespread adoption and implementation of HIV vaccines. Our study aimed to identify and evaluate target antigens for inclusion in new serological tests that can be used to identify HIV infections without interference from vaccine-induced antibodies but also fit within existing platforms for HIV diagnostics.

Whole blood transcriptome analysis in onchocerciasis.

Identifying the molecular mechanisms controlling the host's response to infection with Onchocerca volvulus is important to understand how the human host controls such parasitic infection. Little is known of the cellular immune response upon infection with O. volvulus. We performed a transcriptomic study using PAXgene-preserved whole blood from 30 nodule-positive individuals and 21 non-endemic controls. It was found that of the 45,042 transcripts that were mapped to the human genome, 544 were found to be upregulated and 447 to be downregulated in nodule-positive individuals (adjusted P-value < 0.05). Pathway analysis was performed on this set of differentially expressed genes, which demonstrated an impact on oxidative phosphorylation and protein translation. Upstream regulator analysis showed that the mTOR associated protein RICTOR appears to play an important role in inducing the transcriptional changes in infected individuals. Functional analysis of the genes affected by infection indicated a suppression of antibody response, Th17 immune response and proliferation of activated T lymphocytes. Multiple regression models were used to select 22 genes that could contribute significantly in the generation of a classifier to predict infection with O. volvulus. For these 22 genes, as well as for 8 reference target genes, validated RT-qPCR assays were developed and used to re-analyze the discovery sample set. These data were used to perform elastic net regularized logistic regression and a panel of 7 genes was found to be the best performing classifier. The resulting algorithm returns a value between 0 and 1, reflecting the predicted probability of being infected. A validation panel of 69 nodule-positive individuals and 5 non-endemic controls was used to validate the performance of this classifier. Based on this validation set only, a sensitivity of 94.2% and a specificity of 60.0% was obtained. When combining the discovery test set and validation set, a sensitivity of 96.0% and a specificity of 92.3% was obtained. Large-scale validation approaches will be necessary to define the intended use for this classifier. Besides the use as marker for infection in MDA efficacy surveys and epidemiological transmission studies, this classifier might also hold potential as pharmacodynamic marker in macrofilaricide clinical trials.

Affordable artificial intelligence-based digital pathology for neglected tropical diseases: A proof-of-concept for the detection of soil-transmitted helminths and Schistosoma mansoni eggs in Kato-Katz stool thick smears.

BACKGROUND: With 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 FINDINGS: We 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/SIGNIFICANCE: We 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.

Multimodal biomarker discovery for active Onchocerca volvulus infection.

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.

Identification of antigenic linear peptides in the soil-transmitted helminth and Schistosoma mansoni proteome.

The scientific community identified non stool-based biomarkers as the way forward to support soil-transmitted helminth (STH; Ascaris lumbricoides, Trichuris trichiura and the hookworms Ancylostoma duodenale and Necator americanus) and schistosome (S. mansoni and S. haematobium) deworming programs. This support is needed in making the decision of whether or not to stop preventive chemotherapy intervention efforts and to ultimately transition towards a post-intervention surveillance phase. We applied a two-step micro-array approach to identify antigenic linear epitopes in the STH and S. mansoni proteomes. In a first experiment, we identified antigenic peptides by applying sera from 24 STH and/or S. mansoni infected Ethiopian children on a high-density peptide microarray containing 3.3 million peptides derived from the complete STH and S. mansoni proteomes. A second array experiment with 170,185 peptides that were recognized in the first array was designed to identify non-specific antibody reactivity by applying sera from 24 healthy individuals from Belgium (a non-endemic country). From this array testing cascade, several peptides were identified for STH but none of them appeared to be unique for one species. We therefore concluded that for STH, none of the peptides revealed to be sufficiently sensitive or species specific. For S. mansoni, some promising peptides were identified prompting future investigation. Based on these results, it is unlikely that linear epitopes would be highly useful in detecting species-specific antibody responses to STH in endemic communities. For S. mansoni, one particular peptide of the micro-exon gene 12 (MEG-12) protein deserves further research. In addition, this study emphasizes the need of well-characterized biobanks for biomarker discovery, particularly when the integration of multiple disease programs is envisioned.

Detection of Ascaris lumbricoides infection by ABA-1 coproantigen ELISA.

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.

2-Methyl-pentanoyl-carnitine (2-MPC): a urine biomarker for patent Ascaris lumbricoides infection.

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 < 0.001). This report demonstrates that urinary 2-MPC can be considered an A. lumbricoides-specific biomarker that can be used to monitor infection intensity.

Assessment of multiplex Onchocerca volvulus peptide ELISA in non-endemic tropical regions.

BACKGROUND: Currently, serodiagnosis of infection with the helminth parasite Onchocerca volvulus is limited to the Ov-16 IgG4 test, a test that has limited sensitivity and suboptimal specificity. In previous studies, we identified several linear epitopes that have the potential to supplement the diagnostic toolbox for onchocerciasis. METHODS: In this study three peptides, bearing in total six linear epitopes were transferred to a multiplex ELISA platform. This multiplex ELISA was used to assess the clinical utility of the peptide serology markers by analyzing sample sets from both O. volvulus endemic and non-endemic regions. RESULTS: The multiplex platform was shown to be reproducible and data obtained on the multiplex platform were comparable to the singleplex ELISA data. The clinical utility assessment showed that in a population of school-aged children from western Kenya, a virtually O. volvulus-free area, significant cross-reactivity with an as-yet to be determined immunogen was detected. CONCLUSIONS: The observations made in this study invalidate the usefulness of the peptide serology markers for onchocerciasis detection. We discuss what could be the origin of this unexpected serological response, but also highlight the need for better characterized biobanks for biomarker discovery activities.

Development of a Lab-on-a-Disk Platform with Digital Imaging for Identification and Counting of Parasite Eggs in Human and Animal Stool.

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.

Performance evaluation of 3 serodiagnostic peptide epitopes and the derived multi-epitope peptide OvNMP-48 for detection of Onchocerca volvulus infection.

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.

Patent infections with soil-transmitted helminths and Schistosoma mansoni are not associated with increased prevalence of antibodies to the Onchocerca volvulus peptide epitopes OvMP-1 and OvMP-23.

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.

Linear epitopes in Onchocerca volvulus vaccine candidate proteins and excretory-secretory proteins.

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.

Proof-of-Concept Rapid Diagnostic Test for Onchocerciasis: Exploring Peptide Biomarkers and the Use of Gold Nanoshells as Reporter Nanoparticles.

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.

Droplet digital PCR is an accurate method to assess methylation status on FFPE samples.

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.

Evaluation of the Diagnostic Performance of Onchocerca volvulus Linear Epitopes in a Peptide Enzyme-Linked Immunosorbent Assay.

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.

2-methyl butyramide, a previously identified urine biomarker for Ascaris lumbricoides, is not present in infected Indonesian individuals.

ᅟ: Previous reports suggest that the 2-methyl butyramide and 2-methyl valeramide metabolites of Ascaris lumbricoides in urine of infected individuals could be considered as urinary biomarkers for active infection. We have developed an LC-MS method with a detection limit of 10 ng/mL using synthetic chemicals as reference material. Urine samples (n = 21) of infected individuals were analyzed for the presence of these metabolites, but they were not detected in any of the samples. Furthermore, the recorded 1H-NMR spectrum for reference 2-methyl butyramide did not match with the spectrum that was described for the Ascaris metabolite. Based on these two observations, we concluded that the urinary biomarkers that were detected for A. lumbricoides infection are not 2-methyl butyramide nor 2-methylvaleramide. New discovery efforts will be required to identify the structure of these metabolite biomarkers in urine of infected individuals. TRIAL REGISTRATION: Urine samples used in this study were collected as part of a clinical trial with trial number ISRCTN75636394 (12 November 2013).

Plasma-derived parasitic microRNAs have insufficient concentrations to be used as diagnostic biomarker for detection of Onchocerca volvulus infection or treatment monitoring using LNA-based RT-qPCR.

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.

Identification of three immunodominant motifs with atypical isotype profile scattered over the Onchocerca volvulus proteome.

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.

An isothermal DNA amplification method for detection of Onchocerca volvulus infection in skin biopsies.

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.