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.

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.

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.

JC virus quasispecies analysis reveals a complex viral population underlying progressive multifocal leukoencephalopathy and supports viral dissemination via the hematogenous route.

UNLABELLED: Opportunistic infection of oligodendrocytes by human JC polyomavirus may result in the development of progressive multifocal encephalopathy in immunocompromised individuals. Neurotropic JC virus generally harbors reorganized noncoding control region (NCCR) DNA interspersed on the viral genome between early and late coding genes. By applying 454 sequencing on NCCR DNA amplified from body fluid samples (urine, plasma, and cerebrospinal fluid [CSF]) from 19 progressive multifocal leukoencephalopathy (PML) patients, we attempted to reveal the composition of the JC polyomavirus population (the quasispecies, i.e., the whole of the consensus population and minor viral variants) contained in different body compartments and to better understand intrapatient viral dissemination. Our data demonstrate that in the CSF of PML patients, the JC viral population is often a complex mixture composed of multiple viral variants that contribute to the quasispecies. In contrast, urinary JC virus highly resembled the archetype virus, and urine most often did not contain minor viral variants. It also appeared that archetype JC virus could sporadically be identified in PML patient brain, although selection of rearranged JC virus DNA was favored. Comparison of the quasispecies from different body compartments within a given patient suggested a strong correlation between the viral population in plasma and CSF, whereas the viral population shed in urine appeared to be unrelated. In conclusion, it is shown that the representation of viral DNA in the CSF following the high-level DNA replication in the brain underlying PML has hitherto been much underestimated. Our data also underscore that the hematogenous route might play a pivotal role in viral dissemination from or toward the brain. IMPORTANCE: For the first time, the JC polyomavirus population contained in different body compartments of patients diagnosed with progressive multifocal encephalopathy has been studied by deep sequencing. Two main findings came out of this work. First, it became apparent that the complexity of the viral population associated with PML has been highly underestimated so far, suggestive of a highly dynamic process of reorganization of the noncoding control region of JC polyomavirus in vivo, mainly in CSF and blood. Second, evidence showing viral dissemination from and/or toward the brain via the hematogenous route was provided, confirming a hypothesis that was recently put forward in the field.

Viral miRNAs in plasma and urine divulge JC polyomavirus infection.

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 < 0.001). No correlation was observed between urinary and plasma miRNAs. CONCLUSION: These data indicate that analysis of circulating viral miRNAs divulge the presence of latent JCPyV infection allowing further stratification of seropositive individuals. Also, our data indicate higher infection rates than would be expected from serology alone.

Circulating human microRNAs are not linked to JC polyomavirus serology or urinary viral load in healthy subjects.

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.

Antibodies reacting with JCPyV_VP2 _167-15mer as a novel serological marker for JC polyomavirus infection.

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.

Sampling variability between two mid-turbinate swabs of the same patient has implications for influenza viral load monitoring.

BACKGROUND: With the clinical development of several antiviral intervention strategies for influenza, it becomes crucial to explore viral load shedding in the nasal cavity as a biomarker for treatment success, but also to explore sampling strategies for sensible and reliable virus collection. FINDINGS: In this study, 244 patients suffering from Influenza like Illness and/or acute respiratory tract infection were enrolled. Sampling was done using mid-turbinate flocked swabs and two swabs per patient were collected (one swab per nostril). The influenza A viral loads of two mid-turbinate flocked swabs (one for each nostril) per patient were compared and we have also assessed whether normalization for human cellular DNA in the swabs could be useful. The Influenza mid-turbinate nasal swab testing resulted in considerable sampling variability that could not be normalized against co-isolated human cellular DNA. CONCLUSIONS: Influenza viral load monitoring in nasal swabs could be very valuable as virological endpoints in clinical trials to monitor treatment efficacy, in analogy to HIV, HBV & HCV viral load monitoring. However, the differences between left and right nostrils, as observed in this study, highlight the importance of proper sampling and the need for standardized sampling procedures.

Developing biomarker assays to accelerate tuberculosis drug development: defining target product profiles.

Drug development for tuberculosis is hindered by the methodological limitations in the definitions of patient outcomes, particularly the slow organism growth and difficulty in obtaining suitable and representative samples throughout the treatment. We developed target product profiles for biomarker assays suitable for early-phase and late-phase clinical drug trials by consulting subject-matter experts on the desirable performance and operational characteristics of such assays for monitoring of tuberculosis treatment in drug trials. Minimal and optimal criteria were defined for scope, intended use, pricing, performance, and operational characteristics of the biomarkers. Early-stage trial assays should accurately quantify the number of viable bacilli, whereas late-stage trial assays should match the number, predict relapse-free cure, and replace culture conversion endpoints. The operational criteria reflect the infrastructure and resources available for drug trials. The effective tools should define the sterilising activity of the drug and lower the probability of treatment failure or relapse in people with tuberculosis. The target product profiles outlined in this Review should guide and de-risk the development of biomarker-based assays suitable for phase 2 and 3 clinical drug trials.

The miRNA world of polyomaviruses.

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.

An antibody response to human polyomavirus 15-mer peptides is highly abundant in healthy human subjects.

BACKGROUND: Human polyomaviruses (HPyV) infections cause mostly unapparent or mild primary infections, followed by lifelong nonpathogenic persistence. HPyV, and specifically JCPyV, are known to co-diverge with their host, implying a slow rate of viral evolution and a large timescale of virus/host co-existence. Recent bio-informatic reports showed a large level of peptide homology between JCPyV and the human proteome. In this study, the antibody response to PyV peptides is evaluated. METHODS: The in-silico analysis of the HPyV proteome was followed by peptide microarray serology. A HPyV-peptide microarray containing 4,284 peptides was designed and covered 10 polyomavirus proteomes. Plasma samples from 49 healthy subjects were tested against these peptides. RESULTS: In-silico analysis of all possible HPyV 5-mer amino acid sequences were compared to the human proteome, and 1,609 unique motifs are presented. Assuming a linear epitope being as small as a pentapeptide, on average 9.3% of the polyomavirus proteome is unique and could be recognized by the host as non-self. Small t Ag (stAg) contains a significantly higher percentage of unique pentapeptides. Experimental evidence for the presence of antibodies against HPyV 15-mer peptides in healthy subjects resulted in the following observations: i) antibody responses against stAg were significantly elevated, and against viral protein 2 (VP2) significantly reduced; and ii) there was a significant correlation between the increasing number of embedded unique HPyV penta-peptides and the increase in microarray fluorescent signal. CONCLUSION: The anti-peptide HPyV-antibodies in healthy subjects are preferably directed against the penta-peptide derived unique fraction of the viral proteome.

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.

Comparison of phenotypic and genotypic tropism determination in triple-class-experienced HIV patients eligible for maraviroc treatment.

BACKGROUND: Determination of HIV-1 tropism is a pre-requisite to the use of CCR5 antagonists. This study evaluated the potential of population genotypic tropism tests (GTTs) in clinical practice, and the correlation with phenotypic tropism tests (PTTs) in patients accessing routine HIV care. METHODS: Forty-nine consecutive plasma samples for which an original Trofile(TM) assay was performed were obtained from triple-class-experienced patients in need of a therapy change. Viral tropism was defined as the consensus of three or more tropism calls obtained from the combination of two independent population PTT assays (Trofile Biosciences, San Francisco, CA, USA, and Virco, Beerse, Belgium), population GTTs and GTTs based on ultra-deep sequencing. If no consensus was reached, a clonal PTT was performed in order to finalize the tropism call. This two-step approach allowed the definition of a reference tropism call. RESULTS: According to the reference tropism result, 35/49 samples were CCR5 tropic (R5) (patients eligible for maraviroc treatment) and 14/49 were assigned as non-R5 tropic. The non-R5 samples [patients not eligible for maraviroc treatment according to the FDA/European Medicines Agency (EMEA) label] group included both the CXCR4 (X4) samples and the dual and mixed CCR5/CXCR4 (R5/X4) samples. Compared with Trofile(TM) population PTTs, population GTTs showed a higher sensitivity (97%) and a higher negative predictive value (91%), but almost equal specificity and an equal positive predictive value. CONCLUSIONS: In line with recent reports from clinical trial data, our data support the use of population genotypic tropism testing as a tool for tropism determination before the start of maraviroc.

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.

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.

Secondary integrase resistance mutations found in HIV-1 minority quasispecies in integrase therapy-naive patients have little or no effect on susceptibility to integrase inhibitors.

The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.

Mutation Q95K enhances N155H-mediated integrase inhibitor resistance and improves viral replication capacity.

OBJECTIVES: The genetic barrier to development of raltegravir resistance is considered to be low, requiring at least one primary integrase mutation: Y143C, Q148H/K/R or N155H to confer raltegravir therapy failure. However, during continued raltegravir treatment failure, additional mutations may be selected. In a patient failing raltegravir therapy, we investigated the impact of multiple integrase mutations on resistance and viral replication. Furthermore, in vivo fitness was investigated during failure of raltegravir-containing highly active antiretroviral therapy and after raltegravir was discontinued from the regimen. METHODS: Patient-derived viral integrase genes were cloned into a reference strain. These recombinant viruses were used to determine the contribution of individual integrase mutations to raltegravir resistance and replication capacity in vitro. To determine in vivo fitness, the relative proportion of specific integrase mutations was monitored over time by in-depth clonal analysis of the viral integrase at baseline, during and after raltegravir treatment. RESULTS: Raltegravir therapy failure was associated with the initial selection of primary resistance mutation N155H. This mutation conferred a 3.8-fold reduction in raltegravir susceptibility and a severe reduction in viral replication. Acquisition of integrase mutation Q95K increased resistance (6.2-fold) and partly restored viral replication. Selection of a third mutation, V151I, further increased raltegravir resistance (20-fold), but decreased viral replication. After prolonged raltegravir interruption, raltegravir resistance mutations were lost, demonstrating the reduced replication capacity of the resistant virus. CONCLUSIONS: We describe selection of Q95K as a secondary resistance mutation during raltegravir therapy failure. In the background of N155H, Q95K enhances raltegravir and elvitegravir resistance and improves the impaired replication of the virus.

Development and evaluation of an automated hepatitis C virus NS5B sequence-based subtyping assay.

BACKGROUND: Hepatitis C virus (HCV) genotyping and accurate subtype determination is becoming increasingly important to better understand viral evolution, the development of resistance to STAT-C, and possibly even for the treatment and management of chronic HCV-infected patients. METHODS: A subtyping assay based on a 329-bp sequence of the NS5B region, together with an automated subtype interpretation tool was developed. Clinical samples of the six major genotypes were used to assess assay performance characteristics. RESULTS: The NS5B BLAST-based subtyping assay showed clinical sensitivity for amplification of 89% (n=603 random samples). Assessment of analytical sensitivity of amplification for genotypes 1, 2, 3 and 4 revealed a suitable performance for high viral load samples and decreased only with low viral loads. The results were 100% and 99% accurate at the genotype and subtype level, respectively. A concordance of 97% on genotype level and 62% on subtype level was observed by comparison with subtype results from 5' non-coding-based assays with a panel of 276 isolates. CONCLUSIONS: The HCV NS5B subtyping assay has been validated for research use. Based on its performance, it is the method of choice in cases where subtype rather than genotype information is needed.

A comparative analysis of HIV drug resistance interpretation based on short reverse transcriptase sequences versus full sequences.

BACKGROUND: As second-line antiretroviral treatment (ART) becomes more accessible in resource-limited settings (RLS), the need for more affordable monitoring tools such as point-of-care viral load assays and simplified genotypic HIV drug resistance (HIVDR) tests increases substantially. The prohibitive expenses of genotypic HIVDR assays could partly be addressed by focusing on a smaller region of the HIV reverse transcriptase gene (RT) that encompasses the majority of HIVDR mutations for people on ART in RLS. In this study, an in silico analysis of 125,329 RT sequences was performed to investigate the effect of submitting short RT sequences (codon 41 to 238) to the commonly used virco®TYPE and Stanford genotype interpretation tools. RESULTS: Pair-wise comparisons between full-length and short RT sequences were performed. Additionally, a non-inferiority approach with a concordance limit of 95% and two-sided 95% confidence intervals was used to demonstrate concordance between HIVDR calls based on full-length and short RT sequences.The results of this analysis showed that HIVDR interpretations based on full-length versus short RT sequences, using the Stanford algorithms, had concordance significantly above 95%. When using the virco®TYPE algorithm, similar concordance was demonstrated (>95%), but some differences were observed for d4T, AZT and TDF, where predictions were affected in more than 5% of the sequences. Most differences in interpretation, however, were due to shifts from fully susceptible to reduced susceptibility (d4T) or from reduced response to minimal response (AZT, TDF) or vice versa, as compared to the predicted full RT sequence. The virco®TYPE prediction uses many more mutations outside the RT 41-238 amino acid domain, which significantly contribute to the HIVDR prediction for these 3 antiretroviral agents. CONCLUSIONS: This study illustrates the acceptability of using a shortened RT sequences (codon 41-238) to obtain reliable genotype interpretations by virco®TYPE and Stanford algorithms. Implementation of this simplified protocol could significantly reduce the cost of both resistance testing and ARV treatment monitoring in RLS.