Antiretroviral drug resistant HIV-1 in women and children living in Honduras

Antiretroviral therapy (ART) in HIV infected pregnant women contributes to the prevention of HIV transmission to the newborn. However, as ART can also induce HIV drug resistance during suboptimal levels of virological suppression a major concern is the subsequent risk for transmitted drug resistant (TDR) virus to the child. In Honduras and Belize, like in many other countries around the world, mono-therapy was used to prevent mother-to-child transmission of HIV-1 (MTCT) until relatively recently when it was changed to a more effective combination antiretroviral therapy (cART). Prior to this study there was no information about antiretroviral drug resistance in HIV-1 infected women and children in Honduras and Belize, and limited data other from parts of Latin America. The first aim (Paper I) was to evaluate the prevalence of drug resistance in HIV-1 infected infants born in Honduras and Belize between 2001 to 2004, before cART was implemented for prevention of MTCT. Genotypic resistance was performed by sequencing of the HIV pol region and was successfully in dried blood spots from 66 HIV-1-infected infants (55 from Honduras and 11 from Belize). Mutations associated with antiretroviral drug resistance were detected in sequences from 13% of the Honduran infants and 27% of Belizean infants. Thus the study documented, for first time, the presence of drug resistance in HIV-1 infected Honduran and Belizean infants. Resistance probably was transmitted from the mothers since none of the infants had received antiretroviral drugs as prophylaxis or therapy. The second aim (Paper II) was to evaluate antiretroviral drug resistance in pregnant HIV-1-infected women in Honduras and risk for MTCT subsequent to ART prophylaxis. In addition, we investigated changes in immune activation during pregnancy by evaluating LPS levels. A total of 50 mother-child pairs and 95 HIV-negative pregnant women were enrolled. The presence of antiretroviral drug resistance was monitored in samples drawn during pregnancy and shortly after delivery. Twenty-nine women (58%) were treatment-naïve at study entry and started antiretroviral prophylaxis against MTCT during pregnancy while 21 women were already identified as HIV-1 infected and on ART at study entry. Antiretroviral drug resistance was detected in 20% of the samples obtained from the mothers at baseline; 10% among treatment-naïve patients and 29% among treatment-experienced patients. Furthermore, despite ART prophylaxis 22 of 50 (44%) women were viremic. No MTCT were observed, but still the high prevalence of resistance and viremia indicated that there was a significant risk for MTCT. The LPS levels declined between pregnancy and after delivery in the HIV-1 infected women indicates that pregnancy might influence the LPS levels, a novel finding that merits further investigation. This study demonstrated for the first time a high prevalence of antiretroviral drug resistance and viremia in pregnant Honduran women, which could limit the effectiveness of antiretroviral prophylaxis against MTCT. Taken together the studies indicate that there is a need for improvements of prevention against MTCT in Belize and Honduras. This includes better access to monitoring of plasma HIV-1 RNA levels and antiretroviral drug resistance testing

Capturing sequence diversity in metagenomes with comprehensive and scalable probe design.

Metagenomic sequencing has the potential to transform microbial detection and characterization, but new tools are needed to improve its sensitivity. Here we present CATCH, a computational method to enhance nucleic acid capture for enrichment of diverse microbial taxa. CATCH designs optimal probe sets, with a specified number of oligonucleotides, that achieve full coverage of, and scale well with, known sequence diversity. We focus on applying CATCH to capture viral genomes in complex metagenomic samples. We design, synthesize, and validate multiple probe sets, including one that targets the whole genomes of the 356 viral species known to infect humans. Capture with these probe sets enriches unique viral content on average 18-fold, allowing us to assemble genomes that could not be recovered without enrichment, and accurately preserves within-sample diversity. We also use these probe sets to recover genomes from the 2018 Lassa fever outbreak in Nigeria and to improve detection of uncharacterized viral infections in human and mosquito samples. The results demonstrate that CATCH enables more sensitive and cost-effective metagenomic sequencing

Genome sequencing reveals Zika virus diversity and spread in the Americas

Although the recent Zika virus (ZIKV) epidemic in the Americas and its link to birth defects have attracted a great deal of attention, much remains unknown about ZIKV disease epidemiology and ZIKV evolution, in part owing to a lack of genomic data. Here we address this gap in knowledge by using multiple sequencing approaches to generate 110 ZIKV genomes from clinical and mosquito samples from 10 countries and territories, greatly expanding the observed viral genetic diversity from this outbreak. We analysed the timing and patterns of introductions into distinct geographic regions; our phylogenetic evidence suggests rapid expansion of the outbreak in Brazil and multiple introductions of outbreak strains into Puerto Rico, Honduras, Colombia, other Caribbean islands, and the continental United States. We find that ZIKV circulated undetected in multiple regions for many months before the first locally transmitted cases were confirmed, highlighting the importance of surveillance of viral infections. We identify mutations with possible functional implications for ZIKV biology and pathogenesis, as well as those that might be relevant to the effectiveness of diagnostic tests

Prevalencia de la resistencia genotípica a los fármacos antirretrovirales en pacientes VIH positivos de Tegucigalpa

The objective of this study was to determine the prevalence of HIV drug-resistance in treated and untreated patients. One-hundred samples of HIV-positive patients of Tegucigalpa were analyzed, 50 samples from patients prior to antiretroviral therapy and 50 samples from patients on antiretroviral therapy with treatment failure.HIV-1 pol sequences were generated to determine the presence of drug resistance mutations using the Stanford calibrated population resistance tool according to the WHO Surveillance Drug Resistance Mutations for untreated patients and the Stanford Database list of mutations for treated patients.The results shows that drug-resistance mutation prevalence in patients before treatment initiation was 19 % [IC 95 %: 9-33%], 8.3 % for NRTI, 12.5 % for NNRTI and 6.25 % for PI; the most common mutation founded were: M184V, K103N, M46I. The prevalence of drug-resistance mutations in patients on therapy was 82 % [IC 95 %:60-90%], 58 % for NRTI, 74 % for NNRTI, and 22 % for PI.In conclusions, we observed a significance increase in the prevalence of drug-resistance mutations in untreated patients compared with previous studies, which is of great concern because it limits the effectiveness of first-line treatment. Although the prevalence of acquired resistance in patients with therapeutic failure remains slightly constant remains high, requiring treatment changes to second or third line in these patients, causing an economic impact on public health.Revista Ciencia y Tecnología, N° 15, December 2014: 147-160El objetivo de la investigación fue determinar la prevalencia de resistencia del VIH a las drogas antirretrovirales en pacientes tratados y no tratados. Para tal efecto, se analizaron 100 muestras de pacientes VIH positivos, 50 previos a iniciar tratamiento y 50 bajo tratamiento con evidencia de falla terapéutica, de Tegucigalpa.Secuencias del gen pol del VIH-1 fueron generadas para determinar la presencia de mutaciones asociadas a resistencia, utilizando la herramienta calibrada para la vigilancia de la resistencia propuesta por la OMS para pacientes pretratados y la lista de mutaciones de la base de datos de Stanford para pacientes bajo tratamiento.Los resultados muestran que la prevalencia de farmacorresistencia en pacientes previos a iniciar tratamiento fue del 19 % [IC 95 %: 9-33 %], el 8.3 % presentaron mutaciones contra los NRTI, el 12.5 % contra los NNRTI y el 6.25 % contra los PI. Las mutaciones con mayor frecuencia fueron: M184V, K103N, M46I. La prevalencia de mutaciones asociadas a resistencia en los pacientes en falla terapéutica fue del 82 % [IC 95 %:60-90 %], el 58 % contra los NRTI, 74 % contra los NNRTI y el 22 % contra los PI.En conclusión, se observó un marcado aumento en la prevalencia de mutaciones asociadas a resistencia en pacientes no tratados, comparada con estudios anteriores, lo cual es de mucha preocupación, ya que limita la eficacia del tratamiento de primera línea. Aunque la prevalencia de resistencia adquirida en pacientes con falla terapéutica se mantiene ligeramente constante, sigue siendo alta, requiriendo de cambios de tratamiento a segunda o tercera línea en estos pacientes, ocasionando un impacto económico en la salud pública.Revista Ciencia y Tecnología, N° 15, diciembre 2014: 147-16

HIV Drug Resistance Surveillance in Honduras after a Decade of Widespread Antiretroviral Therapy.

We assessed HIV drug resistance (DR) in individuals failing ART (acquired DR, ADR) and in ART-naïve individuals (pre-ART DR, PDR) in Honduras, after 10 years of widespread availability of ART.365 HIV-infected, ART-naïve, and 381 ART-experienced Honduran individuals were enrolled in 5 reference centres in Tegucigalpa, San Pedro Sula, La Ceiba, and Choluteca between April 2013 and April 2015. Plasma HIV protease-RT sequences were obtained. HIVDR was assessed using the WHO HIVDR mutation list and the Stanford algorithm. Recently infected (RI) individuals were identified using a multi-assay algorithm.PDR to any ARV drug was 11.5% (95% CI 8.4-15.2%). NNRTI PDR prevalence (8.2%) was higher than NRTI (2.2%) and PI (1.9%, p500 vs. <350 CD4+ T cells/μL. PDR in recently infected individuals was 13.6%, showing no significant difference with PDR in individuals with longstanding infection (10.7%). The most prevalent PDR mutations were M46IL (1.4%), T215 revertants (0.5%), and K103NS (5.5%). The overall ADR prevalence in individuals with <48 months on ART was 87.8% and for the ≥48 months on ART group 81.3%. ADR to three drug families increased in individuals with longer time on ART (p = 0.0343). M184V and K103N were the most frequent ADR mutations. PDR mutation frequency correlated with ADR mutation frequency for PI and NNRTI (p<0.01), but not for NRTI. Clusters of viruses were observed suggesting transmission of HIVDR both from ART-experienced to ART-naïve individuals and between ART-naïve individuals.The global PDR prevalence in Honduras remains at the intermediate level, after 10 years of widespread availability of ART. Evidence of ADR influencing the presence of PDR was observed by phylogenetic analyses and ADR/PDR mutation frequency correlations

Development and validation of a rapid lateral flow E1/E2-antigen test and ELISA in patients infected with emerging Asian strain of Chikungunya virus in the Americas

Since its 2013 emergence in the Americas, Chikungunya virus (CHIKV) has posed a serious threat to public health. Early and accurate diagnosis of the disease, though currently lacking in clinics, is integral to enable timely care and epidemiological response. We developed a dual detection system: a CHIKV antigen E1/E2-based enzyme-linked immunosorbent assay (ELISA) and a lateral flow test using high-affinity anti-CHIKV antibodies. The ELISA was validated with 100 PCR-tested acute Chikungunya fever samples from Honduras. The assay had an overall sensitivity and specificity of 51% and 96.67%, respectively, with accuracy reaching 95.45% sensitivity and 92.03% specificity at a cycle threshold (Ct) cutoff of 22. As the Ct value decreased from 35 to 22, the ELISA sensitivity increased. We then developed and validated two lateral flow tests using independent antibody pairs. The sensitivity and specificity reached 100% for both lateral flow tests using 39 samples from Colombia and Honduras at Ct cutoffs of 20 and 27, respectively. For both lateral flow tests, sensitivity decreased as the Ct increased after 27. Because CHIKV E1/E2 are exposed in the virion surfaces in serum during the acute infection phase, these sensitive and specific assays demonstrate opportunities for early detection of this emerging human pathogen.U.S. Public Health Service (award AI100190

Evaluation of ELISA-Based Multiplex Peptides for the Detection of Human Serum Antibodies Induced by Zika Virus Infection across Various Countries

Zika virus (ZIKV) is a mosquito-borne Flavivirus with a positive-sense RNA genome, which are generally transmitted through the bite of an infected Aedes mosquito. ZIKV infections could be associated with neurological sequelae that, and otherwise produces similar clinical symptoms as other co-circulating pathogens. Past infection with one member of the Flavivirus genus often induces cross-reactive antibodies against other flaviruses. These attributes complicate the ability to differentially diagnose ZIKV infection from other endemic mosquito-borne viruses, making it both a public health issue as well as a diagnostic challenge. We report the results from serological analyses using arbovirus-specific peptides on 339 samples that were previously collected from 6 countries. Overall, we found that our multiplexed peptide-based ELISA was highly efficient for identifying ZIKV antibodies as early as 2 weeks post infection, and that it correlates with microneutralization, plaque reduction neutralization tests (PRNTs) and commercial tests for ZIKV in previously characterized samples. We observed that seropositivity varied by patient cohort, reflecting the sampling period in relation to the 2015–2016 ZIKV outbreak. This work evaluates the accuracy, specificity, and sensitivity of our peptide-based ELISA method for detecting ZIKV antibodies from geographically diverse regions. These findings can contribute to ongoing serological methods development and can be adapted for use in future studies

Field-deployable viral diagnostics using CRISPR-Cas13

Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in <2 hours. We further demonstrate that SHERLOCK can distinguish the four DENV serotypes, as well as region-specific strains of ZIKV from the 2015–2016 pandemic. Finally, we report the rapid (<1 week) design and testing of instrument-free assays to detect clinically relevant viral single-nucleotide polymorphisms.NIH (Grants AI-100190, 1R01-HG009761, 1R01-MH110049, and 1DP1-HL141201