Transcriptomic Analysis Reveals Early Alterations Associated with Intrinsic Resistance to Targeted Therapy in Lung Adenocarcinoma Cell Lines.

Lung adenocarcinoma is the most prevalent form of lung cancer, and drug resistance poses a significant obstacle in its treatment. This study aimed to investigate the overexpression of long non-coding RNAs (lncRNAs) as a mechanism that promotes intrinsic resistance in tumor cells from the onset of treatment. Drug-tolerant persister (DTP) cells are a subset of cancer cells that survive and proliferate after exposure to therapeutic drugs, making them an essential object of study in cancer treatment. The molecular mechanisms underlying DTP cell survival are not fully understood; however, long non-coding RNAs (lncRNAs) have been proposed to play a crucial role. DTP cells from lung adenocarcinoma cell lines were obtained after single exposure to tyrosine kinase inhibitors (TKIs; erlotinib or osimertinib). After establishing DTP cells, RNA sequencing was performed to investigate the differential expression of the lncRNAs. Some lncRNAs and one mRNA were overexpressed in DTP cells. The clinical relevance of lncRNAs was evaluated in a cohort of patients with lung adenocarcinoma from The Cancer Genome Atlas (TCGA). RT-qPCR validated the overexpression of lncRNAs and mRNA in the residual DTP cells and LUAD biopsies. Knockdown of these lncRNAs increases the sensitivity of DTP cells to therapeutic drugs. This study provides an opportunity to investigate the involvement of lncRNAs in the genetic and epigenetic mechanisms that underlie intrinsic resistance. The identified lncRNAs and CD74 mRNA may serve as potential prognostic markers or therapeutic targets to improve the overall survival (OS) of patients with lung cancer.

Unveiling ecological/evolutionary insights in HIV viral load dynamics: Allowing random slopes to observe correlational changes to CpG-contents and other molecular and clinical predictors.

In the context of infectious diseases, the dynamic interplay between ever-changing host populations and viral biology demands a more flexible modeling approach than common fixed correlations. Embracing random-effects regression models allows for a nuanced understanding of the intricate ecological and evolutionary dynamics underlying complex phenomena, offering valuable insights into disease progression and transmission patterns. In this article, we employed a random-effects regression to model an observed decreasing median plasma viral load (pVL) among individuals with HIV in Mexico City during 2019-2021. We identified how these functional slope changes (i.e. random slopes by year) improved predictions of the observed pVL median changes between 2019 and 2021, leading us to hypothesize underlying ecological and evolutionary factors. Our analysis involved a dataset of pVL values from 7325 ART-naïve individuals living with HIV, accompanied by their associated clinical and viral molecular predictors. A conventional fixed-effects linear model revealed significant correlations between pVL and predictors that evolved over time. However, this fixed-effects model could not fully explain the reduction in median pVL; thus, prompting us to adopt random-effects models. After applying a random effects regression model-with random slopes and intercepts by year-, we observed potential "functional changes" within the local HIV viral population, highlighting the importance of ecological and evolutionary considerations in HIV dynamics: A notably stronger negative correlation emerged between HIV pVL and the CpG content in the pol gene, suggesting a changing immune landscape influenced by CpG-induced innate immune responses that could impact viral load dynamics. Our study underscores the significance of random effects models in capturing dynamic correlations and the crucial role of molecular characteristics like CpG content. By enriching our understanding of changing host-virus interactions and HIV progression, our findings contribute to the broader relevance of such models in infectious disease research. They shed light on the changing interplay between host and pathogen, driving us closer to more effective strategies for managing infectious diseases. SIGNIFICANCE OF THE STUDY: This study highlights a decreasing trend in median plasma viral loads among ART-naïve individuals living with HIV in Mexico City between 2019 and 2021. It uncovers various predictors significantly correlated with pVL, shedding light on the complex interplay between host-virus interactions and disease progression. By employing a random-slopes model, the researchers move beyond traditional fixed-effects models to better capture dynamic correlations and evolutionary changes in HIV dynamics. The discovery of a stronger negative correlation between pVL and CpG content in HIV-pol sequences suggests potential changes in the immune landscape and innate immune responses, opening avenues for further research into adaptive changes and responses to environmental shifts in the context of HIV infection. The study's emphasis on molecular characteristics as predictors of pVL adds valuable insights to epidemiological and evolutionary studies of viruses, providing new avenues for understanding and managing HIV infection at the population level.

Metagenomic analysis reveals differences in the co-occurrence and abundance of viral species in SARS-CoV-2 patients with different severity of disease.

BACKGROUND: SARS-CoV-2 infections have a wide spectrum of clinical manifestations whose causes are not completely understood. Some human conditions predispose to severe outcome, like old age or the presence of comorbidities, but many other facets, including coinfections with other viruses, remain poorly characterized. METHODS: In this study, the eukaryotic fraction of the respiratory virome of 120 COVID-19 patients was characterized through whole metagenomic sequencing. RESULTS: Genetic material from respiratory viruses was detected in 25% of all samples, whereas human viruses other than SARS-CoV-2 were found in 80% of them. Samples from hospitalized and deceased patients presented a higher prevalence of different viruses when compared to ambulatory individuals. Small circular DNA viruses from the Anneloviridae (Torque teno midi virus 8, TTV-like mini virus 19 and 26) and Cycloviridae families (Human associated cyclovirus 10), Human betaherpesvirus 6, were found to be significantly more abundant in samples from deceased and hospitalized patients compared to samples from ambulatory individuals. Similarly, Rotavirus A, Measles morbillivirus and Alphapapilomavirus 10 were significantly more prevalent in deceased patients compared to hospitalized and ambulatory individuals. CONCLUSIONS: Results show the suitability of using metagenomics to characterize a broader peripheric virological landscape of the eukaryotic virome in SARS-CoV-2 infected patients with distinct disease outcomes. Identified prevalent viruses in hospitalized and deceased patients may prove important for the targeted exploration of coinfections that may impact prognosis.

Clinical and Virological Features of Patients Hospitalized with Different Types of COVID-19 Vaccination in Mexico City.

Coronavirus disease 2019 (COVID-19) vaccines effectively protect against severe disease and death. However, the impact of the vaccine used, viral variants, and host factors on disease severity remain poorly understood. This work aimed to compare COVID-19 clinical presentations and outcomes in vaccinated and unvaccinated patients in Mexico City. From March to September 2021, clinical, demographic characteristics, and viral variants were obtained from 1014 individuals with a documented SARS-CoV-2 infection. We compared unvaccinated, partially vaccinated, and fully vaccinated patients, stratifying by age groups. We also fitted multivariate statistical models to evaluate the impact of vaccination status, SARS-CoV-2 lineages, vaccine types, and clinical parameters. Most hospitalized patients were unvaccinated. In patients over 61 years old, mortality was significantly higher in unvaccinated compared to fully vaccinated individuals. In patients aged 31 to 60 years, vaccinated patients were more likely to be outpatients (46%) than unvaccinated individuals (6.1%). We found immune disease and age above 61 years old to be risk factors, while full vaccination was found to be the most protective factor against in-hospital death. This study suggests that vaccination is essential to reduce mortality in a comorbid population such as that of Mexico.

Phylodynamics of HIV in the Mexico City Metropolitan Region.

Evolutionary analyses of viral sequences can provide insights into transmission dynamics, which in turn can optimize prevention interventions. Here, we characterized the dynamics of HIV transmission within the Mexico City metropolitan area. HIV pol sequences from persons recently diagnosed at the largest HIV clinic in Mexico City (between 2016 and 2021) were annotated with demographic/geographic metadata. A multistep phylogenetic approach was applied to identify putative transmission clades. A data set of publicly available sequences was used to assess international introductions. Clades were analyzed with a discrete phylogeographic model to evaluate the timing and intensity of HIV introductions and transmission dynamics among municipalities in the region. A total of 6,802 sequences across 96 municipalities (5,192 from Mexico City and 1,610 from the neighboring State of Mexico) were included (93.6% cisgender men, 5.0% cisgender women, and 1.3% transgender women); 3,971 of these sequences formed 1,206 clusters, involving 78 municipalities, including 89 clusters of ≥10 sequences. Discrete phylogeographic analysis revealed (i) 1,032 viral introductions into the region, over one-half of which were from the United States, and (ii) 354 migration events between municipalities with high support (adjusted Bayes factor of ≥3). The most frequent viral migrations occurred between northern municipalities within Mexico City, i.e., Cuauhtémoc to Iztapalapa (5.2% of events), Iztapalapa to Gustavo A. Madero (5.4%), and Gustavo A. Madero to Cuauhtémoc (6.5%). Our analysis illustrates the complexity of HIV transmission within the Mexico City metropolitan area but also identifies a spatially active transmission area involving a few municipalities in the north of the city, where targeted interventions could have a more pronounced effect on the entire regional epidemic. IMPORTANCE Phylogeographic investigation of the Mexico City HIV epidemic illustrates the complexity of HIV transmission in the region. An active transmission area involving a few municipalities in the north of the city, with transmission links throughout the region, is identified and could be a location where targeted interventions could have a more pronounced effect on the entire regional epidemic, compared with those dispersed in other manners.

Dominance of Three Sublineages of the SARS-CoV-2 Delta Variant in Mexico.

In this study, we analyzed the sequences of SARS-CoV-2 isolates of the Delta variant in Mexico, which has completely replaced other previously circulating variants in the country due to its transmission advantage. Among all the Delta sublineages that were detected, 81.5 % were classified as AY.20, AY.26, and AY.100. According to publicly available data, these only reached a world prevalence of less than 1%, suggesting a possible Mexican origin. The signature mutations of these sublineages are described herein, and phylogenetic analyses and haplotype networks are used to track their spread across the country. Other frequently detected sublineages include AY.3, AY.62, AY.103, and AY.113. Over time, the main sublineages showed different geographical distributions, with AY.20 predominant in Central Mexico, AY.26 in the North, and AY.100 in the Northwest and South/Southeast. This work describes the circulation, from May to November 2021, of the primary sublineages of the Delta variant associated with the third wave of the COVID-19 pandemic in Mexico and highlights the importance of SARS-CoV-2 genomic surveillance for the timely identification of emerging variants that may impact public health.

The Alpha Variant (B.1.1.7) of SARS-CoV-2 Failed to Become Dominant in Mexico.

During the coronavirus disease 2019 (COVID-19) pandemic, the emergence and rapid increase of the B.1.1.7 (Alpha) lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first identified in the United Kingdom in September 2020, was well documented in different areas of the world and became a global public health concern because of its increased transmissibility. The B.1.1.7 lineage was first detected in Mexico during December 2020, showing a slow progressive increase in its circulation frequency, which reached its maximum in May 2021 but never became predominant. In this work, we analyzed the patterns of diversity and distribution of this lineage in Mexico using phylogenetic and haplotype network analyses. Despite the reported increase in transmissibility of the B.1.1.7 lineage, in most Mexican states, it did not displace cocirculating lineages, such as B.1.1.519, which dominated the country from February to May 2021. Our results show that the states with the highest prevalence of B.1.1.7 were those at the Mexico-U.S. border. An apparent pattern of dispersion of this lineage from the northern states of Mexico toward the center or the southeast was observed in the largest transmission chains, indicating possible independent introduction events from the United States. However, other entry points cannot be excluded, as shown by multiple introduction events. Local transmission led to a few successful haplotypes with a localized distribution and specific mutations indicating sustained community transmission. IMPORTANCE The emergence and rapid increase of the B.1.1.7 (Alpha) lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) throughout the world were due to its increased transmissibility. However, it did not displace cocirculating lineages in most of Mexico, particularly B.1.1.519, which dominated the country from February to May 2021. In this work, we analyzed the distribution of B.1.1.7 in Mexico using phylogenetic and haplotype network analyses. Our results show that the states with the highest prevalence of B.1.1.7 (around 30%) were those at the Mexico-U.S. border, which also exhibited the highest lineage diversity, indicating possible introduction events from the United States. Also, several haplotypes were identified with a localized distribution and specific mutations, indicating that sustained community transmission occurred in the country.

Genomic Characterization of SARS-CoV-2 Isolated from Patients with Distinct Disease Outcomes in Mexico.

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has shown a wide spectrum of clinical manifestations ranging from asymptomatic infections to severe disease and death. Pre-existing medical conditions and age have been mainly linked to the development of severe disease; however, the potential association of viral genetic characteristics with different clinical conditions remains unclear. SARS-CoV-2 variants with increased transmissibility were detected early in the pandemics, and several variants with potential relevance for public health are currently circulating around the world. In this study, we characterized 57 complete SARS-CoV-2 genomes during the exponential growth phase of the early epidemiological curve in Mexico, in April 2020. Patients were categorized under distinct disease severity outcomes: mild disease or ambulatory care, severe disease or hospitalized, and deceased. To reduce bias related to risk factors, the patients were less than 60 years old and with no diagnosed comorbidities A trait-association phylogenomic approach was used to explore genotype-phenotype associations, represented by the co-occurrence of mutations, disease severity outcome categories, and clusters of Mexican sequences. Phylogenetic results revealed a higher genomic diversity compared to the initial viruses detected during the early stage of the local epidemic. We identified a total of 90 single nucleotide variants compared to the Wuhan-Hu-1 genome, including 54 nonsynonymous mutations. We did not find evidence for the co-occurrence of mutations associated with specific disease outcomes. Therefore, in the group of patients studied, disease severity was likely mainly driven by the host genetic background and other demographic factors. IMPORTANCE The genetic association of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) with different clinical conditions remains unclear and needs further investigation. In this study, we characterized 57 complete SARS-CoV-2 genomes from patients in Mexico with distinct disease severity outcomes: mild disease or ambulatory care, severe disease or hospitalized, and deceased. To reduce bias related to risk factors the patients were less than 60 years old and with no diagnosed comorbidities. We did not find evidence for the co-occurrence of mutations associated with specific disease outcomes. Therefore, in the group of patients studied, disease severity was likely mainly driven by the host genetic background and other demographic factors.

Immune Milieu and Genomic Alterations Set the Triple-Negative Breast Cancer Immunomodulatory Subtype Tumor Behavior.

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous disease. Seven subtypes have been described based on gene expression patterns. Herein, we characterized the tumor biology and clinical behavior of the immunomodulatory (IM) subtype. METHODS: Formalin-fixed paraffin-embedded tumor samples from 68 high-risk (stage III-IV) TNBC patients were analyzed through microarrays, immunohistochemistry, and DNA sequencing. RESULTS: The IM subtype was identified in 24% of TNBC tumor samples and characterized by a higher intratumoral (intT) and stromal (strml) infiltration of FOXP3+ TILs (Treg) compared with non-IM subtypes. Further, PD-L1+ (>1%) expression was significantly higher, as well as CTLA-4+ intT and strml expression in the IM subtype. Differential expression and gene set enrichment analysis identified biological processes associated with the immune system. Pathway analysis revealed enrichment of the ß-catenin signaling pathway. The non-coding analysis led to seven Long Intergenic Non-Protein Coding RNAs (lincRNAs) (6 up-regulated and 1 down-regulated) that were associated with a favorable prognosis in the TNBC-IM subtype. The DNA sequencing highlighted two genes relevant to immune system responses: CTNNB1 (Catenin ß-1) and IDH1. CONCLUSION: the IM subtype showed a distinct immune microenvironment, as well as subtype-specific genomic alterations. Characterizing TNBC at a molecular and transcriptomic level might guide immune-based therapy in this subgroup of patients.

Role of HMGB1 in Cisplatin-Persistent Lung Adenocarcinoma Cell Lines.

Significant advances have been made recently in the development of targeted therapy for lung adenocarcinoma. However, platinum-based chemotherapy remains as the cornerstone in the treatment of this neoplasm. This is the treatment option for adenocarcinomas without EGFR gain-of-function mutations or tumors that have developed resistance to targeted therapy. The High-Mobility Group Box 1 (HMGB1) is a multifunctional protein involved in intrinsic resistance to cisplatin. HMGB1 is released when cytotoxic agents, such as cisplatin, induce cell death. In the extracellular milieu, HMGB1 acts as adjuvant to induce an antitumor immune response. However, the opposite effect favoring tumor progression has also been reported. In this study, the effects of cisplatin in lung adenocarcinoma cell lines harboring clinically relevant mutations, such as EGFR mutations, were studied. Subcellular localization of HMGB1 was detected in the cell lines and in viable cells after a single exposure to cisplatin, which are designated as cisplatin-persistent cells. The mRNA expression of the receptor for advanced glycation end products (RAGE), TLR-2, and TLR-4 receptors was measured in parental cell lines and their persistent variants. Finally, changes in plasma HMGB1 from a cohort of lung adenocarcinoma patients without EGFR mutation and treated with cisplatin-based therapy were analyzed. Cisplatin-susceptible lung adenocarcinoma cell lines died by apoptosis or necrosis and released HMGB1. In cisplatin-persistent cells, nuclear relocalization of HMGB1 and overexpression of HMGB1 and RAGE, but not TLR-2 or TLR-4, were observed. In tumor cells, this HMGB1-RAGE interaction may be associated with the development of cisplatin resistance. The results indicate a direct relationship between the plasma levels of HMGB1 and overall survival. In conclusion, HMGB1 may be an effective biomarker associated with increased overall survival of lung adenocarcinoma patients.

HIV Pretreatment Drug Resistance Trends in Mexico City, 2017-2020.

In response to increasing pretreatment drug resistance (PDR), Mexico changed its national antiretroviral treatment (ART) policy, recommending and procuring second-generation integrase strand-transfer inhibitor (INSTI)-based regimens as preferred first-line options since 2019. We present a four-year observational study describing PDR trends across 2017-2020 at the largest HIV diagnosis and primary care center in Mexico City. A total of 6688 baseline protease-reverse transcriptase and 6709 integrase sequences were included. PDR to any drug class was 14.4% (95% CI, 13.6-15.3%). A significant increasing trend for efavirenz/nevirapine PDR was observed (10.3 to 13.6%, p = 0.02). No increase in PDR to second-generation INSTI was observed, remaining under 0.3% across the study period. PDR was strongly associated with prior exposure to ART (aOR: 2.9, 95% CI: 1.9-4.6, p < 0.0001). MSM had higher odds of PDR to efavirenz/nevirapine (aOR: 2.0, 95% CI: 1.0-3.7, p = 0.04), reflecting ongoing transmission of mutations such as K103NS and E138A. ART restarters showed higher representation of cisgender women and injectable drug users, higher age, and lower education level. PDR to dolutegravir/bictegravir remained low in Mexico City, although further surveillance is warranted given the short time of ART optimization. Our study identifies demographic characteristics of groups with higher risk of PDR and lost to follow-up, which may be useful to design differentiated interventions locally.

Characteristics and growth of the genetic HIV transmission network of Mexico City during 2020.

INTRODUCTION: Molecular surveillance systems could provide public health benefits to focus strategies to improve the HIV care continuum. Here, we infer the HIV genetic network of Mexico City in 2020, and identify actively growing clusters that could represent relevant targets for intervention. METHODS: All new diagnoses, referrals from other institutions, as well as persons returning to care, enrolling at the largest HIV clinic in Mexico City were invited to participate in the study. The network was inferred from HIV pol sequences, using pairwise genetic distance methods, with a locally hosted, secure version of the HIV-TRACE tool: Seguro HIV-TRACE. Socio-demographic, clinical and behavioural metadata were overlaid across the network to design focused prevention interventions. RESULTS: A total of 3168 HIV sequences from unique individuals were included. One thousand and one-hundred and fifty (36%) sequences formed 1361 links within 386 transmission clusters in the network. Cluster size varied from 2 to 14 (63% were dyads). After adjustment for covariates, lower age (adjusted odds ratio [aOR]: 0.37, p<0.001; >34 vs. <24 years), being a man who has sex with men (MSM) (aOR: 2.47, p = 0.004; MSM vs. cisgender women), having higher viral load (aOR: 1.28, p<0.001) and higher CD4+ T cell count (aOR: 1.80, p<0.001; ≥500 vs. <200 cells/mm3 ) remained associated with higher odds of clustering. Compared to MSM, cisgender women and heterosexual men had significantly lower education (none or any elementary: 59.1% and 54.2% vs. 16.6%, p<0.001) and socio-economic status (low income: 36.4% and 29.0% vs. 18.6%, p = 0.03) than MSM. We identified 10 (2.6%) clusters with constant growth, for prioritized intervention, that included intersecting sexual risk groups, highly connected nodes and bridge nodes between possible sub-clusters with high growth potential. CONCLUSIONS: HIV transmission in Mexico City is strongly driven by young MSM with higher education level and recent infection. Nevertheless, leveraging network inference, we identified actively growing clusters that could be prioritized for focused intervention with demographic and risk characteristics that do not necessarily reflect the ones observed in the overall clustering population. Further studies evaluating different models to predict growing clusters are warranted. Focused interventions will have to consider structural and risk disparities between the MSM and the heterosexual populations.

Genetic Analysis of SARS-CoV-2 Variants in Mexico during the First Year of the COVID-19 Pandemic.

During the first year of the SARS-CoV-2 pandemic in Mexico, more than two million people were infected. In this study, we analyzed full genome sequences from 27 February 2020 to 28 February 2021 to characterize the geographical and temporal distribution of SARS-CoV-2 lineages and identify the most common circulating lineages during this period. We defined six different geographical regions with particular dynamics of lineage circulation. The Northeast and Northwest regions were the ones that exhibited the highest lineage diversity, while the Central south and South/Southeast regions presented less diversity with predominance of a certain lineage. Additionally, by late February 2021, lineage B.1.1.519 represented more than 89% of all circulating lineages in the country.

Dysbiosis and structural disruption of the respiratory microbiota in COVID-19 patients with severe and fatal outcomes.

The COVID-19 outbreak has caused over three million deaths worldwide. Understanding the pathology of the disease and the factors that drive severe and fatal clinical outcomes is of special relevance. Studying the role of the respiratory microbiota in COVID-19 is especially important as the respiratory microbiota is known to interact with the host immune system, contributing to clinical outcomes in chronic and acute respiratory diseases. Here, we characterized the microbiota in the respiratory tract of patients with mild, severe, or fatal COVID-19, and compared it to healthy controls and patients with non-COVID-19-pneumonia. We comparatively studied the microbial composition, diversity, and microbiota structure between the study groups and correlated the results with clinical data. We found differences in the microbial composition for COVID-19 patients, healthy controls, and non-COVID-19 pneumonia controls. In particular, we detected a high number of potentially opportunistic pathogens associated with severe and fatal levels of the disease. Also, we found higher levels of dysbiosis in the respiratory microbiota of patients with COVID-19 compared to the healthy controls. In addition, we detected differences in diversity structure between the microbiota of patients with mild, severe, and fatal COVID-19, as well as the presence of specific bacteria that correlated with clinical variables associated with increased risk of mortality. In summary, our results demonstrate that increased dysbiosis of the respiratory tract microbiota in patients with COVID-19 along with a continuous loss of microbial complexity structure found in mild to fatal COVID-19 cases may potentially alter clinical outcomes in patients. Taken together, our findings identify the respiratory microbiota as a factor potentially associated with the severity of COVID-19.

Emergence and spread of the potential variant of interest (VOI) B.1.1.519 of SARS-CoV-2 predominantly present in Mexico.

SARS-CoV-2 variants emerged in late 2020, and at least three variants of concern (B.1.1.7, B.1.351, and P1) have been reported by WHO. These variants have several substitutions in the spike protein that affect receptor binding; they exhibit increased transmissibility and may be associated with reduced vaccine effectiveness. In the present work, we report the identification of a potential variant of interest, harboring the mutations T478K, P681H, and T732A in the spike protein, within the newly named lineage B.1.1.519, that rapidly outcompeted the preexisting variants in Mexico and has been the dominant virus in the country during the first trimester of 2021.

Near point-of-care, point-mutation test to detect drug resistance in HIV-1: a validation study in a Mexican cohort.

OBJECTIVE: Pretreatment HIV-drug resistance (PDR, HIVDR) to non-nucleoside reverse transcriptase inhibitors (NNRTIs) is increasing globally. NNRTIs continue to be used as first-line antiretroviral therapy (ART) in some communities due to the cost of dolutegravir-based ART or dolutegravir-associated adverse events. A simplified version of the oligonucleotide ligation assay (OLA) - 'OLA-Simple' - is a low-cost, near point-of-care assay that provides ready-to-use lyophilized reagents and reports HIVDR mutations as colored lines on lateral flow strips. Our objective was to design and validate OLA-Simple for a Mexican cohort. DESIGN: OLA-Simple probes to detect K65R, K103N/S, Y181C, M184V, and G190A were optimized for HIV Mexican sequences. Sixty clinical plasma specimens were analyzed by OLA-Simple by technicians blinded to Illumina-MiSeq sequences, and HIVDR results were compared. METHODS: Plasma RNA was tested using OLA-Simple kits. OLA-Simple lateral flow strips were read by in-house software, and were classified as mutant or wild-type at each codon. The comparison of results by OLA-Simple and Miseq was used to generate receiver-operating characteristic curves. RESULTS: OLA-Simple PCR amplified 59 of 60 specimens and successfully genotyped 287 of 295 codons, with eight of 295 (2.7%) indeterminate results. Compared to MiSeq, OLA-Simple gave five of 295 (1.7%) false-positive and four of 295 (1.4%) false-negative results. Excluding indeterminate results, OLA-Simple classified mutant with an accuracy of 97.4 and 98.8% when using thresholds at 10 and 25% mutant within an individual's HIV quasispecies, respectively. CONCLUSIONS: Compared to MiSeq, OLA-Simple detected HIVDR with high sensitivity and accuracy. OLA-Simple could expand access to affordable and rapid HIVDR testing to guide appropriate ART choices in populations using NNRTI-based ART.

Pretreatment HIV drug resistance spread within transmission clusters in Mexico City.

BACKGROUND: Pretreatment HIV drug resistance (HIVDR) to NNRTIs has consistently increased in Mexico City during the last decade. OBJECTIVES: To infer the HIV genetic transmission network in Mexico City to describe the dynamics of the local HIV epidemic and spread of HIVDR. PATIENTS AND METHODS: HIV pol sequences were obtained by next-generation sequencing from 2447 individuals before initiation of ART at the largest HIV clinic in Mexico City (April 2016 to June 2018). Pretreatment HIVDR was estimated using the Stanford algorithm at a Sanger-like threshold (≥20%). Genetic networks were inferred with HIV-TRACE, establishing putative transmission links with genetic distances <1.5%. We examined demographic associations among linked individuals with shared drug resistance mutations (DRMs) using a ≥ 2% threshold to include low-frequency variants. RESULTS: Pretreatment HIVDR reached 14.8% (95% CI 13.4%-16.2%) in the cohort overall and 9.6% (8.5%-10.8%) to NNRTIs. Putative links with at least one other sequence were found for 963/2447 (39%) sequences, forming 326 clusters (2-20 individuals). The inferred network was assortative by age and municipality (P < 0.001). Clustering individuals were younger [adjusted OR (aOR) per year = 0.96, 95% CI 0.95-0.97, P < 0.001] and less likely to include women (aOR = 0.46, 95% CI 0.28-0.75, P = 0.002). Among clustering individuals, 175/963 (18%) shared DRMs (involving 66 clusters), of which 66/175 (38%) shared K103N/S (24 clusters). Eight municipalities (out of 75) harboured 65% of persons sharing DRMs. Among all persons sharing DRMs, those sharing K103N were younger (aOR = 0.93, 95% CI 0.88-0.98, P = 0.003). CONCLUSIONS: Our analyses suggest age- and geographically associated transmission of DRMs within the HIV genetic network in Mexico City, warranting continuous monitoring and focused interventions.

Longitudinal variation in human immunodeficiency virus long terminal repeat methylation in individuals on suppressive antiretroviral therapy.

BACKGROUND: Persistence of latent, replication-competent provirus in CD4+ T cells of human immunodeficiency virus (HIV)-infected individuals on antiretroviral treatment (ART) is the main obstacle for virus eradication. Methylation of the proviral 5' long terminal repeat (LTR) promoter region has been proposed as a possible mechanism contributing to HIV latency; however, conflicting observations exist regarding its relevance. We assessed 5'-LTR methylation profiles in total CD4+ T cells from blood of 12 participants on short-term ART (30 months) followed up for 2 years, and a cross-sectional group of participants with long-term ART (6-15 years), using next generation sequencing. We then looked for associations between specific 5'-LTR methylation patterns and baseline and follow-up clinical characteristics. RESULTS: 5'-LTR methylation was observed in all participants and behaved dynamically. The number of 5'-LTR variants found per sample ranged from 1 to 13, with median sequencing depth of 16270× (IQR 4107×-46760×). An overall significant 5'-LTR methylation increase was observed at month 42 compared to month 30 (median CpG Methylation Index: 74.7% vs. 0%, p = 0.025). This methylation increase was evident in a subset of participants (methylation increase group), while the rest maintained fairly high and constant methylation (constant methylation group). Persons in the methylation increase group were younger, had higher CD4+ T cell gain, larger CD8% decrease, and larger CD4/CD8 ratio change after 48 months on ART (all p < 0.001). Using principal component analysis, the constant methylation and methylation increase groups showed low evidence of separation along time (factor 2: p = 0.04). Variance was largely explained (21%) by age, CD4+/CD8+ T cell change, and CD4+ T cell subpopulation proportions. Persons with long-term ART showed overall high methylation (median CpG Methylation Index: 78%; IQR 71-87%). No differences were observed in residual plasma viral load or proviral load comparing individuals on short-term (both at 30 or 42 months) and long-term ART. CONCLUSIONS: Our study shows evidence that HIV 5'-LTR methylation in total CD4+ T cells is dynamic along time and that it can follow different temporal patterns that are associated with a combination of baseline and follow-up clinical characteristics. These observations may account for differences observed between previous contrasting studies.

HIV Drug Resistance in Antiretroviral Treatment-Naïve Individuals in the Largest Public Hospital in Nicaragua, 2011-2015.

BACKGROUND: Increasing HIV pre-treatment drug resistance (PDR) levels have been observed in regions with increasing antiretroviral treatment (ART) coverage. However, data is lacking for several low/middle-income countries. We present the first PDR survey in Nicaragua since ART introduction in the country in 2003. METHODS: HIV-infected, ART-naïve Nicaraguan individuals were enrolled at Roberto Calderón Hospital, the largest national HIV referral center, from 2011 to 2015. HIV pol sequences were obtained at a WHO-accredited laboratory in Mexico by Sanger and next generation sequencing (NGS). PDR was assessed using the WHO surveillance drug resistance mutation (SDRM) list and the Stanford HIVdb tool. RESULTS: 283 individuals were enrolled in the study. The overall PDR prevalence based on the list of SDRMs was 13.4%. Using the Stanford HIVdb tool, overall PDR reached 19.4%; with both nucleoside and non-nucleoside reverse transcriptase inhibitor (NRTI and NNRTI) PDR levels independently reaching moderate levels (6.7% and 11.3% respectively). Protease inhibitor PDR was low (2.8%). Using NGS with 2% threshold to detect SDRMs, PDR increased to 25.3%. K103N and M41L were the most frequent SDRMs and were present mostly in proportions >20% in each individual. A significant temporal increase in NNRTI PDR was observed (p = 0.0422), with no apparent trends for other drug classes. Importantly, PDR to zidovudine + lamivudine + efavirenz and tenofovir + emtricitabine + efavirenz, the most widely used first-line regimens in Nicaragua, reached 14.6% and 10.4% respectively in 2015. Of note, a higher proportion of females was observed among individuals with PDR compared to individuals without PDR (OR 14.2; 95% CI: 7.1-28.4; p<0.0001). CONCLUSIONS: Overall PDR in the Nicaraguan cohort was high (19.4%), with a clear increasing temporal trend in NNRTI PDR. Current HIVDR to the most frequently used first-line ART regimens in Nicaragua reached levels >10%. These observations are worrisome and need to be evidenced to strengthen the national HIV program. Also, our observations warrant further nationally representative HIVDR surveillance studies and encourage other countries to perform national surveys. Cost-effectiveness studies are suggested to analyze the feasibility of implementation of baseline HIV genotyping as well as to review the choice of first-line ART regimens in Nicaragua.

Pretreatment HIV-drug resistance in Mexico and its impact on the effectiveness of first-line antiretroviral therapy: a nationally representative 2015 WHO survey.

BACKGROUND: WHO has developed a global HIV-drug resistance surveillance strategy, including assessment of pretreatment HIV-drug resistance. We aimed to do a nationally representative survey of pretreatment HIV-drug resistance in Mexico using WHO-recommended methods. METHODS: Among 161 Ministry of Health antiretroviral therapy (ART) clinics in Mexico, the largest, including 90% of ART initiators within the Ministry of Health (66 in total), were eligible for the survey. We used a probability-proportional-to-size design method to sample 25 clinics throughout the country. Consecutive ART-naive patients with HIV about to initiate treatment were invited to participate in the survey; individuals with previous exposure to ART were excluded. We assessed pretreatment HIV-drug resistance by Sanger sequencing and next-generation sequencing of viruses from plasma specimens from eligible participants with Stanford University HIV Drug Resistance Database methods. We obtained follow-up data for a median of 9·4 months (range 6-12) after enrolment. We investigated possible relations between demographic variables and pretreatment drug resistance with univariate and multivariate logistic regression. FINDINGS: Between Feb 3 and July 30, 2015, we screened 288 patients in 25 clinics, from whom 264 provided successfully sequenced viruses with no evidence of current exposure to antiretroviral drugs. With the Sanger method, of these 264 participants, 41 (15·5%, 95% CI 11·4-20·5) had pretreatment resistance to any antiretroviral drug and 28 (10·6%, 7·2-15·0) had pretreatment resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs). At least low-level pretreatment resistance (Stanford penalty score ≥15) was noted in 13 (4 ·â€ˆ9%) of participants to efavirenz and in 23 (8·7%) to the combination tenofovir plus emtricitabine plus efavirenz. With next-generation sequencing, of 264 participants, 38 (14·4%, 95% CI 10·4-19·2) had pretreatment resistance to any antiretroviral drug and 26 (9·8%, 6·5-14·1) had pretreatment resistance to NNRTIs. After median follow-up of 8 months (IQR 6·5-9·4, range 5-11) after ART initiation, 97 (72%) of 135 NNRTI initiators achieved viral suppression (<50 copies per mL) compared with ten (40%) of 25 individuals who started with protease inhibitor-based regimens (p=0·0045). After multivariate regression considering pretreatment resistance and initial ART regimen as composite variables, people starting NNRTIs with pretreatment drug resistance achieved significantly lower viral suppression (odds ratio 0·24, 95% CI 0·07-0·74; p=0·014) than patients without NNRTI resistance. INTERPRETATION: High levels of pretreatment drug resistance were noted in Mexico, and NNRTI pretreatment drug resistance significantly reduced the effectiveness of first-line ART regimens based on these drugs. Baseline HIV-drug resistance testing for initial ART follow-up and decision making should be considered. FUNDING: The Mexican Government and Consejo Nacional de Ciencia y Tecnología.