Current sampling and sequencing biases of Lassa mammarenavirus limit inference from phylogeography and molecular epidemiology in Lassa fever endemic regions.

Lassa fever (LF) is a potentially lethal viral haemorrhagic infection of humans caused by Lassa mammarenavirus (LASV). It is an important endemic zoonotic disease in West Africa with growing evidence for increasing frequency and sizes of outbreaks. Phylogeographic and molecular epidemiology methods have projected expansion of the Lassa fever endemic zone in the context of future global change. The Natal multimammate mouse (Mastomys natalensis) is the predominant LASV reservoir, with few studies investigating the role of other animal species. To explore host sequencing biases, all LASV nucleotide sequences and associated metadata available on GenBank (n = 2,298) were retrieved. Most data originated from Nigeria (54%), Guinea (20%) and Sierra Leone (14%). Data from non-human hosts (n = 703) were limited and only 69 sequences encompassed complete genes. We found a strong positive correlation between the number of confirmed human cases and sequences at the country level (r = 0.93 (95% Confidence Interval = 0.71-0.98), p < 0.001) but no correlation exists between confirmed cases and the number of available rodent sequences (r = -0.019 (95% C.I. -0.71-0.69), p = 0.96). Spatial modelling of sequencing effort highlighted current biases in locations of available sequences, with increased sequencing effort observed in Southern Guinea and Southern Nigeria. Phylogenetic analyses showed geographic clustering of LASV lineages, suggestive of isolated events of human-to-rodent transmission and the emergence of currently circulating strains of LASV from the year 1498 in Nigeria. Overall, the current study highlights significant geographic limitations in LASV surveillance, particularly, in non-human hosts. Further investigation of the non-human reservoir of LASV, alongside expanded surveillance, are required for precise characterisation of the emergence and dispersal of LASV. Accurate surveillance of LASV circulation in non-human hosts is vital to guide early detection and initiation of public health interventions for future Lassa fever outbreaks.

High seroprevalence of Leishmania infantum is linked to immune activation in people with HIV: a two-stage cross-sectional study in Bahia, Brazil.

Visceral leishmaniasis is an opportunistic disease in HIV-1 infected individuals, unrecognized as a determining factor for AIDS diagnosis. The growing geographical overlap of HIV-1 and Leishmania infections is an emerging challenge worldwide, as co-infection increases morbidity and mortality for both infections. Here, we determined the prevalence of people living with HIV (PWH) with a previous or ongoing infection by Leishmania infantum and investigated the virological and immunological factors associated with co-infection. We adopted a two-stage cross-sectional cohort (CSC) design (CSC-I, n = 5,346 and CSC-II, n = 317) of treatment-naïve HIV-1-infected individuals in Bahia, Brazil. In CSC-I, samples collected between 1998 and 2013 were used for serological screening for leishmaniasis by an in-house Enzyme-Linked Immunosorbent Assay (ELISA) with SLA (Soluble Leishmania infantum Antigen), resulting in a prevalence of previous or ongoing infection of 16.27%. Next, 317 PWH were prospectively recruited from July 2014 to December 2015 with the collection of sociodemographic and clinical data. Serological validation by two different immunoassays confirmed a prevalence of 15.46 and 8.20% by anti-SLA, and anti-HSP70 serology, respectively, whereas 4.73% were double-positive (DP). Stratification of these 317 individuals in DP and double-negative (DN) revealed a significant reduction of CD4+ counts and CD4+/CD8+ ratios and a tendency of increased viral load in the DP group, as compared to DN. No statistical differences in HIV-1 subtype distribution were observed between the two groups. However, we found a significant increase of CXCL10 (p = 0.0076) and a tendency of increased CXCL9 (p = 0.061) in individuals with DP serology, demonstrating intensified immune activation in this group. These findings were corroborated at the transcriptome level in independent Leishmania- and HIV-1-infected cohorts (Swiss HIV Cohort and Piaui Northeast Brazil Cohort), indicating that CXCL10 transcripts are shared by the IFN-dominated immune activation gene signatures of both pathogens and positively correlated to viral load in untreated PWH. This study demonstrated a high prevalence of PWH with L. infantum seropositivity in Bahia, Brazil, linked to IFN-mediated immune activation and a significant decrease in CD4+ levels. Our results highlight the urgent need to increase awareness and define public health strategies for the management and prevention of HIV-1 and L. infantum co-infection.

A global analysis of One Health Networks and the proliferation of One Health collaborations.

There has been a renewed focus on threats to the human-animal-environment interface as a result of the COVID-19 pandemic, and investments in One Health collaborations are expected to increase. Efforts to monitor the development of One Health Networks (OHNs) are essential to avoid duplication or misalignment of investments. This Series paper shows the global distribution of existing OHNs and assesses their collective characteristics to identify potential deficits in the ways OHNs have formed and to help increase the effectiveness of investments. We searched PubMed, Google, Google Scholar, and relevant conference websites for potential OHNs and identified 184 worldwide for further analysis. We developed four case studies to show important findings from our research and exemplify best practices in One Health operationalisation. Our findings show that, although more OHNs were formed in the past 10 years than in the preceding decade, investment in OHNs has not been equitably distributed; more OHNs are formed and headquartered in Europe than in any other region, and emerging infections and novel pathogens were the priority focus area for most OHNs, with fewer OHNs focusing on other important hazards and pressing threats to health security. We found substantial deficits in the OHNs collaboration model regarding the diversity of stakeholder and sector representation, which we argue impedes effective and equitable OHN formation and contributes to other imbalances in OHN distribution and priorities. These findings are supported by previous evidence that shows the skewed investment in One Health thus far. The increased attention to One Health after the COVID-19 pandemic is an opportunity to focus efforts and resources to areas that need them most. Analyses, such as this Series paper, should be used to establish databases and repositories of OHNs worldwide. Increased attention should then be given to understanding existing resource allocation and distribution patterns, establish more egalitarian networks that encompass the breadth of One Health issues, and serve communities most affected by emerging, re-emerging, or endemic threats at the human-animal-environment interface.

Detection of carbapenemases blaOXA48-blaKPC-blaNDM-blaVIM and extended-spectrum-ß-lactamase blaOXA1-blaSHV-blaTEM genes in Gram-negative bacterial isolates from ICU burns patients.

BACKGROUND AND OBJECTIVES: Burn patients are highly susceptible to invasion by multidrug-resistant Gram-negative bacteria (MDR-GNB) through post-burn damage. The prevalence of MDR-GNB isolated from burns patients has increased dramatically in the last decade, representing a serious risk to patients admitted to burns units worldwide. The challenges of managing infected burns patients are exacerbated in poor resource settings. This study was designed to develop a pathway for the rapid diagnosis of multidrug-resistant (MDR) Gram-negative infections and identify the bacterial genes including blaOXA1, blaTEM, and blaSHV encoding ESBLs and blaOXA48, blaKPC, blaNDM, and blaVIM encoding carbapenemases from the patient of post burns infection.  METHODS: Clinical isolates were collected (August 2017 to August 2018) from Intensive care unit (ICU) of Burn Centre. Antibiotic susceptibility testing and phenotypic detection of ESBLs and carbapenemases was performed by disk diffusion, double disk synergy test (DDST), combination disk test (CDT), and Imipenem + EDTA combined disk test (IMP + EDTA CDT). Polymerase chain reaction (PCR) detection was performed for ESBLs blaOXA1-blaSHV-blaTEM and carbapenemases genes blaOXA48-blaKPC-blaNDM-blaVIM RESULTS: In total, of 170 Gram-negative isolates, 104 (61.2%) were confirmed as multidrug-resistant (MDR); Pseudomonas aeruginosa was found to be the most prevalent 43/104 (41.4%), followed by Klebsiella pneumoniae 17/104 (16.4%), Acinetobacter baumannii12/104 (11.5%), and 6/104 Proteus mirabilis (5.8%). All isolates (100%) were resistant to cefotaxime and ceftazidime, while the meropenem resistance was 58.7%. ESBL and carbapenemase genotypes were found to be associated with higher MAR index (0.65-0.88) and MIC (> 32 µg/ml) values P. aeruginosa was the major ESBL and carbapenemase producer as determined by phenotypic testing and PCR. blaTEM positive isolates among ESBLs producers were predominant 81.8% (27/33), followed by 27.3% blaOXA1 and blaSHV, respectively. blaVIM positive isolates among carbapenemase producers were predominant 47.7% (21/44), followed by 27.3% blaKPC, 20.5% blaOXA48, and 11.4% blaNDM positive isolates. CONCLUSIONS: The predominant organism causing burn infections was ESBL and carbapenemase-producing Pseudomonas aeruginosa. There are only limited effective antibiotics against such strains. blaVIM and blaTEM individually and in co-existence with blaKPC, blaOXA48, blaSHV, and blaOXA1 confer antimicrobial resistance in burns patients. Rapid detection of ESBL and carbapenemase genes will inform treatment strategies improving the outcome for post-burn patients in ICU.

Age-associated phenotypic imbalance in TCD4 and TCD8 cell subsets: comparison between healthy aged, smokers, COPD patients and young adults.

BACKGROUND: COPD is associated with an abnormal lung immune response that leads to tissue damage and remodeling of the lung, but also to systemic effects that compromise immune responses. Cigarette smoking also impacts on innate and adaptative immune responses, exerting dual, pro- and anti-inflammatory effects. Previously, we showed that COPD patients presented accelerated telomere shortening and decreased telomerase activity, while, paradoxically, cigarette-smokers exhibited preserved telomerase activity and slower rate of telomere shortening. RESULTS: Here, we evaluated the naive, CM, EM and TEMRA subsets of TCD4 and TCD8 cells according to the expression of CCR7/CD45RA. We compared age-matched COPD patients, cigarette-smokers without clinical-laboratory evidence of pulmonary compromise, and healthy individuals. They were additionally compared with a group of young adults. For each subset we analysed the expression of markers associated with late differentiation, senescence and exhaustion (CD27/CD28/CD57/KLRG1/PD1). We show that COPD patients presented a drastically reduced naive cells pool, and, paradoxically, increased fractions of naive cells expressing late differentiation, senescence or exhaustion markers, likely impacting on their immunocompetence. Pronounced phenotypic alterations were also evidenced in their three memory T-cell subsets compared with the other aged and young groups, suggesting an also dysfunctional memory pool. Surprisingly, our smokers showed a profile closer to the Healthy aged than COPD patients. They exhibited the usual age-associated shift of naive to EM TCD4 and TCD8 cells, but not to CM or TEMRA T-cells. Nonetheless, their naive T-cells phenotypes were in general similar to those of the Youngs and Healthy aged, suggesting a rather phenotypically preserved subset, while the memory T-cells exhibited increased proportions of cells with the late-differentiation or senescence/exhaustion markers as in the Healthy aged. CONCLUSION: Our study extends previous findings by showing that COPD patients have cells expressing a full range of late differentiated, senescent or exhausted phenotypes encompassing all TCD4 and TCD8 subsets, consistent with a premature immunosenescence phenotype. Surprisingly, the smokers group's results suggest that moderate to heavy chronic cigarette smoking did not accelerate the pace of immunosenescence as compared with the Healthy aged.

Long-term tobacco exposure and immunosenescence: Paradoxical effects on T-cells telomere length and telomerase activity.

Immunosenescence are alterations on immune system that occurs throughout an individual life. The main characteristic of this process is replicative senescence, evaluated by telomere shortening. Several factors implicate on telomere shortening, such as smoking. In this study, we evaluated the influence of smoking and Chronic Obstructive Pulmonary Disease (COPD) on cytokines, telomere length and telomerase activity. Blood samples were collected from subjects aged over 60 years old: Healthy (never smokers), Smokers (smoking for over 30 years) and COPDs (ex-smokers for ≥15 years). A young group was included as control. PBMCs were cultured for assessment of telomerase activity using RT-PCR, and cytokines secretion flow cytometry. CD4+ and CD8+ purified lymphocytes were used to assess telomere length using FlowFISH. We observed that COPD patients have accelerated telomere shortening. Paradoxically, smokers without lung damage showed preserved telomere length, suggesting that tobacco smoking may affect regulatory mechanisms, such as telomerase. Telomerase activity showed diminished activity in COPDs, while Smokers showed increased activity compared to COPDs and Healthy groups. Extracellular environment reflected this unbalance, indicated by an anti-inflammatory profile in Smokers, while COPDs showed an inflammatory prone profile. Further studies focusing on telomeric maintenance may unveil mechanisms that are associated with cancer under long-term smoking.

The niche of One Health approaches in Lassa fever surveillance and control.

Lassa fever (LF), a zoonotic illness, represents a public health burden in West African countries where the Lassa virus (LASV) circulates among rodents. Human exposure hinges significantly on LASV ecology, which is in turn shaped by various parameters such as weather seasonality and even virus and rodent-host genetics. Furthermore, human behaviour, despite playing a key role in the zoonotic nature of the disease, critically affects either the spread or control of human-to-human transmission. Previous estimations on LF burden date from the 80s and it is unclear how the population expansion and the improvement on diagnostics and surveillance methods have affected such predictions. Although recent data have contributed to the awareness of epidemics, the real impact of LF in West African communities will only be possible with the intensification of interdisciplinary efforts in research and public health approaches. This review discusses the causes and consequences of LF from a One Health perspective, and how the application of this concept can improve the surveillance and control of this disease in West Africa.

Zoonotic disease preparedness in sub-Saharan African countries.

BACKGROUND: The emergence of high consequence pathogens such as Ebola and SARS-CoV-2, along with the continued burden of neglected diseases such as rabies, has highlighted the need for preparedness for emerging and endemic infectious diseases of zoonotic origin in sub-Saharan Africa (SSA) using a One Health approach. To identify trends in SSA preparedness, the World Health Organization (WHO) Joint External Evaluation (JEE) reports were analysed. JEEs are voluntary, collaborative processes to assess country's capacities to prevent, detect and rapidly respond to public health risks. This report aimed to analyse the JEE zoonotic disease preparedness data as a whole and identify strengths and weaknesses. METHODS: JEE zoonotic disease preparedness scores for 44 SSA countries who had completed JEEs were analysed. An overall zoonotic disease preparedness score was calculated as an average of the sum of all the SSA country zoonotic disease preparedness scores and compared to the overall mean JEE score. Zoonotic disease preparedness indicators were analysed and data were collated into regions to identify key areas of strength. RESULTS: The mean 'Zoonotic disease' preparedness score (2.35, range 1.00-4.00) was 7% higher compared to the mean overall JEE preparedness score (2.19, range 1.55-3.30), putting 'Zoonotic Diseases' 5th out of 19 JEE sub-areas for preparedness. The average scores for each 'Zoonotic Disease' category were 2.45 for 'Surveillance Systems', 2.76 for 'Veterinary Workforce' and 1.84 for 'Response Mechanisms'. The Southern African region scored highest across the 'Zoonotic disease' categories (2.87).A multisectoral priority zoonotic pathogens list is in place for 43% of SSA countries and 70% reported undertaking national surveillance on 1-5 zoonotic diseases. 70% of SSA countries reported having public health training courses in place for veterinarians and 30% had veterinarians in all districts (reported as sufficient staffing). A multisectoral action plan for zoonotic outbreaks was in place for 14% countries and 32% reported having an established inter-agency response team for zoonotic outbreaks. The zoonotic diseases that appeared most in reported country priority lists were rabies and Highly Pathogenic Avian Influenza (HPAI) (both 89%), anthrax (83%), and brucellosis (78%). CONCLUSIONS: With 'Zoonotic Diseases' ranking 5th in the JEE sub-areas and a mean SSA score 7% greater than the overall mean JEE score, zoonotic disease preparedness appears to have the attention of most SSA countries. However, the considerable range suggests that some countries have more measures in place than others, which may perhaps reflect the geography and types of pathogens that commonly occur. The category 'Response Mechanisms' had the lowest mean score across SSA, suggesting that implementing a multisectoral action plan and response team could provide the greatest gains.

Cost-Effective Trap qPCR Approach to Evaluate Telomerase Activity: an Important Tool for Aging, Cancer, and Chronic Disease Research.

OBJECTIVES: Telomeres are a terminal "DNA cap" that prevent chromosomal fusion and degradation. However, aging is inherent to life, and so is the loss of terminal sequences. Telomerase is a specialized reverse transcriptase encoded by self-splicing introns that counteract chromosome erosion. Telomerase activity is observed during early embryonic development, but after the blastocyst stage, the expression of telomerase reduces. The consequences of either insufficient or unrestrained telomerase activity underscore the importance of ongoing studies aimed at elucidating the regulation of telomerase activity in humans. In the present study, we aimed to standardize a simplified telomerase repeat-amplification protocol (TRAP) assay to detect telomerase activity in unstimulated and PHA-stimulated mononuclear cells. METHODS AND RESULTS: Our optimized qPCR-based can efficiently evaluate telomerase activity. Quantification of protein and DNA between unstimulated and PHA-stimulated peripheral blood mononuclear cells revealed cellular activation and cell-cycle entry. The assay also showed that relative telomerase activity is significantly different between these two conditions, supporting the applicability of the assay. Furthermore, our findings corroborated that telomerase activity decreases with age. CONCLUSIONS: Telomeres and telomerase are implicated in aging and development of chronic diseases and cancer; however, difficulty in accessing commercial kits to investigate these aspects is a critical constraint in health surveillance studies. Our optimized assay was successfully used to differentiate telomerase activity between unstimulated and stimulated cells, clearly showing the reactivation of telomerase upon cell activation. This assay is affordable, reproducible, and can be executed in resource-limited settings.

Cost-Effective Trap qPCR Approach to Evaluate Telomerase Activity: an Important Tool for Aging, Cancer, and Chronic Disease Research

OBJECTIVES: Telomeres are a terminal "DNA cap" that prevent chromosomal fusion and degradation. However, aging is inherent to life, and so is the loss of terminal sequences. Telomerase is a specialized reverse transcriptase encoded by self-splicing introns that counteract chromosome erosion. Telomerase activity is observed during early embryonic development, but after the blastocyst stage, the expression of telomerase reduces. The consequences of either insufficient or unrestrained telomerase activity underscore the importance of ongoing studies aimed at elucidating the regulation of telomerase activity in humans. In the present study, we aimed to standardize a simplified telomerase repeat-amplification protocol (TRAP) assay to detect telomerase activity in unstimulated and PHA-stimulated mononuclear cells. METHODS and RESULTS: Our optimized qPCR-based can efficiently evaluate telomerase activity. Quantification of protein and DNA between unstimulated and PHA-stimulated peripheral blood mononuclear cells revealed cellular activation and cell-cycle entry. The assay also showed that relative telomerase activity is significantly different between these two conditions, supporting the applicability of the assay. Furthermore, our findings corroborated that telomerase activity decreases with age. CONCLUSIONS: Telomeres and telomerase are implicated in aging and development of chronic diseases and cancer; however, difficulty in accessing commercial kits to investigate these aspects is a critical constraint in health surveillance studies. Our optimized assay was successfully used to differentiate telomerase activity between unstimulated and stimulated cells, clearly showing the reactivation of telomerase upon cell activation. This assay is affordable, reproducible, and can be executed in resource-limited settings.

COVID-19-Zoonosis or Emerging Infectious Disease?

The World Health Organization defines a zoonosis as any infection naturally transmissible from vertebrate animals to humans. The pandemic of Coronavirus disease (COVID-19) caused by SARS-CoV-2 has been classified as a zoonotic disease, however, no animal reservoir has yet been found, so this classification is premature. We propose that COVID-19 should instead be classified an "emerging infectious disease (EID) of probable animal origin." To explore if COVID-19 infection fits our proposed re-categorization vs. the contemporary definitions of zoonoses, we reviewed current evidence of infection origin and transmission routes of SARS-CoV-2 virus and described this in the context of known zoonoses, EIDs and "spill-over" events. Although the initial one hundred COVID-19 patients were presumably exposed to the virus at a seafood Market in China, and despite the fact that 33 of 585 swab samples collected from surfaces and cages in the market tested positive for SARS-CoV-2, no virus was isolated directly from animals and no animal reservoir was detected. Elsewhere, SARS-CoV-2 has been detected in animals including domesticated cats, dogs, and ferrets, as well as captive-managed mink, lions, tigers, deer, and mice confirming zooanthroponosis. Other than circumstantial evidence of zoonotic cases in mink farms in the Netherlands, no cases of natural transmission from wild or domesticated animals have been confirmed. More than 40 million human COVID-19 infections reported appear to be exclusively through human-human transmission. SARS-CoV-2 virus and COVID-19 do not meet the WHO definition of zoonoses. We suggest SARS-CoV-2 should be re-classified as an EID of probable animal origin.

Antimicrobial resistance preparedness in sub-Saharan African countries.

BACKGROUND: Antimicrobial resistance (AMR) is of growing concern globally and AMR status in sub-Saharan Africa (SSA) is undefined due to a lack of real-time data recording, surveillance and regulation. World Health Organization (WHO) Joint External Evaluation (JEE) reports are voluntary, collaborative processes to assess country capacities and preparedness to prevent, detect and rapidly respond to public health risks, including AMR. The data from SSA JEE reports were analysed to gain an overview of how SSA is working towards AMR preparedness and where strengths and weaknesses lie. METHODS: SSA country JEE AMR preparedness scores were analysed. A cumulative mean of all the SSA country AMR preparedness scores was calculated and compared to the overall mean SSA JEE score. AMR preparedness indicators were analysed, and data were weighted by region. FINDINGS: The mean SSA AMR preparedness score was 53% less than the overall mean SSA JEE score. East Africa had the highest percentage of countries reporting having AMR National Action Plans in place, as well as human and animal pathogen AMR surveillance programmes. Southern Africa reported the highest percentage of countries with training programmes and antimicrobial stewardship. CONCLUSIONS: The low mean AMR preparedness score compared to overall JEE score, along with the majority of countries lacking implemented National Action Plans, suggests that until now AMR has not been a priority for most SSA countries. By identifying regional and One Health strengths, AMR preparedness can be fortified across SSA with a multisectoral approach.

Bacteria arise at the border of mycoplasma-infected HeLa cells, containing cytoplasm with either malformed cytosol, mitochondria and endoplasmic reticulum or tightly adjoined smooth vacuoles.

A study with transmission electron microscopy of mycoplasma-contaminated HeLa cells using five cell donors referred to as donors A, B, C, D and E, observations are herein presented. Experiments performed with cells from donors B, C and D, revealed the presence of Mycoplasma hyorhinis after PCR and sequencing experiments. Bacteria probably originated from a cytoplasm with compacted tiny granular particles replacing the normal cytosol territories, or from the contact with the cytoplasm through a clear semi-solid material. The compact granularity (CG) of the cytoplasm was crossed by stripes of smooth and rough endoplasmic reticulum cisternae. Among apparently normal mitochondria, it was noted, in variable proportions, mitochondria with crista-delimited lucent central regions that expand to and occupied the interior of a crista-less organelle, which can undergo fission. Other components of the scenarios of mycoplasma-induced cell demolition are villus-like structures with associated 80-200 nm vesicles and a clear, flexible semi-solid, process-sensitive substance that we named jam-like material. This material coated the cytoplasmic surface, its recesses, irregular protrusions and detached cytoplasmic fragments. It also cushioned forming bacteria. Cyst-like structures were often present in the cytoplasm. Cells, mainly apoptotic, exhibiting ample cytoplasmic sectors with characteristic net-like profile due to adjoined vacuoles, as well as ovoid or elongated profiles, consistently appeared in all cells from the last four cell donors. These cells were named "modified host cells" because bacteria arose in the vacuoles. The possibility that, in some samples, there was infection and/or coinfection of the host cell by another organism(s) cannot be ruled out.

Bacteria arise at the border of mycoplasma-infected HeLa cells, containing cytoplasm with either malformed cytosol, mitochondria and endoplasmic reticulum or tightly adjoined smooth vacuoles

A study with transmission electron microscopy of mycoplasma-contaminated HeLa cells using five cell donors referred to as donors A, B, C, D and E, observations are herein presented. Experiments performed with cells from donors B, C and D, revealed the presence of Mycoplasma hyorhinis after PCR and sequencing experiments. Bacteria probably originated from a cytoplasm with compacted tiny granular particles replacing the normal cytosol territories, or from the contact with the cytoplasm through a clear semi-solid material. The compact granularity (CG) of the cytoplasm was crossed by stripes of smooth and rough endoplasmic reticulum cisternae. Among apparently normal mitochondria, it was noted, in variable proportions, mitochondria with crista-delimited lucent central regions that expand to and occupied the interior of a crista-less organelle, which can undergo fission. Other components of the scenarios of mycoplasma-induced cell demolition are villus-like structures with associated 80-200 nm vesicles and a clear, flexible semi-solid, process-sensitive substance that we named jam-like material. This material coated the cytoplasmic surface, its recesses, irregular protrusions and detached cytoplasmic fragments. It also cushioned forming bacteria. Cyst-like structures were often present in the cytoplasm. Cells, mainly apoptotic, exhibiting ample cytoplasmic sectors with characteristic net-like profile due to adjoined vacuoles, as well as ovoid or elongated profiles, consistently appeared in all cells from the last four cell donors. These cells were named "modified host cells" because bacteria arose in the vacuoles. The possibility that, in some samples, there was infection and/or coinfection of the host cell by another organism(s) cannot be ruled out.

Determination of viral tropism by genotyping and phenotyping assays in Brazilian HIV-1-infected patients.

The clinical application of CCR5 antagonists involves first determining the coreceptor usage by the infecting viral strain. Bioinformatics programs that predict coreceptor usage could provide an alternative method to screen candidates for treatment with CCR5 antagonists, particularly in countries with limited financial resources. Thus, the present study aims to identify the best approach using bioinformatics tools for determining HIV-1 coreceptor usage in clinical practice. Proviral DNA sequences and Trofile results from 99 HIV-1-infected subjects under clinical monitoring were analyzed in this study. Based on the Trofile results, the viral variants present were 81.1% R5, 21.4% R5X4 and 1.8% X4. Determination of tropism using a Geno2pheno[coreceptor] analysis with a false positive rate of 10% gave the most suitable performance in this sampling: the R5 and X4 strains were found at frequencies of 78.5% and 28.4%, respectively, and there was 78.6% concordance between the phenotypic and genotypic results. Further studies are needed to clarify how genetic diversity amongst virus strains affects bioinformatics-driven approaches for determining tropism. Although this strategy could be useful for screening patients in developing countries, some limitations remain that restrict the wider application of coreceptor usage tests in clinical practice.

Determination o viral tropism by genotyping an phenotyping assays in Braziliaan hiv-1-infected patients / Determinação do tropismo viral por ensaios genotípicos e fenotípicos em pacientes brasileiros infectados por HIV-1

The clinical application of CCR5 antagonists involves first determining the coreceptor usage by the infecting viral strain. Bioinformatics programs that predict coreceptor usage could provide an alternative method to screen candidates for treatment with CCR5 antagonists, particularly in countries with limited financial resources. Thus, the present study aims to identify the best approach using bioinformatics tools for determining HIV-1 coreceptor usage in clinical practice. Proviral DNA sequences and Trofile results from 99 HIV-1-infected subjects under clinical monitoring were analyzed in this study. Based on the Trofile results, the viral variants present were 81.1% R5, 21.4% R5X4 and 1.8% X4. Determination of tropism using a Geno2pheno[coreceptor] analysis with a false positive rate of 10% gave the most suitable performance in this sampling: the R5 and X4 strains were found at frequencies of 78.5% and 28.4%, respectively, and there was 78.6% concordance between the phenotypic and genotypic results. Further studies are needed to clarify how genetic diversity amongst virus strains affects bioinformatics-driven approaches for determining tropism. Although this strategy could be useful for screening patients in developing countries, some limitations remain that restrict the wider application of coreceptor usage tests in clinical practice.

A aplicação clínica dos antagonistas de CCR5 envolve em primeiro lugar determinar o uso de co-receptor pela cepa viral infectante. Programas de bioinformática que prevêem o uso co-receptor poderiam fornecer um método alternativo para selecionar candidatos para o tratamento com os antagonistas do CCR5, particularmente em países com poucos recursos financeiros. Assim, o presente estudo teve por objetivo identificar a melhor abordagem utilizando ferramentas de bioinformática para determinar qual o tipo de co-receptor do HIV-1 que poderia ser usado na prática clínica. Sequências de DNA proviral e Trofile resultados a partir de 99 pacientes infectados pelo HIV-1 sob monitorização clínica foram avaliadas. Com base nos resultados do Teste Trofile, as variantes virais presentes eram R5 (81,1%), R5X4 (21,4%) e X4 (1,8%). Determinação do tropismo pela análise do Geno2pheno, com taxa de falso positivos de 10% apresentou desempenho mais adequado para esta amostragem: as cepas R5 e X4 foram encontradas em frequências de 78,5% e 28,4%, respectivamente, e foi de 78,6% a concordância entre os resultados fenotípicos e genotípicos. Mais estudos são necessários para esclarecer como a diversidade genética entre as cepas do vírus afeta abordagens baseadas na determinação do tropismo pelas ferramentas de bioinformática. Embora esta estratégia possa ser útil para o rastreio de pacientes em países em desenvolvimento, permanecem algumas limitações que restringem a aplicação mais ampla para utilização de testes de co-receptor na prática clínica.

Testing a subtype-specific gp41 amplification method for genotyping individuals infected by human immunodeficiency virus type-1 in the Brazilian population of Itajaí, South Brazil.

The method used by YAGYU et al. for the subtype-specific polymerase chain reaction (PCR) amplification of the gp41 transmembrane region of the human immunodeficiency virus type-1 (HIV-1) env gene, was tested. HIV-1 proviral DNA from 100 infected individuals in Itajaí, South Brazil was used to analyze this method. Seventy individuals were determined according to this method as having PCR products at the expected size for subtypes B, C, D and F. Of these individuals, 26 (37.1%) were observed as having the expected amplification for subtype C, and 42 (60%) were observed as having the expected products for subtypes B and D. Of the subtype B and D amplicons, 16 (22.9%) were classified as subtype D, and 26 (37.1%) were classified as subtype B. Two individuals (2.9%) had amplicons that were observed after subtype F-specific amplification was performed. Sequencing and comparing the patient sequences to reference sequences confirmed the classification of sequences of subtypes C and B. However, sequences that were falsely determined as being D and F in the PCR assay were determined as being subtypes C and B, respectively, by sequence analysis. For those individuals from whom no amplified products were obtained, a low viral load that was indicated in their patient history may explain the difficulty in subtyping by PCR methods. This issue was demonstrated by the results of ANOVA when testing the effect of viral load on the success of PCR amplification. The alignment of the obtained sequences with HIV-1 reference sequences demonstrated that there is high intra-subtype diversity. This indicates that the subtype-specific primer binding sites were not conserved or representative of the subtypes that are observed in the Brazilian populations, and that they did not allow the correct classification of HIV-1 subtypes. Therefore, the proposed method by YAGYU et al. is not applicable for the classification of Brazilian HIV-1 subtypes.