Production of antigens expressed in Nicotiana benthamiana plant and Escherichia coli for the SARS-CoV-2 IgG antibody detection by ELISA.

The recent COVID-19 pandemic disclosed a critical shortage of diagnostic kits worldwide, emphasizing the urgency of utilizing all resources available for the development and production of diagnostic tests. Different heterologous protein expression systems can be employed for antigen production. This study assessed novel SARS-CoV-2 proteins produced by a transient expression system in Nicotiana benthamiana utilizing an infectious clone vector based on pepper ringspot virus (PepRSV). These proteins included the truncated S1-N protein (spike protein N-terminus residues 12-316) and antigen N (nucleocapsid residues 37-402). Two other distinct SARS-CoV-2 antigens expressed in Escherichia coli were evaluated: QCoV9 chimeric antigen protein (spike protein residues 449-711 and nucleocapsid protein residues 160-406) and QCoV7 truncated antigen (nucleocapsid residues 37-402). ELISAs using the four antigens individually and the same panel of samples were performed for the detection of anti-SARS-CoV-2 IgG antibodies. Sensitivity was evaluated using 816 samples from 351 COVID-19 patients hospitalized between 5 and 65 days after symptoms onset; specificity was tested using 195 samples collected before 2018, from domiciliary contacts of leprosy patients. Our findings demonstrated consistent test sensitivity, ranging from 85 % to 88 % with specificity of 97.5 %, regardless of the SARS-CoV2 antigen and the expression system used for production. Our results highlight the potential of plant expression systems as useful alternative platforms to produce recombinant antigens and for the development of diagnostic tests, particularly in resource-constrained settings.

Late relapses in leprosy patients in Brazil: 10-year post-trial of uniform multidrug therapy (U-MDT/CT-BR).

BACKGROUND: Leprosy is a neglected dermato-neurologic, infectious disease caused by Mycobacterium leprae or M. lepromatosis. Leprosy is treatable and curable by multidrug therapy/MDT, consisting of 12 months rifampicin, dapsone and clofazimine for multibacillary/MB patients and for 6 months for paucibacillary/PB patients. The relapse rate is considered a crucial treatment outcome. A randomized Controlled Clinical Trial (U-MDT/CT-BR) conducted from 2007‒2012 compared clinical outcomes in MB patients after 12 months regular MDT/R-MDT and 6 months uniform MDT/U-MDT in two highly endemic Brazilian areas. OBJECTIVES: To estimate the 10 years relapse rate of MB patients treated with 6 months U-MDT. METHODS: The statistical analyses treated the data as a case-control study, sampled from the cohort generated for the randomized trial. Analyses estimated univariate odds ratio and applied logistic regression for multivariate analysis, controlling the confounding variables. RESULTS: The overall relapse rate was 4.08 %: 4.95 % (16 out of 323) in the U-MDT group and 3.10 % (9 out of 290) in the regular/R-MDT group. The difference in relapse proportion between U-MDT and R-MDT groups was 1.85 %, not statistically significant (Odds Ratio = 1.63, 95 % CI 0.71 to 3.74). However, misdiagnosis of relapses, may have introduced bias, underestimating the force of the association represented by the odds ratio. CONCLUSIONS: The relapse estimate of 10 years follow-up study of the first randomized, controlled study on U-MDT/CT-BR was similar to the R-MDT group, supporting strong evidence that 6 months U-MDT for MB patients is an acceptable option to be adopted by leprosy endemic countries worldwide. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00669643.

Development and evaluation of a Lateral flow immunoassay (LFIA) prototype for the detection of IgG anti-SARS-CoV-2 antibodies.

Lateral flow immunoassays (LFIA) for antibody detection represent cost-effective and user-friendly tools for serology assessment. This study evaluated a new LFIA prototype developed with a recombinant chimeric antigen from the spike/S and nucleocapsid/N proteins to detect anti-SARS-CoV-2 IgG antibodies. The evaluation of LFIA sensitivity and specificity used 811 serum samples from 349 hospitalized, SARS-CoV-2 RT-qPCR positive COVID-19 patients, collected at different time points and 193 serum samples from healthy controls. The agreement between ELISA results with the S/N chimeric antigen and LFIA results was calculated. The LFIA prototype for SARS-CoV-2 using the chimeric S/N protein demonstrated 85 % sensitivity on the first week post symptoms onset, reaching 94 % in samples collected at the fourth week of disease. The agreement between LFIA and ELISA with the same antigen was 92.7 %, 0.827 kappa Cohen value (95 % CI [0.765-0.889]). Further improvements are needed to standardize the prototype for whole blood use. The inclusion of the novel chimeric S + N antigen in the COVID-19 IgG antibody LFIA demonstrated optimal agreement with results from a comparable ELISA, highlighting the prototype's potential for accurate large-scale serologic assessments in the field in a rapid and user-friendly format.

Challenges and advances in serological and molecular tests to aid leprosy diagnosis.

Leprosy is a neglected chronic infectious disease caused by obligate intracellular bacilli, Mycobacterium leprae and Mycobacterium lepromatosis. Despite multidrug therapy (MDT) success, leprosy accounts for more than 200,000 new cases yearly. Leprosy diagnosis remains based on the dermato-neurologic examination, but histopathology of skin biopsy and bacilloscopy of intradermal scraping are subsidiary diagnostic tests that require expertise and laboratory infrastructure. This minireview summarizes the state of the art of serologic tests to aid leprosy diagnosis, highlighting enzyme-linked immunosorbent assay (ELISA) and point-of-care tests (POCT) biotechnologies. Also, the impact of the postgenomic era on the description of new recombinantly expressed M. leprae-specific protein antigens, such as leprosy Infectious Disease Research Institute (IDRI) diagnostic (LID)-1 is summarized. Highly specific and sensitive molecular techniques to detect M. leprae DNA as the quantitative polymerase chain reaction (qPCR) and the loop-mediated isothermal amplification (LAMP) are briefly reviewed. Serology studies using phenolic glycolipid-I (PGL-I) semi-synthetic antigens, LID-1 fusion antigen, and the single fusion complex natural disaccharide-octyl (NDO)-LID show high sensitivity in multibacillary (MB) patients. However, serology is not applicable to paucibacillary patients, as they have weak humoral response and robust cell-mediated response, requiring tests for cellular biomarkers. Unlike ELISA-based tests, leprosy-specific POCT based on semi-synthetic PGL-I antigens and NDO-LID 1 antigen is easy to perform, cheaper, equipment-free, and can contribute to early diagnosis avoiding permanent incapacities and helping to interrupt M. leprae transmission. Besides its use to help diagnosis of household contacts or at-risk populations in endemic areas, potential applications of leprosy serology include monitoring MDT efficacy, identification of recent infection, especially in young children, as surrogate markers of disease progression to orient adult chemoprophylaxis and as a predictor of type 2 leprosy reactions. Advances in molecular biology techniques have reduced the complexity and execution time of qPCR confirming its utility to help diagnosis while leprosy-specific LAMP holds promise as an adjunct test to detect M. leprae DNA.

Prediction of the occurrence of leprosy reactions based on Bayesian networks.

Introduction: Leprosy reactions (LR) are severe episodes of intense activation of the host inflammatory response of uncertain etiology, today the leading cause of permanent nerve damage in leprosy patients. Several genetic and non-genetic risk factors for LR have been described; however, there are limited attempts to combine this information to estimate the risk of a leprosy patient developing LR. Here we present an artificial intelligence (AI)-based system that can assess LR risk using clinical, demographic, and genetic data. Methods: The study includes four datasets from different regions of Brazil, totalizing 1,450 leprosy patients followed prospectively for at least 2 years to assess the occurrence of LR. Data mining using WEKA software was performed following a two-step protocol to select the variables included in the AI system, based on Bayesian Networks, and developed using the NETICA software. Results: Analysis of the complete database resulted in a system able to estimate LR risk with 82.7% accuracy, 79.3% sensitivity, and 86.2% specificity. When using only databases for which host genetic information associated with LR was included, the performance increased to 87.7% accuracy, 85.7% sensitivity, and 89.4% specificity. Conclusion: We produced an easy-to-use, online, free-access system that identifies leprosy patients at risk of developing LR. Risk assessment of LR for individual patients may detect candidates for close monitoring, with a potentially positive impact on the prevention of permanent disabilities, the quality of life of the patients, and upon leprosy control programs.

Combined antibodies against internalins A and B proteins have potential application in immunoassay for detection of Listeria monocytogenes.

Listeria monocytogenes is a food-borne bacterium that causes listeriosis upon the ingestion of contaminated food. Traditional methods to detect L. monocytogenes require pre-enrichment broths to increase its concentration. To improve the screening of contaminated food and prevent listeriosis outbreaks, rapid, specific and sensitive assays are needed to detect L. monocytogenes. This study developed a prototype lateral flow immunochromatographic assay (LFIA) employing antibodies against L. monocytogenes Internalin A (InlA) and Internalin B (InlB) proteins, that are involved in non-phagocytic cell invasion. The following antibodies were used to capture L. monocytogenes antigenic targets: mouse anti-Internalin A monoclonal antibody (MAb-2D12) conjugated to colloidal gold nanoparticles and a mouse anti-Internalin B polyclonal antibody. This test was able to detect pure L. monocytogenes from culture with a limit of detection (LOD) ranging from 5.9 × 103 to 1.5 × 104 CFU/mL. In milk artificially contaminated with L. monocytogenes, the LOD was 1 × 105 CFU/mL. This prototype test discriminated L. monocytogenes from other bacterial species (Listeria innocua, Enterobacter cloacae, Bacillus cereus). Results indicate that this LFIA developed using antibodies against L. monocytogenes InlA and InlB proteins is a sensitive and specific tool that can be potentially useful to rapidly detect L. monocytogenes in contaminated food. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05597-9.

HIV-1 genetic diversity and drug resistance mutations in the northern Brazilian region.

Brazil is a huge continental country with striking geographic differences which are well illustrated in the HIV/AIDS epidemic. Contrasting with the significant decline in the national AIDS detection rate in the last decade, a linear growth has been reported in the Northern region. Despite its public health and epidemiologic importance, there is scarce HIV-1 molecular data from Northern Brazil. This scoping review summarizes recent epidemiologic data with special emphasis on HIV-1 genetic diversity and antiretroviral drug resistance mutations in patients from the seven Northern states of Brazil. Studies from the Northern Brazil on different HIV-1 genomic regions, mostly pol (protease/reverse transcriptase) sequences of naïve/antiretroviral treated adults/children were retrieved from PubMed/MEDLINE electronic database. These studies indicate a consistent molecular profile largely dominated by HIV-1 subtype B with minor contribution of subtypes F1 and C and infrequent detection of other subtypes (A1, D, K), recombinants (BF1, BC), circulating recombinant forms (CRF) as the new CRF90_BF1 and CRF02_AG-like, CRF28-29_BF-like, CRF31_BC-like, and a potential new CRF_BF1. This pattern indicates a founder effect of subtype B and the introduction of non-B-subtypes and recombinants probably generated in the Southern/Southeastern regions. In naïve populations transmitted drug resistance (TDR) can impact the outcome of first-line antiretroviral treatment and prophylactic/preventive regimens. In the Northern region TDR rates are moderate while patients failing highly active antiretroviral therapy (HAART) showed high prevalence of acquired drug resistance mutations. The limited HIV-1 molecular data from Northern Brazil reflects the great challenges to generate comprehensive scientific data in isolated, underprivileged areas. It also highlights the need to invest in local capacity building which supported by adequate infrastructure and funding can promote robust research activities to help reduce the scientific asymmetries in the Northern region. Currently the impacts of the overwhelming COVID-19 pandemic on the expanding HIV/AIDS epidemic in Northern Brazil deserves to be closely monitored.

Human immunodeficiency virus infection and syphilis among homeless people in a large city of Central-Western Brazil: prevalence, risk factors, human immunodeficiency virus-1 genetic diversity, and drug resistance mutations

ABSTRACT Homeless people are at high risk for sexually transmitted infections (STIs), such as human immunodeficiency virus (HIV) infection and syphilis. We investigated the epidemiology of HIV-1 infection and syphilis among homeless individuals in a large city in Central-Western Brazil. In this cross-sectional study, we interviewed and tested 355 individuals from September 2014 to August 2015. Rapid test samples positive for syphilis were retested using the Venereal Disease Research Laboratory (VDRL) test. Blood samples from HIV-infected participants were collected for POL sequencing using HIV-1 RNA extracted from plasma, reverse transcription, and nested polymerase chain reaction. Anti-HIV-1-positive samples were subtyped by sequencing the nucleotides of HIV-1 protease and part of the HIV-1 reverse transcriptase genes. Transmitted and acquired drug resistance mutations and susceptibility to antiretroviral drugs were also analyzed. Anti-HIV was positive in 14 patients (3.9%; 95% confidence interval [CI]: 2.3-6.4). HIV-1 RNA was detected in 8 of the 14 samples. Two of the eight (25%) isolates showed HIV-1 drug resistance mutations. Furthermore, 78 (22%; 95% CI: 17.9-26.5) and 29 (8.2%; 95% CI: 5.6-11.4) homeless individuals tested positive for syphilis using the rapid test and VDRL test, respectively. Two individuals were anti-HIV-1 and VDRL test positive. Daily alcohol use (adjusted odds ratio [AOR]: 3.2, 95% CI: 1.0-10.4), sex with people living with HIV (PLWH) infection (AOR: 6.8, 95% CI: 1.9-25.0), and sex with people of the same sex (AOR: 5.4, 95% CI: 1.7-17.5) were predictors of HIV infection. Age ≤35 years (AOR: 3.8, 95% CI: 1.4-10.8), previous syphilis testing (AOR: 3.5, 95% CI: 1.4-8.4), history of genital lesions (AOR: 4.9, 95% CI: 1.3-19.1), and crack use in the last six months (AOR: 3.1, 95% CI: 1.3-7.6) were predictors of syphilis. Our findings highlight the importance of STI prevention and control strategies among the homeless.

Human immunodeficiency virus infection and syphilis among homeless people in a large city of Central-Western Brazil: prevalence, risk factors, human immunodeficiency virus-1 genetic diversity, and drug resistance mutations.

Homeless people are at high risk for sexually transmitted infections (STIs), such as human immunodeficiency virus (HIV) infection and syphilis. We investigated the epidemiology of HIV-1 infection and syphilis among homeless individuals in a large city in Central-Western Brazil. In this cross-sectional study, we interviewed and tested 355 individuals from September 2014 to August 2015. Rapid test samples positive for syphilis were retested using the Venereal Disease Research Laboratory (VDRL) test. Blood samples from HIV-infected participants were collected for POL sequencing using HIV-1 RNA extracted from plasma, reverse transcription, and nested polymerase chain reaction. Anti-HIV-1-positive samples were subtyped by sequencing the nucleotides of HIV-1 protease and part of the HIV-1 reverse transcriptase genes. Transmitted and acquired drug resistance mutations and susceptibility to antiretroviral drugs were also analyzed. Anti-HIV was positive in 14 patients (3.9%; 95% confidence interval [CI]: 2.3-6.4). HIV-1 RNA was detected in 8 of the 14 samples. Two of the eight (25%) isolates showed HIV-1 drug resistance mutations. Furthermore, 78 (22%; 95% CI: 17.9-26.5) and 29 (8.2%; 95% CI: 5.6-11.4) homeless individuals tested positive for syphilis using the rapid test and VDRL test, respectively. Two individuals were anti-HIV-1 and VDRL test positive. Daily alcohol use (adjusted odds ratio [AOR]: 3.2, 95% CI: 1.0-10.4), sex with people living with HIV (PLWH) infection (AOR: 6.8, 95% CI: 1.9-25.0), and sex with people of the same sex (AOR: 5.4, 95% CI: 1.7-17.5) were predictors of HIV infection. Age ≤35 years (AOR: 3.8, 95% CI: 1.4-10.8), previous syphilis testing (AOR: 3.5, 95% CI: 1.4-8.4), history of genital lesions (AOR: 4.9, 95% CI: 1.3-19.1), and crack use in the last six months (AOR: 3.1, 95% CI: 1.3-7.6) were predictors of syphilis. Our findings highlight the importance of STI prevention and control strategies among the homeless.

HIV-1 genetic diversity and drug resistance mutations in the northern Brazilian region

ABSTRACT Brazil is a huge continental country with striking geographic differences which are well illustrated in the HIV/AIDS epidemic. Contrasting with the significant decline in the national AIDS detection rate in the last decade, a linear growth has been reported in the Northern region. Despite its public health and epidemiologic importance, there is scarce HIV-1 molecular data from Northern Brazil. This scoping review summarizes recent epidemiologic data with special emphasis on HIV-1 genetic diversity and antiretroviral drug resistance mutations in patients from the seven Northern states of Brazil. Studies from the Northern Brazil on different HIV-1 genomic regions, mostly pol (protease/reverse transcriptase) sequences of naïve/antiretroviral treated adults/children were retrieved from PubMed/MEDLINE electronic database. These studies indicate a consistent molecular profile largely dominated by HIV-1 subtype B with minor contribution of subtypes F1 and C and infrequent detection of other subtypes (A1, D, K), recombinants (BF1, BC), circulating recombinant forms (CRF) as the new CRF90_BF1 and CRF02_AG-like, CRF28-29_BF-like, CRF31_BC-like, and a potential new CRF_BF1. This pattern indicates a founder effect of subtype B and the introduction of non-B-subtypes and recombinants probably generated in the Southern/Southeastern regions. In naïve populations transmitted drug resistance (TDR) can impact the outcome of first-line antiretroviral treatment and prophylactic/preventive regimens. In the Northern region TDR rates are moderate while patients failing highly active antiretroviral therapy (HAART) showed high prevalence of acquired drug resistance mutations. The limited HIV-1 molecular data from Northern Brazil reflects the great challenges to generate comprehensive scientific data in isolated, underprivileged areas. It also highlights the need to invest in local capacity building which supported by adequate infrastructure and funding can promote robust research activities to help reduce the scientific asymmetries in the Northern region. Currently the impacts of the overwhelming COVID-19 pandemic on the expanding HIV/AIDS epidemic in Northern Brazil deserves to be closely monitored.

Epidemiological and Molecular Characteristics of HIV-1 Infection in a Sample of Men Who Have Sex With Men in Brazil: Phylogeography of Major Subtype B and F1 Transmission Clusters.

This study describes human immunodeficiency virus 1 (HIV-1) prevalence, associated factors, viral genetic diversity, transmitted drug resistance (TDR), and acquired drug resistance mutations (DRM) among a population of 522 men who have sex with men (MSM) recruited by the respondent-driven sampling (RDS) method, in Goiânia city, the capital of the State of Goiás, Central-Western Brazil. All serum samples were tested using a four-generation enzyme-linked immunosorbent assay (ELISA), and reactive samples were confirmed by immunoblotting. Plasma RNA or proviral DNA was extracted, and partial polymerase (pol) gene including the protease/reverse transcriptase (PR/RT) region was amplified and sequenced. HIV-1 subtypes were identified by phylogenetic inference and by bootscan analysis. The time and location of the ancestral strains that originated the transmission clusters were estimated by a Bayesian phylogeographic approach. TDR and DRM were identified using the Stanford databases. Overall, HIV-1 prevalence was 17.6% (95% CI: 12.6-23.5). Self-declared black skin color, receptive anal intercourse, sex with drug user partner, and history of sexually transmitted infections were factors associated with HIV-1 infection. Of 105 HIV-1-positive samples, 78 (74.3%) were sequenced and subtyped as B (65.4%), F1 (20.5%), C (3.8%), and BF1 (10.3%). Most HIV-1 subtype B sequences (67%; 34 out of 51) branched within 12 monophyletic clusters of variable sizes, which probably arose in the State of Goiás between the 1980s and 2010s. Most subtype F1 sequences (n = 14, 88%) branched in a single monophyletic cluster that probably arose in Goiás around the late 1990s. Among 78 samples sequenced, three were from patients under antiretroviral therapy (ART); two presented DRM. Among 75 ART-naïve patients, TDR was identified in 13 (17.3%; CI 95%: 9.6-27.8). Resistance mutations to non-nucleoside reverse transcriptase inhibitors (NNRTI) predominated (14.7%), followed by nucleoside reverse transcriptase inhibitor (NRTI) mutations (5.3%) and protease inhibitor (PI) mutations (1.3%). This study shows a high prevalence of HIV-1 associated with sexual risk behaviors, high rate of TDR, and high genetic diversity driven by the local expansion of different subtype B and F1 strains. These findings can contribute to the understanding about the dissemination and epidemiological and molecular characteristics of HIV-1 among the population of MSM living away from the epicenter of epidemics in Brazil.

Leprosy surveillance study in a highly endemic Brazilian area using leprosy specific serologic tests and IFNγ whole blood assay.

This surveillance study evaluated leprosy-serologic tests and the IFNγ whole-blood-assay/WBA as adjunct diagnostic tools. Previously diagnosed leprosy index cases, intradomiciliary, peridomiciliary contacts from a Brazilian endemic area were enrolled during domiciliary visits. Physical evaluation was performed by trained nurses and leprosy diagnosis confirmed by expert dermatologist. ELISA detected IgM anti-PGL-I, IgG anti-LID-1, and IgM/IgG anti-ND-O-LID antibodies. Heparinized WBA plasma stimulated with LID-1, 46f + LID-1, ML0276 + LID-1 (24 h, 37 °C, 5% CO2) was tested for human IFNγ (QuantiFERON®-TB Gold/QFT-G; Qiagen). The survey included 1731 participants: 44 leprosy index cases, 64 intradomiciliary, 1623 peridomiciliary contacts. Women represented 57.7%, median age was 32 years, 72.2% had BCG scar. Leprosy prevalence was higher in intradomiciliary (8.57%) versus peridomiciliary contacts (0.67%), p < 0.001. Among 23 suspects, five leprosy cases were confirmed: 4 multibacillary/MB and 1 paucibacillary/PB. Leprosy incidence was 0.30%: 1.56% in intradomiciliary versus 0.25% in peridomiciliary (p = 0.028). Seropositivity rates were 1.9% to PGL-I, 4.9% to LID-1, and 1.0% to ND-O-LID. LID-1 positivity was higher in all groups; incident cases were LID-1 seropositive. ND-O-LID positivity was higher in intra- versus peridomiciliary contacts (p = 0.022). IFNγ WBA (40 index cases, 19 suspects, 35 intradomiciliary, 74 peridomiciliary contacts) showed higher LID-1/WBA positivity in peridomiciliary contacts (p > 0.05); significant differences among groups were seen with 46f + LID-1 but 0276 + LID-1 induced higher IFNγ levels. Incident cases were LID-1 seropositive, while IFNγ-WBA had marginal diagnostic application. As seropositivity indicates exposed individuals at higher risk of disease development, the utility of serologic screening for surveillance and prophylactic measures remains to be demonstrated.

Homogenous HIV-1 subtype B from the Brazilian Amazon with infrequent diverse BF1 recombinants, subtypes F1 and C among blood donors.

In the last decade a growing HIV/AIDS epidemic with increased incidence and AIDS-related mortality has been reported in Northern Brazil from which molecular data are scarce. Also, apparently healthy, adult blood donors, recently diagnosed with HIV-1 represent important sentinel populations for molecular studies. This cross-sectional study describes HIV-1 subtypes in blood donors from three reference public blood centers located in three States in Northern Brazil. HIV-1 pol sequencing (protease/PR, reverse transcriptase/RT) was performed on plasma samples of HIV-1 positive donors from HEMOAM, Manaus, Amazonas (n = 198), HEMERON, Porto Velho, Rondônia (n = 20) and HEMORAIMA, Boa Vista, Roraima (n = 9) collected from 2011-2017. HIV-1 subtypes were identified by REGA, phylogenetic inference; recombinant viruses were characterized by SIMPLOT. Young, single, males predominated, around half was first-time donors. Syphilis co-infection was detected in 17% (39 out of 227), 8% (18 out of 227) was anti-HBc positive. Subtype B represented ≥ 90% in Amazonas, Rondônia and Roraima, subtype C (3.1%) was found in Amazonas and Rondônia; subtype F1 (0.9%) and BF1 recombinants (5.3%) were only detected in Amazonas. Subtype B sequences from Amazonas (n = 179), Rondônia (n = 18) and Roraima (n = 9) were combined with viral strains representative of the BPANDEMIC (n = 300) and BCARIBBEAN/BCAR (n = 200) lineages. The BPANDEMIC lineage predominated (78%) although BCAR lineages were frequent in Roraima (56%) and Amazonas (22%). Subtype C and subtype F1 sequences identified here clustered within Brazilian CBR and F1BR lineages, respectively. Twelve BF1 mosaics showed 11 different recombination profiles: six were singleton unique-recombinant-forms/URFs, one displays a CRF28/29_BF-like recombinant pattern and the remaining four BF1 isolates branched with other Brazilian BF1 viruses previously described and may represent putative new CRF_BF1 from Northern Brazil. Our study shows a highly homogeneous molecular pattern with prevalent subtype B, followed by BF1, and sporadic subtype C and F1 in blood donors from the Northern region. Surveillance studies are important to monitor HIV-1 diversity which can reveal patterns of viral dissemination, especially in a highly endemic, remote and geographically isolated region as Northern Brazil.

Detection of human immunodeficiency virus Type 1 phylogenetic clusters with multidrug resistance mutations among 2011 to 2017 blood donors from the highly endemic Northern Brazilian Amazon.

BACKGROUND: This study describes transmitted drug resistance (TDR) in blood donors diagnosed with human immunodeficiency virus Type 1 (HIV-1) infection from 2011 to 2017 in three reference public blood centers from the Northern Brazilian Amazon. STUDY DESIGN AND METHODS: This was a cross-sectional study on HIV-positive blood donors from HEMOAM, Manaus, Amazonas, AM (n = 198); HEMERON, Porto Velho, Rondônia, RO (n = 20); and HEMORAIMA, Boa Vista, Roraima, RR (n = 9). HIV-1 pol sequences (protease, reverse transcriptase) were analyzed for drug resistance mutations (DRMs) using the Calibrated Population Resistance tool (Stanford). TDR/DRM clusters were investigated by phylogenetic analysis after removing positions associated with drug resistance of Subtype B sequences from untreated and treated subjects from Northern Brazil. RESULTS: Transmitted drug resistance/DRM in blood donors was 11% (25 of 227), all of them from HEMOAM. Most blood donors with TDR/DRM had multiple and similar DRMs. Nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations predominated (10.1%), followed by nucleoside reverse transcriptase inhibitor (NRTI) mutations (5.3%) and protease inhibitor mutations (0.4%). Dual-class NNRTI/NRTI mutations represented 4.8%. Three highly supported Subtype B monophyletic clades mostly composed by individuals from Amazonas with TDR/DRM mutations were identified. The largest transmission cluster contained 10 sequences, eight from HEMOAM and two sequences described previously (one from a treated subject from Amazonas and the other one from Roraima). This cluster was characterized by NRTI (D67N, T69D, T215S/F/L, K219Q) and NNRTI (K101H, K103 N, G190A) mutations. The other two transmission clades comprised only three and two sequences from HEMOAM sharing the E138A NNRTI mutation. CONCLUSIONS: The identification of transmission clusters of multidrug-resistant viruses in blood donors from Amazonas highlight the need of continued monitoring of TDR/DRM and the importance of pretreatment genotyping in the highly endemic Amazonas state.

Leprosy survey among rural communities and wild armadillos from Amazonas state, Northern Brazil.

There is evidence that in southern US, leprosy is a zoonosis infecting wild Dasypus novemcinctus armadillos but the extent of this finding is unknown. This ecological study investigated leprosy in rural communities and in wild armadillos from the Brazilian Amazon. The study area was the Mamiá Lake of Coari municipality, Amazonas State, Northern region, a hyper endemic leprosy area where residents live on subsistence farming, fishing and armadillo hunting and its meat intake are frequent. The leprosy survey was conducted in sixteen communities by a visiting team of specialists. Local partakers provided wild armadillos to investigate M. leprae infection. Volunteers had complete dermato-neurological examination by a dermatologist with expertise in leprosy diagnosis, suspect skin lesions were biopsied for histopathology (Hematoxylin-eosin/HE, Fite-Faraco/FF staining); slit skin smears were collected. Armadillos' tissue fragments (skins, spleens, livers, lymph nodes, adrenal glands, others) were prepared for histopathology (HE/FF) and for M. leprae repetitive element-RLEP-qPCR. Among 176 volunteers, six new indeterminate leprosy cases were identified (incidence = 3.4%). Suspect skin sections and slit skin smears were negative for bacilli. Twelve wild D. novemcinctus were investigated (48 specimens/96 slides) and histopathological features of M. leprae infection were not found, except for one skin presenting unspecific inflammatory infiltrate suggestive of indeterminate leprosy. Possible traumatic neuroma, granuloma with epithelioid and Langhans cells, foreign-body granuloma were also identified. Granulomatous/non-granulomatous dermatitides were periodic-acid-Schiff/PAS negative for fungus. M. leprae-RLEP-qPCR was negative in all armadillos' tissues; no bacillus was found in histopathology. Our survey in rural communities confirmed the high endemicity for leprosy while one armadillo was compatible with paucibacillary M. leprae infection. At least in the highly endemic rural area of Coari, in the Brazilian Amazon region where infectious sources from untreated multibacillary leprosy are abundant, M. leprae infected armadillos may not represent a major source of infection nor a significant public health concern.

Leprosy survey among rural communities and wild armadillos from Amazonas state, Northern Brazil

There is evidence that in southern US, leprosy is a zoonosis infecting wild Dasypus novemcinctus armadillos but the extent of this finding is unknown. This ecological study investigated leprosy in rural communities and in wild armadillos from the Brazilian Amazon. The study area was the Mamia´ Lake of Coari municipality, Amazonas State, Northern region, a hyper endemic leprosy area where residents live on subsistence farming, fishing and armadillo hunting and its meat intake are frequent. The leprosy survey was conducted in sixteen communities by a visiting team of specialists. Local partakers provided wild armadillos to investigate M. leprae infection. Volunteers had complete dermato-neurological examination by a dermatologist with expertise in leprosy diagnosis, suspect skin lesions were biopsied for histopathology (Hematoxylin-eosin/HE, Fite-Faraco/FF staining); slit skin smears were collected. Armadillos' tissue fragments (skins, spleens, livers, lymph nodes, adrenal glands, others) were prepared for histopathology (HE/FF) and for M. leprae repetitive elementRLEP-qPCR. Among 176 volunteers, six new indeterminate leprosy cases were identified (incidence = 3.4%). Suspect skin sections and slit skin smears were negative for bacilli. Twelve wild D. novemcinctus were investigated (48 specimens/96 slides) and histopathological features of M. leprae infection were not found, except for one skin presenting unspecific inflammatory infiltrate suggestive of indeterminate leprosy. Possible traumatic neuroma, granuloma with epithelioid and Langhans cells, foreign-body granuloma were also identified. Granulomatous/non-granulomatous dermatitides were periodic-acid-Schiff/ PAS negative for fungus. M. leprae-RLEP-qPCR was negative in all armadillos' tissues; no bacillus was found in histopathology. Our survey in rural communities confirmed the high endemicity for leprosy while one armadillo was compatible with paucibacillary M. leprae infection. At least in the highly endemic rural area of Coari, in the Brazilian Amazon region where infectious sources from untreated multibacillary leprosy are abundant, M. leprae infected armadillos may not represent a major source of infection nor a significant public health concern.

HIV-1 infection among crack cocaine users in a region far from the epicenter of the HIV epidemic in Brazil: Prevalence and molecular characteristics.

Brazil has the largest cocaine market in South America, and crack cocaine use is closely associated with HIV-1 infection. This study investigated the prevalence, risk factors, and HIV-1 subtypes, including recombinant forms and mutations associated with drug resistance, among crack cocaine users in Central-West Brazil. We recruited 600 crack cocaine users admitted to a referral hospital in Goiânia for psychiatric disorders. The participants were interviewed; blood samples were collected for anti-HIV-1/2 serological screening. HIV-1 pol gene sequences (entire protease [PR] and partial reverse transcriptase [RT]) were obtained from plasma RNA. HIV-1 subtypes, recombinant viruses, transmitted drug resistance (TDR), and secondary drug resistance mutations were investigated. The median participant age was 30 years (range, 18-68 years); most were male, single, unemployed, and of mixed races. Among them, 2.8% (17/600) were HIV-1 positive: 2.2% of men (11/507) and 6.5% of women (6/93). The main predictors of HIV-1 seropositivity were a sexual partner with HIV infection, irregular condom use, and previous homelessness. HIV-1 pol sequences (12/17) indicated the predominance of subtype B (n = 7), followed by recombinant forms FPR/BRT (n = 1) and BPR/FRT (n = 2) and subtypes F1 (n = 1) and C (n = 1). TDR prevalence was 58.3% (7/12). Isolates from two participants showed mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTI) only (M41L, T125C, T125F, M184V), while an isolate from one patient who had received antiretroviral therapy (ART) since 2008 had a mutation associated with resistance to non-NRTI (G190S). Five isolates had secondary mutations to protease inhibitors (K20M, L10V, L33I, A71T, A71V). In conclusion, the findings of HIV-1 circulation, TDR to NRTI, and secondary mutations to protease inhibitors in ART-naïve crack cocaine users support the importance of monitoring this population in regions far from the epicenter of the HIV epidemic.

Clinical trial for uniform multidrug therapy for leprosy patients in Brazil (U-MDT/CT-BR): adverse effects approach.

BACKGROUND: The Clinical Trial for Uniform Multidrug Therapy for Leprosy Patients in Brazil (U-MDT/CT-BR), designed to evaluate the effectiveness of a six-months regimen, assessed the adverse effects caused by the drugs. OBJECTIVE: Describe adverse effects due to MDT in U-MDT/CT-BR, comparing the uniform regimen (U-MDT) to the current WHO regimen (R-MDT). PATIENTS AND METHODS: After operational classification, patients were randomly allocated to the study groups. U-MDT PB and U-MDT MB groups, received the U-MDT regimen, six doses of MB-MDT (rifampicin, dapsone and clofazimine). R-MDT PB and R-MDT MB groups, received the WHO regimens: six doses (rifampicin and dapsone) for PB and 12 doses (rifampicin, dapsone and clofazimine) for MB. During treatment, patients returned monthly for clinical and laboratorial evaluation. Patients with single lesion were not included in this trial. RESULTS: Skin pigmentation (21.7%) and xerosis (16.9%) were the most frequent complaints among 753 patients. Laboratory exams showed hemoglobin concentration lower than 10g/dL in 23.3% of the patients, glutamic oxaloacetic transaminase (GOT) above 40U/L in 29.5% and glutamic pyruvic transaminase (GPT) above 40U/L in 28.5%. Twenty-four patients (3.2%) stopped dapsone intake due to adverse effects, of whom 16.6% due to severe anemia. One case of sulfone syndrome was reported. STUDY LIMITATIONS: Loss of some monthly laboratory sample collection. CONCLUSIONS: There was no statistical difference regarding adverse effects in the R-MDT and U-MDT groups but anemia was greater in patients from R-MDT/MB group, therefore adverse effects do not represent a constraint to recommend the six-month uniform regimen of treatment for all leprosy patients.

Clinical trial for uniform multidrug therapy for leprosy patients in Brazil (U-MDT/CT-BR): adverse effects approach

Abstract: BACKGROUND: The Clinical Trial for Uniform Multidrug Therapy for Leprosy Patients in Brazil (U-MDT/CT-BR), designed to evaluate the effectiveness of a six-months regimen, assessed the adverse effects caused by the drugs. OBJECTIVE: Describe adverse effects due to MDT in U-MDT/CT-BR, comparing the uniform regimen (U-MDT) to the current WHO regimen (R-MDT). Patients and methods: After operational classification, patients were randomly allocated to the study groups. U-MDT PB and U-MDT MB groups, received the U-MDT regimen, six doses of MB-MDT (rifampicin, dapsone and clofazimine). R-MDT PB and R-MDT MB groups, received the WHO regimens: six doses (rifampicin and dapsone) for PB and 12 doses (rifampicin, dapsone and clofazimine) for MB. During treatment, patients returned monthly for clinical and laboratorial evaluation. Patients with single lesion were not included in this trial. RESULTS: Skin pigmentation (21.7%) and xerosis (16.9%) were the most frequent complaints among 753 patients. Laboratory exams showed hemoglobin concentration lower than 10g/dL in 23.3% of the patients, glutamic oxaloacetic transaminase (GOT) above 40U/L in 29.5% and glutamic pyruvic transaminase (GPT) above 40U/L in 28.5%. Twenty-four patients (3.2%) stopped dapsone intake due to adverse effects, of whom 16.6% due to severe anemia. One case of sulfone syndrome was reported. STUDY LIMITATIONS: Loss of some monthly laboratory sample collection. CONCLUSIONS: There was no statistical difference regarding adverse effects in the R-MDT and U-MDT groups but anemia was greater in patients from R-MDT/MB group, therefore adverse effects do not represent a constraint to recommend the six-month uniform regimen of treatment for all leprosy patients.

Uniform multidrug therapy for leprosy patients in Brazil (U-MDT/CT-BR): Results of an open label, randomized and controlled clinical trial, among multibacillary patients.

BACKGROUND: Leprosy control is based on early diagnosis and multidrug therapy. For treatment purposes, leprosy patients can be classified as paucibacillary (PB) or multibacillary (MB), according to the number of skin lesions. Studies regarding a uniform treatment regimen (U-MDT) for all leprosy patients have been encouraged by the WHO, rendering disease classification unnecessary. METHODOLOGY AND FINDINGS: An independent, randomized, controlled clinical trial conducted from 2007 to 2015 in Brazil, compared main outcomes (frequency of reactions, bacilloscopic index trend, disability progression and relapse rates) among MB patients treated with a uniform regimen/U-MDT (dapsone+rifampicin+clofazimine for six months) versus WHO regular-MDT/R-MDT (dapsone+rifampicin+clofazimine for 12 months). A total of 613 newly diagnosed, untreated MB patients with high bacterial load were included. There was no statistically significant difference in Kaplan-Meyer survival function regarding reaction or disability progression among patients in the U-MDT and R-MDT groups, with more than 25% disability progression in both groups. The full mixed effects model adjusted for the bacilloscopic index average trend in time showed no statistically significant difference for the regression coefficient in both groups and for interaction variables that included treatment group. During active follow up, four patients in U-MDT group relapsed representing a relapse rate of 2.6 per 1000 patients per year of active follow up (95% CI [0·81, 6·2] per 1000). During passive follow up three patients relapsed in U-MDT and one in R-MTD. As this period corresponds to passive follow up, sensitivity analysis estimated the relapse rate for the entire follow up period between 2·9- and 4·5 per 1000 people per year. CONCLUSION: Our results on the first randomized and controlled study on U-MDT together with the results from three previous studies performed in China, India and Bangladesh, support the hypothesis that UMDT is an acceptable option to be adopted in endemic countries to treat leprosy patients in the field worldwide. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00669643.