Ludimila Paula Vaz Cardoso

Use of Protein Antigens for Early Serological Diagnosis of Leprosy

ABSTRACT Leprosy is a chronic and debilitating human disease caused by infection with the Mycobacterium leprae bacillus. Despite the marked reduction in the number of registered worldwide leprosy cases as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains relatively stable. This indicates that M. leprae is still being transmitted and that, without earlier diagnosis, M. leprae infection will continue to pose a health problem. Current diagnostic techniques, based on the appearance of clinical symptoms or of immunoglobulin M (IgM) antibodies that recognize the bacterial phenolic glycolipid I, are unable to reliably identify early-stage leprosy. In this study we examine the ability of IgG within leprosy patient sera to bind several M. leprae protein antigens. As expected, multibacillary leprosy patients provided stronger responses than paucibacillary leprosy patients. We demonstrate that the geographic locations of the patients can influence the antigens they recognize but that ML0405 and ML2331 are recognized by sera from diverse regions (the Philippines, coastal and central Brazil, and Japan). A fusion construct of these two proteins (designated leprosy IDRI diagnostic 1 [LID-1]) retained the diagnostic activity of the component antigens. Upon testing against a panel of prospective sera from individuals who developed leprosy, we determined that LID-1 was capable of diagnosing leprosy 6 to 8 months before the onset of clinical symptoms. A serological diagnostic test capable of identifying and allowing treatment of early-stage leprosy could reduce transmission, prevent functional disabilities and stigmatizing deformities, and facilitate leprosy eradication.

HIV-1 pol phylogenetic diversity and antiretroviral resistance mutations in treatment naïve patients from Central West Brazil

BACKGROUND Primary resistance represents a challenge for the control of HIV-1 because it can reduce the efficacy of first line antiretroviral therapy and may impact clinical outcomes. OBJECTIVES To describe the prevalence of primary HIV-1 drug resistance and subtypes circulating in Central West Brazil. STUDY DESIGN 103 antiretroviral naïve patients were recruited in Goiânia city, Central West Brazil during 2007-2008. Protease and partial reverse transcriptase regions were retrotranscribed from plasma HIV-1 RNA and 97 were sequenced after direct nested PCR. HIV-1 subtype was assigned by phylogenetic analysis. Primary drug resistance was analyzed by the Calibrated Population Resistance (CPR) tool using Stanford Surveillance Drug Resistance Mutation (SDRM) and International AIDS Society-USA (IAS-USA) major mutation list. RESULTS Primary drug resistance mutations ranged from 8% (IAS) to 10% (SDRM). High level resistance to at least one antiretroviral drug was observed. T215D/S revertant mutations were identified in 4/97 patients. HIV-1 subtype B represented 82.5%, subtype F1 6.2%, subtype C 3.1%, B/F1 7.2% and one sample was a F1/C/B mosaic. HIV-1 subtype C sequences formed a monophyletic cluster with other Brazilian subtype C sequences. CONCLUSIONS Our HIV-1 pol sequences from Central West region include the first inland HIV-1 subtype C sequences and help compose the molecular epidemiology map of HIV-1 in Brazil. This data also show that a significant proportion of the naïve patients presented important drug resistance mutations. Therefore naive patients from this setting may benefit from pre-treatment genotypic testing to optimize the choice of antiretroviral drugs and to help control HIV-1 transmission.

Moderate prevalence of transmitted drug resistance and high HIV‐1 genetic diversity in patients from Mato Grosso State, Central Western Brazil

Few reports have described the molecular characteristics of the AIDS epidemic within the interior regions of Brazil, a country of continental dimensions. To help fill this gap, the prevalence of transmitted antiretroviral drug resistance and human immunodeficiency virus type 1 (HIV‐1) diversity in Mato Grosso State, central western Brazil are reported. Drug‐naïve patients (n = 105) were recruited at a reference center in Cuiabá/Mato Grosso State located across the border with Bolivia and considered a southern gate to the Amazon forest. For 92 HIV‐1 isolates, the protease and partial reverse transcriptase fragments were amplified by nested‐PCR and sequenced. Drug resistance was analyzed by the Calibrated Population Resistance tool and the International AIDS Society‐USA database. HIV‐1 subtypes were determined by REGA and phylogenetic analyses. Recombinant viruses were analyzed by SIMPLOT. Drug resistance mutations were observed in 5.4%: nucleoside reverse transcriptase inhibitor mutations M41L (n = 1), D67N (n = 1), and K219E (n = 1), the non‐nucleoside reverse transcriptase inhibitor mutation K103N (n = 1) and the protease inhibitor mutation L90M (n = 1). Around 20% of the isolates were recombinants: different patterns of B/F1 mosaics (n = 11), four B/C, one F1/C/B, one F1/C, and one D/F1. Subtype BPRBRT represented 71.7%, 5.4% were of subtype CPRCRT and 3.3% were of subtype F1PRF1RT. A moderate prevalence of transmitted resistance and the co‐circulation of subtypes B, F1, C, different recombinants, including the first report of subtype D, were found in Mato Grosso State, far from the epicenter of the epidemic. These results highlight the importance of monitoring transmitted drug resistance and HIV‐1 genetic diversity in the interior regions of Brazil. J. Med. Virol. 83:1301–1307, 2011.

HIV-1 primary and secondary antiretroviral drug resistance and genetic diversity among pregnant women from central Brazil

Antiretroviral (ARV) resistance mutations in HIV‐1 may reduce the efficacy of prophylactic therapy to mother‐to‐child transmission and impact future treatment options. ARV resistance mutations and HIV‐1 phylogenetic diversity in protease (PR) and reverse transcriptase (RT) genes were assessed among 77 pregnant women (35 naïve, 42 treated with ARV) from Goiânia/Goiás, central west Brazil. ARV mutations in PR/RT genes were analyzed against the Stanford Database, PR/RT HIV‐1 subtypes were assigned by phylogenetic analysis and env/gag subtypes were identified by heteroduplex mobility analysis (HMA). Naïve patients had accessory mutations in the PR gene [A71T (1/6), L10V (2/6), L10I (3/6)] and in the RT gene [V118I (2/6), V179D (1/6), V106I (1/6), K101Q (1/6), H221Y (1/6)]. Seven patients (16.7%) under ARV presented drug resistance mutations, one of them to three ARV classes. Most isolates (67.5%) were subtype B, 11.7% subtype F1 and 3.9% subtype C. Recombinant BPR/F1RT viruses represented 10.4% while F1PR/BRT viruses made up 6.5%. HIV‐1 envgag/PRRT genes were identified as 66.2% subtype B, 3.9% subtype C, 6.5% subtype F1 and approximately 25% B and F1 viruses. HIV‐1 genetic diversity in envgag/PRRT genes indicates the spread and dissemination of BF1 recombinant viruses among a significant proportion of patients from central west Brazil. Moreover, discovery of HIV‐1 secondary resistance among a considerable number of pregnant women under ARV therapy indicates the importance of genotypic testing during pregnancy for optimal prophylactic intervention. J. Med. Virol. 82:351–357, 2010.

High level of multidrug resistance mutations in HIV type 1 pol gene and resistance-associated mutations to enfuvirtide (T-20) among antiretroviral-experienced patients from Central Brazil.

HIV-1 resistance mutations to antiretroviral (ARV) drugs (nucleoside reverse transcriptase inhibitors, NRTI; nonnucleoside reverse transcriptase inhibitors, NNRTI; and protease inhibitors, PI) represent a challenge for sustainable virologic and immunologic responses. HIV-1 phylogenetic diversity and ARV resistance mutations associated with protease (PR) and reverse transcriptase (RT) were assessed among 48 ARV-experienced patients from Goiania/Goias, central west Brazil. The gp41 resistance mutations to the fusion inhibitor (T-20) were analyzed among multidrug-resistant (MDR) patients. PR, partial RT, and gp41 genes were amplified and sequenced from plasma RNA. HIV-1 subtype was assigned by phylogenetic analysis. ARV mutations in PR/RT were analyzed by the Stanford Database. T-20 resistance mutations in gp41 were identified by Stanford, the Los Alamos Database, and other sources. Subtype B represented 79.2% (38/48), subtype F1 4.2% (2/48), subtype C 2.1% (1/48), F1(PR)/B(RT) 8.3% (4/48), and B(PR)/F1(RT) 6.3% (3/48). Secondary drug resistance was observed in 79% (38/48): NRTI resistance (n = 1), NNRTI resistance (n = 1), PI + NRTI or NRTI + NNRTI resistance (n = 20), and PI + NRTI +NNRTI resistance, considered MDR (n = 16). Almost half of the MDR patients had viral loads below 10,000 copies/ml. The gp41 sequences from 14 MDR revealed one F1(PR)/B(RT)/F1(ENV) recombinant and 13 subtype B(PR)/B(RT)/B(ENV) isolates. G36E, N42T, and N43S T-20 resistance mutations were observed in three MDR patients, two of them previously treated with T-20 and the other who had never used T-20. Our data among ARV-experienced patients showed a high proportion of drug-resistance mutations and MDR. T-20 resistance mutations were detected among MDR patients corroborating the importance of T-20 genotyping for clinical management and salvage therapy.

Multibacillary leprosy patients with high and persistent serum antibodies to leprosy IDRI diagnostic-1/LID-1: higher susceptibility to develop type 2 reactions

Leprosy inflammatory episodes [type 1 (T1R) and type 2 (T2R) reactions] represent the major cause of irreversible nerve damage. Leprosy serology is known to be influenced by the patients bacterial index (BI) with higher positivity in multibacillary patients (MB) and specific multidrug therapy (MDT) reduces antibody production. This study evaluated by ELISA antibody responses to leprosy Infectious Disease Research Institute diagnostic-1 (LID-1) fusion protein and phenolic glycolipid I (PGL-I) in 100 paired serum samples of 50 MB patients collected in the presence/absence of reactions and in nonreactional patients before/after MDT. Patients who presented T2R had a median BI of 3+, while MB patients with T1R and nonreactional patients had median BI of 2.5+ (p > 0.05). Anti-LID-1 and anti-PGL-I antibodies declined in patients diagnosed during T1R (p < 0.05). Anti-LID-1 levels waned in MB with T2R at diagnosis and nonreactional MB patients (p < 0.05). Higher anti-LID-1 levels were seen in patients with T2R at diagnosis (vs. patients with T1R at diagnosis, p = 0.008; vs. nonreactional patients, p = 0.020) and in patients with T2R during MDT (vs. nonreactional MB, p = 0.020). In MB patients, high and persistent anti-LID-1 antibody levels might be a useful tool for clinicians to predict which patients are more susceptible to develop leprosy T2R.

Molecular characteristics of HIV type 1 infection among prisoners from Central Western Brazil.

Abstract This study among antiretroviral-experienced prisoners from central western Brazil investigated mutations associated with secondary resistance to nucleoside/nonnucleoside reverse transcriptase inhibitors (NRTI/NNRTI), protease inhibitors (Stanford HIV-1 Resistance/International Aids Society Databases), and HIV-1 subtypes (REGA/phylogenetic analyses/SimPlot). Twenty-seven prisoners from three prisons (16 males and four females from Mato Grosso do Sul State and seven males from Goiás State) had HIV-1 protease and reverse transcriptase fragments sequenced after nested PCR. Median age was 35 years. Seven males and two females were intravenous drug users, three males referred homosexual practice. Resistance mutations were present in 37% (10/27): NRTI+NNRTI mutations (n=5), NRTI mutations (n=3), multidrug-resistant mutations (n=2). Subtype B (48%), subtype C (11%), B/F1, B/C, and F1/B/C recombinants (40.7%) were detected. Possible intraprison transmissions were identified: two intravenous drug user females (subtype C); two clusters among homosexual males (subtype B and B/F1). High resistance rate and possible intraprison transmission highlight the need for improved prevention, counseling, and treatment strategies for prisoners.

Tracing the Origin and Northward Dissemination Dynamics of HIV-1 Subtype C in Brazil

Previous studies indicate that the HIV-1 subtype C epidemic in southern Brazil was initiated by the introduction of a single founder strain probably originating from east Africa. However, the exact country of origin of such a founder strain as well as the origin of the subtype C viruses detected outside the Brazilian southern region remains unknown. HIV-1 subtype C pol sequences isolated in the southern, southeastern and central-western Brazilian regions (n = 209) were compared with a large number (n ~ 2,000) of subtype C pol sequences of African origin. Maximum-likelihood analyses revealed that most HIV-1 subtype C Brazilian sequences branched in a single monophyletic clade (CBR-I), nested within a larger monophyletic lineage characteristic of east Africa. Bayesian analyses indicate that the CBR-I clade most probably originated in Burundi and was introduced into the Paraná state (southern region) around the middle 1970s, after which it rapidly disseminated to neighboring regions. The states of Paraná and Santa Catarina have been the most important hubs of subtype C dissemination, and routine travel and spatial accessibility seems to have been the major driving forces of this process. Five additional introductions of HIV-1 subtype C strains probably originated in eastern (n = 2), southern (n = 2) and central (n = 1) African countries were detected in the Rio de Janeiro state (southeastern region). These results indicate a continuous influx of HIV-1 subtype C strains of African origin into Brazil and also unveil the existence of unrecognized transmission networks linking this country to east Africa.

Increasing heterosexual transmission of HIV-1 subtype C in Inland Central Western Brazil†

The molecular epidemiology of HIV‐1 in Brazil is complex and heterogeneous because several subtypes co‐circulate with some important regional differences. This study evaluated HIV‐1 subtypes amongst pregnant women living in the metropolitan area and in the interior cities from central western Brazil. From June 2008 to June 2010, 86.9% of confirmed cases of HIV‐1 infection amongst pregnant women (172 out of 198 cases) were recruited in Goiania/Goias state. The HIV‐1 pol gene was sequenced after nested‐PCR. HIV‐1 subtypes were assigned by REGA, phylogenetic, and bootscan analyses. The median age of participants was 26 years (15–41 years range); 58.7% of participants were diagnosed during prenatal care and 51.7% of participants came from >50 interior cities within Goias state. Amongst the 131 HIV‐1 pol sequences, 64.9% were subtype B, 13.0% were BF1 recombinant, 11.4% were subtype C, 7.6% were subtype F1, and 2.3% were BC recombinant. According to the HIV‐1 diagnosis date (1994–2010), a significant increase in subtype C and a decrease of BF1 mosaics were observed over time. All subtype C patients lived in interior cities where the highest prevalence of subtype C outside southern Brazil was observed (18.4%). Phylogenetic analysis revealed multiple independent introductions of the Brazilian subtype C clade from the southern/southeastern regions of Brazil. The HIV‐1 epidemic in women from central western Brazil infected by the heterosexual route is characterized by an unexpectedly high prevalence of subtype C viruses highly related to those circulating in southern/southeastern Brazil. These findings highlight the importance of molecular surveillance programs outside large metropolitan regions in Brazil. J. Med. Virol. 85:396–404, 2013.

HIV-1 transmitted drug resistance and genetic diversity among patients from Piauí State, Northeast Brazil

HIV‐1 transmitted‐drug‐resistance and genetic diversity are dynamic and may differ in distinct locations/risk groups. In Brazil, increased AIDS incidence and related mortality have been detected in the Northeast region, differently from the epicenter in the Southeast. This cross‐sectional study describes transmitted‐dru‐ resistance and HIV‐1 subtypes in protease/PR and reverse transcriptase/RT regions among antiretroviral naïve patients from Piauí State, Northeast Brazil. Among 96 patients recruited 89 (92.7%) had HIV‐1 PR/RT regions sequenced: 44 females and 45 males, 22 self‐declared as men who have sex with men. Transmitted‐drug‐resistance was investigated by CPR tool (Stanford HIV‐1 Drug Resistance/SDRM). HIV‐1 subtypes were assigned by REGA and phylogenetic inference. Overall, transmitted‐drug‐resistance rate was 11.2% (10/89; CI 95%: 5.8–19.1%); 22.7% among men who have sex with men (5/22; CI 95%: 8.8–43.4%), 10% in heterosexual men (2/20; CI 95%: 1.7–29.3%) and 6.8% in women (3/44; CI 95%: 1.8–17.4%). Singleton mutations to protease‐inhibitor/PI, nucleoside‐reverse‐transcriptase‐inhibitor/NRTI or non‐nucleoside‐reverse‐transcriptase‐inhibitor/NNRTI predominated (8/10): PI mutations (M46L, V82F, L90M); NRTI mutations (M41L, D67N) and NNRTI mutations (K103N/S). Dual class resistance mutations to NRTI and NNRTI were observed: T215L (NRTI), Y188L (NNRTI) and T215N (NRTI), F227L (NNRTI). Subtype B prevailed (86.6%; 77/89), followed by subtype F1 (1.1%, 1/89) and subtype C (1.1%, 1/89). B/F1 and B/C intersubtype recombinants represented 11.2% (10/89). In Piauí State extensive testing of incidence and transmitted‐drug‐resistance in all populations with risk behaviors may help control AIDS epidemic locally. J. Med. Virol. 87:798–806, 2015.