Since leprosy is both treated and controlled by multidrug therapy (MDT) it is important to monitor recurrent cases for drug resistance and to distinguish between relapse and reinfection as a means of assessing therapeutic efficacy. All three objectives can be reached with single nucleotide resolution using next generation sequencing and bioinformatics analysis of Mycobacterium leprae DNA present in human skin. DNA was isolated by means of optimized extraction and enrichment methods from samples from three recurrent cases in leprosy patients participating in an open-label, randomized, controlled clinical trial of uniform MDT in Brazil (U-MDT/CT-BR). Genome-wide sequencing of M. leprae was performed and the resultant sequence assemblies analyzed in silico. In all three cases, no mutations responsible for resistance to rifampicin, dapsone and ofloxacin were found, thus eliminating drug resistance as a possible cause of disease recurrence. However, sequence differences were detected between the strains from the first and second disease episodes in all three patients. In one case, clear evidence was obtained for reinfection with an unrelated strain whereas in the other two cases, relapse appeared more probable. This is the first report of using M. leprae whole genome sequencing to reveal that treated and cured leprosy patients who remain in endemic areas can be reinfected by another strain. Next generation sequencing can be applied reliably to M. leprae DNA extracted from biopsies to discriminate between cases of relapse and reinfection, thereby providing a powerful tool for evaluating different outcomes of therapeutic regimens and for following disease transmission. Leprosy, one of the most ancient human infectious diseases, affects skin and nerves and is caused by Mycobacterium leprae infection. Despite the effective use of multidrug therapy/MDT since the 80´s, over 200,000 new cases are reported yearly, indicating active transmission, especially in India and Brazil. Although rare, recurrent clinical manifestations after MDT can occur due to leprosy reactions, relapse by drug resistance, insufficient treatment or reinfection. Relapse and reinfection cannot be differentiated clinically and molecular genotyping of a predefined set of loci have limited resolution due to exceptional M. leprae genome conservation and low sequence diversity between strains from the same geographical area. This is the first report that has compared whole-genome sequences of M. leprae strains from original and recurrent leprosy episodes. M. leprae genome differences were detected between the strains from the first and second episodes in the three patients. In one patient, there was clear evidence for reinfection with an unrelated strain whereas the other two were considered true relapses due to minor strain differences. No known drug resistance mutations were detected, excluding drug resistance as the recurrence cause. Next generation sequencing of M. leprae DNA discriminates relapse from reinfection representing a powerful tool for evaluating different disease outcomes and transmission.

Hepatitis B virus (HBV) and hepatitis D virus (HDV) represent important public health problems in the Western Amazon region with reported cases of fulminant hepatitis. This cross sectional study describes HBV and HDV genotypes circulating in the Brazilian Amazon region.

Leprosy Type-1 Reactions (T1Rs) are pathological inflammatory responses that afflict a sub-group of leprosy patients and result in peripheral nerve damage. Here, we employed a family-based GWAS in 221 families with 229 T1R-affect offspring with stepwise replication to identify risk factors for T1R. We discovered, replicated and validated T1R-specific associations with SNPs located in chromosome region 10p21.2. Combined analysis across the three independent samples resulted in strong evidence of association of rs1875147 with T1R (p = 4.5x10^-8; OR = 1.54, 95% CI = 1.32–1.80). The T1R-risk locus was restricted to a lncRNA-encoding genomic interval with rs1875147 being an eQTL for the lncRNA. Since a genetic overlap between leprosy and inflammatory bowel disease (IBD) has been detected, we evaluated if the shared genetic control could be traced to the T1R endophenotype. Employing the results of a recent IBD GWAS meta-analysis we found that 10.6% of IBD SNPs available in our dataset shared a common risk-allele with T1R (p = 2.4x10^-4). This finding points to a substantial overlap in the genetic control of clinically diverse inflammatory disorders. Leprosy still affects approximately 200,000 new victims each year. A major challenge of leprosy control is the prevention of permanent disability due to nerve damage. Nerve damage occurs if leprosy remains undiagnosed for extended periods or when patients undergo pathological inflammatory responses termed Type-1 Reactions (T1R). T1R is a rare example where beneficial inflammatory responses are temporal separated from host pathological responses. There is strong experimental evidence that supports a role of host genetic factors in T1R susceptibility. Here, we employed a genome-wide association study (GWAS) to investigate susceptibility factors for T1R in Vietnamese families. We followed up the initial GWAS findings in independent population samples from Vietnam and Brazil and identified a set of cis-eQTL genetic variants for the ENSG00000235140 lncRNA as global risk factors for T1R. To test our proposal that T1R is a strong model for pathological inflammatory responses we evaluated if inflammatory bowel disease (IBD) genetic risk-factors were enriched among T1R risk factors. We observed that more than 10% of IBD-risk loci were nominally associated with risk for T1R suggesting a shared mechanism of excessive inflammatory response in the both disease etiologies.

The Brazilian AIDS epidemic is characterized by significant geographic contrasts: a reduction in incidence and mortality in the epicenter (southeast) and an increase in the northeast. HIV-1-transmitted drug resistance (TDR) and genetic diversity were investigated among 106 antiretroviral (ARV)-naive patients from Maranhão State, northeast. The HIV-1 protease (PR) and reverse transcriptase (RT) regions were sequenced; subtypes were assigned by REGA/phylogenetic analysis. TDR to the nucleoside/nonnucleoside reverse transcriptase inhibitor (NRTI/NNRTI) and protease inhibitor (PI) was identified by the Calibrated Population Resistance tool (Stanford). The median age was 31 years (range 18-72), with 54.7% women, 78.3% heterosexual transmission, and 17.9% men who have sex with men (MSM). Around 30% had <350 CD4(+) T cells/μl and 47.2% had plasma viral loads ≤10,000 copies/ml. The TDR rate was 3.8% (4/106; CI 95%, 1.2-8.9%) (three males, two of them MSM). Only single class mutations to NRTI (M184V; T215S) or NNRTI (K103S/N) were detected. Subtype B represented 81.1% (86/106), F1 1.9% (2/106), and C 2.8% (3/106); 14.2% were mosaics: 13 BF1 and 2 BC. Surveillance of TDR and HIV-1 genetic diversity is important to improve control strategies regionally.

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 patient’s 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.

The predictive value of the serology to detection of IgM against the Mycobacterium leprae-derived phenolic glycolipid-I/PGL-I to identify leprosy patients who are at higher risk of developing reactions remains controversial. Whether baseline results of the ML Flow test can predict leprosy reactions was investigated among a cohort of patients enrolled in The Clinical Trial for Uniform Multidrug Therapy for Leprosy Patients in Brazil (U-MDT/CT-BR). This was a descriptive study focusing on the main clinical manifestations of leprosy patients enrolled in the U-MDT/CT-BR from March 2007 to February 2012 at two Brazilian leprosy reference centers. For research purposes, 753 leprosy patients were categorized according to a modified Ridley-Jopling (R&J) classification and according to the development of leprosy reactions (reversal reaction/RR and erythema nodosum leprosum/ENL), and whether they had a positive or negative bacillary index/BI. More than half of the patients (55.5 %) reported leprosy reaction: 18.3 % (138/753) had a RR and 5.4 % (41/753) had ENL. Leprosy reactions were more frequent in the first year following diagnosis, as seen in 27 % (205/753) of patients, while 19 % (142/753) developed reactions during subsequent follow-up. Similar frequencies of leprosy reactions and other clinical manifestations were observed in paucibacillary (PB) and multibacillary (MB) leprosy patients treated with U-MDT and regular MDT (R-MDT) (P = 0.43 and P = 0.61, respectively). Compared with PB patients, leprosy reactions were significantly more frequent in MB patients with a high BI, and more patients developed RR than ENL. However, RR and neuritis were also reported in patients with a negative BI. At baseline, the highest rate of ML Flow positivity was observed in patients with a positive BI, especially those who developed ENL, followed by patients who had neuritis and RR. Among reaction-free patients, 81.9 % were ML Flow positive, however, the differences were not statistically significant compared to reactional patients (P = 0.45). MB and PB patients treated with R-MDT and U-MDT showed similar frequencies of RR and other clinical manifestations. Positive ML Flow tests were associated with MB leprosy and BI positivity. However, ML Flow test results at baseline showed limited sensitivity and specificity for predicting the development of leprosy reactions.