Early detection of Mycobacterium leprae is a key strategy for disrupting the transmission chain of leprosy and preventing the potential onset of physical disabilities. Clinical diagnosis is ...essential, but some of the presented symptoms may go unnoticed, even by specialists. In areas of greater endemicity, serological and molecular tests have been performed and analyzed separately for the follow-up of household contacts, who are at high risk of developing the disease. The accuracy of these tests is still debated, and it is necessary to make them more reliable, especially for the identification of cases of leprosy between contacts. We proposed an integrated analysis of molecular and serological methods using artificial intelligence by the random forest (RF) algorithm to better diagnose and predict new cases of leprosy.
The study was developed in Governador Valadares, Brazil, a hyperendemic region for leprosy. A longitudinal study was performed, including new cases diagnosed in 2011 and their respective household contacts, who were followed in 2011, 2012, and 2016. All contacts were diligently evaluated by clinicians from Reference Center for Endemic Diseases (CREDEN-PES) before being classified as asymptomatic. Samples of slit skin smears (SSS) from the earlobe of the patients and household contacts were collected for quantitative polymerase chain reaction (qPCR) of 16S rRNA, and peripheral blood samples were collected for ELISA assays to detect LID-1 and ND-O-LID.
The statistical analysis of the tests revealed sensitivity for anti-LID-1 (63.2%), anti-ND-O-LID (57.9%), qPCR SSS (36.8%), and smear microscopy (30.2%). However, the use of RF allowed for an expressive increase in sensitivity in the diagnosis of multibacillary leprosy (90.5%) and especially paucibacillary leprosy (70.6%). It is important to report that the specificity was 92.5%.
The proposed model using RF allows for the diagnosis of leprosy with high sensitivity and specificity and the early identification of new cases among household contacts.
Due to multiple hypothesis testing with often limited sample size, microarrays and other—omics technologies can sometimes produce irreproducible findings. Complementary to better experimental design, ...reanalysis and integration of gene expression datasets may help overcome reproducibility issues by identifying consistent differentially expressed genes from independent studies. In this work, after a systematic search, nine microarray datasets evaluating host gene expression in leprosy were reanalyzed and the information was integrated to strengthen evidence of differential expression for several genes. Our results are relevant in prioritizing genes and pathways for further investigation, whether in functional studies or in biomarker discovery. Reanalysis of individual datasets revealed several differentially expressed genes (DEGs) in accordance with original reports. Then, five integration methods (
P
value and effect size based) were tested. In the end, random-effects model and ratio association were selected as the main methods to pinpoint DEGs. Overall, classic pathways were found corroborating previous findings and validating this approach. Also, we identified some novel DEG involved especially with skin development processes (
AQP3, AKR1C3, CYP27B1, LTB, VDR
) and keratinocyte biology (
CSTA, DSG1, KRT14, KRT5, PKP1, IVL
), both still poorly understood in leprosy context. In addition, here we provide aggregated evidence towards some gene candidates that should be prioritized in further leprosy research, as they are likely important in immunopathogenesis. Altogether, these data are useful in better understanding host responses to the disease and, at the same time, provide a list of potential host biomarkers that could be useful in complementing leprosy diagnosis based on transcriptional levels.
In leprosy, classic diagnostic tools based on bacillary counts and histopathology have been facing hurdles, especially in distinguishing latent infection from active disease and diagnosing ...paucibacillary clinical forms. Serological tests and IFN-gamma releasing assays (IGRA) that employ humoral and cellular immune parameters, respectively, are also being used, but recent results indicate that quantitative PCR (qPCR) is a key technique due to its higher sensitivity and specificity. In fact, advances concerning the structure and function of the Mycobacterium leprae genome led to the development of specific PCR-based gene amplification assays for leprosy diagnosis and monitoring of household contacts. Also, based on the validation of point-of-care technologies for M. tuberculosis DNA detection, it is clear that the same advantages of rapid DNA detection could be observed in respect to leprosy. So far, PCR has proven useful in the determination of transmission routes, M. leprae viability, and drug resistance in leprosy. However, PCR has been ascertained to be especially valuable in diagnosing difficult cases like pure neural leprosy (PNL), paucibacillary (PB), and patients with atypical clinical presentation and histopathological features compatible with leprosy. Also, the detection of M. leprae DNA in different samples of the household contacts of leprosy patients is very promising. Although a positive PCR result is not sufficient to establish a causal relationship with disease outcome, quantitation provided by qPCR is clearly capable of indicating increased risk of developing the disease and could alert clinicians to follow these contacts more closely or even define rules for chemoprophylaxis.
Reduction in incidence has been associated with the introduction of novel approaches, like chemo/immune-prophylaxis. Incidence determined through follow-up cohort studies can evaluate the ...implementation of these innovative policies towards control and prevention. We have assessed the incidence in our contacts cohort over past 33 years, considering the effect of demographic and clinical variables. Survival analysis was used to estimate the risk of leprosy. A total of 9024 contacts were evaluated, of which 192 developed leprosy, resulting in an overall incidence of 1.4/1000 person-years. The multivariate analysis showed that the major risk factors were (i) contact from MB index cases and (ii) consanguinity (iii) intra household contact. Lower risk was detected for contacts with BCG scar who were revaccinated. There was a significant decrease in accumulated risk between the 2011-2019 period compared with 1987, probably linked to the improvement in laboratory tools to monitor contacts, thereby providing early diagnosis of contacts at intake and reduction of transmission. Our findings suggest that a combination of contact surveillance and tracing, adequate neurodermatological examination, and availability of molecular tools is highly effective in supporting early diagnosis, while a second dose of the BCG vaccination can exert extra protection.
Leprosy is a chronic infectious disease that depends on the interplay of several factors. Single nucleotide polymorphisms (SNPs) in host immune related genes have been consistently suggested as ...participants in susceptibility towards disease. Interleukin-10 (IL-10) is a crucial immunomodulatory cytokine in mycobacterial pathogenesis and especially the -819C>T SNP (rs1800871) has been tested in several case-control studies indicating association with leprosy risk, although a recent consensus estimate is still missing. In this study, we evaluated the association of the -819C>T SNP and leprosy in two new Brazilian family-based populations. Then, we performed meta-analysis for this polymorphism summarizing published studies including these Brazilian family-based groups. Finally, we also retrieved published studies for other distal and proximal IL10 polymorphisms: -3575 T>A (rs1800890), -2849 G>A (rs6703630), -2763 C>A (rs6693899), -1082 G>A (rs1800896) and -592 C>A (rs1800872). Results from meta-analysis supported a significant susceptibility association for the -819T allele, with pooled Odds Ratio of 1.22 (CI = 1.11-1.34) and P-value = 3x10(-5) confirming previous data. This result remained unaltered after inclusion of the Brazilian family-based groups (OR = 1.2, CI = 1.10-1.31, P-value = 2x10(-5)). Also, meta-analysis confirmed association of -592 A allele and leprosy outcome (OR = 1.24, CI = 1.03-1.50, P-value = 0.02). In support of this, linkage disequilibrium analysis in 1000 genomes AFR, EUR, ASN and AMR populations pointed to r(2) = 1.0 between the -592C>A and -819C>T SNPs. We found no evidence of association for the other IL10 polymorphisms analyzed for leprosy outcome. Our results reinforce the role of the -819C>T as a tag SNP (rs1800871) and its association with leprosy susceptibility.
There are many different studies that contribute to the global picture of the ethnic heterogeneity in Brazilian populations. These studies use different types of genetic markers and are focused on ...the comparison of populations at different levels. In some of them, each geographical region is treated as a single homogeneous population, whereas other studies create different subdivisions: political (e.g., pooling populations by State), demographic (e.g., urban and rural), or ethnic (e.g., culture, self-declaration, or skin colour). In this study, we performed an enhanced reassessment of the genetic ancestry of ~ 1,300 Brazilians characterised for 46 autosomal Ancestry Informative Markers (AIMs). In addition, 798 individuals from twelve Brazilian populations representing the five geographical macro-regions of Brazil were newly genotyped, including a Native American community and a rural Amazonian community. Following an increasing North to South gradient, European ancestry was the most prevalent in all urban populations (with values up to 74%). The populations in the North consisted of a significant proportion of Native American ancestry that was about two times higher than the African contribution. Conversely, in the Northeast, Center-West and Southeast, African ancestry was the second most prevalent. At an intrapopulation level, all urban populations were highly admixed, and most of the variation in ancestry proportions was observed between individuals within each population rather than among population. Nevertheless, individuals with a high proportion of Native American ancestry are only found in the samples from Terena and Santa Isabel. Our results allowed us to further refine the genetic landscape of Brazilians while establishing the basis for the effective application of an autosomal AIM panel in forensic casework and clinical association studies within the highly admixed Brazilian populations.
The high rate of leprosy cases among children under 15 years of age in Brazil indicates ongoing transmission within the community. The identification of the new leprosy cases among contacts can help ...identify the source of infection and interrupt the transmission chain. This study aims to determine the detection rate of previously undiagnosed cases of leprosy among schoolchildren who are under 15 years of age living in Manaus, Amazonas, Brazil, and their possible source of infection by contact tracing.
This was a school-based, cross-sectional study in which the identification of active leprosy cases was conducted in 277 out of 622 randomly selected public schools in Manaus, Amazonas, Brazil. Suspected cases of leprosy were referred to the Alfredo da Matta Foundation, a reference center for leprosy in Manaus. A total of 34,547 schoolchildren were examined, and 40 new leprosy cases were diagnosed. Among new cases, 57.5% were males, and 80.0% demonstrated paucibacillary leprosy. A total of 196 of 206 registered contacts were screened, and 52.5% of the newly diagnosed children's cases had at least one positive household contact. In these contacts, grandparents (52.4%) were the most common co-prevalent cases, while 14.3% were uncles, 9.5% were parents and 9.5% were granduncles. Seven contacts (5.0%), including four siblings of child patients were newly diagnosed. Our data indicate that the prevalence is 11.58 per 10,000, which is 17 times higher than the registered rate.
This study suggests that the detection rate of leprosy among schoolchildren may have remained unchanged over the past thirty years. It also indicates that that active case finding is necessary for reaching the World Health Organization's goals of zero detection among children, especially in endemic areas where the prevalence of leprosy is obscure. Moreover, we assert that all children must have their household contacts examined in order to identify the possible source of infection and interrupt the disease's transmission. Novel strategies to reinforce contact tracing associated with large-scale strategies of chemo- and immune-prophylaxis should be expanded to prevent the perpetuation of the disease cycle.
Pleural tuberculosis (PlTB), the most common site of extrapulmonary TB, is characterized by a paucibacillary nature and a compartmentalized inflammatory response in the pleural cavity, both of which ...make diagnosis and management extremely challenging. Although transcriptional signatures for pulmonary TB have already been described, data obtained by using this approach for extrapulmonary tuberculosis and, specifically, for pleural tuberculosis are scarce and heterogeneous. In the present study, a set of candidate genes previously described in pulmonary TB was evaluated to identify and validate a transcriptional signature in clinical samples from a Brazilian cohort of PlTB patients and those with other exudative causes of pleural effusion.
As a first step, target genes were selected by a random forest algorithm with recursive feature elimination (RFE) from public microarray datasets. Then, peripheral blood (PB) and pleural fluid (PF) samples from recruited patients presenting exudative pleural effusion were collected during the thoracentesis procedure. Transcriptional analysis of the selected top 10 genes was performed by quantitative RT-PCR (RT-qPCR).
Reanalysis of the public datasets identified a set of candidate genes (
, and
) that demonstrated a global accuracy of 89.5% in discriminating pulmonary TB cases from other respiratory diseases. Our validation cohort consisted of PlTB (
= 35) patients and non-TB (
= 34) ones. The gene expressions of
,
, and
in PF at diagnosis were significantly different between the two (PlTB and non-TB) groups (
< 0.0001). It was observed that the gene expressions of
and
were higher in PlTB PF than in non-TB patients.
showed the opposite behavior, being higher in the non-TB PF. After anti-TB therapy, however,
gene expression was significantly reduced in PlTB patients (
< 0.001). Finally, the accuracy of the three above-cited highlighted genes in the PF was analyzed, showing AUCs of 91%, 90%, and 85%, respectively.
was above 80% (sensitivity = 0.89/specificity = 0.81), and
showed significant specificity (Se = 0.69/Sp = 0.95) in its capacity to discriminate the groups.
,
, and
showed promise in discriminating PlTB from other causes of exudative pleural effusion by providing accurate diagnoses, thus accelerating the initiation of anti-TB therapy.
In Mycobacterium tuberculosis (MTB) infection, the complex interaction of host immune system and the mycobacteria is associated with levels of cytokines production that play a major role in ...determining the outcome of the disease. Several single-nucleotide polymorphisms (SNPs) in cytokine genes have been associated with tuberculosis (TB) outcome. The aim of this study was to evaluate the association between previously reported SNPs IL2-330 T>G (rs2069762); IL4-590 C>T (rs2243250); IL6-174 G>C (rs1800795); IL10-592 A>C (rs1800872); IL10-1082 G>A (rs1800896); IL17A -692 C>T (rs8193036); IL17A -197 G>A (rs2275913); TNF -238 G>A (rs361525); TNF -308 G>A (rs1800629) and IFNG +874 T>A (rs2430561) and pulmonary TB (PTB) susceptibility. We conducted a case-control study in individuals from Southern Brazil who were recruited between February 2012 and October 2013 in a high incidence TB city. We performed a multiplex genotyping assay in 191 patients with PTB and 175 healthy subjects. Our results suggest a decreased risk for PTB development associated with the IL17A -197A allele (OR = 0.29; p = 0.04), AA genotype (OR = 0.12; p = 0.04) and A carrier (AG/AA) (OR = 0.29; p = 0.004) and IL6 -174C carrier (CC/CG) (OR = 0.46; p = 0.04). We could not properly analyze IL17A -692 C>T (rs8193036) and IFNG +874T>A due to genotypic inconsistencies and found no evidence of association for the IL2, IL4, IL10 and TNF polymorphisms and PTB. In conclusion, our results show a protective effect of IL17 and IL6 polymorphisms on PTB outcome in Southern Brazilian population.
PDF
