Evaluation of Proinflammatory Chemokines in HIV Patients with Asymptomatic Leishmania Infantum Infection.

Asymptomatic Leishmania infantum, when associated with HIV, can become severe and potentially fatal. In this co-infection, the worst prognosis may be influenced by the host's immunological aspects, which are crucial in determining susceptibility. Chemokines play an important role in this process by influencing the cellular composition at affected sites and impacting the disease's outcome. Therefore, the aim of this study was to evaluate proinflammatory chemokines in HIV patients with the asymptomatic L. infantum infection. In this cross-sectional study, the levels of CCL2, CCL5, CXCL8, MIG, and IP-10 were measured in 160 serum samples from co-infected patients (n = 53), patients with HIV (n = 90), and negative controls (n = 17). Quantification was determined by flow cytometry. The obtained data were statistically analyzed using the Kruskal-Wallis test, followed by the Dunn's post-test and the Spearman's correlation coefficient. Significance was set at p < 0.05. The chemokines CCL2, CCL5, MIG, and IP-10 exhibited higher levels in the HIV group compared to co-infection. However, the elevated levels of all these chemokines and their increased connectivity in co-infected patients appear to be important in identifying proinflammatory immune responses associated with the asymptomatic condition. Furthermore, a weak negative correlation was observed between higher levels of CXCL8 and lower viral loads in co-infected patients.

Evaluation of SmITS1-LAMP performance to diagnosis schistosomiasis in human stool samples from an endemic area in Brazil.

Schistosomiasis is a life-threatening infectious disease categorized by the World Health Organization as a public health issue. New molecular diagnostic alternatives for intestinal schistosomiasis caused by Schistosoma mansoni, such as the loop-mediated isothermal amplification (LAMP), a fast and simple amplification technique, have been proposed for control of this NTD in low-endemicity locations. A LAMP assay was performed to detect the internal transcribed spacer 1 ribosomal gene of S. mansoni (SmITS1-LAMP) in 322 DNA extracted from stool samples from schistosomiasis endemic area in Brazil. Kato-Katz analysis of human stool samples was used as the gold standard test, detecting 144 positive samples. SmITS1-LAMP detection limit achieved a maximum analytical sensitivity of 10 fg/µL using S. mansoni genomic DNA, subsequently detecting 17/144 (11.8%) positive samples. SmITS1-LAMP sensitivity and specificity were 12% (95%CI: 7%-18%) and 93% (95%CI: 89%-96%), respectively. Positive predictive value (PPV) and negative predictive value (NPV) were 59% (95%CI: 39% - 76%); and 57% (95%CI: 51% - 62%), respectively. Most cases involved men (61.8%), predominantly young adults (20-39 years old) in cases diagnosed by Kato-Katz and adults (40-59 years old) in cases diagnosed by LAMP. The low number of eggs per gram of stool (1-99 EPG) was the most frequently identified by both Kato-Katz and LAMP. Further studies are needed to evaluate the applicability of SmMIT-LAMP on Schistosoma mansoni diagnosis and surveillance of schistosome infections.

Lack of Association of Polymorphisms in IL22 and IL22RA1 Genes with Fibrosis Severity in Patients with Chronic Hepatitis C.

The IL-22 pathway has been shown to play an important role in the pathogenesis of liver fibrosis. However, little is known about the role of single-nucleotide polymorphisms (SNPs) in IL-22-related genes in relation to the severity of liver fibrosis. This study aimed to investigate the association of polymorphisms in IL22 and IL22RA1 genes with the severity of liver fibrosis in patients with chronic hepatitis C. A total of 326 patients (165 with mild fibrosis and 161 with severe fibrosis) were included. Four SNPs in IL22 (rs1179251, rs2227473, rs1012356, and rs2227485) and two in IL22RA1 (rs4648936 and rs3795299) were evaluated by real-time PCR. No significant association was observed between the polymorphisms studied and the severity of liver fibrosis. The SNPs rs1179251, rs2227473, rs1012356, and rs2227485 in IL22 and rs4648936 and rs3795299 in IL22RA1 may not be involved in the pathogenesis of liver fibrosis in patients with chronic hepatitis C.

Evaluation of molecular techniques to visceral leishmaniasis detection in asymptomatic patients: a systematic review.

INTRODUCTION: Visceral leishmaniasis (VL) is a systemic and neglected parasitic disease. Its main symptoms are fever, splenomegaly with or without hepatomegaly, and anemia, however, most individuals remain asymptomatic. Due to the lack of a gold standard and the limitations of current diagnostic techniques, where parasitology is ethically unfeasible for individuals without symptoms and serological tests do not differentiate between past and present disease, molecular methodologies are the most suitable. AREAS COVERED: We performed a systematic review analyzing the molecular techniques based on PCR used, so far, to detect asymptomatic cases of VL in humans. Structured searches were carried out on PubMed, LILACS, Scopus, and Web of Science databases without time and language restrictions. Two reviewers evaluated the studies, performed data extraction, and quality assessment by assigning scores. EXPERT OPINION: qPCR using RNA targets can be used in the diagnosis of asymptomatic cases of human VL, due to its characteristics. We recommend further studies to analyze the methodology, mainly observing the use of different rRNA targets. Therefore, we hope that this technique contributed to the construction of public policies that address the diagnosis and handling of asymptomatic patients.

Asymptomatic Leishmania infection in HIV-positive outpatients on antiretroviral therapy in Pernambuco, Brazil.

BACKGROUND: Visceral leishmaniasis (VL) in HIV-positive individuals is a global health problem. HIV-Leishmania coinfection worsens prognosis and mortality risk, and HIV-Leishmania coinfected individuals are more susceptible to VL relapses. Early initiation of antiretroviral therapy can protect against Leishmania infection in individuals living in VL-endemic areas, and regular use of antiretrovirals might prevent VL relapses in these individuals. We conducted a cross-sectional study in Petrolina, Brazil, an VL-endemic area, to estimate the prevalence of asymptomatic Leishmania cases among HIV-positive outpatients. METHODS: We invited any HIV-positive patients, aged ≥ 18-years-old, under antiretroviral therapy, and who were asymptomatic for VL. Patients were tested for Leishmania with enzyme-linked immunosorbent assays (ELISA)-rK39, immunochromatographic test (ICT)-rK39, direct agglutination test (DAT), latex agglutination test (KAtex), and conventional polymerase chain reaction (PCR). HIV-Leishmania coinfection was diagnosed when at least one VL test was positive. RESULTS: A total of 483 patients were included. The sample was predominantly composed of single, < 48-years-old, black/pardo, heterosexual males, with fewer than 8 years of schooling. The prevalence of asymptomatic HIV-Leishmania coinfection was 9.11% (44/483). HIV mono-infected and HIV-Leishmania coinfected groups differed statistically significantly in terms of race (p = 0.045), marital status (p = 0.030), and HIV viral load (p = 0.046). Black/pardo patients, married patients, and those with an HIV viral load up to 100,000 copies/ml presented higher odds for HIV-Leishmania coinfection. CONCLUSIONS: A considerable number of asymptomatic Leishmania cases were observed among HIV-positive individuals in a VL-endemic area. Given the potential impact on transmission and health costs, as well as the impact on these coinfected individuals, studies of asymptomatic Leishmania carriers can be useful for guiding public health policies in VL-endemic areas aiming to control and eliminate the disease.

Identification of nontuberculous mycobacteria species by multiplex real-time PCR with high-resolution melting.

INTRODUCTION: Nontuberculous mycobacteria (NTM) species, as human pathogens, are increasing in the world, as is the difficulty of accurately identifying them. Differential diagnosis, especially between the M. tuberculosis complex and NTM species, and the characterization of NTM species is important. This study aimed to evaluate the performance of a molecular system based on multiplex real-time PCR with high-resolution melting (HRM) for the identification and differentiation of NTM species of clinical importance of an endemic area for tuberculosis in northeastern Brazil. METHODS: The technical protocol of the molecular system was based on multiplex real-time PCR-HRM, and evaluated the sensitivity and specificity of the detection of NTM species in mycobacterial clinical isolates from the studied region. The gold standard method was specific gene sequencing. RESULTS: The sensitivity and specificity of multiplex real-time PCR-HRM modified for differentiation between NTM and M. tuberculosis were 90% and 100%, respectively. The PCR-HRM sensitivities for the characterization of NTM species (M. kansasii, M. abscesses, M. avium, and M. fortuitum) were 94.59%, 80%, 57.14%, and 54%, respectively. CONCLUSIONS: The multiplex real-time PCR-HRM modified assay has the potential to rapidly and efficiently identify nontuberculous mycobacteria of clinical importance, which is crucial for immediate implementation of the appropriate therapy and thus avoiding complications and sequelae in patients.

Influence of LGALS3 and PNPLA3 genes in non-alcoholic steatohepatitis (NASH) in patients undergone bariatric surgery.

AIM: This study evaluated the genesPNPLA3 and LGALS3 in patients who have undergone bariatric surgery. METHODS: Individuals with NAFLD and NASH were evaluated, the DNA was extracted from total blood for genotyping of rs4644, rs4652 from LGALS3 and rs738409 from PNPLA3 genes, the total RNA was obtained from liver biopsy. For the detection of the molecular targets, real-time PCR through Taqman probes was used. RESULTS: From a total of 46 collected patients, of those 21 (456%) were included as NASH and 25 (544%) as steatosis group. This groups showed significant difference to aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Glutamyl transpeptidase (GGT) (p = 0.0108, p = 0.0090 and p = 0.0044). Regarding to gene expression in studied groups, hepatic steatosis vs NASH, we observed a higher expression of the LGALS3 gene in NASH (p = 0.0273). In addition, patients with C allele in homozygous for rs4644 and rs4652 of LGALS3 gene had higher expression, in NASH group (p = 0.0500 and p = 0.0242, respectively), furthermore for rs4644 both alleles in homozygous showed higher expression (AA/CC vs AC) (p = 0.0500), when analyzed PNPLA3 rs738409, NASH patients with G allele in homozygous had higher expression (p = 0.0494). CONCLUSIONS: Therefore, an increased expression of the LGALS3 gene in patients with NASH may be important in the etiopathogenesis of the disease, as well as the presence of rs4652 and rs4644 SNPs in the regulation of transcriptional levels of the gene in patients with NAFLD and NASH.

Loop-mediated isothermal amplification methods for diagnosis of visceral leishmaniasis (kala-azar) - a systematic review.

Introduction: Visceral leishmaniasis (VL) is a life-threatening infection remaining as one of the most neglected tropical diseases around the world. Despite scientific advances, an accurate diagnosis of VL remains a challenge. Loop-mediated isothermal amplification (LAMP) has emerged as a promising diagnostic tool with the possibility of becoming a point-of-care test to guide VL diagnosis and treatment.Areas covered: We conducted a systematic review assessing LAMP systems for diagnosing VL from 2000 to 2019. We performed structured searches in PubMed, LILACS, Scopus, and Web of Science without language restriction. Two reviewers screened articles, completed the data extraction and assessment of the risk of bias. A qualitative summary of the included studies was performed.Expert opinion: LAMP could be used as a screening test for VL diagnosis, so tissue aspiration could be performed only for those who are LAMP negative. We recommend more studies about the performance of the Loopamp™ Leishmania Detection kit and the Brazilian LAMP assay. Thus, we expect in the future the constitution of an international consortium to share experiences, projects, and other LAMP approaches mainly among researchers and institutions located within VL endemic countries.

SLC11A1 (rs3731865) polymorphism and susceptibility to visceral leishmaniasis in HIV-coinfected patients from Northeastern Brazil.

Following the emergence of human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS), the number of visceral leishmaniasis-HIV (VL-HIV) coinfections has increased worldwide, mainly in Brazil. The development of clinical forms of VL can be influenced by nutritional status, age, and host genetic factors, which are important variables determining susceptibility to disease. There are no studies with a candidate gene approach assayed directly in the VL-HIV-coinfected population. Herein, we determined and analyzed the associations of SLC11A1, LECT2, CCL1, CCL16, and IL4 genetic polymorphisms with susceptibility to VL-HIV coinfection in Northeastern Brazil. We analyzed 309 DNA samples extracted from the peripheral blood of HIV patients, and clinical and hematological data were collected from medical records. The diagnosis of VL was confirmed in 110 out of 309 patients; genotyping was carried out by TaqMan assays afterwards. Our results confirmed the association between the SLC11A1 polymorphism (rs3731865) and VL-HIV coinfection (p = 0.0206, OR 1.8126, 95% CI 1.1050-2.9727). In addition, the SLC11A1 genotype GG (p = 0.0050, OR 3.0395, 95% CI 1.4065-6.5789) and CD4+ T lymphocyte count (p = 0.0030, OR 0.9980, 95% CI 0.9970-0.9990) were associated with VL-HIV coinfection in a multivariate model. The polymorphism of the SLC11A1 gene (rs3731865) was associated with VL-HIV coinfection, suggesting a possible genetic mechanism involved in the susceptibility to VL in HIV patients. This finding can suggest new therapeutic targets and genetic markers for the VL-HIV-coinfected population.

Higher levels of TNF and IL-4 cytokines and low miR-182 expression in visceral leishmaniasis-HIV co-infected patients.

AIMS: The aim of the present study was to assess serum cytokine and miRNA expression in visceral leishmaniasis-HIV (VL-HIV) co-infection and HIV mono-infection. METHODS AND RESULTS: We analysed 113 serum samples from HIV patients in areas endemic for leishmaniasis. The diagnosis of VL was confirmed in 65 of these 113 samples. The VL-HIV and HIV groups presented significant differences regarding haemoglobin level (P < .0001), lymphocyte count (P = .0444), white blood cell count (P = .0108), weight loss (P = .0310), HIV load (P < .0001) and CD4+ T-lymphocytes count (P = .0003). Levels of IL-6 and IL-10, IFN-γ and IL-6, IFN-γ and IL-10, TNF and IL-2 were positively correlated in VL-HIV co-infection, indicating higher serum levels of TNF and IL-4 (P < .0001). In addition, miR-182 expression was found to be significantly higher in HIV (P = .009), miR-210 exhibited no statistically significant difference between groups, and nonexpression of miR-122 was found in both groups. CONCLUSION: Together, TNF, IL-4 and miR-182 may represent circulatory biomarkers of VL-HIV co-infection.

Identification of nontuberculous mycobacteria species by multiplex real-time PCR with high-resolution melting

Abstract INTRODUCTION: Nontuberculous mycobacteria (NTM) species, as human pathogens, are increasing in the world, as is the difficulty of accurately identifying them. Differential diagnosis, especially between the M. tuberculosis complex and NTM species, and the characterization of NTM species is important. This study aimed to evaluate the performance of a molecular system based on multiplex real-time PCR with high-resolution melting (HRM) for the identification and differentiation of NTM species of clinical importance of an endemic area for tuberculosis in northeastern Brazil. METHODS: The technical protocol of the molecular system was based on multiplex real-time PCR-HRM, and evaluated the sensitivity and specificity of the detection of NTM species in mycobacterial clinical isolates from the studied region. The gold standard method was specific gene sequencing. RESULTS: The sensitivity and specificity of multiplex real-time PCR-HRM modified for differentiation between NTM and M. tuberculosis were 90% and 100%, respectively. The PCR-HRM sensitivities for the characterization of NTM species (M. kansasii, M. abscesses, M. avium, and M. fortuitum) were 94.59%, 80%, 57.14%, and 54%, respectively. CONCLUSIONS The multiplex real-time PCR-HRM modified assay has the potential to rapidly and efficiently identify nontuberculous mycobacteria of clinical importance, which is crucial for immediate implementation of the appropriate therapy and thus avoiding complications and sequelae in patients.

Urine as a promising sample for Leishmania DNA extraction in the diagnosis of visceral leishmaniasis - a review

ABSTRACT Visceral leishmaniasis is a serious and debilitating infection with high fatality rate in tropical and subtropical countries. As clinical symptoms of visceral leishmaniasis are not so specific, confirmatory diagnostic methods with high sensitivity and specificity are needed. Noninvasive methods have been developed using urine as a clinical sample for visceral leishmaniasis diagnosis. In fact, there is a clear correlation between kidney impairment and Leishmania DNA in urine. However, it has been proved that Leishmania nucleic acid may also be isolated from patients without any sign of renal involvement. Even though urine has become a promissing biological sample, it is still not widely used due to several issues, such as (i) incomprehension of the whole renal pathophysiology process in visceral leishmaniasis, (ii) presence of many amplification inhibitors in urine, and (iii) lack of an efficient urinary DNA extraction method. In this article, we performed a literature review to bring a new perspective for Leishmania DNA isolation in urine.

Análise das regiões polimórficas do gene HASPB (K26) de Leishmania infantum em amostras clínicas positivas para leishmaniose visceral e coinfecção LV/HIV / Analyzing of the polymorphic regions of HASPB (K26) gene from Leishmania infantum in positive clinical samples for visceral leishmaniasis and VL/HIV co-infection

A leishmaniose visceral(LV) é uma doença grave que afeta a população de vários países, onde o Brasil apresenta a maior prevalência da infecção nas Américas. Com o estudo do gene codificante da proteína B de superfície (HASPB ou K26) de Leishmania infantum é possível identificar as variações polimórficas intraespecíficas e, assim, será possível consolidar a descrição de um perfil polimórfico presente no Estado de Pernambuco. O objetivo do trabalho foi analisar as regiões polimórficas do gene HASPB (K26) de Leishmania infantum em amostras clínicas positivas para leishmaniose visceral e coinfecção LV/HIV. O sistema K26 PCR foi otimizado utilizando concentrações variadas de DNA genômico de L. infantum. Foi realizado o screening de amostras clínicas de DNA através de dois sistemas de PCR simples, kDNA e ITS1/RFLP, para ensaios posteriores com a K26 PCR nas amostras positivas. A curva de dissociação de alta definição (qPCR-HRM) foi empregada na localização de temperaturas de melting específicas para L. infantum. Os amplicons do gene K26 foram sequenciados e alinhados as sequencias selecionadas em base de dados. A K26 PCR apresentou limiar de detecção de 1 pg para amplicon de 700 pb. A especificidade dos primers foi avaliada experimentalmente e in silico, apresentando anelamento inespecífico com DNA humano. Em paralelo, foram selecionadas 78 amostras de DNA através dos dois sistemas screening, sendo 17 caracterizadas como L. infantum. Os ensaios com DNA das amostras clínicas para o sistema K26 PCR revelaram bandas espúrias. A análise através qPCR-HRM em DNA genômico do parasita resultou em amplificação com Tm de 88,2 °C, já o ensaio com amostra clínica revelou duas amplificações com distintas temperaturas de melting, 84,6 e 88,2°C. Três amplicons do gene K26 foram sequenciados e alinhados a cinco sequencias da base de dados, indicando 38,2 por cento de similaridade. Pode-se concluir que o sistema K26 PCR é recomendável para análise dos polimorfismos genéticos, contanto que o DNA seja extraído diretamente de espécies isoladas em meio de cultura.

Rapid Tests and the Diagnosis of Visceral Leishmaniasis and Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome Coinfection.

After the emergence of the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS), the number of visceral leishmaniasis (VL)-HIV/AIDS coinfections has increased worldwide. Herein, we assessed the usefulness of an rK39-based immunochromatographic test (rK39 ICT) (DiaMed-IT LEISH(®); DiaMed AG, Cressier-sur-Morat, Switzerland) and a latex agglutination test (KAtex; Kalon Biological, Guildford, United Kingdom) for urinary antigen detection to diagnose VL in 15 HIV/AIDS patients from northeastern Brazil. VL diagnosis was based on clinical findings, cytology, serology, parasite DNA, and/or urinary antigen detection. VL was confirmed in seven out of 15 HIV/AIDS patients. Only three patients were positive in bone marrow cytology, three patients were conventional polymerase chain reaction (PCR) positive, while six were real-time PCR positive. All patients were direct agglutination test (DAT) (Royal Tropical Institute, Amsterdam, The Netherlands) positive; of these, four were positive by rK39 ICT and five by KAtex. Large-scale studies are needed to validate the use of the KAtex in the national public health laboratory network in Brazil, aiming at improving the diagnosis of VL in HIV/AIDS patients in this country.