Performance of IS6110-LAMP assay for detection of Mycobacterium tuberculosis complex in blood and urine samples from patients with extrapulmonary tuberculosis.

BACKGROUND: The diagnosis of extrapulmonary tuberculosis (EPTB) shows numerous difficulties because of non-specific symptomatology and low sensitivity of conventional methods. Loop-mediated isothermal amplification (LAMP) is a fast and low-cost technique, which can amplify under isothermal conditions an amount of target DNA copies into approximately a billion copies. OBJECTIVE: The present study aimed to evaluate a IS6110-LAMP system for Mycobacterium tuberculosis detection in blood and urine samples from patients with EPTB. METHODS: The collected samples (n = 122) were stratified in two groups: Group EPTB - patient samples with confirmed EPTB (n = 61); Group non-TB - patient samples without TB (n = 61). The urine samples underwent decontamination, and the components of blood samples were separated (plasma and PBMC). DNA extractions were performed in all biological samples followed by IS6110-LAMP assay technique. The detection limit was evaluated through dilution curves (1:10) using Mtb reference strain (H37Rv) genomic DNA. FINDINGS: The detection limit of IS6110-LAMP was 10 fg/µL (∼10-20 bacilli/µL). The IS6110-LAMP technique sensitivity and specificity were 95.65 % and 79.25 %, respectively, with a general kappa agreement index of 0.762. MAIN CONCLUSIONS: Based on these results, IS6110-LAMP test showed considerable diagnostic parameters, being able to aid in the speed and accuracy of the final EPTB diagnosis.

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.

Schistosoma mansoni infection decreases IL-33-mRNA expression and increases CXCL9 and CXCL10 production by peripheral blood cells.

Schistosoma mansoni infections, particularly egg antigens, induce Th2-dominant granulomatous responses accompanied by remarkable immunoregulatory mechanisms that avoid intense fibrosis. Interleukin (IL)-33 is a cytokine that stimulates the early activation of Th2 responses, and its soluble ST2 receptor (sST2) avoids granulomatous response, as well as CXCL9 and CXCL10 chemokines that have antifibrotic activity. However, in schistosomiasis, these molecules have not been suitably studied. Therefore, this study aimed to measure IL-33 and sST2 RNA, cytokines, and chemokines in peripheral blood cultures from individuals living in schistosomiasis-endemic areas. Peripheral blood cells from individuals with S. mansoni (n = 34) and non-infected individuals (n = 31) were cultured under mitogen stimulation. Supernatant chemokines and cytokines were evaluated using a cytometric bead array, and IL-33 and sST2 mRNA expression was measured using qPCR. Infected individuals showed higher levels of CXCL8, CXCL9, CXCL10, IFN-γ, TNF-α, IL-6, IL-2, IL-4, and IL-10; there was a lower expression of IL-33 mRNA and similar expression of sST2mRNA in infected than non-infected individuals. In conclusion, for the first time, we demonstrated lower IL-33mRNA expression and high levels of the antifibrotic chemokines CXCL9 and CXCL10 in schistosomiasis mansoni, which could control exacerbations of the disease in individuals from endemic areas.

Cerebral mansoni schistosomiasis: a systematic review of 33 cases published from 1989 to 2019.

BACKGROUND: We investigated the epidemiology, clinical presentation and outcomes of individuals affected by cerebral schistosomiasis. METHODS: This systematic review was planned in accordance with current guidelines for performing comprehensive systematic reviews and meta-analysis, including the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guideline. RESULTS: Most of patients presented with seizures (48.5%), which is a non-specific symptom despite its high prevalence. There was no specific clinical manifestation that could help the diagnosis, which was made in 69.7% by histopathological analysis of brain tissue. CONCLUSIONS: Seizures are a non-specific symptom to diagnose patients with cerebral schistosomiasis and accurate clinical indicators need to be derived through further studies.

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.

Development of a molecular xenomonitoring protocol to assess filariasis transmission.

According to the World Health Organization, lymphatic filariasis (LF), a mosquito-borne neglected tropical disease (NTD), should be eliminated as a public health concern by the end of 2020. To this end, the goals of the Global Programme to Eliminate Lymphatic Filariasis (GPELF) include interrupting transmission through mass drug administration (MDA). After two decades, several countries have implemented MDA and are now ready to confirm whether transmission has been interrupted. The method for detecting the parasites in mosquito vectors known as xenomonitoring is a non-invasive tool for assessing the current transmission status of the filarial nematode Wuchereria bancrofti (which is responsible for 90% of cases) by their vectors. There are several methods available for detection of the worm in mosquito samples, such as dissection or polymerase chain reaction (PCR). However, most of these techniques still produce a considerable number of false-negative results. The present study describes a new duplex PCR protocol, which is an improvement on the traditional PCR methodology, enhanced by introducing the actin gene as an endogenous control gene. After adjusting the mosquito pool size, DNA extraction, and WbCx PCR duplex design, we achieved a reliable and sensitive molecular xenomonitoring protocol. This assay was able to eliminate 5% of false negative samples and detected less than one Wb larvae. This high sensitivity is particularly valuable after MDA, when prevalence declines. This new method could reduce the number of false-negative samples, which will enable us to improve our ability to generate accurate results and aid the monitoring strategies used by LF elimination programmes.

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.

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.

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.

Leishmaniose Tegumentar Americana: uma análise histopatológica e molecular em lesões de dermatites no estado de Pernambuco, Brasil / American Cutaneous Leishmaniasis: a histopathological and molecular analysis of dermatitis lesions in the State of Pernambuco, Brazil

Objetivo: Realizar uma análise histopatológica e molecular em biópsia de pele entre as lesões de dermatites de pacientes com suspeita de Leishmaniose Tegumentar Americana (LTA) no hospital de referência do estado de Pernambuco entre o período de 2016 e 2017. Métodos: Trata-se de um estudo descritivo observacional, no qual todos os pacientes com lesões clinicamente sugestivas para LTA incluídos no estudo foram submetidos à coleta de biópsia de pele das lesões, as quais foram analisadas pela técnica histopatológica e PCR (Reação em Cadeia de Polimerase). Resultados: Foram analisadas 24 amostras de biópsia de pele de pacientes com suspeita clínica de LTA, por testes histopatológicos e confirmação pela PCR. As amostras foram caracterizadas pela busca do DNA de Leishmania braziliensis através da PCR. Das 24 amostras estudadas, em nenhuma foi encontrado DNA de L. braziliensis. Apenas em um caso foi detectada presença de amastigotas de Leishmania pela técnica histopatológica. Outros achados microscópicos observados foram: dermatite granulomatosa (33,33%), úlcera crônica (20,83%), carcinoma basocelular (16,66%), Leishmaniose, dermatite plasmocitária e inflamação granulomatosa (8,33%) e Hanseníase (4,16%). Conclusão: O diagnóstico histopatológico detectou um caso de LTA, porém, a PCR não encontrou DNA do parasito. A análise histopatológica mostrou que as lesões dermatotrópicas dos pacientes são oriundas principalmente de úlceras, tumores de pele e hanseníase.

Objective: Accomplish a histopathological and molecular analysis in skin biopsy between the dermatitis lesions of patients with suspected American Cutaneous Leishmaniasis (ATL) at the Hospital of Reference of the State of Pernambuco between the period of 2016 and 2017. Methods: This is a descriptive, observational study in which all patients with clinically suggestive lesions for ATL included in the study were submitted to skin biopsy of the lesions and analyzed by the histopathological technique and PCR (Polymerase Chain Reaction). Results: Were analyzed 24 skin biopsy samples from patients with clinical suspicion of ATL, by histopathological tests and confirmation by PCR. Samples were characterized by the search of Leishmania braziliensis DNA through PCR. Of the 24 samples studied, no DNA of L. braziliensis was found. Only in one case was detected presence of Leishmania amastigotes by histopathological technique. Other microscopic findings were granulomatous dermatitis (33.33%), chronic ulcer (20.83%), basal cell carcinoma (16.66%), Leishmaniasis, plasmacytoma dermatitis and granulomatous inflammation (8.33%) and leprosy, 16%). Conclusion: The histopathological diagnosis detected a case of ATL, however, the PCR did not find DNA of the parasite. The histopathological analysis showed that the dermatotropic lesions of the patients come mainly from ulcers, skin tumors and leprosy.

Development and Validation of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for Rapid Detection of ZIKV in Mosquito Samples from Brazil.

The rapid spread of Zika virus (ZIKV) represents a global public health problem, especially in areas that harbor several mosquito species responsible for virus transmission, such as Brazil. In these areas, improvement in mosquito control needs to be a top priority, but mosquito viral surveillance occurs inefficiently in ZIKV-endemic countries. Quantitative reverse transcription PCR (qRT-PCR) is the gold standard for molecular diagnostic of ZIKV in both human and mosquito samples. However, the technique presents high cost and limitations for Point-of-care (POC) diagnostics, which hampers its application for a large number of samples in entomological surveillance programs. Here, we developed and validated a one-step reverse transcription LAMP (RT-LAMP) platform for detection of ZIKV in mosquito samples. The RT-LAMP assay was highly specific for ZIKV and up to 10,000 times more sensitive than qRT-PCR. Assay validation was performed using 60 samples from Aedes aegypti and Culex quinquefasciatus mosquitoes collected in Pernambuco State, Brazil, which is at the epicenter of the Zika epidemic. The RT-LAMP had a sensitivity of 100%, specificity of 91.18%, and overall accuracy of 95.24%. Thus, our POC diagnostics is a powerful and inexpensive tool to monitor ZIKV in mosquito populations and will allow developing countries to establish better control strategies for this devastating pathogen.

Prevalence and risk factors of syphilis and human immunodeficiency virus co-infection at a university hospital in Brazil.

INTRODUCTION: The incidence of syphilis has increased since the 1970s. METHODS: This was a descriptive and analytical cross-sectional study with a non-probabilistic sample. RESULTS: Of 973 patients with human immunodeficiency virus, 179 (18.4%) tested positive for both human immunodeficiency virus and syphilis, 84.8% were men, 50.9% were aged between 36 and 50 years, 47.8% with syphilis were diagnosed with human immunodeficiency virus for 10-20 years, and 40.3% received antiretroviral therapy for 10-20 years. CONCLUSIONS: The prevalence of syphilis in patients with human immunodeficiency virus is higher than expected, making it urgent to adopt efficient public health measures.

Prevalence and risk factors of syphilis and human immunodeficiency virus co-infection at a university hospital in Brazil

Abstract INTRODUCTION The incidence of syphilis has increased since the 1970s. METHODS This was a descriptive and analytical cross-sectional study with a non-probabilistic sample. RESULTS: Of 973 patients with human immunodeficiency virus, 179 (18.4%) tested positive for both human immunodeficiency virus and syphilis, 84.8% were men, 50.9% were aged between 36 and 50 years, 47.8% with syphilis were diagnosed with human immunodeficiency virus for 10-20 years, and 40.3% received antiretroviral therapy for 10-20 years. CONCLUSIONS The prevalence of syphilis in patients with human immunodeficiency virus is higher than expected, making it urgent to adopt efficient public health measures.

Expression on hypoxia-inducible factor-1α in human tegumentary leishmaniosis caused by Leishmania braziliensis

Several immune markers have been studied in controlling American tegumentary leishmaniosis based on mouse models. However, there is a lack of studies regarding human tegumentary leishmaniosis caused by Leishmania braziliensis. In this study, hypoxia-inducible factor-1α was found to be an important effector element in the localized control of human cutaneous and mucocutaneous lesions.

Spatial risk analysis on occurrences and dispersal of Biomphalaria straminea in and endemic area for schistosomiasis.

BACKGROUND & OBJECTIVES: : Schistosomiasis is a rural endemic disease that has been expanding to urban and coastal areas in the state of Pernambuco, Brazil. The aim of this study was to characterize the distribution of breeding sites of the causative vector, Biomphalaria straminea in an endemic municipality for schistosomiasis and to present the predictive models for occurrences and dispersal of this vector snail to new areas. METHODS: : A malacological survey was conducted during January to December 2015 in the municipality of São Lourenço da Mata, Pernambuco, Brazil to identify the breeding sites of Biomphalaria. Faecal contamination was determined by means of the Colitag™ diagnostic kit. Rainfall data were collected, and correlated with snail distribution data. Kernel density estimation, kriging and maximum entropy (MaxEnt) modeling were used for spatial data analysis, by means of the spatial analysis software packages. RESULTS: : Out of the 130 demarcated collection points, 64 were classified as breeding sites for B. straminea. A total of 5,250 snails were collected from these sites. Among these 64 sites, four were considered as foci of schistosomiasis transmission and 54 as potential transmission foci. An inverse relationship between rainfall and snail density was observed. Kernel spatial analysis identified three areas at higher risk of snail occurrence, which were also the areas of highest faecal contamination and included two transmission foci. Kriging and MaxEnt modeling simulated the scenarios obtained through the kernel analyses. INTERPRETATION & CONCLUSION: : Use of geostatistical tools (Kriging and MaxEnt) is efficient for identifying areas at risk and for estimating the dispersal of Biomphalaria species across the study area. Occurrence of B. straminea in the study area is influenced by the rainy season, as it becomes more abundant during the period immediately after the rainy season, increasing the risk of dispersal and the appearance of new transmission foci.

Characterization of Leishmania (L.) infantum chagasi in visceral leishmaniasis associated with hiv co-infection in Northeastern Brazil.

Visceral leishmaniasis, associated with HIV/AIDS coinfection, is becoming a more aggressive disease, complicating an accurate prognosis. A 21-year-old HIV-positive female presenting with clinical features of visceral leishmaniasis was enrolled in this study. Bone marrow cytology, Novy-MacNeal-Nicolle culture and kDNA PCR of peripheral blood were all positive. Typing methods, multilocus enzyme electrophoresis and ITS1-RFLP PCR of peripheral blood confirmed infection by Leishmania (L.) infantum chagasi . PCR has proved to be safer and more affordable than other characterization methods; ITS1-RFLP PCR can diagnose and type Leishmania spp. in both endemic and non-endemic areas, favoring the prognosis and allowing the appropriate treatment of patients.

Sand fly population dynamics and cutaneous leishmaniasis among soldiers in an Atlantic forest remnant in northeastern Brazil.

Outbreaks of cutaneous leishmaniasis are relatively common among soldiers involved in nocturnal activities in tropical forests. We investigated the population dynamics of sand flies in a military training camp located in a remnant of Atlantic rainforest in northeastern Brazil, where outbreaks of cutaneous leishmaniasis have sporadically been described. From July 2012 to July 2014, light traps were monthly placed in 10 collection sites, being nine sites located near the forest edge and one near a sheep and goat stable. Light traps operated from 5:00 pm to 6:00 am, during four consecutive nights. Leishmania infection in sand flies was assessed using a fast real-time PCR assay. Cases of cutaneous leishmaniasis among soldiers were also investigated. In total, 24,606 sand flies belonging to 25 species were identified. Males (n = 12,683) predominated over females (n = 11,923). Sand flies were present during all months, being more numerous in March (n = 1,691) and April 2013 (n = 3,324). Lutzomyia choti (72.9%) was the most abundant species, followed by Lutzomyia longispina (13.8%), Lutzomyia complexa (5.3%), representing together >90% of the sand flies collected. Forty cases of cutaneous leishmaniasis were recorded among soldiers from January 2012 to December 2014. Leishmania isolates were obtained from eight patients and were all characterized as Leishmania braziliensis. Soldiers and anyone overnighting in Atlantic rainforest remnants should adopt preventative measures such as the use of repellents on bare skin or clothes and insecticide-treated tents.

Cerebral toxoplasmosis in patients with acquired immune deficiency syndrome in the neurological emergency department of a tertiary hospital.

INTRODUCTION: Cerebral toxoplasmosis is the most common cause of space occupying brain lesion in patients with HIV/AIDS in Brazil. In the post-HAART era, it is responsible for high rates of morbidity and mortality worldwide. MATERIALS AND METHODS: This study consists of a case series of 56 patients diagnosed with cerebral toxoplasmosis whose clinical features, brain imaging and cerebrospinal fluid aspects were analyzed. RESULTS: Cerebral toxoplasmosis led to the diagnosis of infection by the human immunodeficiency virus (HIV) in 27 (48.2%) of the patients, while 29 (51.2%) others already knew to be HIV seropositive. However, at the time of diagnosis of cerebral toxoplasmosis, only 9 (16.6%) reported being under antiretroviral therapy and 5 (8.9%) were receiving primary prophylaxis for toxoplasmosis. Headache, strength deficit and fever were the most frequent signs and symptoms throughout the study. Fifty-three patients showed changes consistent with toxoplasmosis in CT or MRI. Thirty-four (60.7%) CSF samples were positive in the indirect haemagglutination test and for the reaction of Toxoplasma gondii IgG ELISA, while 31 (55.4%) were positive in the direct haemagglutination test. Fifty (89.3%) patients underwent first-line treatment for toxoplasmosis. CONCLUSION: Cerebral toxoplasmosis is still a very relevant neurological disease in individuals with AIDS admitted to neurology emergency departments. Early diagnosis and initiation of empiric treatment and antiretroviral therapy are important for good prognosis.