Dot blot platform as a novel diagnostic kit: rapid, accurate, and on-site detection of Schistosoma mansoni in urine samples of hard to detect individuals.

Rapid diagnostics provide actionable information for patient care at the time and site of an encounter with the health care system. The mainstay of infectious diseases care is early detection (case finding) and treatment completion, but for many, it is hard to identify positive individuals, as is the case of infection with low burden in schistosomiasis, a parasitic disease common in the tropics and subtropics. We developed a new, accurate, and fast Dot blot methodology (iDot) to indirectly detect Schistosoma mansoni in individuals with very low parasite burden using urine samples. Accuracy of 0.74 was obtained with a significant difference between negative and positive patients and a substantial agreement was found when iDot was compared with five available methods. Our analysis also revealed the superiority of iDot in detecting negative individuals from non-endemic sites, thus, presenting the lowest rate of false positives. This new method called iDot is convenient and suitable for qualitative and quantitative detection of schistosomiasis in individuals with low parasite burden.

Toxoplasmosis in pregnancy: a neglected bane but a serious threat in Nigeria.

Toxoplasmosis is a global health threat in which occurrence in pregnant women poses grave consequences to fetal wellbeing. Studies on prenatal Toxoplasma gondii infection are generally limited in sub-Saharan African countries, including Nigeria. The risk of transmission of toxoplasmosis is very high in Nigeria due to the favourable climatic conditions and prevailing behavioural and socio-economic factors that could aid transmission. Currently, there are no systematic and organized procedures for diagnosis and treatment of maternal toxoplasmosis in Nigeria. These conditions forecast possible unabated transmission in many areas and exponential impact on associated adverse events of the disease during pregnancy. This paper highlights the importance of early diagnosis and treatment during pregnancy which may forestall subsequent development of infection in children delivered by infected mothers. Inclusion of toxoplasmosis control policy in the routine antenatal care of pregnant women is therefore strongly recommended.

Diagnosis of Schistosoma mansoni infections: what are the choices in Brazilian low-endemic areas?

The population of Brazil is currently characterised by many individuals harbouring low-intensity Schistosoma mansoni infections. The Kato-Katz technique is the diagnostic method recommended by the World Health Organization (WHO) to assess these infections, but this method is not sensitive enough in the context of low egg excretion. In this regard, potential alternatives are being employed to overcome the limits of the Kato-Katz technique. In the present review, we evaluated the performance of parasitological and immunological approaches adopted in Brazilian areas. Currently, the diagnostic choices involve a combination of strategies, including the utilisation of antibody methods to screen individuals and then subsequent confirmation of positive cases by intensive parasitological investigations.

Does the Capsule Interfere with Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Cryptococcus neoformans and Cryptococcus gattii?

We described the impact of the capsule size for Cryptococcus neoformans and Cryptococcus gattii identification at the species level by Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). After experimental capsule size modulation, we observed that reducing the capsule size resulted in improved identification by Bruker MALDI-TOF MS across all of the reference strains analyzed.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for differentiation of the dimorphic fungal species Paracoccidioides brasiliensis and Paracoccidioides lutzii.

Isolates of Paracoccidioides brasiliensis and Paracoccidioides lutzii, previously characterized by molecular techniques, were identified for the first time by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). All isolates were correctly identified, with log score values of >2.0. Thus, MALDI-TOF MS is a new tool for differentiating species of the genus Paracoccidioides.

Enhancing the epidemiological surveillance of SARS-CoV-2 using Sanger sequencing to identify circulating variants and recombinants.

Since the emergence of SARS-CoV-2 in December 2019, more than 12,000 mutations in the virus have been identified. These could cause changes in viral characteristics and directly impact global public health. The emergence of variants is a great concern due to the chance of increased transmissibility and infectivity. Sequencing for surveillance and monitoring circulating strains is extremely necessary as the early identification of new variants allows public health agencies to make faster and more effective decisions to contain the spread of the virus. In the present study, we identified circulating variants in samples collected in Belo Horizonte, Brazil, and detected a recombinant lineage using the Sanger method. The identification of lineages was done through gene amplification of SARS-CoV-2 by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). By using these specific fragments, we were able to differentiate one variant of interest and five circulating variants of concern. We were also able to detect recombinants. Randomly selected samples were sequenced by either Sanger or Next Generation Sequencing (NGS). Our findings validate the effectiveness of Sanger sequencing as a powerful tool for monitoring variants. It is easy to perform and allows the analysis of a larger number of samples in countries that cannot afford NGS.

Fluorescent quantum dot-based nanotool for targeted identification and evaluation of the schistosomiasis circulating cathodic antigen in tissue samples.

Schistosomiasis represents a serious public health problem, a disease for which the circulating cathodic antigen (CCA) is a relevant biomarker. Quantum dots (QDs) are advantageous fluorescent nanoparticles that can be used as specific nanoprobes. In this study, a nanotool based on QDs and anti-CCA antibodies was developed, which, in association with fluorescence microscopy, was applied to trace and evaluate the CCA profile in schistosomiasis-infected tissue samples. Kidney and liver tissues from mice at different disease phases were used as models. QDs and the conjugates were characterized by absorption and emission spectroscopies. Microscopy analyses were used to map and assess CCA accumulation in infected tissue slices in respect to non-infected control samples. The fluorescent microplate assay (FMA) and Zeta potential (ζ) analyses indicated an effective conjugation, which was corroborated by the absence of labeling in non-infected tissue slices (which lack CCA) after incubation with the nanoprobe. Infected liver and kidney tissues exhibited notable staining by the QDs-anti-CCA conjugate. The CCA accumulation increased as follows: 30 < 60 = 120 days post-infection, with 30, 60, and 120 days corresponding to the pre-patent, acute, and beginning of chronic disease phases, respectively. Therefore, this innovative approach, combining imaging acquisition with the sensitivity and specificity of the QDs-anti-CCA conjugate, demonstrated efficiency in locating and comparatively evaluating CCA deposition in biological samples, thereby opening new possibilities for schistosomiasis research.

Second booster dose improves antibody neutralization against BA.1, BA.5 and BQ.1.1 in individuals previously immunized with CoronaVac plus BNT162B2 booster protocol.

Introduction: SARS-CoV-2 vaccines production and distribution enabled the return to normalcy worldwide, but it was not fast enough to avoid the emergence of variants capable of evading immune response induced by prior infections and vaccination. This study evaluated, against Omicron sublineages BA.1, BA.5 and BQ.1.1, the antibody response of a cohort vaccinated with a two doses CoronaVac protocol and followed by two heterologous booster doses. Methods: To assess vaccination effectiveness, serum samples were collected from 160 individuals, in 3 different time points (9, 12 and 18 months after CoronaVac protocol). For each time point, individuals were divided into 3 subgroups, based on the number of additional doses received (No booster, 1 booster and 2 boosters), and a viral microneutralization assay was performed to evaluate neutralization titers and seroconvertion rate. Results: The findings presented here show that, despite the first booster, at 9m time point, improved neutralization level against omicron ancestor BA.1 (133.1 to 663.3), this trend was significantly lower for BQ.1.1 and BA.5 (132.4 to 199.1, 63.2 to 100.2, respectively). However, at 18m time point, the administration of a second booster dose considerably improved the antibody neutralization, and this was observed not only against BA.1 (2361.5), but also against subvariants BQ.1.1 (726.1) and BA.5 (659.1). Additionally, our data showed that, after first booster, seroconvertion rate for BA.5 decayed over time (93.3% at 12m to 68.4% at 18m), but after the second booster, seroconvertion was completely recovered (95% at 18m). Discussion: Our study reinforces the concerns about immunity evasion of the SARS-CoV-2 omicron subvariants, where BA.5 and BQ.1.1 were less neutralized by vaccine induced antibodies than BA.1. On the other hand, the administration of a second booster significantly enhanced antibody neutralization capacity against these subvariants. It is likely that, as new SARS-CoV-2 subvariants continue to emerge, additional immunizations will be needed over time.

Immunogenicity and safety of inactivated SARS-CoV-2 vaccine (CoronaVac) using two-dose primary protocol in children and adolescents (Immunita-002, Brazil): A phase IV six-month follow up.

Introduction: Vaccines are essential for the prevention and control of several diseases, indeed, monitoring the immune response generated by vaccines is crucial. The immune response generated by vaccination against SARS-CoV-2 in children and adolescents is not well defined regarding to the intensity and medium to long-term duration of a protective immune response, which may point out the need of booster doses and might support the decisions in public health. Objective: The study aims to evaluate the immunogenicity and safety of inactivated SARS-CoV-2 vaccine (CoronaVac) in a two-dose primary protocol in children and adolescent aging from 3 to 17 years old in Brazil. Methods: Participants were invited to participate in the research at two public healthcare centers located in Serrana (São Paulo) and Belo Horizonte (Minas Gerais), Brazil. Participants underwent medical interviews to gather their medical history, including COVID-19 history and medical records. Physical exams were conducted, including weight, blood pressure, temperature, and pulse rate measurements. Blood samples were obtained from the participants before vaccination, 1 month after the first dose, and 1, 3, and 6 months after the second dose and were followed by a virtual platform for monitoring post-vaccination reactions and symptoms of COVID-19. SARS-CoV-2 genome from Swab samples of COVID-19 positive individuals were sequenced by NGS. Total antibodies were measured by ELISA and neutralizing antibodies to B.1 lineage and Omicron variant (BA.1) quantified by PRNT and VNT. The cellular immune response was evaluated by flow cytometry by the quantification of systemic soluble immune mediators. Results: The follow-up of 640 participants showed that the CoronaVac vaccine (Sinovac/Butantan Institute) was able to significantly induce the production of total IgG antibodies to SARS-CoV-2 and the production of neutralizing antibodies to B.1 lineage and Omicron variant. In addition, a robust cellular immune response was observed with wide release of pro-inflammatory and regulatory mediators in the early post-immunization moments. Adverse events recorded so far have been mild and transient except for seven serious adverse events reported on VigiMed. Conclusions: The results indicate a robust and sustained immune response induced by the CoronaVac vaccine in children and adolescents up to six months, providing evidences to support the safety and immunogenicity of this effective immunizer.

Antischistosomal activity of a calcium channel antagonist on schistosomula and adult Schistosoma mansoni worms.

Current schistosomiasis control strategies are largely based on chemotherapeutic agents and a limited number of drugs are available today. Praziquantel (PZQ) is the only drug currently used in schistosomiasis control programs. Unfortunately, this drug shows poor efficacy in patients during the earliest infection phases. The effects of PZQ appear to operate on the voltage-operated Ca2+ channels, which are located on the external Schistosoma mansoni membrane. Because some Ca2+ channels have dihydropyridine drug class (a class that includes nifedipine) sensitivity, an in vitro analysis using a calcium channel antagonist (clinically used for cardiovascular hypertension) was performed to determine the antischistosomal effects of nifedipine on schistosomula and adult worm cultures. Nifedipine demonstrated antischistosomal activity against schistosomula and significantly reduced viability at all of the concentrations used alone or in combination with PZQ. In contrast, PZQ did not show significant efficacy when used alone. Adult worms were also affected by nifedipine after a 24 h incubation and exhibited impaired motility, several lesions on the tegument and intense contractility. These data support the idea of Ca2+ channels subunits as drug targets and favour alternative therapeutic schemes when drug resistance has been reported. In this paper, strong arguments encouraging drug research are presented, with a focus on exploring schistosomal Ca2+ channels.

Schistosoma mansoni in a low-prevalence area in Brazil: the importance of additional methods for the diagnosis of hard-to-detect individual carriers by low-cost immunological assays.

Schistosomiasis diagnosis is based on the detection of eggs in the faeces, which is laborious and lacks sensitivity, especially for patients with a low parasite burden. Immunological assays for specific antibody detection are available, but they usually demonstrate low sensitivity and/or specificity. In this study, two simple immunological assays were evaluated for the detection of soluble Schistosoma mansoni adult worm preparation (SWAP) and egg-specific IgGs. These studies have not yet been evaluated for patients with low parasite burdens. Residents of an endemic area in Brazil donated sera and faecal samples for our study. The patients were initially diagnosed by a rigorous Kato-Katz analysis of 18 thick smears from four different stool samples. The ELISA-SWAP was successful for human diagnosis with 90% sensitivity and specificity, confirming the Kato-Katz diagnosis with nearly perfect agreement, as seen by the Kappa index (0.85). Although the ELISA-soluble S. mansoni egg antigen was 85% sensitive, it exhibited low specificity (80%; Kappa index: 0.75) and was more susceptible to cross-reactivity. We believe that immunological assays should be used in conjunction with Kato-Katz analysis as a supplementary tool for the diagnosis of schistosomiasis for patients with low infection burdens, which are usually hard to detect.

Access to COVID-19 vaccines and testing in Africa: the importance of COVAX - Nigeria as a case study.

Since pandemic declaration, the vulnerability of countries with serious economic challenges and limited health resources became evident. This vulnerability has been put to the test once again with the appearance of Omicron as another variant of concern. Although great efforts have been made to develop effective and safe vaccines, they need to be made available globally at an affordable price to all governments and distributed equitably to maximize immediate and long-term efforts to contain the virus and advance global public health. Potential sources of unfair allocation of COVID-19 vaccines are not hard to find. The COVID-19 Vaccine Global Access Facility (COVAX) has so far shipped over 406 million COVID-19 vaccines to 144 eligible participants. From that batches, about 115 million doses (28%) were allocated to 49 African countries. If proactive measures are not undertaken, Nigeria, pointed here as a case study, and Sub-Saharan Africa countries may not be self-reliant for COVID-19 vaccines. This report raises a discussion on the difficulties in accessing vaccines and diagnostics in sub-Saharan Africa, compared to high- and middle-income countries. Now more than ever, it is crucial to note that there is no overcoming a pandemic without coordinated action for actions that go beyond borders. The coordinated effort to raise vaccination rates in the African continent is not a humanitarian action aimed exclusively at Africa, but more than that, it is an effort for the benefit of global public health.

Variant-dependent oxidative and cytokine responses of human neutrophils to SARS-CoV-2 spike protein and anti-spike IgG1 antibodies.

Introduction: Severe forms of COVID-19, the disease caused by SARS-CoV-2, are characterized by acute respiratory distress syndrome, robust lung inflammation and death in some patients. Strong evidence has been accumulating that polymorphonuclear neutrophilic granulocytes (PMN) play an important role in the pathophysiology of severe COVID-19. SARS-CoV-2 directly induces in vitro PMN activation, mainly the release of neutrophil extracellular traps (NETs). However, the viral components inducing this PMN response remain unclear. Methods: In this work human PMN responses were assessed in vitro in response to the spike (S) protein of two different SARS-CoV-2 variants, anti-S IgG1 antibodies or immune complexes formed by them. Production of reactive oxygen species (ROS) was measured by Diogenes-based chemiluminescence. Release of myeloperoxidase (MPO) was assessed by ELISA while secretion of a list of cytokines and growth factors was determined by high-performance multiplex cytokine assay. Results and discussion: We show that the SARS-CoV-2 Omicron variant S protein and anti-spike IgG1, either alone or together, stimulate ROS production in human PMNs. We also observed that the SARS-CoV-2 Wuhan S protein and anti-S IgG1 antibody together trigger MPO release from PMNs. Based on the relevance of SARS-CoV-2 and influenza co-infections, we have also investigated the impact of influenza virus infection on the previous PMN responses to S proteins or anti-S antibodies. We did not detect any significant effect of influenza co-infection on ROS generation in PMNs. Our data also show that PMN stimulation by S proteins induced the release of different chemokines, growth factors, regulatory and proinflammatory cytokines. Overall, our findings show that the SARS-CoV-2 S protein, an anti-spike IgG1 antibody or their immune complex, promote oxidative responses of PMNs in a variant-dependent manner, contributing to a better understanding of the role of PMN responses during SARS-CoV-2 infection.

COVID-19 in patients with chronic kidney disease: a 2-year study of incidence and mortality in relation to the variants of concern waves in Brazil.

BACKGROUND: Chronic kidney disease (CKD) is a risk factor for severe coronavirus disease (COVID-19). In Brazil, the disease is the 10th highest cause of death. We evaluated the epidemiological impact of COVID-19 in CDK and non-CDK patients. METHODS: Positive patients for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from 2020 to 2022 were classified according to the severity of COVID-19 and the numbers of cases and deaths were correlated to each wave of SARS-CoV-2 variants in Brazil. RESULTS: We compared all variables, and our data show that CDK significantly increased the mortality rate among patients, especially before COVID-19 vaccination, in comparison with non-CKD patients. CONCLUSIONS: CKD patients had a significantly increased mortality rate compared with non-CKD.

Antibody response and soluble mediator profile in the first six months following acute SARS-CoV-2 infection.

The COVID-19 pandemic has caused a severe global health and economic crisis, with significant consequences for human mortality and morbidity. Therefore, there is an urgent need for more studies on the immune response to SARS-CoV-2 infection, both to enhance its effectiveness and prevent its deleterious effects. This study presents the chronology of antibodies during six months after infection in hospitalized patients and the kinetics of serum soluble mediators of the cellular response triggered by SARS-CoV-2. Samples and clinical data from 330 patients hospitalized at the Hospital da Baleia in Belo Horizonte, Brazil, who were suspected of having COVID-19, were collected at the time of hospitalization and during 6 months after infection. The immune response was analyzed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. There was a significant difference in IgM specific antibody titers from the 7th to 60th days after infection between COVID-19 negative and positive patients. Soon after 60 days after infection, antibody levels started to reduce, becoming similar to the antibody levels of the COVID-19 negative patients. IgG specific antibodies started to be detectable after 9 days of infection and antibody levels were comparatively higher in positive patients as soon as after 7 days. Furthermore, IgG levels remained higher in these patients during the complete period of 180 days after infection. The study observed similar antibody profiles between different patient groups. The soluble systemic biomarkers evaluated showed a decrease during the six months after hospitalization, except for CCL11, CXCL8, CCL3, CCL4, CCL5, IL-6, IFN-g, IL-17, IL-5, FGF-basic, PDGF, VEGF, G-CSF, and GM-CSF. The results indicate that IgM antibodies are more prominent in the early stages of infection, while IgG antibodies persist for a longer period. Additionally, the study identified that patients with COVID-19 have elevated levels of biomarkers after symptom onset, which decrease over time.

Immunodiagnostic methods: what is their role in areas of low endemicity?

Worldwide Schistosomiasis mansoni continues to be a serious public health problem. Over the past decades, control programmes have made remarkable progress in reducing S. mansoni infections to a relatively low level in Brazil and African countries. Endemic regions are currently circumscribed in certain core areas where reinfection and repeated chemotherapy are frequent and, consequently, are related to residents with low parasite load. At present, diagnosis is predominately a key step for final disease control although low endemicity area residents are hardly detected by most of the available assays. In this paper, we review the current status and efforts made aiming at the improvement of diagnostic tools for S. mansoni in low endemicity infections. The establishment of diagnostic assays--simple, affordable, sensitive, and specific for field diagnosis of S. mansoni--is essential and should be given high priority.

Diagnostic and therapeutic biomarkers in colorectal cancer: a review.

Colorectal cancer (CRC) is a public health concern and the second most common type of cancer among men and women causing a significant mortality. Biomarkers closely linked to the disease morbidity could holds potential as diagnostic and/or prognostic biomarker for the disease. This review provides an overview of recent advances in the search for colorectal cancer biomarkers through genomics and proteomics according to clinical function and application. Specifically, a number of biomarkers were identified and discussed. Emphasis was placed on their clinical applications relative to the diagnosis and prognosis of CRC. The discovery of more sensitive and specific markers for CRC is an urgent need, and the study of molecular targets is extremely important in this process, as they will allow for a better understanding of colorectal carcinogenesis, identification and validation of potential genetic signatures.

DNA aptamer selection and construction of an aptasensor based on graphene FETs for Zika virus NS1 protein detection.

Zika virus (ZIKV) is a mosquito-borne virus that is phylogenetically close to other medically important flaviviruses with high global public health significance, such as dengue (DENV) and yellow fever (YFV) viruses. Correct diagnosis of a flavivirus infection can be challenging, particularly in world regions where more than one flavivirus co-circulates and YFV vaccination is mandatory. Acid nucleic aptamers are oligonucleotides that bind to a specific target molecule with high affinity and specificity. Because of their unique characteristics, aptamers are promising tools for biosensor development. Aptamers are usually obtained through a procedure called "systematic evolution of ligands by exponential enrichment" (SELEX). In this study, we select an aptamer (termed ZIKV60) by capillary electrophoresis SELEX (CE-SELEX) to the Zika virus non-structural protein 1 (NS1) and counterselection against the NS1 proteins of DENV (serotypes 1, 2, 3, and 4) and YFV. The ZIKV60 dissociation constant (K d) is determined by enzyme-linked oligonucleotide assay (ELONA) and the aptamer specificity is evaluated by quantitative real-time polymerase chain reaction. ZIKV60 shows a high binding affinity to the ZIKV NS1 protein with a K d value of 2.28 ± 0.28 nM. The aptamer presents high specificity for ZIKV NS1 compared to NS1 of DENV and YFV. Furthermore, graphene field-effect transistor devices functionalized with ZIKV60 exhibit an evident identification of NS1 protein diluted in human serum. These results point to the applicability of biosensors based on the ZIKV60 aptamer for the differential diagnosis of the Zika virus.

Long COVID-19 syndrome: a 14-months longitudinal study during the two first epidemic peaks in Southeast Brazil.

BACKGROUND: A growing number of long COVID cases after infection have been reported. By definition, long COVID is the condition whereby affected individuals do not recover for several weeks or months following the onset of symptoms suggestive of COVID-19, the profile and timeline of which remains uncertain. METHODS: In this work, in-home, outpatient and hospitalized COVID-19 positive patients were monitored for up to 14 mo to establish the prevalence of long COVID symptoms and their correlation with age, pre-existing comorbidities and course of acute infection. The longitudinal study included 646 positive patients who were monitored once a month. RESULTS: From the whole population, 50.2% presented with long COVID syndrome. Twenty-three different symptoms were reported. Most frequent were fatigue (35.6%), persistent cough (34.0%), dyspnea (26.5%), loss of smell/taste (20.1%) and frequent headaches (17.3%). Mental disorders (20.7%), change in blood pressure (7.4%) and thrombosis (6.2%) were also reported. Most patients presented with 2-3 symptoms at the same time. Long COVID started after mild, moderate and severe infection in 60, 13 and 27% of cases, respectively, and it was not restricted to specific age groups. CONCLUSIONS: Older patients tended to have more severe symptoms, leading to a longer post-COVID-19 period. The presence of seven comorbidities was correlated with the severity of infection, and severity itself was the main factor that determined the duration of symptoms in long COVID cases.

Booster dose of BNT162b2 after two doses of CoronaVac improves neutralization of SARS-CoV-2 Omicron variant.

Background: The emergence of the new SARS-CoV-2 Omicron variant, which is known to have a large number of mutations when compared to other variants, brought to light the concern about vaccine escape, especially from the neutralization by antibodies induced by vaccination. Methods: Based on viral microneutralization assays, we evaluated in 90 individuals the impact on antibody neutralization induction, against Omicron variant, by a booster dose of BNT162b2 mRNA vaccine after the CoronaVac primary vaccination scheme. Results: Here we show that the percentage of seroconverted individuals 30 and 60 days after CoronaVac scheme was 16.6% and 10%, respectively. After booster dose administration, the seroconvertion rate increased to 76.6%. The neutralization mean titer against Omicron in the CoronaVac protocol decreased over time, but after the booster dose, the mean titer increased 43.1 times. Conclusions: These results indicate a positive impact of this vaccine combination in the serological immune response against SARS-CoV-2 Omicron variant.