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.

Immunogenicity, Effectiveness, and Safety of Inactivated Virus (CoronaVac) Vaccine in a Two-Dose Primary Protocol and BNT162b2 Heterologous Booster in Brazil (Immunita-001): A One Year Period Follow Up Phase 4 Study.

Background: Effective and safe vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are critical to controlling the COVID-19 pandemic and will remain the most important tool in limiting the spread of the virus long after the pandemic is over. Methods: We bring pioneering contributions on the maintenance of the immune response over a year on a real-life basis study in 1,587 individuals (18-90 yrs, median 39 yrs; 1,208 female/379 male) who underwent vaccination with two doses of CoronaVac and BNT162b2 booster after 6-months of primary protocol. Findings: Elevated levels of anti-spike IgG antibodies were detected after CoronaVac vaccination, which significantly decreased after 80 days and remained stable until the introduction of the booster dose. Heterologous booster restored antibody titers up to-1·7-fold, changing overall seropositivity to 96%. Titers of neutralising antibodies to the Omicron variant were lower in all timepoints than those against Delta variant. Individuals presenting neutralising antibodies against Omicron also presented the highest titers against Delta and anti-Spike IgG. Cellular immune response measurement pointed out a mixed immune profile with a robust release of chemokines, cytokines, and growth factors on the first month after CoronaVac vaccination followed by a gradual reduction over time and no increase after the booster dose. A stronger interaction between those mediators was noted over time. Prior exposure to the virus leaded to a more robust cellular immune response and a rise in antibody levels 60 days post CoronaVac than in individuals with no previous COVID-19. Both vaccines were safe and well tolerated among individuals. Interpretation: Our data approach the effectiveness of CoronaVac association with BNT162b2 from the clinical and biological perspectives, aspects that have important implications for informing decisions about vaccine boosters. Funding: Fiocruz, Brazil.

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.

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.

Suitability of commercially available POC-CCA tests for schistosomiasis: Considerations for efficiency, reproducibility and decision making criteria for field application in areas of low endemicity.

BACKGROUND: Point of care tests would be valuable for field diagnosis. However, high sensitivity will likely be required in low endemicity sets where individuals with low schistosome burden are hard to diagnose. METHODS: Commercially available POC tests (POC-CCA® and Urine CCA (Schisto) ECO Teste®) were evaluated to evidence their potential in low endemicity areas. Individuals with 0-76 eggs per gram of feces were selected, and comparison was performed between Kato-Katz, Saline Gradient and POC-CCA® after urine concentration (POC FLT) methods. RESULTS: Both POC-CCA had poor performances, showing low identification of less than half of positive individuals and several undiagnosed cases, revealing an accuracy of 0.44 and 0.46, and a Kappa Index of 0.308 and 0, respectively. Positivity rates of POC-CCA tests were below the one found for a single Kato-Katz slide. POC FLT had a Kappa Index of 0.617, an accuracy of 0.81, 67% of reproducibility, and was shown to have the same sensitivity of 21 Kato-Katz slides when two tests were performed. CONCLUSIONS: POC-CCA® and POC Eco presented exactly the same inadequacy in low endemicity areas. POC FLT significantly improved the performance of POC-CCA®. More accurate methods must be evaluated in low endemicity areas.

Innovative methodology for point-of-care circulating cathodic antigen with rapid urine concentration for use in the field for detecting low Schistosoma mansoni infection and for control of cure with high accuracy.

Background: Prior to eliminating schistosomiasis, efforts must address accurate and fast individual diagnosis. Diagnosis is still inaccurate by parasitological and point-of-care circulating cathodic antigen (POC-CCA) in areas of low endemicity. Methods: Our group has optimized POC-CCA with a 30 min urine concentration step with no need for specialized technicians or equipment and with high accuracy. We evaluated this new method, called POC-CCA filter (FLT), in two Brazilian endemic areas with distinct profiles. Results: At baseline, POC-CCA had a poor performance with several false results and undefined trace readings, revealing a prevalence rate of 10% against a rate of 23% for POC-CCA FLT, which was similar to the parasitological rates. Accuracy increased from as low as 0.36 to 0.96 after urine concentration in one area. POC-CCA properly diagnosed only half of the cases at three post-treatment time points, while POC-CCA FLT was able to diagnose 96, 83 and 100%, respectively. Conclusions: The improvement of conventional POC methodology by a fast and simple urine concentration step provided not only an increase in its accuracy before and after praziquantel treatment, but also preserved its applicability in low-prevalence endemic areas, allowing the definition of trace readings as negative cases.

Vaccine self-assembling immune matrix is a new delivery platform that enhances immune responses to recombinant HBsAg in mice.

Vaccination remains the most effective public health tool to prevent infectious diseases. Many vaccines are marginally effective and need enhancement for immunocompromised, elderly, and very young populations. To enhance immunogenicity, we exploited the biphasic property of the (RADA)4 synthetic oligopeptide to create VacSIM (vaccine self-assembling immune matrix), a new delivery method. VacSIM solution can easily be mixed with antigens, organisms, and adjuvants for injection. Postinjection, the peptides self-assemble into hydrated nanofiber gel matrices, forming a depot with antigens and adjuvants in the aqueous phase. We believe the depot provides slow release of immunogens, leading to increased activation of antigen-presenting cells that then drive enhanced immunogenicity. Using recombinant hepatitis B virus surface antigen (rHBsAg) as a model immunogen, we compared VacSIM delivery to delivery in alum or complete Freund's adjuvant (CFA). Delivery of the rHBsAg antigen to mice via VacSIM without adjuvant elicited higher specific IgG responses than when rHBsAg was delivered in alum or CFA. Evaluating IgG subtypes showed a mixed Th1/Th2 type response following immunization with VacSIM, which was driven further toward Th1 with addition of CpG as the adjuvant. Increased specific IgG endpoint titers were observed in both C57BL/6 and BALB/c mice, representative of Th1 and Th2 environments, respectively. Restimulation of splenocytes suggests that VacSIM does not cause an immediate proinflammatory response in the host. Overall, these results suggest that VacSIM, as a new delivery method, has the potential to enhance immunogenicity and efficacy of numerous vaccines.

Antigenic extracts of Leishmania braziliensis and Leishmania amazonensis associated with saponin partially protects BALB/c mice against Leishmania chagasi infection by suppressing IL-10 and IL-4 production.

This study evaluated two vaccine candidates for their effectiveness in protecting BALB/c mice against Leishmania chagasi infection. These immunogenic preparations were composed of Leishmania amazonensis or Leishmania braziliensis antigenic extracts in association with saponin adjuvant. Mice were given three subcutaneous doses of one of these vaccine candidates weekly for three weeks and four weeks later challenged with promastigotes of L. chagasi by intravenous injection. We observed that both vaccine candidates induced a significant reduction in the parasite load of the liver, while the L. amazonensis antigenic extract also stimulated a reduction in spleen parasite load. This protection was associated with a suppression of both interleukin (IL)-10 and IL-4 cytokines by spleen cells in response to L. chagasi antigen. No change was detected in the production of IFN-γ. Our data show that these immunogenic preparations reduce the type 2 immune response leading to the control of parasite replication.