SARS-CoV-2 intra-host diversity, antibody response, and disease severity after reinfection by the variant of concern Gamma in Brazil.

The rapid spread of the SARS-CoV-2 Variant of Concern (VOC) Gamma in Amazonas during early 2021 fueled a second large COVID-19 epidemic wave and raised concern about the potential role of reinfections. Very few cases of reinfection associated with the VOC Gamma have been reported to date, and their potential impact on clinical, immunological, and virological parameters remains largely unexplored. Here we describe 25 cases of SARS-CoV-2 reinfection in Brazil. SARS-CoV-2 genomic analysis confirmed that individuals were primo-infected with distinct viral lineages between March and December 2020 (B.1.1, B.1.1.28, B.1.1.33, B.1.195, and P.2) and reinfected with the VOC Gamma between 3 to 12 months after primo-infection. We found a similar mean cycle threshold (Ct) value and limited intra-host viral diversity in both primo-infection and reinfection samples. Sera of 14 patients tested 10-75 days after reinfection displayed detectable neutralizing antibodies (NAb) titers against SARS-CoV-2 variants that circulated before (B.1.*), during (Gamma), and after (Delta and Omicron) the second epidemic wave in Brazil. All individuals had milder or no symptoms after reinfection, and none required hospitalization. These findings demonstrate that individuals reinfected with the VOC Gamma may display relatively high RNA viral loads at the upper respiratory tract after reinfection, thus contributing to onward viral transmissions. Despite this, our study points to a low overall risk of severe Gamma reinfections, supporting that the abrupt increase in hospital admissions and deaths observed in Amazonas and other Brazilian states during the Gamma wave was mostly driven by primary infections. Our findings also indicate that most individuals analyzed developed a high anti-SARS-CoV-2 NAb response after reinfection that may provide some protection against reinfection or disease by different SARS-CoV-2 variants.

Low prevalence of influenza A strains with resistance markers in Brazil during 2017-2019 seasons.

The influenza A virus (IAV) is of a major public health concern as it causes annual epidemics and has the potential to cause pandemics. At present, the neuraminidase inhibitors (NAIs) are the most widely used anti-influenza drugs, but, more recently, the drug baloxavir marboxil (BXM), a polymerase inhibitor, has also been licensed in some countries. Mutations in the viral genes that encode the antiviral targets can lead to treatment resistance. Worldwide, a low prevalence of antiviral resistant strains has been reported. Despite that, this situation can change rapidly, and resistant strain surveillance is a priority. Thus, the aim of this was to evaluate Brazilian IAVs antiviral resistance from 2017 to 2019 through the identification of viral mutations associated with reduced inhibition of the drugs and by testing the susceptibility of IAV isolates to oseltamivir (OST), the most widely used NAI drug in the country. Initially, we analyzed 282 influenza A(H1N1)pdm09 and 455 A(H3N2) genetic sequences available on GISAID. The amino acid substitution (AAS) NA:S247N was detected in one A(H1N1)pdm09 strain. We also identified NA:I222V (n = 6) and NA:N329K (n = 1) in A(H3N2) strains. In addition, we performed a molecular screening for NA:H275Y in 437 A(H1N1)pdm09 samples, by pyrosequencing, which revealed a single virus harboring this mutation. Furthermore, the determination of OST IC50 values for 222 A(H1N1)pdm09 and 83 A(H3N2) isolates revealed that all isolates presented a normal susceptibility profile to the drug. Interestingly, we detected one A(H3N2) virus presenting with PA:E119D AAS. Moreover, the majority of the IAV sequences had the M2:S31N adamantanes resistant marker. In conclusion, we show a low prevalence of Brazilian IAV strains with NAI resistance markers, in accordance with what is reported worldwide, indicating that NAIs still remain an option for the treatment of influenza infections in Brazil. However, surveillance of influenza resistance should be strengthened in the country for improving the representativeness of investigated viruses and the robustness of the analysis.

Unusual SARS-CoV-2 intra-host diversity reveals lineages superinfection

Em períodos como o da presente pandemia de SARS-CoV-2, em que diversas linhagens e variantes de um mesmo vírus circulam simultaneamente em uma população, a ocorrência de coinfecções é sempre uma preocupação. Definidas como eventos nos quais uma mesma pessoa ou célula encontra-se infectada por duas ou mais amostras virais de perfil genético distinto, as coinfecções podem representar um risco à saúde coletiva caso tornem possíveis eventos de recombinação, ou seja, novos perfis genéticos virais derivados de uma "mescla" entre as linhagens genéticas que infectam o mesmo paciente. O presente trabalho, desenvolvido por pesquisadores de diversas unidades da Fiocruz vinculados à Rede Genômica e publicado sob a forma de preprint (sem revisão independente por outros pesquisadores), investiga o fenômeno das reinfecções com base em 2.263 amostras de SARS-CoV-2, utilizando métodos de análise com uso de computadores desenvolvidos pela própria Fiocruz. Estes métodos permitiram identificar sinais de alta variabilidade nos dados de sequenciamento do genoma, variabilidade esta associada ao sequenciamento simultâneo de mais de um perfil genético viral.

A Potential SARS-CoV-2 Variant of Interest (VOI) Harboring Mutation E484K in the Spike Protein Was Identified within Lineage B 1 1 33 Circulating in Brazil

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic in Brazil was dominated by two lineages designated as B.1.1.28 and B.1.1.33. The two SARS-CoV-2 variants harboring mutations at the receptor-binding domain of the Spike (S) protein, designated as lineages P.1 and P.2, evolved from lineage B.1.1.28 and are rapidly spreading in Brazil. Lineage P.1 is considered a Variant of Concern (VOC) because of the presence of multiple mutations in the S protein (including K417T, E484K, N501Y), while lineage P.2 only harbors mutation S:E484K and is considered a Variant of Interest (VOI). On the other hand, epidemiologically relevant B.1.1.33 deriving lineages have not been described so far. Here we report the identification of a new SARS-CoV-2 VOI within lineage B.1.1.33 that also harbors mutation S:E484K and was detected in Brazil between November 2020 and February 2021. This VOI displayed four non-synonymous lineage-defining mutations (NSP3:A1711V, NSP6:F36L, S:E484K, and NS7b:E33A) and was designated as lineage N.9. The VOI N.9 probably emerged in August 2020 and has spread across different Brazilian states from the Southeast, South, North, and Northeast regions.

Diet-induced obesity promotes systemic inflammation and increased susceptibility to murine visceral leishmaniasis.

Obesity is the main causal factor for metabolic syndrome and chronic systemic inflammation, which impacts on immune function and increases susceptibility to pathogens. Here, we investigated the effect of obesity on the outcome of visceral leishmaniasis caused by Leishmaniasis infantum chagasi. C57BL/6 mice fed with high-sugar and butter diet (HSB) showed a significant increase in body weight, adiposity index and morphological changes in adipocyte. To investigate the consequences of obesity on the specific immunity against Leishmania, both control and HSB diet groups were infected with 107 L. infantum chagasi promastigotes in the eighth-week after diet started and euthanized 4 weeks later. HSB-diet fed mice exhibited a significantly higher parasite burden in both liver and spleen compared with control- diet group. Gonadal adipocyte tissue from HSB-diet mice showed increased TNF-α, IL-6 and leptin and diminished IL-10 production compared with control. Cytokines production analysis in the spleen and liver from these animals also demonstrated higher production of IFN-γ, TNF-α, IL-6 and nitric oxide and diminished production of IL-10 and TGF-ß, which correlate with inflammatory foci and the cell hyperplasia observed. Taken together, obesity can interfere with responses to pathogen-derived signals and impair the development of protective anti-Leishmania immunity.

Intranasal vaccination with adjuvant-free S. aureus antigens effectively protects mice against experimental sepsis.

Staphylococcus aureus (S. aureus) is a Gram-positive coccal bacterium comprising part of the human skin, nares and gastrointestinal tract normal microbiota. It is also an important cause of nosocomial/community-acquired infections in humans and animals, which can cause a diverse array of infections, including sepsis, which is a progressive systemic inflammation response syndrome that is frequently fatal. The emergence of drug-resistant strains and the high toxicity of the treatments used for these infections point out the need to develop an effective, inexpensive and safe vaccine that can be used prophylactically. In this work, we used an experimental sepsis model to evaluate the effectiveness of whole antigens from S. aureus (SaAg) given by the intranasal route to induce protective immunity against S. aureus infection in mice. BALB/c mice were vaccinated via intranasal or intramuscular route with two doses of SaAg, followed by biocompatibility and immunogenicity evaluations. Vaccinated animals did not show any adverse effects associated with the vaccine, as determined by transaminase and creatinine measurements. Intranasal, but not intramuscular vaccination with SaAg led to a significant reduction in IL-10 production and was associated with increased level of IFN-γ and NO. SaAg intranasal vaccination was able to prime cellular and humoral immune responses and inducing a higher proliferation index and increased production of specific IgG1/IgG2, which contributed to decrease the bacterial load in both liver and the spleen and improve survival during sepsis. These findings present the first evidence of the effectiveness of whole Ag intranasal-based vaccine administration, which expands the vaccination possibilities against S. aureus infection.

Intranasal vaccination with killed Leishmania amazonensis promastigotes antigen (LaAg) associated with CAF01 adjuvant induces partial protection in BALB/c mice challenged with Leishmania (infantum) chagasi.

The CAF01 adjuvant has previously been shown to be safe for human use and to be a potent adjuvant for several vaccine antigens. In the present work, we sought to optimize the Leishmania amazonensis antigens (LaAg) intranasal vaccine in an attempt to enhance the protective immune responses against Leishmania (infantum) chagasi by using the CAF01 association. LaAg/CAF01 vaccinated mice that were challenged 15 days after booster dose with L. (infantum) chagasi showed a significant reduction in their parasite burden in both the spleen and liver, which is associated with an increase in specific production of IFN-γ and nitrite, and a decrease in IL-4 production. In addition, LaAg/CAF01 intranasal delivery was able to increase lymphoproliferative immune responses after parasite antigen recall. These results suggest the feasibility of using the intranasal route for the delivery of crude antigens and of a human-compatible adjuvant against visceral leishmaniasis.

Comparison of interferon-γ release assay to two cut-off points of tuberculin skin test to detect latent Mycobacterium tuberculosis infection in primary health care workers.

BACKGROUND: An interferon-γ release assay, QuantiFERON-TB (QFT) test, has been introduced an alternative test for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). Here, we compared the performance of QFT with tuberculin skin test (TST) measured at two different cut-off points among primary health care work (HCW) in Brazil. METHODS: A cross-sectional study was carried out among HCWs in four Brazilian cities with a known history of high incidence of TB. Results of the QFT were compared to TST results based on both ≥5 mm and ≥10 mm as cut-off points. RESULTS: We enrolled 632 HCWs. When the cut-off value of ≥10 mm was used, agreement between QFT and TST was 69% (k = 0.31), and when the cut-off of ≥5 mm was chosen, the agreement was 57% (k = 0.22). We investigated possible factors of discordance of TST vs QFT. Compared to the TST-/QFT- group, risk factors for discordance in the TST+/QFT- group with TST cut-off of ≥5 mm included age between 41-45 years [OR = 2.70; CI 95%: 1.32-5.51] and 46-64 years [OR = 2.04; CI 95%: 1.05-3.93], BCG scar [OR = 2.72; CI 95%: 1.40-5.25], and having worked only in primary health care [OR = 2.30; CI 95%: 1.09-4.86]. On the other hand, for the cut-off of ≥10 mm, BCG scar [OR = 2.26; CI 95%: 1.03-4.91], being a household contact of a TB patient [OR = 1.72; CI 95%: 1.01-2.92] and having had a previous TST [OR = 1.66; CI 95%: 1.05-2.62], were significantly associated with the TST+/QFT- group. No statistically significant associations were found among the TST-/QFT+ discordant group with either TST cut-off value. CONCLUSIONS: Although we identified BCG vaccination to contribute to the discordance at both TST cut-off measures, the current Brazilian recommendation for the initiation of LTBI treatment, based on information gathered from medical history, TST, chest radiograph and physical examination, should not be changed.

AIDS-associated paracoccidioidomycosis in a patient with a CD4+ T-cell count of 4 cells/mm³.

We describe a case of a patient presenting with HIV and paracoccidioidomycosis co-infection. At the time of diagnosis total CD4+ T-cell count was 4 cells/mm3. Histopathology revealed tuberculoid granulomas, scarce CD4+ T cells, a moderate number of CD8+ cells and the absence of Foxp3+ cells. Most of the cutaneous lesions healed after two weeks of treatment with amphotericin B. After 14 months the patient is still under antiretroviral therapy and no clinical evidence of recurrence of the mycosis has been observed.

AIDS-associated paracoccidioidomycosis in a patient with a CD4+ T-cell count of 4 cells/mm³ / Paracoccidioidomicose associada a Aids em paciente com a contagem total de quatro células T-CD4+

We describe a case of a patient presenting with HIV and paracoccidioidomycosis co-infection. At the time of diagnosis total CD4+ T-cell count was 4 cells/mm3. Histopathology revealed tuberculoid granulomas, scarce CD4+ T cells, a moderate number of CD8+ cells and the absence of Foxp3+ cells. Most of the cutaneous lesions healed after two weeks of treatment with amphotericin B. After 14 months the patient is still under antiretroviral therapy and no clinical evidence of recurrence of the mycosis has been observed.

Neste trabalho apresenta-se paciente com coinfecção paracoccidioidomicose/Aids. No momento do diagnóstico, a contagem de células T CD4 + era 4 células. No exame histopatológico, observou-se a presença de granulomas tuberculóides bem formados e na imunohistoquímica, ausência de células Foxp3, raros linfócitos T CD4+ e presença de células T CD8+ em moderada quantidade. Com duas semanas de uso da anfotericina B, verificou-se a regressão de grande parte das lesões cutâneas. Após 14 meses, o paciente encontra-se em uso de terapia antiretroviral e sem evidências de atividade da micose.

[Accuracy of inpatient and outpatient gastric lavage in the diagnosis of pulmonary tuberculosis in children]. / Acurácia do lavado gástrico realizado em ambiente hospitalar e ambulatorial no diagnóstico da tuberculose pulmonar em crianças.

OBJECTIVE: To compare gastric lavage (GL) performed in inpatients with GL performed in outpatients in terms of its accuracy in diagnosing pulmonary tuberculosis (TB) in children. METHODS: A prospective study was carried out in the state of Espírito Santo, Brazil, from 1999 to 2003. A total of 230 children suspected of having TB (103 inpatients and 127 outpatients) were selected to undergo GL. Those thus diagnosed with TB (n = 53) were divided into two groups: inpatient GL (n = 30) and outpatient GL (n = 23). All 53 children were monitored for 6 months in order to evaluate the accuracy of the diagnosis. Accuracy was determined based on any change in diagnosis, cure rate, and the percentage of positive cultures in the two groups studied. RESULTS: The cure rate was 100% in both groups, and there was no change in diagnosis in the 53 children studied. No significant difference was found between the two groups studied in terms of detection of Mycobacterium tuberculosis (RR = 1.47; 95% CI: 0.95-2.27; p = 0.095), although the outpatient group presented a greater rate of positive cultures. CONCLUSIONS: Our results show that the accuracy of GL performed in an inpatient setting is similar to that of GL performed in an outpatient setting. This indicates that hospitalization is required only in more severe cases in which GL cannot be performed as an outpatient procedure.

Acurácia do lavado gástrico realizado em ambiente hospitalar e ambulatorial no diagnóstico da tuberculose pulmonar em crianças / Accuracy of inpatient and outpatient gastric lavage in the diagnosis of pulmonary tuberculosis in children

OBJETIVO: Comparar a acurácia do lavado gástrico (LG) realizado em ambiente hospitalar e ambulatorial no diagnóstico da tuberculose (TB) pulmonar em crianças. MÉTODOS: Estudo prospectivo realizado no Estado do Espírito Santo, Brasil, de 1999 a 2003. Um total de 230 crianças com suspeita de TB foi selecionado para realizar exame de LG em ambiente hospitalar (n = 103) ou em ambiente ambulatorial (n = 127). Desse total, 53 foram diagnosticadas como casos de TB e divididas em dois grupos: LG hospitalar (n = 30) e LG ambulatorial (n = 23). Todas as 53 crianças foram monitoradas por 6 meses para avaliação da acurácia do diagnóstico. A acurácia foi determinada com base na mudança do diagnóstico, na taxa de cura e no percentual de culturas positivas nos dois grupos estudados. RESULTADOS: A taxa de cura foi de 100 por cento nos dois grupos, e não houve mudança de diagnóstico nas 53 crianças estudadas. Nenhuma diferença significativa foi encontrada entre os dois grupos estudados em relação ao achado do Mycobacterium tuberculosis (RR = 1,47; IC95 por cento: 0,95-2,27; p = 0,095), apesar de o grupo LG ambulatorial ter apresentado o maior índice de cultura positivas. CONCLUSÕES: Nossos resultados mostram que a acurácia do LG realizado em ambiente hospitalar é semelhante à do realizado em ambiente ambulatorial, o que indica que a internação é necessária apenas em casos mais graves nos quais não se pode realizar o procedimento em ambiente ambulatorial.

OBJECTIVE: To compare gastric lavage (GL) performed in inpatients with GL performed in outpatients in terms of its accuracy in diagnosing pulmonary tuberculosis (TB) in children. METHODS: A prospective study was carried out in the state of Espírito Santo, Brazil, from 1999 to 2003. A total of 230 children suspected of having TB (103 inpatients and 127 outpatients) were selected to undergo GL. Those thus diagnosed with TB (n = 53) were divided into two groups: inpatient GL (n = 30) and outpatient GL (n = 23). All 53 children were monitored for 6 months in order to evaluate the accuracy of the diagnosis. Accuracy was determined based on any change in diagnosis, cure rate, and the percentage of positive cultures in the two groups studied. RESULTS: The cure rate was 100 percent in both groups, and there was no change in diagnosis in the 53 children studied. No significant difference was found between the two groups studied in terms of detection of Mycobacterium tuberculosis (RR = 1.47; 95 percent CI: 0.95-2.27; p = 0.095), although the outpatient group presented a greater rate of positive cultures. CONCLUSIONS: Our results show that the accuracy of GL performed in an inpatient setting is similar to that of GL performed in an outpatient setting. This indicates that hospitalization is required only in more severe cases in which GL cannot be performed as an outpatient procedure.

The importance of the opossum (Didelphis albiventris) as a reservoir for Trypanosoma cruzi in Bambuí, Minas Gerais state

In a survey realized on the sylvatic and peridomestic environment at Bambuí county, Minas Gerais State, 44 (37.9%) out of 116 opossums (Didelphis albiventris) captured were found to be naturally infected with Trypanosoma cruzi. One handred and forty three parasite samples were obtanied from 43 infected opossums using simultaneously hemoculture, xenodiagnosis (Triatoma infestans, Panstrongylus megistus and Rhodnius neglectus) and examination of anal glands contents. The parasite samples were characterized according to six isoenzyme patterns. All samples, independently of the method of isolation, presented an isoenzyme pattern similar to the standard T. cruzi Z1, showing that either xenodiagnosis or hemoculture can used without selecting parasite subpopulation from naturally infected opossums. Preveous isoenzyme patterns reported for human T.cruzi isolates from same region were completely different. This isoenzyme dissimilarity between sylvatic and domiciliar environments suggests the exidstence of two independent T. cruzi transmission cycles in Bambuí. The epidemiological implicatinos of these results are discussed