M.Tech. (Homoeopathy) / The common cold and influenza (flu) are upper respiratory tract conditions; the former characterized by nasal or throat discomfort, sneezing, rhinorrhoea, and malaise, and the latter by fever, coryza, anorexia, cough, headache, malaise and myalgia (Beers et al., 2006). The common cold can be caused by over 200 rhinoviruses while influenza is mostly due to influenza virus A or B. Cold symptoms usually clear after 7-10 days, influenza which is more severe lasts for 2-3 weeks (Eccles, 2005). Athletes are susceptible to common colds and influenza infections due to intensive physical activities and stress, which lowers their immune systems and increases the risk of spread among team-mates. To avoid missing competitions and practice, it would be advantageous for athletes to receive prophylactic treatment during the high risk period for colds and flu such as in winter (Brukner and Khan, 2006). Pelargonium sidoides, which is an ingredient of Linctagon® spray, is a well-researched plant extract which has antiviral, antibacterial and immune-modulator effects (Nativa, 2011). Its effect as a prophylactic for athletes has not been researched. The aim of this study was to determine the efficacy of Linctagon® spray in preventing common colds and influenza in female soccer teams of the University of Johannesburg. This was a double-blind, placebo-controlled study, which took place over 63 days. Thirty female participants aged between 18-30 years were recruited from the University of Johannesburg female soccer teams via direct recruitment during training sessions on the University of Johannesburg Bunting campus. Participants meeting the inclusion criteria completed the Participant Information, Profile and Consent Forms. On day 1, the participants underwent a physical examination (vitals, ear, nose, throat and chest examinations). Participants either received a 20 ml bottle of Linctagon® spray or a placebo spray. Five squirts were taken orally twice daily for nine weeks. Participants also received three Wellness Questionnaires and Health Questionnaires to complete at home weekly and returned these at the following visits. On day 21 and day 42, the researcher collected the completed Questionnaires, performed a physical examination, dispensed and gave participants additional medication, and three additional Wellness and Health Questionnaires. At the final consultation, day 63, the researcher collected the questionnaires, and did a fourth and final physical examination. The data was collected from the participants and analyzed according to group frequencies and independent T-tests. The outcome indicated that the placebo was equally as effective as Linctagon® spray in the prophylaxis of common cold and influenza infections in female soccer team players of the University of Johannesburg for the duration of the study.

Please read the abstract in the section 00front of this document / Thesis (DPhil (Medical Immunology))--University of Pretoria, 2006. / Immunology / unrestricted

Streptococcus pneumoniae é o agente causador de diversas doenças, tais como meningite e pneumonia. PspC (Pneumococcal surface protein C) foi descrito como um importante candidato vacinal proteico de ampla cobertura e com baixo custo de produção. Trata-se de um fator de virulência, capaz de ligar-se ao Fator H (FH) e a IgA secretório (sIgA). Como PspC é um antígeno polimórfico, é crucial a avaliação da sua variabilidade. Foi determinado o grupo de PspC de treze linhagens de pneumococo isoladas no Hospital Universitário da Universidade de São Paulo. Soros contra diferentes grupos de PspC foram produzidos e PspC do grupo 3 (PspC3) foi capaz de induzir anticorpos que reconhecem diferentes grupos de PspC. Foi observada ainda uma pequena redução na ligação de FH e sIgA por anticorpos anti-PspC3 em ensaios in vitro. No entanto, não foi possível observar proteção contra um modelo de colonização da nasofaringe de camundongos através da imunização com PspC3, possivelmente por deficiências no modelo experimental. / Streptococcus pneumoniae is the causative agent of several diseases, such as meningitis and pneumonia. PspC (Pneumococcal surface protein C) has been described as an important vaccine candidate protein as it could provide wide coverage with low cost of production. PspC is a virulence factor capable of binding to Factor H (FH) and secretory IgA (sIgA). PspC is polymorphic antigen, and therefore it is crucial to evaluate its variability. In the present work we have determined the PspC group of 13 pneumococcal isolates obtained at the University Hospital of the University of São Paulo. Antisera against different PspC groups were produced and PspC group 3 (PspC3) was able to induce antibodies that recognized different groups of PspC. Antibodies to PspC3 reduced binding of FH and sIgA to pneumococcus in in vitro assays. However, no protection was observed against a murine model of nasopharyngeal colonization by immunization with PspC3. This was possibly due to deficiencies in the experimental model.

Thesis (Ph.D.(Microbiology)) -- University of Limpopo, 2022 / Respiratory tract infections (RTIs) are frequent ailments among humans and are a high burden to public health. One strategy for the development of new therapies against pathogenic bacteria such as Mycobacterium tuberculosis is to target essential biosynthetic pathways of its metabolism. The aim of this study was to evaluate and target the biosynthesis of aromatic amino acids (shikimate pathway) of Mycobacterial spp using medicinal plant extracts. The selection of the plants in this study was based on their ethnopharmacological use for the treatment of tuberculosis infections and related symptoms. The leaves were dried at ambient temperatures and ground to fine powder. The powdered material was extracted with hexane, dichloromethane, acetone, methanol and water. Phytochemical screening was done using standard protocols that tested for tannins, saponins, terpenoids, alkaloids, flavonoids, steroids, anthraquinones, phlobatannins, quinones, and betacynins. Phytochemical fingerprints were established using thin layer chromatography (TLC) where three mobile phases varying in polarity were used to develop the chromatograms. Total Phenolics, flavonoids, flavonols, tannins, alkaloids and proanthocyanidin contents were quantified using UV/Vis spectrometry. Spectrometric quantification of the free radical (DPPH) scavenging activity and ferric (potassium ferricyanide) reducing power were performed. The heat-dependent bovine serum albumin and egg albumin denaturation assays were used to evaluate anti-inflammatory activity. Antimycobacterial activity was screened using bioautography assay in qualitative analysis. Quantitatively, broth microdilution assay was used to determine the minimal inhibitory concentrations. The Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 interference genetic editing technique was used to evaluate and validate the essentiality of the aromatic amino acids in Mycobacteria to further determine the vulnerability and draggability of the transketolase (tkt) and DAHPs (aroG) genes. Plasmid, PLJR962, was used for the CRISPRi/dCas9 gene knockdown experiments. The integrating CRISPRi plasmid expressed both sgRNA with the targeting region (for tkt or aroG) and the dCas9 handle which is under control of the anhydrotetracycline (ATC) inducible promoters. The spot assay and growth curves were used to for phenotypic characterisation and gene knockdown experiments. RNA microarray (qPCR) was used to evaluate the level of expression inhibition of tkt gene . Mechanism of action of plants extracts bioactive components were predicted based on synergy between gene knockdown, shikimate inhibitors and the plant extracts. To evaluate whether the shikimate intermediates may rescue gene depleted M. smegmatis hypomorphs, the cultures were grown in L-tryptophan, L-phenylalanine, L-tyrosine and shikimic acid and growth curves constructed. Cytotoxicity of the extracts was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on Vero cell lines and phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 macrophages. Phytochemical analysis showed that the various extracts had various polar and non-polar compounds which belonged to phenolics, saponins, steroids, terpenoids, alkaloids, cardiac glycosides and resins. Numerous non-polar compounds from Gardernia volkensii, Senna petersiana, Ficus sur had antimycobacterial activity against M. smegmatis in bioautography. Remarkably, acetone extracts from S. petersiana, Acacia senegal, Carissa bispinosa, P. africanum and C. gratissimus that had moderate to low antimycobacterial activity against wild type M. smegmatis (mc2 155) demonstrated improved inhibitory activity against the tkt PAM1 M. smegmatis CRISPRi mutant. Only the acetone Clerodendrum glabrum, Croton gratissimus, Peltophorum africanum and Gardenia volkensii demonstrated activity against M. tuberculosis H37Rv. These results suggest that the employment of CRISPRi in M. tuberculosis to develop screening models may increase changes of obtaining bioactive chemical species because the tkt gene knockdown was showed to possess the ability to potentiate the antimycobacterial activity of the plant extracts. An added advantage of the plant extracts is their antioxidant and anti-inflammatory activities which may benefit the host immune system during treatment of infection by reducing free radicals and pro-inflammatory agents that perpetuate the infection. Non polar compounds were found to generally have higher anti-inflammatory activity than the polar counterpart for all the plant extracts. These results suggest that the non-polar compounds from the tested extracts may not only confer antimycobacterial effects, but also anti-inflammatory activities. A. senegal, G. volkensii, F. sur, S. petersiana and C. glabrum were found to be toxic to the Vero cell line. However, purification techniques may circumvent their toxic effects. This study demonstrated that the amino acid biosynthesis is a potential antimycobacterial drug target because it was found to be essential, vulnerable and druggable by medicinal plant extracts / University of Limpopo and National Research Foundation (NRF-DAAD In-Country Doctoral Scholarship Programme)

Levels of Immunoglobulin G (IgG)-subclasses are analyzed when patients have reoccurring infections and in order to follow the treatment of IgG4 related disease. The measured quantity of IgG-total can be within the reference interval even if the patient has a deficiency in one of the IgG-subclasses. Due to this Sundsvall’s hospital plans to begin analyzing IgG-subclasses. The aim was to verify the performance level of the analysis IgG-subclasses (IgG1-4) with Siemens Atellica NEPH 630. The method was verified by evaluating the method’s precision and by comparing the sum of IgG-subclasses with the quantity of IgG-total analyzed on Cobas c502. In addition, the reference intervals provided by Siemens were evaluated using samples from blood donors. The study evaluated whether there was a correlation between infection with SARS-CoV2 and a deficiency in IgG-subclasses. The verification began by performing quality control twice daily over a period of four weeks. The IgG-subclasses test was performed on blood donor samples with Siemens Atellica NEPH 630 for evaluation of the reference values. The coefficient of variation was less than 6 % for all four subclasses. The reference values for IgG1, IgG2, IgG3, and IgG4 are all in alliance with the reference values provided by Siemens. The sum of IgG-subclasses corresponded well with IgG-total with a correlation value (R) 0.82. No correlation was found between IgG deficiency and infection with SARS-CoV2. The validation of the analysis of IgG-subclasses was successful with quality measurements within the supplier’s intervals. No adjustments will be needed for the reference intervals.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has proved to be the greatest global crisis of the 21st century and has led to a devastating state of human health and societal infrastructure. Such calamity was met with immense determination from the scientific community to uncover the immunological and virological basis of its accompanying disease and resulted in remarkable feats of public health response and therapeutic design. As SARS-CoV-2 continues to evolve and elicits a heterogenous disease presentation across different demographics, we aimed to define the circulating and tissue-localized immune memory generated following SARS-CoV-2 infection, as well as determine the immunological properties governing severe disease. Using human tissues from seropositive organ donors, we showed that SARS-CoV-2-specific immune memory was present in circulation, lymphoid, and mucosal sites up to 6 months post infection. B and T cell populations mounted against SARS-CoV-2 showed significant correlations between circulating and tissue-resident memory lymphocytes, suggesting local and systemic tissue coordination of cellular and humoral immunity against SARS-CoV-2, set for optimal protection against future infectious challenges. Next, we presented a comprehensive, longitudinal study of the peripheral blood immune system following pediatric SARS-CoV-2 infection and provided new insight on the immunological underpinnings of multisystem inflammatory syndrome in children (MIS-C). Acute MIS-C and pediatric COVID-19 differ in their effector module elicitation, activating opposing type 1 and type 2 immune responses respectively. We reveal that MIS-C presents with a unique peripheral T cell signature marked by activation, exhaustion, and tissue-residency at the proteomic and transcriptional level, along with a major Vβ-biased clonal expansion. Despite the considerable immune dysregulation during acute disease, children recovered from MIS-C maintain stable humoral immunity up to 18 months post hospitalization at comparable levels to seropositive groups, and generate robust, functional T cell memory in greater magnitude than seropositive children. Together, we report a near-complete restoration in global T cell phenotype and function in children following MIS-C, as well as the robust production of competent SARS-CoV-2 specific memory. Finally, following our queries into SARS-CoV-2-specific antiviral immunity, we sought to delineate the dynamics of human follicular immune responses and its role in generating and maintaining humoral immunity across a lifespan. Using healthy pediatric and adult donor tissues to examine blood, lymphoid, and mucosal tissues, our results reveal that TFH cells predominate the CD4+ T-cell memory pool in lymphoid sites in early life and decline in frequency with age. Further, pediatric and adult TFH cells differ in their functional capacities, with pediatric TFH cells expressing higher levels of markers associated with signal regulation and germinal center function, while adult TFH cells demonstrate a TH17-like identity. Further, early life TFH cells in lymphoid exhibit marked TCR repertoire overlap. Together, these results indicate a differential propensity for follicular responses in early life and adulthood, with important implications in considering immunomodulatory strategies in different life stages.

Rhinoviruses (RVs) constitute a substantial public health burden. To evaluate their abundance and genetic diversity in adult patients, RV RNA in respiratory samples was assessed using real-time RT-PCR and the partial nucleic acid sequencing of viral genomes. Additionally, clinical data were retrieved from patient charts to determine the clinical significance of adult RV infections. In total, the respiratory specimens of 284 adult patients (18–90 years), collected from 2013 to 2017, were analyzed. Infections occurred throughout the entire year, with peaks occurring in fall and winter, and showed a remarkably high intra- and interseasonal diversity of RV genotypes. RV species were detected in the following ratios: 60.9% RV-A 173, 12.7% RV-B, and 26.4% RV-C. No correlations between RV species and underlying comorbidities such as asthma (p = 0.167), COPD (p = 0.312) or immunosuppression (p = 0.824) were found. However, 21.1% of the patients had co-infections with other pathogens, which were associated with a longer hospital stay (p = 0.024), LRTI (p < 0.001), and pneumonia (p = 0.01). Taken together, this study shows a pronounced genetic diversity of RV in adults and underlines the important role of co-infections. No correlation of specific RV species with a particular clinical presentation could be deduced.

Rhinoviruses (RVs) constitute a substantial public health burden. To evaluate their abundance and genetic diversity in pediatric patients, RV RNA in respiratory samples was assessed using real-time RT-PCR and partial nucleic acid sequencing of viral genomes. Additionally, clinical data were retrieved from patient charts to determine the clinical significance of pediatric RV infections. In total, the respiratory specimens of 776 patients (<18 years), collected from 2013 to 2017, were analyzed. Infections occurred throughout the entire year, with peaks occurring in fall and winter, and showed remarkably high intra- and interseasonal diversity for RV genotypes. RV species were detected in the following frequencies: 49.1% RV-A, 5.9% RV-B, and 43.6% RV-C. RV-C was found to be more frequently associated with asthma (p = 0.04) and bronchiolitis (p < 0.001), while RV-A was more frequently associated with fever (p = 0.001) and pneumonia (p = 0.002). Additionally, 35.3% of the patients had co-infections with other pathogens, which were associated with a longer hospital stay (p < 0.001), need for ventilation (p < 0.001), and pneumonia (p < 0.001). Taken together, this study shows pronounced RV genetic diversity in pediatric patients and indicates differences in RV-associated pathologies, as well as an important role for co-infections.

Thesis (PhD (Microbiology))--University of Stellenbosch, 2009. / Multidrug resistant strains of Staphylococcus aureus is presenting an increasing threat, especially immune compromised individuals. Many of these strains have developed resistance to newly approved drugs such as quinupristin-dalfopristin, linezolid and daptomycin. The search for alternative treatment, including bacteriocins (ribosomally synthesized antimicrobial peptides) of lactic acid bacteria is increasing . Lactococcus lactis subsp. lactis F10, isolated from freshwater catfish, produced a new nisin variant active against clinical strains of S. aureus. The operon encoding nisin F is located on a plasmid and the structural gene has been sequenced. The lantibiotic is closely related to nisin Z, except at position 30 where valine replaced isoleucine. The antimicrobial activity of nisin F against S. aureus was tested in the respiratory tract of Wistar rats. Non-immunosuppressed and immunosuppressed rats were intranasally infected with S. aureus K and then treated with either nisin F or sterile physiological saline. Nisin F protected immunosuppressed rats against S. aureus, as symptoms of an infection were only detected in the trachea and lungs of immunosuppressed rats treated with saline. The safety of intranasally administered nisin F was also evaluated and proved to have no adverse side effects. The potential of nisin F as an antimicrobial agent to treat subcutaneous skin infections was evaluated by infecting C57BL/6 mice with a bioluminescent strain of S. aureus (Xen 36). Immunosuppressed mice were treated with either nisin F or sterile physiological saline 24 h and 48 h after infection with subcutaneously injected S. aureus Xen 36. Histology and bioluminescence flux measurements revealed that nisin F was ineffective in the treatment of deep dermal staphylococcal infections. Non-infected and infected mice treated with nisin F had an influx of polymorphonuclear cells in the deep stroma of the skin tissue. This suggested that nisin F, when injected subcutaneously, may have modulated the immune system. Nisin F proved an effective antimicrobial agent against S. aureus-related infections in the respiratory tract, but not against subcutaneous infections. The outcome of nisin F treatment thus depends on the route of administration and site of infection.

INTRODUÇÃO: As infecções respiratórias agudas de etiologia viral são um fator de morbimortalidade em todo o mundo. Devido a novas epidemias por vírus respiratórios e avanços no diagnóstico, em especial, com técnicas moleculares, novos agentes têm sido identificados nos últimos anos. Os vírus respiratórios (VR) são responsáveis por cerca de 5% das infecções nosocomiais. Em adultos internados em Unidade de Terapia Intensiva a presença de infecção por vírus respiratórios é reportada em poucas publicações. Os objetivos deste estudo foram avaliar a prevalência de infecções por vírus respiratórios em pacientes internados em Unidade de Terapia Intensiva, a freqüência de infecções virais de origem nosocomial, os fatores de risco associados e seu impacto na morbimortalidade destes pacientes. MÉTODOS: No período de maio de 2003 a junho de 2004, amostras de lavado de nasofaringe foram coletadas de pacientes internados em unidade de terapia intensiva do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, independentemente de sintomas respiratórios. O diagnóstico de vírus respiratórios (Vírus Respiratório Sincicial, vírus da Influenza A e B, parainfluenza, adenovírus, rinovírus, coronavírus e metapneumovírus) foi feito pelas técnicas de imunofluorescência direta e reação em cadeia pela polimerase. Dados clínico-epidemiológicos foram prospectivamente coletados em ficha específica. RESULTADOS: Foram avaliados pacientes em 228 internações, sendo o diagnóstico de 57 infecções por vírus respiratórios estabelecido em cinqüenta e duas admissões (23%). Rinovírus foi diagnosticado em 22 casos, coronavírus em 15, adenovirus em sete, influenza A em seis, vírus sincicial respiratório em cinco, Parainfluenza em um e metapneumovirus em um caso. Trinta e quatro episódios (59,6%) de infecções virais foram considerados de origem nosocomial. Foram investigados os fatores de risco associados à ocorrência de infecções por VR, à necessidade de ventilação mecânica e ao óbito na UTI. Na análise univariada a ocorrência de infecção por vírus respiratório associou-se à presença de quadro respiratório à admissão na UTI, à presença de co-morbidades (hipertensão arterial sistêmica e AIDS), à gravidade do quadro clínico admissional definido pelo valor de APACHE e ao uso de ventilação mecânica invasiva. Na análise multivariada, através de regressão logística, as variáveis que permaneceram significantemente associadas a ocorrência de infecção por VR foram a idade (OR 0,96), a hipertensão arterial (OR 3,95), a presença de quadros respiratórios à admissão na UTI (OR 2,22) e o valor de APACHE (OR 1,06). Os fatores de risco associados à necessidade de ventilação mecânica invasiva foram as infecções por vírus respiratórios (OR 1,98), o tempo de internação na UTI (OR 1,16), e valor do APACHE (OR 1,07). Os fatores de risco para óbito nesta série foram doença cardíaca ou neoplasia, infecções fúngicas, uso de ventilação mecânica, e o valor de APACHE na admissão. Não houve associação entre o diagnóstico de infecção por vírus respiratórios e a ocorrência de óbito (p=0,118). CONCLUSÃO: As infecções virais respiratórias foram freqüentes em pacientes adultos internados em Unidade de Terapia Intensiva, sendo a maioria de origem nosocomial. Os pacientes com diagnóstico de infecção por vírus respiratórios tiveram maior necessidade de ventilação mecânica nesta série. O diagnóstico das viroses respiratórias deveria ser insistentemente buscado em pacientes com quadros respiratórios à admissão para que medidas de controle da transmissão nosocomial fossem implementadas. A associação com hipertensão arterial mereceria ser objeto de novos estudos. / INTRODUCTION: Acute respiratory infections of viral etiology are a factor of morbidity and mortality in the all world. Advances in the diagnosis, in special, with molecular techniques, new agents have been identified in the last years. Respiratory viruses (RV) are responsible for about 5% of the nosocomial infections. Few studies have addressed the incidence, morbidity and mortality of viral respiratory infection in adults admitted in Intensive Care Units (ICU). We evaluated the prevalence of viral respiratory infections in ICU adult patients, the ratio of these infections that were of nosocomial origin, the risk factors and the impact of viral respiratory infections in the morbidity and mortality of these patients. METHODS: From May 2003 to June 2004, nasopharyngeal aspirates were taken twice a week, irrespective of respiratory symptoms, from 228 patients admitted at the ICU of the Hospital of Clinics, Faculty of Medical Sciences, University of São Paulo. Respiratory viruses (Respiratory Syncytial Virus, influenza virus, parainfluenza virus, adenovirus, rhinovirus, coronaviruses and metapneumovirus) were diagnosed by direct immunofluorescent assay (DFA) or polymerase chain reaction. (PCR). Medical and epidemiological data were prospectively collected. RESULTS: Fifty seven RV was diagnosed in 52 of the 228 ICU admissions (23%). Rhinovirus was the RV more frequently diagnosed (22 cases), followed by Coronaviruses (15 cases), Adenoviruses (7 cases), Influenza A viruses (6 cases), Respiratory Syncytial Virus (5 cases), Parainfluenza virus (one case) and Metapneumovirus (one case). Thirty and four episodes (59.6%) were considered of nosocomial origin. We evaluated the risk factors associated with the occurrence of RV infections, the need of invasive mechanical ventilation and death at the ICU. Univariate analysis showed that RV infections were associated with respiratory tract involvement at admission, some comorbidities (arterial hypertension and AIDS), to APACHE score at admission, and to the need of invasive mechanical ventilation. In multivariate analysis, age (OR 0.96), arterial hypertension (OR 3.95), respiratory tract involvement at admission (OR 2.22) and APACHE value (OR 1.06) were significantly associated with the occurrence of RV infection. Risk factors associated with the need of invasive mechanical ventilation were RV infections (OR 1.98), longer time at the ICU (OR 1.16) and APACHE value (OR 1.07). Death at the ICU was significantly associated with heart disease or neoplasia, fungal infection, mechanical ventilation and APACHE value. RV infection was not associated with ICU death (p=0.118). CONCLUSIONS: Respiratory Virus infections were frequent in adult ICU patients in the present series; the majority of them were of nosocomial origin. Patients with RV infections were more likely to need mechanical ventilation during ICU admission. Diagnosis of RV infections should be included in the diagnostic assessment of ICU patients, especially those with respiratory tract involvement at admission. This policy will certainly favor the implementation of preventive measures to control nosocomial transmission. The association with arterial hypertension deserves further studies.