http://www.repositorio.uem.mz/handle258/7 Thu, 23 Apr 2026 07:06:35 GMT 2026-04-23T07:06:35Z http://www.repositorio.uem.mz/handle258/848 Title: Avaliação da malato desidrogenase citoplasmática para o diagnóstico de Trypanosoma congolense Authors: Ferreira, Raquelina Ângela Abstract: African trypanosomiasis is a hemoparasitosis transmitted by the tsetse fly that affects man and animals, causing great economic losses. This disease is caused by protozoa belonging to the genus Trypanosoma, with T. congolense, T. vivax and T. brucei spp. the most important species. Animal trypanosomiasis in Africa is responsible for high losses in livestock and in the economy in general. The main measure of control is chemotherapy and chemoprophylaxis, although limited by the number of trypanocides available, and the always increasing existence of resistance. Thus, diagnosis becomes an important tool because it allows the design of adequate treatment; however, the existing methods are not satisfactory. Mostly based on the detection of circulating antibodies, they cannot differenciate between active infection and recent but passed infection following treatment or self-cure. In this context, room for the development of a circulating antigen capture ELISA exists, which would detect active infections, with the added advantage to be open to adaptation into a dipstick format. Cytoplasmic malate dehydrogenase (cMDH) was chosen as candidate antigen due to its assumed abundance in host bloodstream after trypanosome lysis, and the possibility of enzymatic detection. In order to express the recombinant form of cMDH, the coding sequence of the protein was amplified by PCR and cloned into the T-vector pTZ57R/T, sequenced, and subcloned into the expression vector pET32. Expression was realized in the bacteria Escherichia coli strain BL21 (DH3), under the inducible promoter T7. After induction by IPTG a 50 kDa soluble protein was produced, fused to a His.Tag. The recombinant protein was purified by affinity chromatography based on Nickel chelation and used to immunize two rabbits and two chickens to raise polyclonal antibodies, which were used to develop a capture ELISA test. Antibodies raised in rabbit and chicken showed on an immunoblot the ability to recognize native T. congolense cMDH present in a trypanosome lysate, indicating a potential to also detect the presence of circulating cMDH in sera from infected cattle. Indeed, a sandwich-ELISA was developed, using the IgY as capture antibodies, and the rabbit serum as revelating antibodies that 6Avaliação do potencial antigénico da enzima malato desidrogenase citoplasmática para uso em testes de captura para o diagnóstico de Trypanosoma congolense not only could capture circulating cMDH in the serum of T. congolense-infected cattle, but also showed a positive correlation between the intensity of the test and the level of parasitaemia. This study demonstrates that the polyclonal antibodies produced are able to capture native cMDH in serum from T. congolense infected animals with different levels of parasitemia. These results suggest that the cMDH capture system that we developed can be validated as a test for the field diagnosis of T. congolense infections Wed, 01 Feb 2017 00:00:00 GMT http://www.repositorio.uem.mz/handle258/848 2017-02-01T00:00:00Z http://www.repositorio.uem.mz/handle258/790 Title: Detecção molecular e genotipagem de Helicobacter Pylori em pacientes dispépticos atendidos no Hospital Central de Maputo Authors: Majaliwa, Nashon Dussin Abstract: Background: Helicobacter pylori (H. pylori) is a human pathogenic bacterium responsible for a variety of gastric diseases, including dyspepsia. The global prevalence of its infection is approximately 50%, but can reach 90% in developing countries, such as those in sub-Saharan Africa, where Mozambique is included. H. pylori strains show a high level of genotypic diversity and express several genes that contribute to their pathogenicity and resistance. In Mozambique, there is lack of information on H. pylori infection, its resistance to antibiotics, its virulence factors, and its phylogeny. Therefore, we aimed to investigate the occurrence of H. pylori infection and its genomic characterization in dyspeptic patients seen at the Department of Gastroenterology, Hospital Central de Maputo, in Mozambique. Methodology: This is a cross-sectional descriptive study conducted between June 2017 and June 2020, in which 171 dyspeptic patients were recruited, and through Upper GI endoscopy, gastric biopsies were collected from those patients. To perform molecular analysis, bacterial genomic DNA was directly extracted from gastric biopsies, and PCR was performed for the detection of H. pylori and its resistance mechanisms to clarithromycin (23S rRNA), fluoroquinolones (gyrA) and metronidazole (rdxA) and, mutations conferring resistance to these antibiotics were investigated by sequencing these genes (23S rRNA, gyrA and rdxA). Additionally, virulence genes (vacA, dupA and cagA) were detected, and MLST typing was also performed for the phylogenetic characterization of H. pylori strains infecting patients Results: Of the 171 samples tested, H. pylori was detected in 56.1% (96/171). The clarithromycin resistance rate was 10.4% (the responsible mutations were: A2141G and A2142G), the fluoroquinolones resistance rate was 20% (the responsible mutations were: N87I and D91G) and, the metronidazole resistance rate was 55.2% (4 types of mutations responsible for metronidazole resistance were identified which include, D59N, R90K, H97T and A118T. However, in many cases, they appeared in combination, with D59N+R90K+A118T being the most frequent combination). Regarding virulence genes, vacA was detected in all samples (100%), dupA in 61.5% and cagA was detected only in 16.7% of samples and, among the combinations of vacA genotype, vacA s1/m2 was the most frequent. Characterization of phosphorylation sites in cagA- positive samples revealed that 93.8% were EPIYA-ABC and only 6.2% were EPIYA-ABCCC. Based on MLST typing, a diversity was found among H. pylori strains infecting patients and, they were classified as hpAfrica2 (50%), hpAfrica1 (33.3%) and hpEurope (16.7%). Conclusion: More than half of the dyspeptic patients seen at the Department of Gastroenterology, Hospital Central de Maputo, are infected with H. pylori strains resistant to metronidazole and fluoroquinolones. However, their virulence factors suggest moderate virulence of these strains Fri, 01 Jul 2022 00:00:00 GMT http://www.repositorio.uem.mz/handle258/790 2022-07-01T00:00:00Z