Alexandre Dias Tavares Costa

Necrotizing enterocolitis (NEC) is a severe inflammatory disease of the immature intestine involving complex interactions between intestinal inflammation, microvascular dysfunction, and innate immune activation. Therapeutic strategies targeting nitric oxide-related pathways and Toll-like receptor (TLR) signaling have been proposed, although their effects remain incompletely understood. This study evaluated the effects of L-arginine, sildenafil, and glycine, administered alone or in combination, in an experimental model of NEC. Ninety-two neonatal Wistar rats were allocated into nine groups: negative control (Breastfeeding group), positive control (NEC induction), and intervention groups receiving oral glycine (10 mg), L-arginine (10 mg), sildenafil (0.3 mg), or their combinations. NEC was induced by formula feeding, hypoxia, and hypothermia. Outcomes included histological intestinal injury, mortality, body weight variation, and TLR4 and TLR9 gene expression assessed by Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR). Overall mortality following NEC induction was 17%. Histological injury scores differed significantly among groups ( p < 0.001), with the highest mean scores observed in the L-arginine monotherapy and glycine plus sildenafil groups. TLR4 expression did not differ significantly among groups ( p = 0.236) but showed a moderate positive correlation with histological severity (r = 0.42). TLR9 expression differed among treatment groups but showed a weak correlation with histological injury (r = 0.18). Treatments targeting nitric oxide-related pathways and inflammatory signaling produced heterogeneous effects in experimental NEC. Glycine monotherapy was associated with lower histological injury scores, whereas L-arginine monotherapy was associated with higher mortality and greater histological severity. These findings suggest that modulation of individual therapeutic targets alone may not consistently reduce intestinal injury in experimental NEC. Given the limitations of the experimental model and the absence of direct assessment of nitric oxide pathway activity, these findings should be interpreted with caution and require confirmation in future studies.

Candida auris (also known as Candidozyma auris) relies on extensive transporter networks to maintain cellular homeostasis and adapt to environmental stress, yet the broader regulatory roles of individual transporters remain poorly understood. Qdr3, a member of the major facilitator superfamily (MFS), contributes to multidrug resistance in several pathogenic yeasts, but its function in C. auris has not been defined. To investigate the role of Qdr3, we generated Δqdr3 mutants in C. auris strain B8441 and performed integrated phenotypic, ultrastructural, transcriptomic, and proteomic analyses. Deletion of qdr3 produced pronounced sensitivity to ionic stress without affecting basal growth or antifungal susceptibility. Transcriptomic profiling revealed extensive transcriptional remodeling characterized by reduced expression of heat-shock and cell-surface maintenance pathways alongside strong induction of mitochondrial translation and biogenesis programs. These transcriptional shifts were supported by proteomic signatures and transmission electron microscopy, which demonstrated increased mitochondrial abundance and enhanced cytoplasmic granularity. Qdr3 also influenced multicellular organization, as Δqdr3 biofilms exhibited reduced cellular aggregation and increased extracellular matrix production. Despite increased stress sensitivity, Δqdr3 mutants displayed enhanced virulence in a Galleria mellonella infection model, revealing an unexpected disconnect between stress adaptation and pathogenic potential. Together, these findings identify Qdr3 as a key regulator of cellular homeostasis that links membrane transport, mitochondrial activity, and cell-surface architecture in C. auris. These results demonstrate how disruption of a single MFS transporter can drive extensive physiological and regulatory remodeling, ultimately altering virulence in this WHO critical priority pathogen.

Real-time polymerase chain reaction (qPCR) is a molecular technique that has transformed clinical diagnostics by enabling the amplification and quantification of specific nucleic acid sequences with high sensitivity, specificity, and speed. This approach supports the detection of pathogens, genetic mutations, and biomarkers across clinical samples. qPCR is widely employed in fields, such as infectious diseases, oncology, parasitology, and medical genetics, playing a pivotal role in the rapid identification of infectious agents, including those responsible for COVID-19, tuberculosis, leprosy, leishmaniasis, and arboviral infections. Compared with conventional diagnostic methods, qPCR offers substantial advantages, including reduced turnaround times, increased analytical accuracy, and compatibility with automated workflows. Recent technological innovations, including portable thermocyclers, simplified workflows, and ready-to-use commercial kits, along with integration into digital platforms, have expanded the applicability of qPCR to low-infrastructure environments and point-of-care settings. Despite these advances, the technique faces challenges, such as high costs of equipment and reagents, reliance on trained personnel, and susceptibility to inhibition by sample-derived compounds. Nevertheless, the adoption of standardized guidelines, such as Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) and Standards for Reporting Diagnostic Accuracy (STARD), has strengthened the reproducibility and reliability of qPCR in clinical research. Future perspectives suggest an increasing role for qPCR in personalized medicine, epidemiological surveillance, and decentralized diagnostic strategies, further consolidating its importance in clinical practice and global public health. Its combined sensitivity, specificity, and adaptability will continue to drive innovation in molecular diagnostics.

Tuberculosis remains a major global public health issue, particularly among vulnerable populations in resource-limited settings. This study evaluated a simplified DNA extraction protocol combined with a portable quantitative Polymerase Chain Reaction (qPCR) device for on-site detection of Mycobacterium tuberculosis DNA in low-resource environments. A total of 54 sputum samples were collected from a remote village and a penitentiary in southern Brazil. The simplified extraction method and qPCR were conducted directly in the field without sample transport. Among all samples, qPCR detected three positives (5.6%), while smear microscopy identified one case (1.9%), and culture yielded no positives. The approach demonstrated operational feasibility under field conditions, though the small number of positive samples precluded diagnostic accuracy analysis. These findings suggest that portable molecular diagnostics can support tuberculosis control in hard-to-reach populations by enabling rapid, on-site screening.

Objetivo- Explorar o ponto de vista dos residentes nas comunidades de Porto Velho e vizinhas sobre testes de malária, riscos e benefícios relacionados. Metodologia- Apreciação rápida e participativa das necessidades da comunidade por meio de uma avaliação etnográfica para explorar crenças/práticas comunitárias relacionadas a intervenções específicas relativas ao diagnóstico e tratamento de uma dada doença. A coleta de dados deu-se por grupos focais e entrevistas individuais face-a-face (no mês de agosto de 2022). Resultados- Participaram 21 mulheres e 17 homens, ambos adultos e escolarizados. Todos os participantes dos grupos focais e 90% dos entrevistados sabiam de casos de malária entre familiares e amigos. Entre os entrevistados, a maioria é de homens adultos, empregados/autônomos, enquanto a maioria dos participantes dos grupos é de mulheres, jovens, empregadas ou estudantes. Os achados revelaram como as endemias anteriores afetaram a percepção da comunidade sobre os testes de malária. A exposição à malária nos círculos sociais reforçou a crença de risco ambiental incontrolável. Revelou-se um espectro de questões críticas: apatia e medo generalizados, maior conscientização sobre recorrência da malária, percepções de sua expansão urbana, importância reconhecida de testes e tratamentos e barreiras aos testes e a tratamento eficazes. Participantes compartilharam e criticaram pensamentos sobre modo de pensar das comunidades em relação aos testes de malária. Conclusão- A preocupação com o meio ambiente-saúde humana requer maior engajamento político ao contexto sociocultural da malária. Recomendações propostas incluem educação e comunicação em massa, ação de agentes comunitários, disponibilidade de técnicos para testes e aumento dos locais de teste.

Tuberculosis, caused by Mycobacterium tuberculosis, is a treatable and curable disease, and yet remains one of the leading causes of death worldwide. Diagnosis is essential to reducing the number of cases and starting treatment, but costly tests and equipments that require complex infrastructure hamper their widespread use as a tool to contain the disease in vulnerable populations as well countries lacking resources. Therefore, it becomes necessary to develop new technological approaches to molecular methods as well as screening tests that can be rapidly conducted among people presenting to a health facility to differentiate those who should have further diagnostic evaluation for TB from those who should undergo further investigation for non-TB diagnoses. The present study aimed to evaluate two experimental DNA extraction methods from clinical samples (FTA card versus sonication) followed by analysis in a portable qPCR instrument (the Q3-plus). The FTA card-based protocol showed 100% sensitivity and specificity, while the sonication protocol showed 80% sensitivity and 89% specificity when compared to the traditional gold standard culture. The portable protocol, comprised by the FTA card method and the portable instrument Q3-Plus, showed sensitivity and specificity of 92% and 61%, respectively, when compared to culture, and 75% and 81%, respectively, when compared to the standard TB case classification. The ROC curve showed an AUC of 0.78 (pMycobacterium tuberculosis (H37Rv strain) detection in spiked samples obtained using the portable protocol (FTA card and Q3-Plus) was 19.3 CFU/mL. As an added benefit, using the FTA card facilitates sample handling, transport, and storage. It is concluded that the use of the FTA card protocol and the Q3-Plus yields similar sensitivity and specificity as the gold standard diagnostic tests and case classification. We suggest that the platform is suitable to use as a point of care tool, assisting in the screening of tuberculosis in hard-to-reach or resource-limited areas.

The study aimed to optimize qPCR reactions using oligonucleotides from the first Brazilian molecular diagnostic kit for leprosy on a portable platform (Q3-Plus). In addition, we sought to develop a simplified protocol for DNA extraction that met point-of-care criteria. During optimization on the Q3-Plus, optical parameters, thresholds, and cutoffs for the 16S rRNA and RLEP targets of M. leprae were established using synthetic DNA, purified DNA from M. leprae, and pre-characterized clinical samples. For the simplified extraction protocol, different lysis solutions were evaluated using chaotropic agents, and purification was carried out by transferring the lysed material to FTA cards. The complete protocol (simplified extraction + qPCR on the portable platform) was then evaluated with pre-characterized clinical skin biopsy samples and compared with standard equipment (QuantStudio-5). LOD_95% for the optimized reactions was 113.31 genome-equivalents/μL for 16S rRNA and 17.70 genome-equivalents/μL for RLEP. Among the lysis solutions, the best-performing was composed of urea (2 M), which provided good dissolution of the skin fragment and a lower Ct value, indicating higher concentrations of DNA. The complete technological solution showed a sensitivity of 52% in reactions. Our results highlight the need for additional optimization to deal with paucibacillary samples, but also demonstrate the feasibility of the portable platform for the qPCR detection of M. leprae DNA in low infrastructure settings.

Trachoma is the world-leading infectious cause of preventable blindness and is caused by the bacteria Chlamydia trachomatis. In developing countries, diagnosis is usually based on clinical evaluation. Serological-based tests are cheaper than molecular-based ones, but the latter are more sensitive and specific. The present study developed a new duplex qPCR which concomitantly detects the C. trachomatis cryptic plasmid and the human 18S rRNA gene, with an LOD95% for C. trachomatis DNA of 13.04 genome equivalents per reaction. The new qPCR was tested using 50 samples from an endemic area and 12 from a non-endemic area that were previously characterized using direct immunofluorescence assay (DFA) and clinical evaluation. Among the 50 endemic samples, 3 were found to be positive by clinical evaluation (6%), 18 were found to be positive by DFA (36%), and 48 were found to be positive by qPCR (96%). Next, the new duplex qPCR was validated using 50 samples previously characterized by qPCR. Validation was carried out on a benchtop instrument (ABI7500) or on a portable point-of-care instrument (Q3-Plus), showing 95% specificity and 100% sensitivity. The ubiquitous presence of C. trachomatis DNA in samples from the endemic region confirms that constant monitoring is of paramount importance for the effective measurement of the elimination of trachoma. The newly developed duplex qPCR presented in this study, along with its validation in a portable qPCR system, constitutes important tools toward achieving this goal.

Tuberculosis, caused by Mycobacterium tuberculosis , is a treatable and curable disease, and yet remains one of the leading causes of death worldwide. Diagnosis is essential to reducing the number of cases and starting treatment, but costly tests and equipments that require complex infrastructure hamper their widespread use as a tool to contain the disease in vulnerable populations as well countries lacking resources. Therefore, it becomes necessary to develop new technological approaches to molecular methods as well as screening tests that can be rapidly conducted among people presenting to a health facility to differentiate those who should have further diagnostic evaluation for TB from those who should undergo further investigation for non-TB diagnoses. The present study aimed to evaluate two experimental DNA extraction methods from clinical samples (FTA card versus sonication) followed by analysis in a portable qPCR instrument (the Q3-plus). The FTA card-based protocol showed 100% sensitivity and specificity, while the sonication protocol showed 80% sensitivity and 89% specificity when compared to the traditional gold standard culture. The portable protocol, comprised by the FTA card method and the portable instrument Q3-Plus, showed sensitivity and specificity of 92% and 61%, respectively, when compared to culture, and 75% and 81%, respectively, when compared to the standard TB case classification. The ROC curve showed an AUC of 0.78 (pMycobacterium tuberculosis (H37Rv strain) detection in spiked samples obtained using the portable protocol (FTA card and Q3-Plus) was 19.3 CFU/mL. As an added benefit, using the FTA card facilitates sample handling, transport, and storage. It is concluded that the use of the FTA card protocol and the Q3-Plus yields similar sensitivity and specificity as the gold standard diagnostic tests and case classification. We suggest that the platform is suitable to use as a point of care tool, assisting in the screening of tuberculosis in hard-to-reach or resource-limited areas.