An algorithm for visceral leishmaniasis diagnosis in Brazil from a 10-year analysis of National Reference Laboratory data.

Visceral leishmaniasis (VL) requires diagnostic assays to complement clinical suspicion. However, there is no standardization of a diagnostic flow using available assays. This study aimed to evaluate the performance of parasitological, molecular, and serological assays for diagnosing VL and propose a diagnostic flow based on performance, practicality, and invasiveness. We conducted a study of 10-year (2010-2020) routine diagnoses of VL at the Brazilian National Reference Laboratory. We propose a diagnostic flow where individuals suspected of VL are initially screened for antibodies using an immunochromatographic test (ICT) with rK39 antigen on the nitrocellulose membrane. This is followed by a blood polymerase chain reaction (PCR) for Leishmania sp. kDNA and direct parasitological exam and/or PCR in bone marrow aspirate. A positive result in any of these assays can define a VL case. If clinical suspicion persists in negative individuals, the diagnostic flow should be repeated. The proposed flow has the potential to standardize and improve the diagnosis of VL. It reduces the need for invasive tests without compromising diagnostic accuracy.

A multicenter comparative study of the performance of four rapid immunochromatographic tests for the detection of anti-Trypanosoma cruzi antibodies in Brazil.

Diagnosis of Trypanosoma cruzi (T. cruzi) infection in the chronic phase of Chagas disease (CD) is performed by serologic testing. Conventional tests are currently used with very good results but require time, laboratory infrastructure, and expertise. Rapid diagnostic tests (RDTs) are an alternative as the results are immediate and do not require specialized knowledge, making them suitable for epidemiologic studies and promising as a screening tool. Nevertheless, few studies conducted comparative evaluations of RDTs to validate the results and assess their performance. In this study, we analyzed four trades of rapid tests (OnSite Chagas Ab Combo Rapid Test-United States, SD Bioline Chagas AB-United States, WL Check Chagas-Argentina, and TR Chagas Bio-Manguinhos-Brazil) using a panel of 190 samples, including sera from 111 infected individuals, most of whom had low T. cruzi antibody levels. An additional 59 samples from uninfected individuals and 20 sera from individuals with other diseases, mainly visceral leishmaniasis, were included. All tests were performed by three independent laboratories in a blinded manner. Results showed differences in sensitivity from 92.8 to 100%, specificity from 78.5 to 92.4%, and accuracy from 90.5 to 95.3% among the four assays. The results presented here show that all four RDTs have high overall diagnostic ability. However, WL Check Chagas and TR Chagas Bio-Manguinhos were considered most suitable for use in screening studies due to their high sensitivity combined with good performance. Although these two RDTs have high sensitivity, a positive result should be confirmed with other tests to confirm or rule out reactivity/positivity, especially considering possible cross-reactivity with individuals with leishmaniasis or toxoplasmosis.

Mutant Brucella abortus membrane fusogenic protein induces protection against challenge infection in mice.

Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus Δmfp::kan and Δomp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kan or Δomp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus Δomp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kan strain had a lower virulence in these same mouse models. Furthermore, Δmfp::kan and Δomp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kan mutant may serve as a potential vaccine candidate in future studies.

Brucella cyclic ß-1,2-glucan plays a critical role in the induction of splenomegaly in mice.

Brucella, the etiological agent of animal and human brucellosis, is a bacterium with the capacity to modulate the inflammatory response. Cyclic ß-1,2-glucan (CßG) is a virulence factor key for the pathogenesis of Brucella as it is involved in the intracellular life cycle of the bacteria. Using comparative studies with different CßG mutants of Brucella, cgs (CßG synthase), cgt (CßG transporter) and cgm (CßG modifier), we have identified different roles for this polysaccharide in Brucella. While anionic CßG is required for bacterial growth in low osmolarity conditions, the sole requirement for a successful Brucella interaction with mammalian host is its transport to periplasmic space. Our results uncover a new role for CßG in promoting splenomegaly in mice. We showed that CßG-dependent spleen inflammation is the consequence of massive cell recruitment (monocytes, dendritics cells and neutrophils) due to the induction of pro-inflammatory cytokines such as IL-12 and TNF-α and also that the reduced splenomegaly response observed with the cgs mutant is not the consequence of changes in expression levels of the characterized Brucella PAMPs LPS, flagellin or OMP16/19. Complementation of cgs mutant with purified CßG increased significantly spleen inflammation response suggesting a direct role for this polysaccharide.

Morphological, biochemical, physiological and molecular aspects of the response of Fusobacterium nucleatum exposed to subinhibitory concentrations of antimicrobials.

Subinhibitory concentrations (SICs) of antimicrobials may result in alterations in bacterial biology with implications for its potential aggression. This has considerable importance for the resident microbiota. Our aim was to analyze the effects of SICs of antimicrobials on the morphological, biochemical, physiological and molecular characteristics of the resident anaerobic Fusobacterium nucleatum. Fourteen strains were obtained from F. nucleatum ATCC 25586, selected by culturing on SICs of ampicillin, ampicillin/sulbactam, clindamycin, chloramphenicol, levofloxacin, metronidazole and piperacillin/tazobactam and subsequent culturing in the absence of drugs. Antimicrobial susceptibility, bacterial morphology, biochemical profiles and biofilm formation were evaluated. Genotyping and analysis of protein profiles were also performed. The antimicrobial susceptibility patterns showed that most of the derived strains were less sensitive to the antimicrobials, even after culturing them without drugs. Morphological and cell complexity alterations were observed, mainly in strains grown in SICs of ß-lactam; these strains also expressed a reduced ability for biofilm formation. The other strains showed an increase in biofilm formation but no apparent morphological changes. Alterations were observed in the carbohydrate metabolism patterns and in the activity of microbial enzymes. Several proteins were positively or negatively regulated and there was polymorphism in the DNA from all derived strains. Therefore, SICs of antimicrobials induce alterations in F. nucleatum, which directly impact its biology. These results emphasize the risk of inadequate antibioticotherapy, which may have serious implications for clinical microbiology and infectious diseases and also may interfere with the host-bacteria relationship.