Sustained effect of isoniazid preventive therapy among household contacts in Brazil.

BACKGROUND: Isoniazid preventive therapy (IPT) is highly effective in preventing TB disease; however, its long-term benefit in household contacts (HHCs) of infectious TB cases is unclear.METHODS: We conducted a retrospective analysis of two household contact studies in Vitoria, ES, Brazil, between 2008 and 2015. Households with smear-positive, culture-proven TB disease were enrolled. Eligible HHCs with tuberculin skin test (TST) indurations of ≥10 mm were referred to local TB clinics and IPT was started according to national guidelines. We reviewed the national dataset information system in January 2020 to identify HHCs with a diagnosis of TB disease. Time to event and Cox proportional regression analysis were conducted to identify factors associated with TB disease.RESULTS: Of the 1097 HHCs enrolled, 654 (60%) had TST ≥10 mm; 160 (24%) initiated IPT, of whom 115 (71.9%) completed IPT, which accounts for an overall completion rate of 18% among the population at risk; 42 (6%) TB cases were identified. IPT was associated with a 71% decrease in TB disease rates (HR 0.29, 95% CI 0.10-0.82; P = 0.02) among HHCs with TST ≥10 mm. IPT effect was sustained, as TB cases in HHCs without IPT occurred along the 7.9-year follow-up, whereas all four TB cases in HHCs with IPT were diagnosed within the first 3 years after exposureCONCLUSION: Isoniazid provides long-term protection for TB disease in household contacts of culture-proven TB cases.

Toll-like receptors blocking restores in vitro microbicidal activity in latent tuberculosis-infected subjects.

BACKGROUND: Latent tuberculous infection (LTBI) can function as a 'reservoir' for Mycobacterium tuberculosis. Given that T-regulatory cell (Treg) numbers are augmented in LTBI, it is likely that Toll-like receptors (TLRs) may have a role in Treg function. Elucidation of the immune mechanisms associated with tuberculosis (TB) development may help to control M. tuberculosis spread.OBJECTIVE: To investigate the role of TLR2, TLR4 and TLR9 in hindered in vitro microbicidal activity and increase Treg number during LTBI. DESIGN: Whole blood cell cultures from individuals with LTBI and healthy controls (HCs) infected with live M. tuberculosis H37Rv strain were used to investigate the effect of TLR2, TLR4 and TLR9 on Treg number, microbicidal activity, and interferon-gamma and interleukin (IL)10 production. RESULTS: LTBI subjects were characterised by increased Treg number and impaired microbicidal activity when compared with HCs. Specific blockade of TLR4 and TLR9 led to a significant reduction in Treg number, a decrease in IL-10 production and substantial upregulation of microbicidal activity. CONCLUSION: M. tuberculosis infection may activate TLR4 and TLR9 pathways to suppress M. tuberculosis-specific immune responses. Here, we show that activation of TLR4 and TLR9 hinder microbicidal activity during LTBI.

Immunophenotype of skin lymphocytic infiltrate in patients co-infected with Mycobacterium leprae and human immunodeficiency virus: a scenario dependent on CD8+ and/or CD20+ cells.

BACKGROUND: Leprosy occurs rarely in human immunodeficiency virus (HIV)-positive patients. In contrast to tuberculosis, there has been no report to date of an increase in HIV prevalence among patients with leprosy or of differences in leprosy's clinical spectrum. While several studies describe the systemic immune response profile in patients co-infected with HIV and leprosy, the local immune skin response has been evaluated in only a small number of case reports and limited series of patients. OBJECTIVE: To investigate the interaction between Mycobacterium leprae and HIV infection in the skin. METHODS: We investigated the presence and frequency of cells positive for CD4, CD8, CD20, TIA-1, FOXP3 and CD123 in lymphocytic infiltrates from 16 skin biopsies taken from 15 patients with HIV-leprosy co-infection. RESULTS: CD4+ cells were absent in infiltrates from 6 (38%) skin biopsies and present in 10 (62%) cases at low levels (<1·16%) of the lymphocytic infiltrate. CD8+ was the predominant phenotype in the infiltrate (99·4%), followed by TIA-1, expressed by >75% of CD8+ cells. FOXP3+ cells were also present, representing 3·4% of the lymphocytic infiltrate. CD20+ cells were detected in 75% of the cases; however, in two cases (12%) these cells represented 25-50% of the infiltrate, while in the other 10 cases (62%) they were present only focally (<25% of the infiltrate). CD123+ cells were not observed in any of the studied specimens. CONCLUSIONS: Data presented here suggest that cell-mediated immune responses to M. leprae are preserved at the site of disease and that in the absence of CD4+ cells, CD8+FOXP3+ and CD20+ cells may be involved in granuloma formation.

Presence of intestinal helminths decreases T helper type 1 responses in tuberculoid leprosy patients and may increase the risk for multi-bacillary leprosy.

Resistance to intracellular pathogens such as Mycobacterium leprae is dependent upon an effective T helper type 1 (Th1)-type immune response. On the other hand, intestinal helminths are known to subvert the host's immune response towards to either a Th2-type immune response or a regulatory T cell up-regulation, which may affect the host's ability to mount an effective response to mycobacteria. Here, we report a significant association between intestinal helminth infections and lepromatous leprosy [odds ratio (OR), 10.88; confidence interval (CI) 95%: 4.02-29.4; P<0.001]. We also observed that the frequency of intestinal helminths correlated strongly with the mycobacterial index (r=0.982, P<0.01). Corroborating with our hypothesis, intracellular levels of interferon-gamma were decreased significantly in leprosy patients co-infected with intestinal helminths when compared to leprosy patients without worms. Conversely, lepromatous leprosy patients with intestinal worms produced higher levels of both interleukin (IL)-4 and IL-10. Our results suggest that a pre-existing infection by intestinal helminths may facilitate the establishment of M. leprae infection or its progression to more severe forms of leprosy.

Intestinal helminth co-infection has a negative impact on both anti-Mycobacterium tuberculosis immunity and clinical response to tuberculosis therapy.

The impact of intestinal helminth infection on Mycobacterium tuberculosis (MTB)-specific immune responses during active tuberculosis (TB) is not known. We investigated the role of intestinal helminth infection in anti-MTB immunity by evaluating both cellular phenotype and cytokine profiles in patients with TB and patients with concomitant TB and intestinal helminth infection (TB + Helm) during TB therapy. Twenty-seven per cent of TB patients enrolled for the study were co-infected with at least one intestinal helminth. At baseline, absolute frequencies of leucocytes, monocytes and eosinophils from TB and TB + Helm patients differed from healthy subjects. Concomitant intestinal helminth infection in TB + Helm patients had a negative impact (P < 0.05) on absolute frequencies of CD3(+), CD4(+), CD8(+), natural killer (NK) T and CD4(+) CD25(high) T cell subsets when compared to either TB patients or healthy controls. Differences in CD4(+) T cell frequencies were accompanied by lower interferon (IFN)-gamma and elevated and sustained interleukin (IL)-10 levels in whole blood (WB) cultures from TB + Helm compared to TB patients. In addition to a depressed anti-MTB immunity, TB + Helm patients also presented with more severe radiological pulmonary disease, with a significant difference (P = 0.013) in the number of involved lung zones at the end of TB treatment. The above data may indicate that concomitant intestinal helminth infection in patients with newly diagnosed TB skews their cytokine profile toward a T helper 2 response, which could favour persistent MTB infection and a more protracted clinical course of the disease.

A role for CD4+CD25+ T cells in regulation of the immune response during human tuberculosis.

Active tuberculosis (TB) is associated with prolonged suppression of Mycobacterium tuberculosis (MTB)-specific immune responses, but mechanisms involved are understood incompletely. We investigated a potential role for CD4+CD25+ regulatory T cells in depressed anti-MTB immunity by evaluating serially CD4 cell phenotype and interferon (IFN)-gamma production by mononuclear cells from patients with TB. At diagnosis, frequencies of CD4+CD25+ T cells were increased in blood from TB patients compared to healthy purified protein derivative (PPD)-positive controls (with a history of prior TB exposure), and remained elevated at completion of therapy (6 months). By contrast, expression of another activation marker, CD69, by CD4 T cells was increased at diagnosis, but declined rapidly to control levels with treatment. Among CD4+CD25+ T cells from TB patients at diagnosis those expressing high levels of CD25, probably representing regulatory T cells, were increased 2.9-fold when compared to control subjects, while MTB-stimulated IFN-gamma levels in whole blood supernatants were depressed. A role for CD4+CD25+ T cells in depressed IFN-gamma production during TB was substantiated in depletion experiments, where CD25+-depleted CD4 T cells produced increased amounts of IFN-gamma upon MTB stimulation compared to unseparated T cells. At follow-up, IFN-gamma production improved most significantly in blood from TB patients with high baseline frequencies of CD4+CD25+ T cells (more than threefold higher than controls for both total and CD25hi+ CD4 T cells), who also had a significant drop in frequencies of both total and 'regulatory' CD4+CD25+ T cells in response to treatment. Expansion of CD4+CD25+ regulatory T cells during active TB may play a role in depressed T cell IFN-gamma production.

Short report: do intestinal nematodes increase the risk for multibacillary leprosy?

Intestinal helminths are known to subvert the host's immune response towards a Th2 response, which in turn may lead to both eosinophilia and high immunoglobulin E titers often associated with these parasites. Mycobacterium leprae infection may lead to different clinical and pathological forms. Multibacillary forms are associated with Th2 cytokines, whereas paucibacillary forms are associated with Th1 cytokines. We report a significantly higher frequency of intestinal helminthic infections in patients with the lepromatous form, a multibacillary form of leprosy (odds ratio, 2.99; 95% confidence interval, 1.82-4.95; P = 0.006) when compared with patients with paucibacillary leprosy or to a control group without leprosy. A direct correlation was also found between mycobacterial index and the frequency of intestinal helminths. Our results suggest that the presence of intestinal helminths may facilitate the establishment of M. leprae infection or the progression to more severe forms of leprosy.

Schistosomiasis mansoni: immunoblot analysis to diagnose and differentiate recent and chronic infection.

One hundred seven patients classified into three different groups (11 with acute schistosomiasis, 58 with chronic schistosomiasis, and 38 children with high IgM-specific antibody titers against schistosome gut-associated antigens living in an endemic schistosomiasis area) were studied by immunoblotting for the presence of IgG, IgM, and IgA antibodies against Schistosoma mansoni soluble adult worm antigen preparation. We used sera from 15 individuals infected with various intestinal parasites, as well as sera from 19 uninfected individuals, as controls. An immunogenic fraction with a molecular weight of 31-32 kD (Sm31/32) was the most frequently recognized by the different antibody isotypes. In the group with acute disease, this fraction was recognized by IgG and IgM antibodies of all patients, and by 10 (90.9%) of 11 samples for IgA antibodies. Approximately 98% of the patients with chronic infections had IgG antibodies against Sm31/32, but only about 10% had IgM and IgA antibodies against this fraction. The IgG immunoblot profiles of the children from the endemic area were similar to those obtained for the group with acute schistosomiasis. This observation suggests recent infection of these children. Our data show that the Sm31/32 protein fraction is highly immunogenic and may be a useful serologic marker for diagnosing and differentiating between acute and chronic schistosomiasis infection.

Effects of the antitubulin drug trifluralin on the proliferation and metacyclogenesis of Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi epimastigotes were exposed to varying micromolar concentrations of the dinitroaniline antitubulin trifluralin. The effects of trifluralin on parasite proliferation, metacyclogenesis, morphology, and uptake of horseradish peroxidase (HRP) were investigated. Parasites exposed to the antitubulin showed some ultrastructural alterations, i.e., formation in some parasites of large, membrane-delimited vacuoles and a significant decrease in the number of HRP-positive reservosomes. Whereas there was no perceptible change in the morphology of either subpellicular or flagellar microtubules, there was a significant inhibition of proliferation and metacyclogenesis at trifluralin concentrations in excess of 100 microM. These concentrations were considerably higher than those reported to produce similar results in Leishmania spp. and T. brucei brucei.

Characterization of trypanosomes from the subgenus Schizotrypanum isolated from bats, Eptesicus sp. (Chiroptera: Vespertilionidae), captured in Florianópolis, Santa Catarina State, Brazil.

Seven Trypanosoma spp. isolates obtained from bats (Eptesicus sp.) were characterized using experimental infection in mice, triatomines, and culicines; complement lysis; indirect fluorescence assays; as well as isoenzyme and random-amplified polymorphic DNA (RAPD) profiles. The Trypanosoma sp. isolates were compared with Trypanosoma cruzi, Trypanosoma rangeli. and 2 other bat trypanosomes species, Trypanosoma vespertilionis and Trypanosoma hastatus. Trypanosoma sp. isolates were different from the other species in all experiments, except in complement lysis. Experimental infection of triatomines and culicines with Trypanosoma sp. proved to be transitory. These parasites were noninfective for both normal and immunosuppressed mice. Isoenzyme and RAPD profiles obtained for Trypanosoma sp. were quite distinct from T. cruzi and T. rangeli and closely related to T. vespertilionis and T. hastatus. No cross-reaction was observed between sera from mice infected with Trypanosoma sp. and the other trypanosomatids and vice-versa. Trypanosoma sp. induced no protection against T. cruzi infection in mice. The very low, or nonsimilarity between Trypanosoma sp. isolates and the other species used in this study suggests that they might be members of a distinct bat trypanosome species. However, further studies should be done to prove their affinities with Trypanosoma cruzi-marinkellei, another trypanosome species from bats.

Ultrastructural effects of the chelating agent 1,10-phenanthroline on Trypanosoma cruzi epimastigotes in vitro.

The in vitro effects of the metal chelator 1,10-phenanthroline (OPHEN) on the ultrastructure of Trypanosoma cruzi epimastigotes were investigated. Epimastigotes treated with OPHEN display swelling and electron-dense deposits in the kinetoplast, mitochondrion, and cisternae of the endoplasmic reticulum. These morphological alterations are dose-dependent and first appear at an OPHEN concentration of 5.0 microg/ml. Analytical electron microscope examination indicates that the metallic portion of the electron-dense deposits is predominantly calcium.

Synthesis and in vitro trypanocidal activity of some novel iron chelating agents.

This report describes the syntheses and in vitro trypanocidal activity of a number of iron (III) chelators against epimastigotes of Trypanosoma cruzi. The compounds examined included a number of lipophilic N-alkyl derivatives of 2-ethyl- and 2-methyl-3-hydroxypyrid-4-ones, N,N'-bis(o-hydroxybenzyl)-(+/-)-trans-1,2-diaminocyclohexane, cyclotetrachromotropylene and four commercially available carboxy derivatives of pyridine, pyrazine, and pyarazole. Benznidazole, the drug clinically used in the treatment of Chagas' disease in humans, served as standard. All compounds were screened in vitro against Trypanosoma cruzi epimastigotes at 50 and 100 micrograms/ml for 72 h of exposure. At 100 micrograms/ml dosage, at least 4 compounds exhibited high epimastigote growth inhibition (65-69%) comparable to benznidazole (72%), whereas 9 compounds showed moderate to fair activity (53-64%) in the in vitro assay. At the lower concentration (50 micrograms/ml), the inhibitory activity of the best of these compounds was reduced significantly (39-48%) compared to the standard drug (59%). The activity of all the carboxylic acids remained in the lower range (4-25%). It is hypothesized that the enhanced activity of some of the compounds is due to their increased lipophilicity which enables them to successfully pass through the cellular membrane of Trypanosoma cruzi epimastigotes. The trypanocidal activities of the most effective compounds were significantly reduced when tested in the presence of added ferric ion.

The in vitro effects of extracellular adenosine triphosphate on the ultrastructure of Trypanosoma cruzi epimastigotes.

Incubation for 24 h in culture medium containing 50 mM adenosine triphosphate (ATP) produces distinct alterations in the ultrastructure of Trypanosoma cruzi epimastigotes, most obvious of which is the formation of large membrane-bound vacuoles in the cytosol. These vacuoles become positive following exposure to the macromolecule horseradish peroxidase (HRP). After a 20-min chase in phosphate-buffered saline (PBS) the HRP-positive vacuoles begin to separate into discrete structures such that after a 60-min chase, obvious reservosomes are identifiable. It is hypothesized that extracellular ATP causes increased permeability of the epimastigote's plasma membrane, resulting in ionic fluxes that, in turn, interfere with the normal formation of reservosomes.

In vitro trypanocidal activity of tetraethylthiuram disulfide and sodium diethylamine-N-carbodithioate on Trypanosoma cruzi.

Tetraethylthiuram disulfide (disulfiram) (TETD) and sodium diethylamine-N-carbodithioate (DECD) were examined for their in vitro trypanocidal activity against Trypanosoma cruzi. Benznidazole (BNZ), the drug used clinically for the chemotherapy of Chagas' disease, was used as a positive control. Inhibition assays included evaluation against the epimastigote, trypomastigote, and amastigote forms. Tetraethylthiuram disulfide and its reductive metabolite DECD inhibited 64.6% and 69.7%, respectively, of epimastigotes at a concentration of 50 micrograms/ml after 72 hr and BNZ caused 69.1% inhibition at the same concentration. Both TETD and DECD were not as effective against tissue culture trypomastigotes as BNZ (TETD = 47.7%,; DECD = 46.1%; BNZ = 88.7%) at 50 micrograms/ml after 24 hr. However, TETD and DECD treatment of amastigote-infected 3T3 fibroblasts yielded 60 and 67% inhibition, respectively, as compared to BNZ with an inhibition of 62%. A possible mechanism of action of TETD and DECD is via interference with the essential metal metabolism of T. cruci. Since toxicity data for both TETD and DECD are available and both drugs are active against the parasite, these drugs are candidates for further study to determine if they are of potential clinical interest as a prophylactic or therapeutic agent against Chagas' disease.

Antibodies reactive to Trypanosoma cruzi epimastigotes or amastigotes express different idiotypic patterns if from patients with different clinical forms of Chagas' disease.

Antibodies (Abs) were purified from pooled sera of patients with either indeterminate (IND or I) or cardiac (CARD or C) Chagas' disease, on either epimastigote (EPI or E) or amastigote-enriched (AMAST or A) antigen (Ag) columns and their idiotypic (Id) expression examined. Anti-Id rabbit Abs were raised to the different preparations (E-IdI, E-IdC, A-IdI and A-IdC). Competitive ELISAs using anti-Ids were able to discriminate between IdI and IdC, disregarding Ag reactivity. E-IdI and A-IdI present different inhibitory abilities, as do E-IdC and A-IdC, but IdC always competes with IdI for anti-IdI comparably. In contrast, a 4-8-fold increase of IdI is required to compete in parallel with IdC for anti-IdC. Therefore, Ids from IND patients share only low levels of the Ids that are most characteristic of CARD patients. While some CARD Abs also express Ids in common with IND patients, these studies reveal that CARD Abs express some Ids that are characteristic to only CARD patients, and these Ids are present on Abs purified with either EPI or AMAST.

An ultrastructure study of the in vitro effects of L-leucine methyl ester and ammonium chloride on Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi epimastigotes were subjected to the lysosomotropic agents L-leucine methyl ester and ammonium chloride to determine their effects on the ultrastructure of the parasite. The lysosomotropic agents applied to epimastigotes caused a time-dependent alteration in the morphology of the cells marked by a 5-fold increase in the number of lysosomes. Continued exposure to ammonium chloride caused slight disruption of the reservosomes. The amino acid ester, however, while causing the parasite to swell after prolonged exposure (e.g., 24 h), had little effect on the reservosomes, the kinetoplast, or even the mitochondrion. A specific inhibitor of cysteine proteinases provided some protection for lysosomes from the effects of the amino acid ester. Although it is agreed that reservosomes are similar to endosomes, no lysosomal fusion with the reservosomes was observed. Acid phosphatase activity was observed only in lysosomes.

Biological and isoenzymatic characterization of Trypanosoma cruzi strains isolated from sylvatic reservoirs and vectors from the state of Santa Catarina, Southern Brazil.

Sixty eight Trypanosoma cruzi strains were isolated in the state of Santa Catarina, Southern Brazil, from sylvatic reservoirs or naturally infected vectors and characterized by their biological behaviour in mice, morphology of bloodstream forms and isoenzyme profiles. Twenty eight strains were isolated from the triatomine bug (Panstrongylus megistus), 2 from rodents (Echimys dasythrix and Akodon sp) and 38 from opossums (Didelphis marsupialis). The infectivity in mice of 48 T. cruzi strains showed that 2 (4.2%) were of high virulence, 19 (39.6%) of medium virulence, 15 (31.2%) of low virulence and 12 (25.0%) produced subpatent parasitemia in mice. A morphological study of bloodstream trypomastigotes from 8 T. cruzi strains showed a predominance of stout forms. The isoenzyme pattern of 59 T. cruzi strains showed that 54 (91.5%) belonged to zymodeme Z1, 3 (5.1%) to zymodeme Z2 and 2 (3.4%) to mixed zymodemes, Z1 and Z2. All 34 T. cruzi strains analyzed from opossums were Z1. Three out of 5 strains isolated from adults of P. megistus collected inside houses, belonged to zymodeme Z2 and two strains exhibited mixed zymodemes, Z1 and Z2, in 5 out of 6 enzymes studied. Although the State of Santa Catarina is a non endemic region for human Chagas'disease, the presence of zymodeme Z2 parasites in the sylvatic vector, P. megistus, captured in domiciliary environments suggests the possibility of human and/or domestic mammal infection by T. cruzi.

In vitro effects of mannan and cytochalasin B on the uptake of horseradish peroxidase and [14C]sucrose by Trypanosoma cruzi epimastigotes.

Ultrastructural and quantitative studies were conducted to determine the in vitro effects of mannan and cytochalasin B (CB) on the transport of horseradish peroxidase (HRP) and [14C]sucrose by epimastigotes of Trypanosoma cruzi (strain Y). Time-dependent changes in HRP uptake were observed in cells incubated with the actin inhibitor CB. After 60 min incubation in CB, HRP and sucrose uptakes were inhibited by 48 +/- 15.4% and 16.5 +/- 3.96%, respectively. Morphological changed included HRP reaction product on the cell surface and a reduction in the number of HRP-positive reservosomes when compared to controls. After 120 min incubation, no inhibition was measured for either molecule. However, electron microscopy revealed HRP reaction product on the surface of the cells and in the cytosol. Also, perturbation of the plasma membrane was evident, suggesting that CB compromised the integrity of the plasma membrane, allowing HRP and sucrose to diffuse into the cytosol, giving misleading quantitative results. Mannan displayed a concentration-dependent inhibitory effect on HRP uptake but had little effect on sucrose uptake. Electron microscopic analysis revealed no change in the number of reservosomes per cell but reduction in the amount of HRP in reservosomes concomitant with mannan concentration. These results suggest that T. cruzi epimastigotes transport HRP by receptor-mediated and fluid-phase pinocytosis.

Trypanosoma rangeli (Tejera, 1920) isolated from a sylvatic rodent (Echimys dasythrix) in Santa Catarina Island, Santa Catarina State: first report of this trypanosome in southern Brazil.

A trypanosome strain isolated from a sylvatic rodent (Echimys dasythrix) from Santa Catarina Island (Santa Catarina State, Brazil) was characterized by the following methods: experimental transmission and development in invertebrate and vertebrate hosts, morphometry, cross protection, complement sensitivity, lectin agglutination and isoenzyme profiles. Comparisons were made with standard Trypanosoma cruzi and T. rangeli strains. All methods except isoenzyme analysis led to the identification of the isolate as T. rangeli. The isoenzyme differences found could be explained on the basis of polymorphism. Therefore this is the first report of T. rangeli in southern Brazil, increasing the geographical distribution of this parasite.