Thrombocytopenia and Platelet Dysfunction in Acute Tropical Infectious Diseases.

Thrombocytopenia is a well-known manifestation of acute tropical infectious diseases. The role of platelets in infections has received much attention recently because of their emerging activities in modulation of inflammatory responses, host defense, and vascular integrity. However, while many studies have addressed thrombocytopenia in tropical infections, abnormalities in platelet function have been largely overlooked. This is an important research gap, as platelet dysfunction may contribute to the bleeding tendency that characterizes some tropical infections. The development of novel platelet function assays that can be used in thrombocytopenic conditions (e.g., flow cytometry assays) has contributed to important new insights in recent years. In this review, the importance of platelets in tropical infections is discussed with special emphasis on the underlying mechanisms and consequences of thrombocytopenia and platelet dysfunction in these infections. Special attention is paid to malaria, a disease characterized by microvascular obstruction in which bleeding is rare, and to infections in which bleeding is common, such as dengue, other viral hemorrhagic fevers, and the bacterial infection leptospirosis. Given the importance of platelet function abnormalities in these infections, the development of affordable assays for monitoring of platelet function in low-resource countries, as well as pharmacologic interventions to prevent or reverse platelet function abnormalities, might improve clinical care and the prognosis of these infections.

Interaction of Leptospira interrogans with human proteolytic systems enhances dissemination through endothelial cells and protease levels.

We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date.

Adenosine deaminase activity in serum, erythrocytes and lymphocytes of rats infected with Leptospira icterohaemorrhagiae.

Leptospirosis is a systemic disease of humans and domestic animals, mainly dogs, cattle and swine. The course of human leptospirosis varies from mild to severe fatal forms and the most severe form of human leptospirosis is principally caused by Leptospira interrogans serovar icterohaemorrhagiae (L. icterohaemorrhagiae). The enzyme adenosine deaminase (ADA) plays an important role in the production and differentiation of blood cells. The aim of this study was to evaluate the activity of ADA in serum, erythrocytes and lymphocytes of rats infected with L. icterohaemorrhagiae, as compared with non-infected rats. Twenty-four adult rats, divided into two uniform groups (A and B) were used for the enzymatic assays. The animals in Group B were inoculated intraperitoneally with 2×10(8) leptospires/rat, and the rodents in Group A (control) were not-inoculated. Blood collection was performed on days 5 and 15 post-infection (PI) and the blood used to assess the ADA activity. The infection by L.icterohaemorrhagiae altered erythrocyte count, hemoglobin concentration and hematocrit, causing a decrease in all these parameters on day 15 PI. Lymphocytes decreased significantly on day 15 PI, and ADA activity in serum was inhibited in infected rats on days 5 and 15 PI and its activity in erythrocytes were increased on day 5 PI. On day 5 PI, we found an increase in ADA activity in erythrocytes of infected rats. No correlation was observed between hematocrit and erythrocyte ADA activity on days 5 and 15 PI. The ADA activity was inhibited in rats infected on day 15 PI. A positive correlation (r(2)=60) was also observed between the number of lymphocytes and ADA activity in lymphocytes on day 15 PI (P<0.05). In conclusion, our results showed that the ADA activity is altered in serum, lymphocytes and erythrocytes in experimental infection by L.icterohaemorrhagiae in rats, concomitantly with hematological parameters.

InvA protein is a Nudix hydrolase required for infection by pathogenic Leptospira in cell lines and animals.

Leptospirosis caused by pathogenic species of the genus Leptospira is a re-emerging zoonotic disease, which affects a wide variety of host species and is transmitted by contaminated water. The genomes of several pathogenic Leptospira species contain a gene named invA, which contains a Nudix domain. However, the function of this gene has never been characterized. Here, we demonstrated that the invA gene was highly conserved in protein sequence and present in all tested pathogenic Leptospira species. The recombinant InvA protein of pathogenic L. interrogans strain Lai hydrolyzed several specific dinucleoside oligophosphate substrates, reflecting the enzymatic activity of Nudix in Leptospira species. Pathogenic leptospires did not express this protein in media but temporarily expressed it at early stages (within 60 min) of infection of macrophages and nephric epithelial cells. Comparing with the wild type, the invA-deficient mutant displayed much lower infectivity and a significantly reduced survival rate in macrophages and nephric epithelial cells. Moreover, the invA-deficient leptospires presented an attenuated virulence in hamsters, caused mild histopathological damage, and were transmitted in lower numbers in the urine, compared with the wild-type strain. The invA revertant, made by complementing the invA-deficient mutant with the invA gene, reacquired virulence similar to the wild type in vitro and in vivo. The LD(50) in hamsters was 1000-fold higher for the invA-deficient mutant than for the invA revertant and wild type. These results demonstrate that the InvA protein is a Nudix hydrolase, and the invA gene is essential for virulence in pathogenic Leptospira species.

In vitro evidence for immune evasion activity by human plasmin associated to pathogenic Leptospira interrogans.

Leptospirosis is a widespread re-emerging zoonosis of human and veterinary concern. It has been shown that virulent leptospires protect themselves against the host's innate immune system, a strategy that allows the bacteria to reach immunologically safe environments. Although extensive studies on host-pathogen interactions have been performed, little is known on how leptospires deal with host immune attack. In a previous work, we demonstrated the ability of leptospires to bind human plasminogen (PLG), that after treatment with activators, conferred plasmin (PLA) activity on the bacteria surface. In this study, we show that the PLA activity associated to the outer surface of Leptospira could interfere with the host immune attack by conferring some evasion advantage during infection. We demonstrate that PLA-coated leptospires interfere with complement C3b and IgG depositions on the bacterial surface, probably through the degradation of these components, thus diminishing opsonization process. Similar decrease on the deposition was observed when normal and immune sera from patients diagnosed with leptospirosis were employed as a source of IgG. We believe that decreasing opsonization by PLA generation might be an important aspect of the leptospiral immune escape strategy and survival. To our knowledge, this is the first proteolytic activity of plasmin associated-Leptospira related to anti-opsonic properties reported to date.

Disproportional exaggerated aspartate transaminase is a useful prognostic parameter in late leptospirosis.

AIM: To evaluate the hepatic dysfunction in leptospirosis is usually mild and resolved eventually. However, sequential follow-up of liver biochemical data remained lacking.. METHODS: The biochemistry data and clinical symptoms of 11 sporadic patients were collected and analyzed, focusing on the impacts of leptospirosis upon liver biochemistry tests. RESULTS: The results disclosed that of the 11 cases, 5 or 45% died. The liver biochemistry data in the beginning of the disease course were only mildly elevated. Nevertheless, late exaggerated aspartate transaminase (AST) elevations were noted in three cases who finally died when compared with the typical course. Besides, significant higher AST/alanine transaminase (ALT) ratios (AARs) of the peak levels for transaminase were also noted in the cases who eventually succumbed. The mean+/-SD of AARs for the survival group and dead group were 5.65+/-2.27 (n = 5) and 1.86+/-0.64 (n = 6) respectively (P = 0.006). The ratios of the cases who finally died were all more than 3.0. Conversely, the survival group's ratios were less than 3.0. CONCLUSION: Serial follow-up of transaminase might provide evidence to predict some rare evolutions in leptospirosis. If AST elevated progressively without a concomitant change of ALT, it might indicate an acute disease course with ensuing death. Additionally, AAR is another prognostic parameter for leptospirosis. Once the value was higher than 3.0, a grave prognosis is inevitable.

Alterations in specific activity of glucose-6-phosphatase in laboratory rats after leptospiral exposure followed by triiodothyronine administration.

OBJECTIVE: To determine the effect of leptospirosis on thyroid hormone induction of the specific activity of hepatic microsomal glucose-6-phosphatase in laboratory rats. ANIMALS: Male Fisher 344 rats, 6 and 24 months old, healthy and infected with leptospirosis. PROCEDURE: The maximal velocity of glucose-6-phosphatase in intact and detergent-disrupted hepatic microsomes was assayed in duplicate or triplicate at 5 substrate concentrations, by monitoring the release of inorganic phosphate at 0-, 5-, and 10-minute intervals. The method of least squares was used to determine the velocity of the reactions. The level of statistical significance was determined, using the Student's t-test for unpaired data. Thyroid hormone (40 micrograms of T3/ 100 g of body weight) was administered for 5 consecutive days prior to sacrifice. RESULTS: Leptospirosis significantly increased the specific activity of the translocase component of glucose-6-phosphatase in old, but not young, rats. The activity of the translocase increased more than three-fold in untreated, infected old animals, compared with untreated, healthy old animals. Thyroid hormone induced a two- and threefold increase in the specific activities of the translocase in young and old healthy animals, respectively. Thyroid hormone did not increase the activity of the translocase in old animals infected with leptospirosis. CONCLUSIONS AND CLINICAL RELEVANCE: Leptospirosis alters the specific activity and induction by thyroid hormone of the translocase component of hepatic microsomal glucose-6-phosphatase in old male Fisher 344 rats. It is necessary to be aware of possible alterations in hepatic membrane-bound enzymes after leptospiral infection of older laboratory animals.

Fisiopatologia da leptospirose - hipótese para a insuficiência renal aguda peculiar / Physiopathology of leptospirosis - hypothesis for peculiar acute renal failure

Sao tecidas consideraçoes gerais sobre a insuficiência renal aguda (IRA) da leptospirose e acentuadas as principais características observadas numa série de 109 pacientes, destacando a tendência a hipopotassemia, verificada na metade dos casos, oligúricos ou nao oligúricos. Sobre tal tendência, um dos autores (MY-I) realizou investigaçoes (89-96) que indicaram: baixo potássio intra e extracelular, principalmente nos casos de IRA; presença de um fator plasmático capaz de evitar "in vitro" a ensima Na-K-ATPase de uma preparaçao purificada; a presença de uma endotoxina glicolipoprotéica da Leptospira interrogans cujo alvo parece ser a Na-K-ATPase, que é especificamente inibida no rim de coelhos. Sao feitos comentários clínicos sobre os envolvimentos cardio-vascular, pulmonar, renal, hepático e neuromuscular e sobre as anormalidades eletrolíticas, além de consideraçoes terapêuticas. Por fim é oferecida uma hipótese fisiopatológica para explicar a hipopotassemia na IRA da leptospirose, fundamentada na inibiçao da Na-K-ATPase nos diversos órgaos, provocada pela lise das leptospiras.

Hyperamylasemia and pancreatitis in leptospirosis.

Hyperamylasemia has been documented in up to 65% of our patients with leptospirosis and jaundice. However, pancreatitis is an uncommon complication of leptospirosis. Three patients with leptospirosis and pancreatitis are described and compared with two leptospirosis patients who had hyperamylasemia but in whom the diagnosis of pancreatitis could not be substantiated. The cause of the hyperamylasemia in the latter patients was nonpancreatic. The elevation of the amylase in these latter two patients could not be explained by renal insufficiency, because the level of the amylase was greater than three to four times the normal value, the upper limit to which amylase rises in renal failure. Thus, hyperamylasemia in patients with leptospirosis can be from pancreatic and nonpancreatic sources. Leptospirosis should be considered in the differential diagnosis of hyperamylasemia and pancreatitis.

[Creatine phosphokinase in leptospirosis]. / Creatinfosfocinasa en la leptospirosis.

Leptospirosis is a widely-distributed infectious disease, that usually presents with fever, headache and myalgia. Organ involvement could have very different severity degree. We evaluate 21 patients with leptospirosis looking for prognostic and diagnostic value of myalgia and/or elevated creatine-phosphokinase serum levels. Myalgia was recorded in nearly all patients (91%). Creatine-phosphokinase levels above normal limits were seen in 37% of cases, either in severe forms of leptospirosis with organ involvement or in mild forms of disease. We conclude that creatine-phosphokinase elevated levels could be of early diagnosis interest but they are not solely seen in the more severe forms of the disease.

[Leptospirosis with isolated cardiac manifestations]. / Leptospirose à manifestation cardiaque isolée.

A case of leptospirosis with isolated myocardial involvement is reported. Diagnostic problems set by this uncommon clinical picture are considered. The cause of the increase in CPK and MBCPK concentrations, reflecting myolysis, is discussed.

Serum creatine phosphokinase in leptospirosis.

Serum creatine phosphokinase (CPK) and serum glutamic oxaloacetic and pyruvic transaminase (SGOT, SGPT) levels were determined in 61 patients with leptospirosis and 16 with viral hepatitis during the acute phase of illness. The CPK value was elevated in 29 leptospirosis patients and normal in all 16 hepatitis patients. Conversely, mean SGOT and SGPT levels were lower in leptospirosis patients. The CPK determination is a simple test that may provide diagnostic information in a jaundiced patient, particularly when characteristic manifestations of leptospirosis are absent. The pattern of greatly elevated CPK levels with only modest elevations in transaminase values in an acutely jaundiced patient should strongly suggest a diagnosis of leptospirosis.

[Research of beta-galactosidase in the leptospira]. / Sulla ricerca della beta-galattosidase nelle leptospire

The Authors have investigated on the presence of beta-galactoxidase in the serotypes of Leptospires most frequently employed for the diagnosis of the formes epidemiologic occurrent in Italy. With the two employed methodes it was possible to make evident the enzyme in all the serotypes, but not in L. saxkoebing (Mus 24); L. ballum (Castellon 3), L. doberdò (Percedol 3).