Presence of parasite DNA in clinically unaffected nasal mucosa during cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis.

OBJECTIVES: We aimed to detect Leishmania DNA carriage in nasal mucosa of individuals with cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis. METHODS: A cross-sectional study was performed in all individuals with CL without nasal lesions (n = 153) attended within 2 years in an endemic area of L. (Viannia) braziliensis in Bahia (Brazil). An otorhinolaryngologist assessed the clinical status of the nasal mucosa by anterior rhinoscopy and endoscopic examinations. Swab samples were collected for parasite DNA detection by PCR from all individuals before standard treatment for leishmaniasis. A second evaluation 3 months after treatment was performed to assess clinical outcomes. RESULTS: Parasite DNA was detected in 7.8% (12/153) of clinically healthy nasal mucosa of individuals with CL. Interestingly, DNA was more frequently identified in individuals with more skin lesions (median 1.5, interquartile range (IQR) 1-3.5 versus 1.0, IQR 1-1.5; p 0.044), or larger injuries (median 2.7, IQR 2-3.8 versus 1.6, IQR 1-2.5; p 0.013). Additionally, the disease of those individuals with positive PCR evolved more frequently to unusual forms of leishmaniasis (recidiva cutis and disseminated) (45.5% (5/11) versus 11.5% (14/122); p 0.009), and required more cycles of treatment to reach clinical cure (median 2, IQR 1-4 versus 1, IQR 1-2; p 0.05). CONCLUSION: These findings suggest an early parasite tropism to nasal mucosa in L. (Viannia) braziliensis infection and a clinical phenotype of CL cases associated with parasite DNA in nasal mucosa. Future studies should evaluate whether PCR of nasal swab samples could serve as a prognostic tool for individuals at risk of mucocutaneous leishmaniasis.

Determination of avidity of IgG against protein antigens from Streptococcus pneumoniae: assay development and preliminary application in clinical settings.

The measurement of antibody levels is a common test for the diagnosis of Streptococcus pneumoniae infection in research. However, the quality of antibody response, reflected by avidity, has not been adequately evaluated. We aimed to evaluate the role of avidity of IgG against eight pneumococcal proteins in etiologic diagnosis. Eight pneumococcal proteins (Ply, CbpA, PspA1 and 2, PcpA, PhtD, StkP-C, and PcsB-N) were used to develop a multiplex bead-based avidity immunoassay. The assay was tested for effects of the chaotropic agent, multiplexing, and repeatability. The developed assay was applied to paired samples from children with or without pneumococcal disease (n = 38 for each group), determined by either serology, polymerase chain reaction (PCR), or blood culture. We found a good correlation between singleplex and multiplex assays, with r ≥ 0.94.The assay was reproducible, with mean inter-assay variation ≤ 9% and intra-assay variation < 6%. Children with pneumococcal disease had lower median avidity indexes in the acute phase of disease for PspA1 and 2 (p = 0.042), PcpA (p = 0.002), PhtD (p = 0.014), and StkP-C (p < 0.001). When the use of IgG avidity as a diagnostic tool for pneumococcal infection was evaluated, the highest discriminative power was found for StkP-C, followed by PcpA (area under the curve [95% confidence interval, CI]: 0.868 [0.759-0.977] and 0.743 [0.607-879], respectively). The developed assay was robust and had no deleterious influence from multiplexing. Children with pneumococcal disease had lower median avidity against five pneumococcal proteins in the acute phase of disease compared to children without disease.

Prevalence and risk factors for wheezing in Salvador, Brazil: a population-based study.

BACKGROUND: Wheezing is one of the most frequent causes of visit to emergency rooms among children. However, data on wheezing burden are mostly provided at healthcare setting, and particularly only for infants. AIMS: We sought to estimate the prevalence of wheezing in children under 4 years and to assess potential risk factors in the community. DESIGN: This was a cross-sectional analysis of a population-based cohort study. METHODS: The sample comprised children aged <4 years living in Salvador, Brazil. Data were collected via home visits when the parents/guardians were interviewed. Data were recorded on standardized forms. RESULTS: Of 1534 children, mean age was 21 ± 14 months (minimum 3 days; maximum 47 months; 6% <2 months); 780 (51%) were males and 501 [33%; 95% confidence interval (95% CI): 30-35%] reported wheezing in the last 12 months. Among wheezers, 321 (64%) had occasional wheezing. Overall, 180 (12%; 95% CI: 10-14%) had recurrent wheezing and 157 (10%; 95% CI: 9-12%) had asthma. For children in the first, second, third and fourth year of life wheezing was reported in 23, 41, 34 and 37%, respectively. Mother atopic-related disease was independently associated with recurrent wheezing (AdjPR[95% CI]: 1.54 [1.12-2.11]) and asthma (AdjPR[95% CI]: 1.54 [1.10-2.16]). Smoker at home (AdjPR[95% CI]: 1.34 [1.07-1.67]) and low birth weight (AdjPR[95%CI]: 1.38 [1.05-1.81]) were independently associated with occasional wheezing. CONCLUSIONS: One-third of under 4 years reported wheezing; history of mother's atopic-related disease was an independent risk factor for recurrent wheezing and asthma; smoker at home and low birth weight were independent risk factors for occasional wheezing.

Changes in amounts of total salivary gland proteins of Lutzomyia longipallpis (Diptera: Psychodidae) according to age and diet.

Saliva plays important roles in facilitation of a bloodmeal, lubrication of mouthparts, and parasite transmission for some vector insects. Salivary composition changes during the lifetime of an insect, and differences in the salivary profile may influence its functions. In this report, the amount and profile of salivary gland protein of the American visceral leishmaniasis vector Lutzomyia longipalpis (Lutz & Neiva, 1912) were analyzed at different times of insect development and diet. Protein content from unfed female sand flies increased significantly with age, and a significant difference was observed in sugar-fed females during the first 10 d of adult life. Salivary protein content sharply decreased 1 d after blood feeding, with gradual increase in concentration the following days. SDS-polyacrylamide gel electrophoresis analysis revealed that most polypeptides present in the saliva of sugar-fed also were present in the saliva of blood-fed females. Understanding changes in sand fly's saliva contents at distinct days after emergence and the influence of a bloodmeal in this aspect may reveal the role played by saliva during leishmaniasis transmission.

Role of sand fly saliva in human and experimental leishmaniasis: current insights.

Leishmaniases are wide spread diseases transmitted to their vertebrate host by infected sand fly. The saliva from these arthropods contains a vast repertoire of pharmacologically active molecules that hampers the host's haemostatic, inflammatory and immune responses. The early interactions between Leishmania and the host's immune response are closely linked to disease evolution or protection against the protozoan, and the ectoparasite saliva contributes directly to these interactions. Current studies have depicted these features, and these relations are being widely explored. There are concrete indications that the host response against sand fly saliva influences disease outcome in leishmaniasis. Additionally, there are demonstrations that immunization with whole sand fly saliva, or its components, leads to protection against leishmaniasis in different host species. The combination of these evidences opens up optimistic perspectives for improving vaccine development against Leishmania infection.

Experimental model of chronic osteomyelitis caused by Leishmania (L) amazonensis.

Experimental animal models have been used for the study of the physiopathogenesis of leishmaniasis, on some occasions with success, while in other situations such as bone alterations that accompany tegumentary leishmaniasis, especially in diffuse cutaneous form (DCL), the mechanisms are still unknown. In the present study, we determined these alterations in an animal model susceptible to Leishmania (L) amazonensis. Amastigotes of L. (L) amazonensis isolated from patients with diffuse cutaneous leishmaniasis (DCL) were inoculated into the hind paws of eight BALB/c mice, macroscopic and histopathological aspects were analyzed. After 90 and 120 days of evolution, histopathological analysis demonstrated a mononuclear cell infiltrate rich in plasma cells and intense parasitism of intra- and extra-medullary macrophages, with areas of bone necrosis and discrete involvement of cartilaginous tissue. The results show that the inflammatory process developed during L. (L) amazonensis infection might cause bone tissue destruction and secondarily affect the joints.

Imaging exams of bone lesions in patients with diffuse cutaneous leishmaniasis (DCL).

We studied bone lesion alterations in three patients with diffuse cutaneous leishmaniasis (DCL) by imaging exams (radiography and scintigraphy) and histopathology. Two patients had bone lesions of distal extremities of hands and feet, and one infiltrating plaques in the skin. The study was conducted at three specialized centers (Presidente Dutra Hospital/Nucleus of Tropical Pathology, UFMA-MA; Gonçalo Moniz Research Center-FIOCRUZ-BA; Laboratory of Pathology of Infectious Diseases (LIM-50), University of São Paulo, SP). Three-phase bone scintigraphy demonstrated high sensitivity and specificity for bone lesions, showing increased uptake of the radiopharmaceutical at sites of active lesions. In contrast, radiography demonstrated lytic lesions, cortical destruction and local osteopenia of the bone extremeties in two patients. Histopathological analysis showed sequestration with presence of amastigote forms of Leishmania (osteomyelitis), mononuclear cells and macrophages containing amastigote forms of Leishmania in one patient. These preliminary data indicate that imaging exams (radiography and scintigraphy) are important in the evaluation of bone lesions in diffuse cutaneous leishmaniasis and should be included in the routine histopathological diagnosis of the disease and follow-up of bone lesions.

The susceptibility of domestic cats (Felis catus) to experimental infection with Leishmania braziliensis.

Over the last few years, several cases of feline leishmaniasis (FL) with cutaneous and visceral forms have been reported around the world. Nonetheless, the real susceptibility of cats to infection with Leishmania spp. and the outcome of leishmaniasis in these animals are poorly understood. Experimental studies on feline models will contribute to the knowledge of natural FL. Thus, in order to determine the susceptibility of domestic cats (Felis catus) to experimental infection with Leishmania braziliensis, 13 stray cats were infected with 10(7) promastigotes by the intradermal route in the ear and nose simultaneously and followed up for 72 weeks. Soon after infection, the earliest indication of a lesion was a papule on the ear at 2 weeks post-infection (w.p.i.). The emergence of satellite papules around the primary lesion was observed about 4 w.p.i. Two weeks later these papules coalesced and formed a huge and irregular nodule. Thereafter, there was lesion dissemination to the external and marginal surface of the ipsilateral ear, and later to the contralateral ear. At 10 w.p.i., some nodules became ulcerated. Nose lesions presented a similar evolution. At both sites, the largest lesion sizes occurred at 10 w.p.i. and started to decrease 15 days later. Ear and nose nodules healed at 32 and 40 w.p.i., respectively. Specific L. braziliensis IgG antibody titers (optical density> or = 0.01 as positive result) were detected as early as 2 w.p.i. (0.09 +/- 0.02) in only three animals (23%), and all cats had positive titers at 20 w.p.i. (0.34 +/- 0.06). Only three animals (38%) continued to show positive serology at 72 w.p.i. (0.08 +/- 0.02). Up to that time, none of the cats had lesion recurrence. In a feline model of cutaneous leishmaniasis, it seems that there is no correlation between active lesions and positive serology. The implications of these data are discussed.

Leishmanial infection: analysis of its first steps: a review

The first steps in leishmaniasis are critical in determining the evolution of the disease. Major advances have recently been done in understanding this crucial moment. Fundamental research in parasite-vector interaction, parasite biology, insect saliva, and vertebrate host response have shed new light and uncovered a most fascinating and complex moment in leishmaniasis. We review here some of these aspects and we try to connect them in a logical framework.

Leishmanial infection: analysis of its first steps. A review.

The first steps in leishmaniasis are critical in determining the evolution of the disease. Major advances have recently been done in understanding this crucial moment. Fundamental research in parasite-vector interaction, parasite biology, insect saliva, and vertebrate host response have shed new light and uncovered a most fascinating and complex moment in leishmaniasis. We review here some of these aspects and we try to connect them in a logical framework.

B-cell infiltration and frequency of cytokine producing cells differ between localized and disseminated human cutaneous leishmaniases.

Biopsies from human localized cutaneous lesions (LCL n = 7) or disseminated lesions (DL n = 8) cases were characterized according to cellular infiltration,frequency of cytokine (IFN-gamma, TNF-alpha) or iNOS enzyme producing cells. LCL, the most usual form of the disease with usually one or two lesions, exhibits extensive tissue damage. DL is a rare form with widespread lesions throughout the body; exhibiting poor parasite containment but less tissue damage. We demonstrated that LCL lesions exhibit higher frequency of B lymphocytes and a higher intensity of IFN-gamma expression. In both forms of the disease CD8+ were found in higher frequency than CD4+ T cells. Frequency of TNF-alpha and iNOS producing cells, as well as the frequency of CD68+ macrophages, did not differ between LCL and DL. Our findings reinforce the link between an efficient control of parasite and tissue damage, implicating higher frequency of IFN-gamma producing cells, as well as its possible counteraction by infiltrated B cells and hence possible humoral immune response in situ.

B-cell infiltration and frequency of cytokine producing cells differ between localized and disseminated human cutaneous leishmaniases

Biopsies from human localized cutaneous lesions (LCL n = 7) or disseminated lesions (DL n = 8) cases were characterized according to cellular infiltration,frequency of cytokine (IFN-g, TNF-alpha) or iNOS enzyme producing cells. LCL, the most usual form of the disease with usually one or two lesions, exhibits extensive tissue damage. DL is a rare form with widespread lesions throughout the body; exhibiting poor parasite containment but less tissue damage. We demonstrated that LCL lesions exhibit higher frequency of B lymphocytes and a higher intensity of IFN-gamma expression. In both forms of the disease CD8+ were found in higher frequency than CD4+ T cells. Frequency of TNF-alpha and iNOS producing cells, as well as the frequency of CD68+ macrophages, did not differ between LCL and DL. Our findings reinforce the link between an efficient control of parasite and tissue damage, implicating higher frequency of IFN-gamma producing cells, as well as its possible counteraction by infiltrated B cells and hence possible humoral immune response in situ

Risk factors associated with asymptomatic infection by Leishmania chagasi in north-east Brazil.

Various factors have been associated with a predisposition to the development of clinical American visceral leishmaniasis (AVL). However, little information is available about the factors that predispose to asymptomatic infection. To identify the risk factors associated with asymptomatic infection, a study was carried out between July 1997 and June 1998 on children aged 0-5 years in the districts of Vila Nova and Bom Viver in the municipality of Raposa in the island of São Luís, State of Maranhão, Brazil. A questionnaire containing socioeconomic, demographic and epidemiological data was used. The delayed-type hypersensitivity (DTH) test was carried out on 639 children in the first phase, and on 572 in the second, 7 months after the first survey, using Leishmania amazonensis antigen. Infection was determined by enzyme-linked immunosorbent assay (ELISA) in 638 children during the first phase, and in 572 during the second. Six outcome measures were used: initial prevalence, final prevalence and incidence, each determined by DTH and ELISA. The incidence of infection was 10.8% when determined by DTH and 28.5% when determined by ELISA. After adjustment for confounding variables using Cox regression, infection by L. chagasi was associated with child's age (> or = 2 years), location of the dwellings (Vila Nova) and reporting of relatives with AVL. Bathing outside the house and playing outdoors between 18:00 and 20:00 were identified as risk factors in some analyses but not in others. Presence of intra- and peridomestic Lutzomyia sandflies and animals such as dogs or chickens in the house or in the neighbourhood appeared as risk factors in some analyses but in others they unexpectedly seemed to protect from infection. Malnutrition was not found to be associated with infection.

Differences in gamma interferon production in vitro predict the pace of the in vivo response to Leishmania amazonensis in healthy volunteers.

The initial encounter of Leishmania cells and cells from the immune system is fundamentally important in the outcome of infection and determines disease development or resistance. We evaluated the anti-Leishmania amazonensis response of naive volunteers by using an in vitro priming (IVP) system and comparing the responses following in vivo vaccination against the same parasite. In vitro stimulation allowed us to distinguish two groups of individuals, those who produced small amounts of gamma interferon (IFN-gamma) (n = 16) (low producers) and those who produced large amounts of this cytokine (n = 16) (high producers). IFN-gamma production was proportional to tumor necrosis factor alpha and interleukin 10 (IL-10) levels but did not correlate with IL-5 production. Volunteers who produced small amounts of IFN-gamma in vitro remained low producers 40 days after vaccination, whereas high producers exhibited increased IFN-gamma production. However, 6 months after vaccination, all individuals tested produced similarly high levels of IFN-gamma upon stimulation of their peripheral blood mononuclear cells with Leishmania promastigotes, indicating that low in vitro producers respond slowly in vivo to vaccination. In high IFN-gamma producers there was an increased frequency of activated CD8(+) T cells both in vitro and in vivo compared to the frequency in low producers, and such cells were positive for IFN-gamma as determined by intracellular staining. Such findings suggest that IVP responses can be used to predict the pace of postvaccination responses of test volunteers. Although all vaccinated individuals eventually have a potent anti-Leishmania cell-mediated immunity (CMI) response, a delay in mounting the CMI response may influence resistance against leishmaniasis.

[Leishmania (Leishmania) chagasi infection in children from an endemic area of visceral leishmaniasis in the São Luís Island-MA, Brazil]. / Infecção por Leishmania (Leishmania) chagasi em crianças de uma área endêmica de leishmaniose visceral americana na Ilha de São Luis-MA, Brasil.

A prospective study was undertaken in 648 children with less than 6 years of age in the municipality of Raposa, Maranhão, Brazil, from June 1997 to June 1998, to evaluate the characteristics of the infection by L.(L.)chagasi and verify if there is an association between malnutrition and asymptomatic infection. A standardized questionnaire was used containing socioeconomic, environmental and behavioral data. Montenegro skin reaction (IDRM) with L. amazonensis and Enzyme Linked Immunosorbent Assay (ELISA) test to detect infection, and anthropometric examination were performed. Initial and final prevalence and incidence of infection were 18.6%, 20.6% and 10.8% as measured by IDRM and 13.5%, 34.4% and 28% according to ELISA. The prevalence of chronic malnutrition was 26%. No association was detected between malnutrition and asymptomatic infection by L. (L.) chagasi. More effective control measures are needed in these areas since asymptomatic infection seems to be on the increase.

Interleukin 8 as a vaso-occlusive marker in Brazilian patients with sickle cell disease.

Sickle cell disease has a worldwide distribution and is a public health problem in Brazil. Although vaso-occlusive crisis (VOC) is one of the most important clinical features of the disease, there are still several steps of its pathogenesis which are unknown. The increase of the chemotactic factor interleukin 8 (IL-8) has been reported to be involved in sickle cell disease crisis, but this has not been demonstrated conclusively. In the present study we analyzed serum IL-8 levels by ELISA and hematological parameters and hemoglobin patterns by standard techniques in 23 (21 SS and 2 SC) Brazilian patients with sickle cell syndromes during VOC caused by different inducing factors, 22 (21 SS and 1 SC) sickle cell patients out of crisis, and 11 healthy controls. Increased IL-8 levels were observed in 19 of 23 VOC patients (79.2%), 3 of them with more than 1,000 pg/ml. Seventeen of 22 (77.3%) non-crisis patients showed low IL-8 levels (less than 15 pg/ml). Healthy controls had low IL-8 levels. A significant difference in serum IL-8 levels was observed between crisis and non-crisis sickle cell patients (P<0.0001). There was no correlation between IL-8 levels and hematological data or hemoglobin patterns. High serum IL-8 levels were observed in VOC patients independently of the crisis-inducing factor. We conclude that in the studied population, IL-8 concentration may be a useful VOC marker, although the mechanism of the pathogenic process of sickle cell VOC syndromes remains unclear.

Interleukin 8 as a vaso-occlusive marker in Brazilian patients with sickle cell disease

Sickle cell disease has a worldwide distribution and is a public health problem in Brazil. Although vaso-occlusive crisis (VOC) is one of the most important clinical features of the disease, there are still several steps of its pathogenesis which are unknown. The increase of the chemotactic factor interleukin 8 (IL-8) has been reported to be involved in sickle cell disease crisis, but this has not been demonstrated conclusively. In the present study we analyzed serum IL-8 levels by ELISA and hematological parameters and hemoglobin patterns by standard techniques in 23 (21 SS and 2 SC) Brazilian patients with sickle cell syndromes during VOC caused by different inducing factors, 22 (21 SS and 1 SC) sickle cell patients out of crisis, and 11 healthy controls. Increased IL-8 levels were observed in 19 of 23 VOC patients (79.2 percent), 3 of them with more than 1,000 pg/ml. Seventeen of 22 (77.3 percent) non-crisis patients showed low IL-8 levels (less than 15 pg/ml). Healthy controls had low IL-8 levels. A significant difference in serum IL-8 levels was observed between crisis and non-crisis sickle cell patients (P<0.0001). There was no correlation between IL-8 levels and hematological data or hemoglobin patterns. High serum IL-8 levels were observed in VOC patients independently of the crisis-inducing factor. We conclude that in the studied population, IL-8 concentration may be a useful VOC marker, although the mechanism of the pathogenic process of sickle cell VOC syndromes remains unclear

Flow cytometric determination of cellular sources and frequencies of key cytokine-producing lymphocytes directed against recombinant LACK and soluble Leishmania antigen in human cutaneous leishmaniasis.

Leishmaniasis, caused by infection with the protozoan parasite Leishmania, affects millions of individuals worldwide, causing serious morbidity and mortality. This study directly determined the frequency of cells producing key immunoregulatory cytokines in response to the recombinant antigen Leishmania homolog of receptors for activated kinase C (LACK) and soluble leishmania antigen (SLA), and it determined relative contributions of these antigens to the overall cytokine profile in individuals infected for the first time with Leishmania braziliensis. All individuals presented with the cutaneous clinical form of leishmaniasis and were analyzed for proliferative responses to LACK antigen and SLA, frequency of lymphocyte subpopulations (analyzed ex vivo), and antigen-induced (LACK and SLA) cytokine production at the single-cell level (determined by flow cytometry). The following were determined. (i) The Th1-type response previously seen in patients with cutaneous leishmaniasis is due to gamma interferon (IFN-gamma) production by several different sources, listed in order of contribution: CD4(+) T lymphocytes, CD4(-), CD8(-) lymphocytes, and CD8(+) T lymphocytes. (ii) SLA induced a higher frequency of lymphocytes producing IFN-gamma and tumor necrosis factor alpha (TNF-alpha) than did LACK. (iii) LACK induced an activation of monocyte populations as reflected by an increased percentage of CD14-positive cells. (iv) Neither SLA nor LACK induced detectable frequencies of cells producing interleukin-4 (IL-4) or IL-5. These data demonstrated a multifaceted immune response to SLA in human leishmaniasis involving Th1 CD4(+) T lymphocytes (IFN-gamma(+) and IL-10(-)/IL-4(-)), Tc1 CD8(+) T cells (IFN-gamma(+), and IL-10(-)/IL-4(-)), and a high frequency of TNF-alpha-producing lymphocytes. Moreover, it was determined that the recombinant antigen LACK acts as a weak inducer of Th1-type lymphocyte responses compared to SLA.

A simple method for human peripheral blood monocyte isolation.

We describe a simple method using percoll gradient for isolation of highly enriched human monocytes. High numbers of fully functional cells are obtained from whole blood or buffy coat cells. The use of simple laboratory equipment and a relatively cheap reagent makes the described method a convenient approach to obtaining human monocytes.

Mucosal leishmaniasis: quantitative nasal cytology as a marker of disease activity and indicator of healing.

Mucosal leishmaniasis (ML) follow-up is based on subjective parameters. Using simplified quantitative cytology of nasal lavages (QNCs), we studied 20 ML patients, 10 patients with cutaneous leishmaniasis (CL), and 10 healthy subjects. Patients with ML were treated with antimony and followed up with otolaryngological examination plus QNCs for 6 months. At the first evaluation, the median total number of cells in ML patients (1,540,000) was greater than that in CL patients (215,000) or that in healthy subjects (250,000). Neutrophils were predominant in ML patients, in contrast to both sets of controls, in whom epithelial cells were more frequent. During treatment, we found a significant reduction in total nasal cell counts in ML patients who were cured, and encountered a switch in predominant cell type. The cytology of 2 patients who did not respond to antimony remained the same. It is therefore possible to detect nasal inflammation in ML patients through QNCs, which may indicate extension of mucosal involvement, providing an objective parameter to monitor therapy.