The single recombinant M. tuberculosis protein DPPD provides enhanced performance of skin testing among HIV-infected tuberculosis patients.

Diagnostic testing for M. tuberculosis infection has advanced with QuantiFERON and GeneXpert, but simple cost-effective alternatives for widespread TB screening has remained elusive and purified protein derivative (PPD)-based tuberculin skin testing (TST) remains the most widely used method. PPD-based tests have reduced performance, however, in BCG vaccinees and in individuals with immune deficiencies. We compared the performance of skin testing with the recombinant DPPD protein against that of a standard PPD-based skin test. Our data indicates similar performance of DPPD and PPD (r2 = 0.7689) among HIV-negative, active TB patients, all of whom presented greater than 10 mm induration following administration. In contrast to results demonstrating that PPD induced indurations greater than 5 mm (i.e., the recommended threshold for positive results in this population) in only half (19 of 38) of the HIV positive TB patients, 89.5% (34 of 38) of these participants developed indurations greater than 5 mm when challenged with DPPD. Importantly, none of the patients that were positive following PPD administration were negative following DPPD administration, indicating markedly improved sensitivity of DPPD among HIV-infected individuals. Our data indicate that DPPD has superior performance in skin testing than the current TST standard.

Utility of recombinant proteins LID-1 and PADL in screening for Mycobacterium leprae infection and leprosy.

BACKGROUND: Despite the widespread use of multidrug therapy, leprosy remains an important public health concern in many regions. Detection is generally limited to clinical exam. METHODS: As a two-tiered active case finding strategy, we used the LID-1 (leprosy IDRI diagnostic-1), and PADL (protein advances for the diagnosis of leprosy) antigens for serological examination of 2526 individuals randomly selected from 10 472 residents in a leprosy hyperendemic area (Cajazeiras, Paraiba, Brazil). Almost all seropositive (95%) and a subset of seronegative (17%) subjects then underwent clinical evaluations. RESULTS: Prevalence of clinically apparent leprosy was 2.3% (19 cases among 834 fully examined individuals). LID-1 and PADL demonstrated a high sensitivity for supporting leprosy diagnosis at 89% and 87%, with positive predictive values (PPV) of 3.5% and 3.7%. The specificity for clinically apparent leprosy was low at 42% and 38%, respectively, and was likely reduced due to the presence of many asymptomatic individuals infected with Mycobacterium leprae. CONCLUSIONS: Our data indicate the utility of the LID-1 and PADL antigens as primary screening tools for the detection of M. leprae infection and identification of leprosy patients. The follow-up of seropositive subjects could clarify the predictive value and utility of detecting anti-LID-1 and PADL antibodies within leprosy control programs.

Absorption and safety of alendronate, a nitrogen-containing bisphosphonate, after intrapulmonary administration in rats.

Alendronate, a nitrogen-containing bisphosphonate, has been used as a first-choice drug for the treatment of hypercalcemia and osteoporosis. In the present study, we examined the absorption and safety of alendronate after intrapulmonary administration in rats. The bioavailability (BA) of alendronate after intrapulmonary administration was 47% at a dose of 5 mg/kg, while the BA after oral administration was only 2.9% at a dose of 50 mg/kg in rats. Plasma calcium level, an index of the pharmacological effect of alendronate, was effectively reduced after intrapulmonary administration of alendronate. Furthermore, alendronate continuously reduced the increase in plasma calcium levels for 9 days after a single intrapulmonary administration in rats with 1α-hydroxyvitamin-D(3)-induced hypercalcemia. Intrapulmonary administration of alendronate also effectively suppressed the decrease in bone mass in a rat model of osteoporosis. Alendronate significantly increased the activity of lactate dehydrogenase (LDH) in bronchoalveolar lavage fluid (BALF), indicating that pulmonary mucosal damage was induced by intrapulmonary administration of alendronate. However, co-administration of superoxide dismutase (SOD) with alendronate completely suppressed the alendronate-induced increase in LDH activity in BALF, while maintaining sufficient pulmonary absorption and therapeutic effects of alendronate in rats with 1α-hydroxyvitamin-D(3)-induced hypercalcemia. These findings indicated that the lung is a promising, noninvasive alternative route for the delivery of alendronate in the treatment of bone diseases.

Treatment of canine visceral leishmaniasis by the vaccine Leish-111f+MPL-SE.

Immunotherapy of canine visceral leishmaniasis (CVL) may provide an alternative to both marginally effective chemotherapy and undesired euthanasia of infected dogs and could have a great impact not only on animal welfare, but also on control of human disease. Therefore, we examined the potential immunotherapeutic efficacy of the subunit vaccine Leish-111f+MPL-SE, which has undergone rigorous preclinical testing and been demonstrated safe in human clinical trials. Two separate trials were performed in Salvador, Brazil, to evaluate the vaccine for therapeutic efficacy against CVL caused by natural infection: an Open Trial and a Blinded Trial. In the Open Trial 59 dogs with clinically active CVL were sequentially allocated to four groups: group 1 received Leish-111f+MPL-SE; group 2 was treated with Glucantime; group 3 received a combination of the vaccine and Glucantime; and group 4 was given no treatment. At the 6-month assessment, the 13 non-treated dogs had either died or showed no clinical improvement. In contrast, most dogs in groups 1-3 showed initial improvement (100%, 80%, and 92%, respectively). Upon evaluation for a mean of 36 months after therapy, the following cure rates were observed: 75% for group 1 dogs (exact 95% confidence interval [CI] 43-95%), 64% for group 2 dogs (exact 95% CI 31-89%), and 50% for group 3 dogs (exact 95% CI 19-81%). Therapeutic efficacy of the Leish-111f+MPL-SE vaccine was reconfirmed in a subsequent Blinded Trial. The vaccine was effective for mild cases of CVL and was compromised in dogs with severe disease. Although further studies are required to understand mechanisms of action, the Leish-111f+MPL-SE vaccine is a promising tool to control VL in both dogs and humans.

Distinct antigen recognition pattern during zoonotic visceral leishmaniasis in humans and dogs.

Leishmania infantum is a causative agent of endemic zoonotic visceral leishmaniasis (VL) in regions of South America and the Mediterranean. Dogs are the major reservoirs for L. infantum in these regions, and control of disease in dogs could have a significant impact on human disease. Although dogs share many symptoms of VL with humans as a result of L. infantum infection, they also show some unique clinical manifestations, which are often a combination of visceral and cutaneous leishmaniasis, suggesting different mechanisms of disease development in dogs and humans. Here, we compare antibody responses of dogs and humans with VL to various defined leishmanial antigens. Parasite lysate and K39, the two most commonly used antigens for serodiagnosis of VL, detected the highest levels of antibodies in both humans and dogs with VL, whereas the recognition patterns of these antigens were distinct between the hosts. Among other defined antigens tested, LmSTI1 and CPB detected higher levels of antibodies in dogs and humans, respectively. These results indicate there is a difference between humans and dogs in antigen recognition patterns during VL. We infer that different strategies may need to be used in development of vaccines and diagnostics for humans and for dogs. In addition, we show a correlation between antibody titers to several antigens and severity of clinical symptoms during canine VL.

Use of protein antigens for early serological diagnosis of leprosy.

Leprosy is a chronic and debilitating human disease caused by infection with the Mycobacterium leprae bacillus. Despite the marked reduction in the number of registered worldwide leprosy cases as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains relatively stable. This indicates that M. leprae is still being transmitted and that, without earlier diagnosis, M. leprae infection will continue to pose a health problem. Current diagnostic techniques, based on the appearance of clinical symptoms or of immunoglobulin M (IgM) antibodies that recognize the bacterial phenolic glycolipid I, are unable to reliably identify early-stage leprosy. In this study we examine the ability of IgG within leprosy patient sera to bind several M. leprae protein antigens. As expected, multibacillary leprosy patients provided stronger responses than paucibacillary leprosy patients. We demonstrate that the geographic locations of the patients can influence the antigens they recognize but that ML0405 and ML2331 are recognized by sera from diverse regions (the Philippines, coastal and central Brazil, and Japan). A fusion construct of these two proteins (designated leprosy IDRI diagnostic 1 [LID-1]) retained the diagnostic activity of the component antigens. Upon testing against a panel of prospective sera from individuals who developed leprosy, we determined that LID-1 was capable of diagnosing leprosy 6 to 8 months before the onset of clinical symptoms. A serological diagnostic test capable of identifying and allowing treatment of early-stage leprosy could reduce transmission, prevent functional disabilities and stigmatizing deformities, and facilitate leprosy eradication.

Domestic swine in a visceral leishmaniasis endemic area produce antibodies against multiple Leishmania infantum antigens but apparently resist to L. infantum infection.

In order to investigate whether pigs can be infected by Leishmania infantum, a serological and parasitological study was carried out on swine in the Jequié municipality, Northeast of Brazil. Anti-Leishmania infantum antibodies were detected in 37 out of 92 swine (40.2%), by two different assays: an anti-L. infantum lysate and an anti-K39 recombinant protein ELISA. An experimental study was also carried out to verify the susceptibility of domestic pigs to L. infantum infection. Three sows inoculated with 10(8) stationary-phase infective L. infantum promastigotes (26% metacyclic promastigotes) per kilogram of body weight produced anti-Leishmania antibodies until the end of the experiment, 11 months later. No parasites, however, could be visualized through optical microscopy of spleen, liver and bone marrow or by in vitro culture of these organs. Homogenates of these organs were also inoculated in hamsters, without producing infection. No Leishmania DNA was detected by polymerase chain reaction (PCR) in sand flies fed on these animals. The results indicate that domestic pigs bitten by L. infantum-infected vectors in the endemic area do not display a full infection pattern, and the positive association in endemic areas between the presence of swine and infection in canines may not be ascribable to the former acting as a parasite reservoir.

Heat therapy for cutaneous leishmaniasis elicits a systemic cytokine response similar to that of antimonial (Glucantime) therapy.

Controlled heat delivered as radio waves has been used successfully in the treatment of cutaneous leishmaniasis (CL). Here we investigated whether local heat therapy has systemic effects, as measured by the modulation of cytokine production following heat therapy of CL lesions compared with antimonial (Glucantime) treatment. Patients with CL were randomly assigned into this study. Heat (50 degrees C for 30s) was applied once. The control group received Glucantime therapy for 20 d. Cytokine production by peripheral blood mononuclear cells was assayed on days 0, 14 and 28 after onset of treatment. At the end of 28 d, 75% of lesions were healing or healed in the heat therapy group and 90% in the control group (P=0.1261). There was a decrease in IFN-gamma, IL-5 and TNF-alpha levels comparing day 0 with day 28 in both groups, but no difference between the two therapy groups. In patients with only one of several lesions treated with heat therapy, the untreated lesions also healed. Local heat therapy in CL lesions leads to systemic cytokine responses similar to that induced by systemic Glucantime therapy.

Inoculação experimental de Equus asinus com Leishmania chagasi Cunha & Chagas, 1937 / Experimental infection of Equus asinus with Leishmania chagasi Cunha & Chagas, 1937

Quatro Equus asinus foram inoculados com promastigotas de Leishmania chagasi Cunha & Chagas, 1937 e acompanhados durante 12 meses através de: pesquisa de amastigotas em esfregaços e culturas de sangue periférico em fragmentos de tecido do lábio inferior, medula óssea, baço e fígado e de testes de ELISA e TRALd. Estes foram positivos nos 8º, 10º e 12º meses após a inoculação. O exame histopatológico pós necropsia, demonstrou discreto número de amastigotas no fígado de dois dos eqüídeos inoculados. Apesar de desafiados com elevado número de promastigotas, os animais não desenvolveram infecções patentes e não infectaram experimentalmente a vetora Lutzomya longipalpis. Os resultados induzem a acreditar que os eqüídeos são desprovidos de importância como reservatórios na cadeia de transmissão da leishmaniose visceral, embora sirvam como boa fonte de alimentação sangüínea e proliferação da vetora Lutzomyia longipalpis.

[Experimental infection of Equus asinus with Leishmania chagasi Cunha & Chagas, 1937]. / Inoculação experimental de Equus asinus com Leishmania chagasi Cunha & Chagas, 1937.

Four Equus asinus were challenged with promastigotes of Leishmania chagasi Cunha & Chagas, 1937, and followed up for 12 months. They were observed by means of direct testing for promastigotes in smears and culture of peripheral blood, fragments from inferior lip, bone marrow, spleen and liver and the immunological assays ELISA and TRALd. The post-necropsy histological examination demonstrated a small number of amastigotes in the liver of two animals. ELISA and TRALd tests were positive at the 8th, 10th and 12th month after inoculation. The results suggest that the donkeys were able to overcome the experimental leishmanial infection and did not infect the vector Lutzomyia longipalpis in the laboratory. Consequently they can not be considered an important reservoir in the epidemiological chain of transmission of visceral leishmaniasis, although they represent an important blood source for the vector and its proliferation.

Sucessful use of a defined antigen/GM-CSF adjuvant vaccine to treat mucosal leishmaniasis refractory to antimony: a case report

Immunotherapy has been proposed as a method to treat mucosal leishmaniosis for many years, but the approach has been hampered by poor definition and variability of antigens used, and results have been inconclusive. We report here a case of antimonial-refractory mucosal leishmaniasis in a 45 year old male who was treated with three single injections (one per month) with a cocktail of lour Leishmania recombinant antigens selected after documented hyporesponsiveness of the patient to these antigens, plus 50µg of GM-CSF as vaccine adjuvant. Three months after treatment, all lesions had resolved completely and the patient remains without relapse after two years. Side effects of the treatment included only moderate erythema and induration at the injection site after the second and third injections. We conclude that carefully selected microbial antigens and cytokine adjuvant can be sucessful as immunotherapy for patients with antimonial-refractory mucosal leishmaniasis.

Efficacy and tolerabiliy of loposomal amphotericin B (Ambisome) in the treatment of visceral leishmaniasis in Brazil

Thirty-two patients were enrolled in an open-label, dose/schedule ranging clinical trial to evaluate the efficacy and tolerability of loposomal amphotericin B (Ambisome) in the treatment of visceral leishmaniasis. All the patients received a dose of 2mg/kg daily for the first 4 days, followed by a single repeat dose of 2mg/kg at day 10 in 4 patients (total dose 10mg/kg); repeat doses on days 5, 6, and 10 in 13 patients (total dose 14/kg); or daily doses were continued on days 5 through 10 in 15 patients (total dose 20mg/kg). Patients had a mean age of 9 years, ranging between 3 and 26 years. Their mean weight was 25.9kg, ranging between 9.5kg and 75kg. All patients had splenomegaly,31/32 hepatomegaly, and 20 patients tested had leishmania documented on splenic aspirate. Six of the 32 patients were treated after relapse following antimony therapy. The duration of illness prior to therapy was a mean of 2 months, ranging between 2 weeks and 23 months. During and after treatment, there were significant reductions in liver and spleen size, and significant increases in body weight, hemoglobin levels and white blood cell counts. All patients showed initial cure at the 1 mouth follow-up. Seven patients relapsed between 2 and 6 months after the start of treatment. There was no dose relationship to the occurrence of relapse. The relapse rate in children 5 years of age or less was 7/15 (47 percent). Associated causes of relapse were refractory disease (i.e., previous relapses) in 2, severe malnutrition in 1, and concurrent disease (meningococcal meningitis) in 1. In the other 2 cases, no associated event was observed except young age (ages 3 and 5 years). One relapsed patient was treated successfully with 14 days of lipid amphotericin B, and the others were cured by use of antimony for 20 to 30 days. There were no dose related adverse events. The most common event was fever which occurred in 13/32 patients (41 percent); 3/4 patients in the 10mg dose group, 7/13 in the 14mg dose group, and 3/15 in the 20mg dose group. Three patients had cardiac arrhythmia, one also with myocarditis diagnosed 2 weeks after therapy was discontinued. One patient developed hepatitis after dose 3 and the drug was discontinued. We concluded that liposomal amphotericin B is effective in a daily dose of 2mk/kg given for 5-10 doses as an initial cure, but that relapse occurs in young children, particularly those with documented treatment resistant disease or concurrent malnutrition...

Aspectos epidemiológicos determinantes na manutençäo da leishmaniose visceral no estado do Maranhäo - Brasil / The epidemiological determinant aspects in the maintenance of visceral leishmaniasis in the state of Maranhäo, Brazil

Analisou-se o comportamento da leishmaniose visceral (LV) no Estado do Maranhao-Brasil, no período de 1982 a 1993. A enfermidade vem ocorrendo predominantemente na Ilha de Sao Luís-MA em áreas periurbanas, destacando, no período epidêmico, a capital Sao Luís como principal área endêmica. A maior freqüênciade casos ocorreu em 1993, apesar do uso de inseticidas e controle dos caes. Houve predomínio na faixa etária de 0 a 4 anos de idade com 58,4 por cento dos casos. Nem a doença humana nem o índice pluviométrico apresentaram variaçoes sazonais significativas entretanto estiveram moderadamente correlacionados, havendo quase sempre elevaçao do número de casos após o período de maior precipitaçao chuvosa. A partir deste estudo, poderao ser levantadas questoes para o controle mais eficaz, consoante à urbanizaçao da doença, aliada aos fatores da dinâmica de trasmissao em áreas endêmicas do Estado.