Montenegro Skin Test Research Articles

The influence of copper, selenium and zinc on the response to the Montenegro skin test in subjects vaccinated against American cutaneous leishmaniasis

We evaluated the relationship between the trace elements copper, zinc and selenium and the response to the Montenegro skin test (MST) in 172 volunteers vaccinated against American cutaneous leishmaniasis. The MST diameter was categorized as negative and in quartiles of positive response, constituting five groups. Trace element serum levels were analyzed by coupled plasma atomic emission spectrometry and hydride generation atomic absorption spectrometry, with study subjects classified into two groups depending on low or high levels of trace elements observed. MST-positive subjects had an MST diameter (mean+/-SD) of 10.35+/-4.64mm, with copper, selenium and zinc serum levels of 1433.7+/-665.7microg/l, 88.6+/-39microg/l and 999.2+/-366microg/l, respectively. The MST diameter was significantly different in the selenium groups only. The selenium levels also differed with the quartiles of the MST diameters of individuals testing positive (P<0.05). Our findings suggest that trace elements, particularly selenium, should be measured in future vaccine trials so that vaccine immunogenicity and response can be assessed and compared between different studies.

Use of kDNA-based Polymerase Chain Reaction as a Sensitive and Differentially Diagnostic Method of American Tegumentary Leishmaniasis in Disease-Endemic Areas of Northern Argentina

We evaluated the polymerase chain reaction (PCR) for the diagnosis of endemic American Tegumentary Leishmaniasis in Salta, Argentina. Diverse Leishmania species, coexistence of mycotic and varicose ulcers, and high endemicity T. cruzi, represent diagnostic challenges in the region. We performed a simplified PCR using sensitive, generic primers on samples obtained by a non-invasive method. We tested different culture types and clinical specimens with other microorganisms that induce leishmaniasis-like lesions. The PCR had a sensitivity and specificity of 100%. Forty-five patients with presumptive leishmaniasis were compared to the PCR, smears, and the Montenegro skin test (MST). In the same population, the PCR had an increased sensitivity, detecting 25 of 45 cases compared with 16 of 45 for smears and had a higher sensitivity in detecting mucocutaneous lesions. Diagnosis by PCR was supported by clinical presentation, positive MST results, compatible epidemiology, and in some cases histopathologic results or isolation of parasites by culture. These findings indicate the convenience of incorporating this PCR into diagnostic strategies for detecting leishmaniasis in northern Argentina.

Factors associated to Montenegro skin test positivity in Teresina, Brazil

Visceral leishmaniasis (VL) is an emerging infectious disease of urban areas in Brazil. To better understand this process, we have studied the association between reaction to the Montenegro skin test (MST), and demographic, socioeconomic and environmental factors in an urban area with high force of transmission. Associations between variables were expressed by prevalence ratios and their respective 95% confidence intervals estimated by using Poisson regression models with robust variance. Higher prevalence of positivity to MST was detected among male and older participants, and among subjects who owned dogs for 3 or more years. Individuals with higher literacy and living in households with three or more persons showed lower prevalence of MST reaction. These results suggest that the identification of high-risk groups might be feasible and useful for targeting interventions.

Mixed cytokine profile during active cutaneous leishmaniasis and in natural resistance

Most studies on immune response in human cutaneous leishmaniasis evaluate patients with active disease in comparison with healthy uninfected controls or patients that have had the lesions healed, however, little is known about the immune response associated with natural resistance. In this paper we evaluate the cytokine expression patterns of T-cells and the plasmatic levels of nitrite and nitrate in patients with localized cutaneous leishmaniasis (LCL) as well as endemic non-infected individuals with positive (MST) and negative (NI) Montenegro skin test, without previous history of leishmanial lesions. Our results demonstrated an increased number of IFN-gamma+ and TNF-alpha+ T-cells and high level of plasma nitrite and nitrate in LCL patients. Moreover, we have observed that early in infection (LCL equal/less than 60 days of lesion evolution), Leishmania patients present predominance in IL-4+ and IL-10+ T-lymphocytes. However, this is a transitory phenomenon, since patients with older lesions (LCL more than 60 days of lesion evolution) show a predominant Type-1 immune profile, suggesting that disease development may depend on a transient deregulation of T-cell response, during the initial phase of infection. Interestingly MST displayed a basal mixed Type-0 cytokines profile. However, the low frequency of IL-4+ T-cells, high IFN-gamma+/IL-10+ cell ratio as well as elevated nitrite and nitrate plasma levels observed in MST, suggested that despite basal levels of cytokines, a high proportion of Type-1 over Type-2 cytokines would count to prevent parasite growth and lesion development.

Comparação entre imunofluorescência indireta e aglutinação direta para o diagnóstico sorológico da leishmaniose tegumentar americana em cães errantes

Estudaram-se 92 cães errantes com o objetivo de comparar a imunofluorescência indireta (IFI) e a aglutinação direta (AD) na investigação da leishmaniose tegumentar americana (LTA) canina, no período de outubro de 1999 a novembro de 2001. Os animais foram examinados quanto à presença de lesões e submetidos à intradermorreação de Montenegro (IDRM) e à pesquisa de anticorpos anti-Leishmania por IFI e AD. A IFI apresentou sensibilidade de 78,9% e especificidade de 93,8% (título &gt;40), e a AD sensibilidade de 57,9% e especificidade de 68,8% (título &gt;80). Dois cães apresentavam lesão, mas a pesquisa do parasito foi negativa. A IDRM, realizada em 89 cães, foi positiva em três (3,4%). Trinta e quatro cães (37,0%) foram positivos para a IFI, 61 (66,3%) para a AD e 69 (75,0%) para a IFI e/ou a AD. Dos cães com a IFI positiva, sete eram de Floresta, 13 de Maringá, 12 de Leópolis, um de São Jorge do Ivaí e um (50,0%) de Campo Mourão. Os resultados sugerem o contato prévio desses animais com o parasito.

Analysis of the CC chemokine receptor 5 delta32 polymorphism in a Brazilian population with cutaneous leishmaniasis

Patients with mucocutaneous leishmaniasis (MCL) show a vigorous T-cell immune response against Leishmania braziliensis. Because the Th response is associated with inflammation, the non-functional CC chemokine receptor 5 (CCR5) may rely in a less severe inflammatory state. The aim of this study was to investigate the CCR5 gene in a Brazilian population with leishmaniasis compared with healthy control subjects and to determine the progression from cutaneous to MCL in the Delta32 allele carriers. Among 100 patients with Montenegro skin test and indirect immunofluorescence assay (IIF) values positive for leishmaniasis, there were 32% women and 68% men. The patients were 89% CCR5/CCR5, 10% CCR5/Delta32, and 1% Delta32/Delta32, while healthy subjects showed a 91% incidence of CCR5/CCR5, 8% of CCR5/Delta32, and 1% of Delta32/Delta32. The CCR5/CCR5 patients (89%) showed a large spectrum of clinical manifestations, where 22.47% had active mucous lesions and 77.53% had cutaneous lesions. In this work, the Delta32 allele carriers (10%) showed only cutaneous manifestations when compared with wild-type individuals. Finally, with regard to the Delta32 allele carriers, a less severe spectrum of clinical manifestations was observed in comparison with wild-type individuals. Although a lack of mucocutaneous lesions was evident among Delta32 allele carriers, the number of individuals studied was small. Therefore, further investigations are needed to elucidate the role of CCR5 in the clinical aspects of leishmaniasis.

IMMUNOLOGIC TESTS IN PATIENTS AFTER CLINICAL CURE OF VISCERAL LEISHMANIASIS

The results of five serologic tests (ELISA using promastigote antigen [ELISAp] and recombinant K39 [ELISArK39] and K26 [ELISArK26] antigens, indirect immunofluorescence test using promastigote antigen [IIFT], and immunochromatographic tests using the rK39 antigen [TRALd]) and of the Montenegro skin test (MST) were analyzed in 41 individuals treated for kala-azar and living in Porteirinha, Minas Gerais, Brazil. The tests were carried out 1 week to 12 years after specific treatment. All MSTs during the 8 months after treatment were negative, whereas after 1 year, 28 (84.8%) were positive. Negativity in all serologic tests was observed for 11 (26.8%) of the 41 individuals, whereas positivity in at least one test was observed for 70.3% of subjects evaluated > or = 2 years after treatment. With respect to each exam, positivity was 38.0% for TRALd, 61.9% for ELISA rK39, 47.6% for ELISA rK26, 38.0% for ELISAp, and 40.5% for IIFT. None of the individuals presented recurrence of the disease during the 4 years of follow-up. The tests were repeated in 24 of the 41 individuals, after some time, and the results were the same in 33.3% of the cases. We conclude that serological tests for kala-azar might continue to be positive after treatment of the disease, although this does not indicate a poor prognosis or a poor therapeutic response.

Sensitivity of an immunoenzymatic test for detection of ant-L. brasiliensis antibodies compared to other tests used for the diagnosis of American cutaneous leishmaniasis

The diagnosis of American cutaneous leishmaniasis (ACL) is frequently based on clinical and epidemiological data associated with the results of laboratory tests. Some laboratory methods are currently being applied for the diagnosis of ACL, among them the indirect immunofluorescence reaction (IIFR), the Montenegro skin test (MST), histopathological examination, and the polymerase chain reaction (PCR). The performance of these methods varies in a considerable proportion of patients. After the standardization of an immunoenzymatic test (ELISA) for the detection of IgG in the serum of patients with ACL using a crude Leishmania braziliensis antigen, the results obtained were compared to those of other tests routinely used for the diagnosis. The tests revealed the following sensitivity, when analyzed separately: 85% for ELISA IgG, 81% for PCR, 64.4% for MST, 58.1% for IIFR, and 34% for the presence of parasites in the biopsy. ELISA was positive in 75% of patients with ACL presenting a negative MST, in 84.8% of ACL patients with negative skin or mucous biopsies for the presence of the parasite, and in 100% of cases with a negative PCR. Thus, ELISA presented a higher sensitivity than the other tests and was useful as a complementary method for the diagnosis of ACL.

Haematogenous dissemination of Leishmania (Viannia) braziliensis in human American tegumentary leishmaniasis

One of the potential dangers of American tegumentary leishmaniasis (ATL) caused by Leishmania (Viannia) braziliensis is the development of mucosal lesions. Haematogenous dissemination of the parasite is the most likely mechanism to explain this occurrence, but most attempts to isolate the parasite from blood have so far been unsuccessful. The presence of Leishmania in peripheral blood was therefore evaluated by PCR using DNA samples isolated from patients presenting active cutaneous or mucosal disease, and from individuals cured by antimonial treatment as well as individuals without a past history of leishmaniasis but with a positive Montenegro skin test, all living in L. (V.) braziliensis-endemic areas. Leishmania DNA was found not only in those patients presenting active cutaneous (24.8%) or mucosal (35%) lesions, but also in samples isolated from healed individuals (27.3%) as well as in asymptomatic skin-test-positive residents of endemic areas (37.5%). Overall, PCR showed the presence of parasite DNA in the blood of 26.2% of the 225 examined samples. These data suggest that persistence of parasites within the host may last for many years and, rather than being a risk factor, might be important in maintaining the protective response in those living in endemic areas.

Polymerase chain reaction with lesion scrapping for the diagnosis of human American tegumentary leishmaniasis

The objective of this work was to compare the polymerase chain reaction (PCR) using lesion scrapping with other conventional techniques for the diagnosis of the American tegumentary leishmaniasis (ATL). For this, patients with cutaneous lesions suspected to be ATL were studied. The DNA was amplified with the MP1L/MP3H primers. From the 156 studied patients, 79 (50.6%) presented positive parasite direct search (PD), 81 (51.9%) had positive Montenegro skin test (MST), and 90 (57.7%) presented PD and/or MST positive. The PCR was positive in all of the positive-PD patients (100% sensitivity), in 91.1% of the positive PD and/or MST patients, and in 27.3% of the patients that presented negative PD and positive MST. The PCR positivity was similar to the PD (P = 0.2482) and inferior to the MST (P = 0.0455), and to the PD/MST association (P = 0.0133). The high PCR sensitivity, and positivity in those cases where the PD was negative, highlights the importance of this technique as an auxiliary tool for the diagnosis of ATL.

Epidemiology of the American cutaneous leishmaniasis in a military training center in "Zona da Mata", Pernambuco, Brazil

The objective of this study was to characterize aspects of the ecoepidemiology of the American cutaneous leishmaniasis (ACL) caused by Leishmania (Viannia) braziliensis in an area of military training located in the Atlantic rain forest (“Zona da Mata”) region in the Pernambuco State, Brazil, to isolate and identify Leishmania species in patients and sand fly fauna, the expression of infection and disease and vector(s) involved in the transmission. Sand flies were captured between July 2002 and June 2003 in the places where diurnal and nocturnal training activities were realized. Patients were reported to the “Hospital Geral do Exercito”, in Recife, for clinical diagnostic and treatment. Laboratory diagnosis was confirmed by parasite demonstration and detection of immune response to Leishmania. An epidemiological survey by Montenegro skin test (MST) was carried out to identify the prevalence of infection in the population that made training activities in this area. Nine ACL cases were notified during the study period, eight of them from patients with single cutaneous lesions and one from a patient with multiple lesions. A total of 370 individuals were submitted to MST, and 89 presented positive response, representing 25.28% of prevalence to infection. Six Leishmania (Viannia) braziliensis stocks were isolated from patients and one from a sentinel hamster exposed in the area, characterized by monoclonal antibodies and isoenzymatic profiles. A total of 8,406 sand flies specimens of sixteen species were collected and identified. Lutzomyia (Psychodopygus) complexa was the predominant species, with 51.36% of the total, followed by L. choti, L. amazonensis, L. longispina and L. sordelli. One isolation of flagellated form characteristic of Leishmania was obtained from L. complexa, but not identified due to culture contamination. These results support evidences of the enzootic cycle maintenance involved in this area, with periodical ACL outbreaks after training activities carried out in places of local remnant forests where occurs the primary transmission cycle of L. (V.) braziliensis in this area.

Montenegro's skin reactions and antibodies against different Leishmania species in dogs from a visceral leishmaniosis endemic area

In this study, humoral (circulating anti- Leishmania antibodies) and cellular (Montenegro's skin test) immune responses of dogs from an endemic area of visceral leishmaniosis were tested using Leishmania chagasi, Leishmania amazonensis and Leishmania braziliensis antigens. The antibody response was tested in three animal groups, selected according to their anti- L. chagasi antibody activity, as measured by ELISA in the serum: 19 negative (O.D. below 0.30), seven with undefined (O.D. between 0.40 and 0.70) and 12 positive (O.D. above 1.0) ELISA result. In the group of animals with positive ELISA, the antibody activity against L. chagasi antigens (mean O.D. = 1.31) was significantly higher (ANOVA, P < 0.01) than against L. amazonensis (mean O.D. = 0.88) or L. braziliensis (mean O.D. = 0.87) antigens. The Montenegro's skin test results obtained with L. chagasi and L. braziliensis antigens showed a fair agreement ( κ = 0.309). The same was observed when antigens from L. braziliensis and L. amazonensis were compared ( κ = 0.374), whereas a moderate agreement between the results from tests performed with L. chagasi and L. amazonensis antigens was observed ( κ = 0.530). The induration areas obtained with L. braziliensis antigen were smaller than those obtained with the other antigens. The data presented herein indicate that the use of antigens from different Leishmania species may interfere with the results of the immunological tests performed in dogs in an endemic area of visceral leishmaniosis.

Immuno-biochemical evaluations of phenol and thimerosal as antigen preservatives in Montenegro skin test

Montenegro skin test (MST) represents the main complementary diagnostic test for tegumentary leishmaniases (TL) in endemic regions. Most antigen formulations used for the MST contain thimerosal as preservative. The Food and Drug Administration (FDA), however, recommended reducing or eliminating thimerosal from vaccines and other biological reagents and the Agência Nacional de Vigilância Sanitária (ANVISA) in Brazil, prohibited the use of mercurial compounds in immunobiologicals. In the search for an alternative stabilizer, phenol and thimerosal were tested as antigen preservatives in MST. Formulations were tested when fresh and after a 12-month storage at 4 °C in TL confirmed mice and human patients, and were evaluated for protein constitution by SDS-PAGE, Western blot and anti-gp63 ELISA. In mice, a decrease in the diagnostic effectiveness in merthiolate formulation was observed after a 12-month storage. SDS-PAGE, Western blot and anti-gp63 ELISA analyses showed a degradation of antigen proteins in both formulations after 12-month storage and that phenol-preserved antigen was quantitatively and qualitatively better than the merthiolate-preserved one. In patients, the average of induration diameter was larger in fresh antigens ( p < 0.05). However, storage time did not jeopardize their diagnostic capacity. No non-specific reactions produced by phenol or merthiolate were observed neither in humans nor in mice. Phenol could be a good alternative to replace the merthiolate in MST, and despite the proteolytic activity, antigens remain viable for at least 12 months.

Prevalência de infecção por Leishmania chagasi utilizando os métodos de ELISA (rK39 e CRUDE) e intradermorreação de Montenegro em área endêmica do Maranhão, Brasil

A prospective study was undertaken in 1,520 children less than 15 years of age in São José de Ribamar, Maranhão, Brazil, from June 1994 to January 1995, to evaluate the prevalence and characteristics (socioeconomic, environmental, and behavioral) associated with infection by Leishmania chagasi. Montenegro skin test (MST) and enzyme-linked immunosorbent assay (ELISA-rK39 and CRUDE) test were used to detect infection. The statistical analysis used the chi2 test with Yates correction and a p value less than 0.05 was considered statistically significant. Prevalence of infection was 61.7% as measured by MST, 19.4% according to ELISA (rK39), and 19.7% by ELISA (CRUDE). Association was detected between leishmaniasis in the family, water supply, application of insecticide, and infection by L. chagasi using MST. No association with infection by L. chagasi was detected using ELISA rK39 or CRUDE. More effective control measures are needed to reduce prevalence and to detect asymptomatic cases in this high percentage of infected children.

Comparison of polymerase chain reaction with other laboratory methods for the diagnosis of American cutaneous leishmaniasis: Diagnosis of cutaneous leishmaniasis by polymerase chain reaction

An evaluation of 5 laboratory methods for diagnosing American cutaneous leishmaniasis (ACL) was carried out on patients from an endemic area of Brazil. From 164 patients presenting cutaneous lesions, and suspected to have ACL, 133 (81.1%) were confirmed for the disease by Montenegro skin test (MST) and/or parasitologic examination (PE). In both groups of patients, the positivity of polymerase chain reaction (PCR) was similar to that of immunofluorescence assay and enzyme-linked immunosorbent assay, and higher than that of MST and PE ( P < .05). In the group of patients suspected to have ACL, PCR presented the same positivity as PE and MST together. No correlation between positivity of the laboratory methods and clinical or epidemiologic aspects was observed. Our data confirmed the value of PCR as an alternative laboratory method for diagnosing ACL, especially for those patients with negative PE and MST.

Activated inflammatory T cells correlate with lesion size in human cutaneous leishmaniasis

Leishmaniasis is an important parasitic disease affecting millions worldwide. In attempts to understand the clinical relevance of immunological measurements as determined using flow cytometry, several immunological phenotypes were determined for a group of well defined human leishmaniasis patients and correlated with clinical measurements of the disease (Montenegro skin test (MST) and lesion area). The analysis demonstrated a positive correlation between the MST size and the frequency of ex vivo recent activated CD4 + T cells. In contrast, higher frequencies of recent activated CD8 + T cells were correlated with a smaller MST size. Moreover, a positive correlation was observed between the lesion total area and the frequency of activated CD69 + (ex vivo) and CD40L + (cultured with Leishmania soluble antigen (SLA)) T lymphocytes. Finally, larger lesions were also correlated with a higher frequency of SLA specific inflammatory cytokine (IFN-γ or TNF-α) producing lymphocytes. These studies demonstrate that immunological markers are correlated with clinical indicators of human leishmaniasis and serve to better understand the evolution of this important parasitic disease.

American tegumentary leishmaniasis: A quantitative analysis of Langerhans cells presents important differences between L. ( L.) amazonensis and Viannia subgenus

A quantitative study was conducted on the density of Langerhans cells (LCs) CD1a+ in specimens obtained from patients with American tegumentary leishmaniasis (ATL) lesions without previous treatment, as well as from control healthy individuals. LC density was significantly higher among infected patients when compared to controls and also higher in longer term ones. Regarding parasite quantities, these were proportionally inverse and diminished in chronic patients. Localized cutaneous leishmaniasis (LCL) showed an increase in cell population when compared to diffuse cutaneous leishmaniasis (DCL). A tendency towards density increase was observed in LC Leishmania ( Leishmania) amazonensis patients when compared to Leishmania ( Viannia) sp. Regarding the delayed hypersensitivity test (DTH, Montenegro skin test), L. ( L.) amazonensis demonstrated a peculiar behavior because it is a poor cell immune inducer, presenting – among LCL patients – higher density in negative Montenegro patients than in positive ones. Negative DTH responses are usually poor in LC, although this was not evidenced in this study, possibly due to cell reposition, in order to stimulate immune response. Such results confirm the important role of LC in ATL, while suggesting that L. ( L.) amazonensis may be a good model for LC studies as APC in ATL, due to its spectral immunological and clinical behavior.

Leishmaniose tegumentar americana causada por Leishmania (Viannia) braziliensis, em área de treinamento militar na Zona da Mata de Pernambuco

The aim of work was to study the epidemiology of American tegumentary leishmaniasis in a military training unit situated in Zona da Mata region of Pernambuco State. Between 2002 and 2003 twenty-three cases were notified by clinical exam, detection and/or isolation of parasite and Montenegro skin test. Seven stocks of Leishmania (Viannia) braziliensis were obtained from patients, identified by a panel of specific monoclonal antibodies and isoenzymatic electrophoresis profiles. An epidemiologic survey on prevalence of infection was carried out by Montenegro skin test in the population that underwent training activities during the same period, out of which 25.3% were identified as positive. These results in association with previous data from this area, shows the maintenance of a primary transmission cycle and the occurrence of periodical outbreaks after training activities in local areas of remnant Atlantic rain forest.

Polymerase chain reaction (PCR) is highly sensitive for diagnosis of mucosal leishmaniasis

We evaluated the use of polymerase chain reaction (PCR) for diagnosis of mucosal leishmaniasis (ML) in an endemic area in Acre, Brazil, where Leishmania braziliensis is present. Leishmania DNA was detected 34 of 35 cases, yielding a positivity rate of 97.1%, which was higher than the positivity rates for all of the other diagnostic methods studied, namely Montenegro skin test (MST), anti- Leishmania serological testing and microscopic examination of lesion biopsy specimens. These findings have led us to propose guidelines for the diagnosis of ML that use PCR as the principal method of parasitological confirmation of cases.

Positive Montenegro skin test among patients with sporotrichosis in Rio De Janeiro

We studied 52 patients with sporotrichosis confirmed by isolation of Sporothrix schenckii and reactivity to the Montenegro skin test (MST) during an ongoing outbreak of this mycosis in Rio de Janeiro. The objective was to emphasize the importance of parasitological confirmation and the possibility of incorrect diagnosis based on the lesion's appearance, epidemiological information, and immunological tests. The antigen used for the MST was conserved in either thimerosal 1:10,000 (group 1) or 0.4% phenol (group 2). Nineteen patients (39%) in group 1 and seven (12%) in group 2 presented an induration ≥10 mm ( p < 0.001). Sera from three patients (6.7%) reacted to indirect immunofluorescence (IIF) for leishmaniasis, while sera from 10 patients (22.2%) reacted to enzyme-linked immunosorbent assay (ELISA). Fifteen patients (28.8%) presented up to two lesions, with a predominance of ulcers. Forty-four patients (84.6%) were treated with itraconazole. In the differential diagnosis between sporotrichosis and cutaneous leishmaniasis, the possibility of co-infection, allergy to the reagent diluent, and cross-reactions should be further investigated, especially in regions with limited laboratory facilities.