Investigation of asymptomatic visceral leishmaniasis cases using western blot in an endemic area in Turkey

In Turkey, Leishmania infantum is responsible for human visceral leishmaniasis (VL), which is seen mainly in the Aegean, Mediterranean, and Central Anatolia Regions. This study aimed to determine asymptomatic infections in an endemic area of VL in Turkey using the western blot technique. A total of 82 persons including children and adults were chosen randomly in Denizli province which is one of the endemic sites for VL. Serum samples were collected and screened using indirect immunofluorescent test (IFAT), enzyme-linked immunosorbent assay (ELISA) and western blot (WB). One year later, 35 of the 82 persons were sampled and screened serologically for the second time. Seven out of 82 samples were found to be positive by western blot analysis with the presence of 14 and/or 18 kDa bands. Two of these seven sera were also positive by IFAT, but only one of these two was positive by ELISA. Only one person showing seropositivity with all three tests had clinical symptoms and was diagnosed as VL with the presence of amastigotes in bone marrow aspirate. Because six people, including the one found to be seropositive in all two tests, had no clinical symptoms, they were accepted as asymptomatic carriers. The ratio of asymptomatic infection was calculated as 7.41% (6/81) in the region. In the second sampling, the western blot revealed antibodies against the same antigens in all seven subjects. Our findings showed that the presence of antibodies against 14 and 18 kDa antigens are important for the diagnosis of symptomatic and asymptomatic infections. Western blot was found to be effective in the detection of asymptomatic persons in the epidemiological studies in endemic areas.     

Diagnostic value of rK39 dipstick in zoonotic visceral leishmaniasis in Turkey

K39 is a repetitive immunodominant epitope in a kinesin-related protein expressed predominantly in the amastigotes of visceral Leishmania spp. Enzyme immunoassays of patient's sera with recombinant K39 (rK39) proved to be highly specific and sensitive for diagnosis of active visceral leishmaniasis (VL, kala-azar). The same assays in dipstick format were also found effective for diagnosis of both human VL (HVL) and canine VL (CanVL) in most endemic areas of these diseases. Fifty-eight human patients and 22 dogs, clinically suspected of kala-azar, were screened with rK39 dipstick in comparison with the conventional methods of diagnosis, i.e., microscopic examinations of bone marrow and lymph node aspirates and immunofluorescent antibody tests (IFAT), respectively. Sixteen patients and 12 dogs were found to be rK39 dipstick positive. The results were corroborated with those of parasitological examinations, except 1, rK39-positive but smear-negative, case in each group. IFAT of the 2 discordant cases gave positive results. The rK39 dipstick is thus reliable for diagnosis of both HVL and canVL cases in Turkey.     

A new stable focus of canine leishmaniasis in northern Italy

A new stable focus of canine leishmaniasis (CanL) was identified in a coastal Adriatic area of the Emilia-Romagna Region in Northern Italy. Following the first clinical cases observed starting from 1998, a seroepidemiological survey was carried out on owned dogs from two communes and on animals housed in dog pounds of the Rimini province. Sixteen out of 612 dogs (2.6%) resulted positive to the IFA test. The 16 positive dogs all came from the two communes, with seroprevalences of 3 and 6%, respectively. The autochthonous origin of the infection was confirmed in all the cases. The parasitological investigation led to the isolation and identification of the parasite as Leishmania infantum Zymodeme MON 1. An entomological survey showed that Phlebotomus perniciosus and P. perfiliewi are present in this area and that P. perfiliewi was very abundant in one collection site. The risk of the establishment of a permanent transmission of the infection in the area, previously considered CanL-free, must be analysed in view of further investigations to be extended also to neighbouring areas.     

A survey for American cutaneous and visceral leishmaniasis among 1,342 dogs from areas in Rio de Janeiro (Brazil) where the human diseases occur

There are areas in the periphery of Rio de Janeiro city where human cases of Visceral and/or Cutaneous Leishmaniasis occur. The parasites have been identified as Leishmania donovani and Leishmania braziliensis braziliensis respectively. A survey for Leishmaniasis was done among 1,342 dogs from those areas using an indirect immunofluorescent test. From the dogs, 616 came from areas where only human cases of Visceral Leishmaniasis occurred, 373 from an area where all human cases were of Cutaneous Leishmaniasis and 353 from a third area (Campo Grande) where both visceral and cutaneous human cases were detected. The prevalence of parasite antibody titers among dogs from areas of Cutaneous Leishmaniasis was significantly higher than that of Visceral Leishmaniasis (8.6% vs. 4.3%, p less than 0.02). The highest prevalence was observed among dogs from the area where both diseases are present (12.7%).     

Canine leishmaniasis in the Rif mountains (Moroccan Mediterranean coast): a seroepidemiological survey

A sero-epidemiological survey has been conducted in several localities of the province of Nador to investigate canine leishmaniasis in the North-Eastern slope of the Rif mountains (Mediterranean coast of Morocco). Serum samples collected from 257 dogs were analysed using indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) to detect anti-Leishmania infantum antibodies. Forty eight (18.7%) of the screened dogs were IFAT positive and 54 (21.0%) were ELISA positive; the concordance of the two methods was 96.1%. The prevalence of infection is significantly higher in dogs more than four years of age whereas no significant difference in prevalence of infection was seen between males and females. The frequent symptoms observed in seropositive dogs were the enlargement of lymph nodes (57.4%), emaciation (51.9%) and skin involvement (25.9%). However, 38.9% of those dogs showed no one of the major symptoms of visceral leishmaniasis. Leishmania isolated from three of the examined dogs was identified as L. infantum MON-1. These results show that the North-Eastern slope of the Rif mountains is one of the most active Mediterranean areas of visceral leishmaniasis and confirm that the dog is the main reservoir of L. infantum.     

Nitrogen uptake by crops,soil distribution and recovery of urea-N in a sorghum—wheat rotation in different soils under Mediterranean conditions

The N uptake by crops, soil distribution and recovery of 15N labelled urea-N (100 kg N ha-1) were investigated in a sorghum-wheat rotation in two silty clay soils (Foggia and Rieti Casabianca) and one silt loam soil (Rieti Piedifiume) under different mediterranean conditions. Non-exchangeable labelled NH4-N represented an important pool at both Rieti sites with higher values (p<0.05) under sorghum (14.0 and 24.6% of the urea N in the 0-20 cm layer at the end of the cropping season) than wheat whereas it was much less important in the Foggia soil (10.0% in the surface soil under sorghum). This is probably related to the clay minerals composition of the three soils; because vermiculite was present in both Rieti sites but not in the Foggia soil. At harvest from 4.4 to 5.3% of the urea N initially applied was present as microbial biomass N in the surface soil layer with no generally significant differences due to location and type of crops. Both sorghum and wheat N yields were higher in the driest site (Foggia) probably due to better light conditions, higher temperatures and irrigation during summer of the sorghum cropping period. The recovery of plant fertilizer N (about 21% for sorghum and 27% for wheat) and the percentage of N in the plant derived from the fertilizer (NDFF) were the lowest at Rieti-Casabianca probably as the result of the protection of immobilized fertilizer N against microbial mineralization by the swelling clays. The fertilizer N unaccounted for was nil or very low (10.8% at Rieti-Casabianca under wheat and 11.8 and 4.9% at Rieti-Piedifiume under sorghum and wheat, respectively). Urea-N losses occurred when Rieti Piedifiume and Rieti Casabianca soils were kept bare. In this case the urea N unaccounted for ranged from 12 to 56% of the urea N with higher losses in Rieti-Piedifiume than in Rieti-Casabianca. The higher recoveries in the latter soil were probably confirmed by the stabilizing effect of clays on the immobilized urea N. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytotoxic and suppressor activity of human peripheral blood lymphocytes stimulated by interleukin-2 and phytohemagglutinin before and after fractionation in the percoll gradient

The cultivation of peripheral blood lymphocytes (PBL) obtained from patients with colorectal or bladder carcinoma and melanoma and from healthy donors in the presence of interleukin-2 (IL-2) and PHA resulted in the induction of cytotoxic activity against autologous and/or allogeneic tumour cells in 12 out of 13 patients and in 10 out of 10 donors. A higher level of cytolytic activity was achieved when PBL were separated by means of Percoll density gradient (1.077; 1.067 and 1.056 g/ml) centrifugation and the cells of fraction II (1.077-1.067 g/ml) were employed in the experiment, the level of cytotoxicity being elevated in all cases (1.7-fold elevation in donors and 2-fold elevation in patients on the average). The addition of fraction I (1.067-1.056 g/ml) to fraction II prevented (PHA + IL-2)-mediated induction of cytotoxic activity in all the patients, but in 4 out of 10 donors, i.e. cells of fraction I expressed a suppressor activity. The immunofluorescent analysis has shown that fraction II was enriched by T cells (92%) and depleted of monocytes (7%), as compared to unseparated PBL (66% and 27%, respectively). On the contrary, fraction I was characterized by a decreased T cell ratio (36%) and an increased monocyte level (up to 69%).     

Infantile visceral leishmaniasis in the Campania region, Italy: experience from a Paediatric Referral Centre

In the first half of the 20th century, visceral leishmaniasis (VL) was a common infantile syndrome in coastal territories of the Campania region of Italy. After World War II, the incidence dropped to a few cases/year for three decades; in late 1980s the disease reemerged among both children and adults. To face the VL recrudescence, a Paediatric Reference Centre was established at the Santobono-Pausilipon hospital in Naples, for the clinical diagnosis, care and drug treatment of all infantile VL cases occurred in the Campania region. Rapid laboratory diagnosis was secured by a Diagnostic Reference Centre established at the Istituto Superiore di Sanità. Here, we report on the epidemiological and parasitological features of all cases referred to the Centre in the past 15 years. From 1990 to March 2004, a total of 255 cases were diagnosed and treated at the Centre. The Figure shows the yearly trend of patients (min. 3 cases in 1990 and 1991, max. 30 cases in 2000). There were 135 males (52.9%); the age ranged 4 months-14 years, but 189 patients (74.1%) were < or = 3 years old. The majority of the patients (189, 74.1%) were from the Naples province, with a cluster of 102 cases (40% of total patients) from the towns and districts surrounding Vesuvius. Twenty-seven cases (10.6%) were from the town of Maddaloni, Caserta province, whereas 15 cases (5.9%) were from coastal villages of the Salerno province. Only 1 and 2 cases were from Benevento and Avellino provinces, respectively. All patients but seven, who have been treated with antimonial drugs in the 1990-1993 period, were successfully treated with a liposomal amphotericin B regimen. From bone-marrow aspirate samples, 138 Leishmania cultures were obtained in EMTM and Sloppy Evans' media, of which 134 have been typed by the electrophoretic analysis of 13 isoenzymes. Two zymodemes (Z) of L. infantum were routinely identified over the study period, ZMON-1 (the commonest zymodeme in the Mediterranean area) and ZMON-72, variant from MON-1 in PGM mobility and detected only in our region. The latter, identified in 61 patients (45.5%), was found exclusively distributed in towns of the Vesuvius area and in Maddaloni until 1996, but in recent years it appears to have spread to other areas of the Naples (including the island of Ischia) and Caserta provinces, but not to Salerno province. In conclusion, the VL macrofocus of the Naples-Caserta area is probably responsible for the highest number of infantile cases among any VL macrofoci described in southern Europe. Considering the limited efforts paid to control the canine reservoir, rapid diagnosis and appropriate treatment of patients still remain the first-line control measures aimed at reducing the health impact of the disease.     

Serology of paracoccidioidomycosis

The purposes of the present work were: i) to study the positivity indices and compare titers obtained with the indirect immunofluorescence (II), tube precipitation (TP), complement fixation (CF) and double immunodiffusion on agar gel (ID) tests in the sera of 196 patients with paracoccidioidomycosis before treatment, and ii) to compare the initial titers of II with those obtained 1 year or more after treatment. II was the most sensitive serologic reaction (85.2%), and the positivity indices for CF, ID and TP were 67.7%, 66.0% and 50.0%, respectively. The sera tended to show parallel mean titers in II, CF and TP tests. One year after treatment there was a fall in titers of II in 66.2% of patients. The data, taken as a whole, demonstrate the usefulness of the indirect immunofluorescent test and the importance of using 2 or more serologic tests for the diagnosis and monitoring of patients with paracoccidioidomycosis.     

Diagnosis of human visceral leishmaniasis by PCR using blood samples spotted on filter paper

The polymerase chain reaction (PCR) is a simple, rapid procedure that has been adapted for the diagnosis of leishmaniasis. In the present study, 85 blood samples and seven bone marrow aspirates from 85 patients with clinical symptoms suggestive of visceral leishmaniasis from the metropolitan region of Belo Horizonte in the Brazilian State of Minas Gerais were screened using molecular and serological techniques. Samples that were negative (N = 12) and positive (N = 19) in parasitological and serological tests were used as controls. Of the 85 samples analyzed by PCR, 61 (71.7%) showed the expected amplification products in agarose gels. However, when the technique was combined with molecular hybridization, 72 samples (83.5%) gave a positive signal on film. Nineteen patients with Leishmania parasites in bone marrow cultures (positive controls) showed PCR hybridization in whole-blood samples, as did the seven bone marrow aspirates positive for Leishmania. None of the negative controls reacted in PCR or in an indirect immunofluorescent assay. These results indicate that PCR could replace the conventional parasitological examination in the diagnosis of leishmaniasis since it provides very satisfactory results with blood samples spotted on filter paper.     

Indirect Fluorescent-Antibody and Quantitative Agar-Gel Immunodiffusion Tests for the Serological Diagnosis of Paracoccidioidomycosis

The value of various serological tests in the diagnosis of paracoccidioidomycosis was studied. Quantitative agar-gel immunodiffusion and indirect immunofluorescent tests were performed, and the results were compared with those of complement fixation and qualitative agar-gel procedures. The quantitative immunodiffusion procedure was found to serve as the simplest and safest quantitative test that could be performed for evaluation purposes, whereas the indirect fluorescent-antibody test gave nonspecific reactions and, as such, proved unsuitable.     

Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.     

Identification of the causative agent of cutaneous leishmaniasis in Chichaoua province, Morocco

Cutaneous leishmaniasis (CL) in Morocco is caused by three species, Leishmania major, L. tropica and L. infantum. CL has been known in Chichaoua province since 2000. Using DNA extracted from microscopic slides and parasite cultures, collected in the years 2006 and 2009, we identified for the first time L. tropica as the causative agent of CL in this region. Species identification was achieved by performing the ITS1-PCR-RFLP approach. By using this method it was possible to identify parasites in Giemsa stained slides containing less than five parasites per oil-immersion field even they were conserved for up to four months.     

The Lab-to-Field (LTF) Rating Scheme: A New Method of Investigating the Relationships between Laboratory Sublethal Toxicity Tests and Field Measurements in Environmental Effects Monitoring Studies

The Lab-to-Field (LTF) rating scheme is a straightforward method of relating the results of effluent toxicity tests to the field survey measurements and has proven to be a useful tool for interpretation of Environmental Effects Monitoring studies for pulp and paper mills in the province of Ontario. The LTF method uses the same five-level scale (level 1 for no or low response to level 5 for severe response) for rating the toxicity and field survey results. Regression analysis of LTF scores has revealed that the relationship between the Ceriodaphnia reproduction test and benthic invertebrate field survey measurements was significant (p < 0.001, r = 0.79). However, there were not sufficient data to determine if this can be used as a predictive tool. Nonetheless, Ceriodaphnia-to-benthic survey, Selenastrum-to-benthic survey and fathead-to-fish survey relationships were qualitatively rated strong or moderately strong in 94%, 75%, and 60% of the 16 studies, respectively. The LTF rating scheme would benefit from the use of a more sensitive species or life stage of fish to strengthen the sublethal test-to-fish survey relationship. Further validation of the lab-to-field relationship could be accomplished through the synoptic collection of effluent samples for sublethal tests and field measurements.     

Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis

The direct agglutination test (DAT) based on a freeze-dried antigen and the rK39 dipstick test were evaluated for the sero-diagnosis of visceral leishmaniasis (VL). The sensitivity and specificity of both tests were determined using sera from confirmed VL patients (n = 21), healthy controls (n = 19) and from patients with other confirmed infectious diseases (n = 42). The DAT had a sensitivity and a specificity of 100%. The rK39 had a sensitivity of 85.7% and a specificity of 82%. Both tests were also used to screen blood samples of confirmed VL patients (n = 15) and serum samples of VL suspects (n = 61). The DAT found all blood samples of confirmed VL patients positive and tested 98.4% of the serum samples of the VL suspects positive. In contrast, rK39 detected in 9/15 blood samples (60%) antibodies against Leishmania chagasi and found 85.3% of the serum samples of the suspected patients positive. Although the rK39 dipstick is more rapid and user friendlier than the DAT, the latter has a superior sensitivity and specificity. Furthermore, the reagents used for DAT do not require cold storage, whereas the buffer of the rK39 must be stored at 4oC. Therefore, the DAT is the most suitable test for the sero-diagnosis of VL under field conditions.     

A Bark Necrosis of Grapevine Caused by Pseudomonas syringae V. Hall, in Sardinia

An unusual disorder occurred on ‘Vernaccia’ vine, particularly in the typical cultivation area of the variety, in the province of Oristano. Main symptom is the decaying of wood and bark. This causes the loss of the bark on the circumference of the trunk and causes the plants to die. Degradation can develop very deep in the wood rings. Affected rings can detach from the new formed ones. The infectious process is very serious in the younger plants and can slow their evolution in the years. Furthermore it does not allow the recovery of the affected plants. In some vineyards 4 % of affected plants occur. Mortality can reach up to 20%, of the affected plants and is very high during the early years after infection. A bacterium has been constantly isolated from decaying tissues; for its morphological and physiological characters and the response to serological tests it has been included in the Pseudomonas syringae Van Hall group. Pathogenicity tests carried out on ‘Vernaccia’ vines and other varieties resulted always positive; the bacterium has always been reisolated from artificially affected areas. The latter were much diffused and, sometimes, reproduced the typical symptomatology of the disease observed in nature.     

The infection risk of visceral leishmaniasis among household members of active patients

Human visceral leishmaniasis (HVL), caused by Leishmania infantum is mainly observed as sporadic cases in Turkey and dogs are considered as the main reservoir of the disease. The incidence of visceral leishmaniasis among members of households where a HVL infection has already been diagnosed was studied in clusters around the diagnosed cases in different regions in Turkey. A total of 47 serum samples collected from the households of 11 proven visceral leishmaniasis patients were screened for anti-Leishmania antibodies by indirect immunofluorescent antibody test (IFAT). Three and one such household members belonging to the different families were found to be seropositive and borderline, respectively. Diagnosis was confirmed with the presence of amastigotes in bone marrow aspiration samples in all seropositives while the borderline case with slight and indefinitive symptoms of VL was followed only serologically at 3-month intervals and improved spontaneously in 1 year. Household members of individuals with previously confirmed visceral leishmaniasis were found to have higher frequency of the disease suggesting the household members should be included in the risk group for visceral leishmaniasis and serological screening should be performed for the detection of possible infection.     

Seroepidemiologic study of arbovirus infections in the north-east and south of Thailand

Blood specimens were collected from villagers in Khon-Kaen province, in the north-eastern region, and in Songkhla province, in the southern region of Thailand in the pre- and post-epidemic seasons of hemorrhagic fever. The hemagglutination inhibition antibody titers of the specimens were measured against dengue virus type 2, type 4, Japanese encephalitis virus and chikungunya virus. In general, Khon-Kaen villagers showed higher levels of HI antibodies against these arboviruses than Songkhla villagers and in both areas the antibody level against chikungunya virus, a group A arbovirus, was usually much lower than those against group B arboviruses. Specimens from Khon-Kaen province had such high antibody positive rates for group B arboviruses (80-100%) from an early age (3-4 years), in the pre-epidemic season, that scarcely any increase in the positive rate was observed in the post-epidemic season. The positive rate in specimens from Songkhla province was not so high in the pre-epidemic season and some increase was seen in the post-epidemic season, especially in lower age groups (3-4 to 7-9 years). The patterns of HI antibody titers in different age groups in the Khon-Kaen and Songkhla provinces were also compared. The patterns changed with increase of ages and were also different with different viruses. The patterns for chikungunya virus were quite different from those for dengue type 2, type 4 and Japanese encephalitis virus. These results suggest that people in Khon-Kaen are exposed to group B arbovirus infections more often in childhood and attain high antibody levels more rapidly than people in Songkhla.     

从云南省蝙蝠脑组织中分离出乙型脑炎病毒

为进一步阐明蝙蝠在保存乙脑病毒中的作用,于1997年7月,在云南省耿马县捕捉蝙蝠64只,取脑组织作病毒分离,从一只金管鼻蝠脑组织中分离出1株病毒。该毒株能引起BHK21细胞病变和乳鼠发病死亡,在pH5.75～7.4时能凝集鸽红血球,经用单克隆抗体血凝抑制和免疫荧光试验鉴定,证实为乙型脑炎病毒。进一步证明蝙蝠在乙型脑炎病毒保存和扩散中具有重要作用。从金管鼻蝠体内分离出乙型脑炎病毒属国内外首次报道。     

Cutaneous leishmaniasis caused by Leishmania infantum transmitted by Phlebotomus tobbi

Transmission of cutaneous leishmaniasis (CL) caused by Leishmania infantum was studied in South Anatolia, Turkey. Small, non-ulcerating lesions prevailed and patients were negative in rK39 tests for antibody detection for human visceral leishmaniasis (VL). The most abundant sand fly species, Phlebotomus tobbi, was found positive for Leishmania promastigotes with a prevalence of 1.4% (13 out of 898 dissected females). The isolated strains were identical with those obtained from patients with CL and were typed as L. infantum. Phylogenetic analysis revealed similarity to MON-188 and a clear difference from the MON-1 clade. Blood-meal identification showed that P. tobbi feeds preferentially on cattle and humans. This finding, the high number of CL patients and relative scarcity of dogs in the focus, suggests that the transmission cycle could be anthroponotic.