Persistence of Leishmania donovani antibodies in past visceral leishmaniasis cases in India

The persistence of anti-Leishmania donovani antibodies in past visceral leishmaniasis (VL) cases was retrospectively assessed by means of the direct agglutination test (DAT) and the rK39 enzyme-linked immunosorbent assay (ELISA). Antibody titers remained high for an extended period of time in past cases of VL. These results highlight the need to carefully elicit the history of patients with VL symptoms.     

Genetic heterogeneity in clinical isolates of Leishmania donovani from India

Genetic diversity within 45 Indian Leishmania donovani isolates was analyzed using seven genetic markers. While kinetoplast DNA (kDNA) analysis revealed 15 genotypes, 8 genotypes were obtained by analysis of other markers. In contrast to earlier reports, our data suggest that significant genetic polymorphisms exist in L. donovani strains in Bihar, India. Our results confirm the presence of 2 zymodemes in India.     

Parasite detection in patients with post kala-azar dermal leishmaniasis in India: a comparison between molecular and immunological methods

AIMS: To evaluate the sensitivity and specificity of serological, immunohistochemical, and molecular methods in the diagnosis of post kala-azar dermal leishmaniasis (PKDL). METHODS: Twenty five patients with confirmed PKDL and 25 controls were included in the study. G2D10, a monoclonal antibody against Leishmania, was used for the immunohistochemical (IHC) staining of lesion sections to visualise anti-Leishmania donovani antibodies. The diagnostic usefulness of IHC was compared with enzyme linked immunosorbent assay (ELISA) with a recombinant (rk39) antigen, and a species specific polymerase chain reaction (PCR) assay, amplifying a kinetoplast minicircle DNA sequence. RESULTS: IHC detected 22 of 25 PKDL cases, giving a sensitivity of 88%. The diagnostic sensitivity of both the ELISA and PCR tests was higher (96%). All of the 25 controls examined were negative in PCR, indicating 100% specificity of the test, whereas ELISA showed 96% specificity. CONCLUSIONS: IHC with G2D10 significantly enhances the sensitivity of detection of PKDL over routine haematoxylin and eosin staining. ELISA with a recombinant antigen is an economical and practical assay. PCR is the most sensitive and specific diagnostic method for PKDL. The tests described would facilitate the recognition of patients with PKDL, enabling timely treatment, which would contribute greatly to the control of kala-azar.     

DNA polymorphism assay distinguishes isolates of Leishmania donovani that cause kala-azar from those that cause post-kala-azar dermal Leishmaniasis in humans

Leishmania donovani in India causes visceral infection (kala-azar) and dermal infection (post-kala-azar dermal leishmaniasis). We report here the identification of polymorphism in a well-defined genetic locus among the Leishmania parasites causing the visceral and dermal manifestations, in a comparison of 15 post-kala-azar dermal leishmaniasis and 12 kala-azar patient isolates.     

Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis

We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae, and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples.     

Immunoblot analysis of the humoral immune response to Leishmania donovani polypeptides in cases of human visceral leishmaniasis: its usefulness in prognosis

Sera from Indian patients with parasitologically confirmed visceral leishmaniasis were studied by immunoblot analysis in order to identify a specific pattern for Leishmania infection. A soluble extract of Leishmania donovani was used as antigen. At diagnosis the sera from patients with visceral leishmaniasis specifically recognized fractions represented by bands of 201 kDa (50% of serum samples), 193 kDa (60%), 147 kDa (50%), 120 kDa (60%), 100 kDa (50%), 80 kDa (80%), 70 kDa (70%), 65 kDa (100%), 50 kDa (50%), 36 kDa (50%), 20 kDa (70%), and 18 kDa (50%). The 65-kDa band, common to all patients infected with Leishmania parasites, was found at the time of diagnosis. However, the immunoblot pattern changed after patients were treated and cured with sodium antimony gluconate (SAG; n =10) or miltefosine (n =10), as was evident from blots of sera obtained pretreatment and at 1, 3, and 6 months posttreatment. At 6 months posttreatment, immunoblots of sera from patients on the SAG regimen showed the disappearance of all bands except the 70-kDa band. Similarly, sera from those on the miltefosine regimen showed the disappearance of all bands except the 65- and 70-kDa bands. This study shows that Western blot analysis is a sensitive test for detection of anti-Leishmania antibodies. Moreover, the persistence of reactivity with the 65- and 70-kDa bands in the sera of all groups shows its promise as a diagnostic and prognostic tool.     

Identification of new antigens in visceral leishmaniasis by expression cloning and immunoblotting with sera of kala-azar patients from Bihar, India

Sera of kala-azar patients from Bihar, India, were used to identify Leishmania donovani antigens encoded by a phage expression library. Ten antigens were identified, five of which have not been described as leishmania antigens before. The antigens specifically react with sera of leishmania-infected patients but not of toxoplasma- or plasmodium-infected patients.     

Relationship between canine visceral leishmaniosis and the Leishmania (Leishmania) chagasi burden in dermal inflammatory foci

The skin is the first point of contact with organisms of the genus Leishmania from sand fly vectors, and apparently normal skin of sick dogs harbours amastigote forms of Leishmania chagasi. In relation to canine visceral leishmaniosis (CVL), the ear skin was examined in 10 uninfected dogs (UDs) and in 31 dogs dogs naturally infected with L. chagasi. The infected animals consisted of 10 symptomless dogs (SLDs), 12 mildly affected dogs (MADs) and nine affected dogs (ADs). A higher parasite burden was demonstrated in ADs than in SLDs by anti-Leishmania immunohistochemistry (P<0.01), and by Leishman Donivan Unit (LDU) indices (P=0.0024) obtained from Giemsa-stained impression smears. Sections stained with haematoxylin and eosin demonstrated a higher intensity of inflammatory changes in ADs than in SLDs (P<0.05), and in the latter group flow cytometry demonstrated a correlation (P=0.05/r=0.7454) between the percentage of CD14(+) monocytes in peripheral blood and chronic dermal inflammation. Extracellular matrix assessment for reticular fibres by staining of sections with Masson trichrome and Gomori ammoniacal silver demonstrated a decrease in collagen type I and an increase in collagen type III as the clinical signs increased. The data on correlation between cellular phenotypes and histological changes seemed to reflect cellular activation and migration from peripheral blood to the skin, mediated by antigenic stimulation. The results suggested that chronic dermal inflammation and cutaneous parasitism were directly related to the severity of clinical disease.     

Eco-epidemiology of visceral and cutaneous leishmaniasis in the Yemen Arab Republic. I. Presence, in sympatric condition, of Leishmania infantum and Leishmania donovani complexes

In complement to a previous survey, the authors proceed to the analysis of strains isolated from visceral human and canine leishmaniasis. Finally, among eight human strains isolated and identified with an enzymatic method, seven belong to the Leishmania donovani complex and one to the L. infantum complex. The L. donovani complex is represented by the MON-31 and MON-83 zymodem. The first one is also present in Saudi Arabia and Ethiopia. The second one, corresponding to a small variant, pleads for an intrafocal polymorphism phenomenon which was until now unknown in the L. donovani complex. The L. infantum complex is observed: 1) in sympatria with L. donovani in mountainous areas; 2) alone in the Tihama coastal plain. As for human cutaneous leishmaniasis present in the same focuses it is caused by L. tropica MON-71 and not by the above mentioned complexes.     

Fine‐needle sampling provides appreciable diagnostic yield in lesions of post kala azar dermal leishmaniasis: Analysis of four cases from North Eastern India

Post‐kala‐azar dermal leishmaniasis (PKDL) is a sequel of visceral leishmaniasis (VL), usually occurring 6 months to 3 years after VL. Spectrum of cutaneous lesions in PKDL can be hypopigmented macules, nodules, plaques, or erythema. It is usually diagnosed clinically, supplemented by ancillary techniques like skin smear examination, histopathology, polymerase chain reaction, and monoclonal antibody test. Literature on the role of cytology in the diagnosis of PKDL is extremely limited. Here we highlight the appreciable yield of fine‐needle sampling in four cases of PKDL, which may be considered as a useful diagnostic aid. Diagn. Cytopathol. 2014;42:525–529. © 2013 Wiley Periodicals, Inc.     

Type I signal peptidase from Leishmania is a target of the immune response in human cutaneous and visceral leishmaniasis

The gene encoding type I signal peptidase (Lmjsp) has been cloned from Leishmania major. Lmjsp encodes a protein of 180 amino residues with a predicted molecular mass of 20.5 kDa. Comparison of the protein sequence with those of known type I signal peptidases indicates homology in five conserved domains A-E which are known to be important, or essential, for catalytic activity. Southern blot hybridisation analysis indicates that there is a single copy of the Lmjsp gene. A recombinant SPase protein and a synthetic peptide of the L. major signal peptidase were used to examine the presence of specific antibodies in sera from either recovered or active individuals of both cutaneous and visceral leishmaniasis. This evaluation demonstrated that sera from cutaneous and visceral forms of leishmaniasis are highly reactive to both the recombinant and synthetic signal peptidase antigens. Therefore, the Leishmania signal peptidase, albeit localised intracellularly, is a significant target of the Leishmania specific immune response and highlights its potential use for serodiagnosis of cutaneous and visceral leishmaniasis.     

Immunization with Leishmania donovani protein disulfide isomerase DNA construct induces Th1 and Th17 dependent immune response and protection against experimental visceral leishmaniasis in Balb/c mice

In the present study, the efficacy of Leishmania donovani protein disulfide isomerase (LdPDI) as a DNA vaccine was evaluated in BALB/C mice. Mice immunized with the LdPDI-DNA construct were found to be the most immuno-reactive, as the construct induced higher T-cell proliferation. The increased T-cell proliferation was associated with a substantial rise in Th1 and Th17+ CD4 cell response and triggered a higher proportion of CD8+ T cells for the release of interferon-gamma along with a reduced splenic parasite load on Days20 and 60 post challenge (PC). Furthermore, the vaccine construct triggered increased interferon (IFN)-γ, interleukin(IL)-17A, and IL-22 release accompanied by decreased extracellular signal-regulated kinases (ERK) 1/2 signaling and increased mitogen-activated protein kinase (MAPK) signaling coinciding with an increase in the amount of nitrite and reactive oxygen species (ROS)in vaccinating the splenocyts. We summarize from our data that the PDI-DNA construct of Leishmania donovani has the potential to elicit protective immunity through the pro-inflammatory cytokines of CD8+ and CD4+(Th1 and Th17) following an intervention in the downstream signaling event of ERK1/2 (probably through p38MAPK signaling). Therefore, the study suggests a new control against visceral leishmaniasis in the future.     

Complete protection against experimental visceral leishmaniasis with complete soluble antigen from attenuated Leishmania donovani promastigotes involves Th1‐immunity and down‐regulation of IL‐10

Compared with cutaneous leishmaniasis, vaccination against visceral leishmaniasis has received limited attention. Most available drugs are toxic, and relapse after cure remains a chronic problem. Growing limitations in available chemotherapeutic strategies due to emerging resistant strains and lack of an effective vaccine strategy against visceral leishmaniasis deepens the crisis. Complete soluble antigen (CSA), from a β1‐4 galactosyltransferase expressing attenuated Leishmania donovani parasite, induced protection against subsequent challenge and during active infections. CSA immunization was effective against both pentavalent antimony sensitive and resistant strains of L. donovani. Majority (～85%) of the immunized animals showed sterile protection. Resolution of the disease required the presence of T cells, and the recovered animals remained immune to re‐challenge. Control of the parasites was dependent on type 1 CD4⁺ helper cells, which evolved in the presence of IL‐12 and activated macrophages through the production of IFN‐γ. Immunity was adoptively transferable and was dependent on both CD4⁺ and CD8⁺ cells. CSA immunization led to enhanced IFN‐γ production, while suppressing the IL‐10 production. However, CSA immunization did not abrogate IL‐4 production. Our results accentuate the need to establish a favorable cellular immunity while intervening with the development of Th2 cells during leishmania infection.     

Genetic and phylogenetic characterization of polycistronic dsRNA segment-10 of bluetongue virus isolates from India between 1985 and 2011

Virus Genes - The smallest polycistronic dsRNA segment-10 (S10) of bluetongue virus (BTV) encodes NS3/3A and putative NS5. The S10 sequence data of 46 Indian BTV field isolates obtained between...     

Genetic characterization of Aino and Peaton virus field isolates reveals a genetic reassortment between these viruses in nature

Sequence determination and phylogenetic analysis were conducted using the S, M and L RNA segments of the 10 Aino, 6 Peaton and 1 Sango virus (AINOV, PEAV and SANV) field isolates of the genus Orthobunyavirus in the family Bunyaviridae, respectively. The Japanese AINOV strains were genetically stable, but the sequence differences between the Japanese and Australian AINOV strains were considerably larger than those among the Japanese AINOV strains. A similar result was found in the genetic relationship among Japanese and Australian PEAVs, and SANV which was isolated in Nigeria and was thought as a synonym of PEAV, suggesting that geographic separation contributed significantly to the evolution of those viruses. The Australian AINOV strain B7974 is more closely related to the Australian PEAV strain CSIRO110 than to the Japanese AINOV strains in the S and L RNA segments, while the phylogenetic position of the M RNA segment of the B7974 strain was clustered with those of the Japanese AINOV strains. Our findings indicate that the B7974 strain is a reassortment with the M RNA segment derived from AINOV and the S and L RNA segments derived from an Australian PEAV.     

Dynamics of the antibodies in cohorts of cured cases of visceral leishmaniasis: its implication on the validity of serological test, value in prognosis and in post therapeutic assessment

The major disadvantage of a Serological test like Direct Agglutination Test (DAT) for Visceral Leishmaniasis (also called Kala-azar) is its inability to distinguish between recent and past infection. The objective of our study was to look at rate of decline of antibodies in fully cured cases of Kala-azar and length of time it takes for DAT to become negative. Cohort Study involving completely treated Kala-azar cases from Government Hospital during one calendar year of study. Cases were selected on the basis of treatment cohorts 0, 3, 6, 9 & 12 mo after completion of treatment.. Phase I--The cases were traced and after obtaining the informed consent they were subjected to Direct Agglutination Test (DAT). Phase II--The five treatment cohorts, constituting 82 cured cases (average of 15 cured cases per each treatment cohort) were tested again with DAT three months after the first test. The titers of Phase-I and phase-II tests were analyzed for the dynamics of the antibodies for the period. Cutoff-Values of DAT below 1:800 are considered negative. Values of 1:800, 1:1200, 1:1600 and so on are considered positive. The mean titer [Geometric Mean Titer (GMT)] at the start of treatment was 1:1120, which showed steady decline up to six months, plummeting below the cutoff titer for the DAT (1:800) at the ninth month. Antibodies continue to linger for about one year in cured Kala-azar cases even after correct and complete treatment. Single DAT results may be misleading due to high false positivity up to one year after the cure. Paired test defined as two tests 3 mo apart on the same subject. Paired test is highly recommended for diagnosis and prognosis. DAT is still a very useful tool for diagnosis if used along with clinical correlation.     

Eco-epidemiology of visceral and cutaneous leishmaniasis in the Arab Republic of Yemen. II. A survey using intradermal reaction to leishmanin in a zone of mixed infestation with Leishmania tropica, L. donovani and L. infantum

Frequency distribution of leishmanin test survey in Dhamran valley around Taez (Yemen Arab Republic) is reported. It was carried out on 174 school children from 6 to 12 years old. Three schools located at 950 m, 1,100 m and 1,430 m of altitude were visited. The maximum of positivity is observed in the lower range where L. tropica, L. donovani and L. infantum are rife. In the upper valley, where cutaneous leishmaniasis is rare and visceral leishmaniasis absent, the rate of positivity is a little bit lower. The conjugated influence of the three parasites is suggested.     

An international multicenter study of antimicrobial resistance and typing of hospital Staphylococcus aureus isolates from 21 laboratories in 19 countries or states

During 1996, 4065 consecutive Staphylococcus aureus strains from different patients were collected in 21 worldwide hospital laboratories. The strains, their resistance pattern, and hospital demographic data were forwarded to Statens Serum Institut where the strains were typed and data analyzed. Resistance patterns varied by region and resistance to other antibiotics than methicillin were mainly related to the occurrence of methicillin resistance, except for mupirocin, rifampicin, and fusidic acid. Methicillin-resistant S. aureus (MRSA) occurred with low levels in hospitals in Northern Europe (<1%), increasing levels in middle-European countries, United States, New Zealand, and Australia (6-22%), and very high levels in Southern European countries as well as in parts of the United States, Asia, and South Africa (28-63%). MRSA found in large hospitals were more resistant to other antibiotics than MRSA found in smaller hospitals serviced by the same laboratory. No difference in resistance levels was seen for methicillin-susceptible S. aureus (MSSA) isolated in large or small hospitals. Intensive Care Units had the highest level of MRSA. Strains from the lower respiratory tract showed the highest resistance levels and blood isolates the lowest. A dominating MRSA clone was found in hospitals with an MRSA frequency of more than 10%. Pulsed-field gel electrophoresis (PFGE) typing recognized several of these clones as international epidemic MRSA (E-MRSA). All MSSA isolates were phage typed (typeability 85.4%) and divided in seven major phage patterns. Isolates of all patterns were found in all hospitals except one, indicating that the MSSA seldom represented the spread of clones within the hospital. The comparison should evaluate the prevalence of community-acquired MRSA and identify internationally E-MRSA. The present study gives a snapshot of the MRSA situation, but it is important to build up a continuous national and international surveillance, because MRSA is a global socioeconomic problem. Global infection control procedures, including rational antibiotic use, should be agreed on. The accompanying paper will address the issue of antibiotic consumption and MRSA.