Clinico-Epidemiological Patterns of Cutaneous Leishmaniasis Patients Attending the Anuradhapura Teaching Hospital, Sri Lanka

Cutaneous leishmaniasis (CL) caused by Leishmania donovani is an endemic vector-borne disease in Sri Lanka. Over 2,500 cases have been reported since 2000 and the number of CL cases has dramatically increased annually. Total 57 clinically suspected CL patients attending the dermatology clinic in Anuradhapura Teaching Hospital were recruited from January to June 2015. Slit skin smears and skin biopsies were taken from each of the subjects. Clinical and epidemiological data were obtained using interviewer administered questionnaire. Forty-three (75.4%) patients among 57 were confirmed positive for L. donovani. The majority of infected patients was males (P=0.005), and the most affected age group was 21–40 years. Soldiers in security forces, farmers, and housewives were identified as high risk groups. The presence of scrub jungles around the residence or places of occupation (P=0.003), the presence of sandflies (P=0.021), and working outsides more than 6 hr per day (P=0.001) were significantly associated with CL. The number of lesions ranged from 1–3, and the majority (76%) of the patients had a single lesion. Upper and lower extremities were the prominent places of lesions, while the wet type of lesions were more prevalent in females (P=0.022). A nodular-ulcerative type lesion was common in both sexes. The presence of sandflies, scrub jungles, and outdoor activities contributed to spread of Leishmania parasites in an endemic pattern. Implementation of vector control programs together with health education with regard to transmission and prevention of CL are necessary to control the spread of this infection.

Arginine kinase in Toxocara canis: Exon-intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors

OBJECTIVES@#To determine exon/intron organization of the Toxocara canis (T. canis) AK (TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants.@*METHODS@#Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame (1200 bp) of TCAK (wild type) was cloned into the BamH1/SalI site of pMAL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pMAL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25 °C for the forward reaction (phosphagen synthesis).@*RESULTS@#Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K value of the mutant (Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The K value of the mutant (Serine to Glycine) increased to 0.19 mM. The K value (0.19 mM) of the double mutant (Alanine-Serine to Serine-Glycine) was slightly greater than in the wild-type (0.12 mM). In addition, several other chemicals were tested; including plant extract Azadiracta indica (A. indica), an aminoglycoside antibiotic (aminosidine), a citrus flavonoid glycoside (rutin) and a commercially available catechin mixture against TCAK. Green and black tea (1:10 dilution) produced 15% and 25% inhibition of TCAK, respectively. The extract of A. indica produced 5% inhibition of TCAK. Moreover, green and black tea produced a non-competitive type of inhibition and A. indica produced a mixed-type of inhibition on TCAK.@*CONCLUSIONS@#Arginine kinase in T. canis has a seven-exon/six-intron gene structure. However, further studies are needed to identify a specific compound within the extract causing the inhibitory effect and also to determine the molecular mechanisms behind inhibition of arginine kinase in T. canis.

Arginine kinase in Toxocara canis: Exon–intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors

Objectives To determine exon/intron organization of the Toxocara canis (T. canis) AK (TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants. Methods Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame (1 200 bp) of TCAK (wild type) was cloned into the BamH1/SalI site of pMAL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pMAL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25 °C for the forward reaction (phosphagen synthesis). Results Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K

Human ocular dirofilariasis due to Dirofilaria repens in Sri Lanka

OBJECTIVE@#To identify worms obtained from patients with eye lesions and to describe the demographic factors of patients with ocular dirofilariasis.@*METHODS@#A retrospective descriptive study was conducted in 31 worm samples from 30 patients referred by consultant ophthalmologists between 2006 and February 2014. Data on age, sex and site of the lesion were ascertained from the details given in the referral letters. Morphological identification of the worm was based on the maximum width, length and appearance of the cuticle. The sex of the worm was determined by the width, length and presence or absence of vulva opening. PCR was performed using Dirofilaria repens specific primers to confirm the species of worms which couldnot be identified morphologically.@*RESULTS@#Most of the patients belonged to the age group of 40-49 years (mean age = 42 years). Majority of them were females (70%). Subconjunctival lesions were the most frequent presentation, while the rest (n = 4) were found on eyelids. Female worms were extracted from 18 cases, and 11 had male worms. One individual had both male and female worms in a single nodule. Adults were the most commonly affected. This pattern was different from the previous studies in Sri Lanka where the most common age group affected was younger than 9 years old.@*CONCLUSIONS@#The present study showed a considerably high incidence of ocular dirofilariasis, stressing the importance of implementing preventive measures to reduce the transmission of this zoonotic filarial disease.

Human ocular dirofilariasis due to Dirofilaria repens in Sri Lanka

Objective: To identify worms obtained from patients with eye lesions and to describe the demographic factors of patients with ocular dirofilariasis. Methods: A retrospective descriptive study was conducted in 31 worm samples from 30 patients referred by consultant ophthalmologists between 2006 and February 2014. Data on age, sex and site of the lesion were ascertained from the details given in the referral letters. Morphological identification of the worm was based on the maximum width, length and appearance of the cuticle. The sex of the worm was determined by the width, length and presence or absence of vulva opening. PCR was performed using Dirofilaria repens specific primers to confirm the species of worms which couldnot be identified morphologically. Results: Most of the patients belonged to the age group of 40-49 years (mean age = 42 years). Majority of them were females (70%). Subconjunctival lesions were the most frequent presentation, while the rest (n = 4) were found on eyelids. Female worms were extracted from 18 cases, and 11 had male worms. One individual had both male and female worms in a single nodule. Adults were the most commonly affected. This pattern was different from the previous studies in Sri Lanka where the most common age group affected was younger than 9 years old. Conclusions: The present study showed a considerably high incidence of ocular dirofilariasis, stressing the importance of implementing preventive measures to reduce the transmission of this zoonotic filarial disease.

Aureobasidium-Derived Soluble Branched (1,3-1,6) beta-Glucan (Sophy beta-glucan) Enhances Natural Killer Activity in Leishmania amazonensis-Infected Mice

The beta-glucans derived from yeast cell walls have been reported for having many immunomodulatory activities in vivo and in vitro. In this study, Aureobasidium-derived soluble branched (1,3-1,6) beta-glucan (Sophy beta-glucan) was checked for natural killer (NK) activity and for the production of IFN-gamma and IL-4 in Leishmania amazonensis infection. The main experiment was performed with a group of female C57BL/6 and BALB/c mice, orally supplemented with 5% of Sophy beta-glucan and infected with promastogotes of L. amazonensis (1 x 10(7)) into the footpad. Increase in the footpad thickness with time was observed in BALB/c mice in spite of the oral Sophy beta-glucan supplement, but it was less in C57BL/6 mice. The difference in overall mean footpad thickness between 'infection only' versus 'infection + glucan' groups was statistically significant (P < 0.001). High NK activity in C57BL/6 than BALB/c mice was observed in 'glucan only' group compared to the control group and also in 'infection + glucan' group compared to 'infection only' group. The difference in the NK activity among these groups was significant (P < 0.05). The IFN-gamma level increased at weeks 7 and 8 post-infection in C57BL/6 mice and was significantly high in 'infection + glucan' group compared to the 'infection only' group (P < 0.05). IL-4 levels did not increase up to detectable levels throughout the study. The results led a conclusion that Sophy beta-glucan enhances NK activity and cellular immunity in L. amazonensis-infected mice.

Morphological and molecular studies on Sri Lankan Leishmania

Cutaneous leishmaniasis (CL) is an emerging disease in Sri Lanka, more than 400 cases having been reported since 2001. However, the morphology and taxonomic status of the Sri Lankan strain of <I>Leishmania</I> is not known yet. Therefore, it is important to study the morphology and to analyze the phylogenetic position to predict the risk and expansion of the disease and thereby to develop an effective control programme. Morphology of the amastigote of the Sri Lankan isolate was checked by light microscopy and electron microscopic observation. Presence of amastigotes within macrophages was confirmed in skin biopsy samples. The promastigote had the characteristic appearance of a kinetoplastid cell in cultures. The kinetoplast minicircle DNA has been used for diagnosis of <I>Leishmania</I> for a long time and also for phylogenetic studies on trypanosomatid flagellates. The kinetoplast minicircle was amplified using PCR and subsequently sequenced from samples obtained from Sri Lankan patients with cutaneous lesions. Mitochondrial cytochrome b gene has been recently shown to be useful for identification and phylogenetic analysis of the genus <I>Leishmania</I>. The nucleotide sequence of the cytochrome b gene of Sri Lankan <I>Leishmania</I> was determined using the semi-nested PCR and 620 bp of this gene obtained. Phylogenetic analysis using these sequences unambiguously indicated that Sri Lankan isolate of <I>Leishmania</I> belongs to <I>L. donovani</I> complex. However, the Sri Lankan isolate forms a distinct lineage within the complex and probably represents a new branch.