msp1, msp2, and glurp genotyping to differentiate Plasmodium falciparum recrudescence from reinfections during prevention of reestablishment phase, Sri Lanka, 2014-2019.

BACKGROUND: Sri Lanka after eliminating malaria in 2012, is in the prevention of re-establishment (POR) phase. Being a tropical country with high malariogenic potential, maintaining vigilance is important. All malaria cases are investigated epidemiologically and followed up by integrated drug efficacy surveillance (iDES). Occasionally, that alone is not adequate to differentiate Plasmodium falciparum reinfections from recrudescences. This study evaluated the World Health Organization and Medicines for Malaria Venture (MMV) recommended genotyping protocol for the merozoite surface proteins (msp1, msp2) and the glutamate-rich protein (glurp) to discriminate P. falciparum recrudescence from reinfection in POR phase. METHODS: All P. falciparum patients detected from April 2014 to December 2019 were included in this study. Patients were treated and followed up by iDES up to 28 days and were advised to get tested if they develop fever at any time over the following year. Basic socio-demographic information including history of travel was obtained. Details of the malariogenic potential and reactive entomological and parasitological surveillance carried out by the Anti Malaria Campaign to exclude the possibility of local transmission were also collected. The msp1, msp2, and glurp genotyping was performed for initial and any recurrent infections. Classification of recurrent infections as recrudescence or reinfection was done based on epidemiological findings and was compared with the genotyping outcome. RESULTS: Among 106 P. falciparum patients, six had recurrent infections. All the initial infections were imported, with a history of travel to malaria endemic countries. In all instances, the reactive entomological and parasitological surveillance had no evidence for local transmission. Five recurrences occurred within 28 days of follow-up and were classified as recrudescence. They have not travelled to malaria endemic countries between the initial and recurrent infections. The other had a recurrent infection after 105 days. It was assumed a reinfection, as he had travelled to the same malaria endemic country in between the two malaria attacks. Genotyping confirmed the recrudescence and the reinfection. CONCLUSIONS: The msp1, msp2 and glurp genotyping method accurately differentiated reinfections from recrudescence. Since reinfection without a history of travel to a malaria endemic country would mean local transmission, combining genotyping outcome with epidemiological findings will assist classifying malaria cases without any ambiguity.

Humoral immune response and changes in peritoneal cell populations in rats immunized against two Leptospira serovars; serovar patoc and serovar pyrogenes.

BACKGROUND: Leptospirosis is a zoonotic disease caused by Leptospira species. Variations in lipopolysaccharide (LPS) structure in Leptospira are known to be associated with the serovar diversity and antigenicity. Development of immunodiagnostics for early detection of leptospirosis based on immune responses against different pathogenic antigens as well as development of vaccines are important. Hence, this study has assessed the immune response generated against leptospiral LPS and whole antigen preparations of pathogenic and saprophytic Leptospira and specific changes in peritoneal cells was also studied to elucidate the cellular responses associated with immune response of Wistar rats. METHODS: During the study, immune response induced by two types of Leptospira antigen preparations of two selected serovars was compared. Changes in the specific peritoneal cell subpopulations following immunizations of rats were analyzed using flow cytometry. RESULTS: Of the two antigen preparations tested, the LPS extract induced a higher IgM immune response as opposed to the sonicated antigen preparation. Of the two serovars tested, L. interrogans serovar Pyrogenes had induced a higher IgM response compared to that by L. biflexa serovar Patoc. Considering the IgG titers, equivalent responses were observed with all four antigen preparations. Significant increases in lymphocytes were observed following immunization with LPS of both serovars. Interestingly, the B2 cell percentages increased significantly during the immunization period. Further, significant correlations were observed with both IgM and IgG responses and percentage of B2 cells in the peritoneal cavity (PC). CONCLUSION: LPS extract of L. interrogans serovar Pyrogenes induced higher IgM response while the IgG response was equivalent among the four antigen preparations tested. Significant increase of B2 cell percentage in the peritoneal cavity during the immunization reflects the accumulation of B2 cells in the PC which may play considerable role in generating humoral response against Leptospira antigens.

Identification of allergens in Artocarpus heterophyllus, Moringa oleifera, Trianthema portulacastrum and Syzygium samarangense.

BACKGROUND: It is clinically important to identify allergens in Artocarpus heterophyllus (jackfruit), Moringa oleifera (moringa), Trianthema portulacastrum (horse purslane) and Syzygium samarangense (rose apple). This study included 7 patients who developed anaphylaxis to jackfruit (1), moringa (2), horse purslane (3) and rose apple (1). We sought to determine allergens in the edible ripening stages of jackfruit (tender, mature, and ripened jackfruit) and seeds, edible parts of moringa (seeds, seedpod, flesh inside seedpod, and leaves), horse purslane leaves and ripened rose apple fruit. The persistence of the allergens after cooking was also investigated. METHODS: Allergens were identified by clinical history followed by a skin prick test. Protein profiles of plant/fruit crude protein extracts were determined by SDS-PAGE. Molecular weights of the allergens were determined by immunoblotting with patient sera. RESULTS: A heat-stable allergen of 114 kDa in A. heterophyllus which is shared among different ripening stages and seeds was identified. Additionally, 101 kDa allergen in boiled tender jackfruit, 86 kDa allergen in boiled seeds and 80 kDa allergen in boiled mature jackfruit were identified. Five heat-stable allergens of 14, 23, 35, 43, and 48 kDa in M. oleifera, 1 heat-stable allergen of 97 kDa in T. portulacastrum, and 4 allergens of 26, 31. 60, and 82 kDa in S. samarangense were identified. CONCLUSION: Novel IgE-sensitive proteins of A. heterophyllus, M. oleifera, T. portulacastrum and S. samarangense were identified which would be especially useful in the diagnosis of food allergies. The identified allergens can be used in Component Resolved Diagnostics (CRD).

Comparative virome analysis of individual shedding routes of Miniopterus phillipsi bats inhabiting the Wavul Galge cave, Sri Lanka.

Bats are described as the natural reservoir host for a wide range of viruses. Although an increasing number of bat-associated, potentially human pathogenic viruses were discovered in the past, the full picture of the bat viromes is not explored yet. In this study, the virome composition of Miniopterus phillipsi bats (formerly known as Miniopterus fuliginosus bats in Sri Lanka) inhabiting the Wavul Galge cave, Sri Lanka, was analyzed. To assess different possible excretion routes, oral swabs, feces and urine were collected and analyzed individually by using metagenomic NGS. The data obtained was further evaluated by using phylogenetic reconstructions, whereby a special focus was set on RNA viruses that are typically associated with bats. Two different alphacoronavirus strains were detected in feces and urine samples. Furthermore, a paramyxovirus was detected in urine samples. Sequences related to Picornaviridae, Iflaviridae, unclassified Riboviria and Astroviridae were identified in feces samples and further sequences related to Astroviridae in urine samples. No viruses were detected in oral swab samples. The comparative virome analysis in this study revealed a diversity in the virome composition between the collected sample types which also represent different potential shedding routes for the detected viruses. At the same time, several novel viruses represent first reports of these pathogens from bats in Sri Lanka. The detection of two different coronaviruses in the samples indicates the potential general persistence of this virus species in M. phillipsi bats. Based on phylogenetics, the identified viruses are closely related to bat-associated viruses with comparably low estimation of human pathogenic potential. In further studies, the seasonal variation of the virome will be analyzed to identify possible shedding patterns for particular viruses.

Cardioprotective Effects of Vitex negundo: A Review of Bioactive Extracts and Compounds.

There has been accumulating interest in the application of medicinal plants as alternative medicine to treat various diseases and/or to develop modern medicines. Vitex negundo is one of such medicinal plants that has been of interest to many researchers and has been of use in traditional medicine. V. negundo is found in Sri Lanka, Madagascar, Malaysia, India, China, The Philippines and East Africa. Therapeutic properties of V. negundo have previously been reviewed. Different parts, preparations and bioactive components of V. negundo possess potential protective and therapeutic effects against cardiovascular disease and related conditions as demonstrated in previous studies. We review the present state of scientific knowledge on the potential use of V. negundo and some of its bioactive components in protecting against cardiovascular diseases and related pathologies. Previous studies in animal and non-animal experimental models, although limited in number and vary in design, seem to support the cardioprotective effect of V. negundo and some of its active components. However, there is need for further preclinical and clinical studies to validate the use of V. negundo and its active constituents in protection and treatment of cardiovascular diseases. Additionally, since only a few V. negundo compounds have been evaluated, specific cardioprotective effects or mechanisms and possible side effects of other V. negundo compounds need to be extensively evaluated.

Identification of allergens in coconut milk and oil with patients sensitized to coconut milk in Sri Lanka.

BACKGROUND: Despite the low prevalence of IgE sensitivity to fresh or boiled coconut milk and coconut oil, those may contain allergens of which the clinical significance remains undetermined. This study aimed to identify and compare allergens in fresh coconut milk (FCM), boiled coconut milk (BCM), unrefined wet-processed coconut oil (WPCO), and dry-processed coconut oil (DPCO) using sera from patients with allergy to coconut milk. METHODS: The study included 18 patients with immediate hypersensitivity to coconut milk, including five who developed anaphylaxis. Sensitization was assessed by skin prick test and ImmunoCAPs using commercially available coconut extracts. Immunoblotting was performed to identify and compare allergen profiles. RESULTS: Total sIgE levels and overall IgE reactivity of patients with anaphylaxis were higher compared to patients with allergy. Twelve allergens ranging from 5 to 128 kDa including six novel allergens with 5, 12, 47, 87, 110, and 128 kDa were visualized in immunoblots with FCM. Similarly, nine allergens of 5, 12, 17, 32, 35, 47, 87, 110, and 128 kDa were detected in BCM. One allergen (110 kDa) was discerned in all four extracts. Higher IgE prevalence was detected with three allergens of 55, 87, and 110 kDa. CONCLUSIONS: Allergens of BCM and unrefined coconut oil (WPCO and DPCO) were determined for the first time. Novel allergens of 87 and 110 kDa and the 55 kDa allergen have the highest potential to be used in Component Resolved Diagnostics. Further, these findings demonstrate that, patients who have an allergy to coconut milk could also react to boiled coconut milk and unrefined coconut oil.

Molecular characterization of BK virus detected in renal transplant patients in Sri Lanka: a preliminary study.

Background & objectives: BK virus (BKV) is a polyomavirus and cause of a common infection after renal transplantation which could be preceded to BKV-associated nephropathy. It has four main subtypes (I-IV). BKV subtypes II and III are rare, whereas subtype I shows a ubiquitous distribution. The objective of the present study was to investigate the prevailing BKV subtypes and subgroups in renal transplant patients in Sri Lanka. Methods: The presence of BKV in urine was tested through virus load quantification by real-time PCR from 227 renal transplant patients who were suspected to have BKV infection. Of these patients only 41 were found to be BKV infected (>103 copies/ml) and those were subjected to conventional PCR amplification of VP1 gene followed by BKV genotyping via phylogenetic analysis based on DNA sequencing data. Results: Persistent BK viral loads varied from 1×103 to 3×108 copies/ml. Of the 41 patient samples, 25 gave positive results for PCR amplification of subtyping region of VP1 gene of BKV. BKV genotyping resulted in detecting subtype I in 18 (72%) and subtype II in seven (28%) patients. BKV subgroups of Ia, Ib-1 and Ib-11, and Ic were identified with frequencies of 6/18 (33.3%), 6/18 (33.3%), 5/18 (27.8%), and 1/18 (5.6%), respectively. Interpretation & conclusions: Findings from this preliminary study showed a high occurrence of subtype I, while the presence of subtype II, which is rare and less prevalent, was a novel finding for this Asian region. This emphasizes the need for further molecular and serological studies to determine the prevalence of different BKV subtypes in Sri Lanka.

Characterization of antigens of Enterobius vermicularis (pinworm) eggs.

Enterobiasis (pinworm infection) caused by Enterobius vermicularis is a common parasitic infection prevalent worldwide especially in children. Infection is diagnosed by microscopic detection of E. vermicularis eggs on perianal swabs. This study aimed to characterize the antigens of E. vermicularis eggs as a preliminary step towards identifying diagnostic targets for detection in infected individuals. The study was conducted between October 2019 and February 2020, following approval from Ethics Review Committee of the Faculty of Medicine, University of Colombo (EC-19-034). E. vermicularis eggs were harvested from perianal swabs using acetone and purified with 1× PBS (pH 7.2). A portion of eggs was used for preparing antigen slides, while the rest were sonicated and vortexed with glass beads and inoculated subcutaneously (with weekly booster doses) into a Wistar rat for developing antibodies. Blood drawing from rat was done weekly for 5 weeks. Confirmation of the presence of antibodies was done by surface immunofluorescence against eggs on the antigen slides. Protein bands were determined using SDS-PAGE assay and immunogenic antigen bands were determined by reacting with antiserum after immunoblotting. The band sizes of the proteins were determined against corresponding bands of a protein ladder. Surface immunofluorescence was positive with serum obtained from day 14 post-inoculation from the Wistar rat as well as that obtained from a person with chronic enterobiasis. The most prominent and immunogenic protein bands identified from egg antigens were 21 kDa, 66 kDa, 83 kDa, 96 kDa, 112 kDa, 121 kDa, 140 kDa and 151 kDa. Methods used in this study were effective in obtaining E. vermicularis egg antigens which were immunogenic. Furthermore, surface antigens of intact eggs reacted with antibodies developed against crushed egg antigens. These findings may pave the way for the development of effective immunodiagnostics.

First Complete Cytochrome B Sequences and Molecular Taxonomy of Bat Species from Sri Lanka.

This is the first report on the molecular identification and phylogeny of the Rousettus leschenaultii Desmarest, 1810, Rhinolophus rouxii Temminck, 1835, Hipposideros speoris Schneider, 1800, Hipposideros lankadiva Kelaart, 1850, and Miniopterus fuliginosus Kuhl, 1817, bat species in Sri Lanka, inferred from analyses by mitochondrially encoded cytochrome b gene sequences. Recent research has indicated that bats show enormous cryptic genetic diversity. Moreover, even within the same species, the acoustic properties of echolocation calls and morphological features such as fur color could vary in different populations. Therefore, we have used molecular taxonomy for the accurate identification of five bat species recorded in one of the largest cave populations in Sri Lanka. The bats were caught using a hand net, and saliva samples were collected non-invasively from each bat by using a sterile oral swab. Nucleic acids were extracted from the oral swab samples, and mitochondrial DNA was amplified by using primers targeting the mitochondrially encoded cytochrome b gene. This study reports the first molecular evidence for the identification of five bat species in Sri Lanka. Our findings will contribute to future conservation and systematic studies of bats in Sri Lanka. This study will also provide the basis for a genetic database of Sri Lankan bats which will contribute significantly to the investigation of potentially zoonotic bat viruses.

Paramyxovirus Diversity within One Population of Miniopterus fuliginosus Bats in Sri Lanka.

Bats are known as typical reservoirs for a number of viruses, including viruses of the family Paramyxoviridae. Representatives of the subfamily Orthoparamyxovirinae are distributed worldwide and can cause mild to fatal diseases when infecting humans. The research on Paramyxoviruses (PMVs) from different bat hosts all over the world aims to understand the diversity, evolution and distribution of these viruses and to assess their zoonotic potential. A high number of yet unclassified PMVs from bats are recorded. In our study, we investigated bat species from the families Rhinolophidae, Hipposiderae, Pteropodidae and Miniopteridae that are roosting sympatrically in the Wavul Galge cave (Koslanda, Sri Lanka). The sampling at three time points (March and July 2018; January 2019) and screening for PMVs with a generic PCR show the presence of different novel PMVs in 10 urine samples collected from Miniopterus fuliginosus. Sequence analysis revealed a high similarity of the novel strains among each other and to other unclassified PMVs collected from Miniopterus bats. In this study, we present the first detection of PMVs in Sri Lanka and the presence of PMVs in the bat species M. fuliginosus for the first time.

Full Genome of batCoV/MinFul/2018/SriLanka, a Novel Alpha-Coronavirus Detected in Miniopterus fuliginosus, Sri Lanka.

Coronaviruses (CoV) are divided into the genera α-CoVs, ß-CoVs, γ-CoVs and δ-CoVs. Of these, α-CoVs and ß-CoVs are solely capable of causing infections in humans, resulting in mild to severe respiratory symptoms. Bats have been identified as natural reservoir hosts for CoVs belonging to these two genera. Consequently, research on bat populations, CoV prevalence in bats and genetic characterization of bat CoVs is of special interest to investigate the potential transmission risks. We present the genome sequence of a novel α-CoV strain detected in rectal swab samples of Miniopterus fuliginosus bats from a colony in the Wavul Galge cave (Koslanda, Sri Lanka). The novel strain is highly similar to Miniopterus bat coronavirus 1, an α-CoV located in the subgenus of Minunacoviruses. Phylogenetic reconstruction revealed a high identity of the novel strain to other α-CoVs derived from Miniopterus bats, while human-pathogenic α-CoV strains like HCoV-229E and HCoV-NL63 were more distantly related. Comparison with selected bat-related and human-pathogenic strains of the ß-CoV genus showed low identities of ~40%. Analyses of the different genes on nucleotide and amino acid level revealed that the non-structural ORF1a/1b are more conserved among α-CoVs and ß-CoVs, while there are higher variations in the structural proteins known to be important for host specificity. The novel strain was named batCoV/MinFul/2018/SriLanka and had a prevalence of 50% (66/130) in rectal swab samples and 58% (61/104) in feces samples that were collected from Miniopterus bats in Wavul Galge cave. Based on the differences between strain batCoV/MinFul/2018/SriLanka and human-pathogenic α-CoVs and ß-CoVs, we conclude that there is a rather low transmission risk to humans. Further studies in the Wavul Galge cave and at other locations in Sri Lanka will give more detailed information about the prevalence of this virus.

Increased expression of co-stimulatory molecules and enhancement of the IgG response in rats orally administered with a polyherbal formulation.

BACKGROUND: Link Samahan® (LS) is a standardized modern formulation of a polyherbal preparation used in the indigenous system of medicine in Sri Lanka. OBJECTIVE: Evaluation of the immunostimulatory activity of LS and the molecular mechanisms that modulate the humoral immune response. MATERIAL AND METHODS: Immunostimulatory activity of LS was tested in rats following oral administration on days 1-5 and 15-19 and immunization with bovine serum albumin (BSA) on day 1 and 15. Anti-BSA IgM and IgG response in rats treated with LS, water and sugar (as controls) were compared on days 0-35, using ELISA. The expression of co-stimulatory molecules on lymphocytes was assessed on days 0-8 and days 14-22 using RT-qPCR. RESULTS: IgM and IgG levels of LS-treated rats were increased significantly by day 7 and 21 respectively compared to controls (p < 0.05). IgG response of LS-treated group reached a higher magnitude compared to its IgM response. Gene expression of CD28 and CD40L on T cells (4.9-5.1 fold) and CD80, CD86 and CD40 on APCs (2.4-3.1 fold) were induced significantly by day 2 compared to their expression on day 0 (p < 0.05). The expression levels of CD28 and CD40L on day 2-4 and 16-18 were similar while the expression of CD80, CD86 and CD40 on day 16-18 was higher (3.7-5.1 folds) compared to their levels on day 2-4 (2.4-3.2). CONCLUSIONS: These findings support an adjuvant effect of LS contributing to its immunostimulatory activity and increased expression of co-stimulatory molecules that contribute to boosting immune response.

Dynamics of maternally transferred antibodies against measles, mumps, and rubella in infants in Sri Lanka.

BACKGROUND: Determining the dynamics of maternally transferred antibodies against measles, mumps, and rubella infections in infants is important for making evidence-based policy decisions regarding the timing of vaccination. METHODS: The levels of serum immunoglobulin G (IgG) developed against measles, mumps, and rubella infections were assessed using commercial ELISA kits in mother-newborn pairs (n = 294) and 6-12-month-old infants (n = 280) recruited from Colombo District, Sri Lanka. Antibody levels of mothers and their newborns were assessed with respect to sex and parity. Antibody levels and the protection conferred were assessed in a sample of infants who completed 6-12 months of age in relation to their age and sex. Antibody levels were compared between different age and sex groups using the Mann-Whitney U-test, and correlations of antibody titers were performed using the Spearman correlation test. RESULTS: The prevalence rates of seropositivity for measles, mumps, and rubella were 91.5%, 89%, and 88%, respectively, in mothers, and 95%, 91.5%, and 93%, respectively, in their newborns. The newborns had mean IgG levels exceeding those of the mothers (P < 0.001). Mothers with natural infections had higher antibody levels compared to vaccinated mothers, which resulted in a higher level of maternal transfer. All of the infants who were 9-10 months of age or older were seronegative for measles, all of those who were 10-11 months of age or older were seronegative for rubella, and all of those who were 11-12 months old were seronegative for mumps. CONCLUSIONS: The maternal transfer of antibodies to newborns is efficient and renders protection until the infants are 6-7 months old in the case of mumps and rubella and 7-8 months old in the case of measles. Hence infants remain vulnerable to infections before the first dose of the MMR vaccine.

Ion Transport Modulators Differentially Modulate Inflammatory Responses in THP-1-Derived Macrophages.

Ion transport modulators are most commonly used to treat various noncommunicable diseases including diabetes and hypertension. They are also known to bind to receptors on various immune cells, but the immunomodulatory properties of most ion transport modulators have not been fully elucidated. We assessed the effects of thirteen FDA-approved ion transport modulators, namely, ambroxol HCl, amiloride HCl, diazoxide, digoxin, furosemide, hydrochlorothiazide, metformin, omeprazole, pantoprazole, phenytoin, verapamil, drug X, and drug Y on superoxide production, nitric oxide production, and cytokine expression by THP-1-derived macrophages that had been stimulated with ethanol-inactivated Mycobacterium bovis BCG. Ambroxol HCl, diazoxide, digoxin, furosemide, hydrochlorothiazide, metformin, pantoprazole, phenytoin, verapamil, and drug Y had an inhibitory effect on nitric oxide production, while all the test drugs had an inhibitory effect on superoxide production. Amiloride HCl, diazoxide, digoxin, furosemide, phenytoin, verapamil, drug X, and drug Y enhanced the expression of IL-1ß and TNF-α. Unlike most immunomodulatory compounds currently in clinical use, most of the test drugs inhibited some inflammatory processes while promoting others. Ion pumps and ion channels could therefore serve as targets for more selective immunomodulatory agents which do not cause overt immunosuppression.

Anti-inflammatory effect of leaves of Vernonia zeylanica in lipopolysaccharide-stimulated RAW 264.7 macrophages and carrageenan-induced rat paw-edema model.

ETHNOPHARMACOLOGICAL RELEVANCE: Vernonia zeylanica (L.) Less (Family: Compositae) is a medicinal plant used as external applications for boils, bone fractures, eczema and internally for asthma in traditional medicine in Sri Lanka. Anti-nociceptive, anti-bacterial and anti-proliferative activities have been reported previously. AIM OF THE STUDY: To investigate the anti-inflammatory activity of methanol/dichloromethane extract (MDE) of leaves of V. zeylanica by assessing in vivo inhibition of rat paw-edema, in vitro inhibition of the production of nitric oxide (NO) and superoxide and inhibitory effect on inducible nitric oxide synthase (iNOS) gene expression. MATERIALS AND METHODS: In vivo anti-inflammatory activity of MDE was tested at the dose of 1500 mg/kg using rat paw-edema model. Indomethacin and Gum acacia was used as the positive and vehicle control respectively. In vitro NO inhibitory activity of 7.8-250 µg/ml MDE was tested using lipopolysaccharide (LPS)-stimulated (1 µg/ml) mouse macrophages (RAW264.7 cells) and rat peritoneal cells (RPC) obtained following carrageenan-induction (5 mg/Kg). Griess method was used to quantify the nitrite levels in culture supernatants. In vitro inhibition of superoxide production of Phorbal 12-myristate 13-acetate (PMA)-stimulated RAW cells was determined by quantitative Nitroblue Tetrazolium (NBT) assay. N-monomethyl-L-arginine acetate (NMMA) (1 mM) and Diphenyleneiodonium chloride (DPI) (10 µM) were used as the positive controls for inhibitory activity of NO and superoxide production respectively. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was carried out to test the inhibitory effect on mRNA expression of iNOS gene. RESULTS: Treatment with MDE of V. zeylanica at 1500 mg/kg showed significant inhibition of paw-edema from 1st-5th hour (P < 0.01) compared with the control. The reference drug, indomethacin showed a biphasic pattern and its highest inhibition was (98.3 ± 7.1%) at 4th h (P < 0.01). MDE of V. zeylanica showed similar inhibition of paw-edema with highest inhibition recorded as 94.5 ± 5.28%, at 5th h (P < 0.01). The inhibitory concentration (IC50) of MDE for in vitro NO inhibitory activity was 105 µg/ml for RAW cells and 80 µg/ml for RPCs. Both NO inhibitory activities showed significant dose-dependency (r = 0.998 and r = 0.915 respectively; p < 0.05). MDE concentration of 250 µg/ml showed 55% inhibition of ROS production in RAW cells. NMMA showed 78% and 70.1% inhibition of NO production with RAW cells and RPCs whereas DPI showed 61% superoxide inhibitory activity with RAW cells. NO inhibitory activity of MDE on RAW cells was confirmed by the significant reduction (99.1%) in iNOS gene expression. CONCLUSION: These results demonstrated potent anti-inflammatory activity of MDE of V. zeylanica reflected by its significant in vivo inhibition of rat paw-edema, in vitro inhibition of NO and superoxide production, and the reduction of iNOS gene expression. Thus, further purification and isolation of bioactive compounds from V. zeylanica are emphasized.

Development of in-house ELISAs as an alternative method for the serodiagnosis of leptospirosis.

BACKGROUND: Leptospirosis is most often diagnosed clinically, and a laboratory test with high diagnostic accuracy is required. METHODS: IgM and IgG ELISAs using Leptospira antigens were established and evaluated in relation to the microscopic agglutination test (MAT). Antigen preparation consisted of saprophytic Leptospira biflexa to detect genus-specific antibodies (genus-specific ELISA) and a pool of the five most prevalent Leptospira interrogans serovars in Sri Lanka to detect serovar-specific antibodies (serovar-specific ELISA). IgM and IgG immune responses were studied in severe and mild leptospirosis patients (n = 100 in each group). RESULTS: The ELISAs showed high repeatability and reproducibility. The serovar-specific IgM-ELISA showed a sensitivity of 80.2% and specificity of 89%; the genus-specific IgM-ELISA showed a sensitivity of 83.3% and specificity of 91%. The serovar- and genus-specific IgG-ELISAs showed sensitivities of 73.3% and 81.7%, respectively, and specificities of 83.3% and 83.3%, respectively. The commercial IgM-ELISA showed a sensitivity of 79.2% and specificity of 93%. The commercial IgG-ELISA showed a sensitivity of 50% and specificity of 96.7%. IgM levels observed in mild and severe leptospirosis patients were significantly higher than in the healthy control group, with mean absorbance values of 0.770, 0.778, and 0.163, respectively. Severe leptospirosis patients had significantly higher mean anti-leptospiral IgG levels compared to both mild leptospirosis patients and healthy control group subjects (0.643, 0.358, and 0.116, respectively; ANOVA, p < 0.001). The presence of anti-leptospiral IgG above an optical density of 0.643 at 1:100 could predict a high risk of severe disease. CONCLUSION: The serovar-specific in-house ELISA could be used for the laboratory diagnosis of leptospirosis in endemic settings. The high levels of anti-leptospiral IgG observed suggest its value as a predictor of disease severity.

Hemostatic derangement in leptospirosis: A prospective cross-sectional study.

CONTEXT: Coagulation abnormalities have been observed among leptospirosis patients. However, coagulopathy in severe leptospirosis has not been further characterized. AIMS: The aim of this study was to evaluate conventional coagulation and rotational thromboelastometry (ROTEM®) parameters in leptospirosis patients. SETTINGS AND DESIGN: This prospective cross-sectional comparative study included patients presenting to a tertiary hospital in Sri Lanka with clinically and serologically confirmed leptospirosis (14 severe and 6 mild), dengue (6), sepsis (5), and 6 healthy individuals. SUBJECTS AND METHODS: Blood samples were collected between the 3rd and 10th days of illness for prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), fibrinogen, lupus anticoagulant, factors VII and VIII, D-dimer, platelet count, and ROTEM. STATISTICAL ANALYSIS USED: ANOVA post hoc comparison using Bonferroni was applied to compare groups. RESULTS: PT and aPTT were prolonged in leptospirosis patients and were corrected with normal plasma. TT was not significantly prolonged in leptospirosis. Fibrinogen was significantly elevated in severe leptospirosis (P = 0.001) and sepsis (P = 0.001) compared with healthy controls and dengue. Thirty percent of leptospirosis patients had thrombocytopenia (17% in mild and 36% in severe). No significant differences were seen in inTEM clotting time (CT) and exTEM CT in leptospirosis when compared to the other three groups. inTEM clot formation time (CFT) and exTEM CFT in dengue were significantly higher compared to severe (P = 0.001) and mild (P = 0.005) leptospirosis. inTEM maximum clot firmness (MCF) (P = 0.001) and exTEM MCF (P = 0.001) were significantly lower in dengue than in leptospirosis. Only one patient with leptospirosis had bleeding manifestations. CONCLUSIONS: Abnormalities in conventional coagulation parameters occur in leptospirosis. However, ROTEM parameters in leptospirosis are not significantly altered.

Ion Transport Modulators as Antimycobacterial Agents.

There is an urgent need for better and safer therapeutic interventions for tuberculosis (TB). We assessed the effects of FDA-approved ion transport modulators, namely, ambroxol HCl, amiloride HCl, diazoxide, digoxin, furosemide, hydrochlorothiazide (HCTZ), metformin, omeprazole, pantoprazole, phenytoin, verapamil, and drug X and Y on the growth of free and intracellular Mycobacterium bovis BCG. Free and intracellular M. bovis BCG were cultured in the presence or absence of the test drugs for 3 to 9 days and then quantified. For both free and intracellular bacteria, cultures that were exposed to furosemide, phenytoin, or drug Y yielded lower bacteria counts compared to drug-free controls (p < 0.05). The same was observed with diazoxide, HCTZ, verapamil, and drug X, but only for intracellular M. bovis BCG (p < 0.05). To assess the effects of the drugs on bactericidal activity of rifampicin, free and intracellular M. bovis BCG were treated with rifampicin alone or in combination with each of the thirteen test drugs for 3 to 9 days. For extracellular bacteria, higher bacteria clearance rates were observed in cultures exposed to rifampicin in combination with amiloride HCl, diazoxide, digoxin, furosemide, HCTZ, metformin, pantoprazole, phenytoin, drug X, or drug Y than those exposed to rifampicin alone, indicating that rifampicin had a synergistic effect with these test drugs. Rifampicin was also synergistic with ambroxol HCl, diazoxide, digoxin, furosemide, HCTZ, omeprazole, pantoprazole, phenytoin, verapamil, and drug X against intracellular M. bovis BCG. The antimycobacterial properties exhibited by the ion transport modulators in this study make them viable candidates as adjuncts to the current anti-TB regimens.

Immunomodulatory Activity of a Traditional Sri Lankan Concoction of Coriandrum sativum L. and Coscinium fenestratum G.

OBJECTIVE: To investigate the immunomodulatory activity of a traditional Sri Lankan concoction of Coriandrum sativum L. and Coscinium fenestratum (Gaertn.) Colebr., which is a Sri Lankan traditional medicine used to relieve inflammation and cold. METHODS: In vivo anti-inflammatory activity was tested using carrageenan-induced rat paw-edema model. Mechanism of anti-inflammatory activity was assessed by investigating the production of nitric oxide (NO), expression of iNOS enzyme, and reactive oxygen species (ROS) by rat peritoneal cells. The membrane stabilizing activity was also tested. The antibody response was determined by assessing the specific haemagglutination antibodies raised against sheep red blood cells. RESULTS: The three doses of freeze-dried concoction used ((human equivalent dose (HED)-183 mg/kg) 2 × HED and 1/2HED; n = 6 rats/group) showed significant inhibition of paw edema compared to water control at 3rd-5th hours (p < 0.05). Both HED and 1/2HED exhibited marked anti-inflammatory activity (72-83% inhibition at 4th-5th hours; p < 0.05). The HED of the concoction showed significant inhibition of NO (77.5 ± 0.73%, p < 0.001) and ROS production (26.9 ± 2.55%; p < 0.01) by rat peritoneal cells. Inhibition of NO production in the concoction treated rat peritoneal cells was confirmed by the lack of iNOS expression. The concoction also exhibited significant membrane stabilizing activity (IC50 = 0.0006 µg/ml; p = 0.001). HED resulted in a significantly high induction of specific antibody production against SRBC antigens as detected by SRBC haemagglutination assay (mean day 14 titers 253.3 compared to control: 66.7) (p < 0.01). CONCLUSIONS: The traditional Sri Lankan concoction of C. sativum and C. fenestratum demonstrated potent in vivo anti-inflammatory activity, significant reduction of ROS, and NO production by rat peritoneal cells and the lack of iNOS expression confirmed the low NO production. The increased membrane stability also supports the anti-inflammatory activity of the concoction. Further, this concoction induced a significantly high antibody response reflecting its immunostimulatory activity. Together these results scientifically validate the therapeutic use of the concoction of C. sativum and C. fenestratum in Sri Lankan traditional medicinal system for immunomodulatory effects.

Differences in serum markers of oxidative stress in well controlled and poorly controlled asthma in Sri Lankan children: a pilot study.

BACKGROUND: Asthma is a disease characterised by hyper responsiveness and bronchoconstriction of airways, and is a major health burden globally. A dysfunction of the oxidant-antioxidant balance, termed oxidative stress, has been implicated in the pathophysiology of asthma. The present study aims to assess the changes in oxidative stress markers, namely nitric oxide metabolites and antioxidant capacity, in children with poorly controlled and well controlled asthma, in comparison to healthy controls. METHODS: The present study enrolled 72 children (ages 5-15 years) classified into three groups: (1) poorly controlled asthma (n = 20), (2) well controlled asthma (n = 24) and (3) healthy controls (n = 27). An interviewer-administered questionnaire was used to record socio-demographic data of the participants. The serum concentrations of the oxidant markers (nitrite, nitrate and total nitric oxide metabolites [NOx]) were determined using the Griess test, and the total antioxidant capacity (TAOC) was determined using the ABTS decolorisation method. The concentrations of these markers were compared across the three groups. RESULTS: The three study groups were similar in terms of socio-demographic data. The differences across the three groups were statistically significant for serum concentrations of nitrate and NOx (but not nitrite) and serum TAOC. Further analyses showed that the disparity for nitrate and NOx concentrations was greatest between poorly controlled asthma and healthy controls (p = 0.001 and p < 0.001) compared to the well-controlled asthmatics and healthy controls (p = 0.036 and p = 0.049). A significant difference in serum nitrate and NOx concentrations was not observed between the two asthma groups (p = 0.311 and 0.203). The TAOC were significantly lower in poorly controlled asthmatics as compared to well-controlled asthmatics (p = 0.003) and healthy controls (p < 0.001). However, there was no significant difference in the serum TAOC between healthy controls and well-controlled asthmatics (p = 0.496). These findings may indicate that it is perhaps the higher TAOC that contributes to the well controlled state of asthma. CONCLUSIONS: The present study indicated that an imbalance of oxidants and antioxidants in the serum may have an underlying role in asthma pathophysiology, and how these markers may be effective in asthma management.