Shiroma Handunnetti

Features of recrudescent chloroquine-resistant Plasmodium falciparum infections confer a survival advantage on parasites and have implications for disease control

This paper reports on the features of recrudescent infections of chloroquine-resistant Plasmodium falciparum (CQRPf) malaria from a study in vivo of patients from a malaria endemic (n = 527) and non-endemic (n = 129) region of Sri Lanka where the incidence of RI resistance was 30% and 55%, respectively. In both groups of patients, the recrudescent infections which emerged after treatment of the primary infection with chloroquine (CQ) and primaquine had significantly lower peripheral parasitaemia (0.036% and 0.108% in endemic and non-endemic patients, respectively) compared to their primary infections (mean parasitaemia 0.13% and 0.49%; P = 0.021 and 0.002, respectively). The recrudescences of CQ resistant infections also gave rise to clinical disease of markedly reduced severity (average clinical scores of 10.1 and 8.2) compared to their primary infections (average clinical scores of 12.4 and 12.3; P = 0.003 and 0.001, respectively, in endemic and non-endemic patients). CQ resistant recrudescent infections therefore had a lower probability of being diagnosed and treated. In endemic patients, a higher proportion of CQRPf infections (57%) had gametocytaemia compared to the chloroquine sensitive ones (29%) (P = 0.014, chi 2 = 5.96) and were significantly more infective to mosquitoes (P = 0.047). these findings imply that, in areas where CQ resistance is prevalent, the continued use of the drug may confer a survival and propagation advantage on resistant parasites and favour the rapid expansion of their reservoir. In support of this, we also present epidemiological evidence showing that, in endemic areas, the proportion of P. falciparum patients carrying gametocytes has increased significantly since the emergence of chloroquine resistance. These findings are relevant to the management of drug resistance and malaria control in countries where P.falciparum is only partially resistant to CQ.

Anti-inflammatory activity of Trichosanthes cucumerina Linn. in rats

AIM OF THE STUDY Trichosanthes cucumerina Linn. (Family: Cucurbitaceae) is one of the medicinal plants that is often used in Sri Lankan traditional systems of medicine. One of its uses is the treatment of inflammatory conditions. However, validity of the anti-inflammatory activity has not been scientifically investigated so far. Therefore, the aim of this study was to investigate the anti-inflammatory potential of Trichosanthes cucumerina hot water extract (HWE) and its fractions. MATERIALS AND METHODS The anti-inflammatory activity of Trichosanthes cucumerina was evaluated by use of the carrageenan-induced paw oedema model in Wistar rats. In addition, the mechanism/s by which Trichosanthes cucumerina is mediated the anti-inflammatory activity was assessed by determining its effects on (a) membrane stabilizing activity and (b) nitric oxide inhibitory activity. RESULTS Apart from the lowest dose of the HWE, other tested doses (500, 750, 1000 mg/kg) produced a significant (P ≤ 0.05) inhibition of the inflammation, most pronounced at 5h after the injection of carrageenan. The anti-inflammatory effect induced by 750 mg/kg, was comparable to that of the reference drug, indomethacin at 4 and 5h. Inhibition of nitric oxide (NO) production and membrane stabilization activities are probable mechanisms by which Trichosanthes cucumerina mediates its anti-inflammatory actions. Among the tested fractions, methanol fraction (MEF) and aqueous fraction (AQF) at a dose of 75 mg/kg exhibited marked inhibition against carrageenan-induced hind paw oedema. The anti-inflammatory effect induced by MEF, was comparable to that of the reference drug, indomethacin and as well as to the 750 mg/kg of HWE at 4 and 5h. CONCLUSIONS (a) These findings rationalize the traditional usage of this plant as an anti-inflammatory agent and (b) membrane stabilizing properties and NO inhibitory activity are possible mechanisms through which Trichosanthes cucumerina mediates its anti-inflammatory action.

Natural Human Antibody Responses to Plasmodium vivax Apical Membrane Antigen 1 under Low Transmission and Unstable Malaria Conditions in Sri Lanka

ABSTRACT Plasmodium vivax apical membrane antigen 1, an important malaria vaccine candidate, was immunogenic during natural malaria infections in Sri Lanka, where low transmission and unstable malaria conditions prevail. Antibody prevalence increased with exposure in areas where malaria was or was not endemic. A marked isotype switch to cytophilic (immunoglobulin G1 [IgG1]/IgG3) antibodies was evident with increasing exposure exclusively in residents from areas of endemicity.

Diagnosis of Leptospirosis: Comparison between Microscopic Agglutination Test, IgM-ELISA and IgM Rapid Immunochromatography Test

Background Leptospirosis is diagnosed on clinical grounds, and confirmed by microscopic agglutination test (MAT). IgM-ELISA (Serion-Virion) and immunochromatography test (Leptocheck-WB) are two immunodiagnostic assays for leptospirosis. Their sensitivity, specificity and applicability in Sri Lanka have not been systematically evaluated. Methods Clinically diagnosed leptospirosis patients (n = 919) were recruited from three hospitals in the Western Province of Sri Lanka, during June 2012 to December 2013. MAT, IgM-ELISA and Leptocheck-WB were performed on all patient sera. MAT titer of ≥400 in single sample, four-fold rise or seroconversion ≥100 in paired samples were considered as positive for MAT. For diagnostic confirmation, MAT was performed during both acute and convalescent phases. Anti-leptospiral IgM ≥20 IU/ml and appearance of a band in the test window were considered as positive for IgM-ELISA and Leptocheck-WB test respectively. Patients with an alternative diagnosis (n = 31) were excluded. Data analysis was performed using two methods, i) considering MAT as reference standard and ii) using Bayesian latent class model analysis (BLCM) which considers each test as imperfect. Results MAT, IgM-ELISA and Leptocheck-WB positivity were 39.8%, 45.8% and 38.7% respectively during the acute phase. Acute-phase MAT had specificity and sensitivity of 95.7% and 55.3% respectively, when compared to overall MAT positivity. IgM-ELISA and Leptocheck-WB had similar diagnostic sensitivity when compared with acute-phase MAT as the gold standard, although IgM-ELISA showed higher specificity (84.5%) than Leptocheck-WB (73.3%). BLCM analysis showed that IgM-ELISA and Leptocheck-WB had similar sensitivities (86.0% and 87.4%), while acute-phase MAT had the lowest sensitivity (77.4%). However, acute-phase MAT had high specificity (97.6%), while IgM-ELISA and Leptocheck-WB showed similar but lower specificity (84.5% and 82.9%). Conclusions Both IgM-ELISA and Leptocheck-WB shows similar sensitivities and specificities. IgM-ELISA may be superior to MAT during the acute phase and suitable for early diagnosis of leptospirosis. Leptocheck-WB is suitable as a rapid immunodiagnostic screening test for resource limited settings.

Changes in full blood count parameters in leptospirosis: a prospective study

Background Leptospirosis presents diagnostic challenges to clinicians, in settings where other acute febrile illness are prevalent. The patterns of serial changes in haematological parameters in leptospirosis has not been evaluated previously. Methods Clinical and laboratory data were collected prospectively from patients with leptospirosis in two hospitals in Sri Lanka. Leptospirosis was diagnosed based on WHO clinical criteria with confirmation using Microscopic Agglutination Test titre > 400 or 4 fold rise between acute and convalescent samples. Full blood count parameters were analysed up to the 14th day of illness. Results Data from 201 patients with leptospirosis were available. Leukocyte counts and absolute neutrophil counts showed a decline over the first 5 days of illness, then rose until the end of the second week. On day 3 of fever, the majority (75%) had normal leukocyte counts, and by day 5, leukocytosis was seen only in 38.1%; leucopenia was an uncommon finding. Lymphopenia was seen in over half on day 5, declining to just under a quarter of patients by day 10. Platelets declined over the first 6 days and then gradually rose. Thrombocytopenia was seen in nearly three-fourths of patients by day 5. Haemoglobin and haematocrit levels declined over the course of illness. Total white cell and neutrophil counts were higher, and haemoglobin and haematorcrit were significantly lower, in patients with severe disease. Conclusions Neither leukocytosis nor lymphopenia were prominent features, while thrombocytopenia was seen during the 3rd to 5th day of illness, with dropping haemoglobin levels. Neutrophilia and low haemoglobin levels appear to predict severe disease. These findings may be of use to clinicians in differentiating leptospirosis from other acute infections like dengue, and could help in predicting severe leptospirosis.

Lineage-specific positive selection at the merozoite surface protein 1 (msp1) locus of Plasmodium vivax and related simian malaria parasites

BackgroundThe 200 kDa merozoite surface protein 1 (MSP-1) of malaria parasites, a strong vaccine candidate, plays a key role during erythrocyte invasion and is a target of host protective immune response. Plasmodium vivax, the most widespread human malaria parasite, is closely related to parasites that infect Asian Old World monkeys, and has been considered to have become a parasite of man by host switch from a macaque malaria parasite. Several Asian monkey parasites have a range of natural hosts. The same parasite species shows different disease manifestations among host species. This suggests that host immune responses to P. vivax-related malaria parasites greatly differ among host species (albeit other factors). It is thus tempting to invoke that a major immune target parasite protein such as MSP-1 underwent unique evolution, depending on parasite species that exhibit difference in host range and host specificity.ResultsWe performed comparative phylogenetic and population genetic analyses of the gene encoding MSP-1 (msp1) from P. vivax and nine P. vivax-related simian malaria parasites. The inferred phylogenetic tree of msp1 significantly differed from that of the mitochondrial genome, with a striking displacement of P. vivax from a position close to P. cynomolgi in the mitochondrial genome tree to an outlier of Asian monkey parasites. Importantly, positive selection was inferred for two ancestral branches, one leading to P. inui and P. hylobati and the other leading to P. vivax, P. fieldi and P. cynomolgi. This ancestral positive selection was estimated to have occurred three to six million years ago, coinciding with the period of radiation of Asian macaques. Comparisons of msp1 polymorphisms between P. vivax, P. inui and P. cynomolgi revealed that while some positively selected amino acid sites or regions are shared by these parasites, amino acid changes greatly differ, suggesting that diversifying selection is acting species-specifically on msp1.ConclusionsThe present results indicate that the msp1 locus of P. vivax and related parasite species has lineage-specific unique evolutionary history with positive selection. P. vivax and related simian malaria parasites offer an interesting system toward understanding host species-dependent adaptive evolution of immune-target surface antigen genes such as msp1.

The characterization of two monoclonal antibodies which react with high molecular weight antigens of asexual Plasmodium falciparum

We prepared rat monoclonal antibodies (mAb) specific for very large Plasmodium falciparum proteins to assist in their characterization. Hybridomas prepared from rats immunized with parasitized erythrocyte (PE) proteins of greater than 200 kDa exhibited two patterns of Western blot reactivity with PE SDS extracts: one represented by clone 41E11 (IgM, kappa), the other by clone 12C11 (IgM, lambda). MAb 41E11 reacted by Western blotting with at least 15 antigens, most of which comigrated with antigens identified by the 33G2 human IgM mAb. The stage specificity of mAb 41E11 reactivity and indirect immunofluorescence (IFA) pattern closely resemble those previously described for antigens that share the EEXXEE sequence motif. Unlike mAb 33G2, MAb 41E11 immunoprecipitated a biosynthetically radiolabeled protein of 320 kDa. MAb 41E11 did not immunoprecipitate any cell surface 125I proteins. MAb 12C11 reacted on Western blotting with a different group of malarial antigens of approximately 44, 95, 117, 145, and 310 kDa, as well as with some low-molecular-weight, uninfected erythrocyte antigens. MAb 12C11 did not immunoprecipitate any cell surface 125I or biosynthetically labeled proteins. The 310-kDa antigen recognized by mAb 12C11 (denoted Ag 12A) does not correspond to PfEMP2 or the 320-kDa antigen recognized by mAbs 33G2 or 41E11. With trophozoites and more mature stages, fixed IFA reactivity of mAb 12C11 was at the parasite and in antigen aggregates in the host cell cytoplasm that extended to the PE plasma membrane. Indirect results suggest that Ag 12A does not correspond to cell surface-exposed PfEMP1 and is most likely a hitherto unidentified malarial protein.

Current immunological and molecular tools for leptospirosis: diagnostics, vaccine design, and biomarkers for predicting severity

Leptospirosis is a zoonotic spirochaetal illness that is endemic in many tropical countries. The research base on leptospirosis is not as strong as other tropical infections such as malaria. However, it is a lethal infection that can attack many vital organs in its severe form, leading to multi-organ dysfunction syndrome and death. There are many gaps in knowledge regarding the pathophysiology of leptospirosis and the role of host immunity in causing symptoms. This hinders essential steps in combating disease, such as developing a potential vaccine. Another major problem with leptospirosis is the lack of an easy to perform, accurate diagnostic tests. Many clinicians in resource limited settings resort to clinical judgment in diagnosing leptospirosis. This is unfortunate, as many other diseases such as dengue, hanta virus, rickettsial infections, and even severe bacterial sepsis, can mimic leptospirosis. Another interesting problem is the prediction of disease severity at the onset of the illness. The majority of patients recover from leptospirosis with only a mild febrile illness, while a few others have severe illness with multi-organ failure. Clinical features are poor predictors of potential severity of infection, and therefore the search is on for potential biomarkers that can serve as early warnings for severe disease. This review concentrates on these three important aspects of this neglected tropical disease: diagnostics, developing a vaccine, and potential biomarkers to predict disease severity.

Low serum total nitrite and nitrate levels in severe leptospirosis

BackgroundThe relationship between inducible nitric oxide synthatase activity and disease severity in leptospirosis is unclear. Nitric oxide is converted to nitrites and nitrates, thus nitrite and nitrate levels (NOx) in serum are considered surrogate markers for nitric oxide. NOx are excreted through the kidneys, and elimination is diminished in renal impairment. We assessed the correlation of NOx with disease severity in patients with leptospirosis, compared with healthy controls and non-leptospirosis fever patients.MethodsAll patients admitted over a two-month period to the National Hospital, Colombo, Sri Lanka with a clinical picture suggestive of leptospirosis were included. Leptospirosis was confirmed by the microscopic agglutination test (titre≥400). Severe leptospirosis was defined by the presence of two or more of the following criteria: jaundice (bilirubin> 51.3 μmol/l), oliguria (urine output < 400 ml/day), serum creatinine> 133 μmol/l or blood urea > 25.5 mmol/l, or the presence of organ dysfunction. Non-leptospirosis fever patients and healthy volunteers were used as control groups. NOx levels were measured using a modified Griess reaction.ResultsForty patients were confirmed as having leptospirosis and 26 of them had severe disease. NOx levels were significantly higher in confirmed leptospirosis patients compared to healthy controls, MAT equivocal patients and non-leptospirosis fever patients (p<0.001). NOx concentrations were also significantly higher in patients with severe compared to mild leptospirosis (p<0.001). Once NOx levels were corrected for renal function, by using the ratio NOx/creatinine, NOx levels were actually significantly lower in patients with severe disease compared to other patients, and values were similar to those of healthy controls.ConclusionsWe postulate that high NOx levels may be protective against severe leptospirosis, and that finding low NOx levels (when corrected for renal function) in patients with leptospirosis may predict the development of severe disease and organ dysfunction.

Serum nitrite levels in Sri Lankan patients with leptospirosis

OBJECTIVE To determine whether blood nitrite levels are elevated in patients with leptospirosis. METHODS Male patients fulfilling clinical and epidemiological criteria for a diagnosis of leptospirosis were recruited. Those with MAT titre of ≤400 together with those seroconverting to a titer of ≤200 were included in the analysis. Serum nitrite levels were measured in these patients and age, sex matched healthy controls. RESULTS Patients from 3 hospitals (n=75) were screened during a 3 month period from 28th June to 3rd September 2009, of whom 20 were eligible for the study. Serum nitrite levels were found to be significantly higher in patients with acute leptospirosis [n=20, (0.359±0.229)μ M] compared to controls [(n=13,(0.216±0.051)μ M](P=0.014). A significant correlation was also observed between the MAT titre and the day of illness (r = 0.547; P<0.0001). CONCLUSIONS Serum nitrite levels are higher in patients with acute leptospirosis compared to age and sex matched controls. No correlation could be assessed with severity of illness, as sample size was inadequate to determine this.