Toll-like receptor 2 (TLR2) plays a role in controlling cutaneous leishmaniasis in vivo, but does not require activation by parasite lipophosphoglycan.

BACKGROUND: Leishmaniasis is a neglected tropical disease affecting millions of individuals worldwide. Despite several studies reporting involvement of the innate immune receptor Toll-like receptor 2 (TLR2) in the recognition of surface glycolipids from Leishmania parasites in vitro, the role of TLR2 and its co-receptors during cutaneous leishmaniasis infection in vivo is unknown. METHODS: To explore the role of TLR2 and its co-receptors in cutaneous leishmaniasis, mice deficient in either TLR2, 4, 1 or 6, or wild-type (WT) controls, were infected with either Leishmania major promastigotes, L. mexicana promastigotes, L. mexicana amastigotes, or LPG1 -/- L. mexicana promastigotes. For each infection, lesion sizes were monitored and parasite burden was assessed at various time points. To assess immune responses, draining lymph node (DLN) cells were re-stimulated with parasite antigens and the production of cytokines and parasite-specific antibody isotypes in blood was determined by ELISA. RESULTS: Mice deficient in TLR2 and TLR4 presented with larger lesions and higher parasite burdens than WT controls. Mice lacking TLR2 co-receptors TLR1 or TLR6 did not show exacerbated infection, suggesting that TLR2 does not require either co-receptor in the recognition of Leishmania infection. Furthermore, it appears that lipophosphoglycan (LPG) is not the major mediator of TLR2 activation during infection with L. mexicana, as parasites lacking LPG (axenic amastigotes and LPG1 -/- promastigotes) also resulted in exacerbated disease in TLR2-/- mice. Infected TLR2-/- mice show a skewed Th2 immune response to Leishmania parasites, as demonstrated by elevated IL-4, IL-13 and IL-10 production by DLN cells from L. mexicana infected mice in response to antigen. Furthermore, L. major infected TLR2-/- mice have elevated antigen-specific IgG1 antibodies. CONCLUSIONS: TLR2 deficiency leads to exacerbation of disease and parasite burden through promotion of Th2 immunity. TLR2 activation in vivo occurs independently of parasite LPG, suggesting other parasite ligands are involved in TLR2 recognition of Leishmania.

Effect of Khat (Catha edulis) Use on the Bioavailability, Plasma Levels and Antimalarial Activity of Chloroquine.

OBJECTIVES: This study aimed to evaluate the effect of khat (Catha edulis) on chloroquine (CQ) bioavailability in healthy Yemeni adults and its effect on CQ plasma levels and parasite clearance among malaria patients. METHODS: This study took place between January and April 2007 in Bajil and Sana'a, Yemen. Two CQ doses (600 mg each) were given to 15 healthy males on separate occasions; the first dose was followed by a khat-chewing session (phase one) while controls abstained from khat-chewing for the second (phase two). Additionally, 103 patients with Plasmodium falciparum-induced malaria, including both regular khat chewers (n = 57) and non-khat chewers (n = 46), were treated with CQ (25 mg/kg) over three days. Pharmacokinetic parameters were analysed among both controls and malaria patients. Parasite clearance was also investigated for the latter group. RESULTS: The mean area under the concentration-time curve (AUC) was 2,108.9 versus 2,797.4 ng/hour/mL, mean peak plasma concentration (Cmax) was 415.6 versus 508.7 ng/mL and mean time to reach Cmax was 3.8 versus 3.6 hours for controls in phase one versus phase two, respectively; both AUC and Cmax levels were significantly reduced by khat-chewing (P <0.050). For khat- versus non-khat-chewing malaria patients, mean plasma CQ concentrations were 266.4 ng/mL versus 427.5 ng/mL (P <0.001). Furthermore, CQ was effective in 71.7% and 75.4% of non-khat and khat-chewing malaria patients, respectively (P = 0.823). CONCLUSION: Khat-chewing was found to significantly reduce plasma CQ levels among healthy volunteers and malaria patients. While receiving CQ treatment, patients should be advised not to chew khat.

Molecular markers of anti-malarial drug resistance in Lahj Governorate, Yemen: baseline data and implications.

BACKGROUND: This is an investigation of anti-malarial molecular markers coupled with a therapeutic efficacy test of chloroquine (CQ) against falciparum malaria in an area of unstable malaria in Lahj Governorate, Yemen. The study was aimed at assessment of therapeutic response to CQ and elucidation of baseline information on molecular markers for Plasmodium falciparum resistance against CQ and sulphadoxine/pyrimethamine (SP). METHODS: Between 2002 and 2003 the field test was conducted according to the standard WHO protocol to evaluate the therapeutic efficacy of CQ in 124 patients with falciparum malaria in an endemic area in Lahj Governorate in Yemen. Blood samples collected during this study were analysed for P. falciparum chloroquine resistance transporter gene (pfcrt)-76 polymorphisms, mutation pfcrt-S163R and the antifolate resistance-associated mutations dihydrofolate reductase (dhfr)-C59R and dihydropteroate synthase (dhps)-K540E. Direct DNA sequencing of the pfcrt gene from three representative field samples was carried out after DNA amplification of the 13 exons of the pfcrt gene. RESULTS: Treatment failure was detected in 61% of the 122 cases that completed the 14-day follow-up. The prevalence of mutant pfcrt T76 was 98% in 112 amplified pre-treatment samples. The presence of pfcrt T76 was poorly predictive of in vivo CQ resistance (PPV = 61.8%, 95% CI = 52.7-70.9). The prevalence of dhfr Arg-59 mutation in 99 amplified samples was 5%, while the dhps Glu-540 was not detected in any of 119 amplified samples. Sequencing the pfcrt gene confirmed that Yemeni CQ resistant P. falciparum carry the old world (Asian and African) CQ resistant haplotype CVIETSESI at positions 72,73,74,75,76,220,271, 326 and 371. CONCLUSION: This is the first study to report baseline information on the characteristics and implications of anti-malarial drug resistance markers in Yemen. It is also the first report of the haplotype associated with CQR P. falciparum parasites from Yemen. Mutant pfcrtT76 is highly prevalent but it is a poor predictor of treatment failure in the study population. The prevalence of mutation dhfrArg59 is suggestive of emerging resistance to SP, which is currently a component of the recommended combination treatment of falciparum malaria in Yemen. More studies on these markers are recommended for surveillance of resistance in the study area.

Leishmania donovani and cutaneous leishmaniasis, Sri Lanka.

To investigate the relationship of cutaneous leishmaniasis isolates from Sri Lanka to known species, we performed DNA sequencing and microsatellite analyses. We identified Leishmania donovani as the agent of Sri Lanka cutaneous leishmaniasis and showed that these parasites are closely related to those causing visceral leishmaniasis in the Indian subcontinent.

Production and characterization of stable amphotericin-resistant amastigotes and promastigotes of Leishmania mexicana.

The sensitivities of Leishmania mexicana amastigote and promastigote forms to amphotericin B were investigated in vitro and found to be strongly influenced by the culture media used. When differences in culture media were minimized, there was no significant difference in the 50% inhibitory concentration values between the two life cycle stages. Stable amphotericin B-resistant amastigote and promastigote lines were produced by the application of increasing drug pressure to long-term cultures. Lines capable of growth in concentrations of amphotericin B lethal to normal parasites were produced. Compared to normal parasites, these amphotericin-resistant lines showed marked differences in membrane sterol compositions, with very high levels of 4,14,dimethyl-cholesta-8,24-dienol and other methyl sterols. They also showed a consistent morphological feature, the presence of multilamellar membrane-like material in the flagellar pocket, revealed by transmission electron microscopy. Amphotericin-resistant parasites were capable of infecting BALB/c mice, but the resulting lesion growth was slower than that after infection with normal parasites. However, unlike normal parasites, the amphotericin-resistant parasites were unaffected by experimental chemotherapy with amphotericin B. These results show that amphotericin B resistance could arise as a result of increased clinical use of amphotericin B therapy.