An estimated 2.3 million disability-adjusted life years are lost globally from leishmaniasis. In Peru's Amazon region, the department of Madre de Dios (MDD) rises above the rest of the country in terms of the annual incidence rates of human leishmaniasis. Leishmania (Viannia) braziliensis is the species most frequently responsible for the form of disease that results in tissue destruction of the nose and mouth. However, essentially nothing is known regarding the reservoirs of this vector-borne, zoonotic parasite in MDD. Wild rodents have been suspected, or proven, to be reservoirs of several Leishmania spp. in various ecosystems and countries. Additionally, people who live or work in forested terrain, especially those who are not regionally local and whose immune systems are thus naïve to the parasite, are at most risk for contracting L. (V.) braziliensis. Hence, the objective of this study was to collect tissues from wild rodents captured at several study sites along the Amazonian segment of the newly constructed Transoceanic Highway and to use molecular laboratory techniques to analyze samples for the presence of Leishmania parasites. Liver tissues were tested via polymerase chain reaction from a total of 217 rodents; bone marrow and skin biopsies (ear and tail) were also tested from a subset of these same animals. The most numerous rodent species captured and tested were Oligoryzomys microtis (40.7%), Hylaeamys perenensis (15.7%), and Proechimys spp. (12%). All samples were negative for Leishmania, implying that although incidental infections may occur, these abundant rodent species are unlikely to serve as primary reservoirs of L. (V.) braziliensis along the Transoceanic Highway in MDD. Therefore, although these rodent species may persist and even thrive in moderately altered landscapes, we did not find any evidence to suggest they pose a risk for L. (V.) braziliensis transmission to human inhabitants in this highly prevalent region.

Human African trypanosomiasis or sleeping sickness is a vector-borne parasitic disease that has a major impact on human health and welfare in sub-Saharan countries. Based mostly on data from animal models, it is currently thought that trypanosome entry into the brain occurs by initial infection of the choroid plexus and the circumventricular organs followed days to weeks later by entry into the brain parenchyma. However, Trypanosoma brucei bloodstream forms rapidly cross human brain microvascular endothelial cells in vitro and appear to be able to enter the murine brain without inflicting cerebral injury. Using a murine model and intravital brain imaging, we show that bloodstream forms of T. b. brucei and T. b. rhodesiense enter the brain parenchyma within hours, before a significant level of microvascular inflammation is detectable. Extravascular bloodstream forms were viable as indicated by motility and cell division, and remained detectable for at least 3 days post infection suggesting the potential for parasite survival in the brain parenchyma. Vascular inflammation, as reflected by leukocyte recruitment and emigration from cortical microvessels, became apparent only with increasing parasitemia at later stages of the infection, but was not associated with neurological signs. Extravascular trypanosomes were predominantly associated with postcapillary venules suggesting that early brain infection occurs by parasite passage across the neuroimmunological blood brain barrier. Thus, trypanosomes can invade the murine brain parenchyma during the early stages of the disease before meningoencephalitis is fully established. Whether individual trypanosomes can act alone or require the interaction from a quorum of parasites remains to be shown. The significance of these findings for disease development is now testable.

Background

A considerable challenge in quantification of the antimalarial piperaquine in plasma is carryover of analyte signal between assays. Current intensive pharmacokinetic studies often rely on the merging of venous and capillary sampling. Drug levels in capillary plasma may be different from those in venous plasma, Thus, correlation between capillary and venous drug levels needs to be established.

Methods

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to develop the method. Piperaquine was measured in 205 pairs of capillary and venous plasma samples collected simultaneously at ≥24hr post dose in children, pregnant women and non-pregnant women receiving dihydroartemisinin-piperaquine as malaria chemoprevention. Standard three-dose regimen over three days applied to all participants with three 40mg dihydroartemisinin/320mg PQ tablets per dose for adults and weight-based dose for children. Correlation analysis was performed using the program Stata® SE12.1. Linear regression models were built using concentrations or logarithm transformed concentrations and the final models were selected based on maximal coefficient of determination (R2) and visual check.

Results

An LC-MS/MS method was developed and validated, utilizing methanol as a protein precipitation agent, a Gemini C18 column (50x2.0mm, 5μm) eluted with basic mobile phase solvents (ammonium hydroxide as the additive), and ESI+ as the ion source. This method had a calibration range of 10-1000 ng/mL and carryover was negligible. Correlation analysis revealed a linear relationship: Ccap = 1.04×Cven+4.20 (R2 = 0.832) without transformation of data, and lnCcap = 1.01×lnCven+0.0125, (R2 = 0.945) with natural logarithm transformation. The mean ratio (±SD) of Ccap/Cven was 1.13±0.42, and median (IQR) was 1.08 (0.917, 1.33).

Conclusions

Capillary and venous plasma PQ measures are nearly identical overall, but not readily exchangeable due to large variation. Further correlation study accounting for disposition phases may be necessary.