A sensitive and reliable quantitative immunohistochemistry technique to evaluate the percentage of Trypanosoma cruzi-infected tissue area.

Quantification of parasites in the context of Chagas disease is required to monitor the treatment with benznidazole, disease-associated cardiomyopathies and graft rejection after heart transplantation. As parasitological exams lack sensitivity, Real Time Polymerase Chain Reaction (rt-PCR) has emerged to evaluate the parasite load in blood samples and cardiac biopsies. However, despite its higher sensitivity, rt-PCR does not provide information on the location and distribution of amastigote nests within infected tissues, the characterization of inflammatory infiltrates or changes to tissue architecture. On the contrary, a sensitive immunohistochemistry technique (IHC) could fill these gaps. In the present study, a quantitative IHC exam was standardized and validated by testing adipose and cardiac tissues of experimentally infected mice containing variable parasite load levels of T. cruzi assessed by a sensitive Sybr Green rt-PCR with kDNA primers. Tissues were divided into four groups according to the parasite load: group A- 100 parasites/50 ng of DNA; group B -10 parasites; group C - around 1 parasite and group D - less than 1 parasite/50 ng/DNA. IHC was able to detect T. cruzi in the four groups, even in group D tissues containing fractions of a single parasite/50 ng of DNA sample according to rt-PCR. In conclusion, a highly sensitivity and reliable quantitative immunohistochemistry technique was developed and is proposed to estimate the percentage of T. cruzi-infected tissue area in chagasic patients presenting with cardiomyopathies, as a complementary test to rt-PCR.

Quantification of Toxocara canis DNA by qPCR in mice inoculated with different infective doses.

The nematode Toxocara canis is of public health importance and is the main causative agent of toxocariasis in humans. This disease is difficult to diagnose due to several factors, including the possibility of cross-reactions with other nematodes in the ELISA. To overcome this problem, molecular tests have been recommended as an alternative to identify the parasite. The quantitative real-time polymerase chain reaction (qPCR) technique was used in this study to identify and quantify the parasite load of T. canis in the mouse brain. To this end, 24 mice were divided into six groups, five of which were challenged with different infective doses of T. canis larvae (L3) (1000, 500, 250, 100 and 50 larvae), while the sixth group, uninfected, acted as negative control. Forty-five days after infection, the animals were euthanized to collect the brain, from which two portions of 20 mg of tissue were taken for DNA extraction, while the rest of the brain tissue was digested to quantify the number of larvae by microscopy. The number of DNA copies was calculated from the standard DNA quantification curve, showing values of E = 93.4%, R2 = 0.9655 and Y = -3.415. A strong positive correlation (R = 0, 81; p < .001) was found between the number of copies and the recovery of larvae from brain. However, the parasite's DNA was also identified even in animals from whose brain no larvae were recovered after tissue digestion. The results of this study therefore confirm that the qPCR technique can be a valuable tool for the detection and quantification of T. canis DNA in murine hosts, even in animals whose with tissues contain very few parasites.

Evaluating the Role of Host AMPK in Leishmania Burden.

The study of host AMP-activated protein kinase (AMPK) activation during Leishmania infection imposes distinct types of techniques to measure protein expression and activation, as well as to quantify, at transcription and translational levels, its downstream targets. The investigation of host AMPK protein modulation during Leishmania infection should primarily be assessed during in vitro infections using as a host murine bone marrow-derived macrophages (BMMos). The infection outcome is assessed measuring the percentage of infected cells in the context of BMMos. To evaluate AMPK activity during infection, the expression of AMPK phosphorylated at Thr172 as well as the transcription and translational levels of its downstream targets are evaluated by quantitative PCR and immunoblotting. The modulation of AMPK activity in vivo is determined specifically in sorted splenic macrophages harboring Leishmania parasites recovered from infected mice using fluorescent-labeled parasites in the infectious inocolum. The modulation of AMPK activity was assessed by AMPK activators and inhibitors and also using AMPK, SIRT1, or LKB1 KO mice models. The infection outcome in BMMos and in vivo was further determined using these two different approaches. To finally understand the metabolic impact of AMPK during infection, in vitro metabolic assays in infected BMMos were measured in the bioenergetic profile using an extracellular flux analyzer.

A Comparative Study of Four Methods for the Detection of Nematode Eggs and Large Protozoan Cysts in Mandrill Faecal Material.

Coproscopical methods like sedimentation and flotation techniques are widely used in the field for studying simian gastrointestinal parasites. Four parasites of known zoonotic potential were studied in a free-ranging, non-provisioned population of mandrills (Mandrillus sphinx): 2 nematodes (Necatoramericanus/Oesophagostomum sp. complex and Strongyloides sp.) and 2 protozoan species (Balantidium coli and Entamoeba coli). Different coproscopical techniques are available but they are rarely compared to evaluate their efficiency to retrieve parasites. In this study 4 different field-friendly methods were compared. A sedimentation method and 3 different McMaster methods (using sugar, salt, and zinc sulphate solutions) were performed on 47 faecal samples collected from different individuals of both sexes and all ages. First, we show that McMaster flotation methods are appropriate to detect and thus quantify large protozoan cysts. Second, zinc sulphate McMaster flotation allows the retrieval of a higher number of parasite taxa compared to the other 3 methods. This method further shows the highest probability to detect each of the studied parasite taxa. Altogether our results show that zinc sulphate McMaster flotation appears to be the best technique to use when studying nematodes and large protozoa.

Design and fabrication of field-effect biosensors for biochemical detection.

Biochemically sensitive field-effect sensors are fabricated with simplified chip technology. Its fabrication process flow is designed based on metal gate complementary metal-oxide semiconductor technology, in which only six pattern masks are employed. The sensors are measured as field modulation resistors since they are made in its depletion mode. The milliampere magnitude response of conducting currents from certain biochemical materials achieves distinct sensitivity when measured on our fabricated sensors with different sensitive areas of W/L = 4.2 and 20.0. To check the stability of the sensor, up to 20 repeated tests are conducted on the same sensor chip operated in its three states, in which no materials (blank state, called 'blank'), pure water and biochemical materials are coated on its gate dielectric film, respectively. Measured results show that the response currents for certain materials are distributed in certain current range. Taking the response current of blank as a reference value, the response current of pure water is positive but very close to that of blank because of the small electric dipole properties of pure water. However, the response current of biochemical materials are negative and far apart from that of blank, because the biochemical materials have large electric dipole properties and clearly show measurement resolution.

Sensitive detection and accurate monitoring of Plasmodium vivax parasites on routine complete blood count using automatic blood cell analyzer (DxH800(TM)).

INTRODUCTION: Plasmodium vivax malaria is one of the most important infectious diseases plaguing humanity and causes significant mortality and morbidity worldwide. The gold standard of P. vivax malaria diagnosis is the microscopy of blood smears. Although microscopy is a rapid, cost-effective, and readily applicable method, it has many disadvantages, including low sensitivity, specificity, and precision. Therefore, there is a clear need for an effective screening test for P. vivax malaria detection both in high-prevalence areas and developed countries. METHODS: A total of 1761 complete blood count (CBC) samples generated by the automated hematology analyzer (DxH 800™; Beckman Coulter Inc., Miami, FL, USA) were retrospectively analyzed. The sample pool contained 123 samples from 52 P. vivax malaria patients and 1504 nonmalarial samples including 509 patients with leukopenia (white blood cell <2000/µL) and 134 normal subjects. RESULTS: The P. vivax malaria samples exhibited easily recognizable typical malaria signals on the nucleated red blood cell (nRBC) plots (sensitivity 100%) in DxH 800™. All 1504 samples without P. vivax infection were negative for malaria signal (specificity 100%). The size of P. vivax malaria signals correlated roughly with the parasite burden. CONCLUSION: DxH800™ provides very sensitive and specific, easily recognizable P. vivax malaria signals on routine CBC without need for the additional reagents or special procedures.