INsPECT, an Open-Source and Versatile Software for Automated Quantification of (Leishmania) Intracellular Parasites

The major health problems of developing countries constitute infectious diseases caused by intracellular parasites. Leishmania, Toxoplasma and Trypanosoma parasites are smart enough to reside inside the host cells.

Researchers working on the aspect of how such parasites proliferate intracellularly, have to do some laborious parasite counting, analyze drug [natural/synthetic] susceptibility or compare virulence properties of modified strains? High Content Screening (HCS) Technology has emerged recently and is a powerful tool for screening new compounds or for testing susceptibility of parasites to drugs. However the laborious procedure and cost of the equipments makes HCS poorly accessible for academics and research.

In this paper novel open-source, Java based software has been introduced that is quite helpful in automating the measurement of infection levels. This method is based on fluorescent DNA staining and has been named as INsPECT (Intracellular ParasitE CounTer). This software is believed to be versatile enough by the authors; to precisely detect intracellular parasites and to be the best one for parasite quantification.

The image based INsPECT is more fast, user friendly and open-source based software for quantification of parasites. The fluorescent images taken are used by the software to calculate the required data automatically by using image processing framework. The software can run based on DNA fluorescent images alone or in combination with phase contrast/Differential Interference Contrast. Such combination makes the intra- and extracellular parasites to be discriminated without any use of fluorescent membrane marker.

The out-put of the software provides one with annotated images, a report table, total cell count, total infected cell count, parasite index etc. The processing pipeline used by INsPECT software can be seen in the image below:

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Citation:

INsPECT, an Open-Source and Versatile Software for Automated Quantification of (Leishmania) Intracellular Parasites. Ehsan Yazdanparast, Antonio Dos Anjos, Deborah Garcia, Corinne Loeuillet,  Hamid Reza Shahbazkia mail, Baptiste Vergnes mail. Published: May 15, 2014 DOI: 10.1371/journal.pntd.0002850

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Parasitic Infections and Mutilation of T-Cell Function

Recently a review has been published in PLOS Neglected Tropical Diseases (http://www.plosntds.org/). This review focuses mainly on the development of the four intracellular parasite species (Plasmodium spp., Trypanosoma cruzi, Toxoplasma gondii and Leishmania spp.) in the mammalian hosts they infect, with special emphasis on T lymphocyte function. These parasites after invading the host, blight T cell function and augment their apoptosis. Such impairments lead the host unresponsive for the parasite because of the collapse of the T cell number. This weakening of T-cells aids the parasites to survive throughout the infection or become persistent. All of such process follows a particular tier system as:

1: Invading/breaching the host barriers and integument or epidermis.

2: Down-regulate T cell function and lead to their exhaustion.

3: Apoptosis of T-cells or T-cell contraction.

4: Stay persistently in the host.

These parasites can accomplish apoptosis of host T-cells by activation-induced cell death (AICD) involving death ligands and caspase-8 or activated T cell autonomous death (ACAD) involving Bcl-2 family. This course of action respectively results in the formation of the death-inducing signalling complex (DISC) or apoptosome. Several studies have shown that patients infected with Plasmodium falciparum, Trypanosoma cruzi and Leishmania donovani have elevated levels of death ligand FasL. Also some studies have proven that engulfment of apoptotic cells stimulates expansion of parasites like Trypanosoma cruzi and Leishmania major inside host macrophages.

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This work is Licensed under Creative Commons Licenses.

Reference: Vasco Rodrigues,  Anabela Cordeiro-da-Silva, Mireille Laforge, Ali Ouaissi, Khadija Akharid, Ricardo Silvestre mail, Jérôme Estaquier mail.. DOI: 10.1371/journal.pntd.0002567

rK39-ICT Is Less Sensitive in Africa as Compared to Asia.

Leishmaniasis is a fatal systemic disease that occurs due to bite of Leishmania donovani carrier sandfly.  In Asia, Africa and Brazil Viseral Leishmaniasis is most prevalent and is caused by Leishmania donovani complex (L.infantum+L.donovani).

An immunochromatographic test, rK39 (an antibody based test) is used to detect the disease. This test is based on dual 39 amino acid repeats of a Sudanese L. donovani-obtained kinesin homologue of rK39, flanked by HASPB sequences.  However this test has less sensitivity in Africa than in Asia. Possible reasons for such variation could be:

  1. Molecular diversity that is continent specific
  2. Variable immunological response due to different IgG anti-Leish patient levels.

Immunoglobulin G or IgG titers were determined for VL patients from India and Sudan. It was found that IgG titers of VL patients from Sudan were less than Indian VL patients. About 46-61 folds higher mean ELISA titers were found for Indian VL patients as compared to Sudanese patients. Higher titers occurred in adults (both sexes) and children less than 16 years old. Possible cause for such lower titers could be Zinc deficiency or either variable antigencity. Malnutrition of zinc, iron and protein are known to trim down immune responses in experimental models.  Extensive research on zinc deficient models has established earlier that reduced B-cell responses and impairment of memory cells occurs. This could explain as why rK39-ICT has lower sensitivity in African areas or a lower titer of IgG.Picture1 Picture2

Since VL is not the only version of disease caused by Leishmania, other types like (post kala-azar dermal leishmaniasis) PKDL could be of curiosity. Assessment of PKDL patient antibody levels might be the future study of the authors. Such efforts could really speed up the process of disease detection in future and save many lives by earlier and easier diagnosis.

Reference:  Significantly Lower Anti-Leishmania IgG Responses in Sudanese versus Indian Visceral Leishmaniasis. Tapan Bhattacharyya, Duncan E. Bowes, Sayda El-Safi, Shyam Sundar, Andrew K. Falconar, Om Prakash Singh, Rajiv Kumar, Osman Ahmed, Marleen Boelaert, Michael A. Miles. (2014) DOI: 10.1371/journal.pntd.0002675

Lundep increases leishmania parasite survival inside neutrophils

Leishmaniasis or kala azar is a disease caused by the parasite Leishmania, which belongs to lower eukaryotes. The bite of an insect Lutzomyia longipalpis aids in the transmission of the parasite to complete its life cycle.

In earlier studies it has been shown that the components of saliva (like hyaluronidase) from this arthropod aid in the transmission of Leishmania in host systems. However authors here have shown that another protein Lundep is also an active constituent of saliva for enhancing the parasite infection.

Since the first line of defense is by neutrophils and it has been established in case of Leishmaniasis that the parasites dodge this line of defense by entering into the compartments that are nonlytic or by dodging neutrophil extracellular traps (NETs).  Salivary Gland Extracts (SGE) from Lutzomyia longipalpis are known to support parasite survival inside the neutrophils, however still less is known about the NETosis or NET formation in respose to Leishmania parasite.

Lundep (Lutzomyia NET destroying protein), a female specific endonuclease has been shown in this study to have enhancing effect on the infectivity as well as inhibitory effect on intrinsic coagulation pathway. It has been demonstrated that catalytic activity of the salivary endonuclease is accountable for enhancing infectivity or in aiding parasites escape from NETs.

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This study has shown that Lundep:-

  1. Aids in degradation of the DNA scaffold of NETs. [Lundep has a DNase activity of about 300000 Kunitz units per mg of protein and can hydrolise both single stranded and double-stranded DNA.]
  2. Protects parasites from leishmanicidal activity of NETs.
  3. Promotes survival of promastigotes.
  4. Prevents blood coagulation while insect biting. [ Lundep is shown to have DNase activity that promotes antithrombotic effects]
  5. Assists in taking blood meal by decreasing the viscosity at the site of bite. [At the site of bite viscosity augments due to host DNA release]

So far no vaccine is available for Leishmaniasis (kala azar) and the authors consider Lundep as a potential target for vaccine generation.

Reference Used:

Lundep, a Sand Fly Salivary Endonuclease Increases Leishmania Parasite Survival in Neutrophils and Inhibits XIIa Contact Activation in Human Plasma. Andrezza C. Chagas, Fabiano Oliveira, Alain Debrabant, Jesus G. Valenzuela, José M. C. Ribeiro, Eric Calvo.  DOI: 10.1371/journal.ppat.1003923