title: ARAM: an automated image analysis software to determine rosetting parameters and parasitaemia in Plasmodium samples.
abstract: BACKGROUND
  Rosetting is associated with severe malaria and a primary cause of death in Plasmodium falciparum infections. Detailed understanding of this adhesive phenomenon may enable the development of new therapies interfering with rosette formation. For this, it is crucial to determine parameters such as rosetting and parasitaemia of laboratory strains or patient isolates, a bottleneck in malaria research due to the time consuming and error prone manual analysis of specimens. Here, the automated, free, stand-alone analysis software automated rosetting analyzer for micrographs (ARAM) to determine rosetting rate, rosette size distribution as well as parasitaemia with a convenient graphical user interface is presented.
  
  
  METHODS
  Automated rosetting analyzer for micrographs is an executable with two operation modes for automated identification of objects on images. The default mode detects red blood cells and fluorescently labelled parasitized red blood cells by combining an intensity-gradient with a threshold filter. The second mode determines object location and size distribution from a single contrast method. The obtained results are compared with standardized manual analysis. Automated rosetting analyzer for micrographs calculates statistical confidence probabilities for rosetting rate and parasitaemia.
  
  
  RESULTS
  Automated rosetting analyzer for micrographs analyses 25 cell objects per second reliably delivering identical results compared to manual analysis. For the first time rosette size distribution is determined in a precise and quantitative manner employing ARAM in combination with established inhibition tests. Additionally ARAM measures the essential observables parasitaemia, rosetting rate and size as well as location of all detected objects and provides confidence intervals for the determined observables. No other existing software solution offers this range of function. The second, non-malaria specific, analysis mode of ARAM offers the functionality to detect arbitrary objects.
  
  
  CONCLUSIONS
  Automated rosetting analyzer for micrographs has the capability to push malaria research to a more quantitative and statistically significant level with increased reliability due to operator independence. As an installation file for Windows © 7, 8.1 and 10 is available for free, ARAM offers a novel open and easy-to-use platform for the malaria community to elucidate resetting.
authors.affiliation: Experimental Physics I, University of Augsburg, Universitätsstraße 1, Augsburg, Germany.
authors.forename: Patrick Wolfgang
authors.initials: PW
authors.lastname: Kudella
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Box 280, 171 77, Stockholm, Sweden.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Moll
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Box 280, 171 77, Stockholm, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Wahlgren
authors.affiliation: Experimental Physics I, University of Augsburg, Universitätsstraße 1, Augsburg, Germany.|Nanosystems Initiative Munich, Schellingstraße 4, Munich, Germany.
authors.forename: Achim
authors.initials: A
authors.lastname: Wixforth
authors.affiliation: Experimental Physics I, University of Augsburg, Universitätsstraße 1, Augsburg, Germany. christoph.westerhausen@gmail.com.|Nanosystems Initiative Munich, Schellingstraße 4, Munich, Germany. christoph.westerhausen@gmail.com.
authors.forename: Christoph
authors.initials: C
authors.lastname: Westerhausen
mesh_terms: D004912:Erythrocytes / Q000502:physiology; D007090:Image Interpretation, Computer-Assisted; D016778:Malaria, Falciparum / Q000097:blood / Q000000981:diagnostic imaging* / Q000503:physiopathology; D008853:Microscopy / Q000295:instrumentation; D018512:Parasitemia / Q000000981:diagnostic imaging* / Q000469:parasitology; D010963:Plasmodium falciparum / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012397:Rosette Formation / Q000295:instrumentation; D012984:Software
keywords: Automatic analysis; Cell detection; Image analysis; Malaria; Parasitaemia; Plasmodium falciparum; Rosetting; Software