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      The buffy coat method: a tool for detection of blood parasites without staining procedures

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          Abstract

          Background

          Blood parasites belonging to the Apicomplexa, Trypanosomatidae and Filarioidea are widespread in birds and have been studied extensively. Microscopical examination (ME) of stained blood films remains the gold standard method for the detection of these infections in birds, particularly because co-infections predominate in wildlife. None of the available molecular tools can detect all co-infections at the same time, but ME provides opportunities for this to be achieved. However, fixation, drying and staining of blood films as well as their ME are relatively time-consuming. This limits the detection of infected hosts during fieldwork when captured animals should be released soon after sampling. It is an obstacle for quick selection of donor hosts for parasite experimental, histological and other investigations in the field. This study modified, tested and described the buffy coat method (BCM) for quick diagnostics (~ 20 min/sample) of avian blood parasites.

          Methods

          Blood of 345 birds belonging to 42 species was collected, and each sample was examined using ME of stained blood films and the buffy coat, which was examined after centrifugation in capillary tubes and after being transferred to objective glass slides. Parasite detection using these methods was compared using sensitivity, specificity, positive and negative predictive values and Cohen’s kappa index.

          Results

          Haemoproteus, Leucocytozoon, Plasmodium, microfilariae, Trypanosoma and Lankesterella parasites were detected. BCM had a high sensitivity (> 90%) and specificity (> 90%) for detection of Haemoproteus and microfilariae infections. It was of moderate sensitivity (57%) and high specificity (> 90%) for Lankesterella infections, but of low sensitivity (20%) and high specificity (> 90%) for Leucocytozoon infections. Trypanosoma and Plasmodium parasites were detected only by BCM and ME, respectively. According to Cohen’s kappa index, the agreement between two diagnostic tools was substantial for Haemoproteus (0.80), moderate for Lankesterella (0.46) and fair for microfilariae and Leucocytozoon (0.28) infections.

          Conclusions

          BCM is sensitive and recommended as a quick and reliable tool to detect Haemoproteus, Trypanosoma and microfilariae parasites during fieldwork. However, it is not suitable for detection of species of Leucocytozoon and Plasmodium. BCM is a useful tool for diagnostics of blood parasite co-infections. Its application might be extended to studies of blood parasites in other vertebrates during field studies.

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          Most cited references76

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          Understanding diagnostic tests 1: sensitivity, specificity and predictive values.

          The usefulness of diagnostic tests, that is their ability to detect a person with disease or exclude a person without disease, is usually described by terms such as sensitivity, specificity, positive predictive value and negative predictive value. In this article, the first of the series, a simple, practical explanation of these concepts is provided and their use and misuse discussed. It is explained that while sensitivity and specificity are important measures of the diagnostic accuracy of a test, they are of no practical use when it comes to helping the clinician estimate the probability of disease in individual patients. Predictive values may be used to estimate probability of disease but both positive predictive value and negative predictive value vary according to disease prevalence. It would therefore be wrong for predictive values determined for one population to be applied to another population with a different prevalence of disease. Sensitivity and specificity are important measures of the diagnostic accuracy of a test but cannot be used to estimate the probability of disease in an individual patient. Positive and negative predictive values provide estimates of probability of disease but both parameters vary according to disease prevalence.
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            A comparative analysis of microscopy and PCR-based detection methods for blood parasites.

            We compared information obtained by both microscopy and nested mitochondrial cytochrome b PCR in determining prevalence of haemosporidian infections in naturally infected birds. Blood samples from 472 birds of 11 species belonging to 7 families and 4 orders were collected in Europe, Africa and North America. Skilled investigators investigated them using the PCR-based screening and microscopic examination of stained blood films. The overall prevalence of haemosporidian infections, which was determined combining results of both these methods, was 60%. Both methods slightly underestimated the overall prevalence of infection, which was 54.2% after the PCR diagnostics and 53.6% after microscopic examination. Importantly, both these tools showed the same trends of prevalence of Haemoproteus spp. (21% by PCR and 22% by microscopy), Plasmodium spp. (17% and 22%) and Leucocytozoon spp. (30% and 25%) in the same sample, testifying that microscopy is a reliable tool in determining patterns of distribution of blood haemosporidian parasites in naturally infected birds. We encourage using optical microscopy in studies of blood parasites in parallel to the now widely employed molecular methods. Microscopy is relatively inexpensive and provides valuable information about directions how molecular methods can be further improved and most effectively applied, especially in the field studies of parasites. Importantly, blood films, which are used for microscopic examination, should be of good quality; they should be examined properly by skilled investigators. In spite of relatively long duration of microscopy of each sample, such examination provides opportunities for simultaneous determination and verification of taxonomically different parasites. Presently, different PCR protocols must be used for the detection of parasites belonging to different genera; this is expensive and time-consuming.
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              Nested cytochrome b polymerase chain reaction diagnostics underestimate mixed infections of avian blood haemosporidian parasites: microscopy is still essential.

              Numerous polymerase chain reaction (PCR)-based methods have been developed and used increasingly to screen vertebrate blood samples for the diagnosis of haemosporidian blood parasites (Sporozoa, Haemosporida), but a rigorous evaluation of the sensitivity of these methods for detecting mixed infections of different haemosporidian species belonging to the same and different genera and subgenera is lacking. This study links the information obtained by nested cytochrome b PCR and traditional microscopy in determining mixed haemosporidian infections in naturally infected birds. Samples from 83 individual passerine birds with single infections of Haemoproteus or Plasmodium spp., as determined by mitochondrial DNA amplification, also were investigated by microscopic examination of stained blood films. Thirty-six samples (43%) were found to harbor mixed Haemoproteus, or Plasmodium spp. infections, or both. Thus, the PCR assays alone underestimate the occurrence of mixed infections of haemosporidian parasites in naturally infected birds. To determine the true species composition of the haemosporidians in each individual host, PCR diagnostics need to be improved. Specific primers for Haemoproteus spp. and Plasmodium spp. should be developed. Ideally, a combination of the approaches of both microscopy and PCR-based methods is recommended for this purpose.
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                Author and article information

                Contributors
                crfchagas@gmail.com
                rasa.binkiene@gamtc.lt
                ilgunasmikas@gmail.com
                tatjana.jezova@gamtc.lt
                gediminas.valkiunas@gamtc.lt
                Journal
                Parasit Vectors
                Parasit Vectors
                Parasites & Vectors
                BioMed Central (London )
                1756-3305
                27 February 2020
                27 February 2020
                2020
                : 13
                : 104
                Affiliations
                ISNI 0000 0004 0522 3211, GRID grid.435238.b, Nature Research Centre, ; Akademijos 2, 08412 Vilnius, Lithuania
                Author information
                http://orcid.org/0000-0002-7884-9085
                Article
                3984
                10.1186/s13071-020-3984-8
                7045512
                32103784
                77715899-d223-4652-83c2-a1161f33bb3b
                © The Author(s) 2020

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                History
                : 2 December 2019
                : 18 February 2020
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100004504, Lietuvos Mokslo Taryba;
                Award ID: DOTSUT-137-09.3.3-LMT-K-712-02-0004
                Award ID: S-MIP-17-27
                Award Recipient :
                Categories
                Methodology
                Custom metadata
                © The Author(s) 2020

                Parasitology
                blood parasites,parasite diagnostics,birds,haemoproteus,plasmodium,leucocytozoon,trypanosoma,lankesterella,microfilaria,fieldwork

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