SAMPLE COMPOSITION: PARASITE ECOLOGY'S DIRTY LITTLE SECRET.

In comparative studies, the advantage of increased sample sizes might be outweighed by detrimental effects on sample homogeneity and comparability when small numbers of hosts from a different demographic of the same species are included in samples. A mixed sample of sunfishes (Lepomis spp.) was subdivided in different ways and examined using cumulative performance curves to determine whether the exclusion of larger hosts from a single-species sample and/or the inclusion of hosts of the same size demographic from closely related host species would produce more homogeneous samples. The exclusion of larger hosts from the single-species samples tended to reduce the aggregation of the infrapopulation samples, and mixed-species samples of smaller fishes tended to have lower degrees of aggregation for a given sample size relative to the single-species sample. Cumulative performance curves for diversity and richness, in concert with nonmetric multidimensional scaling of the infracommunities, demonstrated sunfish size to be a more reliable determinant of infracommunity similarity than sunfish species in this particular sample. The results demonstrate that cumulative aggregation curves can be an effective tool for delineating homogeneous and comparable subsamples and that, under some circumstances, it is possible to offset the smaller sample sizes that result from the exclusion of older/larger hosts by the addition of congeneric or confamilial hosts within the same size/age classes as the stratified sample.

A MISIDENTIFICATION CRISIS PLAGUES SPECIMEN-BASED RESEARCH: A CASE FOR GUIDELINES WITH A RECENT EXAMPLE (ALI ET AL., 2020).

A recent paper in this journal concerning parasites of rock pigeons (Columba livia) published by Ali and colleagues exemplifies a growing trend of misidentified parasites in the literature, despite increased online resources that should help facilitate accurate identification. In the Ali et al. paper, a pigeon louse in the genus Columbicola (Phthiraptera: Ischnocera) is misidentified as Menopon gallinae, which is a parasite of chickens (Gallus gallus) and their relatives; moreover, this louse is from an entirely different suborder of lice (Phthiraptera: Amblycera). Another louse is misidentified as Goniodes dissimilis, another parasite of chickens and junglefowl. In addition, photographs of cestodes from pigeons in the same paper are not sufficient to confirm identification. Misidentifications are fueled, in part, by increasing pressure to publish coupled with a decrease in taxonomic expertise. We consider the downstream consequences of misidentification and suggest guidelines for authors, reviewers, and editors that could help to improve the reliability of specimen-based research.

A Beginner's Guide to Collecting Questing Hard Ticks (Acari: Ixodidae): A Standardized Tick Dragging Protocol.

Tick-borne diseases are emerging globally, necessitating increased research and coordination of tick surveillance practices. The most widely used technique for active collection of host-seeking, human-biting tick vectors is 'tick dragging', by which a cloth is dragged across the top of the vegetation or forest floor and regularly checked for the presence of ticks. Use of variable dragging protocols limits the ability of researchers to combine data sets for comparative analyses or determine patterns and trends across different spatial and temporal scales. Standardization of tick drag collection and reporting methodology will greatly benefit the field of tick-pathogen studies. Based on the recommendations of the Center for Disease Control and Prevention and other ecological considerations, we propose that tick dragging should be conducted to sample at least 750 m2 along linear transects when habitat allows in a manner that reduces bias in the sampled area, and report density of each tick species and life stage separately. A protocol for constructing a standard drag cloth, establishing linear transects, and drag sampling is presented, along with a downloadable datasheet that can be modified to suit the needs of different projects. Efforts to align tick surveillance according to these standard best practices will help generate robust data on tick population biology.

[Subtype Distribution of Blastocystis spp. with DNA Barcoding and Evaluation of Diagnostic Methods]. / DNA Barkotlama Yöntemiyle Blastocystis Alt Tiplerinin Belirlenmesi ve Tani Yöntemlerinin Degerlendirilmesi.

Blastocystis spp. is one of the most common protozoa in Turkey and throughout the world; laboratory diagnosis, genetic diversity and clinical features are among the most controversial topics related to the parasite. The aims of the present study were to investigate the subtype distribution of Blastocystis spp. Isolates from Aydin, Turkey, to evaluate the efficiency of some diagnostic methods and to evaluate the relationship between Blastocystis spp. infection with demographic factors and clinical findings. According to the direct microscopy results, 100 stool samples with and without Blastocystis spp. were selected by simple random sampling method. All were directly subjected to DNA isolation and cultured in Jones medium. DNA isolation was also carried out in Blastocystis spp. positive cultures with a different kit. Genomic DNA samples were analysed by PCR targeting the Blastocystis spp. small subunit ribosomal RNA (SSU rRNA) gene and subtypes (ST) were determined according to the sequence analyses. Moreover, the samples with undetected ST were further studied with sequence tagged site-PCR (STS-PCR). In addition, the patients with and without Blastocystis spp. were compared in terms of demographic characteristics (gender, age, residence) and clinical findings (itching, diarrhoea, abdominal pain, dyspepsia, nausea, vomiting, constipation and weight loss)., Among 100 stool positive samples diagnosed with direct microscopic examination 81 (81%) and 86 (86%) were found as positive with culture and PCR, retrospectively. Additionally, among 100 Blastocystis spp. negative stool samples five (5%) and seven (7%) samples were found positive with the same methods, respectively. The results of the analysis of Blastocystis spp. with SSU rRNA gene sequencing and STS-PCR methods revealed the subtype distribution of 95 Blastocystis spp. isolates as follows: ST3 (n= 50, 52.6%), ST2 (n= 21, 22.1%), ST1 (n= 17, 17.9%), ST7 (n= 4, 4.2%), ST2 + ST3 (n= 2, 2.1%) and ST1 + ST3 (n= 1, 1.1%). In addition, a complete accordance was observed in subtype distribution between direct DNA isolation from stools and 35 randomly selected isolates from the culture. In our study, the comparison of 107 Blastocystis spp. positive (by any of the methods) cases and 93 negative cases showed that there was no correlation in terms of demographic characteristics and clinical findings. Similarly, there was no significant relationship between symptoms and subtypes. In conclusion, it is recommended that in addition to direct microscopic examination, the use of additional methods such as culture and PCR will be useful in routine laboratory diagnosis of Blastocystis spp. The distribution of Blastocystis subtype in Aydin is mainly in accordance with the global findings. Lack of a relationship between Blastocystis spp. Infection and symptoms in our study was supported the idea that Blastocystis spp. infection is mostly asymptomatic in humans and it may be a member of healthy microbiota.

[Evaluation of Pre-analytical Process with Quality Indicators and Six Sigma Methodology in the Parasitology Laboratory of a Tertiary Healthcare Center]. / Üçüncü Basamak Saglik Merkezinin Parazitoloji Laboratuvarinda Analiz Öncesi Sürecin Kalite Belirteçleri ve Alti Sigma Yöntemi ile Degerlendirilmesi.

Laboratories have important role in decisions related to the patient. Laboratory performance needs to be evaluated to ensure accurate and sustainable laboratory results. Total test process consists of pre-analytical, analytical and post-analytical sub-processes. Most of the laboratory errors occur in pre-analytical process, which is mostly outside the laboratory, and this important situation has to be monitored by laboratory specialists. Although the standard statistical methods in which the frequency is evaluated can reveal which error is more than the others, they cannot determine which error is needed due to the absence of accepted target values. The decision to intervene in errors can only be made according to the targets by evaluating with methods such as six-sigma and quality indicators. Six-sigma method; is a quality management tool that provides information about process performance. Low sigma level indicates variability or errors in the relevant process. Quality indicators have been developed to measure quality and efficiency of laboratory processes. Use of quality indicators is effective in reducing errors, increasing patient safety and helping to meet ISO-15189 requirements. In this study, it was aimed to evaluate pre-analytical process performance in Parasitology Direct Diagnosis Laboratory of Ege University Faculty of Medicine according to the quality targets of International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Laboratory Errors and Patient Safety (IFCC WG-LEPS) and the six-sigma method. The data of rejected samples in our laboratory during the period 2014-2017 were obtained retrospectively from laboratory information system. Errors were classified using laboratory errors classification system. Quality indicators were calculated for each error category and assessed according to IFCC WG-LEPS quality targets. Pre-analytical sigma level was calculated for each year. Our pre-analytical process sigma goal was 4.6. Sigma levels were calculated according to the reasons of rejection and Pareto analysis was performed. All of the rejected samples were pre-analytical process errors. Unacceptable quality indicators according to the IFCC WG-LEPS targets were found as "insufficient sample" in 2015; "insufficient sample" and "inappropriate sample tube" in 2016 and 2017. Our pre-analytical process sigma levels according to the rejection reasons were found to be 4.39, 4.31, 4.11, 4.17, respectively in 2014- 2017. "Improper test request" in 2014, and "insufficient sample" in 2015-2017 had sigma levels below 4.6. In addition "improper test request" in 2014, and "insufficient sample" in 2015, 2016 and 2017 were noticeable in Pareto analysis. In this study, pre-analysis process was evaluated with six sigma method and quality indicators and the areas open for improvement were determined quantitatively. We found "insufficient sample", "improper test request" and "inappropriate sample tube" indicators as inappropriate according to our target values with both quality indicators and six-sigma methods. For this reason, we have planned video conference training focused on error sources for all employees. We consider that risk and number of errors will be reduced and efficiency of whole test process can be increased by evaluating pre-analytical process with accepted methods and monitoring the results. Process evaluation studies with six-sigma and quality indicators are limited in microbiology and parasitology laboratories. We think that laboratory quality is indispensable and this study will be an example for the laboratory specialists who want to evaluate pre-analytical process of their laboratories.

Toolbox for In Vivo Imaging of Host-Parasite Interactions at Multiple Scales.

Animal models have for long been pivotal for parasitology research. Over the last few years, techniques such as intravital, optoacoustic and magnetic resonance imaging, optical projection tomography, and selective plane illumination microscopy developed promising potential for gaining insights into host-pathogen interactions by allowing different visualization forms in vivo and ex vivo. Advances including increased resolution, penetration depth, and acquisition speed, together with more complex image analysis methods, facilitate tackling biological problems previously impossible to study and/or quantify. Here we discuss advances and challenges in the in vivo imaging toolbox, which hold promising potential for the field of parasitology.

Best practice guidelines for studies of parasite community ecology.

In recent decades, parasite community ecology has produced hundreds of studies on an ever-growing number of host species, and developed into an active sub-discipline of parasitology. However, this growth has been characterized by a lack of standards in the practices used by researchers, with many common approaches being flawed, unjustified or misleading. Here, in the hope of promoting advances in the study of parasite community ecology, I identify some of the most common errors or weaknesses in past studies, and propose ten simple rules for best practice in the field. They cover issues including, among others, taxonomic resolution, proper and justifiable analytical methods, higher-level replication, controlling for sampling effort or species richness, accounting for spatial distances, using experimental approaches, and placing raw data in the public domain. While knowledge of parasite communities has expanded in breadth, with more and more host species being studied, true progress has been very limited with respect to our understanding of fundamental general processes shaping these communities. It is hoped that the guidelines presented here can direct researchers away from the entrenched use of certain approaches flawed in design, analysis or interpretation, by offering a more rigorous and standardized set of practices, and, hopefully, a way forward.

Assessors assemble: the need for harmonised external quality assessment schemes for emerging diagnostic methodologies in the field of parasitology.

Global travel and migration trends have meant a huge increase in the numbers of people exposed to tropical parasitic diseases. Thus, there is an increasing need for robust, reproducible and reliable diagnostic techniques in the field. Advanced molecular and lateral flow techniques have pushed the boundaries of clinical parasite diagnostics with their enhanced sensitivities and specificities. These emerging technologies are, however, not without their challenges, and recently there has been multiple evidence of a lack of consensus among protocols and results obtained by quality assessment of these novel technologies. This commentary discusses findings from some recent quality assessment studies in the field of blood and faecal parasitology. The article also makes recommendations for a unified and harmonised approach towards delivering high-quality clinical parasitology diagnoses, especially through the use of proficiency testing.

Ten Events That Defined Anthelmintic Resistance Research.

Fifty years after anthelmintic resistance in livestock parasites was first reported, the prevalence of resistance has increased globally, and is of increasing significance in animal industries. It is now timely to reflect on what we have learnt, how research has unfolded, and what we hope to learn in the future. This Opinion paper examines ten important research events that were pivotal in resistance research. The moments include the discovery, description, and diagnosis of parasite resistance, as well as important physiological and genetic findings, and the development of online tools to help manage resistance. Despite our efforts, resistance remains the greatest challenge in parasite control. The future directions for research, including people and funding, are discussed.

Parasite Collections: Overlooked Resources for Integrative Research and Conservation.

Parasite natural history collections form vital scientific infrastructure that play a substantial role in increasing awareness of the importance of parasites to ecosystems, conservation assessments, science, and society. These collections support novel investigations that integrate across taxa, time, and space, and should be cultivated to advance organismal-based science. Promoting and supporting parasite collections will ensure their ongoing stability and accessibility.

Plasmodium species differentiation by non-expert on-line volunteers for remote malaria field diagnosis.

BACKGROUND: Routine field diagnosis of malaria is a considerable challenge in rural and low resources endemic areas mainly due to lack of personnel, training and sample processing capacity. In addition, differential diagnosis of Plasmodium species has a high level of misdiagnosis. Real time remote microscopical diagnosis through on-line crowdsourcing platforms could be converted into an agile network to support diagnosis-based treatment and malaria control in low resources areas. This study explores whether accurate Plasmodium species identification-a critical step during the diagnosis protocol in order to choose the appropriate medication-is possible through the information provided by non-trained on-line volunteers. METHODS: 88 volunteers have performed a series of questionnaires over 110 images to differentiate species (Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, Plasmodium malariae, Plasmodium knowlesi) and parasite staging from thin blood smear images digitalized with a smartphone camera adapted to the ocular of a conventional light microscope. Visual cues evaluated in the surveys include texture and colour, parasite shape and red blood size. RESULTS: On-line volunteers are able to discriminate Plasmodium species (P. falciparum, P. malariae, P. vivax, P. ovale, P. knowlesi) and stages in thin-blood smears according to visual cues observed on digitalized images of parasitized red blood cells. Friendly textual descriptions of the visual cues and specialized malaria terminology is key for volunteers learning and efficiency. CONCLUSIONS: On-line volunteers with short-training are able to differentiate malaria parasite species and parasite stages from digitalized thin smears based on simple visual cues (shape, size, texture and colour). While the accuracy of a single on-line expert is far from perfect, a single parasite classification obtained by combining the opinions of multiple on-line volunteers over the same smear, could improve accuracy and reliability of Plasmodium species identification in remote malaria diagnosis.

[THE ITERATIVE APPROACH TO PRICING LABORATORY SERVICES FOR THE PROVISION OF MEDICAL AID (COPROPROCYCOSOPHICAL EXAMINATION ON LYAMBLIOSIS)].

The purpose of the study was to substantiate the theoretical and methodical principles of pricing for laboratory services in the diagnosis of giardiasis, taking into account their iterability and peculiarities of parasitic research methods. The methods of laboratory study of gum disease of native smear, treated with Lyulol solution, and ether-formalin enrichment on the criteria of their quality and effectiveness are analyzed. On the basis of the study of the effectiveness of the first and repeated analyzes, the conclusion on the iterative nature of laboratory studies of giardiasis and the effectiveness of the use of an iterative approach to the determination of prices for laboratory services is substantiated. The approaches to pricing laboratories providing diagnostic services for giardiasis in Ukraine are analyzed. The necessity of applying the price trajectory for laboratory diagnosis of giardiasis on the basis of multiplicity of researches (interactive approach) and the determination of the minimum and maximum price levels (the minimax approach) is proved. The main factors of pricing for laboratory diagnostics of giardiasis are identified and characterized: iterative research, economic efficiency, social value, value for the patient, competitiveness and reputation.

Reestablishing rigor in archaeological parasitology.

Archaeological parasitology originated in the mid-twentieth century with interdisciplinary teams of specialists directed by archaeologists. The goals of such studies were detailed analyses of dietary, medicinal, and environmental factors that shaped the patterns of infection. By the 1970s, a cadre of unique coprolite analysts was trained to analyze macroscopic and microscopic remains for integrated reconstructions of the cultural determinants of parasitism. During these first phases of research, diagnostic rigor was maintained by direct training of specialists in parasitology and archaeology sub-disciplines including archaeobotany and archaeopalynology. Near the end of the twentieth century, however, "paleoparasitology" was defined as a separate field focusing on defining parasite distribution through time and space. Ironically, this focus resulted in an increase in misdiagnosis, especially prominent after 2000. Paleoparasitology does not explicitly include other specialized studies in it research design. Thus, dietary, environmental and medicinal inferences have been neglected or lost as samples were destroyed solely for the purpose of parasitological analysis. Without ancillary archaeological studies, paleoparasitology runs the risk of separation from archaeological context, thereby reducing its value to the archaeologists who recover samples for analysis.

Identification, Validation, and Application of Molecular Diagnostics for Insecticide Resistance in Malaria Vectors.

Insecticide resistance is a major obstacle to control of Anopheles malaria mosquitoes in sub-Saharan Africa and requires an improved understanding of the underlying mechanisms. Efforts to discover resistance genes and DNA markers have been dominated by candidate gene and quantitative trait locus studies of laboratory strains, but with greater availability of genome sequences a shift toward field-based agnostic discovery is anticipated. Mechanisms evolve continually to produce elevated resistance yielding multiplicative diagnostic markers, co-screening of which can give high predictive value. With a shift toward prospective analyses, identification and screening of resistance marker panels will boost monitoring and programmatic decision making.

Survival analysis of diagnostic assays in Plasmodium falciparum malaria.

BACKGROUND: Rapid diagnostic tests (RDT) and real-time PCR (qPCR) assays are sensitive for diagnosing malaria, but because they detect antigen and DNA, respectively, positivity may not reflect active infection. Performance characteristics of RDT and qPCR in Plasmodium falciparum positive specimens were evaluated over time to elucidate duration of positivity following conversion to microscopy negative. METHODS: Specimens from patients with at least one specimen that was positive for P. falciparum by microscopy, and at least one specimen that was negative for P. falciparum within a 1-month period were identified. Survival distributions of the diagnostic tests over time were compared. Performance characteristics for each test were calculated. RESULTS: Ninety specimens were included, with 48 initially positive for P. falciparum, and 42 subsequently negative. Of 42 specimens that converted to microscopy-negative following an initial positive, 26 (61.9 %) and 41 (97.6 %) were positive by qPCR and RDT, respectively. Survival curves of microscopy versus qPCR, as well as microscopy vs RDT differed significantly (p = 0.0002 and p < 0.0001, respectively). Compared to microscopy, sensitivity of qPCR was 100.0 % (95 % CI 90.8-100.0 %), and that of RDT was 100.0 % (95 % CI 90.8-100.0 %). CONCLUSIONS: Due to slow clearance of circulating antigen and DNA from bloodstream, RDT and qPCR have low positive predictive value for clinically relevant asexual parasitaemia in post-treatment specimens. Thus, microscopy remains the only available malaria diagnostic that can reliably distinguish true asexual parasitaemia from prolonged clearance of antigen and nucleic acid in a convalescing patient.

[THE FUNCTIONAL CONSTITUENT OF A BIOLOGICAL COMPONENT IN PROGRAMS FOR TRAINING SPECIALISTS IN THE AREA OF PARASITOLOGY FOR ACCREDITATION].

The paper considers the functional aspects of a biological component in programs for training specialists in the area of Parasitology for accreditation within the current enactments, including those on modernization of public health and additional professional education. The working program of the module "Fundamental Disciplines" has been used as an example to outline approaches to molding a medical parasitologist's capacity and readiness to solve professional tasks on the basis of knowledge of fundamental disciplines: biology, immunology, and medical geography. Education fundamentalization is shown to suggest more unsupervised work of a learner in the teaching process. The fundamental constituent of a biological component of the 'programs for training learners in the specialty of Parasitology for accreditation is shown in the interaction of all sections of this area with special and allied subjects.

An integrated parasitology: revealing the elephant through tradition and invention.

The field of parasitology contributes to the elucidation of patterns and processes in evolution, ecology, and biogeography that are of fundamental importance across the biosphere, leading to a thorough understanding of biodiversity and varied responses to global change. Foundations from taxonomic and systematic information drive biodiversity discovery and foster considerable infrastructure and integration of research programs. Morphological, physiological, behavioral, life-history, and molecular data can be synthesized to discover and describe global parasite diversity, in a timely manner. In fully incorporating parasitology in policies for adaptation to global change, parasites and their hosts should be archived and studied within a newly emergent conceptual universe (the 'Stockholm Paradigm'), embracing the inherent complexity of host-parasite systems and improved explanatory power to understand biodiversity past, present, and future.

The quality of methods reporting in parasitology experiments.

There is a growing concern both inside and outside the scientific community over the lack of reproducibility of experiments. The depth and detail of reported methods are critical to the reproducibility of findings, but also for making it possible to compare and integrate data from different studies. In this study, we evaluated in detail the methods reporting in a comprehensive set of trypanosomiasis experiments that should enable valid reproduction, integration and comparison of research findings. We evaluated a subset of other parasitic (Leishmania, Toxoplasma, Plasmodium, Trichuris and Schistosoma) and non-parasitic (Mycobacterium) experimental infections in order to compare the quality of method reporting more generally. A systematic review using PubMed (2000-2012) of all publications describing gene expression in cells and animals infected with Trypanosoma spp was undertaken based on PRISMA guidelines; 23 papers were identified and included. We defined a checklist of essential parameters that should be reported and have scored the number of those parameters that are reported for each publication. Bibliometric parameters (impact factor, citations and h-index) were used to look for association between Journal and Author status and the quality of method reporting. Trichuriasis experiments achieved the highest scores and included the only paper to score 100% in all criteria. The mean of scores achieved by Trypanosoma articles through the checklist was 65.5% (range 32-90%). Bibliometric parameters were not correlated with the quality of method reporting (Spearman's rank correlation coefficient <-0.5; p>0.05). Our results indicate that the quality of methods reporting in experimental parasitology is a cause for concern and it has not improved over time, despite there being evidence that most of the assessed parameters do influence the results. We propose that our set of parameters be used as guidelines to improve the quality of the reporting of experimental infection models as a pre-requisite for integrating and comparing sets of data.

Variability of the egg hatch assay to survey benzimidazole resistance in nematodes of small ruminants under field conditions.

The egg hatch assay (EHA) is one of the main in vitro methods for detection of benzimidazole resistance in nematode parasites of small ruminants. However, although the EHA has been standardised at the laboratory level, the diagnostic performance of this method has not been fully characterised for field screenings. In the present work, monthly variation of benzimidazole resistance estimated by EHA was surveyed over two years in three sheep flocks and in one goat and an additional sheep flock sharing the same pastures. Resistance was measured by calculating both the effective dose of thiabendazole (TBZ) that inhibited hatching of ≥50% of parasite eggs (ED50) and the proportion (Pdd) of eggs hatching at a discriminating dose of 0.1 µg/ml TBZ. Pdd exhibited higher variability than ED50, in agreement with the higher sensitivity of Pdd to changes in resistance levels. Both resistance parameters, however, were highly correlated, and their variation was similarly related to the same factors. Resistance levels differed among sheep flocks, and the resistance level of the goat flock was higher than that measured for the sheep flock sharing the same pasture. Moreover, monthly variation of resistance in goats did not mirror that recorded in sheep. Resistance levels varied seasonally, with the highest values recorded in the spring, and they were inversely related to the number of days that samples were stored under anaerobic conditions. In addition, they were directly associated with the relative abundance of Teladorsagia spp. but inversely related to the relative abundance of Haemonchus spp. After controlling for the effects of these identified factors for variation, inter-monthly sampling variation due to unknown factors was the main source of variability, accounting for more than 60-70% of variance for both resistance parameters and yielding absolute estimation errors higher than 0.06 for ED50 or 0.2 for Pdd when resistance was estimated from a single sampling. Optimum sample size, estimated from variance components, suggested that at least 4-5 samplings would be needed to halve this absolute error, whereas additional samplings would slightly increase precision but at the cost of substantially increasing sampling effort. More research is needed to identify the main factors involved in this inter-sampling variation to standardise the implementation of EHA under field conditions.