Detection Of Parasites Research Articles

A deep architecture based on attention mechanisms for effective end-to-end detection of early and mature malaria parasites in a realistic scenario.

Malaria is a critical and potentially fatal disease caused by the Plasmodium parasite and is responsible for more than 600,000 deaths globally. Early and accurate detection of malaria parasites is crucial for effective treatment, yet conventional microscopy faces limitations in variability and efficiency. We propose a novel computer-aided detection framework based on deep learning and attention mechanisms, extending the YOLO-SPAM and YOLO-PAM models. Our approach facilitates the detection and classification of malaria parasites across all infection stages and supports multi-species identification. The framework was evaluated on three publicly available datasets, demonstrating high accuracy in detecting four distinct malaria species and their life stages. Comparative analysis against state-of-the-art methodologies indicates significant improvements in both detection rates and diagnostic utility. This study presents a robust solution for automated malaria detection, offering valuable support for pathologists and enhancing diagnostic practices in real-world scenarios.

Identification and molecular characterization of Dirofilaria sp. "hongkongensis" (Rhabditida: Onchocercidae) in wild-caught mosquitoes (Diptera: Culicidae): first report from India.

Dirofilariasis is a globally significant emerging-zoonotic-disease caused by nematode parasites belonging to the genus Dirofilaria (Rhabditida: Onchocercidae) and is transmitted by mosquitoes (Diptera: Culicidae) of the Culicidae family. A recent study on molecular prevalence of Dirofilaria sp. "hongkongensis" To, 2012 (nomen nudum) among the dog population in Kerala indicated a high infection rate. However, no data are currently available on the vectors involved in the transmission of this disease. In this study, an entomological survey was conducted to detect natural infection and to characterize the Dirofilaria parasite from Kani tribal settlements in Thiruvananthapuram, India. A total of 356 mosquitoes were collected, morphologically identified, and analyzed for natural infection of Dirofilaria from 4 different villages in the study area. The species composition of the collected mosquito specimens comprised 10 species belonging to 6 genera, namely Anopheles, Aedes, Armigeres, Culex, Uranotaenia, and Heizmannia. Aedes albopictus (Skuse, 1895) was found to be the predominant species, followed by Armigeres subalbatus Coquillett, 1898. Out of these, 200 female mosquitoes were grouped by species into 31 pools, for genomic DNA extraction. Detection of the nematode parasite was performed using nematode-specific primers via conventional PCR, and the species was subsequently confirmed by sanger sequencing. Natural-infection of Dirofilaria sp. "hongkongensis" was identified in Ae. albopictus and Ar. subalbatus, with a minimum infection rate of 17.6% and 13.3%, respectively. This study represents the first report of natural-infection of Dirofilaria sp. among field-collected mosquitoes in India. These findings underscore the need for robust entomological-surveillance system to prevent potential future outbreaks.

Comparative performance of microscopy, rapid diagnostic tests, and multiplex real-time PCR for detection of malaria parasites among pregnant women in northwest Ethiopia

BackgroundLow malaria parasitaemia is a diagnostic challenge in pregnancy, leading to false negative microscopy and rapid diagnostic test (RDT) results. However, these submicroscopic or subpatent infections could cause adverse pregnancy outcomes. Thus, evaluating the diagnostic performance of microscopy, RDT, and multiplex qPCR in pregnancy is vital for informed decisions.MethodsA total of 835 peripheral blood and 372 placental blood samples were collected from 835 pregnant women attending first antenatal care or admitted for delivery at selected health facilities in northwest Ethiopia between November 2021 and July 2022. In multiplex qPCR, all microscopy and/or RDT positive samples were extracted and amplified individually, whereas all samples negative by both RDT and microscopy were extracted after pooling ten samples together and tested for Plasmodium genus. The diagnostic performance of microscopy, RDT, and multiplex qPCR in pregnancy was compared and evaluated against each other.ResultsUsing multiplex qPCR as a reference test, microscopy had a sensitivity of 73.8% (95% confidence interval (CI): 65.9–80.7) and 62.2% (95% CI: 46.5–76.2) to detect Plasmodium parasites in peripheral and placental blood samples, respectively, with a 100% (95% CI: 98.9–100) specificity in both samples. Similarly, the RDT had a sensitivity of 67.6% (95% CI: 59.3–75.1) and a specificity of 96.5% (95% CI: 94.9–97.8) for Plasmodium infection diagnosis in peripheral blood and a sensitivity of 62.2% (95% CI: 46.5–76.2) and a specificity of 98.8% (95% CI: 96.9–99.7) in placental blood samples. Considering microscopy as a reference test, multiplex qPCR showed a sensitivity of 100% (95% CI: 96.6–100) and a specificity of 94.8% (95% CI: 93.0–96.3) to diagnose Plasmodium infections in both peripheral and placental blood samples. Pooled multiplex qPCR detected 34 peripheral and 12 placental blood Plasmodium infections from microscopy and RDT negative samples. The pooled assay obviated about half of the reactions and its testing costs. Microscopy showed almost perfect agreement (κ = 0.823) with multiplex qPCR for detecting malaria parasites in pregnancy, whereas the RDT showed a substantial agreement (κ = 0.684).ConclusionMultiplex qPCR had a better performance for Plasmodium infection diagnosis in pregnancy compared to microscopy and RDT. Pooled multiplex qPCR could be a sensitive and resource-efficient strategy for epidemiological surveillance of Plasmodium infections in pregnancy.

AN EVALUATION OF ONSITE TRAINING AND SUPPORTIVE SUPERVISION PLUS (OTSS+) TO IMPROVE MALARIA DIAGNOSTIC SERVICE DELIVERY IN 22 HOSPITALS IN SIERRA LEONE

Malaria, a formidable global health issue, disproportionately impacts sub-Saharan Africa, including Sierra Leone, where a substantial burden persists with a significant number of reported cases annually. This global health challenge necessitates a comprehensive approach to address its impact on public health, emphasizing the crucial role of accurate and prompt diagnosis in effective case management. Laboratory technicians play a pivotal role in malaria diagnosis, conducting microscopy examinations of blood samples for the detection of malaria parasites. The competency and reliability of these technicians are paramount for appropriate treatment and surveillance, as malaria diagnosis forms the cornerstone of case management. However, studies in sub-Saharan Africa highlight the challenges associated with maintaining and enhancing the skills of laboratory technicians over time. Factors such as inadequate training, inconsistent adherence to standard operating procedures, and limited access to necessary equipment contribute to the variable and often poor quality of microscopy. The primary aim of this research is to assess the impact of supportive supervision on malaria diagnosis service delivery among laboratory technicians in Sierra Leone The study employs a quasi-experimental design to assess the impact of supportive supervision on malaria diagnosis service delivery among laboratory technicians. A pre-designed checklist of four thematic area (Lab evaluation, Proficiency testing, External Quality Assurance and Microscopy observation) was used to collect data from 22 hospitals in three rounds of supervisory visits. Laboratory Evaluation: There was a significant improvement from R1 (64%) to R2 (93%), with a slight decrease in R3 (81%). Stockouts of consumables for microscope microscopy and stockouts of malaria RDT showed moderate improvement from R1 to R3. For example, stockouts of malaria RDT improved from 35% to 59%. Scores related to microscopes, spare parts, and maintenance showed consistent improvement, particularly from R1 (29%) to R3 (47%). Both internal and external quality assurance scores showed an upward trend. Internal Quality Assurance increased from 49% in R1 to 81% in R3. There was a consistent improvement in this category, with scores rising from 50% in R1 to 90% in R3. The significant improvement across the rounds in all categories, particularly in the later rounds, suggests that supportive supervision played a crucial role. implying that continuous oversight and guidance significantly enhanced both accuracy and timeliness in malaria diagnosis. KEY WORDS: Microscope, Malaria Diagnosis, Laboratory Evaluation

Seroepidemiology, molecular detection, and isolation attempts of Toxoplasma gondii in dairy goats from Rio de Janeiro, Brazil.

Goats are the one of the most susceptible domestic species to toxoplasmosis affecting animal health and production. The present study aimed to determine the seroprevalence of T. gondii infection in dairy goats from Rio de Janeiro, Brazil, as well as to evaluate associated risk factors, parasitic DNA detection in raw goat milk samples, and attempts to isolate the parasite from raw goat milk samples. Between October 2020 and December 2022, blood samples were collected from 460 goats, and 220 milk samples were taken from goats raised in the Rio de Janeiro state, Brazil. The serum samples were tested for anti-T. gondii IgG antibodies using IFAT. The milk samples were tested for parasitic DNA detection using nPCR, and attempts were made to isolate the protozoan in mice. For risk factor analysis, the farmers answered a semi-structured questionnaire covering various epidemiological variables. Overall, 24.1% tested positive for anti-T. gondii IgG in serum samples. Risk factors included farm location, age group, rearing system, presence of cats, grazing, and access of cats to water sources. In nPCR, 38.6% were positive, but none were positive in the bioassay. However, 3.2% of the mice inoculated with goat milk seroconverted. This study found that the exposure of dairy goats in Rio de Janeiro to T. gondii is driven by ineffective management practices in preventing and controlling the parasite in these animals. Moreover, despite the detection of parasitic DNA in raw goat milk, the shedding of viable parasites in milk from naturally exposed goats appears to be low.

Image-Based Detection and Classification of Malaria Parasites and Leukocytes with Quality Assessment of Romanowsky-Stained Blood Smears.

Malaria remains a global health concern, with 249 million cases and 608,000 deaths being reported by the WHO in 2022. Traditional diagnostic methods often struggle with inconsistent stain quality, lighting variations, and limited resources in endemic regions, making manual detection time-intensive and error-prone. This study introduces an automated system for analyzing Romanowsky-stained thick blood smears, focusing on image quality evaluation, leukocyte detection, and malaria parasite classification. Using a dataset of 1000 clinically diagnosed images, we applied feature extraction techniques, including histogram bins and texture analysis with the gray level co-occurrence matrix (GLCM), alongside support vector machines (SVMs), for image quality assessment. Leukocyte detection employed optimal thresholding segmentation utility (OTSU) thresholding, binary masking, and erosion, followed by the connected components algorithm. Parasite detection used high-intensity region selection and adaptive bounding boxes, followed by a custom convolutional neural network (CNN) for candidate identification. A second CNN classified parasites into trophozoites, schizonts, and gametocytes. The system achieved an F1-score of 95% for image quality evaluation, 88.92% for leukocyte detection, and 82.10% for parasite detection. The F1-score-a metric balancing precision (correctly identified positives) and recall (correctly detected instances out of actual positives)-is especially valuable for assessing models on imbalanced datasets. In parasite stage classification, CNN achieved F1-scores of 85% for trophozoites, 88% for schizonts, and 83% for gametocytes. This study introduces a robust and scalable automated system that addresses critical challenges in malaria diagnosis by integrating advanced image quality assessment and deep learning techniques for parasite detection and classification. This system's adaptability to low-resource settings underscores its potential to improve malaria diagnostics globally.

VL-M2C: Leveraging deep learning approach for stage detection of malaria parasites

VL-M2C: Leveraging deep learning approach for stage detection of malaria parasites

Molecular detection of parasites and host preference in wild-caught Culicoides biting midges (Diptera: Ceratopogonidae) in Chiang Mai and Nakhon Si Thammarat Provinces, Thailand.

Culicoides biting midges (Diptera: Ceratopogonidae) have been reported as potential vectors for haemoparasites. Information about host-vector-parasite specificity is required to confirm their status. Here, molecular detection of haemosporidians, Leishmania, trypanosomatids, and filarial nematodes in biting midges was conducted to understand their potential role as vectors, and their host preference was determined. Wild-caught biting midges were collected from six different localities of Chiang Mai and Nakhon Si Thammarat provinces, Thailand. A total of 6,578 individual Culicoides (170 males, 6,408 females) comprising 15 species of six sub-genera and two groups were collected. Also, 738 parous females and 29 engorged females were examined for parasites and host blood meals, respectively. Culicoides arakawae, C. mahasarakhamense, C. peregrinus and C. innoxius in Chiang Mai province, and C. innoxius and C. peregrinus in Nakhon Si Thammarat province were the most dominant species. Leucocytozoon spp., Leucocytozoon caulleryi and Plasmodium juxtanucleare were identified in five Culicoides species including C. mahasarakhamense, C. arakawae, C. oxystoma, C. fulvus, and C. guttifer. This study is the first record of L. caulleryi in the biting midge C. arakawae in Thailand. Blood meal analysis revealed that Culicoides primarily fed on cattle (17/29, 58.6%), followed by chickens (10/29, 34.5%), and humans (2/29, 6.9%). Our findings confirmed the existence of several Culicoides species in Thailand, which might be potential vectors for transmission of haemosporidians (Leucocytozoon and Plasmodium). Information from host blood meal analyses underlined their preference for large mammals, followed by domestic chickens. More anthropophilic Culicoides species remain to be discovered.

Malaria among febrile patients suspected of Yellow fever during an outbreak in Ghana.

Introduction Between October 2021 and February 2022, there was an outbreak of Yellow fever that spread within several districts in the northern part of Ghana. Febrile illnesses such as Yellow fever are often misdiagnosed as malaria and vice versa, which delays appropriate management and treatment. Hence, the true burden of Yellow fever and malaria are mostly underestimated. This study investigated the epidemiology of malaria in febrile patients suspected of Yellow fever in and around the epicenter of a Yellow fever outbreak in Ghana. Methods The study was a cross-sectional study conducted in two outbreak sites (Wenchi and Damongo) and two non-outbreak sites (Kumbungu and Tamale). A total of 498 febrile patients from healthcare facilities were recruited in the rainy and dry seasons. A structured questionnaire was administered to collect patients' demographic information. Venous blood was collected from consented study participants for malaria parasite detection via microscopy and PCR. Total RNA was extracted from serum samples for the detection of yellow fever virus using Reverse Transcriptase PCR. Results None of the patients tested positive for Yellow fever virus. Out of the 498 participants tested for plasmodium parasites, 98 (19.7%) were microscopy positive while 92 (29.7%) were PCR positive. Plasmodium prevalence via microscopy was significantly higher in the dry season, 42 (18.67%) compared to the rainy season, 12 (4.4%) ( P < 0.001). However, the difference in malaria prevalence via PCR in the rainy and dry seasons was not significant ( P > 0.05). Kumbungu had the highest parasite prevalence by PCR during the dry (78.38%) and rainy (27.18%) seasons. Higher Plasmodium falciparum prevalence was observed using PCR compared to microscopy across all age groups. The age groups with the highest prevalence of P. falciparum were under five years (34.69%) and 5 to 9 years (35.56%). The differences in P. falciparum prevalence across the age groups were significant ( P < 0.05). Conclusion High malaria prevalence was found in the study sites, affecting preschool and the school-aged children the most. Although Yellow fever was not detected, its overlap with malaria in Ghana, suggests the importance of enhancing surveillance for both diseases to better prevent and control.

Current status of intestinal parasitosis and microsporidiosis in industrialized countries: Results from a prospective study in France and Luxembourg.

Human intestinal parasitosis and microsporidiosis are a global health concern, mostly in endemic areas but should not be neglected elsewhere. Recent nationwide epidemiological data are scarce, especially from primary health care and developed countries. Diagnosis by molecular tools are increasing and several commercial gastrointestinal panel assays including protozoans and/or helminths are now available. These news tools improve the knowledge into real human parasite epidemiology. This study provides an epidemiological update on intestinal parasites found in primary health care in France and Luxembourg. Two thousand fifty-six stools from primary health care patients were analyzed for the presence of intestinal parasites (IPs) during two different seasons of 2022, the winter and the summer, corresponding to more than 1500 patients from all over France and Luxembourg. Parasite detection was performed combining standard microscopy (merthiolate-iodine-formaldehyde and Bailenger concentration procedures) with two molecular panel assays (AMPLIQUICK Fecal Pretreatment, AMPLIQUICK Protozoans and AMPLIQUICK Helminths, BIOSYNEX, France). The prevalence of IPs in primary care patients reached 33.2%. Blastocystis sp. and Dientamoeba fragilis were the most frequently detected parasites in 20.5% and 13.1% of patients, respectively. Coinfection with two or more parasites was detected in 9.9% of patients. For some parasites, patterns according to gender, age, geography or season have been observed. The high prevalence of pathogenic IPs (about 7%) underlines the importance of investigating gastrointestinal disorders through parasite examination, even in developed countries. The detection of parasites, pathogenic or not, remains a marker of the faecal-oral route of transmission and results should be interpreted accordingly. Parasites molecular characterization give new insights and should encourage further research as industrialized countries are not exempt of parasitic circulation and a better survey is necessary.

Enhanced Detection of Leishmania Parasites in Microscopic Images Using Machine Learning Models.

Cutaneous leishmaniasis is a parasitic disease that poses significant diagnostic challenges due to the variability of results and reliance on operator expertise. This study addresses the development of a system based on machine learning algorithms to detect Leishmania spp. parasite in direct smear microscopy images, contributing to the diagnosis of cutaneous leishmaniasis. Starting with acquiring and labeling 500 images, an experimental design was implemented, including preprocessing and segmentation techniques such as Otsu, local thresholding, and Iterative Global Minimum Search (IGMS) to improve parasite detection. The phenotypic features of the parasites were extracted, focusing on morphology, texture, and color. Machine learning models (ANN, SVM, and RF) optimized through Grid Search were applied for classification. The model with the best results was a Support Vector Machine (SVM), achieving a sensitivity of 91.87% and a specificity of 89.21% at the crop level. Compared with previous studies, these results highlight the relevance and consistency of the methodology used, supporting the initial hypothesis. This suggests that machine learning techniques offer a promising path toward improving the diagnosis of cutaneous leishmaniasis.

Pan African Vivax and Ovale Network (PAVON) Malaria Diagnostic Competency Training: Offering Training Opportunities to Impact Malaria Elimination Strategies in Sub-Saharan Africa.

PAVON has developed a malaria microscopy competency training scheme to augment competency in malaria microscopy. Here, data accrued from training activities between 2020 and 2023 in Botswana are presented. Three trainings were done for 37 central and peripheral level technicians for a two-week period. The scheme consisted of basic theory on Plasmodium parasites, malaria epidemiology and diagnosis. The practicals focused on standard slide preparation, staining, parasite detection, speciation and counting. Scores were assessed by the Wilcoxon signed rank test. Participants who excelled joined the WHO External Competency Assessment for Malaria Microscopy (ECAMM). The median competency scores for the three trainings were detection: 100 (IQR = 94-100), 100 (IQR = 94-100) and 92 (IQR = 92-100), respectively, from pre-test scores of 40 (IQR = 27-54), 44 (IQR = 32-52) and 20 (IQR = 10-40) (z = 2.937, p < 0.003, z = 3.110, p = 0.002 and (z = 2.251, p = 0.024), respectively. Speciation: 93 (IQR = 86-96), 81 (IQR = 73-96) and 88, (IQR = 88-100) from pre-test scores of 50 (IQR = 30-50), 36 (IQR = 20-45) and 17 (IQR = 17-50) (z = 2.936, p < 0.003, z = 3.152, p = 0.002 and z = 3.237, p = 0.001). The competency scores achieved in the ECAMM were 98/98 and 97/98 for sensitivity and specificity pre- and post-tests detection. The training was effective in raising the competency skills of participants and is open to NMPs (National Malaria Programs) for critical capacity building.

Novel Haemocystidium sp. Intraerythrocytic Parasite in the Flatback (Natator depressus) and Green (Chelonia mydas) Turtle in Western Australia.

Malaria and other haemosporidian parasites are common in reptiles. During baseline health surveys of sea turtles in Western Australia (WA), haemosporidian parasites were detected in flatback (Natator depressus) and green (Chelonia mydas) turtle erythrocytes during routine blood film examination. 130 blood samples were screened via polymerase chain reaction (PCR), including 105 N. depressus, 20 C. mydas, and 5 olive ridley turtles (Lepidochelys olivacea). A novel Haemocystidium sp. was identified, detected exclusively in foraging turtles and not in nesting turtles. The combined prevalence by microscopic and molecular methods was 16.9% (22/130), primarily affecting immature C. mydas (77.3%; 17/22). Mature N. depressus were also affected (22.7%; 5/22). DNA sequencing of a partial fragment of the mitochondrial cytochrome b (cytb) gene together with phylogenetic analysis identified two different Haemocystidium sp. genotypes, A and B, with genotype A being most prevalent. The phylogenetic analysis showed close genetic relationships to Haemocystidium sp. in freshwater and terrestrial turtles, suggesting a shared evolutionary lineage despite ecological differences. Preliminary analysis indicates that this parasite is incidental, as no association between health and parasite presence or grade was detected. This study provides the first formal detection of haemosporidian parasites in sea turtles, contributing essential baseline data while highlighting their evolutionary significance and host-parasite ecological relationships.

Non-Invasive Malaria Detection in Sub-Saharan Africa Using a DNA-Based Sensor System.

Malaria poses a serious global health problem, with half the world population being at risk. Regular screening is crucial for breaking the transmission cycle and combatting the disease spreading. However, current diagnostic tools relying on blood samples face challenges in many malaria-epidemic areas. In the present study, we demonstrate the detection of the malaria-causing Plasmodium parasite in non-invasive saliva samples (N = 61) from infected individuals by combining a DNA-based Rolling-circle-Enhanced-Enzyme-Activity-Detection (REEAD) sensor system with a chemiluminescence readout that could be detected with an in-house-developed affordable and battery-powered portable reader. We successfully transferred the technology to sub-Saharan Africa, where the malaria burden is high, and demonstrated a proof of concept in a small study (N = 40) showing significant differences (p < 0.00001) between malaria-positive individuals (N = 33) and presumed asymptomatic negative individuals (N = 7) all collected in Gabon. This is the first successful application of the REEAD sensor system for the detection of malaria in saliva in a high-epidemic area and holds promise for the potential future use of REEAD for malaria diagnosis or surveillance based on non-invasive specimens in sub-Saharan Africa.

Preliminary Evidence of Human Plasmodium in Domestic Animals from a Malaria-Endemic Region in Indonesia

Background In Indonesia, malaria incidence is at a high rate despite maximum preventive efforts. Therefore, this study aims to determine the possibility of a Plasmodium reservoir among domestic animals in malaria-endemic areas. Methods Animal blood was collected using EDTA tubes, then smeared and stained with Giemsa for Plasmodium microscopic identification. About 10 μl of blood was dropped on to a filter paper to capture Plasmodium DNA. Nested PCR was used for parasite molecular detection, while Plasmodium species were identified using the sequenced DNA. Results A total of 208 and 62 animal blood samples were collected from Gaura village, West Sumba and Fakfak village, West Papua, Indonesia respectively. In total, 32 samples from Gaura contained P. falciparum or P. vivax, while the Plasmodium percentage in buffalo, horse, goat, and dogs were 20.7%, 14.3%, 5.8%, 16.7%, respectively. P. knowlesi was not found in any of the samples, and no other species were detected in 18 pig blood samples. Conclusion The human Plasmodium DNA in domestic animals within malaria-endemic regions suggests a potential link to the persistence and high prevalence of malaria in these areas. While the findings suggest a potential role of domestic animals in malaria transmission, they remain preliminary and do not definitively establish domestic animals as reservoirs. Further research is necessary to confirm these findings and to better understand the contribution of domestic animals to the transmission dynamics of malaria.

Molecular Detection and Characterization of Haemonchus spp. in Cattle in Nigeria.

Haemonchosis is a major parasitic infestation in ruminant livestock, causing significant economic losses annually. The causative organisms are helminths of the genus Haemonchus spp. Detection of the causative agent is important for effective management and control of the disease. Molecular detection and characterization of parasites is a very dependable approach for parasite identification, especially where morphological characterization is unreliable. To detect and characterize Haemonchus species in cases of haemonchosis at a Municipal abattoir in Ibadan, Nigeria; abomasal samples were collected from cattle at the abattoir. Polymerase chain reaction (PCR) was used to detect and amplify 320 bp internal transcribed spacer-2 (ITS-2) and 400 bp external transcribed spacer (ETS) genes of the adult worms in the samples. Multiple sequence alignment and phylogenetic tree reconstruction were carried out to further confirm the presence of the worms. PCR, multiple sequence alignment, and phylogenetic reconstruction confirmed the presence of H. placei in the abomasal samples and further confirmed the species as a distinct species of bovine worms at the abattoir. Multiple sequence alignment also revealed genetic sites that can be employed to distinguish H. placei from H. contortus and H. similis. Molecular techniques; PCR and sequence analysis are very important and reliable in the diagnosis of parasitic diseases. This will help to formulate effective control measures for eradication of the parasite.

Disentangling the effects of intermittent faecal shedding and imperfect test sensitivity on the microscopy-based detection of gut parasites in stool samples.

Gut-parasite transmission often involves faecal shedding, and detecting parasites in stool samples remains the cornerstone of diagnosis. However, not all samples drawn from infected hosts contain parasites (because of intermittent shedding), and no test can detect the target parasites in 100% of parasite-bearing samples (because of imperfect sensitivity). Disentangling the effects of intermittent shedding and imperfect sensitivity on pathogen detection would help us better understand transmission dynamics, disease epidemiology, and diagnostic-test performance. Using paediatric Giardia infections as a case-study, here we illustrate a hierarchical-modelling approach to separately estimating the probabilities of host-level infection (Ψ); stool-sample-level shedding, given infection (θ); and test-level detection, given infection and shedding (p). We collected 1-3 stool samples, in consecutive weeks, from 276 children. Samples (413 overall) were independently examined, via standard sedimentation/optical microscopy, by a senior parasitologist and a junior, trained student (826 tests overall). Using replicate test results and multilevel hierarchical models, we estimated per-sample Giardia shedding probability at [Formula: see text] and observer-specific test sensitivities at [Formula: see text] and [Formula: see text]. Gender-specific infection-frequency estimates were [Formula: see text] and [Formula: see text]. Had we used a (hypothetical) Perfect Test with 100% narrow-sense sensitivity (pPT = 1.0), the average probability of detecting Giardia in a sample drawn from an infected child (Ψ = 1.0) would have been Pr(d|i,PT) = Ψ×θ×pPT≈1.0×0.44×1.0≈0.44. Because no test can be >100% sensitive, Pr(d|i) (which measures clinical sensitivity) can only be brought above ~ 0.44 by tinkering with the availability of Giardia in stool samples (i.e., θ); for example, drawing-and-pooling 3 replicate samples would yield [Formula: see text]. By allowing separate estimation (and modelling) of pathogen-shedding probabilities, the approach we illustrate provides a means to study pathogen transmission cycles and dynamics in unprecedented detail. Separate estimation (and modelling) of true test sensitivity, moreover, may cast new light on the performance of diagnostic tests and procedures, whether novel or routine-practice.

Instrument-Free Point-of-Care Diagnostic for Leishmania Parasites.

Leishmaniasis is the second deadliest parasitic disease in the world, after malaria, with an estimated 1.6 million new cases each year. While cutaneous leishmaniasis can result in permanent scars from lesions after treatment, the mucocutaneous and visceral diseases can result in life-altering and life-threatening complications. Accurate species diagnosis is critical for treatment and follow-up, and while PCR-based diagnostics can provide sensitive parasite detection and species identification, they are slow, expensive, and not suitable for low-resource settings. In this publication, we describe our efforts to develop a simple, affordable, and instrument-free Leishmania DNA diagnostic that can be used in both high-tech settings and the field. Computational biology was utilized to design region-targeted RPA oligos and the corresponding CRISPR guides for the detection of all Leishmania species as well as the specific identification of L. (V.) panamensis as a predictor of mucocutaneous disease. Then, we executed systematic approaches for parasite lysis, RPA amplification of DNA, and fluorescent CRISPR crRNA detection. We have demonstrated the ability to detect single-digit parasites without compromising the specificity in identifying single species as the proof of concept for a point-of-care diagnostic. Individual assays were carried out in succession, culminating in an unquenched fluorescent signal quantifiable over negative control. The described work is the foundation which will be implemented into a three-track [all Leishmania, mucocutaneous or visceral only, and a human positive control] assay that we plan to utilize in a Funnel Adapted Sensing Tube (FAST) single use, instrument-free, and affordable diagnostic.

Prevalence and seasonal patterns of vector borne diseases in patients presenting with acute febrile illness in a tertiary care hospital in Puducherry- a prospective observational study.

Vector-borne diseases (VBD) are a major public health concern. Globalization, urbanization & climate change are reasons for the emergence and re-emergence of VBDs. In our study, we looked into the prevalence of VBD infections around our tertiary care hospital in South India. The objective was to determine the prevalence of common VBDs like Malaria, Dengue, Japanese encephalitis (JE), Chikungunya and Scrub typhus in patients with acute febrile illness (AFI). This was a prospective laboratory based observational study. Blood samples from patients with AFI were tested for Dengue NS1 Antigen, IgM and IgG; and IgM antibodies for JE, Chikungunya and Scrub typhus using ELISA tests. Peripheral blood smear examination was performed for malarial parasite detection. Total 802 samples were analysed. The sample positivity rate for VBD was 63.6% (510/802 samples) On diving the positive results across seasons in the study period, the VBD positivity rates were 66.3%, 49.1%, 61.2% and 67.3% for the first post-monsoon, summer, monsoon and the second post-monsoon seasons respectively- a trend of increased rates noted during the post-monsoon seasons. 192 samples (23.9%) were positive for scrub typhus alone, 189 samples (23.6%) were positive for dengue infection alone, six samples (0.7%) were positive for chikungunya infection alone, 121 samples (15.1%) were positive for dengue plus scrub typhus co-infection, two samples (0.2%) were positive for dengue plus chikungunya co-infection, while 292 samples (36.4%) showed negative results. None of the samples were positive for malaria and Japanese encephalitis. Scrub typhus and dengue were the most prevalent VBDs in concordance with the prevalence pattern noted in other studies in South India. Increasing awareness and surveillance of the VBDs, developing stringent control policies, easy access to testing and initiating early appropriate therapy can help reduce the incidence of VBDs.

Systemic Granulomatosis in the Meagre Argyrosomus regius: Fishing for a Plausible Etiology.

Meagre (Argyrosomus regius) is one of the fast-growing species considered for sustainable aquaculture development along the Mediterranean and Eastern Atlantic coasts. The emergence of Systemic Granulomatosis (SG), a disease marked by multiple granulomas in various tissues, poses a significant challenge in meagre aquaculture. In the current study, we investigate the association of Mycobacterium spp. and SG in offshore aquaculture facilities in Sardinia, Italy. A total of 34 adult seemingly healthy meagre were arbitrarily collected and analyzed, combining histological, microbiological, molecular, metagenomics, and in situ techniques to investigate the presence of pathogens. Ziehl-Neelsen (ZN), periodic acid-schiff (PAS), and Giemsa stains were performed for the detection of acid-fast bacteria, common parasites, and fungi within granulomas, respectively. Granulomas were detected in 91% (31/34) of fish. The affected organs were kidney (88%), liver (47%), heart (41%), intestine (17.6%), and brain (5%). Acid-fast staining, along with Mycobacterium spp. specific quantitative PCR (qPCR), in situ hybridization (ISH) assay, and microbiological analyses showed negative results for the detection of Mycobacterium spp. and other bacteria implicated in granuloma formation. However, PCR amplification and sequencing of the 65-kDa heat shock protein gene revealed the presence of M. chelonae in 13% of both formalin-fixed and frozen liver tissues. Bacterial isolation failed to detect nontuberculous mycobacteria (NTM) and other bacteria typically associated with granulomas. Consistently, the use of an M. chelonae-specific probe in ISH failed to identify this bacterial species in granulomas. Collectively, results do not support the role of M. chelonae in the development of granulomas and suggest rejecting the hypothesis of a potential link between NTM and SG.