Rapid Detection of Leishmania infantum Infection in Dogs: Comparative Study Using an Immunochromatographic Dipstick Test, Enzyme-Linked Immunosorbent Assay, and PCR

Current zoonotic visceral leishmaniasis (ZVL) control programs in Brazil include the culling of Leishmania infantum-infected reservoir dogs, a strategy that has failed to prevent a rise of canine and human ZVL cases over the past decade. One of the main reasons this strategy has failed is because of a long delay between sample collection, sample analysis, and control implementation. A rapid, sensitive, and specific diagnostic tool would be highly desirable, because it would allow control interventions to be implemented in situ. We compared an immunochromatographic dipstick test to enzyme-linked immunosorbent assay (ELISA) and PCR for detecting L. infantum infections in dogs from an area of ZVL endemicity in Brazil. The dipstick test was shown to have 61 to 75% specificity and 72 to 77% sensitivity, compared to 100% specificity for both ELISA and PCR and 71 to 88% and 51 to 64% sensitivity for ELISA and PCR, respectively. Of the field samples tested, 92 of 175 (53%), 65 of 175 (37%), and 47 of 175 (27%) were positive by dipstick, ELISA, and PCR, respectively. The positive and negative predictive values for the tested dipstick were 58 to 77% and 75%, respectively. Efforts should be made to develop a more specific dipstick test for diagnosis of leishmaniasis, because they may ultimately prove more cost-effective than currently used diagnostic tests when used in mass-screening surveys

Approaches to study and conceptions of biology: Differential outcomes for generalist and vocational degree students

KEYWORDS: learning in biology, vocational learning, generalist science degree, Learner Profiling BACKGROUND: Students have diverse learning styles and a raft of instruments have been created and validated to examine learner characteristics such as approaches to study (Biggs, 1987; Biggs, Kember & Leung, 2001) and conception of discipline in various science-based courses, including maths (Crawford, Gordon, Nicholas & Prosser, 1998), physics (Prosser, Trigwell, Hazel & Waterhouse, 2000) and biology (Quinnell, May, Peat & Taylor, 2005). Student survey response data can be analysed statistically in a number of ways: for example, students returning similar responses (i.e. students who adopt similar orchestrations) can be characterised using hierarchical cluster analysis (see Trigwell, Hazel & Prosser, 1996; Trigwell Prosser & Waterhouse, 1999; Prosser et al., 2000). Such analysis has allowed us to monitor changes in these learning orchestrations over the course of a semester by extending the work of Prosser et al. (2000) and employing sequential hierarchical cluster analyses in a process we refer to as ‘Learner Profiling’ (Quinnell, May & Peat, 2012). We have demonstrated that 48% of students in an introductory university biology course changed their learning orchestrations from the start to the end of their first semester at university, with some orchestrations being more persistent than others (Quinnell et al., 2012). Biology, like other enabling science courses at first year level, involves service teaching to some extent, and we were interested to see whether students enrolled in vocational or professional degrees engaged with our curriculum differently from students enrolled in generalist degrees. With this in mind we are beginning to explore the notion of differences in learning agendas of our students and if this has an impact on how students engage with our biology curriculum. AIM: Our aim was to evaluate our learner profiling method as a means to inform curriculum design which must, by necessity, be suitable for students across a broad range of degree programs, i.e. generalist and vocational degrees. DESIGN AND METHODS: We profiled biology students as described previously (Quinnell et al., 2012) and employed post-hoc analyses to see how elements of the curriculum (good teaching, clear goals, independence, assessment, workload; as defined by Ramsden, 1991) correlate with the changes in Learner Profile. We also identified students are ‘generalist’ or ‘vocational’ based on their degree program. RESULTS: Interestingly, although perhaps not surprisingly, students enrolled in generalist degrees demonstrated greater engagement with our biology curriculum than those enrolled in vocational degrees. Our data provide some evidence that our curriculum: 1. supports generalist degree students whose conception of biology is sound and whose study approach is intrinsic, 2. is less than ideal for meeting the needs of students in vocational degrees, and 3. has failed to engage students who demonstrated dissonance at the start of semester. CONCLUSIONS: Our findings suggest that a course in biology literacy would be more suitable to students in vocational degrees and a course that is biology content-rich would suit our generalist-degree students. REFERENCES: Biggs, J. (1987). Student approaches to learning and studying. Melbourne, Australian Council for Educational Research. Biggs, J., Kember D., & Leung D. Y. P. (2001). The revised two-factor Study Process Questionnaire: R-SPQ-2F. British Journal of Educational Psychology 71: 133-149. Crawford, K., Gordon, S., Nicholas, J., & Prosser, M. (1988). Qualitatively different experiences of learning mathematics at university. Learning and Instruction, 8, 455–468. Prosser, M., Trigwell, K., Hazel, E., & Waterhouse, F. (2000). Students’ experiences of studying physics concepts: the effects of disintegrated perceptions and approaches, European Journal of Psychology of Education, 15, 61-74. Quinnell, R., May, E., & Peat, M. (2012). Conceptions of Biology and Approaches to Learning of First Year Biology Students: Introducing a technique for tracking changes in learner profiles over time. International Journal of Science Education, 34(7), 1053-1074. Quinnell, R., May, E., Peat, M., & Taylor, C. (2005). Creating a reliable instrument to assess students’ conceptions of studying biology at tertiary level. Proceedings of the Uniserve Science Conference: Blended Learning in Science Teaching and Learning, 30 September 2005 (pp. 87-92) Sydney: Uniserve Science, The University of Sydney. http://science.uniserve.edu.au/pubs/procs/wshop10/2005Quinnell.pdf Ramsden, P. (1991). A performance indicator of teaching quality in Higher Education: The Course Experience Questionnaire. Studies in Higher Education, 16(2), 129-150. Trigwell, K., Hazel, E., & Prosser, M. (1996). Perceptions of the learning environment and approaches to learning university science at the topic level. Different Approaches: Theory and Practice in Higher Education. Proceedings HERDSA Conference 1996. Perth, Western Australia, 8-12 July. (Retrieved 24 March 2011 from http://www.herdsa.org.au/confs/1996/trigwell2.html) Trigwell, K., Prosser, M., & Waterhouse F. (1999). Relations between teachers' approaches to teaching and students' approaches to learning. Higher Education, 37(1), 57-70

Profiling our students' learning orchestrations to evaluate the biology curriculum

© 2018, Institute for Innovation in Science and Mathematics Education. We have identified the major shifts in individual student study orchestrations over the first semester of a university biology course. We offer evidence that our curriculum, designed and taught by generalist biologists, has engaged generalist degree students. Professional degree students have not engaged with this course to the same level and many were demonstrably dissonant. At the end of semester, dissonant students, from both generalist and professional degrees, demonstrated little engagement with the curriculum, which is consistent with previous reports of the high degree of disengagement of first year students. The challenge to improve the engagement of students in professional degrees and to address the tendency towards dissonance and disengagement by our first year students is discussed and improvements in engagement are likely to be aided by systems that allow students to assess for themselves their approaches to study and conceptions of discipline development over the course of their degree

Heterogeneities in leishmania infantum infection : using skin parasite burdens to identify highly infectious dogs

Background: The relationships between heterogeneities in host infection and infectiousness (transmission to arthropod vectors) can provide important insights for disease management. Here, we quantify heterogeneities in Leishmania infantum parasite numbers in reservoir and non-reservoir host populations, and relate this to their infectiousness during natural infection. Tissue parasite number was evaluated as a potential surrogate marker of host transmission potential. Methods: Parasite numbers were measured by qPCR in bone marrow and ear skin biopsies of 82 dogs and 34 crab-eating foxes collected during a longitudinal study in Amazon Brazil, for which previous data was available on infectiousness (by xenodiagnosis) and severity of infection. Results: Parasite numbers were highly aggregated both between samples and between individuals. In dogs, total parasite abundance and relative numbers in ear skin compared to bone marrow increased with the duration and severity of infection. Infectiousness to the sandfly vector was associated with high parasite numbers; parasite number in skin was the best predictor of being infectious. Crab-eating foxes, which typically present asymptomatic infection and are non-infectious, had parasite numbers comparable to those of non-infectious dogs. Conclusions: Skin parasite number provides an indirect marker of infectiousness, and could allow targeted control particularly of highly infectious dogs

Evaluation of rK39 rapid diagnostic tests for canine visceral leishmaniasis : longitudinal study and meta-analysis

Canine visceral leishmaniasis is a vector-borne disease caused by the intracellular parasite Leishmania infantum. It is an important veterinary disease, and dogs are also the main animal reservoir for human infection. The disease is widespread in the Mediterranean area, and parts of Asia and South and Central America, and is potentially fatal in both dogs and humans unless treated. Diagnosis of canine infections requires serological or molecular tests. Detection of infection in dogs is important prior to treatment, and in epidemiological studies and control programmes, and a sensitive and specific rapid diagnostic test would be very useful. Rapid diagnostic tests (RDTs) have been developed, but their diagnostic performance has been reported to be variable. We evaluated the sensitivity of a RDT based on serological detection of the rK39 antigen in a cohort of naturally infected Brazilian dogs. The sensitivity of the test to detect infection was relatively low, but increased with time since infection and the severity of infection. We then carried out a meta-analysis of published studies of rK39 RDTs, evaluating the sensitivity to detect disease and infection. The results suggest that rK39 RDTs may be useful in a veterinary clinical setting, but the sensitivity to detect infection is too low for operational control programmes

Efficacy of Thermotherapy to Treat Cutaneous Leishmaniasis Caused by Leishmania tropica in Kabul, Afghanistan: A Randomized, Controlled Trial

BACKGROUND: Pentavalent antimony is the agent recommended for treatment of cutaneous leishmaniasis (CL). Its use is problematic, because it is expensive and because of the potential for drug-associated adverse effects during a lengthy and painful treatment course. METHODS: We tested the efficacy of thermotherapy for the treatment of CL due to Leishmania tropica in a randomized, controlled trial in Kabul, Afghanistan. We enrolled 401 patients with a single CL lesion and administered thermotherapy using radio-frequency waves (1 treatment of ≥1 consecutive application at 50°C for 30 s) or sodium stibogluconate (SSG), administered either intralesionally (a total of 5 injections of 25 mL every 57 days, depending on lesion size) or intramuscularly (20 mg/kg daily for 21 days). RESULTS: Cure, defined as complete reepithelialization at 100 days after treatment initiation, was observed in 75 (69.4%) of 108 patients who received thermotherapy, 70 (75.3%) of 93 patients who received intralesional SSG, and 26 (44.8%) of 58 patients who received intramuscular SSG. The OR for cure with thermotherapy was 2.80 (95% confidence interval [CI], 1.455.41), compared with intramuscular SSG treatment (P = .002). No statistically significant difference was observed in the odds of cure in comparison of intralesional SSG and thermotherapy treatments. The OR for cure with intralesional SSG treatment was 3.75 (95% CI, 1.867.54), compared with intramuscular SSG treatment (P 100 days, respectively; P = .003). CONCLUSIONS: Thermotherapy is an effective, comparatively well-tolerated, and rapid treatment for CL, and it should be considered as an alternative to antimony treatment

Antibody response to sand fly saliva is a marker of transmission intensity but not disease progression in dogs naturally infected with Leishmania infantum

BACKGROUND: Antibody responses to sand fly saliva have been suggested to be a useful marker of exposure to sand fly bites and Leishmania infection and a potential tool to monitor the effectiveness of entomological interventions. Exposure to sand fly bites before infection has also been suggested to modulate the severity of the infection. Here, we test these hypotheses by quantifying the anti-saliva IgG response in a cohort study of dogs exposed to natural infection with Leishmania infantum in Brazil. METHODS: IgG responses to crude salivary antigens of the sand fly Lutzomyia longipalpis were measured by ELISA in longitudinal serum samples from 47 previously unexposed sentinel dogs and 11 initially uninfected resident dogs for up to 2 years. Antibody responses were compared to the intensity of transmission, assessed by variation in the incidence of infection between seasons and between dogs. Antibody responses before patent infection were then compared with the severity of infection, assessed using tissue parasite loads and clinical symptoms. RESULTS: Previously unexposed dogs acquired anti-saliva antibody responses within 2 months, and the rate of acquisition increased with the intensity of seasonal transmission. Over the following 2 years, antibody responses varied with seasonal transmission and sand fly numbers, declining rapidly in periods of low transmission. Antibody responses varied greatly between dogs and correlated with the intensity of transmission experienced by individual dogs, measured by the number of days in the field before patent infection. After infection, anti-saliva antibody responses were positively correlated with anti-parasite antibody responses. However, there was no evidence that the degree of exposure to sand fly bites before infection affected the severity of the infection. CONCLUSIONS: Anti-saliva antibody responses are a marker of current transmission intensity in dogs exposed to natural infection with Leishmania infantum, but are not associated with the outcome of infection

Of cattle, sand flies and men : a systematic review of risk factor analyses for South Asian visceral leishmaniasis and implications for elimination

Background: Studies performed over the past decade have identified fairly consistent epidemiological patterns of risk factors for visceral leishmaniasis (VL) in the Indian subcontinent. Methods and Principal Findings: To inform the current regional VL elimination effort and identify key gaps in knowledge, we performed a systematic review of the literature, with a special emphasis on data regarding the role of cattle because primary risk factor studies have yielded apparently contradictory results. Because humans form the sole infection reservoir, clustering of kala-azar cases is a prominent epidemiological feature, both at the household level and on a larger scale. Subclinical infection also tends to show clustering around kala-azar cases. Within villages, areas become saturated over a period of several years; kala-azar incidence then decreases while neighboring areas see increases. More recently, post kalaazar dermal leishmaniasis (PKDL) cases have followed kala-azar peaks. Mud walls, palpable dampness in houses, and peridomestic vegetation may increase infection risk through enhanced density and prolonged survival of the sand fly vector. Bed net use, sleeping on a cot and indoor residual spraying are generally associated with decreased risk. Poor micronutrient status increases the risk of progression to kala-azar. The presence of cattle is associated with increased risk in some studies and decreased risk in others, reflecting the complexity of the effect of bovines on sand fly abundance, aggregation, feeding behavior and leishmanial infection rates. Poverty is an overarching theme, interacting with individual risk factors on multiple levels. Conclusions: Carefully designed demonstration projects, taking into account the complex web of interconnected risk factors, are needed to provide direct proof of principle for elimination and to identify the most effective maintenance activities to prevent a rapid resurgence when interventions are scaled back. More effective, short-course treatment regimens for PKDL are urgently needed to enable the elimination initiative to succeed

Loss of top-down biotic interactions changes the relative benefits for obligate mutualists

The collapse of mutualisms owing to anthropogenic changes is contributing to losses of biodiversity. Top predators can regulate biotic interactions between species at lower trophic levels and may contribute to the stability of such mutualisms, but they are particularly likely to be lost after disturbance of communities. We focused on the mutualism between the fig tree Ficus microcarpa and its host-specific pollinator fig wasp and compared the benefits accrued by the mutualists in natural and translocated areas of distribution. Parasitoids of the pollinator were rare or absent outside the natural range of the mutualists, where the relative benefits the mutualists gained from their interaction were changed significantly away from the plant's natural range owing to reduced seed production rather than increased numbers of pollinator offspring. Furthermore, in the absence of the negative effects of its parasitoids, we detected an oviposition range expansion by the pollinator, with the use of a wider range of ovules that could otherwise have generated seeds. Loss of top-down control has therefore resulted in a change in the balance of reciprocal benefits that underpins this obligate mutualism, emphasizing the value of maintaining food web complexity in the Anthropocene

Association between canine leishmaniosis and Ehrlichia canis co-infection: a prospective case-control study

Abstract Background In the Mediterranean basin, Leishmania infantum is a major cause of disease in dogs, which are frequently co-infected with other vector-borne pathogens (VBP). However, the associations between dogs with clinical leishmaniosis (ClinL) and VBP co-infections have not been studied. We assessed the risk of VBP infections in dogs with ClinL and healthy controls. Methods We conducted a prospective case-control study of dogs with ClinL (positive qPCR and ELISA antibody for L. infantum on peripheral blood) and clinically healthy, ideally breed-, sex- and age-matched, control dogs (negative qPCR and ELISA antibody for L. infantum on peripheral blood) from Paphos, Cyprus. We obtained demographic data and all dogs underwent PCR on EDTA-blood extracted DNA for haemoplasma species, Ehrlichia/Anaplasma spp., Babesia spp., and Hepatozoon spp., with DNA sequencing to identify infecting species. We used logistic regression analysis and structural equation modelling (SEM) to evaluate the risk of VBP infections between ClinL cases and controls. Results From the 50 enrolled dogs with ClinL, DNA was detected in 24 (48%) for Hepatozoon spp., 14 (28%) for Mycoplasma haemocanis, 6 (12%) for Ehrlichia canis and 2 (4%) for Anaplasma platys. In the 92 enrolled control dogs, DNA was detected in 41 (45%) for Hepatozoon spp., 18 (20%) for M. haemocanis, 1 (1%) for E. canis and 3 (3%) for A. platys. No Babesia spp. or “Candidatus Mycoplasma haematoparvum” DNA was detected in any dog. No statistical differences were found between the ClinL and controls regarding age, sex, breed, lifestyle and use of ectoparasitic prevention. A significant association between ClinL and E. canis infection (OR = 12.4, 95% CI: 1.5–106.0, P = 0.022) was found compared to controls by multivariate logistic regression. This association was confirmed using SEM, which further identified that younger dogs were more likely to be infected with each of Hepatozoon spp. and M. haemocanis, and dogs with Hepatozoon spp. were more likely to be co-infected with M. haemocanis. Conclusions Dogs with ClinL are at a higher risk of co-infection with E. canis than clinically healthy dogs. We recommend that dogs diagnosed with ClinL should be tested for E. canis co-infection using PCR