Evaluation of Cytopathological Techniques for the Diagnosis of Canine Visceral Leishmaniosis with Lymph Node Samples

Summary

The identification of the parasite in cytological smears of lymph node aspirates is a widely applied technique for the direct diagnosis of Leishmania spp. infection, especially in endemic areas. Although very specific, this method has limited sensitivity, and improving the technique would be highly desirable. This study aimed to evaluate the efficacy of conventional smear cytology (SC), liquid-based cytology (LBC), cell block (CB) stained with haematoxylin and eosin (HE) and immunocytochemistry (ICC), and formalin-fixed paraffin wax-embedded tissue immunohistochemistry (FFPE–IHC) compared with serology and polymerase chain reaction for the diagnosis of canine visceral leishmaniosis (CVL) in lymphoid tissue. The use of a preservative medium and centrifugation for cytological samples reduced the number of unsatisfactory artefacts/background. Moreover, LBC allowed excellent cellular preservation and the application of ancillary techniques, such as CB and ICC. SC was the most accurate morphological diagnostic method (45.0%). CB–ICC alone or associated with SC demonstrated significantly higher sensitivity (70.0% and 72.0%, respectively) when compared with SC alone (34.00%). CB–ICC was found to be more effective in the detection of infected animals with mild clinical signs, similar to FFPE–IHC. The specificity and positive predictive value were similar between all methods. Finally, the detection limit for CB–ICC and SC + CB–ICC was identical (18.46 amastigotes/mm²). Our study suggests that CB–ICC is a promising tool for improvement of the cytopathological diagnosis of CVL and may be applied in routine epidemiological screening.

Introduction

Zoonotic visceral leishmaniosis, caused by the protozoan Leishmania infantum, is an important public health issue in the Mediterranean basin, Central and South America and parts of Asia, due to the large number of cases and the clinical severity of the disease. Dogs (Canis lupus familiaris) are considered the main domestic reservoir of the parasite in urban and peri-urban areas, and have a role in the epidemiological cycle of human transmission since an increase in cases of canine visceral leishmaniosis (CVL) usually precedes human infection (Alvar et al., 2012).

The diagnosis of CVL often demands an integrated approach due to the wide clinical spectrum of the disease and presence of non-specific clinicopathological abnormalities (Solano-Gallego et al., 2009). Classical laboratory methods for diagnosis of CVL include direct parasitological identification and serological and molecular assays (Tavares Veras et al., 2014). Direct microscopical detection of the parasite in air-dried stained smears made from aspirates of the lymph nodes, spleen and bone marrow have high specificity, which allows the confirmation of Leishmania spp. infection. In vaccinated dogs, the application of direct diagnostic methodologies is recommended, since no test for discrimination between antibodies due to vaccination or to L. infantum natural infection are described (Solano-Gallego et al., 2017). However, the sensitivity of conventional smear cytology (SC) is generally <50% due to the limited parasite identification, especially in those dogs that are mildly symptomatic or asymptomatic and those having a low parasitic load, which could be implicated in false-negative results (Barrouin-Melo et al., 2004, Reis et al., 2006, Gomes et al., 2008, Maia and Campino, 2008). The lymph nodes are a frequent site of infection by the parasite, both in symptomatic and asymptomatic dogs, and fine needle aspiration is easier and safer to perform from lymph nodes than from bone marrow (Lima et al., 2004, Saridomichelakis et al., 2005).

In the last 10 years, several new technologies have been introduced in human medicine, attempting to reduce the occurrence of false-negative results in cytology due to sampling, screening and interpretation problems (Gibb and Martens, 2011). Techniques that use a preservative fluid medium, such as liquid-based cytology (LBC), for slide preparation, producing thin layers of immediately-fixed cell smears, have been developed to overcome these limitations (Pawar et al., 2014). Even so, LBC has largely replaced conventional cytological preparations for female cervical screening worldwide (Kavatkar et al., 2008). Moreover, this method allows samples to be used for ancillary techniques, such as cell block (CB) and immunocytochemistry (ICC) (Bhatia et al., 2008, Kavatkar et al., 2008, Khan et al., 2012). The CB technique enables the concentration of cells in the suspension, and the resulting pellet is processed to form a paraffin wax block, which improves diagnostic accuracy and increases the cellular yield (Khan et al., 2012). From the CBs, it is also possible to obtain paraffin wax-embedded sections for histopathology, multiple immunolabelling and molecular studies (Bhatia et al., 2008).

Despite the importance of cytological diagnosis for clinical practice (Solano-Gallego et al., 2011) and epidemiological monitoring (Ciaravolo et al., 2015) of CVL, few reports of LBC, CB and ICC can be found in veterinary medicine (Fernandes et al., 2016, Menezes et al., 2016). In this context, the present study aimed to apply these techniques to aspirate samples taken from the popliteal lymph node of dogs with CVL and to compare the performance with conventional SC and histopathology.