Abstract
Cutaneous leishmaniasis (CL) remains a globally spreading public health problem. Among Latin America countries, Brazil has the greatest number of recorded CL cases with several Leishmania species being associated with human cases. Laboratory diagnosis is one of the major challenges to disease control due to the low accuracy of parasitological techniques, the restricted use of molecular techniques, and the importance of differential diagnosis with regard to several dermatological and systemic diseases. In response, we have developed and validated an immunohistochemistry (IHC) technique for CL diagnosis using anti-mTXNPx monoclonal antibody (mAb). Recombinant Leishmania–mTXNPx was produced and used as an immunogen for mAb production through the somatic hybridization technique. The viability of mAb labeling of Leishmania amastigotes was tested by IHC performed with skin biopsies from hamsters experimentally infected with Leishmania amazonensis, Leishmania braziliensis, and Leishmania guyanensis. The enzymes horseradish peroxidase (IHC-HRP) and alkaline phosphatase (IHC-AP), both biotin-free polymer detection systems, were used in the standardization step. The IHC was further validated with skin biopsies from 49 CL patients diagnosed by clinical examination and quantitative real-time polymerase chain reaction and from 37 patients presenting other dermatological infectious diseases. Other parasitological techniques, such as direct examination and culture, were also performed for confirmed CL patients. Histopathology and IHC were performed for all included patients. Overall, the highest sensitivity was observed for IHC-AP (85.7%), followed by IHC-HRP (79.6%), direct examination (77.6%), histopathological examination (HE; 65.3%), and in vitro culture (49%). Only IHC and HE presented specificity over 90% and were able to detect CL patients regardless of parasite burden (odds ratio > 1.94; 95%CI: 0.34–11.23). A significant increase in positivity rates was observed when IHC-AP was combined with direct examination (95.9%) and HE (93.9%). The IHC techniques evaluated in here detected the main Leishmania species causing CL in Brazil and can support diagnostic strategies for controlling this neglected disease, especially if used in combination with other approaches for an integrative laboratorial diagnosis.
Highlights
Cutaneous leishmaniasis (CL) is a neglected global disease that is often prevalent in the poorest and marginalized communities
The fragment of 226aa corresponding to L. infantum–mitochondrial tryparedoxin peroxidase (mTXNPx) was used to obtain the DNA sequence by reverse translation, which was optimized for expression in prokaryotic systems (Supplementary Figure 2)
Protein expression was confirmed through the difference between the bacterial lysate before and after IPTG induction in 15% SDS-PAGE, and mTXNPx purification was confirmed by reactivity with 6x-His-tag antibody (Supplementary Figure 3)
Summary
Cutaneous leishmaniasis (CL) is a neglected global disease that is often prevalent in the poorest and marginalized communities. Immunological methods are not currently used in clinical practice, different antigens have been evaluated to improve the restricted scenario for CL diagnosis (Freire et al, 2021), including peroxidoxin (MenezesSouza et al, 2014), renamed as mitochondrial tryparedoxin peroxidase (mTXNPx) in trypanosomatids (Teixeira et al, 2015). This member of an antioxidant protein family from Leishmania is highly expressed in amastigote forms and has been detected in the immunochromatographic assay CL DetectTM Rapid Test (InBios International Inc., Seattle, WA, United States), with sensitivity of around 65% in Old World countries (Bennis et al, 2018; Vink et al, 2018)
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