Mayara Ingrid Sousa Lima

The role of the human papillomavirus (HPV) in the establishment of cervical cancer has driven studies to find more effective methods of viral detection so that early intervention strategies can be performed. However, the methods still have limitations, especially regarding detecting the different genotypes simultaneously. We have developed a high-throughput system using a single-tube nested-multiplex polymerase chain reaction (NMPCR) for the detection of 40 HPV genotypes using capillary electrophoresis. The NMPCR assay was compared to the Hybrid Capture 2 assay (HC2) with 40 women from the Northeast of Brazil (São Luis, MA), a high endemic region, where the HPV positivity was 75% and 37.5%, respectively. These results were validated by performing a molecular epidemiological study on 5223 Brazilian women undergoing gynecological examinations from 2009 to 2017, who presented with an HPV prevalence of 59%. Multiple infections were found in 62.5% and 58% of the patients from the endemic region and from the Brazilian women population, respectively, mostly presenting high-risk genotypes (90.5% and 60%, respectively). Considering cervical intraepithelial neoplasia and adenocarcinomas, the sensitivity and specificity were 97.5% and 100%, respectively. The NMPCR assay was also capable of identifying viral subtypes in cases of multiple infections, even with low viral loads (10⁻⁶ ng/µL of HPV DNA). The NMPCR test is a promising and robust tool for HPV diagnostics and a screening tool for prevention of cervical cancer.

The early diagnosis of leprosy serves as an important tool to reduce the incidence of this disease in the world. Phage display (PD) technology can be used for mapping new antigens to the development of immunodiagnostic platforms. Our objective was to identify peptides that mimic Mycobacterium leprae proteins as serological markers using phage display technology. The phages were obtained in the biopanning using negative and positive serum from household contacts and leprosy patients, respectively. Then, the peptides were synthesized and validated in silico and in vitro for detection of IgG from patients and contacts. To characterize the native protein of M. leprae, scFv antibodies were selected against the synthetic peptides by PD. The scFv binding protein was obtained by immunocapture and confirmed using mass spectrometry. We selected two phase-fused peptides, MPML12 and MPML14, which mimic the HSP60 protein from M. leprae. The peptides MPML12 and MPML14 obtained 100% and 92.85% positivity in lepromatous patients. MPML12 and MPM14 detect IgG, especially in the multibacillary forms. The MPML12 and MPML14 peptides had positivity of 11.1% and 16.6% in household contacts, respectively. There was no cross-reaction in patient’s samples with visceral leishmaniasis, tuberculosis and other mycobacteriosis for both peptides. Given these results and the easy obtainment of mimetic antigens, our peptides are promising markers for application in the diagnosis of leprosy, especially in endemic and hyperendemic regions.

Leishmaniasis is a disease caused by protozoa of the genus Leishmania. Treatment options are limited, and there are frequent cases of treatment failure and clinical relapse. To understand these phenomena better, a systematic review was conducted, considering studies published between 1990 and 2021 in Portuguese, English, and Spanish. The review included 64 articles divided into three categories. Case reports (26 articles) focused on treatment failure and clinical relapse in cutaneous leishmaniasis patients (47.6%), primarily affecting males (74%) and children (67%), regardless of the clinical manifestation. Experimental studies on the parasite (19 articles), particularly with L. major (25%), indicated that alterations in DNA and genic expression (44.82%) played a significant role in treatment failure and clinical relapse. Population data on the human host (19 articles) identified immunological characteristics as the most associated factor (36%) with treatment failure and clinical relapse. Each clinical manifestation of the disease presented specificities in these phenomena, suggesting a multifactorial nature. Additionally, the parasites were found to adapt to the drugs used in treatment. In summary, the systematic review revealed that treatment failure and clinical relapse in leishmaniasis are complex processes influenced by various factors, including host immunology and parasite adaptation.

The parasitic protozoan Leishmania (Leishmania) infantum is the etiological agent of human visceral leishmaniasis in South America, an infectious disease associated with malnutrition, anemia, and hepatosplenomegaly. In Brazil alone, around 2700 cases are reported each year. Treatment failure can occur as a result of drug, host, and/or parasite-related factors. Here, we isolated a Leishmania species from a pediatric patient with visceral leishmaniasis that did not respond to chemotherapy, experiencing a total of nine therapeutic relapses and undergoing a splenectomy. The parasite was confirmed as L. (L.) infantum after sequencing of the ribosomal DNA internal transcribed spacer, and the clinical isolate, in both promastigote and amastigote forms, was submitted to in vitro susceptibility assays with all the drugs currently used in the chemotherapy of leishmaniasis. The isolate was susceptible to meglumine antimoniate, amphotericin B, pentamidine, miltefosine, and paromomycin, similarly to another strain of this species that had previously been characterized. These findings indicate that the multiples relapses observed in this pediatric patient were not due to a decrease in the drug susceptibility of this isolate; therefore, immunophysiological aspects of the patient should be further investigated to understand the basis of treatment failure in this case.

Diffuse cutaneous leishmaniasis (DCL) is a rare type of leishmaniasis characterized by diffuse skin lesions. In Brazil, Leishmania (L.) amazonensis is the main etiological agent of this clinical form. The state of Maranhão has the highest prevalence of this disease in the country, as well as a high rate of HIV infection. Here, we report the first case of DCL/HIV of Brazil. A 46-year-old man from the Amazonian area of Maranhão state presented atypical lesion in the left upper limb and dissemination of diffuse erythematous nodules over his entire body. Histopathological examination confirmed the presence of intracellular amastigotes of Leishmania, and a polymerase chain reaction and molecular identification by restriction fragment profile identified L. (L.) amazonensis as the causative agent of the disease. The patient was also diagnosed with HIV virus after the leishmaniasis diagnosis. The initial treatments for leishmaniasis were liposomal amphotericin B (AmB-L) (4 mg/kg) for 10 days and prophylactic use of Glucantime® (10 mg/Sb+5/kg) for 2 months. After unsuccessful initial treatments, he was treated with a combination of AmB-L (4 mg/kg) alternated with pentamidine (4 mg/kg) for 10 days but failed in the first therapeutic cycle. Subsequently, he had a good response to treatment with pentamidine (4 mg/kg).

Phenolic glycolipid I (PGL-I) is an abundant antigen on the Mycobacterium leprae cell wall, commonly used for operational classification of leprosy patients. Our aim was to develop PGL-I mimotopes with similar characteristics and functions of the native antigen. We have used a random peptide phage display (PD) library for selections against the monoclonal antibody anti-PGL-I. After three selection cycles, six peptides were identified. All sequences were interspersed by a spacer generating a chimeric peptide (PGLI-M3) that was artificially synthesized. The highly reactive peptide was submitted to a reverse PD selection with a single-chain Fv (scFv) antibody fragment combinatorial library. The most reactive scFv was then validated by enzyme-linked immunosorbent assay (ELISA) against both native PGL-I and two derived synthetic (NDO and ND-O-HSA). We have further proved the scFv specificity by detecting M. leprae bacilli in leprosy lesions through immunohistochemistry. We then described its applicability in ELISA for all clinical forms and household contacts (HC). Afterward, we showed differential binding affinities of PGLI-M3 to sera (anti-PGL-I IgM) from all leprosy clinical forms through surface plasmon resonance (SPR). ELISA IgM detection showed 89.1% sensitivity and 100% specificity, considering all clinical forms. Positivity for anti-PGL-I IgM was twofold higher in both HC and patients with paucibacillary forms in hyperendemic regions than in endemic ones. The SPR immunosensor was able to differentiate clinical forms with 100% accuracy. This is the first time that a PGL-I mimotope has efficiently mimicked the carbohydrate group of the M. leprae antigen with successful immunoassay applications and may become a substitute for the native antigen.