High seroprevalence of Rickettsia spp. and molecular detection of Rickettsia amblyommatis in human-biting ticks from the eastern Amazon, Brazil Mayra F. F. R. Ferreira, Lina de Campos Binder, Rafael M. S. Nogueira, Ruth M. M. e Silva, Carlos C. M. Ramos, et al. Parasites and Vectors, 2025 Background In Brazil, spotted fever (SF) is caused by the bacteria Rickettsia parkeri and Rickettsia rickettsii. Seroepidemiological data on Rickettsia spp. in humans are rare in Brazil and nonexistent in the Amazon biome. We sought to quantify antibodies reactive to Rickettsia spp. in serum samples collected from humans in the Amazonia biome, and to detect Rickettsia spp. in ticks parasitizing these hosts. Methods Human blood samples were collected from three different locations within the eastern Amazon in Maranhão State, northeastern Brazil between 2010 and 2018. Sera generated from those samples were tested for the presence of antibodies reactive to Rickettsia by immunofluorescence assay (IFA) using crude antigens from five Rickettsia isolates from Brazil: R. rickettsii strain Taiaçu, R. amblyommatis strain Ac37, Rickettsia rhipicephali strain HJ5, R. parkeri strain At24, and Rickettsia bellii strain Mogi. Between 2020 and 2025, ticks were manually collected while attached to the skin of humans in seven municipalities in Maranhão State. Adult ticks were randomly selected and individually processed for DNA extraction and examined using a real-time PCR (qPCR) assay targeting a fragment of the rickettsial gltA gene. The qPCR-positive samples were subsequently examined by conventional PCR (cPCR) targeting the ompA gene of SFG rickettsiae. The cPCR amplicons were purified and sequenced bidirectionally using the amplification primers. The resulting sequences were compared with those in GenBank using BLASTn to identify related Rickettsia spp. Results A total of 341 human serum samples were analyzed, and 145 (42.5%) were recorded as reactive with at least one species of Rickettsia. Among the reactive samples, 68 (47%) were from Imperatriz, 45 (31%) from Açailândia, and 32 (22%) from the municipality of São Luís. Rickettsia rhipicephali was recorded as the possible antigen involved in a homologous reaction (PAIHR) in one individual, R. amblyommatis in five, and R. bellii in three. A total of 187 ticks were collected parasitizing humans in the Amazon biome. Molecular analyses revealed that Rickettsia DNA was present in 44.4% (4/9) of A. cajennense s.s. from Açailândia, 52.4% (22/42) from Centro Novo do Maranhão, and 0% (0/8) from Imperatriz. Among the adults of A. coelebs from Centro Novo do Maranhão, 36.8% (7/19) tested positive. In contrast, all adults of A. oblongoguttatum from the same location tested negative (0/20). Conclusions This is the first study to detect anti-Rickettsia antibodies in humans and to identify R. amblyommatis in ticks parasitizing humans in the Amazon biome. The detection of R. amblyommatis in human-biting ticks, together with concurrent seropositivity in human sera from the same region, supports the hypothesis that this agent is actively circulating in the Amazon biome and may be responsible for undiagnosed cases of nonlethal spotted fever in the area. Graphical abstract
Expanded HPV Genotyping by Single-Tube Nested-Multiplex PCR May Explain HPV-Related Disease Recurrence Luiz Ricardo Goulart, Bruna França Matias Colombo, Mayara Ingrid Sousa Lima, Maria Socorro A. de Andrade, Juliana São Julião, et al. Microorganisms, 2024 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.
Amphotericin B resistance in Leishmania amazonensis: In vitro and in vivo characterization of a Brazilian clinical isolate Bianca A. Ferreira, Elizabeth M. Coser, Stephane de la Roca, Juliana I. Aoki, Nilson Branco, et al. Plos Neglected Tropical Diseases, 2024 In Brazil, Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The state of Maranhão in the Northeast of Brazil is prevalent for these clinical forms of the disease and also has high rates of HIV infection. Here, we characterized the drug susceptibility of a L. amazonensis clinical isolate from a 46-year-old man with diffuse cutaneous leishmaniasis coinfected with HIV from this endemic area. This patient underwent several therapeutic regimens with meglumine antimoniate, liposomal amphotericin B, and pentamidine, without success. In vitro susceptibility assays against promastigotes and intracellular amastigotes demonstrated that this isolate had low susceptibility to amphotericin B, when compared with the reference strain of this species that is considered susceptible to antileishmanial drugs. Additionally, we investigated whether the low in vitro susceptibility would affect the in vivo response to amphotericin B treatment. The drug was effective in reducing the lesion size and parasite burden in mice infected with the reference strain, whereas those infected with the clinical isolate and a resistant line (generated experimentally by stepwise selection) were refractory to amphotericin B treatment. To evaluate whether the isolate was intrinsically resistant to amphotericin B in animals, infected mice were treated with other drugs that had not been used in the treatment of the patient (miltefosine, paromomycin, and a combination of both). Our findings demonstrated that all drug schemes were able to reduce lesion size and parasite burden in animals infected with the clinical isolate, confirming the amphotericin B-resistance phenotype. These findings indicate that the treatment failure observed in the patient may be associated with amphotericin B resistance, and demonstrate the potential emergence of amphotericin B-resistant L. amazonensis isolates in an area of Brazil endemic for cutaneous leishmaniasis.
Factors associated with relapse and hospital death in patients coinfected with visceral leishmaniasis and HIV: a longitudinal study Larissa D. L. N. Costa, Uiara S. Lima, Vandilson Rodrigues, Mayara I. S. Lima, Lucilene A. Silva, et al. BMC Infectious Diseases, 2023 Objective Visceral leishmaniasis (VL) is an endemic parasitic disease in Latin America, and its clinical picture is aggravated in coinfections with the human immunodeficiency virus (HIV). The objective of this study was to investigate clinical factors and laboratory variables associated with VL relapse and death in VL/HIV coinfected patients. Methods A prospective longitudinal study was conducted from January 2013 to July 2020 among 169 patients coinfected with VL and HIV. The outcomes investigated were the occurrence of VL relapse and death. Chi-square test, Mann–Whitney test and logistic regression models were used for statistical analysis. Results The occurrence rates were 41.4% for VL relapse and 11.2% for death. Splenomegaly and adenomegaly were associated with the increased risk of VL relapse. Patients with VL relapse had higher levels of urea (p = .005) and creatinine (p < .001). Patients who died had lower red blood cell counts (p = .012), hemoglobin (p = .017) and platelets (p < .001). The adjusted model showed that antiretroviral therapy for more than 6 months was associated with a decrease in VL relapse, and adenomegaly was associated with an increase in VL relapse. In addition, edema, dehydration, poor general health status, and paleness were associated with an increase in hospital death. Conclusion The findings suggest that adenomegaly, antiretroviral therapy, and renal abnormalities can be associated with VL relapse, while hematological abnormalities, and clinical manifestations like paleness, and edema can be associated with an increased odds of hospital death. Trial registration number: The study was submitted to the Ethics and Research Committee of the Federal University of Maranhão (Protocol: 409.351).
HSP60 mimetic peptides from Mycobacterium leprae as new antigens for immunodiagnosis of Leprosy Mayara Ingrid Sousa Lima, Meydson Benjamim Carvalho Corrêa, Emilly Caroline dos Santos Moraes, Jaqueline das Dores Dias Oliveira, Paula de Souza Santos, et al. AMB Express, 2023 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.
Systematic Review of Treatment Failure and Clinical Relapses in Leishmaniasis from a Multifactorial Perspective: Clinical Aspects, Factors Associated with the Parasite and Host Gustavo de Almeida Santos, Juliana Mendes Sousa, Antônio Henrique Braga Martins de Aguiar, Karina Cristina Silva Torres, Ana Jessica Sousa Coelho, et al. Tropical Medicine and Infectious Disease, 2023 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.
In Vitro Drug Susceptibility of a Leishmania (Leishmania) infantum Isolate from a Visceral Leishmaniasis Pediatric Patient after Multiple Relapses Bianca A. Ferreira, Gustavo de A. Santos, Elizabeth M. Coser, Juliana M. Sousa, Mônica E. A. Gama, et al. Tropical Medicine and Infectious Disease, 2023 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.
