MiniPCR as a portable equipment for the molecular diagnosis of american cutaneous leishmaniasis.

There is an urgent need to improve the diagnostic capacity of cutaneous leishmaniasis (CL) in rural health centers to improve the management of the disease in patients from remote regions where the infection is endemic. Microscopy of Giemsa-stained lesion smears is the standard-of-care diagnostic test in virtually all health centers, but its sensitivity is suboptimal (50-70%) and prone to false negative results. We evaluated the performance of a low-cost DNA extraction buffer (LAB) using a portable miniPCR™ equipment coupled with an inexpensive fluorescence viewer to detect Leishmania DNA with the naked eye or using a commercial photo app. Using ten-fold serial dilutions of Leishmania (V.) panamensis promastigotes the miniPCR-F test detected 10 parasites per µL, which was comparable to real-time PCR. Utilization of DNA from retrospective clinical samples preserved at -80 °C from Colombia (n = 28) or lesion exudate preserved in filter papers from Peru (n = 48) showed that the miniPCR-fluorescent test had a 100% sensitivity and > 90% specificity compared to real-time PCR. This study demonstrated the utility of LAB DNA extraction method for direct amplification of Leishmania using the miniPCR and reading of P51 results with the naked eye or via digital reading with a photo app. These preliminary results indicated that the miniPCR-F test workflow could be amenable to implementation in resource-limited health centers.

Diagnostic performance of a Recombinant Polymerase Amplification Test-Lateral Flow (RPA-LF) for cutaneous leishmaniasis in an endemic setting of Colombia.

BACKGROUND: Control of cutaneous leishmaniasis by public health systems in the Americas relies on case identification and treatment. Point-of-care diagnostics that can be performed by health workers within or near affected communities could effectively bring the health system to the resource-limited sites providing early diagnosis and treatment, reducing morbidity and the burden of disease. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional study was undertaken to evaluate the diagnostic test performance of Isothermal Recombinase Polymerase Amplification (RPA) targeting Leishmania kinetoplast DNA, coupled with a lateral flow (LF) immunochromatographic strip, in a field setting and a laboratory reference center. Minimally invasive swab and FTA filter paper samples were obtained by community health workers and highly trained technicians from ulcerated lesions of > 2 weeks' evolution from 118 patients' ≥ 2 years of age in the municipality of Tumaco, Nariño. Extracted DNA was processed by RPA-LF at a reference center or in a primary health facility in the field. Evaluation was based on a composite "gold standard" that included microscopy, culture, biopsy and real-time polymerase chain reaction detection of Leishmania 18S rDNA. Standard of care routine diagnostic tests were explored as comparators. Sensitivity and specificity of RPA-LF in the reference lab scenario were 87% (95%CI 74-94) and 86% (95%CI 74-97), respectively. In the field scenario, the sensitivity was 75% (95%CI 65-84) and specificity 89% (95%CI 78-99). Positive likelihood ratios in both scenarios were higher than 6 while negative likelihood ratios ranged to 0.2-0.3 supporting the usefulness of RPA-LF to rule-in and potentially to rule-out infection. CONCLUSIONS/SIGNIFICANCE: The low complexity requirements of RPA-LF combined with non-invasive sampling support the feasibility of its utilization by community health workers with the goal of strengthening the diagnostic capacity for cutaneous leishmaniasis in Colombia. TRIAL REGISTRATION: ClinicalTrials.gov NCT04500873.

Immune Profile of the Nasal Mucosa in Patients with Cutaneous Leishmaniasis.

Localized skin lesions are characteristic of cutaneous leishmaniasis (CL); however, Leishmania (Viannia) species, which are responsible for most CL cases in the Americas, can spread systemically, sometimes resulting in mucosal disease. Detection of Leishmania has been documented in healthy mucosal tissues (conjunctiva, tonsils, and nasal mucosa) and healthy skin of CL patients and in individuals with asymptomatic infection in areas of endemicity of L (V) panamensis and L (V) braziliensis transmission. However, the conditions and mechanisms that favor parasite persistence in healthy mucosal tissues are unknown. In this descriptive study, we compared the cell populations of the nasal mucosa (NM) of healthy donors and patients with active CL and explored the immune gene expression signatures related to molecular detection of Leishmania in this tissue in the absence of clinical signs or symptoms of mucosal disease. The cellular composition and gene expression profiles of NM samples from active CL patients were similar to those of healthy volunteers, with a predominance of epithelial over immune cells, and within the CD45+ cell population, a higher frequency of CD66b+ followed by CD14+ and CD3+ cells. In CL patients with molecular evidence of Leishmania persistence in the NM, genes characteristic of an anti-inflammatory and tissue repair responses (IL4R, IL5RA, POSTN, and SATB1) were overexpressed relative to NM samples from CL patients in which Leishmania was not detected. Here, we report the first immunological description of subclinically infected NM tissues of CL patients and provide evidence of a local anti-inflammatory environment favoring parasite persistence in the NM.

Parasitological Confirmation and Analysis of Leishmania Diversity in Asymptomatic and Subclinical Infection following Resolution of Cutaneous Leishmaniasis.

BACKGROUND: The contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL) is unknown. Immunological evidence of exposure to Leishmania in residents of endemic areas has been the basis for defining the human population with asymptomatic infection. However, parasitological confirmation of subclinical infection is lacking. METHODS: We investigated the presence and viability of Leishmania in blood and non-invasive mucosal tissue samples from individuals with immunological evidence of subclinical infection in endemic areas for CL caused by Leishmania (Viannia) in Colombia. Detection of Leishmania kDNA was conducted by PCR-Southern Blot, and parasite viability was confirmed by amplification of parasite 7SLRNA gene transcripts. A molecular tool for genetic diversity analysis of parasite populations causing persistent subclinical infection based on PCR amplification and sequence analysis of an 82bp region between kDNA conserved blocks 1 and 2 was developed. PRINCIPAL FINDINGS: Persistent Leishmania infection was demonstrated in 40% (46 of 114) of leishmanin skin test (LST) positive individuals without active disease; parasite viability was established in 59% of these (27 of 46; 24% of total). Parasite burden quantified from circulating blood monocytes, nasal, conjunctival or tonsil mucosal swab samples was comparable, and ranged between 0.2 to 22 parasites per reaction. kDNA sequences were obtained from samples from 2 individuals with asymptomatic infection and from 26 with history of CL, allowing genetic distance analysis that revealed diversity among sequences and clustering within the L. (Viannia) subgenus. CONCLUSIONS: Our results provide parasitological confirmation of persistent infection among residents of endemic areas of L. (Viannia) transmission who have experienced asymptomatic infection or recovered from CL, revealing a reservoir of infection that potentially contributes to the endemicity and transmission of disease. kDNA genotyping establishes proof-of-principle of the feasibility of genetic diversity analysis in previously inaccessible and unexplored parasite populations in subclinically infected individuals.

The molecular diagnosis of pelvic tuberculosis: a case report.

Tuberculosis is a re-emerging infectious disease. A retrospective analysis was made of the clinical history of a 48-year-old woman in April 2009; she was a secretary at a third-level hospital living in an urban area. Pelvic tubercular infection was suggested as a possible diagnosis; spoligotyping molecular methodology was used on a peritoneal secretion sample to confirm such diagnosis and confirmed the presence of Mycobacterium tuberculosis (octal code 777777777760771, SIT 53, Family T1).

The molecular diagnosis of pelvic tuberculosis: a case report / Diagnóstico molecular de tuberculosis pélvica: reporte de caso

Tuberculosis is a re-emerging infectious disease. A retrospective analysis was made of the clinical history of a 48-year-old woman in April 2009; she was a secretary at a third-level hospital living in an urban area. Pelvic tubercular infection was suggested as a possible diagnosis; spoligotyping molecular methodology was used on a peritoneal secretion sample to confirm such diagnosis and confirmed the presence of Mycobacterium tuberculosis (octal code 777777777760771, SIT 53, Family T1).

La tuberculosis es una enfermedad infecciosa reemergente. Durante el mes de abril de 2009 se realizó el análisis retrospectivo de la historia clínica de una mujer de 48 años de edad, residente en área urbana, secretaria de un hospital de tercer nivel. Se determinó infección tuberculosa pélvica como diagnóstico presuntivo. Con el fin de confirmar dicho diagnóstico, se realizó la metodología molecular de spoligotyping en muestra de secreción peritoneal y confirmó la presencia de Mycobacterium tuberculosis, código octal 777777777760771, SIT 53, familia T1.