Non-Invasive Cytology Brush PCR Diagnostic Testing in Mucosal Leishmaniasis: Superior Performance to Conventional Biopsy with Histopathology

Traditional methods of diagnosing mucosal leishmaniasis (ML), such as biopsy with histopathology, are insensitive and require collection of an invasive diagnostic specimen. species identification was performed by PCR-based assays of positive specimens. (n = 3).Use of commercial grade cytology brush PCR for diagnosis of ML is sensitive, rapid, well tolerated, and carries none of the risks of invasive diagnostic procedures such as biopsy. Further optimization is required for adequate species identification. Further evaluation of this method in field and other settings is warranted

An estimate of the number of tropical tree species

The high species richness of tropical forests has long been recognized, yet there remains substantial uncertainty regarding the actual number of tropical tree species. Using a pantropical tree inventory database from closed canopy forests, consisting of 657,630 trees belonging to 11,371 species, we use a fitted value of Fisher’s alpha and an approximate pantropical stem total to estimate the minimum number of tropical forest tree species to fall between ∼40,000 and ∼53,000, i.e. at the high end of previous estimates. Contrary to common assumption, the Indo-Pacific region was found to be as species-rich as the Neotropics, with both regions having a minimum of ∼19,000–25,000 tree species. Continental Africa is relatively depauperate with a minimum of ∼4,500–6,000 tree species. Very few species are shared among the African, American, and the Indo-Pacific regions. We provide a methodological framework for estimating species richness in trees that may help refine species richness estimates of tree-dependent taxa

HOST AND PATHOGEN GENETIC DETERMINANTS OF SEVERITY AND OUTCOME OF ACUTE VIRAL INFECTIONS

Acute viral infections are associated with manifestations ranging from brief and minimally symptomatic illnesses to severe conditions. Genetic variations in host and pathogen genomes and environmental and co-morbid conditions explain this diversity. This thesis aimed to 1) analyse the interactions of single nucleotide polymorphisms (SNPs) in inflammatory gene candidate pathways and the severity and duration of manifestations in a prospective acute infection cohort (Dubbo Infection Outcomes Study - DIOS); 2) analyse the viral determinants of plasma leakage and other severe dengue phenotypes in a prospective dengue virus (DENV) cohort (Dengue Colombo Study - DCS); and 3) examine the host immunological footprint on dengue virus genomes using pathogen and host genome-wide characterisation. Clinical and laboratory data from DIOS (n=484) and CDS (n=347) were used in principal components (PC) analyses to construct indices of severity (endophenotypes). Nine SNPs in the NLRP3 inflammasome pathway were genotyped to test for associations with severe illness in DIOS. To assess the pathogen contribution to severity, full-length DENV genomes were sequenced to identify viral SNPs and to reduce them as viral PC. Logistic and linear regressions were conducted using plasma leakage or DENV severity endophenotypes as outcomes and viral PCs, previous DENV infection, age, and gender as covariates. Finally, after a genome-wide characterisation of DCS participants using the UK Biobank array, viral SNPs were used as outcomes, including covariates (previous DENV, viral and host PCs, and each human SNP) in a logistic regression analysis. The frequency of rs35829419, a missense mutation causing an overactive NRLP3 inflammasome, was associated with severe fatigue in DIOS. In DCS, non-synonymous mutations - G754U and A7538G in the DENV Membrane protein and non-structural protein NS5 were associated with DENV severity. Finally, the genome-to-genome association analysis provided preliminary identification of associations between synonymous mutations in the viral proteins NS5 (G7725A, G7845A, and C8268T) and NS4B (C7011T) and intronic regions in chromosomes 11, 14, 16 and 18 within host genes including CHST9, SBF2, NRXN3, and SRL. Viral and host SNPs influence the severity of diverse acute infections in the DIOS and DCS studies. Further investigation is warranted to confirm and extend these findings to enhance our understanding of host and pathogen determinants of illness phenotype

Non-invasive cytology brush PCR for the diagnosis and causative species identification of American cutaneous leishmaniasis in Peru.

BACKGROUND: Traditional methods of detecting Leishmania from cutaneous lesions involve invasive diagnostic procedures, such as scrapings, which cause discomfort, require technical expertise, and carry risks of invasive procedures. We compared the performance of 2 novel, molecular-based non-invasive methods for the diagnosis of cutaneous leishmaniasis (CL). METHODS: Consecutive patients presenting to the Leishmania Clinic at the Hospital Nacional Cayetano Heredia were enrolled. PCR was performed on filter paper lesion impressions (FPLIs), cytology brushes, and lancets for detection of Leishmania DNA. Smears from lesion scrapings and leishmanin skin test were also performed. Outcome measures were sensitivity and specificity. Composite reference standard was any 2 of 5 tests positive. Species identification was performed by PCR assays of positive specimens. RESULTS: Ninety patients with 129 lesions were enrolled, 117 of which fulfilled reference criteria for a diagnosis of CL. Of these 117 lesions, 113 were positive by PCR of lancets used for lesion scrapings versus 116 by PCR of FPLIs (p=0.930) or 116 by PCR of cytology brushes (p=0.930). Sensitivity and specificity of PCR on lancets were 96.6% [95% CI 93.3-99.9%] and 100%, respectively. Sensitivity and specificity of FPLI PCR were 99.1% [95% CI 97.4-100%] and 100%, respectively. Sensitivity and specificity of cytology brush PCR were 99.1% [95% CI 97.4-100%] and 100%, respectively. Giemsa-stained lesion smear and leishmanin skin test had inferior sensitivities at 47.9% [95% CI 38.9-57.0%] and 82.3% [95% CI 73.9-90.7%], respectively, compared to PCR of invasive or non-invasive specimens (p<0.001). CONCLUSIONS: Cytology brush PCR constitutes a sensitive and specific alternative to traditional diagnostic assays performed on invasive specimens such as lesion scrapings. It performs comparatively to non-invasive FPLI PCR. This novel, rapid, and well-tolerated method has the potential for widespread use in the field and in pediatric populations where traditional specimen collection is difficult

Clinical and Public Health Implications of Human T-Lymphotropic Virus Type 1 Infection

International audienceHuman T-lymphotropic virus type 1 (HTLV-1) is estimated to affect 5 to 10 million people globally and can cause severe and potentially fatal disease, including adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The burden of HTLV-1 infection appears to be geographically concentrated, with high prevalence in discrete regions and populations. While most high-income countries have introduced HTLV-1 screening of blood donations, few other public health measures have been implemented to prevent infection or its consequences. Recent advocacy from concerned researchers, clinicians, and community members has emphasized the potential for improved prevention and management of HTLV-1 infection. Despite all that has been learned in the 4 decades following the discovery of HTLV-1, gaps in knowledge across clinical and public health aspects persist, impeding optimal control and prevention, as well as the development of policies and guidelines. Awareness of HTLV-1 among health care providers, communities, and affected individuals remains limited, even in countries of endemicity. This review provides a comprehensive overview on HTLV-1 epidemiology and on clinical and public health and highlights key areas for further research and collaboration to advance the health of people with and at risk of HTLV-1 infection

Species identification of 91 out of 118 kDNA PCR-positive lesions subsequently tested with PCR targeting the mannose phosphate isomerase, cysteine proteinase B and heat shock protein 70 genes and subsequent RFLP.

*<p>Only specimens with sufficient amplifiable DNA from the kDNA PCR assay were selected for species identification PCR assays. These 9 specimens had a positive cytology brush kDNA PCR but insufficient genomic DNA concentration for species identification based on weak banding pattern.</p

Filter paper lesion impression (FPLI) sampling method in an ulcer suspected to be cutaneous leishmaniasis.

<p>A, uninoculated filter paper; B, filter paper pressed gently onto ulcer base; C, lesion exudates wicked onto filter paper; D, filter paper with several lesion impressions and wicked exudates ready for air drying.</p

Species identification from 2 sets of specimens out of 16 PCR-positive lesions subsequently tested with PCR-based assays targeting the mannose phosphate isomerase, cysteine proteinase B, heat shock protein 70, and gp63 genes.

<p>*Only specimens with sufficient amplifiable DNA from the kDNA PCR of cytology brushes were selected for direct clinical specimen PCR.</p

Analysis of 5 Diagnostic Tests used in the Evaluation of 129 Lesions Suspected to be Cutaneous Leishmaniasis in 90 Peruvian patients.

*<p>per patient analysis.</p><p>Abbreviations: FPLI, filter paper lesion impression; LST, leishmanin skin test; NPV, negative predictive value; PPV, positive predictive value.</p

CerviSoft® cytology brush sampling method in an ulcer suspected to be cutaneous leishmaniasis.

<p>A, CerviSoft® cytology brush package and brush tip; CerviSoft® cytology brush held by health care worker in preparation for specimen collection; C, CerviSoft® cytology brush being rolled across ulcer base in order to collect lesion cellular and exudative material; D, CerviSoft® cytology brush tip broken off into a microcentrifuge tube containing 70% ethanol.</p