Cervical cytology brushes.

<p>A, sterile wrapped CerviSoft® and Histobrush® cervical cytology brushes; B, CerviSoft® and Histobrush® cytology brush tips.</p

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

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

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

Clinical and Demographic Characteristics of 28 Patients with Suspected Mucosal Leishmaniasis.

<p>*Intercurrent CL confirmed by smear or culture of cutaneous lesion specimens.</p><p>**All 5 control volunteers had negative cytology brush specimens.</p>†<p>CerviSoft® and Histobrush® PCR demonstrated 97% concordance; in 1 patient, CerviSoft® PCR was positive and Histobrush® PCR was negative.</p>§<p>Patient with known previous CL on history.</p>¶<p>Patient with scars suspicious to be healed CL but no definitive history of diagnosis.</p><p>Abbreviations: CL, cutaneous leishmaniasis; F, female; LST, leishmanin skin test; M, male; Neg, negative; NS, nasal septum; Pos, positive.</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

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

Analysis of 5 Diagnostic Tests used in the Evaluation of 28 Patients with Suspected Mucosal Leishmaniasis.

<p>*includes nasal and buccal specimens from 5 healthy control volunteers.</p><p>Abbreviations: LST, leishmanin skin test; NPV, negative predictive value; PPV, positive predictive value.</p

Quantification of <i>Leishmania</i> (<i>Viannia</i>) Kinetoplast DNA in Ulcers of Cutaneous Leishmaniasis Reveals Inter-site and Inter-sampling Variability in Parasite Load

<div><p>Background</p><p>Cutaneous leishmaniasis (CL) is a skin disease caused by the protozoan parasite <i>Leishmania</i>. Few studies have assessed the influence of the sample collection site within the ulcer and the sampling method on the sensitivity of parasitological and molecular diagnostic techniques for CL. Sensitivity of the technique can be dependent upon the load and distribution of <i>Leishmania</i> amastigotes in the lesion.</p><p>Methodology/Principal Findings</p><p>We applied a quantitative real-time PCR (qPCR) assay for <i>Leishmania</i> (<i>Viannia</i>) minicircle kinetoplast DNA (kDNA) detection and parasite load quantification in biopsy and scraping samples obtained from 3 sites within each ulcer (border, base, and center) as well as in cytology brush specimens taken from the ulcer base and center. A total of 248 lesion samples from 31 patients with laboratory confirmed CL of recent onset (≤3 months) were evaluated. The kDNA-qPCR detected <i>Leishmania</i> DNA in 97.6% (242/248) of the examined samples. Median parasite loads were significantly higher in the ulcer base and center than in the border in biopsies (<i>P</i><0.0001) and scrapings (<i>P</i> = 0.0002). There was no significant difference in parasite load between the ulcer base and center (<i>P</i> = 0.80, 0.43, and 0.07 for biopsy, scraping, and cytology brush specimens, respectively). The parasite load varied significantly by sampling method: in the ulcer base and center, the descending order for the parasite load levels in samples was: cytology brushes, scrapings, and biopsies (<i>P</i><0.0001); in the ulcer border, scrapings had higher parasite load than biopsies (<i>P</i><0.0001). There was no difference in parasite load according to <i>L</i>. <i>braziliensis</i> and <i>L</i>. <i>peruviana</i> infections (<i>P</i> = 0.4).</p><p>Conclusion/Significance</p><p>Our results suggest an uneven distribution of <i>Leishmania</i> amastigotes in acute CL ulcers, with higher parasite loads in the ulcer base and center, which has implications for bedside collection of diagnostic specimens. The use of scrapings and cytology brushes is recommended instead of the more invasive biopsy.</p></div

<i>Leishmania</i> parasite load levels per skin lesion site and sampling method.

<p><b>Note.</b> IQR, interquartile range (25^th percentile–75^th percentile).</p><p>Data shown are the quantified paired parasite load results (8 specimens per lesion) corresponding to 28 patients.</p><p>^‡Number of parasites per μg of tissue DNA.</p><p>^¶<i>P</i><0.0001, for the comparison of parasite loads between biopsy and scraping specimens (Wilcoxon signed rank test).</p><p>*<i>P</i><0.0001, for the comparison of parasite loads among biopsy, scraping, and cytology brush specimens (Friedman test with Dunn’s post hoc test).</p><p>^¥<i>P</i><0.0001, for the comparison of parasite loads among biopsy specimens of the ulcer border, base, and center (Friedman test with Dunn’s post hoc test).</p><p>^†<i>P</i> = 0.0002, for the comparison of parasite loads among scraping specimens of the ulcer border, base, and center (Friedman test with Dunn’s post hoc test).</p><p>^§<i>P</i> = 0.07, for the comparison of parasite loads between cytology brush specimens of the ulcer base and center (Wilcoxon signed rank test).</p><p><i>Leishmania</i> parasite load levels per skin lesion site and sampling method.</p