Web-Based Virtual Microscopy for Parasitology: A Novel Tool for Education and Quality Assurance

Here, we describe a novel tool to observe parasites by virtual microscopy on the Internet. Microscopy-based identification of parasites is the basis for both diagnostics and epidemiological assessment of parasite burden globally. Yet, quality assessment of diagnostic parasitology laboratories is difficult, as delivering identical educational specimens has been impossible. In this study, a series of parasite specimens on ordinary glass slides were digitized using a recently developed microscope scanner technique. Up to 50,000 images captured at high magnification are digitally stitched together to form a representation of the entire glass slide. These “virtual slides” digitized at a thousand-fold magnification can hold more than 60 gigabytes of data. Handling such large amounts of data was made possible because of efficient compression techniques and a viewing system adopted from the geospatial imaging industry. Viewing the samples on the Internet very much resembles, for example, the use of Google Maps, and puts only modest requirements on the viewer's computer. In addition, we captured image stacks at different focal planes, and developed a web-based viewing system for three-dimensional navigation in the specimens. This novel technique is especially valuable for detailed visualization of large objects such as helminth eggs in stool specimens

Giemsa-stained thick blood films as a source of DNA for Plasmodium species-specific real-time PCR

<p>Abstract</p> <p>Background</p> <p>This study describes the use of thick blood films (TBF) as specimens for DNA amplification with the <it>Plasmodium </it>species-specific real-time PCR that was recently validated on whole blood samples.</p> <p>Methods</p> <p>The panel of 135 Giemsa-stained clinical TBFs represented single infections of the four <it>Plasmodium </it>species with varying parasite densities or only gametocytes, mixed infections, and negative samples and was stored for up to 12 years. Half of the Giemsa-stained TBF was scraped off by a sterile scalpel and collected into phosphate buffered saline. DNA was extracted with the Qiagen DNA mini kit with minor modifications. DNA was amplified with the 18S rRNA real-time PCR targeting the four <it>Plasmodium </it>species with four species-specific primers and probes in combination with one genus-specific reverse primer. Results of the PCR on TBF were compared to those of the PCR on whole blood and to microscopy.</p> <p>Results</p> <p>Correct identification for single species infections was obtained for all TBF samples with <it>Plasmodium falciparum </it>(n = 50), <it>Plasmodium vivax </it>(n = 25), <it>Plasmodium ovale </it>(n = 25) and in all but one samples with <it>Plasmodium malariae </it>(n = 10). Compared to whole blood samples, higher Ct-values were observed by PCR on TBF with a mean difference of 5.93. Four out of five mixed infections were correctly identified with PCR on TBF. None of the negative samples (n = 20) gave a PCR signal. PCR on TBF showed a detection limit of 0.2 asexual parasites/μl compared to 0.02/μl for whole blood. Intra-run variation was higher for PCR on TBF (%CV 1.90) compared to PCR on whole blood (%CV 0.54). Compared to microscopy, PCR on TBF generated three more species identifications in samples containing a single species and detected the same four mixed-infections.</p> <p>Conclusions</p> <p>Giemsa-stained TBFs are a reliable source of DNA for <it>Plasmodium </it>real-time PCR analysis, allowing applications in reference and research settings in case whole blood samples are not available.</p

External quality assessment on the use of malaria rapid diagnostic tests in a non-endemic setting

<p>Abstract</p> <p>Background</p> <p>Malaria rapid diagnostic tests (RDTs) are increasingly used as a tool for the diagnosis of malaria, both in endemic and in non-endemic settings. The present study reports the results of an external quality assessment (EQA) session on RDTs in a non-endemic setting.</p> <p>Methods</p> <p>After validation of antigen stability during shipment at room temperature, three clinical samples and a questionnaire were sent to clinical laboratories in Belgium and the Grand Duchy of Luxembourg using malaria RDTs. Participants were asked to report the results of the RDTs as observations (visibility of the RDT control and test lines) and interpretations (report as formulated to the clinician). In addition, participants were invited to fill in a questionnaire on the place of RDTs in the diagnostic strategy of malaria.</p> <p>Results</p> <p>A total of 128/133 (96.2%) of clinical laboratories using RDTs participated. Six three-band and one four-band RDT brands were used. Analytical errors were rare and included (i) not recognizing invalid RDT results (1.6%) and (ii) missing the diagnosis of <it>Plasmodium falciparum </it>(0.8%). Minor errors were related to RDT test result interpretation and included (i) reporting "RDT positive" without species identification in the case of <it>P. falciparum </it>and non-<it>falciparum </it>species (16.9% and 6.5% respectively) and (ii) adding incorrect comments to the report (3.2%). Some of these errors were related to incorrect RDT package insert instructions such as (i) not reporting the possibility of mixed species infection in the case of <it>P. falciparum </it>and <it>Plasmodium vivax </it>(35.5% and 18.5% respectively) and (ii) the interpretation of <it>P. vivax </it>instead of non-falciparum species at the presence of a pan-species antigen line (4.0%). According to the questionnaire, 48.8% of participants processed ≤20 requests for malaria diagnosis in 2009. During opening hours, 93.6% of 125 participants used RDTs as an adjunct to microscopy but outside opening hours, nearly one third of 113 participants relied on RDTs as the primary (4.4%) or the single tool (25.7%) for malaria diagnosis.</p> <p>Conclusion</p> <p>In this non-endemic setting, errors in RDT performance were mainly related to RDT test line interpretations, partly due to incorrect package insert instructions. The reliance on RDTs as the primary or the single tool for the diagnosis of malaria outside opening hours is of concern and should be avoided.</p