Misclassification of Plasmodium infections by conventional microscopy and the impact of remedial training on the proficiency of laboratory technicians in species identification.

BACKGROUND: Malaria diagnosis is largely dependent on the demonstration of parasites in stained blood films by conventional microscopy. Accurate identification of the infecting Plasmodium species relies on detailed examination of parasite morphological characteristics, such as size, shape, pigment granules, besides the size and shape of the parasitized red blood cells and presence of cell inclusions. This work explores misclassifications of four Plasmodium species by conventional microscopy relative to the proficiency of microscopists and morphological characteristics of the parasites on Giemsa-stained blood films. CASE DESCRIPTION: Ten-day malaria microscopy remedial courses on parasite detection, species identification and parasite counting were conducted for public health and research laboratory personnel. Proficiency in species identification was assessed at the start (pre) and the end (post) of each course using known blood films of Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax infections with densities ranging from 1,000 to 30,000 parasites/μL. Outcomes were categorized as false negative, positive without speciation, P. falciparum, P. malariae, P. ovale, P. vivax and mixed infections. DISCUSSION AND EVALUATION: Reported findings are based on 1,878 P. falciparum, 483 P. malariae, 581 P. ovale and 438 P. vivax cumulative results collated from 2008 to 2010 remedial courses. Pre-training false negative and positive misclassifications without speciation were significantly lower on P. falciparum infections compared to non-falciparum infections (p < 0.0001). Post-training misclassifications decreased significantly compared to pre- training misclassifications which in turn led to significant improvements in the identification of the four species. However, P. falciparum infections were highly misclassified as mixed infections, P. ovale misclassified as P. vivax and P. vivax similarly misclassified as P. ovale (p < 0.05). CONCLUSION: These findings suggest that the misclassification of malaria species could be a common occurrence especially where non-falciparum infections are involved due to lack of requisite skills in microscopic diagnosis and variations in morphological characteristics within and between Plasmodium species. Remedial training might improve reliability of conventional light microscopy with respect to differentiation of Plasmodium infections

Stabilization of RDT target antigens present in dried Plasmodium falciparum-infected samples for validating malaria rapid diagnostic tests at the point of care

Abstract Background Malaria rapid diagnostic tests (RDTs) are a great achievement in implementation of parasite based diagnosis as recommended by World Health Organization. A major drawback of RDTs is lack of positive controls to validate different batches/lots at the point of care. Dried Plasmodium falciparum-infected samples with the RDT target antigens have been suggested as possible positive control but their utility in resource limited settings is hampered by rapid loss of activity over time. Methods This study evaluated the effectiveness of chemical additives to improve long term storage stability of RDT target antigens (HRP2, pLDH and aldolase) in dried P. falciparum-infected samples using parasitized whole blood and culture samples. Samples were treated with ten selected chemical additives mainly sucrose, trehalose, LDH stabilizer and their combinations. After baseline activity was established, the samples were air dried in bio-safety cabinet and stored at room temperatures (~ 25 °C). Testing of the stabilized samples using SD Bioline, BinaxNOW, CareStart, and First Response was done at intervals for 53 weeks. Results Stability of HRP2 at ambient temperature was reported at 21–24 weeks while that of PAN antigens (pLDH and aldolase) was 2–18 weeks of storage at all parasite densities. The ten chemical additives increased the percentage stability of HRP2 and PAN antigens. Sucrose alone and its combinations with Alsever’s solution or biostab significantly increased stability of HRP2 by 56% at 2000 p/µL (p < 0.001). Trehalose and its combinations with biostab, sucrose or glycerol significantly increased stability of HRP2 by 57% (p < 0.001). Unlike sucrose, the stability of the HRP2 was significantly retained by trehalose at lower concentrations (500, and 200 p/µL). Trehalose in combination biostab stabilizer increased the percentage stability of PAN antigens by 42, and 32% at 2000 and 500 p/µL respectively (p < 0.01). This was also the chemical combination with the shortest reconstitution time (~ < 20 min). Conclusions These findings confirm that stabilizing RDT target antigens in dried P. falciparum-infected samples using chemical additives provides field-stable positive controls for malaria RDTs

Scrub Typhus Outbreak in Chonburi Province, Central Thailand, 2013

Investigation of a scrub typhus outbreak in Thailand during September 2013 found that 9.1% of Thai soldiers and 11.1% of residents living in areas surrounding training sites had antibodies against the causative agent, Orientia tsutsugamushi. Sequence analysis of O. tsutsugamushi from rodents and chiggers identified 7 genogroups and 3 genotypes

Comparative evaluation of fluorescent in situ hybridization and Giemsa microscopy with quantitative real-time PCR technique in detecting malaria parasites in a holoendemic region of Kenya

Abstract Background Early and accurate diagnosis of malaria is important in treatment as well as in the clinical evaluation of drugs and vaccines. Evaluation of Giemsa-stained smears remains the gold standard for malaria diagnosis, although diagnostic errors and potential bias estimates of protective efficacy have been reported in practice. Plasmodium genus fluorescent in situ hybridization (P-Genus FISH) is a microscopy-based method that uses fluorescent labelled oligonucleotide probes targeted to pathogen specific ribosomal RNA fragments to detect malaria parasites in whole blood. This study sought to evaluate the diagnostic performance of P-Genus FISH alongside Giemsa microscopy compared to quantitative reverse transcription polymerase chain reaction (qRT-PCR) in a clinical setting. Method Five hundred study participants were recruited prospectively and screened for Plasmodium parasites by P-Genus FISH assay, and Giemsa microscopy. The microscopic methods were performed by two trained personnel and were blinded, and if the results were discordant a third reading was performed as a tie breaker. The diagnostic performance of both methods was evaluated against qRT-PCR as a more sensitive method. Results The number of Plasmodium positive cases was 26.8% by P-Genus FISH, 33.2% by Giemsa microscopy, and 51.2% by qRT-PCR. The three methods had 46.8% concordant results with 61 positive cases and 173 negative cases. Compared to qRT-PCR the sensitivity and specificity of P-Genus FISH assay was 29.3 and 75.8%, respectively, while microscopy had 58.2 and 93.0% respectively. Microscopy had a higher positive and negative predictive values (89.8 and 68.0% respectively) compared to P-Genus FISH (56.0 and 50.5%). In overall, microscopy had a good measure of agreement (76%, k = 0.51) compared to P-Genus FISH (52%, k = 0.05). Conclusion The diagnostic performance of P-Genus FISH was shown to be inferior to Giemsa microscopy in the clinical samples. This hinders the possible application of the method in the field despite the many advantages of the method especially diagnosis of low parasite density infections. The P-Genus assay has great potential but application of the method in clinical setting would rely on extensive training of microscopist and continuous proficiency testing

Characterization of <i>Plasmodium ovale curtisi</i> and <i>P. ovale wallikeri</i> in Western Kenya Utilizing a Novel Species-specific Real-time PCR Assay

<div><p>Background</p><p><i>Plasmodium ovale</i> is comprised of two genetically distinct subspecies, <i>P. ovale curtisi</i> and <i>P. ovale wallikeri</i>. Although <i>P. ovale</i> subspecies are similar based on morphology and geographical distribution, allelic differences indicate that <i>P. ovale curtisi</i> and <i>P. ovale wallikeri</i> are genetically divergent. Additionally, potential clinical and latency duration differences between <i>P. ovale curtisi</i> and <i>P. ovale wallikeri</i> demonstrate the need for investigation into the contribution of this neglected malaria parasite to the global malaria burden.</p><p>Methods</p><p>In order to detect all <i>P. ovale</i> subspecies simultaneously, we developed an inclusive <i>P. ovale</i>-specific real-time PCR assay based on conserved regions between <i>P. ovale curtisi</i> and <i>P. ovale wallikeri</i> in the reticulocyte binding protein 2 (rbp2) gene. Additionally, we characterized the <i>P. ovale</i> subspecies prevalence from 22 asymptomatic malaria infections using multilocus genotyping to discriminate <i>P. ovale curtisi</i> and <i>P. ovale wallikeri</i>.</p><p>Results</p><p>Our <i>P. ovale rbp2</i> qPCR assay validation experiments demonstrated a linear dynamic range from 6.25 <i>rbp2</i> plasmid copies/microliter to 100,000 rbp2 plasmid copies/microliter and a limit of detection of 1.5 <i>rbp2</i> plasmid copies/microliter. Specificity experiments showed the ability of the <i>rbp2</i> qPCR assay to detect low-levels of <i>P. ovale</i> in the presence of additional malaria parasite species, including <i>P. falciparum</i>, <i>P. vivax</i>, and <i>P. malariae</i>. We identified <i>P. ovale curtisi</i> and <i>P. ovale wallikeri</i> in Western Kenya by DNA sequencing of the tryptophan-rich antigen gene, the small subunit ribosomal RNA gene, and the <i>rbp2</i> gene.</p><p>Conclusions</p><p>Our novel <i>P. ovale rbp2</i> qPCR assay detects <i>P. ovale curtisi</i> and <i>P. ovale wallikeri</i> simultaneously and can be utilized to characterize the prevalence, distribution, and burden of <i>P. ovale</i> in malaria endemic regions. Using multilocus genotyping, we also provided the first description of the prevalence of <i>P. ovale curtisi</i> and <i>P. ovale wallikeri</i> in Western Kenya, a region holoendemic for malaria transmission.</p></div

GenBank accession numbers used for DNA alignment of the <i>P. ovale curtisi</i> and <i>P. ovale wallikeri tra, rbp2</i>, and ssrRNA DNA sequences.

<p>GenBank accession numbers used for DNA alignment of the <i>P. ovale curtisi</i> and <i>P. ovale wallikeri tra, rbp2</i>, and ssrRNA DNA sequences.</p

<i>P. ovale rbp2</i> plasmid standard curve.

<p>A representative standard curve demonstrates linearity based on 10-fold serial dilutions (1 to 100,000 copies/μl) of <i>rbp2</i> plasmid.</p

Primer and probe sequences utilized for conventional PCR and qPCR experiments.

<p>Primer and probe sequences utilized for conventional PCR and qPCR experiments.</p