Buffer substitution in malaria rapid diagnostic tests causes false-positive results

<p>Abstract</p> <p>Background</p> <p>Malaria rapid diagnostic tests (RDTs) are kits that generally include 20 to 25 test strips or cassettes, but only a single buffer vial. In field settings, laboratory staff occasionally uses saline, distilled water (liquids for parenteral drugs dilution) or tap water as substitutes for the RDT kit's buffer to compensate for the loss of a diluent bottle. The present study assessed the effect of buffer substitution on the RDT results.</p> <p>Methods</p> <p>Twenty-seven RDT brands were run with EDTA-blood samples of five malaria-free subjects, who were negative for rheumatoid factor and antinuclear antibodies. Saline, distilled water and tap water were used as substitute liquids. RDTs were also run with distilled water, without adding blood. Results were compared to those obtained with the RDT kit's buffer and <it>Plasmodium </it>positive samples.</p> <p>Results</p> <p>Only eight cassettes (in four RDT brands) showed no control line and were considered invalid. Visible test lines occurred for at least one malaria-free sample and one of the substitutes in 20/27 (74%) RDT brands (saline: n = 16; distilled water: n = 17; and tap water: n = 20), and in 15 RDTs which were run with distilled water only. They occurred for all <it>Plasmodium </it>antigens and RDT formats (two-, three- and four-band RDTs). Clearance of the background of the strip was excellent except for saline. The aspects (colour, intensity and crispness) of the control and the false-positive test lines were similar to those obtained with the RDT kits' buffer and <it>Plasmodium </it>positive samples.</p> <p>Conclusion</p> <p>Replacement of the RDT kit's dedicated buffer by saline, distilled water and tap water can cause false-positive test results.</p

Evaluation of the rapid diagnostic test SDFK40 (Pf-pLDH/pan-pLDH) for the diagnosis of malaria in a non-endemic setting

<p>Abstract</p> <p>Background</p> <p>The present study evaluated the SD Bioline Malaria Ag 05FK40 (SDFK40), a three-band RDT detecting <it>Plasmodium falciparum</it>-specific parasite lactate dehydrogenase (Pf-pLDH) and pan <it>Plasmodium</it>-specific pLDH (pan-pLDH), in a reference setting.</p> <p>Methods</p> <p>The SDFK40 was retrospectively and prospectively tested against a panel of stored (n = 341) and fresh (n = 181) whole blood samples obtained in international travelers suspected of malaria, representing the four <it>Plasmodium </it>species as well as <it>Plasmodium </it>negative samples, and compared to microscopy and PCR results. The prospective panel was run together with OptiMAL (Pf-pLDH/pan-pLDH) and SDFK60 (histidine-rich protein-2 (HRP-2)/pan-pLDH).</p> <p>Results</p> <p>Overall sensitivities for <it>P. falciparum </it>tested retrospectively and prospectively were 67.9% and 78.8%, reaching 100% and 94.6% at parasite densities >1,000/μl. Sensitivity at parasite densities ≤ 100/μl was 9.1%. Overall sensitivities for <it>Plasmodium vivax </it>and <it>Plasmodium ovale </it>were 86.7% and 80.0% (retrospectively) and 92.9% and 76.9% (prospectively), reaching 94.7% for both species (retrospective panel) at parasite densities >500/μl. Sensitivity for <it>Plasmodium malariae </it>was 21.4%. Species mismatch occurred in 0.7% of samples (3/411) and was limited to non-<it>falciparum </it>species erroneously identified as <it>P. falciparum</it>. None of the <it>Plasmodium </it>negative samples in the retrospective panel reacted positive. Compared to OptiMAL and SDFK60, SDFK40 showed lower sensitivities for <it>P. falciparum</it>, but better detection of <it>P. ovale</it>. Inter-observer agreement and test reproducibility were excellent, but lot-to-lot variability was observed for pan-pLDH results in case of <it>P. falciparum</it>.</p> <p>Conclusion</p> <p>SDFK40 performance was poor at low (≤ 100/μl) parasite densities, precluding its use as the only diagnostic tool for malaria diagnosis. SDFK40 performed excellent for <it>P. falciparum </it>samples at high (>1,000/μl) parasite densities as well as for detection of <it>P. vivax </it>and <it>P. ovale </it>at parasite densities >500/μl.</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

Malaria rapid diagnostic kits: quality of packaging, design and labelling of boxes and components and readability and accuracy of information inserts

<p>Abstract</p> <p>Background</p> <p>The present study assessed malaria RDT kits for adequate and correct packaging, design and labelling of boxes and components. Information inserts were studied for readability and accuracy of information.</p> <p>Methods</p> <p>Criteria for packaging, design, labelling and information were compiled from Directive 98/79 of the European Community (EC), relevant World Health Organization (WHO) documents and studies on end-users' performance of RDTs. Typography and readability level (Flesch-Kincaid grade level) were assessed.</p> <p>Results</p> <p>Forty-two RDT kits from 22 manufacturers were assessed, 35 of which had evidence of good manufacturing practice according to available information (<it>i.e</it>. CE-label affixed or inclusion in the WHO list of ISO13485:2003 certified manufacturers). Shortcomings in devices were (i) insufficient place for writing sample identification (n = 40) and (ii) ambiguous labelling of the reading window (n = 6). Buffer vial labels were lacking essential information (n = 24) or were of poor quality (n = 16). Information inserts had elevated readability levels (median Flesch Kincaid grade 8.9, range 7.1 - 12.9) and user-unfriendly typography (median font size 8, range 5 - 10). Inadequacies included (i) no referral to biosafety (n = 18), (ii) critical differences between depicted and real devices (n = 8), (iii) figures with unrealistic colours (n = 4), (iv) incomplete information about RDT line interpretations (n = 31) and no data on test characteristics (n = 8). Other problems included (i) kit names that referred to <it>Plasmodium vivax </it>although targeting a pan-species <it>Plasmodium </it>antigen (n = 4), (ii) not stating the identity of the pan-species antigen (n = 2) and (iii) slight but numerous differences in names displayed on boxes, device packages and information inserts. Three CE labelled RDT kits produced outside the EC had no authorized representative affixed and the shape and relative dimensions of the CE symbol affixed did not comply with the Directive 98/79/EC. Overall, RDTs with evidence of GMP scored better compared to those without but inadequacies were observed in both groups.</p> <p>Conclusion</p> <p>Overall, malaria RDTs showed shortcomings in quality of construction, design and labelling of boxes, device packages, devices and buffers. Information inserts were difficult to read and lacked relevant information.</p

Prozone in malaria rapid diagnostics tests: how many cases are missed?

<p>Abstract</p> <p>Background</p> <p>Prozone means false-negative or false-low results in antigen-antibody reactions, due to an excess of either antigen or antibody. The present study prospectively assessed its frequency for malaria rapid diagnostic tests (RDTs) and <it>Plasmodium falciparum </it>samples in an endemic field setting.</p> <p>Methods</p> <p>From January to April 2010, blood samples with <it>P. falciparum </it>high parasitaemia (≥ 4% red blood cells infected) were obtained from patients presenting at the Provincial Hospital of Tete (Mozambique). Samples were tested undiluted and 10-fold diluted in saline with a panel of RDTs and results were scored for line intensity (no line visible, faint, weak, medium and strong). Prozone was defined as a sample which showed no visible test line or a faint or weak test line when tested undiluted, and a visible test line of higher intensity when tested 10-fold diluted, as observed by two blinded observers and upon duplicate testing.</p> <p>Results</p> <p>A total of 873/7,543 (11.6%) samples showed <it>P. falciparum</it>, 92 (10.5%) had high parasitaemia and 76 were available for prozone testing. None of the two Pf-pLDH RDTs, but all six HRP-2 RDTs showed prozone, at frequencies between 6.7% and 38.2%. Negative and faint HRP-2 lines accounted for four (3.8%) and 15 (14.4%) of the 104 prozone results in two RDT brands. For the most affected brand, the proportions of prozone with no visible or faint HRP-2 lines were 10.9% (CI: 5.34-19.08), 1.2% (CI: 0.55-2.10) and 0.1% (CI: 0.06-0.24) among samples with high parasitaemia, all positive samples and all submitted samples respectively. Prozone occurred mainly, but not exclusively, among young children.</p> <p>Conclusion</p> <p>Prozone occurs at different frequency and intensity in HRP-2 RDTs and may decrease diagnostic accuracy in the most affected RDTs.</p

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

Laboratory demonstration of a prozone-like effect in HRP2-detecting malaria rapid diagnostic tests: implications for clinical management

Background: Malaria rapid diagnostic tests (RDTs) are now widely used for prompt on-site diagnosis in remote endemic areas where reliable microscopy is absent. Aberrant results, whereby negative test results occur at high parasite densities, have been variously reported for over a decade and have led to questions regarding the reliability of the tests in clinical use. Methods. In the first trial, serial dilutions of recombinant HRP2 antigen were tested on an HRP2-detectiing RDT. In a second trial, serial dilutions of culture-derived Plasmodium falciparum parasites were tested against three HRP2-detecting RDTs. Results: A prozone-like effect occurred in RDTs at a high concentration of the target antigen, histidine-rich protein-2 (above 15,000 ng/ml), a level that corresponds to more than 312000 parasites per L. Similar results were noted on three RDT products using dilutions of cultured parasites up to a parasite density of 25%. While reduced line intensity was observed, no false negative results occurred. Conclusions: These results suggest that false-negative malaria RDT results will rarely occur due to a prozone-like effect in high-density infections, and other causes are more likely. However, RDT line intensity is poorly indicative of parasite density in high-density infections and RDTs should, therefore, not be considered quantitative. Immediate management of suspected severe malaria should rely on clinical assessment or microscopy. Evaluation against high concentrations of antigen should be considered in malaria RDT product development and lot-release testing, to ensure that very weak or false negative results will not occur at antigen concentrations that might be seen clinically