Performance of the hematology analyzer XN-31 prototype in the detection of Plasmodium infections in an endemic region of Colombia.

Early and accurate diagnosis is critical in reducing the morbidity and mortality associated with malaria. Microscopy (MI) is the current diagnostic gold standard in the field; however, it requires expert personnel, is time-consuming, and has limited sensitivity. Although rapid diagnostic tests for antigen detection (RDTs) are an alternative to diagnosis, they also have limited sensitivity and produce false positive results in detecting recent past infection. The automated hematology analyzer XN-31 prototype (XN-31p) (Sysmex Corporation, Kobe, Japan) is able to identify plasmodium-infected erythrocytes, count parasitemia and perform complete blood-cell counts within one minute. The performance of the XN-31p in diagnosing malaria was evaluated and compared with real-time polymerase chain reaction (qPCR), MI and RDT in an endemic area of Colombia where Plasmodium falciparum and Plasmodium vivax are present. Acute febrile patients were enrolled from July 2018 to April 2019 in Quibdó, Colombia. Malaria diagnoses were obtained from MI and RDT in the field and later confirmed by qPCR. Venous blood samples in EDTA were processed with an XN-31p in the field. Sensitivity, specificity, positive/negative predictive values, and the likelihood ratios of positive and negative tests were calculated with respect to the results from qPCR, MI and RDT. The intraclass correlation coefficient (ICC) and Bland-Altman plot were used to evaluate the concordance in the parasitemia with respect to MI. A total of 1,754 subjects were enrolled. The mean age was 27.0 years (IQR 14-44); 89.6% were Afro-Colombians, 94.3% lived in urban areas and 0.91% were pregnant. With respect to qPCR, the XN-31p showed a sensitivity of 90% (95% CI 87.24-92.34) and a specificity of 99.83% (95% CI 99.38-99.98) in detecting Plasmodium spp.; both parameters were equivalent to those for MI and RDT. Using MI as the reference, the XN-31p showed a sensitivity of 98.09% (95% CI 96.51-99.08), a specificity of 99.83% (95% CI 99.4-99.98), an ICC of 0.85 (95% CI 0.83-0.87) and an average difference of - 3096 parasites/µL when compared with thick-smear MI and an ICC of 0.98 (95% CI 0.97-0.98) and an average difference of - 0.0013% when compared with thin-smear MI. The XN-31p offers a rapid and accurate alternative method for diagnosing malaria in clinical laboratories in areas where P. falciparum and P. vivax cocirculate.

Lysercell M enhances the detection of stage-specific Plasmodium-infected red blood cells in the automated hematology analyzer XN-31 prototype.

The automated hematology analyzers XN-30 (for research) and XN-31 prototype (for diagnosis support) can easily and rapidly detect Plasmodium-infected red blood cells (iRBCs) and distinguish the developmental stages of the parasite in approximately 1 min. Two dedicated reagents, Lysercell M and Fluorocell M, are available with the analyzers. Lysercell M plays an indispensable role in enhancing the fluorescence intensity of the nucleic acid staining dye in Fluorocell M and altering cell morphology. These effects of Lysercell M have been empirically determined but insufficiently analyzed. In this study, the properties of Lysercell M were analyzed using two flow cytometers and a fluorescence microscope. First, the fluorescence intensity emitted by iRBCs treated with Lysercell M or phosphate-buffered saline (PBS) was evaluated. Second, the size of RBCs treated with Lysercell M or PBS was measured. Finally, the morphology of individual parasites was observed after reconstruction of an M scattergram, a cytogram of the XN-31 prototype system, using an imaging flow cytometer. These analyses showed that treatment of iRBCs with Lysercell M increased the fluorescence intensity of stained parasite nucleic acids by approximately 10-fold and reduced the size of iRBCs in a stage-specific manner, facilitating the identification and quantification of ring form, trophozoite, and schizont stage iRBCs. These properties suggest that Lysercell M is useful for rapidly detecting iRBCs and accurately distinguishing the parasite developmental stages, thereby contributing to the usability of the XN-30 and XN-31 prototype analyzers.

Implementation of a red blood cell-optical (RBO) channel for detection of latent iron deficiency anaemia by automated measurement of autofluorescence-emitting red blood cells.

Iron deficiency is the most common and widespread nutritional disorder worldwide. The automated haematology analyser XN-30 (Sysmex, Kobe, Japan) was developed to detect malaria-infected red blood cells (RBCs) in human blood samples using flow cytometry. The optical system of the analyser detects autofluorescence (AF)-emitting RBCs containing iron-deficient haem groups and would aid in the diagnosis of anaemia resulting from iron deficiency. Here, an RBC-optical (RBO) channel was devised and implemented on the analyser. In vitro analyses showed that the analyser detected AF-emitting RBCs treated with 5-aminolevulinic acid. Furthermore, the analyser detected AF-emitting RBCs in mice fed a low iron diet and infected with a rodent malaria parasite; it could also be effectively used in humans. This study demonstrates that the analyser can quantitatively and reproducibly detect AF-emitting RBCs and measure other haematological parameters, suggesting its usefulness for the initial evaluation of latent iron deficiency anaemia in conjunction with the diagnosis of malaria.

Application of the automated haematology analyzer XN-30 for discovery and development of anti-malarial drugs.

BACKGROUND: The erythrocytic stage of Plasmodium falciparum parasites in humans is clinically important, as the parasites at this growth stage causes malarial symptoms. Most of the currently available anti-malarial drugs target this stage, but the emergence and spread of parasites resistant to anti-malarial drugs are a major challenge to global eradication efforts; therefore, the development of novel medicines is urgently required. In this study, the in vitro anti-malarial activity of five current anti-malarial drugs (artemisinin, atovaquone, chloroquine, mefloquine, and pyrimethamine) and 400 compounds from the Pathogen Box provided by the Medicines for Malaria Venture on P. falciparum parasites was characterized using the XN-30 analyzer. Furthermore, the outcomes obtained using the analyser were classified according to the parasitaemias of total and each developmental stages. RESULTS: The growth inhibition rate and the half-maximal (50%) inhibitory concentration (IC50) of the five current anti-malarial drugs were calculated from the parasitaemia detected using the XN-30 analyzer. Respective strains and drugs presented strongly fitted sigmoidal curves, and the median SD at all tested concentrations was 1.6, suggesting that the variation in values measured with the analyser was acceptably low for the comparison of drug efficacy. Furthermore, the anti-malarial activity of the 400 compounds from the Pathogen Box was tested, and 141 drugs were found to be effective. In addition, the efficacy was classified into 4 types (Type I, parasites were arrested or killed without DNA replication; Type II, parasites were arrested or killed similar to Type I, and the parasitaemia was apparently decreased; Type III, parasites progressed to trophozoite without sufficient DNA replication; and Type IV, parasites were arrested at late trophozoite or schizont after DNA replication). CONCLUSION: The current study demonstrates that the XN-30 analyzer objectively, reproducibly, and easily evaluated and characterized the anti-malarial efficacy of various compounds. The results indicate the potential of the XN-30 analyzer as a powerful tool for drug discovery and development in addition to its use as an important diagnostic tool.

Application of the automated haematology analyzer XN-30 in an experimental rodent model of malaria.

BACKGROUND: The erythrocytic stage, where malaria parasites proliferate in human blood, is clinically significant as this causes the symptoms and illness of malaria. Experimental rodent models of malaria at the erythrocytic stage are used for the development of anti-malarial drugs and for biological analysis. An automated haematology analyzer XN-30 was developed for detection of infected red blood cells (iRBCs) in human blood samples and measurement of their parasitaemia in approximately 1 min through flow cytometry analysis. Additionally, the analyzer simultaneously measured other haematological parameters in these samples. It is inferred that the analyzer would also allow easy and rapid measurement of parasitaemia in mice and provide important clues on the mouse haematological state during infection and treatment. RESULTS: The XN-30 analyzer is a simple and rapid tool to detect iRBCs in mouse blood samples infected with rodent malarial parasites, with three-dimensional analysis permitting the precise measurement of parasitaemia (referred herein as the 'XN-30 system'). The XN-30 analyzer allowed not only the detection of iRBCs but also the monitoring of RBC, white blood cell, and platelet counts, as well as haematocrit, mean corpuscular volume and mean platelet volume values in the mouse blood sample. For anti-malarial drug development, aside from demonstrating possible efficacy in mouse models, XN-30 analyzer could provide a first glimpse of the safety profile of the drug. CONCLUSIONS: The XN-30 system is a powerful tool that can be utilized for the in vivo screening, development, and evaluation of anti-malarial drugs as well as for pre-clinical pharmacology and/or toxicity tests in rodent models.

An automated haematology analyzer XN-30 distinguishes developmental stages of falciparum malaria parasite cultured in vitro.

BACKGROUND: The automated haematology analyzer XN-30 (Sysmex, Kobe, Japan) easily and rapidly detects malarial parasites in clinical blood samples using flow cytometry. The XN-30 analyzer is able to distinguish each developmental stage by measuring DNA content and cell size. Thus, it was expected to be capable of quantifying the developmental stages of cultured falciparum parasite. To achieve this requirement, a modified algorithm was tested for its validity and reliability using in vitro cultured falciparum parasite. RESULTS: The XN-30 analyzer automatically measured each developmental stage as well as total parasitaemia. Comparison of the parasitaemia obtained using the XN-30 analyzer equipped with the modified algorithm with that obtained using microscopy examination of Giemsa-stained smears revealed the greater sensitivity and reproducibility of the former. The XN-30 analyzer also detected free merozoites and purified gametocytes. CONCLUSIONS: The XN-30 analyzer allows the precise recognition and enumeration of total and each developmental stages of cultured falciparum parasites, and permits the sensitive and reproducible calculation of parasitaemia. The results indicate the potential of the XN-30 analyzer for basic research on malarial biology, anti-malarial drug discovery, and evaluation of drug efficacy.