A Novel Immunochromatographic Test Strip Versus A Commercial Rapid Diagnostic Test for the Detection of Imported Plasmodium Species in Guangxi Province, China: A Comparative Evaluation

Background: China has made signicant progress towards malaria elimination by achieving zero reports of indigenous malaria cases over two consecutive years. The diagnosis of malaria, which is based on microscopic examination, still remains a challenge, due to a lack of sensitivity for detecting low-level parasitaemia. This study aimed to evaluate the diagnostic value of a novel immunochromatographic test strip for imported malaria. Methods: After obtaining informed consent, blood samples were collected from migrants returning home from Africa to Shanglin County in Guangxi, PR China, in 2018-2019, and were tested with the novel test strips. The test results were compared with those of microscopic examination, commercial malaria rapid diagnostic test (RDT), and polymerase chain reaction (PCR) assay. Results: A total of 535 samples were tested. Both microscopy and PCR test results showed a total of 162 (30.28%) malaria-positive samples, of which 99 were positive for Plasmodium falciparum (P. falciparum), three for P. vivax, ve for P. malariae, and 50 for P. ovale, whereas ve had mixed infections. The sensitivity, specicity, and eciency of the novel RDT were 74.1%, 93.0%, and 87.3%, respectively, whereas its sensitivity of detecting P. falciparum, P. vivax, P. malariae, and P. ovale was 91.9%, 100%, 20.0%, and 44.0%, respectively. Furthermore, the sensitivity, specicity, and eciency of the commercial RDT were 86.0%, 89.3%, and 88.2%, respectively, whereas its sensitivity of detecting P. falciparum, P. vivax, P. malariae, and P. ovale was 96.0%, 100%, 40.0%, and 70.0% respectively. The differential detection of Plasmodium species using the new RDT was signicantly better compared to that of the commercial RDT (c 2 = 14.73; P = 0.0001), which can detect only P. falciparum. This was consistent with Kappa analysis (Kappa = 0.881).


Background
Malaria is one of the parasitic diseases that endanger human health with an estimated 228 million recorded malaria infection cases, and 405,000 deaths, worldwide, in 2018 [1]. Since the launch of the National Malaria Elimination Action Plan (2010-2020) in 2010, China has made signi cant achievements in malaria elimination [2], with zero reports of local indigenous cases for two consecutive years (2017 and 2018) [3,4]. However, with frequent international trade and Chinese foreign investments, there has been an increase in imported malaria infections. Between 2011 and 2016, nearly 3,000 cases of imported malaria cases were reported each year [5]. This put the entire country at risk for malaria infections, especially in areas which still harbour mosquito Anopheles vectors. Without a timely diagnosis and effective treatment, the appearance of new cases of severe malaria, mainly caused by Plasmodium falciparum (P. falciparum), could be catastrophic [6], presenting a challenge in the malaria elimination efforts in China [7]. Early and accurate detection of malaria infection is important in reducing its associated morbidity and mortality. The gold standard for the detection of malaria infection is microscopic examination, while the most reliable method is genetic examination using polymerase chain reaction (PCR) or deoxyribonucleic acid (DNA) sequencing [2]. However, rapid diagnostic tests (RDTs) are an alternative to diagnosis based on clinical grounds or microscopy, particularly where good quality microscopy services cannot be readily provided. RDTs detect different target antigens for P. falciparum identi cation, such as histidine rich protein 2 (HRP2) [3][4][5] or P. falciparum-speci c lactate dehydrogenase (PfLDH) [5][6][7], whereas P. vivax-speci c lactate dehydrogenase (Pv-pLDH) [8], and aldolase [3,5,6] or Plasmodium lactate dehydrogenase (pLDH) [5,7,9,10] are detected for P. vivax, P. ovale and P. malariae identi cation. Although the usefulness of RDTs for P. falciparum identi cation is well recognised [11,12], few studies have described the performance of RDTs in the identi cation of imported Plasmodium species, particularly that of P. ovale and P. malariae.
The purpose of this article was to determine the diagnostic performance of a recently developed immunochromatographic test strip by comparing the test results with those of microscopic examination, PCR, and commercial malaria RDT, using blood samples collected from Chinese migrants returning from Africa to Shanglin County in Guangxi during 2018-2019.

Study area and population
Shanglin County is located in the southeast of Guangxi. It is home to the largest population of migrant workers from Africa and Southeast Asia, where the imported malaria cases account for 80.0% of the total number of cases in Guangxi each year. The study was conducted in Shanglin County in Guangxi, China between 2018 and 2019. Chinese migrants returning from Africa, with or without fever, willing to sign an informed consent form were included in the study. Migrants who failed to sign the consent form or were unable to provide a blood sample were excluded from the study. A total of 535 participants were enrolled in the study.

Malaria diagnosis using microscopy
Three blood smears were prepared for each study participant. Brie y, three Giblet-stained blood smears were prepared for each participant, including thick and thin smears, and then examined under a microscope by two experienced microscopists, according to the microscopic examination of blood smear for Plasmodium detection guide, (WS/T 569-2017) [10]. When no Plasmodium parasites were found in at least 200 elds, or the entire blood smear, the test result was declared negative. Any observation of Plasmodium parasite was considered positive, and was followed by the determination of Plasmodium speciation. When there was a discrepancy in the interpretation of the microscopy results, a third microscopist with a level 1 World Health Organization (WHO) external competency assessment certi cate evaluated the controversial results and made the nal decision. The RDTs were performed and the results were recorded immediately. The malaria diagnosis reference laboratory in the Guangxi Zhuang Autonomous Region used microscopy and PCR to check all the positive cases, and randomly screened 30% of the negative blood samples for blind check. When either the microscopy or PCR test result was positive, a malaria diagnosis was provided. The workers returning from Africa with negative test results were allocated to the control group. Furthermore, 100 blood samples were collected randomly from examinees at Guangxi Centre Disease Control and Prevention and used as negative controls.
Extraction of DNA using a DNA extraction kit (Qiagen, Hilden, Germany) was conducted according to the manufacturer's instructions. The 18SSU r RNA gene was chosen as the target for malaria detection. Primers used for the study. Nested PCR reactions in a total volume of 25 µL for each reaction were performed. In the rst round of PCR ampli cation the following components were included in the samples: template DNA 2 µL, upstream and downstream primers 0.5 µL, Premix Taq enzyme 12.5 µL, ddH 2 O 8.5 µL. The reaction conditions were as follows: 94 ℃ 3 min, 94 ℃ 30 sec, 58 ℃ 30 sec, 72 ℃ for 1 min, 35 cycles; and nally 72 ℃ for 5 min. In the second round of PCR ampli cation the following components were included in the samples: template DNA 2 µL, upstream and downstream primers 0.5 µL, Premix Taq enzyme 12.5 µL, ddH 2 O 8.5 µL. The reaction conditions were as follows: 94 ℃ 3 min, 94 ℃ 30 sec, 58 ℃ 30 sec, 72 ℃ for 1 min, 35 cycles, and nally 72 ℃ for 5 min. The PCR products from the second round were then analysed by 2.5% agarose gel electrophoresis and observed with a gel imager.

Malaria diagnosis using RDTs
Two types of RDTs were used to test the blood samples. The commercial RDT was used according to the manufacturer's instructions ( Table 1). The novel malaria RDT is an immunochromatographic test strip that uses antibodies to detect malaria. Brie y, 5 µL of whole blood sample was rst added to a reaction which was then stopped by adding four drops of buffer solution. The results were interpreted within 15 minutes. For the novel RDT test strip, rst, 5 µL of whole blood sample was added, followed by four drops of cell dissociation buffer solution. The results were read within 15 minutes. The novel malaria RDT test strip has a control line (C), a detection line "1" indicating a P. falciparum infection (T1), a detection line "2" indicating a P. vivax infection (T2), and a detection line "3", indicating an infection by other species of Plasmodium. Visual comparison of the results of the novel and commercial RDT in the Plasmodium blood sample test The novel malaria RDT could differentiate P. falciparum (colour in T1) and P. vivax (colour in T2, no colour in T1). A positive test for P. malariae or P. ovale infection was indicated by colour in T3 (no colour in T1 and T2). However, it was challenging distinguishing between P. ovale and P. malariae infections. The commercial RDT test could differentiate P. falciparum (T1 displaying colour) from other Plasmodium species (P. vivax, P. malariae, or P.ovale). The presence of these three Plasmodium was indicated by colour in T3 (no colour in T1 and T2). However, this test could not distinguish between these three species. (Fig. 1) The detection of different Plasmodium species using the two RDTs Using the microscopy and PCR results as the reference standards (a case was regarded positive when either test result was positive), the novel malaria RDT had a total sensitivity of 74.1%, speci city of 93.0%, and test e ciency of 87.3%, Kappa = 0.690 (P < 0.0001). The sensitivity of detecting P. falciparum, P. vivax, P. malariae, and P. ovale was 91.9%, 100%, 20.0%, and 44.0%, respectively. The commercial RDT had a total sensitivity of 86.0%, speci city of 89.3%, and test e ciency of 88.2%, Kappa = 0.729 (P < 0.0001). The sensitivity of detecting P. falciparum, P. vivax, P. malariae, and P. ovale was 96.0%, 100%, 40.0%, and 70.0%, respectively ( Tables 2 and 3).  The commercial RDT had a speci city of 89.3%, with a P. falciparum-detection sensitivity of 96.0% (Kappa = 0.752; P < 0.0001), while its detection sensitivity for other Plasmodium species was 69.0% (Kappa = 0.502; P < 0.0001). The novel malaria RDT had a speci city of 93.0%, with a P. falciparumdetection sensitivity of 91.9% (Kappa = 0.796;P < 0.0001) and a detection sensitivity of 44.8% for other Plasmodium species (Kappa = 0.396; P < 0.0001) ( Table 4).

Discussion
With the acceleration in the progress towards national malaria elimination in 2020 [11], China needs to ensure timely detection and treatment of active malaria cases to prevent reintroduction. However, as China continues its efforts in the pursuit of malaria elimination and sees no indigenous cases nationwide for many years, plus frequent transfer of the microscopists, it is challenging to maintain the capacity of microscopy [13]. Moreover, the low density of Plasmodium and protozoan morphology within asymptomatic carriers in endemic areas also contribute to di culty in sustaining the capacity of microscopy [14].
In recent years, China has seen a fair number of imported P. vivax and P. ovale malaria cases. These two species are di cult to be detected using the traditional microscopy technique, hindering the accomplishment of malaria control [15]. RDTs based on immunochromatographic analysis technology have developed rapidly recently, with nearly one hundred commercial kits on the domestic and foreign markets. RDTs are simple and convenient to operate and time-saving, as the test results are straightforward and easy to interpret, and no other instruments are needed. After training, healthcare professionals can skilfully use RDTs. Following continuous improvement, RDTs now show good sensitivity and speci city [16].
RDTs have been recommended by the WHO as a convenient visual way of diagnosing malaria [17]. A study in Sudan by Osman et al., [18] showed that the RDTs they used produced a relatively high number of false positive samples. The RDT was compared to PCR and an agreement of only 81.2 and k = 0.48 (sensitivity 69%, speci city 84%) was reported. Herrera et al., [16] in a study in endemic areas of Colombia, showed that the percentage concordance between visual and device interpretation of RDT was 98.5% and 99.0% for P. vivax and P. falciparum, respectively. Megnekou et al. [19] used RDTs to test individuals at the Marie Reine Health Centre in Etoudi, a peri-urban area of Yaoundé, Cameroon, from August 2013 to January 2015. The speci city of these RDTs was 96.49% and 100%. Studies have shown differences in the ability of different RDT products to detect malaria. In recent years, it has also become a common way for routine or active malaria surveillance of four Plasmodium species differentiation among migrants [20,21]. To maximise the test outcomes, it is imperative to further study and optimise the RDT products.
A total of 162 malaria positive cases were detected using the novel RDT product in Guangxi, China. The results showed that the novel RDT had a general sensitivity of 74.1%, a speci city of 93.0%, and a test e ciency of 87.3%, (Kappa = 0.690; P < 0.0001), among which the speci city and sensitivity for P. falciparum detection were 93.0% and 91.9%, respectively. Collectively, our data showed that the two RDTs had a similar capacity for P. falciparum detection, speci city, and test e ciency. Both test strips could detect the three samples of P. vivax, but statistical signi cance was not observed due to the low sample number.
The on-site test result showed that the novel malaria RDT products had potential advantages over the commercial RDT products. For example, the novel RDT could distinguish P. falciparum from P. vivax. This could help local medical institutions adopt a timely and appropriate treatment. Furthermore, due to the wide distribution of Anopheles sinensis in most endemic provinces of China, an accurate and quick identi cation of P. vivax will help the local Centres for Disease Control and Prevention carry out a timely foci response during the transmission season [22]. However, the e ciency of this novel test for P. malariae and P. ovale detection needs to be further improved for a large-scale application.
One limitation of this study is that the development of the novel RDT test is still in a preliminary stage.
Therefore, the experimental design requires further improvements. As the monoclonal antibody (Mab) used in this novel RDT was still the pan antibody obtained from immunisation with P. falciparum lactate dehydrogenase and was not sensitive enough, the detection rates of P.vivax, P. malariae, and P. ovale were relatively low. In the future, further studies are required using Mabs with a better sensitivity and speci city for P. vivax, P. malariae, and P. ovale.

Conclusion
The newly developed malaria diagnostic immunochromatographic test strip had high sensitivity and speci city in detecting P. falciparum and P. vivax. However, an improvement of the test e ciency for P. ovale and P. malariae detection may be important for scaling up its application.

Declarations
Ethics approval and consent to participate Written informed consent for participation and blood specimen collection was obtained from all the participants in the study.

Consent for publication
Written informed consent for publication was obtained from all participants.

Availability of data and material
The datasets generated and/or analysed during the current study are not publicly available, due to the fact that they are part of research project that is still ongoing. They are available from the corresponding author, however, on reasonable request.