The Application of Biomedical Engineering Techniques to the Diagnosis and Management of Tropical Diseases: A Review <s> Microfluidics <s> In eastern Africa where 90% of the malaria is due to Plasmodium falciparum,the accuracy of malaria diagnosis at the outpatient level is becoming increasingly important due to problems of drug resistance and use of alternative, costly antimalarial drugs. The quantitative buffy coat (QBC t) technique, acridine orange staining with an interference filter system, and the ParaSightt-F test have been introduced as alternative methods to conventional microscopy for the diagnosis of malaria. Two hundred thirteen outpatients were tested using these alternative methods and conventional microscopy by five experienced technologists; two were randomly allocated to read the results of each test. Paired results showed the highest level of agreement with the ParaSight t-F test (99%), followed by Field stain (92%). The results of the QBCt technique showed the least agreement (73%). Using conventional microscopy as the reference standard, the ParaSightt-F test had a sensitivity range of 90-92% and a specificity of 99%, staining with acridine orange had a sensitivity range of 77-96% and a specificity range of 81-98% and the QBC t technique had a sensitivity range of 88-98% and a specificity range of 58-90%. All microscopic tests showed lower sensitivities (as low as 20% using staining with acridine orange) in detecting low parasitemias (# 320/ml) than the ParaSightt-F test (70%). Due to the high cost of the ParaSightt-F test, Field-stained blood films remain the most appropriate method for diagnosis of P. falciparum in eastern Africa. The ParaSightt-F test may be used in situations where no trained microscopists are available, or where malaria is strongly suspected and the results of microscopy are negative. Malaria is one of the leading causes of morbidity and mortality in eastern Africa. In malaria-endemic areas, most patients with febrile disease receive antimalarial drug treat- ment. However, in semiarid zones, the risk of malaria trans- mission is low and may be subject to seasonal variation. The clinical diagnosis of malaria is usually based on a history or presence of fever; presumptive treatment of fever results in over-administration of antimalarial drugs. 1-3 This practice may result in the erroneous treatment of fever caused by other infections, and may exert a high drug pressure on ma- laria parasite populations, leading to selection of drug-resis- tant parasites. Reliable diagnosis of malaria requires labo- ratory confirmation of the presence of malaria parasites in the blood of a febrile patient. In eastern Africa, where 90% of the malaria is due to Plasmodium falciparum, accuracy of malaria diagnosis at the outpatient level is becoming in- creasingly important due to emerging drug resistance and the use of alternative, costly antimalarial drugs. 4 <s> BIB001 </s> The Application of Biomedical Engineering Techniques to the Diagnosis and Management of Tropical Diseases: A Review <s> Microfluidics <s> Abstract Mouse malaria is often used as a model for drug testing. The results of drug trials are monitored by tedious (and consequently, sometimes inaccurate) microscopic counting of blood smears, or by flow cytometry. We suggest an improved, accurate and time-saving flow cytometric method for determination of parasitaemias in mice infected with Plasmodium vinckei petteri or Plasmodium berghei. The method involves collection of drops of blood from the tail vein, fixation, storage, permeabilisation, staining and analysis with a visible range flow cytometer. Three nucleic acid dyes, YOYO-1, propidium iodide and acridine orange were compared. YOYO-1 was found to be the best stain for the discrimination of parasitised erythrocytes from non-infected ones. A good direct correlation was obtained between parasitaemia determined by conventional microscopy and parasitaemia measured by flow cytometry. Drug effects could be assessed by the cytometric method. For the detection of low level of parasitemia, parasitised cells were treated with RNAse to completely cancel RNA-derived signals originating from host reticulocytes. This procedure also revealed discrete peaks arising from red cells infected with multiple parasites or from parasites with different numbers of nuclei. <s> BIB002 </s> The Application of Biomedical Engineering Techniques to the Diagnosis and Management of Tropical Diseases: A Review <s> Microfluidics <s> The past decade was a milestone in the development of malaria diagnostic technology. Today, a variety of simplified and rapid malaria diagnostic devices, collectively referred to as 'dipsticks', is available. This paper discusses the potential roles of these devices, and obstacles to their use, in supporting malaria control strategies. <s> BIB003 </s> The Application of Biomedical Engineering Techniques to the Diagnosis and Management of Tropical Diseases: A Review <s> Microfluidics <s> We have established a rapid, simple and sensitive flow cytometric system for the detection of Plasmodium falciparum that involves lysing erythrocytes and staining parasites at the same time using a newly developed hemolysing and staining solution containing dodecyl methyl ammonium chloride and acridine orange. In this system, freed parasites of P. falciparum could be plotted separately from erythrocyte ghosts, white blood cells and platelets on the two-dimensional scattergram of forward-angle light scatter and green fluorescence by flow cytometry with an argon laser. It took only 2-3 min per sample to obtain the scattergram and analyze the data, including the time of sample preparation for flow cytometric analysis. Sample preparation with this method does not require any difficult handling procedures. The threshold of parasite detection was almost equal to that of microscopic examination for cultured P. falciparum. The results of drug-susceptibility assays using this system were also almost identical to those obtained using microscopic examination. In this system, parasites at different erythrocytic stages could be easily distinguished. This system must prove useful and practical for basic laboratory studies of P. falciparum including those requiring the differential measurement of parasites at specific erythrocytic stages. <s> BIB004 </s> The Application of Biomedical Engineering Techniques to the Diagnosis and Management of Tropical Diseases: A Review <s> Microfluidics <s> Malaria presents a diagnostic challenge to laboratories in most countries. Endemic malaria, population movements, and travelers all contribute to presenting the laboratory with diagnostic problems for which it may have little expertise available. Drug resistance and genetic variation has altered many accepted morphological appearances of malaria species, and new technology has given an opportunity to review available procedures. Concurrently the World Health Organization has opened a dialogue with scientists, clinicians, and manufacturers on the realistic possibilities for developing accurate, sensitive, and cost-effective rapid diagnostic tests for malaria, capable of detecting 100 parasites/microl from all species and with a semiquantitative measurement for monitoring successful drug treatment. New technology has to be compared with an accepted "gold standard" that makes comparisons of sensitivity and specificity between different methods. The majority of malaria is found in countries where cost-effectiveness is an important factor and ease of performance and training is a major consideration. Most new technology for malaria diagnosis incorporates immunochromatographic capture procedures, with conjugated monoclonal antibodies providing the indicator of infection. Preferred targeted antigens are those which are abundant in all asexual and sexual stages of the parasite and are currently centered on detection of HRP-2 from Plasmodium falciparum and parasite-specific lactate dehydrogenase or Plasmodium aldolase from the parasite glycolytic pathway found in all species. Clinical studies allow effective comparisons between different formats, and the reality of nonmicroscopic diagnoses of malaria is considered. <s> BIB005 </s> The Application of Biomedical Engineering Techniques to the Diagnosis and Management of Tropical Diseases: A Review <s> Microfluidics <s> Recently introduced rapid nonmicroscopic immunocapture assays for the diagnosis of malaria infection are being evaluated for their sensitivity and specificity in various epidemiological settings. A Plasmodium falciparum histidine-rich protein-2 (PfHRP-2)-based assay (ICT malaria Pf assay) was evaluated for its performance and compared to that of Giemsa-stained thick blood film microscopy. Of the 515 patients tested, 163 were positive for malaria parasites on thick blood film microscopy: 87 were infected with P. vivax; 63 with P. falciparum; 1 with P. malariae; and 12 with both P. falciparum and P. vivax. The ICT assay detected 53 P. falciparum infections and, as expected, failed to detect all but one case of P. vivax. Three cases of mixed infections were also not detected by this assay. The performance of the ICT assay in diagnosing P. falciparum infection was comparable to that of microscopy. The sensitivity of the ICT assay was 82% and the specificity 99.0%. The ICT assay also detected 4 false-positive cases. These patients reported treatment with chloroquine in the previous 2-5 weeks. The specificity of the assay was evaluated in different groups of patients, who had tested negative for malaria infection by microscopy. These patients were selected from different disease groups: rheumatoid arthritis; hepatitis C; toxoplasmosis; schistosomiasis; and hydatid disease. Of the 225 patients studied, 133 were positive for rheumatoid factor. Thirty-five (26%) of the 133 patients had false positive-reactions with the ICT assay, while only four had false positive-reactions with the OptiMAL test. After the rheumatoid factor was absorbed 33 of the 35 false-positive specimens were negative when retested with the ICT assay. Our study shows that the PfHRP-2-based ICT assay gave a false positive-reaction in 26% of the patients who had rheumatoid factors, but were negative for malaria by microscopy. We conclude that new rapid nonmicroscopic methods for the diagnosis of malaria that complement or support blood film microscopy would be of great use in the diagnosis and treatment of patients with malaria and also in epidemiological studies. <s> BIB006 </s> The Application of Biomedical Engineering Techniques to the Diagnosis and Management of Tropical Diseases: A Review <s> Microfluidics <s> The replacement of conventional antimalarial drugs with high-cost, artemisinin-based alternatives has created a gap in the successful management of malaria. This gap reflects an increased need for accurate disease diagnosis that cannot be met by traditional microscopy techniques. The recent introduction of rapid diagnostic tests (RDTs) has the potential to meet this need, but successful RDT implementation has been curtailed by poor product performance, inadequate methods to determine the quality of products and a lack of emphasis and capacity to deal with these issues. Economics and a desire for improved case management will result in the rapid growth of RDT use in the coming years. However, for their potential to be realized, it is crucial that high-quality RDT products that perform reliably and accurately under field conditions are made available. In achieving this goal, the shift from symptom-based diagnosis to parasite-based management of malaria can bring significant improvements to tropical fever management, rather than represent a further burden on poor, malaria-endemic populations and their overstretched health services. <s> BIB007 </s> The Application of Biomedical Engineering Techniques to the Diagnosis and Management of Tropical Diseases: A Review <s> Microfluidics <s> Fluorescence and electrochemical microfluidic biosensors were developed for the detection of cholera toxin subunit B (CTB) as a model analyte. The microfluidic devices were made from polydimethylsiloxane (PDMS) using soft lithography from silicon templates. The polymer channels were sealed with a glass plate and packaged in a polymethylmethacrylate housing that provided leakproof sealing and a connection to a syringe pump. In the electrochemical format, an interdigitated ultramicroelectrode array (IDUA) was patterned onto the glass slide using photolithography, gold evaporation and lift-off processes. For CTB recognition, CTB-specific antibodies were immobilized onto superparamagnetic beads and ganglioside GM(1) was incorporated into liposomes. The fluorescence dye sulforhodamine B (SRB) and the electroactive compounds potassium hexacyanoferrate (II)/hexacyanoferrate (III) were used as detection markers that were encapsulated inside the liposomes for the fluorescence and electrochemical detection formats, respectively. Initial optimization experiments were carried out by applying the superparamagnetic beads in microtiter plate assays and SRB liposomes before they were transferred to the microfluidic systems. The limits of detection (LoD) of both assay formats for CTB were found to be 6.6 and 1.0 ng mL(-1) for the fluorescence and electrochemical formats, respectively. Changing the detection system was very easy, requiring only the synthesis of different marker-encapsulating liposomes, as well as the exchange of the detection unit. It was found that, in addition to a lower LoD, the electrochemical format assay showed advantages over the fluorescence format in terms of flexibility and reliability of signal recording. <s> BIB008 </s> The Application of Biomedical Engineering Techniques to the Diagnosis and Management of Tropical Diseases: A Review <s> Microfluidics <s> Nano/Microfluidic technologies are emerging as powerful enabling tools for diagnosis and monitoring of infectious diseases in both developed and developing countries. Miniaturized nano/microfluidic platforms that precisely manipulate small fluid volumes can be used to enable medical diagnosis in a more rapid and accurate manner. In particular, these nano/microfluidic diagnostic technologies are potentially applicable to global health applications, since they are disposable, inexpensive, portable, and easy-to-use for detection of infectious diseases. In this paper, we review recent advances in nano/microfluidic technologies for clinical point-of-care applications at resource-limited settings in developing countries. <s> BIB009
The portability and the small volume of sample required to perform a test are the main advantages of the microfluidic platforms BIB009 . These features, along with the low cost of the microfluidic platform make it ideal to be used on site and in resource-limited areas. Different immunochromatographic methods (dipstick) of malaria infection detection have been described BIB005 BIB003 . These platforms are easy to use, portable, and can be used in the most challenging environments. Most are based on the detection of malaria antigens from a patient's blood sample. The targeted antigen types include histidine-rich protein-2 (HRP-2), aldolase, and plasmodium lactate dehydrogenase (pLDH). The developed methods have the ability to differentiate between the main malaria types through the immunogenic differences in the proteins. Figure 7 shows an example of a microfluidic platform (dipstick) that utilizes immunochromatographic lateral flow to detect malarial proteins (antigen (Ag)) that are extracted from patient blood BIB007 . The disadvantage of the microfluidic platforms is their accuracy which is less than that from gold standard microscopy techniques BIB006 BIB001 . As the disease severity declines, the concentration of malarial antigens quickly decreases. Therefore, these detection techniques are good for detection of recurrent infection and not for observing the response of the patient to treatment. Many platforms were proposed using the flow cytometry approaches for malaria detection have been reported BIB002 BIB004 . Saito-Ito et al. BIB004 presented a fast, high sensitivity, and low error diagnostic platform that is capable of detecting the P. falciparum from a sample that contains erythrocytes and stained parasites. The sensing principle was based on forward-angle light scattering from an argon laser, and green fluorescence was utilized in this process. The author claimed that the required time to perform a full test is 2-3 min including sample preparation. Jiménez-Díaz et al.  proposed a new flow cytometric platform for malaria detection based on observing the differences of infected erythrocytes stained auto-fluorescence and DNA content with that of healthy cells. The author claimed that the proposed platform is rapid, simple to use, sensitive, and can accurately detect malarial pathogens. Bunyakul et al. BIB008 developed a microfluidic platform incorporating fluorescence and electrochemical detection techniques for the detection of cholera toxin subunit B (CTB). The microfluidic platform was fabricated using soft lithography on polydimethylsiloxane (PDMS). The sample with CTB concentrations of up to 1000 ng/mL is injected into the platform towards a capture zone withCTB-antibodies and ganglioside GM1 receptor immobilized onto magnetic beads. The beads are then immobilized using an external magnet for washing and then transferred to a detection zone for either fluorescence detection, or electrochemical detection. The implementation of the microfluidic platform allows for limits of detection of 6.6 and 1.0 ng·mL −1 respectively for the fluorescence and electrochemical techniques.