Immunochromatographic Antigen Detection Tests

The World Health Organization has recognized the need for simple, cost-effective, and accurate diagnostic tests for malaria which could overcome the expertise-associated limitations of microscopy as well as the changing patterns of accepted morphology of malaria species due to drug pressure, strain variation, or approaches to blood collection.[5]

Nonmicroscopic malaria-diagnostic devices rely on the detection of malaria antigen, such as parasite histidine-rich protein 2 (e.g., NOW ICT Pf/Pv™, PATH Falciparum Malaria ICTM, and Parasight FTM), or parasite LDH (e.g., OptiMAL™) in whole blood. The underlying principle of immunochromatographic procedures utilize conjugated monoclonal antibodies against the malaria antigen of choice as an indicator of infection.

Histidine-rich protein 2 (HRP-2) is a P. falciparum-specific antigen, therefore assays limited to the detection of HRP-2 are unable to detect other human Plasmodium species. A newer generation of rapid diagnostic assays (RDA) use HRP-2 as well as aldolase, a pan-malarial antigen, to identify nonfalciparum infections, yet they remain unable to differentiate between the nonfalcipa-rum species.

Parasite LDH (pLDH)-based assays take advantage of the species-specific isomers of this enzyme from the parasite glycolytic pathway. Parasite LDH-based RDAs have a P. falciparum-specific antibody and two pan-malarial antibodies, which recognize nonfalcipa-rum pLDH.

Compared to microscopy and PCR, RDAs range widely in sensitivity and specificity. One of the latest HRP-2-based assays was 96% sensitive for pure P. falciparum and 94% for mixed P. falciparum infections and 84% sensitive for nonfalciparum infections with an overall specificity of 96%.[6] However, for both falciparum and vivax infections, sensitivity fell as parasitemia declined.[6] In a recent evaluation of returning travelers in Berlin, the OptiMal test (a pLDH assay) was found to be 76.2% sensitive and 99.7% specific.[7] In another study, the OptiMAL assay failed to detect malaria infection in 15% of cases.[8] The sensitivity for detection of P. falciparum infection was 87%, and for P. vivax was 79%. Similar to HRP-2 tests, the sensitivity of the pLDH tests decreased significantly with lower parasite densities (<500/pL).[8]

False negative and false positive results do occasionally occur with these devices, even at high parasitemias, possibly due to prozone effect (false negatives), the possible lack of or alteration in the hrp 2 gene (false negatives),[9] or the presence of rheumatoid factor (false positives).[10] Importantly, if the clinical suspicion of malaria remains high despite a negative RDA result the assay should be repeated within 12 to 24 hr[9] and these assays should be accompanied by thick and thin blood smears.

Despite some inherent limitations, evidence suggests that rapid malaria diagnostic devices may represent a useful adjunct diagnostic tool to microscopy in a clinical setting while definitive results are sought from a reference laboratory.

Molecular Methods—Radiolabeled Probes

As an alternative approach to the detection and species identification of malaria, species-specific probes were developed in the late 1980s and early 1990s. Hybridization of radiolabeled oligonucleotide probes to complementary species-specific regions of the abundant and stable parasite small ribosomal subunit RNA (ssRNA)[11] seemed promising for the detection of the four species of human Plasmodia. This method of detection is not commonly used as a diagnostic tool.

Molecular Methods—Polymerase Chain Reaction (PCR)

Nested PCR

Polymerase chain reaction-based diagnostic methods for malaria represent a major advancement, surpassing microscopic methods both in sensitivity and specificity. Polymerase chain reaction can detect as few as 1-5 parasites/pL of blood (<0.0001% of infected red blood cells) compared to 50-100 parasites/pL using microscopy or RDAs.[12-14] Furthermore, PCR can readily detect mixed-species infections and may be automated allowing processing of large number of samples.

The multicopy 18S (small subunit) rRNA genes of Plasmodium spp. that infect humans have been demonstrated to be highly stable and conserved. Assays to detect them have displayed no cross-reactions to human DNA or other human pathogen DNA/RNA including nonhuman Plasmodium spp.[11,13,14] The 18S gene of Plasmodium is an ideal molecular target for malaria parasite identification, both for the abundance of the DNA gene targets and because it is composed of a mosaic of conserved and variable regions, allowing the amplification of sequences from samples using primers that are conserved within every member of the genus Plasmodium.[15,16] Identification to the species level, however, requires an additional round of PCR, a nested PCR, with species-specific primers.

Although this nested-amplification procedures is highly sensitive and specific, it is more cumbersome and expensive than single PCR assays. Also, because it is an open system (i.e., open transfer of amplicons between tubes) there is an inherent risk of contamination. Amplified products can be detected by gel electrophore-sis, Southern or slot blotting followed by hybridization with DNA probes or by a colorimetric detection procedure with an enzyme-labeled antibody and chromogenic substrate in a microtiter format. The detection of amplification products by chemiluminescence hybridization or enzyme immunoassays in microplate format offers better potential for standardized commercial assays and automation.[12,17-19]

Real-time PCR

Despite their superior sensitivity and specificity over microscopy, traditional PCR, and particularly nested PCR, methods[14] are labor intensive with turnaround times that are generally too long for routine clinical application. Moreover, these are open systems that require considerable pre- and postsample handling and therefore special efforts need to be employed in order to prevent false positive assays. Real-time quantitative PCR technology has the potential to overcome these limitations, offering a simple, time-effective, and quantitative diagnostic option. Using DNA binding dyes, such as SYBR green, molecular probes, or hybrids labeled with fluorescent probes, realtime PCR can detect and quantify amplicons in as little as 40 min.

The closed amplification vessels utilized in this system minimize the need for excessive sample handling, particularly when compared to nested reactions, and eliminate additional post-PCR sample handling steps normally required for amplicon detection. These features significantly reduce the potential for sample contamination. When the real-time assay is combined with the available robotic sample preparation/nucleic acid extraction modules, the process may be fully automated. This combined with their ease of use and rapid turnaround times makes these assays well suited to routine diagnostic laboratories.

Although a number of in-house real-time assays for malaria diagnosis have been developed, few meet the good manufacturing practices (GMP) standards for commercialization. One commercially available real-time PCR assay for malaria diagnosis has recently been evaluated.1-201 It contains reagents and enzymes for the specific amplification and detection of a species-conserved 140-bp region of the Plasmodium 18S rRNA genes of all four human malaria species. In addition to the hybridization probe used for amplicon detection, the assay contains a second heterologous amplification system to identify potential PCR inhibition in samples. Four quantification standards with known concentrations of cloned gene copies per microliter (equivalent to 70 to 70,000 genome copies per microliter) are used as known positive controls and to generate a standard curve to assess parasite burden.

Direct correlations between parasitemia, as determined by microscopy, and gene copy number, as determined from the standard curve, are somewhat confounded by the multicopy nature of the rRNA genes, by the variable numbers of these genes within each species, and by the presence of multinucleate schizont stages. However, these two approaches represent alternative methods of quantifying parasite burden within a sample and additional clinical trials will be required in order to determine which method is more predictive of clinical outcome.

One limitation of the current generation of real-time assay is their inability to differentiate between the four Plasmodium species. In its current form, a positive assay would need to be accompanied by a malaria smear or nested PCR in order to ascertain the Plasmodium spp. involved, unless the assay is used primarily as a screening test or to exclude malaria in blood products. The high cost of this state-of the-art technology places real-time PCR-based assays out of reach for developing countries. However, diagnostic laboratories may find the rapid turnaround time, quantitative results, high sensitivity, and the excellent negative predictive value (allowing one to exclude malaria in a patient) to have potential impact on patient care.

Improved real-time PCR malaria diagnostic devices are underway. These utilize fluorescence resonance energy transfer (FRET) technology to differentiate species based on differing melting curve profiles. With a quantification component and an internal control component, this new generation of diagnostic tools will yield sensitive and specific results in under an hour.

Mass Spectrometry

A novel blood-based malaria diagnostic approach has recently been published.1-21-1 Ultraviolet laser desorption mass spectrometry (LDMS) is based on the detection of hemozoin (malaria pigment) formed by the parasites during the intraerythrocytic growth phase. As all four human malaria species catabolize host hemoglobin releasing hemozoin in blood, this by-product becomes a pan-malarial diagnostic biomarker. The LDMS detects the heme within the hemozoin, but not heme bound to intact hemoglobin with a sensitivity of 100 parasites per microliter, comparable to that of an average micros-copist. A semiquantitative relationship seems to exist between heme signal and parasitemia, rendering this a quantitative diagnostic assay. Upon further field validation, this could become a powerful, low-cost tool (<U.S.$0.05) for rapid (<5 min) and high-throughput malaria screening by nonspecialists. Although a field-portable version of this assay is currently under development, this assay is limited by its inability to distinguish between Plasmodium species.

Microarrays

The publication of the Plasmodium genome offers much opportunity in the field of malaria diagnostics. Although at present an expensive technology suited only to reference laboratories, microarrays may play an important role in future infectious disease diagnostics. Species-specific malaria diagnosis will be combined with genetic markers for other febrile infectious diseases to further enhance the diagnostic screening process of patients.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook


Post a comment