Figure 7.14 Flow anti-pLDH Plasmodium monoclonal antibodies rapid malaria test. (Left) Diagram of blood flow on the membrane through the timed test; note the positive control line and positive test line. (Right) Diagram of negative control, a positive P. vivax result, and a positive P. falciparum result. Note that the line for P. vivax is a panspecific antibody against all four species; however, the panspecific antibody has been shown to detect only P. vivax with any degree of consistency. (Photographs adapted from Flow’s website, with permission.) doi:10.1128/9781555819002.ch7.f14
Although the first PCR methods were developed to identify P. falciparum, many can now detect several or all five species. It has been well demonstrated that PCR can detect lower parasitemias than any of the traditional blood film or nontraditional dipstick methods, and it may be preferable to microscopy as the reference standard when evaluating new diagnostic tests (22, 24, 42, 46–48). One advantage of PCR is the ability to confirm false-negative results by microscopy as true positives. Another advantage is the enhanced ability to identify organisms to the species level, particularly in mixed infections, compared with microscopy. However, even PCR may miss some cases as a result of PCR inhibitors, DNA degradation, or genotypic variants. Unfortunately, the time required for PCR presents a problem for the clinical laboratory; this approach does not lend itself to routine use. With the development of automation for PCR, these tests may become much more user-friendly for the routine laboratory; however, currently PCR is reserved for reference centers and special circumstances. As PCR becomes more widely used, it will be important to remember that the mode of collection and storage of blood samples may influence the sensitivity of Plasmodium detection. This may be critical in studies of individuals with low parasitemia or mixed infections and in comparison of data from different settings, including field settings.
Other developments include the post-PCR/ligase detection reaction-fluorescent microsphere assay (LDR-FMA) (49). This assay, which uses Luminex FlexMAP microspheres, provides simultaneous, semiquantitative detection of infection by four human malaria parasite species at a sensitivity and specificity equal to those of other PCR-based assays. In blinded studies using P. falciparum-infected blood from in vitro cultures, the authors identified infected and uninfected samples with 100% concordance. Also, in analyses of P. falciparum in vitro cultures and P. vivax-infected monkeys, comparisons between parasitemia and LDR-FMA signal intensity showed very strong positive correlations (r > 0.95). Application of this multiplex Plasmodium LDR-FMA diagnostic assay will increase the speed, accuracy, and reliability of diagnosing human Plasmodium infections.
Certainly molecular testing offers many advantages over microscopy, including identification to species levels; this capability is becoming much more important with the shortage of experienced microscopists. A recent multiplex quantitative real-time PCR can rapidly and simultaneously identify all five Plasmodium species known to cause malaria in humans (P. vivax, P. ovale, P. malariae, P. falciparum, P. knowlesi) (50). In another recent study, malaria loop-mediated isothermal amplification (LAMP) used in a remote Ugandan clinic achieved sensitivity similar to that achieved by a single-well nested PCR in a United Kingdom reference laboratory (51).
Automated Blood Cell Analyzers
Although the use of automated blood cell analyzers is not yet clinically relevant for the diagnosis of blood parasites, improvements in the systems may offer future information that will supplement the routine microscopy procedures currently in use (52, 53). Unfortunately, when the parasitemia is light, automation tends to have some of the same problems seen with other alternative procedures. In many cases, the changes seen using analyzers are not specific to malaria infection and could occur in many other diseases (increases in the number of large, unstained cells and thrombocytopenia). The accuracy for malaria diagnosis appears to vary according to the Plasmodium species, parasitemia, immunity, and clinical context.
The Cell-Dyn 4000 automated hematology analyzer (Abbott, Chicago, IL) has the ability to detect 91.2% of malaria patients. In one study on day 3 of follow-up posttreatment, the sensitivity was 96.7% that of microscopy. The atypical polarizing events, which indicate the presence of malarial parasites in the analyzer, were highly correlated with the levels of parasitemia in serially diluted samples of the leukocyte-depleted blood; parasites were detected down to the level of 288 ± 17.7/µl). These data suggest that the atypical light depolarization could be influenced by parasitemia and could be used as a screening method for P. vivax malaria patients, as well as for therapeutic monitoring (52). Microscopy is still required for species determination and parasite quantitation.
The potential of automated depolarization analysis in detecting malaria infection as part of the routine full blood count performed by the Cell-Dyn 4000 analyzer has been described previously (52). In these cases, abnormal depolarizing patterns are due to the presence of leukocyte-associated malaria hemozoin, a pigment which depolarizes the laser light. Abnormal polarizing events have also been described for samples from three individual patients infected by the nematode Mansonella perstans. The observed depolarizing pattern consisted of a normal depolarizing eosinophil population plus an abnormal depolarizing population that showed a close “linear” relationship between “granularity” (90° depolarization) and “lobularity” (90° polarization). This atypical population was smaller than that of normal leukocytes and thus clearly different from the patterns associated with malaria infection. Abnormal depolarization patterns of M. perstans clearly do not reflect leukocyte-associated malaria hemozoin. It is possible, however, that the erythrocyte-lysing agent used to facilitate leukocyte analysis by the instrument may have caused microfilaria fragmentation and thus the distinctive straight-line features of the abnormal scatter plots (54).
Diagnosis of Leishmaniasis: Review of Alternatives to Conventional Microscopy
ICT for Detection of Anti-rK-39 Antibodies
In one study, the diagnostic utility of an immunochromatographic test for detection of anti-rK-39 antibodies for the diagnosis of kala azar and post-kala azar dermal leishmaniasis (PKDL) was evaluated (55). Of the 120 samples tested, 57 were positive by ICT; 51 of these were diagnosed as kala azar, and 6 were diagnosed as PKDL. The controls included individuals from areas of endemic (21) and nonendemic infection with malignancies, hemolytic disorders, chronic liver disease, hypersplenism, portal hypertension, metabolic disorders, or sarcoidosis. In addition, 47 sera from patients with confirmed cases of tuberculosis, malaria, typhoid, filariasis, leptospirosis, histoplasmosis, toxoplasmosis, invasive aspergillosis, amebic liver abscess, AIDS, leprosy, cryptococcosis, strongyloidiasis, and cyclosporosis, as well as from patients with collagen vascular diseases and patients with hypergammaglobulinemia, were tested to check the specificity
