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Declining malaria transmission and known difficulties with current diagnostic tools for malaria, such as microscopy and rapid diagnostic tests (RDTs) in particular at low parasite densities, still warrant the search for sensitive diagnostic tests. Molecular tests need substantial simplification before implementation in clinical settings in countries where malaria is endemic. Direct blood PCR (db-PCR), circumventing DNA extraction, to detect Plasmodium was developed and adapted to be visualized by nucleic acid lateral flow immunoassay (NALFIA). The assay was evaluated in the laboratory against samples from confirmed Sudanese patients (n = 51), returning travelers (n = 214), samples from the Dutch Blood Bank (n = 100), and in the field in Burkina Faso (n = 283) and Thailand (n = 381) on suspected malaria cases and compared to RDT and microscopy. The sensitivity and specificity of db-PCR-NALFIA compared to the initial diagnosis in the laboratory were 94.4% (95% confidence interval [CI] = 0.909 to 0.969) and 97.4% (95% CI = 0.909 to 0.969), respectively. In Burkina Faso, the sensitivity was 94.8% (95% CI = 0.88.7 to 97.9%), and the specificity was 82.4% (95% CI = 75.4 to 87.7%) compared to microscopy and 93.3% (95% CI = 87.4 to 96.7%) and 91.4% (95% CI = 85.2 to 95.3%) compared to RDT. In Thailand, the sensitivity and specificity were 93.4% (CI = 86.4 to 97.1%) and 90.9 (95% CI = 86.7 to 93.9%), respectively, compared to microscopy and 95.6% (95% CI = 88.5 to 98.6%) and 87.1% (95% CI = 82.5 to 90.6) compared to RDT. db-PCR-NALFIA is highly sensitive and specific for easy and rapid detection of Plasmodium parasites and can be easily used in countries where malaria is endemic. The inability of the device to discriminate Plasmodium species requires further investigation.

Molecular tools, such as real-time nucleic acid sequence-based amplification (NASBA) and PCR, have been developed to detect Trypanosoma brucei parasites in blood for the diagnosis of human African trypanosomiasis (HAT). Despite good sensitivity, these techniques are not implemented in HAT control programs due to the high cost of the equipment, which is unaffordable for laboratories in developing countries where HAT is endemic. In this study, a simplified technique, oligochromatography (OC), was developed for the detection of amplification products of T. brucei 18S rRNA by NASBA. The T. brucei NASBA-OC test has analytical sensitivities of 1 to 10 parasites/ml on nucleic acids extracted from parasite culture and 10 parasites/ml on spiked blood. The test showed no reaction with nontarget pathogens or with blood from healthy controls. Compared to the composite standard applied in the present study, i.e., parasitological confirmation of a HAT case by direct microscopy or by microscopy after concentration of parasites using either a microhematocrit centrifugation technique or a mini-anion-exchange centrifugation technique, NASBA-OC on blood samples had a sensitivity of 73.0% (95% confidence interval, 60 to 83%), while standard expert microscopy had a sensitivity of 57.1% (95% confidence interval, 44 to 69%). On cerebrospinal fluid samples, NASBA-OC had a sensitivity of 88.2% (95% confidence interval, 75 to 95%) and standard microscopy had a sensitivity of 86.2% (95% confidence interval, 64 to 88%). The T. brucei NASBA-OC test developed in this study can be employed in field laboratories, because it does not require a thermocycler; a simple heat block or a water bath maintained at two different temperatures is sufficient for amplification.

Currently available methods for the diagnosis of cutaneous leishmaniasis (CL) have low sensitivities or are unable to quantify the number of viable parasites. This constitutes a major obstacle for the diagnosis of the disease and for the study of the effectiveness of treatment schedules and urges the development of improved detection methods. In this study, quantitative nucleic acid sequence-based amplification (QT-NASBA) technology was used to detect and quantify Leishmania parasites in skin biopsy samples from CL patients. The assay is based on the detection of a small subunit rRNA (18S rRNA), which may allow for the detection of viable parasites. The QT-NASBA assay was evaluated using in vitro-cultured promastigotes and amastigotes and 2-mm skin biopsy samples from Old and New World CL patients. The study demonstrated that the lower detection limit of the QT-NASBA was two parasites per biopsy sample. Parasites could be quantified in a range of 2 to 11,300,000 parasites per biopsy sample. The QT-NASBA could detect levels of parasites 100-fold lower than those detected by conventional PCR. Test evaluation revealed that the QT-NASBA had a sensitivity of 97.5% and a specificity of 100% in the present study. The QT-NASBA is a highly sensitive and specific method that allows quantification of both Old and New World Leishmania parasites in skin biopsy samples and may provide an important tool for diagnosis as well as for monitoring the therapy of CL patients.

A dipstick assay, based on Leishmania infantum antigen, for the rapid detection of Leishmania-specific antibodies in canine serum samples was developed and evaluated. After determination of optimal dipstick test conditions, test performance was compared with two existing serological tests, i.e., the direct agglutination test (DAT) and the fast agglutination screening test (FAST). In the present study the dipstick test had a sensitivity of 99.2% and a specificity of 87.9%. The DAT had a sensitivity of 97.7% and a specificity of 95.2%, whereas the FAST had also a sensitivity of 97.7% and a specificity of 93.0%. High degrees of agreement were observed between the dipstick test and DAT (93.7%; kappa value, 0.86), between the dipstick test and FAST (91.8%; kappa value, 0.82), and between the DAT and FAST (95.2%; kappa value, 0.90). The high sensitivity and ease of performance make the dipstick test very suitable for surveillance surveys.

A quantitative nucleic acid sequence-based amplification (QT-NASBA) assay for the detection of Plasmodium parasites has been developed. Primers and probes were selected on the basis of the sequence of the small-subunit rRNA gene. Quantification was achieved by coamplification of the RNA in the sample with one modified in vitro RNA as a competitor in a single-tube NASBA reaction. Parasite densities ranging from 10 to 10(8) Plasmodium falciparum parasites per ml could be demonstrated and quantified in whole blood. This is approximately 1,000 times more sensitive than conventional microscopy analysis of thick blood smears. Comparison of the parasite densities obtained by microscopy and QT-NASBA with 120 blood samples from Kenyan patients with clinical malaria revealed that for 112 of 120 (93%) of the samples results were within a 1-log difference. QT-NASBA may be especially useful for the detection of low parasite levels in patients with early-stage malaria and for the monitoring of the efficacy of drug treatment.

The potential of Cooperia oncophora excretory/secretory (ES) proteins as antigens in a serological assay which aims to establish exposure levels in cattle was assessed. ES proteins were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis and immunoblotting. The N-terminal domains of two ES proteins were sequenced, and the corresponding cDNAs were cloned. Two cDNAs, designated CoES14.0 and CoES14.2, were expressed in Escherichia coli. The recombinant proteins were tested in an indirect enzyme-linked immunosorbent assay (ELISA) in which crude worm antigen (CWA) was used as a reference standard. In total, 67 reference serum samples were used: 27 negative serum samples, 29 C. oncophora-specific serum samples, 7 Dictyocaulus viviparus-specific serum samples, and 4 Ostertagia ostertagi-specific serum samples. This showed respective sensitivities and specificities of 17 and 84%, 0 and 100%, and 100 and 100% by the ELISAs with the three different types of proteins (CWA, CoES14.0, and CoES14.2, respectively). Since the CoES14.2 ELISA had the best sensitivity and specificity with reference sera, its specificity was further validated in an antigen inhibition ELISA. In this assay CoES14.2 and CWA preparations of C. oncophora, Cooperia curticei, O. ostertagi, Nematodirus helvetianus, Fasciola hepatica, D. viviparus, Haemonchus placei, and Trichostrongylus colubriformus were used as competitor antigens. This experiment showed that only the homologous antigens C. oncophora CWA and CoEs14.2 resulted in 100% inhibition. The CWA preparations of all other nematodes did not affect the ELISA, even if concentrations of 250 times the 50% inhibitory concentration of C. oncophora CWA were used. These results indicate that CoES14.2 does not share cross-reactive epitopes with heterologous CWAs. Finally, we tested the CoES14.2 ELISA with sequential serum samples from naturally infected groups of animals. The optical density values that were obtained correlated well with exposure levels based on cumulative egg excretion. Thus, the CoES14.2 ELISA seems to be a very sensitive tool for estimating exposure levels in cattle.