Laboratory evaluation of the miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA), a simplified molecular diagnostic test for Plasmodium.

BACKGROUND: Point-of-care diagnosis of malaria is currently based on microscopy and rapid diagnostic tests. However, both techniques have their constraints, including poor sensitivity for low parasitaemias. Hence, more accurate diagnostic tests for field use and routine clinical settings are warranted. The miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative, easy-to-use molecular assay for diagnosis of malaria in resource-limited settings. Unlike traditional molecular methods, mini-dbPCR-NALFIA does not require DNA extraction and makes use of a handheld, portable thermal cycler that can run on a solar-charged power pack. Result read-out is done using a rapid lateral flow strip enabling differentiation of Plasmodium falciparum and non-falciparum malaria infections. A laboratory evaluation was performed to assess the performance of the mini-dbPCR-NALFIA for diagnosis of pan-Plasmodium and P. falciparum infections in whole blood. METHODS: Diagnostic accuracy of the mini-dbPCR-NALFIA was determined by testing a set of Plasmodium-positive blood samples from returned travellers (n = 29), and Plasmodium-negative blood samples from travellers with suspected malaria (n = 23), the Dutch Blood Bank (n = 19) and intensive care patients at the Amsterdam University Medical Centers (n = 16). Alethia Malaria (LAMP) with microscopy for species differentiation were used as reference. Limit of detection for P. falciparum was determined by 23 measurements of a dilution series of a P. falciparum culture. A fixed sample set was tested three times by the same operator to evaluate the repeatability, and once by five different operators to assess the reproducibility. RESULTS: Overall sensitivity and specificity of the mini-dbPCR-NALFIA were 96.6% (95% CI, 82.2%-99.9%) and 98.3% (95% CI, 90.8%-100%). Limit of detection for P. falciparum was 10 parasites per microlitre of blood. The repeatability of the assay was 93.7% (95% CI, 89.5%-97.8%) and reproducibility was 84.6% (95% CI, 79.5%-89.6%). CONCLUSIONS: Mini-dbPCR-NALFIA is a sensitive, specific and robust method for molecular diagnosis of Plasmodium infections in whole blood and differentiation of P. falciparum. Incorporation of a miniature thermal cycler makes the assay well-adapted to resource-limited settings. A phase-3 field trial is currently being conducted to evaluate the potential implementation of this tool in different malaria transmission areas.

Head-to-head validation of six immunoassays for SARS-CoV-2 in hospitalized patients.

BACKGROUND: Detecting SARS-CoV-2 antibodies may help to diagnose COVID-19. Head-to-head validation of different types of immunoassays in well-characterized cohorts of hospitalized patients remains needed. METHODS: We validated three chemiluminescence immunoassays (CLIAs) (Liaison, Elecsys, and Abbott) and one single molecule array assay (SIMOA) (Quanterix) for automated analyzers, one rapid immunoassay RIA (AllTest), and one ELISA (Wantai) in parallel in first samples from 126 PCR confirmed COVID-19 hospitalized patients and 158 pre-COVID-19 patients. Specificity of the AllTest was also tested in 106 patients with confirmed parasitic and dengue virus infections. Specificity of the Wantai assay was not tested due to limitations in sample volumes. RESULTS: Overall sensitivity in first samples was 70.6 % for the Liaison, 71.4 % for the Elecsys, 75.4 % for the Abbott, 70.6 % for the Quanterix, 77.8 % for the AllTest, and 88.9 % for the Wantai assay, respectively. Sensitivity was between 77.4 % (Liaison) and 94.0 % (Wantai) after 10 dpso. No false positive results were observed for the Elecsys and Abbott assays. Specificity was 91.1 % for the Quanterix, 96.2 % for the Liaison, and 98.1 % for the AllTest assay, respectively. CONCLUSION: We conclude that low sensitivity of all immunoassays limits their use early after onset of illness in diagnosing COVID-19 in hospitalized patients. After 10 dpso, the Wantai ELISA has a relatively high sensitivity, followed by the point-of-care AllTest RIA that compares favorably with automated analyzer immunoassays.

Diagnosis and subtype analysis of Blastocystis sp. in 442 patients in a hospital setting in the Netherlands.

BACKGROUND: Blastocystis sp. are among the most commonly observed intestinal parasites in routine clinical parasitology. Blastocystis in humans consists of at least 9 genetic subtypes. Different subtypes of Blastocystis may be associated with differences in pathogenicity and symptomatology. METHODS: Advanced microscopy on two samples and sequence-confirmed PCR on a third sample from the same individual were used for Blastocystis diagnosis and subtype analyses on routine clinical samples in a university hospital. RESULTS: With a combined gold standard of sequence-confirmed PCR and positive advanced microscopy, 107 out of 442 (24.2%) patients were diagnosed with Blastocystis. infection, which is a high frequency of detection in comparison to previous reports from industrialized countries. The sensitivity of microscopy and sequence-confirmed PCR was 99.1% (106/107) and 96.3% (103/107), respectively.Among 103 typable samples, subtype 3 was most abundant (n = 43, 42%), followed by subtypes 1 and 2 (both n = 23, 22%), subtype 4 (n = 12, 12%), and single samples with subtypes 6 (1%) and subtype 7 (1%). The prevalence of Blastocystis infection was 38% in patients from the Department of Tropical Medicine and 18% in patients from other departments. CONCLUSIONS: A high prevalence of Blastocystis infection was found with both advanced microscopy and sequence-confirmed PCR in our patient population. Most cases were caused by subtypes ST1, ST2, ST3 and ST4. A significantly higher prevalence was found among patients with a history of recent travel to tropical countries.

Frequent occurrence of human-associated microsporidia in fecal droppings of urban pigeons in amsterdam, the Netherlands.

Human-associated microsporidia were frequently observed in fecal samples of 331 feral pigeons in Amsterdam, The Netherlands, obtained during high- and low-breeding periods. Thirty-six of 331 samples (11%) contained the human pathogens Enterocytozoon bieneusi (n = 18), Encephalitozoon hellem (n = 11), Encephalitozoon cuniculi (n = 6), and Encephalitozoon intestinalis (n = 1); 5 samples contained other microsporidia. Pigeon feces can be an important source of human microsporidian infection.

Specific cross-reactivity in sera from cystic echinococcosis patients in an enzyme-linked immunoelectrotransfer blot for cysticercosis diagnostics.

A commercially available enzyme-linked immunoelectrotransfer blot originally intended for diagnosis of cysticercosis was evaluated for echinococcosis diagnosis, because a characteristic band pattern--different from the specific cysticercosis pattern--was observed in sera from patients with echinococcosis. This band pattern was observed in 29 (78%) of 37 parasitologically proven cystic echinococcosis patients. Specificity of these bands was 100% for echinococcosis, when tested with 75 control sera.

Reliable serodiagnosis of imported cystic echinococcosis with a commercial indirect hemagglutination assay.

A commercially available indirect hemagglutination assay (IHA) (Echinococcosis Fumouze; Laboratoires Fumouze, Levallois-Perret, France) was evaluated using sera from 52 patients with proven cystic echinococcosis. The specificity was assessed using 247 sera from patients with various parasitic, bacterial, viral, and fungal infectious diseases; sera containing autoimmune antibodies; and sera from healthy blood donors. With a cutoff value for a positive result of 320 (as recommended by the manufacturer), the sensitivity and specificity were 88% and 98.4%; with a cutoff of 160, the sensitivity and specificity were 94% and 95.1%, respectively. The IHA is rapid, easy to perform, and is a very sensitive serodiagnostic test for cystic echinococcosis.

Carbohydrate moieties of microsporidian polar tube proteins are targeted by immunoglobulin G in immunocompetent individuals.

Microsporidia of the Encephalitozoon species are frequently found as opportunistic pathogens of immunocompromised patients, but very little is known about the prevalence and significance of Encephalitozoon infection in immunocompetent individuals. It was reported previously that 8% of Dutch blood donors and 5% of pregnant French women had an immunoglobulin G (IgG) immune response against specific organelles of Encephalitozoon intestinalis. These organelles, the so-called polar tube and anchoring disk, are used to penetrate membranes of host cells during infection. The unexpectedly high percentage of immunocompetent individuals with IgG against these organelles suggested that infection of humans with microsporidia might be more common than previously recognized. In the present study, we analyzed this anti-Encephalitozoon IgG response by using indirect immunofluorescence, Western blotting, two-dimensional gel electrophoresis, and chemical deglycosylation. Our results show that the antibody response is directed against the posttranslational carbohydrate modification of the major polar tube protein (polar tube protein 1) and carbohydrate moieties of proteins in the anchoring region of the polar tube of Encephalitozoon. In addition, the antibodies were found to decrease the infectivity of E. intestinalis in vitro. The significance and possible origin of these prevalent antibodies are discussed.

Detection and identification of Enterocytozoon bieneusi and Encephalitozoon species in stool and urine specimens by PCR and differential hybridization.

Several species of microsporidia can cause disease in humans in both immunocompromised and immunocompetent individuals. Enterocytozoon bieneusi and Encephalitozoon intestinalis are most commonly associated with chronic diarrhea. All Encephalitozoon species, including E. intestinalis, E. hellem, and E. cuniculi, also cause disseminated infections. As distinctive treatment options are available for the different genera, identification is clinically important. We evaluated a PCR with primers directed to a conserved region of the small subunit rRNA gene of microsporidia. Hybridization with a generic microsporidium probe and specific probes for each of the four different species was used for identification. Probes were labeled with ruthenium and detected by electrochemiluminescence. The sensitivity of the assay was tested with plasmids containing the region of interest from each of the four different species and Vittaforma corneae as a control. In addition, the assay was tested with feces spiked with cultured spores from each of the three Encephalitozoon species and V. corneae. An analytical sensitivity of 3.5 x 10(2) to 3.5 x 10(3) spores per g of feces, corresponding to 17 to 170 gene copies per PCR, was found, which is several orders of magnitude more sensitive than microscopy after Uvitex 2B fluorescent staining. Stool samples from 22 microscopically diagnosed patients and from 61 uninfected controls were evaluated, showing a sensitivity of at least 95% and a specificity of 100% compared to microscopy. The method was further tested by spiking urine samples with spores of the different Encephalitozoon species.

Serodiagnostic studies in an immunocompetent individual infected with Encephalitozoon cuniculi.

Little is known about the prevalence and clinical significance of infection with Encephalitozoon species in immunocompetent individuals. In the present study, by using indirect immunofluorescence technique (IFAT), Western blot, and recombinant antigens of the spore wall (SWP1) and polar tube (PTP1, PTP2, and PTP3 ), we analyzed the IgG antibody response of a laboratory worker who was infected with Encephalitozoon cuniculi. Serum samples were analyzed 1, 20, 32, and 38 months after infection. After 1 month, by use of IFAT, only spore-wall antigens were recognized, an antibody reaction that changed toward both the spore wall and polar tube in the following months. By use of Western blot analysis, a characteristic pattern that recognized multiple bands was noticed. Reaction against SWP1 was present in all 4 serum samples. The IgG response against PTP1, PTP2, and PTP3 was not detectable 1 month after infection, but became evident in the follow-up serum samples. Serum samples showed cross-reactivity with the spore wall of Encephalitozoon hellem and Encephalitozoon intestinalis, but only little cross-reactivity with the polar tube of these parasites. This is the first study to our knowledge that provides full details about the antibody response against a specified Encephalitozoon species in an immunocompetent person. The results strongly encourage the development and use of reliable serodiagnostic methods, which will provide information about the prevalence and clinical significance of Encephalitozoon species infection in humans.