False positive circumsporozoite protein ELISA: a challenge for the estimation of the entomological inoculation rate of malaria and for vector incrimination

<p>Abstract</p> <p>Background</p> <p>The entomological inoculation rate (EIR) is an important indicator in estimating malaria transmission and the impact of vector control. To assess the EIR, the enzyme-linked immunosorbent assay (ELISA) to detect the circumsporozoite protein (CSP) is increasingly used. However, several studies have reported false positive results in this ELISA. The false positive results could lead to an overestimation of the EIR. The aim of present study was to estimate the level of false positivity among different anopheline species in Cambodia and Vietnam and to check for the presence of other parasites that might interact with the anti-CSP monoclonal antibodies.</p> <p>Methods</p> <p>Mosquitoes collected in Cambodia and Vietnam were identified and tested for the presence of sporozoites in head and thorax by using CSP-ELISA. ELISA positive samples were confirmed by a <it>Plasmodium </it>specific PCR. False positive mosquitoes were checked by PCR for the presence of parasites belonging to the Haemosporidia, Trypanosomatidae, Piroplasmida, and Haemogregarines. The heat-stability and the presence of the cross-reacting antigen in the abdomen of the mosquitoes were also checked.</p> <p>Results</p> <p>Specimens (N = 16,160) of seven anopheline species were tested by CSP-ELISA for <it>Plasmodium falciparum </it>and <it>Plasmodium vivax </it>(Pv210 and Pv247). Two new vector species were identified for the region: <it>Anopheles pampanai </it>(<it>P. vivax</it>) and <it>Anopheles barbirostris </it>(<it>Plasmodium malariae</it>). In 88% (155/176) of the mosquitoes found positive with the <it>P. falciparum </it>CSP-ELISA, the presence of <it>Plasmodium </it>sporozoites could not be confirmed by PCR. This percentage was much lower (28% or 5/18) for <it>P. vivax </it>CSP-ELISAs. False positive CSP-ELISA results were associated with zoophilic mosquito species. None of the targeted parasites could be detected in these CSP-ELISA false positive mosquitoes. The ELISA reacting antigen of <it>P. falciparum </it>was heat-stable in CSP-ELISA true positive specimens, but not in the false positives. The heat-unstable cross-reacting antigen is mainly present in head and thorax and almost absent in the abdomens (4 out of 147) of the false positive specimens.</p> <p>Conclusion</p> <p>The CSP-ELISA can considerably overestimate the EIR, particularly for <it>P. falciparum </it>and for zoophilic species. The heat-unstable cross-reacting antigen in false positives remains unknown. Therefore it is highly recommended to confirm all positive CSP-ELISA results, either by re-analysing the heated ELISA lysate (100°C, 10 min), or by performing <it>Plasmodium </it>specific PCR followed if possible by sequencing of the amplicons for <it>Plasmodium </it>species determination.</p

Clinical, laboratory and pathological findings in dogs experimentally infected with Angiostrongylus vasorum

The aim of this comparative study was to investigate the development of clinical signs and accompanying haematological, coproscopic and pathological findings as a basis for the monitoring of health condition of Angiostrongylus vasorum infected dogs. Six beagles were orally inoculated with 50 (n = 3) or 500 (n = 3) A. vasorum third stage larvae (L3) obtained from experimentally infected Biomphalaria glabrata snails. Two dogs were treated with moxidectin/imidacloprid spot-on solution and two further dogs with an oral experimental compound 92 days post infection (dpi), and were necropsied 166 dpi. Two untreated control dogs were necropsied 97 dpi. Prepatency was 47-49 days. Dogs inoculated with 500 L3 exhibited earlier (from 42 dpi) and more severe respiratory signs. Clinical signs resolved 12 days after treatment and larval excretion stopped within 20 days in all four treated dogs. Upon necropsy, 10 and 170 adult worms were recovered from the untreated dogs inoculated with 50 and 500 L3, respectively. Adult worms were also found in two treated dogs, in the absence of L1 or eggs. Despite heavy A. vasorum infection load and severe pulmonary changes including vascular thrombosis, only mild haematological changes were observed. Eosinophilia was absent but the presence of plasma cells was observed. Neutrophilic leucocytes showed a transient increase but only after treatment. Signs for coagulopathies were slight; nevertheless coagulation parameters were inoculation dose dependent. Ten weeks after treatment pulmonary fibrosis was still present. Infections starting from 50 L3 of A. vasorum had a massive impact on lung tissues and therefore on the health of affected dogs, particularly after prepatency, although only mild haematological abnormalities were evident

An ELISA for sensitive and specific detection of circulating antigen of Angiostrongylus vasorum in serum samples of naturally and experimentally infected dogs

Canine angiostrongylosis is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We developed a sandwich-ELISA based on a monoclonal antibody (mAb Av 56/1/2) and on polyclonal rabbit antibodies directed against Angiostrongylus vasorum adult excretory/secretory – antigen for the detection of circulating serum antigen of A. vasorum. The sensitivity of the test was 95.7% (78.1–99.9, 95% CI) as determined with sera of 23 dogs naturally infected with A. vasorum. The specificity was 94.0% (83.5–98.7, 95% CI) using 50 dog sera (control group) submitted for reasons other than parasitic infections. Potential cross-reactions were investigated with sera of a group of totally 61 dogs with proven infections with Dirofilaria immitis (n = 23), Crenosoma vulpis (n = 14), Ancylostoma caninum (n=4) or Toxocara canis (n = 20). No significant difference was observed concerning the proportion of positive reactions between the control group and the group with proven helminth infections other than A. vasorum. In experimentally inoculated dogs with proven worm burdens of A. vasorum, the proportion of seropositive dogs increased over the first 3 months of infection, starting from 35 days post inoculation (dpi) which was before the onset of larval excretion. Ten weeks post inoculation, 98.6% of the dogs were seropositive, and circulating antigen persisted in two dogs with long-term follow-up over 286 and 356 days, respectively. In contrast, in dogs with a single treatment with imidacloprid/ moxidectin at four or 32 dpi, no circulating antigen was observed, while in dogs treated at 88–92 dpi, OD values decreased within 13–34 days. The specific detection of circulating A. vasorum antigen by ELISA represents a valid alternative for reliable diagnosis and for follow-up investigations after anthelmintic treatment. Moreover, the test can be used for mass screening in large epidemiological investigations

PCR detection of Angiostrongylus vasorum in faecal samples of dogs and foxes

The cardiovascular nematode Angiostrongylus vasorum is spreading in the fox and dog populations of northern Europe. A. vasorum can result in severe clinical manifestations in dogs; therefore, specific diagnosis is crucial for assessing its prevalence. In the present study, faecal samples from foxes and domestic dogs were tested by a new polymerase chain reaction (PCR) targeting the second internal transcribed region of the ribosomal DNA (ITS2) of A. vasorum. Initial isolation of faecal larvae by sieving facilitated the processing of larger sample volumes and allowed for the recovery of dead larvae from frozen samples. The sieve-PCR method enabled the identification of a single larva per 2 g of faecal sample and did not amplify DNA of a range of canine helminths, thus presenting a non-invasive tool for wildlife surveillance and for confirmative diagnosis in dogs

Detection of specific antibodies in dogs infected with Angiostrongylus vasorum

Canine angiostrongylosis, caused by the nematode Angiostrongylus vasorum, is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We determined the diagnostic value of the specific detection of antibodies against A. vasorum adult somatic antigen, adult excretory/secretory (E/S) antigen and first stage larvae (L1) somatic antigen in ELISAs. Also, A. vasorum adult somatic antigen purified by monoclonal antibodies (mAb) was evaluated in a sandwich-ELISA. Among the crude antigens, the best sensitivities when testing 21 naturally infected dogs were obtained using adult E/S and somatic antigen (85.7% and 76.2%, respectively), which were comparable with the results of the sandwich-ELISA based on mAb-purified antigens (81%). The ELISA performed with L1 antigen had the lowest sensitivity (42.9%). In experimentally inoculated dogs, the sensitivities ranged from 97.7% to 100% with all test settings. The specificity was 98.8% (92.5-99.9%, 95% CI) with all ELISAs using sera of 82 randomly selected dogs. Cross-reactions using adult somatic, adult E/S and L1 somatic antigen were observed in sera of dogs infected with Crenosoma vulpis, Dirofilaria immitis, Dirofilaria repens, and Eucoleus aerophilus. In contrast, using the mAb-purified antigens, the cross-reactions were minimal. Depending on the antigens used, specific antibodies were detected starting between 13 and 21 days post experimental inoculation (dpi), and at latest between 35 and 48 dpi, thus before or around the onset of patency. The serological follow-up of four A. vasorum-infected dogs after anthelmintic treatment at 88 dpi showed a decrease of antibody levels after drug administration, and the animals became seronegative 2-9 weeks later. Two untreated dogs remained seropositive. In four dogs treated 4 dpi, virtually no antibody-reaction was detectable, with the exception of the ELISA performed with L1 antigen. The early detection of specific antibodies against A. vasorum by ELISA represents a valid alternative for a reliable diagnosis and for follow-up investigations after anthelmintic treatment

Cross-reactions of sera from dogs infected with <it>Angiostrongylus vasorum</it> in commercially available <it>Dirofilaria immitis</it> test kits

<p>Abstract</p> <p>Background</p> <p><it>Dirofilaria immitis</it> and <it>Angiostrongylus vasorum</it> are both important potentially fatal canine nematodes with overlapping endemic areas, especially in Europe. The preadult and adult stages of both species are living in the Arteria pulmonalis and the right heart, and diagnostically detectable circulating parasite antigens have been demonstrated for both species. For the detection of <it>D. immitis</it> infections, a variety of commercial tests have been developed, however, they have not been evaluated for cross-reactions against circulating antigens of <it>A. vasorum.</it></p> <p>Methods</p> <p>In this study, potential cross-reactions of sera from 16 dogs, which were experimentally infected with <it>A. vasorum</it> and which had circulating antigens as confirmed by a species-specific ELISA, were evaluated for the detection of <it>A. vasorum</it> antigen in six commercially available <it>D. immitis</it> test kits.</p> <p>Results</p> <p>In three fast tests (Witness® Dirofilaria, SensPERT® Canine Heartworm, SNAP® 4Dx® Plus), all sera were negative. One fast membrane ELISA (SNAP® HTWM RT Test) was positive with four sera (25%), and one serum delivered a non-valid result twice. In the PetChek® HTWM PF Test, depending on the interpretation protocol, 5 or 8 dogs (31.2 – 50%) were positive. With the DiroCHEK®-ELISA, a single <it>A. vasorum</it>-infected dog (6.2%) tested positive.</p> <p>Conclusions</p> <p>Due to potential cross-reactions with <it>A. vasorum</it> in commercially available test kits for the detection of <it>D. immitis</it> antigen, the simultaneous use of highly specific diagnostic methods for the differentiation of these two canine heart worms is recommended.</p