A systematic review and meta-analysis of the validation of serological methods for detecting anti-Toxoplasma gondii antibodies in humans and animals.

Toxoplasma gondii is a paradigmatic zoonotic parasite from the One Health perspective, since it is broadly distributed and virtually infects all warm-blooded species. A wide variety of serological techniques have been developed to detect T. gondii infection in humans and animals. Our aim was to describe and compare the main characteristics of these serological tests and validation processes and to critically analyze whether these tests meet the standards required to ensure an accurate serological diagnosis. The current systematic review and meta-analysis included 134 studies that were published from 2013 to 2023. QUADAS 2 tool was used to evaluate the quality of the included studies. A total of 52 variables related to the characteristics of the techniques and analytical and diagnostic validation parameters were studied. A wider panel of tests was developed for humans, including techniques exclusively developed for humans that involve costly equipment and the measurement of different Ig isotypes that are considered biomarkers of congenital toxoplasmosis. Studies conducted in humans frequently employed commercial techniques as reference tests, measured different immunoglobulin isotypes with a predominance for IgG (>50%) and discriminated between acute and chronic infections. In animals, the most commonly used reference techniques were in-house tests, which almost exclusively detected IgG. Common limitations identified in a large number of studies were some misunderstandings of the terms "gold standard" and "reference test" and the absence of information about the negative and positive control sera used or the exact cutoff employed, which were independent of the quality of the study. There is a lack of analytical validation, with few evaluations of cross-reactivity with other pathogens. Diagnostic odds ratio values showed that indirect ELISA based on native or chimeric antigens performed better than other tests. The reproducibility of serological test results in both humans and animals is not guaranteed due to a lack of relevant information and analytical validation. Thus, several key issues should be considered in the future, including interlaboratory ring trials.

Description of potential vectors of zoonotic filarial nematodes, Brugia pahangi, Setaria digitata, and Setaria labiatopapillosa in Thai mosquitoes.

Filariasis is classified as a vector-borne zoonotic disease caused by several filarial nematodes. The disease is widely distributed in tropical and subtropical regions. Understanding the relationship between mosquito vectors, filarial parasites, and vertebrate hosts is therefore essential for determining the probability of disease transmission and, correspondingly, developing effective strategies for prevention and control of diseases. In this study, we aimed to investigate the infection of zoonotic filarial nematodes in field-caught mosquitoes, observe the potential vectors of filaria parasites in Thailand using a molecular-based survey, conduct a study of host-parasite relationship, and propose possible coevolution of the parasites and their hosts. Mosquitoes were collected around cattle farms in Bangkok, Nakhon Si Thammarat, Ratchaburi, and Lampang provinces from May to December 2021 using a CDC Backpack aspirator for 20-30 minutes in each area (intra-, peri-, and wild environment). All mosquitoes were identified and morphologically dissected to demonstrate the live larvae of the filarial nematode. Furthermore, all samples were tested for filarial infections using PCR and sequencing. A total of 1,273 adult female mosquitoes consisted of five species: 37.78% Culex quinquefasciatus, 22.47% Armigeres subalbatus, 4.71% Cx. tritaeniorhynchus, 19.72% Anopheles peditaeniatus, and 15.32% An. dirus. Larvae of Brugia pahangi and Setaria labiatopapillosa were found in Ar. subalbatus and An. dirus mosquitoes, respectively. All mosquito samples were processed by PCR of ITS1 and COXI genes for filaria nematode species identification. Both genes showed that B. pahangi was found in four mosquitoes of Ar. subalbatus from Nakhon Si Thammarat, S. digitata was detected in three samples of An. peditaeniatus from Lampang, and S. labiatopapillosa was detected in one of An. dirus from Ratchaburi. However, filarial nematodes were not found in all Culex species. This study infers that this is the first data regarding the circulation of Setaria parasites in Anopheles spp. from Thailand. The phylogenetic trees of the hosts and parasites are congruent. Moreover, the data could be used to develop more effective prevention and control strategies for zoonotic filarial nematodes before they spread in Thailand.

A systematic review and meta-analysis of the serological diagnosis of Toxoplasma gondii infection highlight the lack of a One Health integrative research.

Toxoplasma gondii is a globally distributed food-borne zoonotic parasite with numerous infection sources. The control of this zoonosis requires a One Health response that partially depends on serological monitoring in humans and animals. Herein, a systematic review and a meta-analysis were performed to analyse and compare the transdisciplinary and integrative research under the One Health approach. We searched for articles published between January 1st 2014 and September 5th 2022, focused on the development and evaluation of serological techniques for the diagnosis of T. gondii infection in humans and animals. After an exhaustive search on three scientific databases, a quality assessment was performed on 291 articles by QUADAS-2 tool, and 113 articles were finally selected. A total of 18 variables were extracted and analysed, including bibliometric characteristics, study aims and methodology. Remarkably, none of the studies included in the meta-analysis explicitly quoted the words "One Health", and only 23.9% of them alluded to the principles underlying the One Health approach; in particular, none were conducted by physician-only teams, with the majority of these studies involving interdisciplinary research teams, followed by veterinarians and by non-physician or non-veterinarian researchers. The One Health approach followed in the serodiagnosis of T. gondii still needs further integration among scientific disciplines, which is essential to design effective control strategies.

Development and evaluation of a visible reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for the detection of Asian lineage ZIKV in field-caught mosquitoes.

The Zika virus (ZIKV) infection is an emerging and re-emerging arbovirus infection that is transmitted to humans through the bite of infected mosquitoes. Early detection of ZIKV in mosquitoes is one of the prerequisite approaches for tracking the spread of the virus. Therefore, this study aims to develop and validate a visual reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method called ZIKV-RT-LAMP, for detecting ZIKV in field collected mosquito samples from Thailand. A single-tube ZIKV-RT-LAMP assay was developed to detect Asian lineage ZIKV RNA. The detection limit and cross-reactivity of ZIKV were investigated. The hemi-nested RT-PCR (hn-RT-PCR) and the colorimetric LAMP kit (cLAMP kit) were performed as reference assays. The detection limit of the ZIKV-RT-LAMP assay was 10-6 ffu/ml or pfu/ml, making it highly specific and 100 times more sensitive than the hn-RT-PCR and cLAMP kits. The ZIKV-RT-LAMP assay detected the Asian lineage of ZIKV RNA without cross-reactivity with other arthropod-borne viruses. The sensitivity and specificity of the ZIKV-RT-LAMP assay were 92.31% and 100%, respectively. The ZIKV-RT-LAMP is a simple, rapid, and inexpensive method for detecting ZIKV in field-caught mosquitos. In the future, extensive surveys of field-caught mosquito populations should be conducted. Early detection of ZIKV in field-caught mosquitoes provides for prompt and effective implementation of mosquito control strategies in endemic areas.

Detection of Leishmania infantum DNA by real-time PCR in saliva of dogs.

This study developed a real-time quantitative PCR (qPCR) assay to detect L. infantum kinetoplast DNA (kDNA) in canine saliva. The qPCR showed an efficiency of 93.8%, a coefficient of correlation of 0.996 and a detection limit of 0.5 fg/reaction (0.005 parasites), although it detected until 0.25 fg/reaction (0.0025 parasites). When samples from 12 dogs experimentally infected with L. infantum were collected, L. infantum kDNA was detected at 16-weeks post-infection (wpi) in 41.7% and 91.7% of saliva and bone marrow samples, respectively, and at 47-wpi in 75% of both samples. L. infantum kDNA can be detected by qPCR in canine saliva, with lower sensitivity in the early stages of infection and a lower parasite load estimation compared to bone marrow. However, saliva had similar sensitivities to bone marrow in the later stages of the infection and could be used to detect L. infantum kDNA being aware of its limitations.

Clinical leishmaniosis in a captive Eurasian otter (Lutra lutra) in Spain: a case report.

BACKGROUND: Captive and free-ranging wild mammals have been recognized as potential reservoirs of Leishmania infantum infection. The aim of this study was to describe the first clinical case of leishmaniosis in the Eurasian otter. CASE PRESENTATION: A case of clinical leishmaniosis is reported in a 4-year-old male Eurasian otter housed at a wildlife park (Murcia, South Eastern Spain). The Eurasian otter showed bilateral epistaxis, anorexia, apathy, and weight loss. A complete blood cell count and biochemical analyses revealed hyperproteinemia, hyperglobulinemia, decreases of paraoxonase-1, increases of haptoglobin and ferritin, and proteinuria. Bilateral nephropathy with hydronephrosis, mesenteric lymphadenomegaly, and ascites were also observed. L. infantum infection was confirmed by microscopy (amastigotes were detected in macrophages from spleen aspirate), molecular diagnosis (L. infantum DNA was detected by real-time polymerase chain reaction), and serology (anti-Leishmania IgG2 antibodies were detected by time-resolved immunofluorometry). The animal was treated with allopurinol for 3 months and gained weight, the epistaxis disappeared, and the ferritin concentration decreased. CONCLUSIONS: This is the first report of clinical leishmaniosis in the Eurasian otter. Our results suggest that Eurasian otters are susceptible to infection with L. infantum and can develop clinical leishmaniosis in endemic areas.

Evaluation of the circadian rhythm of anti-Leishmania IgG2 and IgA antibodies in serum and saliva of dogs with clinical leishmaniosis.

In this study, the circadian rhythm of IgG2 and IgA specific antibodies in serum and saliva samples of 6 dogs experimentally infected with Leishmania infantum was assessed. Sampling was performed at 8.00, 12.00, 16.00, 20.00, and 00.00 h on two consecutive days. Anti-Leishmania antibody levels in serum were expressed without any correction, whereas in saliva were shown in different ways: without any correction, adjusted by protein concentration and corrected by the salivary flow rate. No significant differences in anti-Leishmania IgG2 antibody levels in serum and saliva samples with or without correction were found. Significant differences were found when anti-Leishmania IgA levels were corrected by the salivary flow rate. In addition, a greater intra-individual variation of antibody levels was observed in saliva than in serum. However, this variation did not modify the serological status of the dogs. Therefore, it could be concluded that there is no circadian rhythm in serum and saliva samples and sampling can be performed at any time of the day.

One-year follow-up of anti-Leishmania antibody concentrations in serum and saliva from experimentally infected dogs.

The quantification of anti-Leishmania antibodies in serum and saliva by a time-resolved immunofluorometric assay is useful for the diagnosis and treatment monitoring of dogs with clinical leishmaniasis. We compared the kinetics of anti-Leishmania IgG2 and IgA antibodies in serum and saliva from 11 Beagle dogs experimentally infected with Leishmania infantum. Most dogs showed detectable concentrations of anti-Leishmania IgG2 earlier in serum (between 3 and 4 months p.i.) than in saliva (between 4 and 6 months p.i.). Overall, a high correlation between concentrations of anti-Leishmania IgG2 in serum and saliva (r = 0.853; P < 0.0001) was observed. The quantification of anti-Leishmania IgA showed less diagnostic value than IgG2, since detectable amounts of IgA were not observed in the saliva of four dogs and in the serum of one dog. In addition, a very low correlation between anti-Leishmania IgA in serum and saliva (r = 0.289; P < 0.001) was observed. Our results indicate that the antibodies against L. infantum in saliva appear approximately 1 month later than in serum, and suggest that there is a threshold for the passing of immunoglobulins from serum to saliva in dogs. These facts should be taken into consideration for a proper interpretation of saliva assays for quantification of antibodies.

Relationship between serum anti-Leishmania antibody levels and acute phase proteins in dogs with canine leishmaniosis.

This study examined the relationship between two serologic assays which quantify anti-Leishmania antibodies (a commercial enzyme-linked immunosorbent assay (ELISA) and a time-resolved immunofluorometric assay (TR-IFMA)) and selected acute phase proteins (APPs) and analytes related to protein concentration. Data were obtained from 205 canine serum samples from different veterinary clinics located in an area in which canine leishmaniosis (CanL) is endemic. The samples were submitted to the Interdisciplinary Laboratory of Clinical Analysis (Interlab-UMU), University of Murcia, Spain, for analysis. The biochemical analytes evaluated were serum ferritin, C-reactive protein (CRP), haptoglobin, paraoxonase-1 (PON-1) and albumin as APPs and total proteins and globulins as indicative analytes of protein concentration. Samples were submitted for the initial diagnosis of CanL, or to monitor the response to treatment in patients with CanL. The evaluation of the biochemical analytes did not show differences between Leishmania-seronegative and Leishmania-seropositive dogs. However, dogs with high antibody titers showed more pronounced clinicopathological abnormalities. Both serological assays had correlations of different significance with the biochemical analytes, showing higher significant correlations with total proteins and globulins than with the rest of the analytes. When the samples submitted for diagnosis and treatment monitoring were analyzed separately, serological assays showed lower correlation in samples for treatment monitoring (r = 0.531, p < 0.0001) than in samples for diagnosis (r = 0.769, p < 0.0001). In addition, higher correlations were found between TR-IFMA and analytes such as serum ferritin and CRP in the treatment monitoring group than with the ELISA. These results may help to clarify the relationship between anti-Leishmania antibody levels and selected biochemical analytes related to inflammation and protein concentration in CanL.

Changes in the concentration of anti-Leishmania antibodies in saliva of dogs with clinical leishmaniosis after short-term treatment.

The aim of this study was to evaluate the possible changes in the concentration of anti-Leishmania antibodies in saliva samples from dogs with clinical leishmaniosis after short-term treatment. Twenty dogs with clinical signs and laboratory abnormalities compatible with canine leishmaniosis (CanL) were diagnosed and treated with a standard antimonial plus allopurinol therapy. The concentration of anti-Leishmania IgG2 and IgA antibodies in saliva was measured at the time of diagnosis (day 0) and after treatment (day 30) by time-resolved immunofluorometric assays (TR-IFMAs) and results were compared with those of serum. In addition, correlations between antibody concentrations in saliva and serum, clinical scores and selected laboratory analytes were calculated. TR-IFMA results were expressed as Units of Fluorometry for Leishmania (UFL). Most dogs that adequately responded to treatment (n = 17) showed a reduction of anti-Leishmania antibodies in saliva [median IgG2: from 678.0 (day 0) to 201.1 UFL (day 30), p < 0.0001; median IgA: from 91.3 (day 0) to 60.2 UFL (day 30), p < 0.01] in accordance with clinical improvement (p < 0.0001). However, two of these dogs showed an increase of anti-Leishmania antibodies in saliva. Among dogs that did not improve after one month of treatment (n = 3), two showed a reduction in serum and saliva antibodies. In these two dogs, clinical recovery was achieved after one additional month of treatment with allopurinol. The other dog that did not respond to treatment showed increases in the concentration of anti-Leishmania antibodies, both in saliva and serum, and did not adequately respond to an additional month of treatment with allopurinol. From this pilot study, it could be concluded that, despite the low number of dogs used, the measurement of anti-Leishmania IgG2 and IgA antibodies in saliva could have a potential use for treatment monitoring of CanL, provided that a sufficient amount of specific antibodies is present at diagnosis. This is because, especially in the case of IgG2, there is a high correlation between the saliva and serum concentrations, and the reduction of antibodies is generally in accordance with the clinical improvement. Further long-term studies with a larger population should be undertaken to confirm this potential.

Changes in serum anti-Leishmania antibody concentrations measured by time-resolved immunofluorometric assays in dogs with leishmaniosis after treatment.

The aim of this study was to evaluate the changes in anti-Leishmania IgG2 and IgA antibodies measured by two time-resolved immunofluorometric assays (TR-IFMAs) recently validated and by means of a commercially available ELISA test in dogs with leishmaniosis after treatment. Serum samples from 16 dogs with clinical leishmaniosis were obtained on days 0, 30 and 180 of treatment. In addition, these serological changes were compared with the clinical signs and selected analytes (total proteins, albumin, globulins and urinary protein:creatinine ratio). Concentrations of IgG2 and IgA by TR-IFMA were significantly lower on days 30 (p < 0.05) and 180 of treatment (p < 0.0001) compared to day 0 in dogs that showed a positive response to treatment. Magnitudes of decrease of IgG2 (1.66 and 20.4-fold) and IgA (1.3 and 11.43-fold) concentrations on days 30 and 180 were greater than those of the commercially available ELISA test (1.29 and 2.06-fold), and that of other analytes (total proteins: 1.11 and 1.25-fold; globulins: 1.22 and 1.74-fold; and albumin: 0.93 and 0.8-fold). This study shows that serum IgG2 and IgA anti-Leishmania antibodies measured by TR-IFMAs were useful for treatment monitoring in dogs with leishmaniosis, showing a significant reduction in antibody concentrations earlier than the commercial ELISA assay. Results suggest that the method used for antibody measurements greatly influences the results and, consequently, the usefulness for measuring anti-Leishmania antibodies to monitor the treatment of canine leishmaniosis.

New wide dynamic range assays for quantification of anti-Leishmania IgG2 and IgA antibodies in canine serum.

The aims of this study were (1) to develop and validate time resolved-immunofluorometric assays for the detection of anti-Leishmania IgG2 and IgA antibodies in canine serum and (2) to evaluate the ability of these assays to quantify different amounts of anti-Leishmania antibodies in Leishmania-seronegative and seropositive dogs, determined by a commercial ELISA assay, and between different clinical stages according to LeishVet guidelines. The analytical validation showed that the assays had a good precision with intra- and inter-assay coefficients of variation lower than 10%. In addition, the assays allowed the quantification of very low concentration of antibodies as well as demonstrated a high level of accuracy, as determined by linearity under dilution (R2=0.99) and recovery tests (>85%). Moreover, no cross-reactions with Ehrlichia canis, Canine Parvovirus Type 2, Anaplasma phagocytophilum, Babesia canis, Dirofilaria immitis and pyometra were found. The assays were able to detect higher values of anti-Leishmania IgG2 and IgA antibodies in seropositive dogs compared with seronegative dogs (p<0.0001), although an overlap between groups existed in the case of IgA. In addition, significantly higher values for both antibodies were detected in LeishVet groups II (p<0.05) and III (p<0.01) when compared with LeishVet group I. From our study, it could be concluded that the immunofluorometric assays developed would be suitable for determination of anti-Leishmania IgG2 and IgA antibodies in serum samples with an adequate precision, analytical sensitivity and accuracy. In addition, these assays showed a wider difference in the concentration of both IgG2 and IgA antibodies between seronegative and seropositive dogs and between different clinical stages of CanL than a current commercial ELISA kit. Further studies would be recommended to evaluate the diagnostic sensitivity and specificity of these new assays as well as their application in monitoring CanL.

Quantification of anti-Leishmania antibodies in saliva of dogs.

Detection of serum anti-Leishmania antibodies by quantitative or qualitative techniques has been the most used method to diagnose Canine Leishmaniosis (CanL). Nevertheless, saliva may represent an alternative to blood because it is easy to collect, painless and non-invasive in comparison with serum. In this study, two time-resolved immunofluorometric assays (TR-IFMAs) for quantification of anti-Leishmania IgG2 and IgA antibodies in saliva were developed and validated and their ability to distinguish Leishmania-seronegative from seropositive dogs was evaluated. The analytical study was performed by evaluation of assay precision, sensitivity and accuracy. In addition, serum from 48 dogs (21 Leishmania-seropositive and 27 Leishmania-seronegative) were analyzed by TR-IFMAs. The assays were precise, with an intra- and inter-assay coefficients of variation lower than 11%, and showed high level of accuracy, as determined by linearity under dilution (R2=0.99) and recovery tests (>88.60%). Anti-Leishmania IgG2 antibodies in saliva were significantly higher in the seropositive group compared with the seronegative (p<0.0001), whereas no significant differences for anti-Leishmania IgA antibodies between both groups were observed. Furthermore, TR-IFMA for quantification of anti-Leishmania IgG2 antibodies in saliva showed higher differences between seropositive and seronegative dogs than the commercial assay used in serum. In conclusion, TR-IFMAs developed may be used to quantify anti-Leishmania IgG2 and IgA antibodies in canine saliva with an adequate precision, analytical sensitivity and accuracy. Quantification of anti-Leishmania IgG2 antibodies in saliva could be potentially used to evaluate the humoral response in CanL. However, IgA in saliva seemed not to have diagnostic value for this disease. For future studies, it would be desirable to evaluate the ability of the IgG2 assay to detect dogs with subclinical disease or with low antibody titers in serum and also to study the antibodies behaviour in saliva during the treatment of CanL.