Comparative Assessment of ELISAs Using Recombinant Saposin-Like Protein 2 and recombinant Cathepsin L-1 from Fasciola hepatica for the Serodiagnosis of Human Fasciolosis

Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2) and cathepsin L-1 (recCL1), were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG) conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory–secretory products (FhES) from adult stage liver flukes was assessed by receiver operator characteristic (ROC) analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n = 20), patients with other parasitic infections (n = 87) and patients with malignancies (n = 121). The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy) employing the threshold (cut-off) to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.     

Seroprevalence of fasciolosis in Hokkaido sika deer (Cervus nippon yesoensis) from Hokkaido Prefecture,Japan revealed by ELISA using recombinant cathepsin L1

Fasciolosis, a zoonotic disease caused by liver flukes of the genus Fasciola, has been reported in Hokkaido (Yezo) sika deer (Cervus nippon yesoensis) in Hokkaido Prefecture, Japan; however, the actual seroprevalence in the animal has not been adequately evaluated. The objective of the present study was to analyze the seroprevalence of the disease among Hokkaido sika deer. Recombinant cathepsin L1 (rCatL1) was used as an antigen for an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola flukes. The sensitivity and specificity of the ELISA were 84.6% and 100%, respectively. The average seroprevalence in 1109 Hokkaido sika deer from 20 locations in Hokkaido Prefecture was 43.9%. Mature deer showed higher seroprevalence than younger individuals; however, even younger animals may act as a reservoir for the disease. Monitoring infection levels in the Hokkaido sika deer population is important not only for the livestock industry, but also for preventing human fasciolosis.     

Development and evaluation of a new lateral flow immunoassay for serodiagnosis of human fasciolosis

Background

Human fasciolosis is a re-emerging disease worldwide and is caused by species of the genus Fasciola (F. hepatica and F. gigantica). Human fasciolosis can be diagnosed by classical coprological techniques, such as the Kato-Katz test, to reveal parasite eggs in faeces. However, although 100% specific, these methods are generally not adequate for detection of acute infections, ectopic infections, or infections with low number of parasites. In such cases immunological methods may be a good alternative and are recommended for use in major hospitals where trained personnel are available, although they are not usually implemented for individual testing.

Methodology/Principal Findings

We have developed a new lateral flow test (SeroFluke) for the serodiagnosis of human fasciolosis. The new test was constructed with a recombinant cathepsin L1 from F. hepatica, and uses protein A and mAb MM3 as detector reagents in the test and control lines, respectively. In comparison with an ELISA test (MM3-SERO) the SeroFluke test showed maximal specificity and sensitivity and can be used with serum or whole blood samples.

Conclusions/Significance

The new test can be used in major hospitals in hypoendemic countries as well as in endemic/hyperendemic regions where point-of-care testing is required.     

Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease

Background

Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden.

Methods

In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities.

Results

The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test).

Conclusions

These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.     

Dot-Blot Immunoassay of Fasciola gigantica Infection using 27 kDa and Adult Worm Regurge Antigens in Egyptian Patients

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.     

Seroprevalence of COVID-19 in Riyadh city during the early increase of COVID-19 infections in Saudi Arabia,June 2020

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) emerged in December 2019 and caused a global pandemic of the Coronavirus Disease 2019 (COVID-19). More than 170 million cases have been reported worldwide with mortality rate of 1–3%. The detection of SARS-CoV-2 by molecular testing is limited to acute infections, therefore serological studies provide a better estimation of the virus spread in a population. This study aims to evaluate the seroprevalence of SARS-CoV-2 in the major city of Riyadh, Saudi Arabia during the sharp increase of the pandemic, in June 2020. Serum samples from non-COVID patients (n = 432), patients visiting hospitals for other complications and confirmed negative for COVID-19, and healthy blood donors (n = 350) were collected and evaluated using an in-house enzyme-linked immunosorbent assay (ELISA). The overall percentage of positive samples was 7.80% in the combined two populations (n = 782). The seroprevalence was lower in the blood donors (6%) than non-COVID-19 patients (9.25%), p = 0.0004. This seroprevalence rate is higher than the documented cases, indicating asymptomatic or mild unreported COVID-19 infections in these two populations. This warrants further national sero-surveys and highlights the importance of real-time serological surveillance during pandemics.     

Measuring the sero-prevalence of Leishmania donovani induced cutaneous leishmaniasis: A method comparison study

An in-house enzyme-linked immunosorbent assay (ELISA) based on crude antigen of Leishmania reported a high sero-prevalence (82.0%) in Leishmania donovani induced cutaneous leishmaniasis (CL) in Sri Lanka. ELISA was further compared with established serological tools to identify a suitable point of care diagnostic tool. Sero-prevalence of 100 CL samples were analyzed using in-house ELISA, Indian dipstick test and rK39 strip test. Results obtained were further compared with direct agglutination test (DAT) for 40 CL. Test performance was evaluated using Kappa index value. Clinico-epidemiological characteristics of patients were analyzed using SPSSv25.0. Cost analysis of tests was carried out. ELISA showed a high sero-positivity of 81.0% (n = 81/100) while DAT (57.5%,n = 23/40), Indian dipstick test (22.0%,n = 22/100) and rK39 test (15.0%,n = 15/100) showed a comparatively less sero-positivity. According to Kappa index values, there were no perfect agreement between tests. Among ELISA positive patients (n = 81/100), DAT, Indian dipstick test and rK39 demonstrated sero-positivity rates of 61.3% (n = 19/31), 25.9% (n = 21/81) and 16.0% (n = 13/81) respectively. Among ELISA negative patients (n = 19/100), three assays demonstrated sero-positivity rates of 44.4% (n = 4/9), 5.3% (n = 1/9) and 10.5% (n = 2/19) respectively. DAT can be used as an alternative test when ELISA is not available or negative. Clinico-epidemiological profiles of patients that showed sero-positivity by each assay were different. Cost per patient was approximately 5.5 USD for DAT and 3.0 USD for each other tests. Established serological tests demonstrated different and relatively lower detection rates. Species, strain and antigen heterogeneity, inconsistency in amount of used antigens, sera, antibody expression patterns and testing methodologies could be responsible. This study highlighted the importance of designing an in-house serological assay based on local parasite.     

Reactivity of purified and axenic amastigotes as a source of antigens to be used in serodiagnosis of canine visceral leishmaniasis

Although there is a great diversity of techniques and antigens used in the serodiagnosis of canine visceral leishmaniasis (CVL), total sensitivity and specificity have not yet been found. Since the use of amastigote forms in the indirect immunofluorescence assay has shown an improvement in the specificity of the test for the diagnosis of CVL, the performance of amastigotes forms of L. (L.) infantum chagasi as antigen source were evaluated in automatized ELISA test using crude antigen of axenic amastigote and purified amastigote from spleen of hamster chronically infected comparing with ELISA using total antigen produced with promastigote forms of L. (L.) infantum chagasi. One hundred and fifteen sera from dogs with positive parasitological diagnosis by PCR were used. The animals were classified into 2 groups: symptomatic (n = 67) and asymptomatic (n = 48) animals, in accordance with the clinical signs and laboratory tests were. As control, ninety-four sera from dogs with negative parasitological diagnosis were included. No significant difference was found in sensitivity, specificity, predictive values and accuracy between ELISA using whole antigens produced with both axenic and purified amastigotes in comparison with promastigotes forms. Correlation and concordance between the three total antigens tested in ELISA was observed. According to the similar performance among antigens, data pointed out to use antigen from promastigote forms for diagnosing canine leishmaniasis, especially due the easily in the production, lower cost and the abundance of correlative literature.     

Novel methods for the molecular discrimination of Fasciola spp. on the basis of nuclear protein-coding genes

Fasciolosis is an economically important disease of livestock caused by Fasciola hepatica, Fasciola gigantica, and aspermic Fasciola flukes. The aspermic Fasciola flukes have been discriminated morphologically from the two other species by the absence of sperm in their seminal vesicles. To date, the molecular discrimination of F. hepatica and F. gigantica has relied on the nucleotide sequences of the internal transcribed spacer 1 (ITS1) region. However, ITS1 genotypes of aspermic Fasciola flukes cannot be clearly differentiated from those of F. hepatica and F. gigantica. Therefore, more precise and robust methods are required to discriminate Fasciola spp. In this study, we developed PCR restriction fragment length polymorphism and multiplex PCR methods to discriminate F. hepatica, F. gigantica, and aspermic Fasciola flukes on the basis of the nuclear protein-coding genes, phosphoenolpyruvate carboxykinase and DNA polymerase delta, which are single locus genes in most eukaryotes. All aspermic Fasciola flukes used in this study had mixed fragment pattern of F. hepatica and F. gigantica for both of these genes, suggesting that the flukes are descended through hybridization between the two species. These molecular methods will facilitate the identification of F. hepatica, F. gigantica, and aspermic Fasciola flukes, and will also prove useful in etiological studies of fasciolosis.     

Molecular identification and genetic-polymorphism analysis of Fasciola flukes in Dali Prefecture,Yunnan Province,China

This study aimed to identify species of Fasciola flukes in Dali Prefecture (Yunnan Province, China) and analyze their genetic diversity. Fasciola flukes (n = 122) were collected from cattle livers in a farmers' market in Xiaguan Town, Dali Prefecture. Nucleotide sequences of ribosomal internal transcribed spacer (ITS) as well as nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) and mitochondrial cytochrome c oxidase subunit 1 (CO1) were amplified, sequenced, and subjected to homology analysis. The heterozygosity ratios of different ITS alleles were determined using the peak-height ratio of heterozygous loci. Multiplex PCR analysis of the nuclear protein coding gene, phosphoenolpyruvate carboxykinase (pepck), was used to identify Fasciola species. Multiple ND1 sequence alignments enabled further genetic diversity analysis of regional Fasciola flukes. Seven ITS sequences belonged to F. hepatica and 115 belonged to Fh/Fg heterozygous flukes. Sequencing analysis of heterozygous flukes revealed 11 heterozygous loci with double peaks, with significantly variable ratios among individuals. ND1 and CO1 results indicated that one specimen was identical to F. hepatica, while 121 specimens were identical to F. gigantica or contained one variable site. Multiplex PCR results for pepck showed that double bands for F. hepatica and F. gigantica were amplified from Dali Fasciola specimens; hence, they were all heterozygous. By combining ITS, ND1, and CO1 sequences with multiplex pepck PCR results, all 122 specimens were identified as Fh/Fg heterozygous Fasciola flukes. Our experimental results preliminarily confirmed a high degree of Fh/Fg heterozygosity among Fasciola flukes in the Dali area. Selecting multiple molecular markers for concurrent analysis will provide more comprehensive and accurate genetic information.     

A Schistosoma haematobium-Specific Real-Time PCR for Diagnosis of Urogenital Schistosomiasis in Serum Samples of International Travelers and Migrants

Background

Diagnosis of urogenital schistosomiasis by microscopy and serological tests may be elusive in travelers due to low egg load and the absence of seroconversion upon arrival. There is need for a more sensitive diagnostic test. Therefore, we developed a real-time PCR targeting the Schistosoma haematobium-specific Dra1 sequence.

Methodology/Principal Findings

The PCR was evaluated on urine (n = 111), stool (n = 84) and serum samples (n = 135), and one biopsy from travelers and migrants with confirmed or suspected schistosomiasis. PCR revealed a positive result in 7/7 urine samples, 11/11 stool samples and 1/1 biopsy containing S. haematobium eggs as demonstrated by microscopy and in 22/23 serum samples from patients with a parasitological confirmed S. haematobium infection. S. haematobium DNA was additionally detected by PCR in 7 urine, 3 stool and 5 serum samples of patients suspected of having schistosomiasis without egg excretion in urine and feces. None of these suspected patients demonstrated other parasitic infections except one with Blastocystis hominis and Entamoeba cyst in a fecal sample. The PCR was negative in all stool samples containing S. mansoni eggs (n = 21) and in all serum samples of patients with a microscopically confirmed S. mansoni (n = 22), Ascaris lumbricoides (n = 1), Ancylostomidae (n = 1), Strongyloides stercoralis (n = 1) or Trichuris trichuria infection (n = 1). The PCR demonstrated a high specificity, reproducibility and analytical sensitivity (0.5 eggs per gram of feces).

Conclusion/Significance

The real-time PCR targeting the Dra1 sequence for S. haematobium-specific detection in urine, feces, and particularly serum, is a promising tool to confirm the diagnosis, also during the acute phase of urogenital schistosomiasis.     

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.     

Molecular characterization and phylogenetic analysis of Fasciola hepatica from Peru

The causative agent of fasciolosis in South America is thought to be Fasciola hepatica. In this study, Fasciola flukes from Peru were analyzed to investigate their genetic structure and phylogenetic relationships with those from other countries. Fasciola flukes were collected from the three definitive host species: cattle, sheep, and pigs. They were identified as F. hepatica because mature sperms were observed in their seminal vesicles, and also they displayed Fh type, which has an identical fragment pattern to F. hepatica in the nuclear internal transcribed spacer 1. Eight haplotypes were obtained from the mitochondrial NADH dehydrogenase subunit 1 (nad1) sequences of Peruvian F. hepatica; however, no special difference in genetic structure was observed between the three host species. Its extremely low genetic diversity suggests that the Peruvian population was introduced from other regions. Nad1 haplotypes identical to those of Peruvian F. hepatica were detected in China, Uruguay, Italy, Iran, and Australia. Our results indicate that F. hepatica rapidly expanded its range due to human migration. Future studies are required to elucidate dispersal route of F. hepatica from Europe, its probable origin, to other areas, including Peru.     

Molecular analyses confirm the coexistence of Fasciola gigantica and parthenogenetic Fasciola in the Philippines

Fasciola flukes collected from domestic buffalos and cattle in the Philippines were confirmed as Fasciola gigantica and parthenogenetic Fasciola based on DNA analyses of nuclear pepck and pold genes, and the mitochondrial ND1 gene. This study is the first to elucidate that F. gigantica and parthenogenetic Fasciola coexist in the Philippines with prevalences of 90.6% and 9.4%, respectively. The F. gigantica population showed a high genetic diversity with 25 ND1 haplotypes, suggesting that F. gigantica has existed in the Philippines for a long time. In contrast, parthenogenetic Fasciola flukes showed a single ND1 haplotype (Fsp-ND1-P1), which was identical to the founder haplotype, Fg-C2 of parthenogenetic Fasciola in China. These results indicate that parthenogenetic Fasciola in the Philippines is a recently introduced population from a neighboring continent.     

Risk factors associated with intestinal pathogenic parasites in schoolchildren

Diseases caused by intestinal parasites impose a substantial burden on population of middle income countries including Pakistan. This research was aimed to assess the risk factors for intestinal parasites in school children of Malakand, Pakistan. Two hundred and eighty eight students were enrolled between February and June 2016. Out of the total enrolled 184 were agreed to collect stool specimens. A questionnaire was also used to collect the data on socio-demographic and socio-economic characteristics of the participants. All the students were guided to collect at least 10gof their own stool specimens. Each of the stool specimens was diagnosed for the presence of any stage of helminth or protozoal parasites. Formal ether concentration method and wet mount techniques were applied. One way ANOVA was used for calculation of P value when it was less than 0.05 which was considered significant. Eighty two percent of the participants were found infected with one species of parasite while 69.9% of the participants were infected with more than one species of intestinal parasites. The most prevalent parasite was hook worm 33.4% (n = 99/296) followed by Taenia saginata 28.7% (n = 85/296), Ascaris lumbricoides 27.7% (n = 82/296), Hymenolepis nana 6.08% (n = 18/296), Entamoeba histolytica 3.37% (n = 10/296) and least for each Enterobius vermicularis and Fasciola hepatica 0.37% (n = 1/296). Previously used drugs, level in school, ages, weight and upper arm circumference were the most significantly (P < 0.05) related factors for the occurrence of intestinal parasite infection. Present research endorsed that risk factors play a key role in the transmission of parasitic diseases. Lack of safe water supply, using raw vegetables, animal keeping, which should be considered for sustainable strategies in the control of these infections preferably in remote parts of the world.     

Field evaluation of the MM3-SERO ELISA for detection of anti-Fasciola IgG antibodies in milk samples from individual cows and bulk milk tanks

We carried out a field evaluation of the MM3-SERO ELISA for the diagnosis of Fasciola hepatica infection, by analysing serum and milk samples from individual cows and samples from bulk milk tanks. The diagnostic performance of the assay was assessed with serum samples from all 257 cows in eight fluke-free herds, and 240 cows with natural fasciolosis (diagnosed in vivo and/or post-mortem). Assay performance for individual milk samples was determined by analysis of paired serum and milk samples from 947 lactating cows from 33 F. hepatica-infected farms. The diagnostic usefulness of the assay for bulk tank milk was evaluated by analysis of bulk milk from infected (33) and non-infected (35) farms. For serum samples, the sensitivity, specificity and diagnostic accuracy of the assay were respectively 99.2% (95% CI: 97.0%–99.9%), 100% (95% CI: 98.6%–100%) and 0.997 (95% CI: 0.987–1.000). The only two infected animals in which serum antibodies were not detected had very low parasitic burdens (with only 2 and 3 flukes observed). The performance of the MM3 SERO ELISA for individual milk samples was similar to that for serum samples, and the stepwise linear regression revealed a strong correlation between the results for the milk samples and the serum samples (R² = 0.84; p < 0.001). The agreement between results obtained with pairs of serum and milk samples was very high: there was matching classification in 96% (910/947) of paired samples (kappa = 0.92; p < 0.001). Individual milk samples may therefore be used, instead of serum samples, in the MM3-SERO ELISA, for reliable detection of seropositive cows. Testing bulk tank milk samples enabled detection of infected herds, even when the within-herd prevalence of infection was as low as 12%. We conclude that the MM3-SERO ELISA is a sensitive and highly specific test for serodiagnosis of bovine fasciolosis, and can be used with individual samples of either serum or milk. Use of the assay with bulk milk samples enables estimation of the within-herd prevalence of infection.     

Phylogenetic Characteristics of Fasciola hepatica Isolated from a Korean Patient

Fascioliasis is a parasitic infection caused by liver flukes. Although several cases have been reported in Korea, phylogenetic analysis of isolates is lacking. In this study, a 66-year-old woman with right upper quadrant (RUQ) abdominal pain was diagnosed as fascioliasis involving abdominal muscle by imaging study. She received praziquantel treatment, but symptoms were not improved. Lateral movement of the abscess lesion was followed. Trematode parasite was surgically removed from the patient’s rectus abdominis muscle. The fluke was identified as Fasciola hepatica based on sequence analysis of 18S rDNA. To determine the phylogenetic position of this Fasciola strain (named Korean Fasciola 1; KF1), the cox1 gene (273 bp) was analyzed and compared with the genes of 17 F. hepatica strains isolated from cows, sheep, goats, and humans from various countries. Phylogenetic analysis showed that KF1 was closely related with the isolates from China goat.     

Quantification of Parasite Load in Clinical Samples of Leishmaniasis Patients: IL-10 Level Correlates with Parasite Load in Visceral Leishmaniasis

A rapid and accurate method to detect and quantify Leishmania parasite is urgently needed to facilitate early diagnosis of Leishmaniasis and monitoring of antileishmania therapy. In this study, real-time assay was applied to estimate parasite load in clinical samples of visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) patients. The mean parasite load in blood of VL patients (n = 31) was 8,372 parasites/ml, while the mean parasite load in bone marrow aspirate (BMA) was 194,962 parasites/million nucleated cells (n = 12). Parasite load was undetectable after treatment with amphotericin B (n = 9) in VL, while a residual parasite burden was detected in 2 of 6 patients following treatment with sodium antimony gluconate. Further, circulating levels of IFN-γ, TNF-α, IL-10, IL-6, IL-4 and IL-2 were analysed in VL patients (n = 29) by Cytometric Bead Array to evaluate correlation with parasitic load. Interestingly, IL-10 levels correlated significantly with parasite load (r = 0.82, P<0.0001). The mean parasite load in dermal lesions of PKDL patients was 9,502 parasites/µg tissue DNA at pre-treatment stage (n = 25), with no detectable parasites after therapy (n = 5). Parasite burden was distinctly higher (P<0.0001) in nodular lesions (n = 12) (19,586 parasites/µg tissue DNA) compared to papular/macular lesions (n = 13, 193 parasites/µg tissue DNA). Further, chronic PKDL lesions showed significantly (P = 0.0166) higher parasite load in comparison with acute lesions. Results indicate that chronic, nodular cases constitute the major parasite reservoir for anthroponotic transmission. Our results establish that the high parasite load in VL is strongly correlated with a high level of IL-10, implicating IL-10 as a marker of disease severity. The assay is applicable for diagnosis as well as prognosis of both VL and PKDL, providing a simple molecular tool to monitor the efficacy of antileishmanial drugs or vaccines.     

Immunodiagnosis of anti-Toxocara vitulorum IgG antibodies by using commercially available bovine ELISA Kit in bovine of Potohar region Pakistan

The Toxocarosis is a common zoonotic parasitic infection caused by three etiological agents T. cati, T. canis and T. vitulorum. It has a severe impact on health and economy especially in countries where it is endemic. The study was conducted from June 2013–2014. The blood samples were collected from (n = 270) cattle grazing in the Potohar region. The sera were separated and subjected to anti-Toxocara ELISA and results revealed an overall seroprevalence of 16.67% (45/270) in cattle. The relationship between age groups and seroprevalence of T. vitulorum indicated a statistically significant difference (p = .01, χ² = 16.81) and non-significant was sex group of cattle and seroprevalence of T. vitulorum (p = .859, χ² = 0.032). The results also revealed a statistically non-significant difference between cattle breed and Toxocara infection (p = .142, χ² = 5.424). From the study, we concluded that indirect ELISA is a highly efficient assay for timely diagnosis of infection in order to adapt control strategies to mitigate the infection. The present study recommended the need to develop ELISA by using excretory-secretory (ES) T. vitulorum antigen in order to reduce the cost of commercial kits for mass screening purposes. Furthermore, the result of the current study also suggested and conducting similar nature of studies in other agroecological zones of Pakistan, which will help to provide baseline epidemiological data of parasitic diseases.