Characterization of Specific RAPD Markers of Virulence in Tri-chomonas vaginalis Isolates

Background: As for human trichomoniasis the host-parasite relationship is very complex, and the broad ranges of clinical symptoms are unlikely be attributable to a single pathogenic mechanism. Specific Random Amplified Polymorphic DNA (RAPD) markers of 490 bp, 720 bp and 460 bp using the primers Tv-5, OPA-6 and OPA-11, respectively, were reported. This was the first description of possible ge­netic virulence markers of the infection by T. vaginalis. The aim of this study was to characterize the specific RAPD markers in order to elucidate their importance on virulence of this illness. Methods: The selected specific RAPD fragments were cloned and sequenced. The obtained sequences were compared by the BLAST algorithm. Results: The nucleotide sequence of the Tv-5490 RAPD marker exhibited signifi­cant similarity to T. vaginalis hypothetical G3 leucine rich repeat (LRR) family pro­tein (e-value: 6e-14) and Giardia lamblia leucine rich repeat protein 1 virus receptor protein (e-value: 6e-14 and 2e-12) ; however, the OPA-6720 and OPA-11460 showed no significant similarity with any coding published sequence. All the evaluated strains showed the presence of the LRR gene. Conclusion: These results demonstrate a possible role of this gene in the viru­lence of T. vaginalis and in the parasite infection with Trichomonas virus as a possible virus receptor. Further analysis of this gene and encoded protein will allow determin­ing the role that they play in the isolates virus susceptible or resistant pheno­types

Development of a nanobody-based amperometric immunocapturing assay for sensitive and specific detection of Toxocara canis excretory-secretory antigen

Introduction Human Toxocariasis (HT) is a zoonosis that, despite of its wide distribution around the world, remains poorly diagnosed. The identification of specific IgG immunoglobulins against the Toxocara canis Excretory-Secretory antigen (TES), a mix of glycoproteins that the parasite releases during its migration to the target organs in infected patients, is currently the only laboratory tool to detect the disease. The main drawbacks of this test are the inability to distinguish past and active infections together with lack of specificity. These factors seriously hamper the diagnosis, follow-up and control of the disease. Aim To develop an amperometric immunocapturing diagnostic assay based on single domain immunoglobulins from camelids (nanobodies) for specific and sensitive detection of TES. Methods After immunization of an alpaca (Vicugna pacos) with TES, RNA from peripheral blood lymphocytes was used as template for cDNA amplification with oligo dT primers and library construction. Isolation and screening of TES-specific nanobodies were carried out by biopanning and the resulting nanobodies were expressed in Escherichia coli. Two-epitopes amperometric immunocapturing assay was designed using paramagnetic beads coated with streptavidin and bivalent nanobodies. Detection of the system was carried out with nanobodies chemically coupled to horseradish peroxidase. The reaction was measured by amperometry and the limit of detection (LOD) was compared to conventional sandwich ELISA. Results We obtained three nanobodies that specifically recognize TES with no-cross reactivity to antigens of Ascaris lumbricoides and A. suum. The LOD of the assay using PBST20 0.05% as diluent was 100 pg/ml, 10 times more sensitive than sandwich ELISA. Conclusion Sensitive and specific detection of TES for discrimination of active and past infections is one of the most difficult challenges of T. canis diagnosis. The main advantage of our system is the use of two different nanobodies that specifically recognize two different epitopes in TES with a highly sensitive and straightforward readout. Considering that the amounts of TES available for detection in clinical samples are in the range of picograms or a few nanograms maximum, the LOD found in our experiments suggests that the test is potentially useful for the detection of clinically relevant cases of HT

Ultrasensitive detection of toxocara canis excretory-secretory antigens by a nanobody electrochemical magnetosensor assay.

peer reviewedHuman Toxocariasis (HT) is a zoonotic disease caused by the migration of the larval stage of the roundworm Toxocara canis in the human host. Despite of being the most cosmopolitan helminthiasis worldwide, its diagnosis is elusive. Currently, the detection of specific immunoglobulins IgG against the Toxocara Excretory-Secretory Antigens (TES), combined with clinical and epidemiological criteria is the only strategy to diagnose HT. Cross-reactivity with other parasites and the inability to distinguish between past and active infections are the main limitations of this approach. Here, we present a sensitive and specific novel strategy to detect and quantify TES, aiming to identify active cases of HT. High specificity is achieved by making use of nanobodies (Nbs), recombinant single variable domain antibodies obtained from camelids, that due to their small molecular size (15kDa) can recognize hidden epitopes not accessible to conventional antibodies. High sensitivity is attained by the design of an electrochemical magnetosensor with an amperometric readout with all components of the assay mixed in one single step. Through this strategy, 10-fold higher sensitivity than a conventional sandwich ELISA was achieved. The assay reached a limit of detection of 2 and15 pg/ml in PBST20 0.05% or serum, spiked with TES, respectively. These limits of detection are sufficient to detect clinically relevant toxocaral infections. Furthermore, our nanobodies showed no cross-reactivity with antigens from Ascaris lumbricoides or Ascaris suum. This is to our knowledge, the most sensitive method to detect and quantify TES so far, and has great potential to significantly improve diagnosis of HT. Moreover, the characteristics of our electrochemical assay are promising for the development of point of care diagnostic systems using nanobodies as a versatile and innovative alternative to antibodies. The next step will be the validation of the assay in clinical and epidemiological contexts

Nanobody-Based Immunosensor Detection Enhanced by Photocatalytic-Electrochemical Redox Cycling

peer reviewedDetection of antigenic biomarkers present in trace amounts is of crucial importance for medical diagnosis. A parasitic disease, human toxocariasis, lacks an adequate diagnostic method despite its worldwide occurrence. The currently used serology tests may stay positive even years after a possibly unnoticed infection, whereas the direct detection of a re-infection or a still active infection remains a diagnostic challenge due to the low concentration of circulating parasitic antigens. We report a time-efficient sandwich immunosensor using small recombinant single-domain antibodies (nanobodies) derived from camelid heavy-chain antibodies specific to Toxocara canis antigens. An enhanced sensitivity to pg/mL levels is achieved by using a redox cycle consisting of a photocatalytic oxidation and electrochemical reduction steps. The photocatalytic oxidation is achieved by a photosensitizer generating singlet oxygen (1O2) that, in turn, readily reacts with p-nitrophenol enzymatically produced under alkaline conditions. The photooxidation produces benzoquinone that is electrochemically reduced to hydroquinone, generating an amperometric response. The light-driven process could be easily separated from the background, thus making amperometric detection more reliable. The proposed method for detection of the toxocariasis antigen marker shows superior performances compared to other detection schemes with the same nanobodies and outperforms by at least two orders of magnitude the assays based on regular antibodies, thus suggesting new opportunities for electrochemical immunoassays of challenging low levels of antigens

SUMOylation of Paraflagellar Rod Protein, PFR1, and Its Stage-Specific Localization in Trypanosoma cruzi

BACKGROUND: The flagellate protozoan parasite, Trypanosoma cruzi, is a causative agent of Chagas disease that is transmitted by reduviid bugs to humans. The parasite exists in multiple morphological forms in both vector and host, and cell differentiation in T. cruzi is tightly associated with stage-specific protein synthesis and degradation. However, the specific molecular mechanisms responsible for this coordinated cell differentiation are unclear. METHODOLOGY/PRINCIPAL FINDINGS: The SUMO conjugation system plays an important role in specific protein expression. In T. cruzi, a subset of SUMOlylated protein candidates and the nuclear localization of SUMO have been shown. Here, we examined the biological roles of SUMO in T. cruzi. Site-directed mutagenesis analysis of SUMO consensus motifs within T. cruzi SUMO using a bacterial SUMOylation system revealed that T. cruzi SUMO can polymerize. Indirect fluorescence analysis using T. cruzi SUMO-specific antibody showed the extra-nuclear localization of SUMO on the flagellum of epimastigote and metacyclic and bloodstream trypomastigote stages. In the short-flagellate intracellular amastigote, an extra-nuclear distribution of SUMO is associated with basement of the flagellum and becomes distributed along the flagellum as amastigote transforms into trypomastigote. We examined the flagellar target protein of SUMO and show that a paraflagellar rod protein, PFR1, is SUMOylated. CONCLUSIONS: These findings indicate that SUMOylation is associated with flagellar homeostasis throughout the parasite life cycle, which may play an important role in differentiation of T. cruzi

Frecuencia de trichomoniasis vaginal en mujeres suspuestamente sanas

Se realizó un estudio de la frecuencia de trichomoniasis en 5 hospitales de diferentes municipios de Ciudad de La Habana, en un total de 427 mujeres sin antecedentes de enfermedades ginecológicas que acudieron a los laboratorios de microbiología de esos centros en el período comprendido entre mayo y septiembre de 1997. Treinta y cinco mujeres del total estudiado resultaron ser portadoras de Trichomonas vaginalis, lo cual se corresponde con una frecuencia de positividad de 8,2 %. De acuerdo con la edad, el mayor porcentaje de positividad (16,1) correspondió al grupo comprendido entre 15 y 20 años. El diagnóstico por el método de cultivo in vitro resultó tener una mayor sensibilidad con respecto al examen directo del exudado vaginal. El tiempo de lectura a las 48 h aportó la mayor positividad diagnóstica.The frequency of trichomoniasis in 5 hospitals from different municipalities of Havana City was studied among 427 women with no history of gynecological diseases that attended the microbiology laboratories of these centers from May to September, 1997. 35 women of the total of women studied proved to be carriers of Trichomonas vaginalis, which accounts for a positivity frequency of 8.2 %. According to age, the highest percentaje of positivity (16.1) corresponded to the group aged 15-20. The diagnosis by culture in vitro had a greater sensitivity compared with the direct examination of the vaginal exudate. The reading obtained at 48 hours showed the highest diagnostic positivity

Nanobody-based sandwich enzyme-linked immunosorbent assay for the detection of Toxocara canis excretory secretory proteins.

IntroductionHuman Toxocariasis (HT) is a neglecteddisease resulting from tissue invasion of L2larva fromToxocaracanis. The only laboratory diagnostic tool currently available is aserological test detecting IgG against the excretory secretoryproteins of the parasite (TES). This method is unable todistinguish between current and past infections. We developed asandwich enzyme-linked immunosorbent assay (ELISA) takingadvantage of the inherent features of specific single variabledomain fragments of camelids (nanobodiesâ).Methods and MaterialsAn alpaca was immunized with125lg of TES 5 times in intervals of 7 days. One week after thelast immunization, peripheral blood was extracted andnanobodyâsequences were amplified through reverse-transcription polymerase chain reaction (RT-PCR) from bloodlymphocytes. Selection of binders was performed by biopanningbased on phage display. A sandwich ELISA was set up with acapturing nanobodyâcloned in pHEN6c vector and a detectionnanobodyâin vector pBAD17 containing an AviTagTMforin vivobiotinylation. Cross-reactivity was tested with excretoryantigens ofAscaris lumbricoidesandA. suum. Immunocapturingwith paramagnetic beads was used to isolate the specific fractionof TES recognized in the sandwich ELISA.ResultsNanobodyâsequences were present in 3x108transformants. 84% of them contained a plasmid encoding 20different nanobody sequences. The combination 1TCE39 and1TCE52 had the best Optical Density (OD) signal in sandwichELISA with no cross-reactivity withA. lumbricoidesorA. suum.The detection limit using ELISA sandwich format in negativesamples spiked with TES was 40 ng/ml. Immunocapturingdemonstrated that the epitopes recognised by the sandwichELISA are located in the 120 kDa fraction of TES.ConclusionsTests to diagnose active HT are currently notavailable, hampering the estimation of the real prevalence of thedisease and its control. Nanobodyâ-based sandwich ELISAprovides an innovative approach to detect TES ofT. canisinclinical samples.DisclosureThis project is funded by the ResearchFoundation Flanders (FWO