Genotyping of toxoplasma gondii isolates from México reveals non-archetypal and potentially virulent strains for mice.

Genotyping and virulence studies of Toxoplasma gondii are essential to investigate the pathogenesis of strains circulating worldwide. In this study, eight T. gondii isolates obtained from a congenitally infected newborn, a calf, two cats, three dogs, and a wallaby from five states of México were genotyped by Mn-PCR-RFLP with 11 typing markers (SAG1, SAG2 5'3', alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico), five virulence markers (CS3, ROP16, ROP17, ROP18 and ROP5), 15 microsatellite markers (TUB-2, W35, TgM-A, B18, B17, M33, IV.1, XI.1, M48, M102, N60, N82, AA, N61, N83), and sequencing. A phylogenetic network was built to determine the relationship between Mexican isolates and those reported worldwide. Six different genotypes were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), ToxoDB #8, #10, #28 (n = 3), #48, #116, and #282. Genotyping by microsatellite analysis differentiated the three PCR-RFLP genotype #28 isolates into two strains, revealing a total of seven microsatellite genotypes. Three different allele combinations of ROP18/ROP5 virulence markers were also found, 3/3, 1/1, and 4/1. The last two combinations are predicted to be highly virulent in the murine model. According to the phylogenetic network, the T. gondii strains studied here are related to archetypal strains I and III, but none are related to the strains previously reported in México. The genotypes identified in this study in different species of animals demonstrate the great genetic diversity of T. gondii in México. The ToxoDB-PCR-RFLP #28 genotype was found in three isolates from different hosts and states. Additionally, four of the isolates are predicted to be highly virulent in mice. The next step will be to perform in vitro and in vivo assays to determine the phenotype of these T. gondii isolates in murine models.

Maternal anti-Toxoplasma gondii antibodies IgG2, IgG3 and IgG1 are markers of vertical transmission and clinical evolution of toxoplasmosis in the offspring.

Toxoplasma gondii can be transmitted vertically during pregnancy and may cause neurological, ocular, and even systemic damage to the offspring. Congenital toxoplasmosis (CT) can be diagnosed during gestation and/or after birth in the postnatal period. The timely diagnosis is highly relevant for efficient clinical management. The most common laboratory methods for diagnosing CT are based on Toxoplasma-specific humoral immune responses. However, these methods are of low sensitivity or specificity. In a previous study with a small number of cases, the comparison of anti-T. gondii IgG subclasses between mothers and their offspring showed promising results for CT diagnosis and prognosis. Thus, in this work, we analyzed specific IgG subclasses and IgA in 40 T. gondii-infected mothers and their children, of which 27 were congenitally infected and 13 uninfected. A higher frequency of anti-Toxoplasma IgG2, IgG3, IgG4, and IgA antibodies was observed in mothers and congenitally infected offspring. Of these, IgG2 or IgG3 were statistically the most conspicuous. In the CT group, maternal IgG3 antibodies were significantly associated with severe disease of the infants and IgG1 and IgG3 with disseminated disease. The results support that maternal anti-T. gondii IgG3, IgG2 and IgG1 are markers of congenital transmission and severity/spread of disease in the offspring.

IgY Antibodies as Biotherapeutics in Biomedicine.

Since the discovery of antibodies by Emil Von Behring and Shibasaburo Kitasato during the 19th century, their potential for use as biotechnological reagents has been exploited in different fields, such as basic and applied research, diagnosis, and the treatment of multiple diseases. Antibodies are relatively easy to obtain from any species with an adaptive immune system, but birds are animals characterized by relatively easy care and maintenance. In addition, the antibodies they produce can be purified from the egg yolk, allowing a system for obtaining them without performing invasive practices, which favors the three "rs" of animal care in experimentation, i.e., replacing, reducing, and refining. In this work, we carry out a brief descriptive review of the most outstanding characteristics of so-called "IgY technology" and the use of IgY antibodies from birds for basic experimentation, diagnosis, and treatment of human beings and animals.

Anti-Toxoplasma gondii IgM Long Persistence: What Are the Underlying Mechanisms?

Diagnosis of Toxoplasma gondii acute infection was first attempted by detection of specific IgM antibodies, as for other infectious diseases. However, it was noted that this immunoglobulin declines slowly and may last for months or even years. Apart from the diagnostic problem imposed on clinical management, this phenomenon called our attention due to the underlying phenomena that may be causing it. We performed a systematic comparison of reports studying IgM antibody kinetics, and the data from the papers were used to construct comparative plots and other graph types. It became clear that this phenomenon is quite generalized, and it may also occur in animals. Moreover, this is not a technical issue, although some tests make more evident the prolonged IgM decay than others. We further investigated biological reasons for its occurrence, i.e., infection dynamics (micro-reactivation-encystment, reinfection and reactivation), parasite strain relevance, as well as host innate, natural B cell responses and Ig class-switch problems inflicted by the parasite. The outcomes of these inquiries are presented and discussed herein.

SAG3 Toxoplasma gondii cloning reveals unexpected fivefold infection in the blood of feral cats in the Mexican Caribbean.

BACKGROUND: Currently, more than 300 genotypes of Toxoplasma gondii (T. gondii) have been described throughout the world, demonstrating its wide genetic diversity. The SAG3 locus is one of the genes included in the genotyping panel of this parasite. It is associated with its virulence since it participates during the invasion process of the host cells. Therefore, cloning, sequencing, and bioinformatic analysis were used to deepen the understanding of the SAG3 locus genetic diversity of T. gondii in blood samples from feral cats. RESULTS: Six different SAG3 sequences were detected, five of which were detected in one feline. Three sequences were first reported here; one of them was an intragenic recombinant. In the cladogram, four out of ten SAG3 sequences did not share nodes with others reported worldwide. CONCLUSIONS: Cloning and sequencing of samples with more than one restriction pattern by PCR-RFLP were very helpful tools to demonstrate the presence of more than three genotypes of T. gondii in the blood of feral cats from southeastern Mexico. This suggests a potential mixed infection of multiple T. gondii strains and high genetic diversity of the parasites in felines in this tropical region of Mexico.

NF-κB and Its Regulators During Pregnancy.

The transcriptional factor NF-κB is a nuclear factor involved in both physiological and pathological processes. This factor can control the transcription of more than 400 genes, including cytokines, chemokines, and their modulators, immune and non-immune receptors, proteins involved in antigen presentation and cell adhesion, acute phase and stress response proteins, regulators of apoptosis, growth factors, other transcription factors and their regulators, as well as different enzymes; all these molecules control several biological processes. NF-κB is a tightly regulated molecule that has also been related to apoptosis, cell proliferation, inflammation, and the control of innate and adaptive immune responses during onset of labor, in which it has a crucial role; thus, early activation of this factor may have an adverse effect, by inducing premature termination of pregnancy, with bad outcomes for the mother and the fetus, including product loss. Reviews compiling the different activities of NF-κB have been reported. However, an update regarding NF-κB regulation during pregnancy is lacking. In this work, we aimed to describe the state of the art around NF-κB activity, its regulatory role in pregnancy, and the effect of its dysregulation due to invasion by pathogens like Trichomonas vaginalis and Toxoplasma gondii as examples.

Autoantibodies Against Ubiquitous and Confined Antigens in Patients With Ocular, Neuro-Ophthalmic and Congenital Cerebral Toxoplasmosis.

Toxoplasma gondii infection can trigger autoreactivity by different mechanisms. In the case of ocular toxoplasmosis, disruption of the blood-retinal barrier may cause exposure of confined retinal antigens such as recoverin. Besides, cross-reactivity can be induced by molecular mimicry of parasite antigens like HSP70, which shares 76% identity with the human ortholog. Autoreactivity can be a determining factor of clinical manifestations in the eye and in the central nervous system. We performed a prospective observational study to determine the presence of autoantibodies against recoverin and HSP70 by indirect ELISA in the serum of 65 patients with ocular, neuro-ophthalmic and congenital cerebral toxoplasmosis. We found systemic autoantibodies against recoverin and HSP70 in 33.8% and 15.6% of individuals, respectively. The presence of autoantibodies in cases of OT may be related to the severity of clinical manifestations, while in cases with CNS involvement they may have a protective role. Unexpectedly, anti-recoverin antibodies were found in patients with cerebral involvement, without ocular toxoplasmosis; therefore, we analyzed and proved cross-reactivity between recoverin and a brain antigen, hippocalcin, so the immunological phenomenon occurring in one immune-privileged organ (e.g. the central nervous system) could affect the environment of another (egg. the eye).

Toxoplasma gondii infection in European mouflons (Ovis musimon) and captive wild felines from Puebla, México.

The presence of Toxoplasma gondii in zoos is cause of alert because many susceptible species kept in captivity die of clinical toxoplasmosis. Moreover, excretion of T. gondii oocysts by infected captive wild felines into the facilities could pose a risk to workers. Herbivores in wild collections can serve as sentinels of local transmission, since they get infected by the consumption of oocysts present in ground or water. Both herbivores and felids may reveal the parasite variants which are circulating in the region. We determined the seroprevalence of T. gondii in European mouflons (n = 55) and wild felines (n = 15) from a private zoological collection located in the Eastern region of México, as well as the incidence in 41 of the mouflons using ELISA. The prevalence of T. gondii in mouflons was 14.5% (n = 55) and 17.1% (n = 41) in 2011 and 19.5% in 2012. The estimated incidence was 9.8%-12.2%. In wild felines the frequency was 80%. Four sero-positive animals (two mouflons and the two oldest African lions) were euthanized. Histopathology, conventional PCR (for B1 and SeqRep529 loci) and molecular characterization were carried out. All euthanized animals were positive to T. gondii by PCR. We identified a triple infection (I + II + III) in the brain of a mouflon. In conclusion, a high infective pressure of T. gondii in the collection was found, supported by changes in its prevalence in European mouflons. A high prevalence of infection in wild felines was determined. At least four genotypes of T. gondii are present in herbivores and carnivores, and one mouflon had a mixed infection.

A Proinflammatory Immune Response Might Determine Toxoplasma gondii Vertical Transmission and Severity of Clinical Features in Congenitally Infected Newborns.

Toxoplasma gondii is the etiological agent of toxoplasmosis. Mother-to-child transmission of this parasite can occur during pregnancy. Newborns with congenital toxoplasmosis may develop central nervous system impairment, with severity ranging from subclinical manifestations to death. A proinflammatory/regulated specific immune profile is crucial in the defense against the parasite; nevertheless, its role in the infected pregnant women and the congenitally infected offspring has been poorly explored, and there is still no consensus about its relation to parasite vertical transmission or to severity and dissemination in the congenitally infected newborns. This work aimed to characterize these relations by means of principal component and principal factor analyses. For this purpose, we determined the specific production of the four immunoglobulin G antibody subclasses, cytokines, and lymphocyte proliferation in the T. gondii-infected pregnant women-10 who transmitted the infection to their offspring and seven who did not-as well as in 11 newborns congenitally infected and grouped according to disease severity (five mild and six moderate/severe) and dissemination (four local and seven disseminated). We found that the immune response of nontransmitter women differed from that of the transmitters, the latter having a stronger proinflammatory response, supporting a previous report. We also found that newborns who developed moderate/severe disease presented higher levels of lymphocyte proliferation, particularly of CD8+ and CD19+ cells, a high proportion of tumor necrosis factor α producers, and reduced expression of the immune modulator transforming growth factor ß, as opposed to children who developed mild clinical complications. Our results suggest that a distinctive, not regulated, proinflammatory immune response might favor T. gondii vertical transmission and the development of severe clinical manifestations in congenitally infected newborns.

Stray dogs in the tropical state of Chiapas, Mexico, harbour atypical and novel genotypes of Toxoplasma gondii.

Genotyping of Toxoplasma gondii remains a relevant topic of study, since genotypes can be related to the presentation and severity of toxoplasmosis. To date, 292 restriction fragment length polymorphism genotypes have been described around the world. Serosurveys in southeastern Mexico have documented exposure in over 70% of people and certain animals. Recently, we have described new genotypes and mixed infections in feral cats from Quintana Roo. Thus, the aim of this study was to genotype T. gondii and to describe its genetic variability, from naturally infected stray dogs of Chiapas, which has different geographical and climatic conditions from those found at the Yucatan Peninsula and the other parts of the country. Eleven stray dogs were captured and bled to obtain DNA, and then they were euthanized to perform necropsies and to collect target tissues. Diagnosis of T. gondii was done by quantitative real-time PCR (qPCR) and endpoint PCR. Genotyping was carried out, amplifying 12 polymorphic markers and 15 microsatellites. Atypical SAG3 gene products were cloned and sequenced. All blood samples of dogs were positive to T. gondii DNA by PCR. Two isolates were obtained from pooled heart and diaphragm tissue of two dogs. Two complete PCR-RFLP genotypes were identified (type BrIII and #28). Four animals had mixed infections. A new RFLP atypical allele for the SAG3 marker was observed; cloning and sequencing analysis of this locus revealed mixed infection by a strain identical to GT1, and one type I × II intragenic recombinant. The microsatellite analysis revealed that both isolates are atypical. Thus, atypical new genotypes of T. gondii and mixed infections were found in dogs of Chiapas. The results found here and in genotyping studies in México suggest that the southeastern region favours wide genetic diversity of T. gondii and the possible presence of virulent genotypes such as those found in central and South America.

Testing New Peptides From Toxoplasma gondii SAG1, GRA6, and GRA7 for Serotyping: Better Definition Using GRA6 in Mother/Newborns Pairs With Risk of Congenital Transmission in Mexico.

Toxoplasma gondii variant influences clinical profile in human congenital and ocular toxoplasmosis. Parasite genotyping represents a challenge due to insufficient amount of genetic material of the protozoan in the host samples, and isolates are hard to obtain, especially from pediatric patients. An alternative is serotyping, which is based on the presence of specific antibodies against polymorphic proteins related to virulence; the more widely used are GRA6 and GRA7, but most works report cross reactions among the classical strains (I, II, and III). We designed new peptides of GRA6, GRA7, and SAG1 proteins, with more SNPs among the three clonal strains than those previously designed. This was done by identifying BcR and polymorphic epitopes by means of bioinformatics; then we designed peptides with linkers joining the specific regions and predicted their 3D structure. With the commercial molecules synthesized on the basis of these designs, we tested 86 serum samples from 42 mother/newborn pairs and two congenitally infected newborns, by indirect ELISA. We implemented a strategy to determine the serotype based on scatter plots and a mathematical formula, using ratios among reactivity indexes to peptides. We found low frequency of samples reactive to GRA7 and SAG1, and cross reactions between GRA6 serotypes I and III; we modified these later peptides and largely improved distinction among the three clonal strains. The chronicity of the infection negatively affected the reactivity index against the peptides. Serotyping both members of the mother/child pair improves the test, i.e., among 26% of them only one member was positive. Serotype I was the most frequent (38%), which was congruent with previous genotyping results in animals and humans of the same area. This serotype was significantly more frequent among mothers who transmitted the infection to their offspring than among those who did not (53 vs. 8%, p = 0.04) and related to disease dissemination in congenitally infected children, although non-significantly. In conclusion, serotyping using the improved GRA6 peptide triad is useful to serotype T. gondii in humans and could be implemented for clinical management and epidemiological studies, to provide information on the parasite type in specific areas.

Mixed Toxoplasma gondii infection and new genotypes in feral cats of Quintana Roo, México.

Toxoplasmosis is a zoonosis caused by Toxoplasma gondii that infects homeothermic animals, including humans. To date, as many as 287 genotypes have been described worldwide. Genetic characterization of the parasite is crucial because the parasite type can determine the presentation and severity of toxoplasmosis. Previously, we reported that the Yucatán Peninsula has a frequency of infection of over 70% in humans and other animals; moreover, there are seven species of felids, including domestic cats; thus, we hypothesized that this might be a region with a high diversity of the parasite. Nevertheless, no genotyping of this protozoan has been performed in this region. Thus, the aim of this study was to genotype T. gondii from naturally infected feral cats of Quintana Roo, within the Yucatán Peninsula, and to describe its genetic variability. Eleven feral cats were captured and bled to obtain the buffy coat; then, they were euthanized to collect target organs or tissues to extract DNA. Samples were processed by PCR for diagnosis, and ten polymorphic markers were genotyped by PCR-RFLP. Atypical GRA6 gene products were cloned and sequenced. Ten of the eleven cats were PCR positive for toxoplasmosis in blood; of these, seven had mixed infections. Also, two isolates were obtained from the heart and diaphragm of two animals. At least 23 different genotypes were detected, from which 18 are new worldwide. From the atypical GRA6 gene cloning and sequencing analysis, a mixed infection was discovered, due to one strain identical to GT1 and another to VAND. In conclusion, T. gondii genetic diversity in the region is high and different from that in other regions, with new genotypes exclusive to México and some others shared with USA and South America.

Maternal Immune Response During Pregnancy and Vertical Transmission in Human Toxoplasmosis.

Toxoplasmosis is a parasitic zoonosis distributed worldwide, caused by the ingestion of contaminated water/food with the parasite Toxoplasma gondii. If a pregnant woman is infected with this parasite, it may be transmitted to the fetus and produce ocular, neurological, or systemic damage with variable severity. The strength and profile of mother's immune response have been suggested as important factors involved in vertical transmission rate and severity of clinical outcome in the congenitally infected fetus. The aim of this work was to evaluate a possible relation between the mother's immune response during pregnancy and congenital transmission to the fetus. We obtained peripheral blood from T. gondii infected pregnant woman and tested it for anti T. gondii (IgG1, IgG2, IgG3, IgG4, and IgA) in serum. Peripheral blood mononuclear cells (PBMCs) were isolated to analyze the in vitro effect of soluble T. gondii antigens on proliferation and production of cytokines. We found that IgG2-4 and IgA antibodies and lymphocytes proliferation, especially CD4+, CD8+, and CD19+ were positive in a higher proportion of cases in transmitter than in non-transmitter women. Furthermore, IgG2-3 and IgA anti-Toxoplasma antibody levels were higher in those mothers who transmitted the infection than in those who did not. Interestingly, a higher proportion of positive cases to IFN-γ and negatives to the immunoregulatory cytokine TGF-ß, were related to T. gondii vertical transmission. Our descriptive results are consistent with the paradoxical previous observations in murine models of congenital toxoplasmosis, which suggest that an increased immune response that protects the mothers from a disseminated or severe disease, and should protect the fetus from infection, is positively related to parasite transmission.

Potential public health benefits from cat eradications on islands.

Cats (Felis catus) are reservoirs of several pathogens that affect humans, including Toxoplasma gondii. Infection of pregnant women with T. gondii can cause ocular and neurological lesions in newborns, and congenital toxoplasmosis has been associated with schizophrenia, epilepsy, movement disorders, and Alzheimer's disease. We compared seroprevalence of T. gondii and risk factors in people on seven islands in Mexico with and without introduced cats to determine the effect of cat eradication and cat density on exposure to T. gondii. Seroprevalence was zero on an island that never had cats and 1.8% on an island where cats were eradicated in 2000. Seroprevalence was significantly higher (12-26%) on the five islands with cats, yet it did not increase across a five-fold range of cat density. Having cats near households, being male and spending time on the mainland were significant risk factors for T. gondii seroprevalence among individuals, whereas eating shellfish was protective. Our results suggest that cats are an important source of T. gondii on islands, and eradicating, but not controlling, introduced cats from islands could benefit human health.

High heterogeneity, mixed infections and new genotypes in human congenital toxoplasmosis cases in the mega-metropolis of Central Mexico.

Mexico presents high prevalence of Toxoplasma gondii infection, including the congenital form, but there are few data about the genetic diversity of the parasite, so we attempted parasite isolation and genotyping in nine mother/children pairs with congenital toxoplasmosis (CT), living in the Valley of Mexico, who were part of a 30 cases cohort that started 12 years ago. They were recruited through research projects which included pre- and postnatal screening of congenital infections or directly CT, and cases referred to INP for management because they had clinical abnormalities. Genotyping was performed by PCR-RFLP of SAG1, SAG2, SAG3, BTUB GRA6, c22-8, c29-2, L358, PK1 and Apico markers, followed by sequencing. Sixty seven percent of samples were typed for the SAG3 locus, 39% for Apico and 33% for BTUB, while Alt. SAG2, GRA6 and c29-2 types could be labelled in less cases. Type I alleles predominated, followed by II and III. We isolated the first strain obtained from humans in Mexico and found three genotypes not previously found in the world. The presence of ToxoDB#10 clonal type was documented in one pair, as well as mixed infections in five mothers. No relation of genotype or parasite load with clinical signs was found. In conclusion, we encountered great genetic diversity and mixed T. gondii infections among mother/children pairs with congenital toxoplasmosis in the mega-metropolis of the Valley of Mexico.

Report of an unsual case of anophthalmia and craniofacial cleft in a newborn with Toxoplasma gondii congenital infection.

BACKGROUND: We present one unusual case of anophthalmia and craniofacial cleft, probably due to congenital toxoplasmosis only. CASE PRESENTATION: A two-month-old male had a twin in utero who disappeared between the 7th and the 14th week of gestation. At birth, the baby presented anophthalmia and craniofacial cleft, and no sign compatible with genetic or exposition/deficiency problems, like the Wolf-Hirschhorn syndrome or maternal vitamin A deficiency. Congenital toxoplasmosis was confirmed by the presence of IgM abs and IgG neo-antibodies in western blot, as well as by real time PCR in blood. CMV infection was also discarded by PCR and IgM negative results. Structures suggestive of T. gondii pseudocysts were observed in a biopsy taken during the first functional/esthetic surgery. CONCLUSIONS: We conclude that this is a rare case of anophthalmia combined with craniofacial cleft due to congenital toxoplasmosis, that must be considered by physicians. This has not been reported before.

Genotyping Toxoplasma gondii with the B1 Gene in Naturally Infected Sheep from an Endemic Region in the Pacific Coast of Mexico.

Toxoplasma gondii is a protozoan parasite with a broad ecological valence, which has been detected in a wide range of hosts and landscapes. Although the genus is considered monospecific, in recent years it has been demonstrated to exhibit more genetic variability than previously known. In Mexico, there are few genotyping studies, which suggest that classical, autochthonous, and atypical strains are circulating. The goal of this study was to describe T. gondii genetic diversity in naturally infected sheep from Colima, Mexico. This is a good site to study ecological aspects of this parasite since it is located between the Nearctic and Neotropical ecozones and it includes domestic and wild risks for transmission. We analyzed 305 tissue samples of semicaptive sheep from six coastal and central zones of Colima and border zones of Michoacán. We used an 803 bp amplicon of the B1 gene to genotype T. gondii and seroprevalence was determined by ELISA. Indexes for genetic diversity and genetic differentiation were calculated and compared with reference strains from North America (NA) and South America (SA). Twenty-three tissue samples were positive for the B1 gene by PCR, which were sequenced. Crude prevalence was 24.4%. The genetic analysis showed 16 variable sites along the 803 bp region that grouped all sequences into 13 haplotypes in the phylogenetic tree. Bayesian and haplotype network analysis showed nine new B1-types, of which three were frequent and six had unique alleles. Comparisons among sequence sets revealed that the Mexican population had lower differentiation than SA and an intermediate genetic variability between South America and North America. The B1 gene analysis showed new T. gondii haplotypes in naturally infected sheep; therefore, this marker could be initially used in molecular screening studies to identify potentially virulent genotypes of this parasite using natural host samples directly.

Vertical transmission and fetal damage in animal models of congenital toxoplasmosis: A systematic review.

In humans, the probability of congenital infection and fetal damage due to Toxoplasma gondii is dependent on the gestation period at which primary infection occurs. Many animal models have been used for vaccine, drug testing, or studies on host or parasite factors that affect transmission or fetal pathology, but few works have directly tested fetal infection and damage rates along gestation. So, the purpose of this work was to perform a systematic review of the literature to determine if there is a model which reflects these changes as they occur in humans. We looked for papers appearing between 1970 and 2014 in major databases like Medline and Scopus, as well as gray literature. From almost 11,000 citations obtained, only 49 papers fulfilled the criteria of having data of all independent variables and at least one dependent datum for control (untreated) groups. Some interesting findings could be extracted. For example, pigs seem resistant and sheep susceptible to congenital infection. Also, oocysts cause more congenitally infected offspring than tissue cysts, bradyzoites or tachyzoites. In spite of these interesting findings, very few results on vertical transmission or fetal damage rates were similar to those described for humans and only for one of the gestation thirds, not all. Moreover, in most designs tissue cysts - with unknown number of bradyzoites - were used, so actual dose could not be established. The meta-analysis could not be performed, mainly because of great heterogeneity in experimental conditions. Nevertheless, results gathered suggest that a model could be designed to represent the increase in vertical transmission and decrease in fetal damage found in humans under natural conditions.

Immunoglobulin Concentrations in Plasma and Saliva During the Neonatal Period.

BACKGROUND: Screening for infectious diseases in newborns using immunoglobulin (Ig)A-, IgM-, and IgE-specific antibodies is expensive and impractical. To determine if total levels of these Igs can be used for screening purposes, thus simplifying the process, their basic levels in the 1(st) month of extrauterine life need to be determined. Additionally, the ability to simplify screening by using saliva also needs to be determined. The aim of this study was to determine IgA, IgM, and IgE concentrations in plasma and saliva in newborns, correlation between the samples, and relationship between Ig levels and newborn age. METHODS: We enrolled 53 apparently healthy newborns, paired samples of plasma and saliva were collected, and total IgA, IgM, and IgE concentrations determined by capture enzyme-linked immunosorbent assay. The correlation between plasma and saliva values was calculated by Spearman's rank correlation coefficient and the IgA, IgM, and IgE distributions were analyzed by the Shapiro-Wilk test. We also determined the level of each Ig concentration according to age. RESULTS: IgA and IgM levels in plasma and IgA levels in saliva increased significantly during 1(st) month of life, especially in the 2(nd) week and 3(rd) week, with a good correlation of IgA between plasma and saliva. IgE levels in both plasma and saliva and IgM levels in saliva were very low or absent. CONCLUSION: These results suggest that Igs in saliva could be good biomarkers for newborn screening programs during the 1(st) week of life. This study established reference values for Igs according to age in the neonatal period.

Successful capture of Toxocara canis larva antigens from human serum samples.

BACKGROUND: Toxocara canis is a nematode that parasitizes dogs, while humans are paratenic hosts. When humans are infected the migrating larvae damage the liver, lungs and even the nervous system. Larva migrans diagnosis is based on immunological techniques; however, the commercial immunodiagnostic kits detect anti-T. canis antibodies which may cross-react with other parasites, mainly nematodes with extra-intestinal migration. Moreover, antibodies do not necessarily reflect an active infection; so detection and quantification of circulating antigens may provide appropriate and timely information for treatment, which prevents irreversible damage. Here we report the standardization of a monoclonal antibody based antigen capture ELISA to diagnose human toxocariasis without cross-reaction. METHODS: We developed anti-T. canis polyclonal antibodies in rabbits and a monoclonal antibody in mouse which did not cross-react with 15 antigens from several parasites. The sandwich ELISA standardization was performed using sera from mice experimentally infected. We tested the method using 29 positive and 58 negative human sera previously typified with a commercial kit, which detects antibodies. RESULTS: Only 5.0 µg/mL and 10 µg/mL polyclonal antibodies and monoclonal antibody, respectively, were needed in the sandwich ELISA standardization, detecting since 440 pg/mL larva antigens. Nine out of 29 antibody-positive sera were also positive for antigens and no false positive were found. Taking the antibody kit as the reference standard, the sensibility and specificity of the antigen test were 31% and 100%, respectively. CONCLUSIONS: With these tools we established a detection threshold as low as 440 pg/mL antigen. Monoclonal antibody is specific, and did not cross-react with antigens from other parasites. Detection of circulating antigens helps provide appropriate and timely treatment and prevents irreversible damage.