Toxoplasma gondii in Capybara (Hydrochaeris hydrochaeris) antibodies and DNA detected by IFAT and PCR

Toxoplasmosis is considered nowadays as one of the most important foodborne diseases in the world. One of the emerging risks in acquiring infection with Toxoplasma gondii is the increasing popularity of wild animals and game meat. Capybara (Hydrochaeris hydrochaeris) is the world’s largest extant rodent and is used for human consumption in many areas of South America, and in case it carries T. gondii cysts, it may act as a source of infection. In the present study, we detected infection with T. gondii in capybaras from the south of Brazil. Antibodies to T. gondii were assayed in the serum of capybaras using the indirect fluorescent antibody test (IFAT ≥ 1:16). Blood, liver, heart, lymph nodes, and spleen tissues were collected and tested by polymerase chain reaction (PCR) for B1 gene and ITS1 region. The results showed that 61.5% (16/26) capybaras were seropositive to T. gondii. Titers of specific antibodies to T. gondii ranged from 1:16 to 1:512. Among the feral rodents studied, 7.7% (2/26) were PCR positive for B1 gene assay and 11.5% (3/26) were positive for ITS1 PCR assay; for both test, the prevalence was 15.4%. Liver, heart, and blood tissues were those which tested positive for the apicomplexan. Our findings show a high percentage of infection with T. gondii in asymptomatic capybaras. Based on those data, we hypothesize that the consumption of raw or undercooked capybara meat could be a source of infection for humans.     

Small reciprocal insertion detected by spectral karyotyping (SKY) and delimited by array-CGH analysis

A 5.4-year-old male propositus is reported with mild dysmorphic features including hypoplasia of the radial part of both hands affecting thenar, thumb and fingers 2-3, incomplete syndactyly of fingers 3-4, single palmar creases, brachymesophalangia of toes 3-5, dissociated retardation of bone age, telecanthus, spina bifida occulta, cryptorchidism, muscular hypotonia, and borderline mental retardation. His karyotype was unbalanced, 46,XY,der(16)ins(4;16)(q26q28.1; q12.1q12.2)pat. In the propositus' father who had brachydactyly of fingers 2-5 and brachymesophalangia of toes 3-5 the insertion was reciprocal, 46,XY,rep ins(4;16)(q26q28.1;q12.1q12.2). Insertions are rare, reciprocal insertions most unusual. The characterization of the insertion in the propositus and the detection of its reciprocity in the father were achieved by the application of spectral karyotyping (SKY). Further examination of the propositus' unbalanced genome by array-CGH analysis delimited the chromosomal locations of the deletion/insertion rearrangement on a 0.5-2 Mb resolution level and allowed to design specific BAC FISH analyses that pinpointed the borders of the affected segments. The rearrangement involved a segment of 7.7 Mb between RP11-1030 g22 and RP11-52k8 at the chromosomal regions 4q26 and 4q28.1, respectively, and a segment of 2.8 Mb between RP11-242n20 at 16q12.1 and RP11-324d17 at 16q12.2. A simple molecular genetic explanation of the phenotype cannot be given. A relation to the Townes Brocks gene (SALL1) located 340 kb proximal of the 16q12 deletion/insertion is unlikely. Possibly more relevant is an overlap of the 16q12 deletion/insertion with a small deletion of the syntenic chromosomal region in the mouse that causes a developmental disorder of digits ("Fused toes").     

Interstrain mitochondrial DNA polymorphism detected in Acanthamoeba by restriction endonuclease analysis

The genus Acanthamoeba includes pathogenic and nonpathogenic strains of amebas with unclear taxonomic and evolutionary relationships. To explore these relationships further, we have examined mitochondrial DNA fragment patterns obtained for 15 Acanthamoeba strains by use of five restriction endonucleases. The mitochondrial DNA molecules were circular, averaging 41.6 +/- 1.5 kilobase pairs. Fragments resulting from endonuclease digestion of the DNA were separated by agarose gel electrophoresis. Ten distinct families of electrophoretic patterns (digestion phenotypes) were observed. Seven phenotypes were found for seven strains considered nonpathogenic or of unknown pathogenicity. Three phenotypes were associated with pathogenic strains. One of these phenotypes included a single pathogenic strain, a second included one pathogen and one strain of unknown pathogenicity, and the third included five pathogenic strains. The latter five were of widespread geographic origin and previously were assigned to two different species. The results suggest that extensive nucleotide sequence diversity occurs among strains from a single species of Acanthamoeba, but that subgroups of strains with similar sequences also occur. Thus, restriction enzyme analysis can identify clusters of strains and may be a useful approach to classification in the genus. Improvements in classification should help clarify relationships among pathogenic and non-pathogenic strains.     

Muscular dystrophies and myopathies: the spectrum of mutated genes in the Czech Republic

Inherited neuromuscular disorder (NMD) is a wide term covering different genetic disorders affecting muscles, nerves, and neuromuscular junctions. Genetic and clinical heterogeneity is the main drawback in a routine gene‐by‐gene diagnostics. We present Czech NMD patients with a genetic cause identified using targeted next‐generation sequencing (NGS) and the spectrum of these causes. Overall 167 unrelated patients presenting NMD falling into categories of muscular dystrophies, congenital muscular dystrophies, congenital myopathies, distal myopathies, and other myopathies were tested by targeted NGS of 42 known NMD‐related genes. Pathogenic or probably pathogenic sequence changes were identified in 79 patients (47.3%). In total, 37 novel and 51 known disease‐causing variants were detected in 23 genes. In addition, variants of uncertain significance were suspected in 7 cases (4.2%), and in 81 cases (48.5%) sequence changes associated with NMD were not found. Our results strongly indicate that for molecular diagnostics of heterogeneous disorders such as NMDs, targeted panel testing has a high‐clinical yield and should therefore be the preferred first‐tier approach. Further, we show that in the genetic diagnostic practice of NMDs, it is necessary to take into account different types of inheritance including the occurrence of an autosomal recessive disorder in two generations of one family.     

Solitary neurocysticercosis case caused by Asian genotype of Taenia solium confirmed by mitochondrial DNA analysis

A Japanese woman presenting with neurologic symptoms was presumptively diagnosed with neurocysticercosis based on imaging findings. Hooklets in the scolex of the resected lesion were not confirmed through histopathological observation. However, the illness was confirmed by mitochondrial DNA analysis to be a solitary neurocysticercosis case caused by the Asian genotype of Taenia solium.     

Molecular typing of Stenotrophomonas (Xanthomonas) maltophilia by DNA macrorestriction analysis and random amplified polymorphic DNA analysis.

Stenotrophomonas (Xanthomonas) maltophilia is a multidrug-resistant, nosocomial pathogen for which optimal typing methods in epidemiologic investigations of nosocomial outbreaks have not been defined. We compared DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) with random amplified polymorphic DNA (RAPD) analysis by arbitrarily primed PCR for molecular typing of 109 multidrug-resistant strains of S. maltophilia from multiple outbreaks at our institution over a 10-month period in 1993. PFGE after digestion with restriction endonuclease DraI revealed 62 unique DNA restriction profiles among the 109 strains, with 23, 11, 6, 6, and 3 strains having concordant profiles in each of five types. There were four concordant profiles among 8 strains (2 strains with each profile), while unique profiles were present in each of the remaining 52 strains. Further RAPD analysis with a decanucleotide primer showed the same number of distinct strain types as PFGE but more subtype diversity within each clonal type. We concluded that DNA macrorestriction analysis and RAPD analysis are sufficiently discriminatory and useful for differentiation of S. maltophilia strains in epidemiologic investigations of nosocomial outbreaks. However, RAPD analysis by arbitrarily primed PCR is faster and less laborious method of molecular typing.     

Evaluation of flow cytometric immunophenotyping and DNA analysis for detection of malignant cells in serosal cavity fluids

The serosal cavities are frequent sites of tumor metastasis. The distinction between carcinoma cells, inflammatory cells, and reactive or malignant mesothelial cells can be difficult in cytology. Multicolor flow cytometry (FCM) provides the opportunity to evaluate multiple antigens simultaneously, making it possible to characterize various cell populations. In this study, we aimed to assess the diagnostic accuracy of FCM immunophenotyping and DNA in comparison with serum tumor markers and classic cytology for detection of malignant cells in pleural and ascitic fluids. One hundred and nineteen samples of body cavity fluids were analyzed. Immunophenotyping was performed by four‐color immunofluorescent staining using monoclonal antibodies against Ber‐EP4, cytokeratin, CD3, and CD45. The DNA analysis by FCM was also performed. In addition, serum CA19‐9, CEA, AFP, and CA125 were analyzed. Ber‐EP4 marker had the highest sensitivity (73%) and specificity (95.5%) in the detection of carcinoma cells in serous fluid and correlated with cytology in most of cases (73%). The mean of DI differed statistically in patients with malignant effusions than in benign one. DI showed no difference in fluids due to infiltration of malignant epithelial cells or hematopoitic malignancy or due to hepatocellular carcinoma developing in cirrhotic liver. Thus, flow cytometry appears to aid not only in the detection of malignant cells but also in the characterization of cell type. On the other hand, although DNA ploidy examination had better sensitivity; it had no advantage over conventional cytopathological examination in identification of malignant cells. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

Development and immunophenotyping of the pharyngeal tonsil (adenoid) in cattle.

Immunoperoxidase staining and electron and light microscopy were used to characterize the development of the pharyngeal tonsil in 98 cattle aged between 30 days of gestation and 12 years. The rugae of the pharyngeal tonsil were poorly formed before 95 days of gestation. Microvillous (M) cells associated with intra-epithelial leucocytes (lympho-epithelium) were scattered among ciliated and goblet cells covering most of the surface in post-natal animals. Intra-epithelial leucocytes were rare in fetuses, but ciliated and M cells could be distinguished. Leucocytes of the lamina propria started to accumulate at approximately 120 days of gestation. A loose accumulation of mononuclear cells progressed into a B-cell rich upper and T-cell rich lower layer, with typical lymphoid tissue organization in post-natal animals and lymphoid involution in aged cattle. Primary lymphoid follicles formed at 5 months of gestation, but germinal centres did not form until 2 to 4 weeks after birth. Except for null cells, the relative number of cells staining for each leucocyte phenotype or MHC class II antigen increased with age, especially during the neonatal period. The early development, strategic location and specialized structure of the pharyngeal tonsil suggest an important role in modulating inhaled antigens in cattle. Fetal and neonatal calves had minimal lymphoid tissue priming, as indicated by lack of secondary follicles, low MHC class II expression and few intra-epithelial leucocytes. The phenotypic differences may be relevant to the increased susceptibility of calves to infectious diseases shortly after birth.     

Evaluation of carrier detection rates for Duchenne and Becker muscular dystrophies using serum creatine-kinase (CK) and pyruvate-kinase (PK) through discriminant analysis

Serum pyruvate-kinase (PK) and creatine-kinase (CK) determinations have been carried out in a sample of 100 obligate carriers for the Duchenne muscular dystrophy (DMD) gene, 23 obligate carriers for the Becker muscular dystrophy (BMD) gene, and 50 normal adult control women. Blood samples were collected from all subjects three times on three independent occasions and the means of these three determinations were considered for both PK and CK activities in the statistical analysis. Discriminant analysis has shown that, in the group of carriers for the DMD gene, the estimated misclassification frequencies (M.F.) using either serum CK, PK, or both enzymes were: 26.5% for CK alone, 19.5% for PK alone, and 19% for both enzymes. In the group of carriers for the BMD gene, the estimated proportions of M.F. were: 31.7% for CK alone, 23.8% for PK alone, and 20.4% for both enzymes. It is concluded that, although a proportion of carries still remains undetected, the use of serum PK determinations enhances the capability of detecting carriers of both DMD and BMD mainly when compared with serum CK alone.     

Analysis of DNA sequence variants detected by high-throughput sequencing

The Undiagnosed Diseases Program at the National Institutes of Health uses high-throughput sequencing (HTS) to diagnose rare and novel diseases. HTS techniques generate large numbers of DNA sequence variants, which must be analyzed and filtered to find candidates for disease causation. Despite the publication of an increasing number of successful exome-based projects, there has been little formal discussion of the analytic steps applied to HTS variant lists. We present the results of our experience with over 30 families for whom HTS sequencing was used in an attempt to find clinical diagnoses. For each family, exome sequence was augmented with high-density SNP-array data. We present a discussion of the theory and practical application of each analytic step and provide example data to illustrate our approach. The article is designed to provide an analytic roadmap for variant analysis, thereby enabling a wide range of researchers and clinical genetics practitioners to perform direct analysis of HTS data for their patients and projects.     

Feline leukaemia virus proviral DNA detected by polymerase chain reaction in antigenaemic but non-viraemic (‘discordant’) cats

Summary. A nested polymerase chain reaction (PCR) was established to detect exogenous feline leukaemia virus (FeLV) proviral DNA in feline peripheral blood leukocytes (PBL). The assay detected a single copy of plasmid DNA of an infectious molecular clone of FeLV subgroup A in the sample by ethidium bromide staining in agarose gels. The utility of the nested PCR in the diagnosis of FeLV infections was compared with the detection of FeLV p27 antigen by enzyme-linked immunosorbent assay (ELISA) and virus isolation (VI). FeLV genomes were detected by PCR in all 4 samples that were positive by ELISA and VI but in none of 7 samples that were negative by the two methods. FeLV genomes were found by PCR in 13 of 39 samples from cats that were antigenaemic but from which no virus was isolated (‘discordant’ cats). These results demonstrated that a proportion of discordant cats harboured FeLV genome in their PBL. Received June 29, 1996 Accepted August 29, 1996     

Familial pericentric inversion (13) detected by antenatal diagnosis.

A recombinant rec (13), dup q chromosome was diagnosed in a 17-week fetus following amniocentesis. Subsequently, a familial pericentric inversion of chromosome 13 was seen to be segregating in the family and the same recombinant 13 was present in a mentally retarded aunt of the fetus. The clinical features of the carriers of the inversion product are discussed with reference to previous cases.     

Critical analysis and diagnostic usefulness of limited immunophenotyping of B-cell non-Hodgkin lymphomas by flow cytometry

We report the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of various immunophenotypes characteristic of each class of B-cell non-Hodgkin lymphoma (NHL) based on analysis of 352 morphologically well-characterized B-cell NHLs and 175 benign lymph nodes (LNs) using 2-color flow cytometry. All B-cell NHLs that exhibited a characteristic immunophenotype (except diffuse large B-cell lymphoma) had a high NPV. The immunophenotypes of small lymphocytic lymphoma and mantle cell lymphoma showed high specificity, but only small lymphocytic lymphoma also showed a high PPV. One third of follicular lymphomas coexpressed CD23 and CD10. Diffuse large B-cell NHL showed no consistent immunophenotype. About 90% of all benign LNs expressed no substantial amounts of CD5, CD10, or CD23. Most benign LNs also failed to express substantial amounts of immunoglobulin heavy chains. In contrast, about 90% of NHLs showed expression of 1 or 2 heavy chains. The expression pattern of immunoglobulin light chains was not found helpful in favoring one lymphoma type over another. The usefulness of each immunophenotype for each lymphoma group is of particular diagnostic importance in limited specimens, such as fine-needle aspiration biopsies, small core biopsies, body effusions, extranodal sites, and nodal tissues with various artifacts.     

Extensive conservation of genomic imbalances in canine transmissible venereal tumors (CTVT) detected by microarray-based CGH analysis

Canine transmissible venereal tumor (CTVT) is an intriguing cancer that is transmitted naturally as an allograft by transplantation of viable tumor cells from affected to susceptible dogs. At least initially, the tumor is able to evade the host's immune response; thus, CTVT has potential to provide novel insights into tumor immunobiology. The nature of CTVT as a “contagious” cancer, originating from a common ancestral source of infection, has been demonstrated previously by a series of studies comparing geographically distinct tumors at the molecular level. While these studies have revealed that apparently unrelated tumors share a striking degree of karyotypic conservation, technological restraints have limited the ability to investigate the chromosome composition of CTVTs in any detail. We present characterization of a strategically selected panel of CTVT cases using microarray-based comparative genomic hybridization analysis at ~one-megabase resolution. These data show for the first time that the tumor presents with an extensive range of non-random chromosome copy number aberrations that are distributed widely throughout the dog genome. The majority of abnormalities detected were imbalances of small subchromosomal regions, often involving centromeric and telomeric sequences. All cases also showed the sex chromosome complement XO. There was remarkable conservation in the cytogenetic profiles of the tumors analyzed, with only minor variation observed between different cases. These data suggest that the CTVT genome demonstrates a vast degree of both structural and numerical reorganization that is maintained during transmission among the domestic dog population.     

Presymptomatic diagnosis of SMA III by genotype analysis

Linkage analysis and prenatal prediction in families segregating autosomal recessive spinal muscular atrophy (SMA) has become feasible since the assignment of the locus responsible for type I-III SMA to region 5q12-q13.3. We have performed a segregation study of SMA in Italian families using molecular probes and highly informative PCR-based polymorphic markers. In one family, a 7-year-old boy affected with type III SMA and an 8-year-old apparently healthy brother had identical haplotypes. These findings prompted us to reexamine the apparently unaffected child. His neurological exam was normal. However, the electromyography (EMG) showed a pattern consistent with chronic SMA. To our knowledge this is the first example of presymptomatic diagnosis of SMA based on genotype analysis. © 1993 Wiley-Liss, Inc.     

Evidence of two genetic entities inBothriocephalus funiculus (Cestoda) detected by arbitrary-primer polymerase chain reaction random amplified polymorphic DNA fingerprinting

The genetic diversity of two samples of Cestoda (Bothriocephalus funiculus, Renaud and Gabrion, 1984) parasitizing two sympatric teleostean species was assessed using random amplified polymorphic DNA (RAPD). A total of 72Bothriocephalus were analyzed individually, and electrophoretic analysis of the amplification products of 65 primers among the 68 tested revealed monomorphic patterns, reflecting the close genetic relatedness within and between the parasites of the two samples. However, 3 primers showed polymorphic patterns at 6 RAPD sites. Analysis of the distribution of these genomic fragments, assuming random mating, showed strong linkage disequilibria (only 8 genetic combinations were observed among the 32 expected). Two genetic entities displaying a high degree of host specificity were evidence within our two samples ofB. funiculus. This powerful molecular technique can be used as a diagnostic tool in studies concerning the biodiversity of related genetic entities and could have broad applications in parasitology.     

A genotype distribution of human papillomaviruses detected by polymerase chain reaction and direct sequencing analysis in a large sample of common warts in Japan

There have been no large-scale epidemiological studies of human papillomavirus (HPV) genotype distribution of common warts in Japan. A total of 213 patients with common warts (104 males and 109 females) in Japan were studied to detect HPV genotype distribution by polymerase chain reaction (PCR) and direct sequencing analysis. The results were as follows: 94 HPV-1a (44.1%), 35 HPV-4 (16.4%), 30 HPV-65 (14.1%), 13 HPV-27 (6.1%), 13 HPV-2a (6.1%), 9 HPV-57b (4.22%), 3 HPV-16 (1.41%), 2 HPV-6a (0.94%), 2 HPV-63 (0.94%), and 1 case for each of HPV-3, -5, -5b, -7, -10, -21, -29, -47, -56, -57, -62, and -92 (0.47%, respectively). Four cases (1.88%) were found in which two different HPV types were detected within the lesions: one case of HPV-1a with HPV-16, one case of HPV-1a with HPV-65, one case of HPV-6a with HPV-8, and one case of HPV-65 with HPV-16. There were seven cases of mucosal types (3.3%), that is, two HPV-6a, three HPV-16, one HPV-56, and one HPV-62, and three cases of epidermodysplasia verruciformis (EV)-related types (1.41%), that is, one HPV-5, one HPV-5b (both of which belonged to a high-risk group), and one HPV-47 (which belonged to a low-risk group). To date, this is the largest sequencing-based study of HPV for common warts in Japan. It is said that common warts are induced predominantly by HPV-2, -27, and -57 in European population. However, the present results showed that in Japan they were induced mostly by HPV-1, -4, and -65. This suggests that regional differences in HPV genotype distribution may exist between European and Japanese populations.