Fecal specimens preparation methods for PCR diagnosis of human taeniosis

Sample preparation and DNA extraction protocols for DNA amplification by PCR, which can be applied in human fecal samples for taeniasis diagnosis, are described. DNA extracted from fecal specimens with phenol/chloroform/isoamilic alcohol and DNAzol® reagent had to be first purified to generate fragments of 170 pb and 600 pb by HDP2-PCR. This purification step was not necessary with the use of QIAmp DNA stool mini kit®. Best DNA extraction results were achieved after eggs disruption with glass beads, either with phenol/chloroform/isoamilic alcohol, DNAzol® reagent or QIAmp DNA stool mini kit®.

Classic and molecular study of Giardia duodenalis in children from a daycare center in the region of Presidente Prudente, São Paulo, Brazil

Epidemiological studies on giardiasis by using molecular techniques such as RAPD (Randomly Amplified Polymorphic DNA) may give information on factors related to the transmission of Giardia duodenalis. The aim of this work was to assess the epidemiology of G. duodenalis in 101 children attended at a daycare center in Presidente Bernardes, SP, Brazil. After parasitological examinations in feces samples, 15 children presented cysts of G. duodenalis. Their respective parents, brothers and pets, besides the daycare center workers, also had their feces submitted to parasitological analysis. Seven mothers and nine brothers also presented G. duodenalis cysts, while fathers, daycare workers and pets (dogs) did not presented the parasite. Besides the 15 cases with G. duodenalis, other 23 children presented other enteroparasites (Entamoeba coli, Endolimax nana, Enterobius vermicularis, Ascaris lumbricoides and Trichuris trichiura). Samples of G. duodenalis cysts from children and their relatives were submitted to molecular typing by RAPD after genomic DNA extraction and amplification of a fragment of the 18S rDNA region by PCR. After examining 31 isolates of G. duodenalis (children and their respective mothers and brothers), it was concluded that the parasite transmission occurred in children, probably during daily cohabitation at the daycare center, but not at home among their relatives or pets.

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

Evaluation of pathogenic fungi occurrence in traumatogenic structures of freshwater fish

INTRODUCTION: Fungal infections in human skin, such as sporotrichosis, can occur after fish induced trauma. This work aimed to identify fungi in freshwater fish that are pathogenic to humans. METHODS: Extraction of dental arches from Serrassalmus maculatus (piranha) and Hoplias malabaricus (wolf fish), stings from Pimelodus maculatus (mandis catfish), dorsal fin rays from Plagioscion spp. (corvina) and Tilapia spp., for culture in Mycosel agar. Some cultures were submitted to DNA extraction for molecular identification by sequencing ITS-5.8S rDNA. RESULTS: Cultures identified most yeast as Candida spp., while sequencing also permitted the identification of Phoma spp. and Yarrowia lipolytica. CONCLUSIONS: While the search for S. schenckii was negative, the presence of fungus of the genera Phoma and Candida revealed the pathogenic potential of this infection route. The genus Phoma is involved in certain forms of phaeohyphomycosis, a subcutaneous mycosis caused by dematiaceous fungi, with reports of infections in human organs and systems. Traumatizing structures of some freshwater fish present pathogenic fungi and this may be an important infection route that must be considered in some regions of Brazil, since there are a large number of a fisherman in constant contact with traumatogenic fish.

cagE as a biomarker of the pathogenicity of Helicobacter pylori

IntroductionHelicobacter pylori infection is associated with gastro-duodenal diseases. Genes related to pathogenicity have been described for H. pylori and some of them appear to be associated with more severe clinical outcomes of the infection. The present study investigates the role of cagE as a pathogenicity biomarker of H. pylori compare it to cagA, vacA, iceA and babA2 genes and correlate with endoscopic diagnoses.MethodsWere collected biopsy samples of 144 dyspeptic patients at the Hospital of the Federal University of Rio Grande, Rio Grande do Sul, Brazil. After collection, the samples were sent for histological examination, DNA extraction and detection of all putative pathogenicity genes by PCR.ResultsOf the 144 patients undergoing endoscopy, 57 (39.6%) presented H. pylori by histological examination and PCR by detection of the ureA gene. Based on the endoscopic diagnoses, 45.6% (26/57) of the patients had erosive gastritis, while 54.4% (31/57) had enanthematous gastritis. The genes cagA, cagE, vacAs1/m1, vacAs1/m2 and iceA1 were related to erosive gastritis, while the genes vacAs2/m2, iceA2 and babA2 were associated to enanthematous gastritis. We found a statistically significant association between the presence of cagE and the endoscopic diagnosis. However, we detect no statistically significant association between the endoscopic diagnosis and the presence of cagA, vacA, iceA and babA2, although a biological association has been suggested.ConclusionsThus, cagE could be a risk biomarker for gastric lesions and may contribute to a better evaluation of the H. pylori pathogenic potential and to the prognosis of infection evolution in the gastric mucosa.

Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil

Introduction Cryptosporidium is an important protozoan cause of waterborne disease worldwide of concern to public health authorities. To prevent outbreaks of cryptosporidiosis, the monitoring of this parasite in drinking water is necessary. In the present work, the polymerase chain reaction (PCR) and nested-PCR techniques were used to detect Cryptosporidium in raw water from catchment points of four water treatment plants (WTP) in Curitiba, Paraná, Brazil. Methods First, DNA extraction techniques were tested in samples containing decreasing amount of oocysts in reagent water, and PCR and nested-PCR with specific primers for 18SSU rDNA of Cryptosporidium were conducted to determine their sensitivity. In reagent water, a commercial extraction kit provided the best analytical sensitivity, and PCR and nested-PCR allowed the detection of five and two oocysts, respectively, with the primers XIAOR/XIAOF and XIAO1F/XIAO2R. Results In the spiking experiments, only the PCR with the primers AWA995F/AWA1206R was successful at detecting concentrations of 0.1 oocysts/mL. Two catchments samples of raw water and/or water sludge from four WTPs were contaminated with Cryptosporidium. Conclusions The application of the techniques to monitor Cryptosporidium in water and detect contamination in water catchments of WTPs in Curitiba are discussed in the present work.

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (∞), NLR (0.017), and Ef (99%).

Molecular tools in the diagnostic and epidemiology of infections caused by members of Mycobacterium avium Complex

Mycobacterium avium Complex (MAC) comprises microorganisms that affect a wide range of animals including humans. The most relevant are Mycobacterium avium subspecies hominissuis (Mah) with a high impact on public health affecting mainly immunocompromised individuals and Mycobacterium avium subspecies paratuberculosis (Map) causing paratuberculosis in animals with a high economic impact worldwide. In this work, we characterized 28 human and 67 porcine Mah isolates and evaluated the relationship among them by Multiple-Locus Variable number tandem repeat Analysis (MLVA). We concluded that Mah population presented a high genetic diversity and no correlations were inferred based on geographical origin, host or biological sample. For the first time in Portugal Map strains, from asymptomatic bovine faecal samples were isolated highlighting the need of more reliable and rapid diagnostic methods for Map direct detection. Therefore, we developed an IS900 nested real time PCR with high sensitivity and specificity associated with optimized DNA extraction methodologies for faecal and milk samples. We detected 83% of 155 faecal samples from goats, cattle and sheep, and 26% of 98 milk samples from cattle, positive for Map IS900 nested real time PCR. A novel SNPs (single nucleotide polymorphisms) assay to Map characterization based on a Whole Genome Sequencing analysis was developed to elucidate the genetic relationship between strains. Based on sequential detection of 14 SNPs and on a decision tree we were able to differentiate 14 phylogenetic groups with a higher discriminatory power compared to other typing methods. A pigmented Map strain was isolated and characterized evidencing for the first time to our knowledge the existence of pigmented Type C strains. With this work, we intended to improve the ante mortem direct molecular detection of Map, to conscientiously aware for the existence of Map animal infections widespread in Portugal and to contribute to the improvement of Map and Mah epidemiological studies.

Investigación de defectos genéticos en el sistema de la fosforilasa hepática en pacientes argentinos. Definición nosológica mediante una estrategia de análisis molecular. Investigation of genetic defects in the hepatic phosphorylase system in Argentinean patients. Nosological definition by molecular analysis strategy.

Las Enfermedades de Atesoramiento de Glucógeno (EAGs) también llamadas Glucogenosis comprenden un grupo de entidades causadas por una deficiencia enzimática específica relacionada con la vía de síntesis o degradación de esta macromolécula. La heterogeneidad fenotípica de los pacientes afectados dificulta la identificación de las diferentes variantes de EAG y por ende la correcta definición nosológica. En el Centro de Estudio de las Metabolopatías Congénitas, CEMECO, se fueron definiendo los diferentes tipos de Glucogenosis a través de una estrategia multidisciplinaria que integra distintos niveles de investigación clínica y complementaria, laboratorio metabólico especializado, enzimático, histomorfológico y de análisis molecular. Sin embargo, en algunos enfermos, entre los que se encuentran aquellos con defectos en el sistema de la fosforilasa hepática (EAG-VI y EAG-IX), la exacta definición nosológica aún no está resulta. La EAG-VI se refiere a un defecto en la fosforilasa hepática, enzima codificada por el gen PYGL, mientras que la EAG-IX es causada por un defecto genético en una de las subunidades de la fosforilasa b quinasa hepática codicadas por los genes PHKA2, PHKB y PHKG2, respectivamente. El objetivo del presente trabajo es propender a la definición nosológica de pacientes con defectos en el sistema de la fosforilasa mediante una estrategia de análisis molecular investigando los genes PYGL, PHKA2, PHKB y PHKG2. Los pacientes incluidos en este estudio deberán ser compatibles de padecer una EAG-VI o EAG-IX sobre la base de síntomas clínicos y hallazgos bioquímicos. La metodología incluirá la determinación de la enzima fosforilasa b quinasa en glóbulos rojos y dentro del análisis molecular la extracción de DNA genómico a partir de sangre entera para la amplificación por PCR de los exones más las uniones exon/intron de los genes PHKG2 y PYGL y la extracción de RNA total y obtención de cDNA para posterior amplificación de los cDNA PHKA2 y PHKB. Todos los fragmentos amplificados serán sometidos a análisis de secuencia de nucleótidos. Resultados esperados. Este trabajo, primero en Argentina, permitirá establecer las bases moleculares de los defectos del sistema de la fosforilasa hepática (EAG-VI y EAG-IX). El poder lograr este nivel de investigación traerá aparejado, una oferta integrativa en el vasto capítulo de las glucogenosis hepáticas, con extraordinaria significación en la práctica asistencial para el manejo, pronóstico y correspondiente asesoramiento genético. Hepatic glycogen storage diseases (GSDs) are a group of disorders produced by a deficiency in a specific protein involved in the metabolism of glycogen causing different types of GSDs. Phenotypic heterogeneity of affected patients difficult to identify the different GSD variants and therefore the correct definition of the disease. In the “Centro de Estudio de las Metabolopatías Congénitas”, CEMECO, were defined the different GSD types by a protocol which included complex gradual levels of clinical, biochemical, enzymatic and morphological investigation. However, in some patients, like those one with defects in the hepatic phosphorylase system (GSD-VI and GSD-IX) the exact definition of the disease has not yet been resolved. The GSD-VI is produced by a defect in the PYGL gen that encode the liver phosphorylase, while the GSD-IX is caused by a genetic defect in one of the Phosphorylase b kinase subunits, encoded by the PHKA2, PHKB and PHKG2 genes, respectively. The aim of the present study is to define the phosphorylase system defects in argentinian patients through a molecular strategy that involve the investigation of PYGL, PHKA2, PHKB and PHKG2 genes. Patients included in the present study must be compatible with a GSD-VI or GSD-IX on the bases of clinical symptoms and biochemical findings. The phosphorylase b kinase activity will be assay on in blood red cells. The molecular study will include genomic DNA extraction for the amplification of PHKG2 and PYGL genes and the total RNA extraction for amplification of the PHKA2 and PHKB cDNA by PCR. All PCR-amplified fragments will be subjected to direct nucleotide sequencing. This work, first in Argentina, will make possible to establish the molecular basis of the defects on the hepatic phosphorylase system (GSD-VI and GSD IX). To achieve this level of research will entail advance in the study of the hepatic glycogen storage disease, with extraordinary significance in the treatment, prognosis and the genetic counselling.

Prevalence of cagA and vacA genes in isolates from patients with Helicobacter pylori-associated gastroduodenal diseases in Recife, Pernambuco, Brazil

Geographical differences in the prevalence of Helicobacter pylori genes and their association with disease severity have been identified. This study analyzes the prevalences of the cagA gene and alleles of the vacA gene in H. pylori-associated gastroduodenal diseases in isolates from Recife, PE, Brazil. Gastric biopsy of 61 H. pylori-positive patients were submitted to DNA extraction and gene amplification by polymerase chain reaction. Among the 61 patients, 21 suffered from duodenal ulcer (DU) and 40 from gastritis (GT). The prevalence of H. pylori strains harbouring the cagA gene was higher in the DU group (90.5%) than in the GT group (60%) (p = 0.02). The vacA gene was amplified in 56 out of 61 biopsies, of which 43 (76.8%) contained bacteria carrying the s1 allele and 13 (23.2%) the s2. However, the prevalence of the vacA s1 genotying was the same in either DU or GT group. The majority of the s1-typed strains, 39 (90.7%) out of 43, were subtype s1b. In resume there was a strong association between the H. pylori cagA+ gene and DU. However, there were no differences between the DU and GT groups in relation to the vacA s1 and s2 alleles distribution, albeit the subtype s1b was predominat.

First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): new perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.

Experimental reinfection of BALB/c mice with different recombinant type I/III strains of Toxoplasma gondii: involvement of IFN-³ and IL-10

To assess reinfection of BALB/c mice with different Toxoplasma gondii strains, the animals were prime infected with the non-virulent D8 strain and challenged with virulent recombinant strains. Thirty days after challenge, brain cysts were obtained from surviving BALB/c mice and inoculated in Swiss mice to obtain tachyzoites for DNA extraction and PCR-RFLP analysis to distinguish the different T. gondii strains present in possible co-infections. Anti-Toxoplasma immune responses were evaluated in D8-primed BALB/c mice by detecting IFN-³ and IL-10 produced by T cells and measuring immunoglobulin levels in serum samples. PCR-RFLP demonstrated that BALB/c mice were reinfected with the EGS strain at 45 days post prime infection (dpi) and with the EGS and CH3 strains at 180 dpi. High levels of IFN-³ were detected after D8 infection, with no significant difference between 45 and 180-day intervals. However, higher IL-10 levels and higher plasmatic IgG1 and IgA were detected from samples obtained 180 days after infection. BALB/c mice were susceptible to reinfection with different recombinant T. gondii strains and this susceptibility correlated with enhancement of IL-10 production.

Helicobacter pylori cagA and vacA genotypes in Cuban and Venezuelan populations

The aim of this study was to determine the presence of Helicobacter pylori cytotoxin-associated gene (cagA)/vacuolating cytotoxin gene (vacA) among patients with chronic gastritis in Cuba and Venezuela. Gastric antrum biopsies were taken for culture, DNA extraction and PCR analysis. Amplification of vacA and cagA segments was performed using two regions of cagA: 349 bp were amplified with the F1/B1 primers and the remaining 335 bp were amplified with the B7629/B7628 primers. The VA1-F/VA1-R set of primers was used to amplify the 259-bp (s1) or 286-bp (s2) product and the VAG-R/VAG-F set of primers was used to amplify the 567-bp (m1) or 642-bp (m2) regions of vacA. cagA was detected in 87% of the antral samples from Cuban patients and 80.3% of those from Venezuelan patients. All possible combinations of vacA regions were found, with the exception of s2/m1. The predominant combination found in both countries was s1/m1. The percentage of cagA+ strains was increased by the use of a second set of primers and a greater number of strains was amplified with the B7629/B7628 primers in the Cuban patients (p = 0.0001). There was no significant difference between the presence of the allelic variants of vacA and cagA in both populations. The predominant genotype was cagA+/s1m1 in both countries. The results support the necessary investigation of isolates circulating among the human population in each region.

Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2% (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85% and 98%, and 94% and 100%, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.

JC polyomavirus infection in candidates for kidney transplantation living in the Brazilian Amazon Region

This study evaluated the relative occurrences of BK virus (BKV) and JC virus (JCV) infections in patients with chronic kidney disease (CKD). Urine samples were analysed from CKD patients and from 99 patients without CKD as a control. A total of 100 urine samples were analysed from the experimental (CKD patients) group and 99 from the control group. Following DNA extraction, polymerase chain reaction (PCR) was used to amplify a 173 bp region of the gene encoding the T antigen of the BKV and JCV. JCV and BKV infections were differentiated based on the enzymatic digestion of the amplified products using BamHI endonuclease. The results indicated that none of the patients in either group was infected with the BKV, whereas 11.1% (11/99) of the control group subjects and 4% (4/100) of the kidney patients were infected with the JCV. High levels of urea in the excreted urine, low urinary cellularity, reduced bladder washout and a delay in analysing the samples may have contributed to the low prevalence of infection. The results indicate that there is a need to increase the sensitivity of assays used to detect viruses in patients with CDK, especially given that polyomavirus infections, especially BKV, can lead to a loss of kidney function following transplantation.