Molecular Epidemiological Survey Research Articles

Simultaneous detection of eight swine reproductive and respiratory pathogens using a novel GeXP analyser-based multiplex PCR assay

A new high-throughput GenomeLab Gene Expression Profiler (GeXP) analyser-based multiplex PCR assay was developed for the detection of eight reproductive and respiratory pathogens in swine. The reproductive and respiratory pathogens include North American porcine reproductive and respiratory syndrome virus (PRRSV-NA), classical swine fever virus (CSFV), porcine circovirus 2 (PCV-2), swine influenza virus (SIV) (including H1 and H3 subtypes), porcine parvovirus (PPV), pseudorabies virus (PRV) and Japanese encephalitis virus (JEV). Nine pairs of specific chimeric primers were designed and used to initiate PCRs, and one pair of universal primers was used for subsequent PCR cycles. The specificity of the GeXP assay was examined using positive controls for each virus. The sensitivity was evaluated using serial ten-fold dilutions of in vitro-transcribed RNA from all of the RNA viruses and plasmids from DNA viruses. The GeXP assay was further evaluated using 114 clinical specimens and was compared with real-time PCR/single RT-PCR methods. The specificity of the GeXP assay for each pathogen was examined using single cDNA/DNA template. Specific amplification peaks of the reproductive and respiratory pathogens were observed on the GeXP analyser. The minimum copies per reaction detected for each virus by the GeXP assay were as follows: 1000 copies/μl for PRV; 100 copies/μl for CSFV, JEV, PCV-2 and PPV; and 10 copies/μl for SIV-H1, SIV-H3 and PRRSV-NA. Analysis of 114 clinical samples using the GeXP assay demonstrated that the GeXP assay had comparable detection to real-time PCR/single RT-PCR. This study demonstrated that the GeXP assay is a new method with high sensitivity and specificity for the identification of these swine reproductive and respiratory pathogens. The GeXP assay may be adopted for molecular epidemiological surveys of these reproductive and respiratory pathogens in swine populations.

Molecular epidemiological survey and genetic analysis of vector-borne infections of cattle in Luzon Island, the Philippines

In the Philippines, vector-borne disease is one of the important problems in the livestock industry. To elucidate the epidemiology of vector-borne diseases in cattle on Luzon Island, the Philippines, the prevalence of five protozoan agents was assessed by polymerase chain reaction. Out of the 339 samples, 324 (95.5%), 154 (45.4%), 209 (61.6%), 140 (41.3%), and 2 (0.6%) were positive for Anaplasma marginale, Babesia bigemina, Babesia bovis, Theileria spp., and Trypanosoma evansi infections, respectively. Mixed infections were detected in 290 (85.5%) samples, of which 115 (33.9%) had two pathogens, 144 (42.5%) had three pathogens, and 31 (9.1%) had four kinds of pathogens. 16S rRNA gene was 100% identical in A. marginale compared with the same lineage across the world. B. bovis RAP-1 and B. bigemina AMA-1 genes were identical with 92.27%–100% and 97.07%–100% sequences, respectively, in the database (Asian isolates). MPSP genes of Theileria spp. were 83.51%–100% identical with the one another. Phylogenetic analysis showed that they belong to the groups of T. sergenti and T. buffeli. Positive rates of the tick-borne pathogens were extremely high in this area. These findings provide vital information that can be used for the planning and execution of effective control measures for vector-borne diseases in the Philippine cattle industry.

Comparative genomics to investigate the emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 clone in Geneva, Switzerland

Molecular epidemiological surveys of CA-MRSA revealed a wide diversity of genetic backgrounds with only sporadic identification of USA300 isolates during the period 1994-2012.

Application of hexon gene sequencing of adenovirus in pathogen detection of epidemic keratoconjunctivitis

Background Epidemic keratoconjunctivitis is a common eye disease, and adenovirus is one of the common pathogens.The hexon protein, one main capsid protein of the virus, is an important target of antibody binding.Thus, sequencing the coding region of the hexon protein is an important way for adenovirus fast typing. Objective This study was to complete a molecular epidemiology survey of epidemic keratoconjunctivitis and investigate its association with adenovirus in Shanghai area by sequencing the coding region of hexon protein. Methods Two hundred and fourteen sacconjunctival swab specimens were collected from 214 patients with suspicious epidemic keratoconjunctivitis who visited Shanghai Eye Disease Prevention and Treatment Center and the clinical sites supervised by the Shanghai Prevention and Monitoring Office of Acute Hemerragic Conjunctivitis under the informed consent from January 2010 to December 2012.DNA was extracted from the specimens and then the 140 bp conserved sequence in hexon protein coding region was amplified by PCR initially to determine an adenovirus pathogen.Furtherly, 956 bp conserved sequence of the hexon codind district was sequencied to clarify the serotype of adenovirus in the adenovirus-positive specimens. Results 50.93% patients (109/214) were detected to be adenovirus-positive by generic PCR, in which AdV1+ was in 4 patiens, AdV2+ was in 33 patients, AdV3+ was in 15 patients, AdV4+ was in 12 patients, AdV8+ was in 19 patiens, AdV19+ was in 15 patients, AdV37+ was in 8 patients.The subgenus D adenoviruses, including AdV8+ , AdV19+ and AdV37+ often resulted in corneal inflammation, pseudomembranous conjunctivitis and preauricular lymph nodes; while subgenus B adenovirus induced much frequent tract infection and less corneal response. Conclusions PCR-sequence of conserved region of hexon protein coding district is applicable for the detection and serotyping of adenovirus in epidemic keratoconjunctivitis. Key words: Adenovirus infections, human; Keratoconjunctivitis, viral/diagnosis; Base sequence; Conserved sequence; PCR/methods; Hexon

Aggressive Emerging Pathovars of Xanthomonas arboricola Represent Widespread Epidemic Clones Distinct from Poorly Pathogenic Strains, as Revealed by Multilocus Sequence Typing.

Deep and comprehensive knowledge of the genetic structure of pathogenic species is the cornerstone on which the design of precise molecular diagnostic tools is built. Xanthomonas arboricola is divided into pathovars, some of which are classified as quarantine organisms in many countries and are responsible for diseases on nut and stone fruit trees that have emerged worldwide. Recent taxonomic studies of the genus Xanthomonas showed that strains isolated from other hosts should be classified in X. arboricola, extending the host range of the species. To investigate the genetic structure of X. arboricola and the genetic relationships between highly pathogenic strains and strains apparently not relevant to plant health, we conducted multilocus sequence analyses on a collection of strains representative of the known diversity of the species. Most of the pathovars were clustered in separate monophyletic groups. The pathovars pruni, corylina, and juglandis, responsible for pandemics in specific hosts, were highly phylogenetically related and clustered in three distinct clonal complexes. In contrast, strains with no or uncertain pathogenicity were represented by numerous unrelated singletons scattered in the phylogenic tree. Depending on the pathovar, intra- and interspecies recombination played contrasting roles in generating nucleotide polymorphism. This work provides a population genetics framework for molecular epidemiological surveys of emerging plant pathogens within X. arboricola. Based on our results, we propose to reclassify three former pathovars of Xanthomonas campestris as X. arboricola pv. arracaciae comb. nov., X. arboricola pv. guizotiae comb. nov., and X. arboricola pv. zantedeschiae comb. nov. An emended description of X. arboricola Vauterin et al. 1995 is provided.

Fishing area and fish size as risk factors of Anisakis infection in sardines (Sardina pilchardus) from Iberian waters, southwestern Europe

The sardine (Sardina pilchardus) is a fish commonly consumed and appreciated in many countries, although they are more likely to be eaten fresh in western Mediterranean countries such as Spain, Portugal, France or Italy. A molecular epidemiological survey of sardines from 5 fishing areas of the Spanish Mediterranean (Málaga, southern Spain) and Atlantic coasts (southern: Cádiz and Isla Cristina; northern: A Coruña and Ondarroa) was carried out to determine the presence of Anisakis spp. larvae. The highest prevalence of these larvae was observed in fish from A Coruña (28.3%), followed by Ondarroa (5%) and Cádiz (2.5%). No Anisakis larvae were found in fish from Málaga and Isla Cristina. Three Anisakis genotypes were identified: Anisakis simplex sensu stricto, Anisakis pegreffii and a hybrid genotype between these two species. A. pegreffii was the most prevalent species in A Coruña (71% of larvae). Only three Anisakis larvae (9% collected larvae) were located in the musculature of sardines: two were identified as A. pegreffii while the other was a hybrid genotype. Sardine infection was associated with fishing area and fish length/weight (length and weight were strongly correlated; Pearson's correlation 0.82; p<0.001). Risk factor multivariate analysis showed that the risk of infection increases 1.6 times for every additional cm in the length of the sardines from the same fishing area. Comparison of fish of equal length showed that in sardines from A Coruña the risk of parasitization is 11.5 times higher than in those from other fishing areas. Although the risk of infection by Anisakis through consumption of sardines is generally low due to the low epidemiological parameter values (prevalence 10%, mean intensity 1.7 (range 1–5) and mean abundance 0.17), as larger fish are more heavily parasitized, there is an increased risk of infection by Anisakis through consumption of large sardines which are raw or have undergone insufficient treatment (undercooked, smoked, marinated, salted, pickled, freezing,…).

Molecular epidemiological survey of the quinolone- and carbapenem-resistant genotype and its association with the type III secretion system in Pseudomonas aeruginosa.

This study evaluated the predictors of mortality and the impact of inappropriate therapy on the outcomes of patients with bacteraemia and ventilator-associated pneumonia (VAP). Additionally, we evaluated the correlation of the type III secretion system (TTSS) effector genotype with resistance to carbapenems and fluoroquinolones, mutations in the quinolone resistance-determining regions (QRDRs), metallo-β-lactamase and virulence factors. A retrospective cohort was conducted at a tertiary hospital in patients with multidrug-resistant (MDR) P. aeruginosa bacteraemia (157 patients) and VAP (60 patients). The genes for blaIMP, blaVIM, blaSIM, blaGIM and blaSPM and virulence genes (exoT, exoS, exoY, exoU, lasB, algD and toxA) were detected; sequencing was conducted for QRDR genes on fluoroquinolone-resistant strains. The multivariate analyses showed that the predictors independently associated with death in patients with bacteraemia were cancer and inappropriate therapy. Carbapenem resistance was more frequent among strains causing VAP (53.3 %), and in blood we observed the blaSPM genotype (66.6 %) and blaVIM genotype (33.3 %). The exoS gene was found in all isolates, whilst the frequency was low for exoU (9.4 %). Substitution of threonine to isoleucine at position 83 in gyrA was the most frequent mutation among fluoroquinolone-resistant strains. Our study showed a mutation at position 91 in the parC gene (Glu91Lys) associated with a mutation in gyrA (Thre83Ile) in a strain of extensively drug-resistant P. aeruginosa, with the exoT(+)exoS(+)exoU(+) genotype, that has not yet been described in Brazil to the best of our knowledge. This comprehensive analysis of resistance mechanisms to carbapenem and fluoroquinolones and their association with TTSS virulence genes, covering MDR P. aeruginosa in Brazil, is the largest reported to date.

Molecular detection of Anaplasma platys and Ehrlichia canis in dogs from Kabylie, Algeria

Ehrlichia canis and Anaplasma platys are bacteria belonging to the Anaplasmataceae family that cause acute, self-limiting and sometimes fatal vector-borne infections in dogs. These bacteria have been reported worldwide and are transmitted mainly by Rhipicephalus sanguineus. Aside from a report on E. canis once in 1935, no other Anaplasmataceae bacteria have been reported in Algeria to date. The aim of this study was to identify the microbial species implicated in ehrlichiosis and anaplasmosis by a molecular epidemiological survey in dogs. The study was carried out in Kabylie, in northeast Algeria. Sampling was performed in 11 municipalities in the province of Tizi Ouzou and 2 municipalities in the province of Béjaïa. Peripheral blood samples from 110 dogs were screened by qPCR, which is capable of identifying most Anaplasmataceae bacteria. Out of 110, a total of 13 samples screened positive (7/110 E. canis and 6/110 A. platys), and two genetic variants of A. platys and one of E. canis were identified. This is the first study to report the presence of A. platys in dogs from Algeria using a molecular investigative method. This survey was conducted in early spring. As tick activity can affect the prevalence of these pathogens in dogs, further investigations are needed to establish the year-round prevalence of these infections.

A molecular epidemiological survey of Babesia, Hepatozoon, Ehrlichia and Anaplasma infections of dogs in Japan.

Tick-borne diseases are often encountered in canine clinical practice. In the present study, a molecular epidemiological survey of dogs in Japan was conducted to understand the prevalence and geographical distribution of Babesia spp., Hepatozoon spp., Ehrlichia spp. and Anaplasma spp. Pathogen-derived DNA in blood samples obtained from 722 dogs with a history of exposure to ticks and/or fleas was examined by PCR. The prevalence of Babesia gibsoni, Babesia odocoilei-like species, Hepatozoon canis and Ehrlichia spp./Anaplasma spp. was 2.4% (16/722), 0.1% (1/722), 2.5% (18/722) and 1.5% (11/722), respectively. While B. gibsoni and Ehrlichia spp./Anaplasma spp. were detected in the western part of Japan, H. canis was detected in Tohoku area in addition to western and central parts of Japan.

Multilocus sequence typing indicates diverse origins of invasive Candida tropicalis isolates in China

Background According to data from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET) 2010, Candida tropicalis (C. tropicalis) is the third most common pathogen causing invasive candidiasis. Moreover, the majority of fluconazole-resistant C. tropicalis isolates were from a single hospital. Therefore, a molecular epidemiological survey is necessary to investigate the genetic relatedness of C. tropicalis isolates in China. Methods In this study, 48 C. tropicalis isolates causing invasive fungal infections from four tertiary hospitals in China were studied. All the isolates were identified by sequencing the internal transcribed spacer region. Antifungal susceptibility to triazoles, amphotericin B, and caspofungin was determined by the Clinical and Laboratory Standards Institute standard broth microdilution method. Multilocus sequence typing (MLST) was performed, and phylogenetic analysis was further performed by the eBURST and maximum parsimony (MP) methods to characterize the genetic relatedness of isolates. Results MLST discriminated 40 diploid sequence types (DSTs) among 48 isolates, including 36 novel DSTs, and the XYR1 gene showed the highest discriminatory power. The DSTs obtained from this study were compared with those of previously reported C. tropicalis isolates, and there was poor type alignment with regional strains. Nine groups and 11 singletons were identified by eBURST, whereas two groups and 10 subgroups were clustered by MP analysis. Generally, there were no obvious correlations between clonal clusters generated and the specimen source or hospital origin. Seven fluconazole-resistant isolates were confirmed and assigned to three distinguishable branches. Conclusions The results suggested diverse origins of invasive C. tropicalis isolates in China. Although most invasive C. tropicalis strains in the mainland of China were clustered with previously characterized Asian isolates, major C. tropicalis clusters identified in this study were genetically distinct from those of other geographic regions.

Prevalence and risk factor assessment of Tropheryma whipplei in a rural community in Gabon: a community-based cross-sectional study

Tropheryma whipplei is the causative agent of Whipple's disease and has been detected in stools of asymptomatic carriers. Colonization has been associated with precarious hygienic conditions. There is a lack of knowledge about the epidemiology and transmission characteristics on a population level, so the aim of this study was to determine the overall and age-specific prevalence of T. whipplei and to identify risk factors for colonization. This molecular epidemiological survey was designed as a cross-sectional study in a rural community in Central African Gabon and inhabitants of the entire community were invited to participate. Overall prevalence assessed by real-time PCR and sequencing was 19.6% (95% CI 16–23.2%, n = 91) in 465 stool samples provided by the study participants. Younger age groups showed a significantly higher prevalence of T. whipplei colonization ranging from 40.0% (95% CI 27.8–52.2) among the 0–4 year olds to 36.4% (95% CI 26.1–46.6) among children aged 5–10 years. Prevalence decreased in older age groups (p <0.001) from 12.6% (95% CI 5.8–19.4%; 11–20 years) to 9.7% (95% CI 5.7–13.6) among those older than 20. Risk factor analysis revealed young age, male sex, and number of people sharing a bed as factors associated with an increased risk for T. whipplei carriage. These results demonstrate that T. whipplei carriage is highly prevalent in this part of Africa. The high prevalence in early life and the analysis of risk factors suggest that transmission may peak during childhood facilitated through close person-to-person contacts.

Detection of Plasmodium knowlesi DNA in the urine and faeces of a Japanese macaque (Macaca fuscata) over the course of an experimentally induced infection.

BackgroundDiagnostic techniques based on PCR for the detection of Plasmodium DNA can be highly sensitive and specific. The vast majority of these techniques rely, however, on the invasive sampling of blood from infected hosts. There is, currently, considerable interest in the possibility of using body fluids other than blood as sources of parasite DNA for PCR diagnosis.MethodsUrine and faeces were obtained from a Plasmodium knowlesi infected-Japanese macaque (Macaca fuscata) over the course of an experimentally induced infection. P. knowlesi DNA (PkDNA) extracted from urine and faeces were monitored by nested PCR targeting the P. knowlesi specific cytochrome b (cytb) gene.ResultsUrinary PkDNA was detected on day 2, but was not amplified using DNA templates extracted from the samples on day 4, day 5 and day 6. Subsequently, urinary PkDNA was detected from day 7 until day 11, and from day 20 until day 30. PkDNA in faeces was detected from day 7 until day 11, and from day 20 until day 37. Moreover, real-time quantitative PCR showed a remarkable increase in the amount of urinary PkDNA following anti-malarial treatment. This might have been due to the release of a large amount of PkDNA from the degraded parasites as a result of the anti-malarial treatment, leading to excretion of PkDNA in the urine.ConclusionsThe cytb-PCR system using urine and faecal samples is of potential use in molecular epidemiological surveys of malaria. In particular, monkey faecal samples could be useful for the detection of zoonotic primate malaria in its natural hosts.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-13-373) contains supplementary material, which is available to authorized users.

Mycobacterium tuberculosis isolates from single outpatient clinic in Panama City exhibit wide genetic diversity.

Understanding Mycobacterium tuberculosis biodiversity and transmission is significant for tuberculosis control. This short report aimed to determine the genetic diversity of M. tuberculosis isolates from an outpatient clinic in Panama City. A total of 62 M. tuberculosis isolates were genotyped by 12 loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and Spoligotyping. Forty-five (72.6%) of the isolates showed unique MIRU-VNTR genotypes, and 13 (21%) of the isolates were grouped into four clusters. Four isolates showed polyclonal MIRU-VNTR genotypes. The MIRU-VNTR Hunter-Gaston discriminatory index reached 0.988. The Spoligotyping analysis revealed 16 M. tuberculosis families, including Latin American-Mediterranean, Harlem, and Beijing. These findings suggest a wide genetic diversity of M. tuberculosis isolates at one outpatient clinic. A detailed molecular epidemiology survey is now warranted, especially following second massive immigration for local Panama Canal expansion activities.

First molecular characterization of Cryptosporidium and Giardia from bovines (Bos taurus and Bubalus bubalis) in Sri Lanka: unexpected absence of C. parvum from pre-weaned calves

BackgroundThe genetic characterization of Cryptosporidium and Giardia has important implications for investigating their epidemiology and underpins their control. We undertook the first molecular epidemiological survey of domestic bovids in selected regions of Sri Lanka to establish whether they excreted Cryptosporidium and/or Giardia with zoonotic potential.MethodsFaecal samples were collected from dairy calves (n = 340; Bos taurus; < 3 months of age; weekly sampling for six weeks) and water buffaloes (n = 297; Bubalus bubalis; <6 months and ≥6 months of age; one sampling) from seven different farms in Sri Lanka. Genomic DNAs were extracted from individual faecal samples and then tested for the presence of parasite DNA using a PCR-based mutation scanning-targeted sequencing-phylogenetic approach, employing genetic markers within the small subunit of nuclear ribosomal RNA and 60 kDa glycoprotein genes (designated pSSU and pgp60, respectively) for Cryptosporidium, and within the triose phosphate isomerise (ptpi) gene for Giardia.ResultsBased on pSSU sequence data, C. bovis, C. ryanae and six new genotypes that were genetically similar but not identical to C. andersoni (n = 1), C. bovis (n = 1), C. ryanae (n = 3) and C. suis (n = 1) were recorded in cattle. For pSSU, two other, new genotypes were defined in water buffalo, which were genetically most similar to Cryptosporidium genotypes recorded previously in this host species in other countries including Australia. Consistent with the findings for pSSU, no species or genotypes of Cryptosporidium with zoonotic potential were detected using pgp60. Based on ptpi sequence data, G. duodenalis assemblages A and E were detected in four and 137 samples from cattle, respectively, and assemblage E in two samples from water buffaloes.ConclusionsThe present study showed that C. parvum, the most commonly reported zoonotic species of Cryptosporidium recognised in bovine calves globally, was not detected in any of the samples from pre-weaned calves tested in the present study. However, eight new genotypes were recorded. Future studies of different host species in various regions are required to investigate the molecular epidemiology of cryptosporidiosis and giardiasis in Sri Lanka and neighbouring countries in South Asia.

Molecular epidemiological survey on quinolone resistance genotype and phenotype of Escherichia coli in septicemic broilers in Hebei, China

In this study, the quinolone-resistant determining region (QRDR) of gyrA of Escherichia coli and plasmid-mediated quinolone resistance (PMQR) genes, qnr(qnrA, qnrB, and qnrS), and aac(6 ')-Ib-cr were detected, sequenced, and analyzed. In addition, antimicrobial susceptibility tests (using the Kirby-Bauer disc diffusion method) were performed for all 111 E. coli isolates from septicemic broilers in Hebei, China. The results show that the resistance rates were as follows: ofloxacin 99.10%, ciprofloxacin 93.69%, levofloxacin 91.89%, norfloxacin 90.09%, and gatifloxacin 76.58%. Of the PMQR genes examined, aac(6 ')-Ib-cr (36.04%) was the most frequently identified gene in all isolates, followed by qnrS (8.11%), qnrB (0.90%), and qnrA (0%). Of the QRDR examined in the 40 phenotypic quinolone-resistant isolates, compared with the gyrA(+) gene of E. coli K-12, 4 amino acid exchanges were found, namely Ser-83→Asp, Asp-87→Asn, Asp-87→Tyr, and Asp-87→Ala, and all 40 isolates had 1 or 2 exchanges in QRDR. It was concluded that quinolone-resistance in E. coli remains a serious problem in Hebei, China. Therefore, there is considerable local surveillance of quinolone resistance. Plasmid-mediated quinolone resistance of the qnr type remains rare in Hebei, China, and mutation in QRDR may be the main problem.

PCR Detection of Anaplasma phagocytophilum and Borrelia burgdorferi in Ixodes persulcatus Ticks in Mongolia

A molecular epidemiological survey was conducted to identify the tick-borne disease agents Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in Selenge Province, Mongolia. The survey was in response to a suspected A. phagocytophilum infection in a patient. In 2012, a total of 129 questing Ixodes persulcatus adult ticks were sampled by flagging vegetation. A. phagocytophilum and Borrelia spp. were detected by PCR, targeting the 16S rDNA (rrs) and 5S-23S intergenic spacer region, respectively. Infection rates for A. phagocytophilum and B. burgdorferi sensu lato spp. were 6.2% and 55.0%, respectively. Six of the 129 ticks (4.9%) were coinfected with A. phagocytophilum and B. burgdorferi sensu lato. Among Borrelia spp., the highest prevalence rate was that for B. garinii 20047 type (26.3%), followed by B. afzelii (7.8%) and B. garinii NT29 type (7.0%). Furthermore, ticks were detected that were dually infected with B. afzelii and B. garinii 20047 type (7.8%) and B. garinii NT29 and 20047 types (6.2%).

Genotoxic effect of exposure to metal(loid)s. A molecular epidemiology survey of populations living and working in Panasqueira mine area, Portugal

Previous studies investigating the exposure to metal(loid)s of populations living in the Panasqueira mine area of central Portugal found a higher internal dose of elements such as arsenic, chromium, lead, manganese, molybdenum and zinc in exposed individuals. The aims of the present study were to evaluate the extent of genotoxic damage caused by environmental and occupational exposure in individuals previously tested for metal(loid) levels in different biological matrices, and the possible modulating role of genetic polymorphisms involved in metabolism and DNA repair. T-cell receptor mutation assay, comet assay, micronucleus (MN) test and chromosomal aberrations (CA) were performed in a group of 122 subjects working in the Panasqueira mine or living in the same region. The modifying effect of polymorphisms in GSTA2, GSTM1, GSTP1, GSTT1, XRCC1, APEX1, MPG, MUTYH, OGG1, PARP1, PARP4, ERCC1, ERCC4, and ERCC5 genes was investigated. Significant increases in the frequency of all biomarkers investigated were found in exposed groups, however those environmentally exposed were generally higher. Significant influences of polymorphisms were observed for GSTM1 deletion and OGG1 rs1052133 on CA frequencies, APEX1 rs1130409 on DNA damage, ERCC1 rs3212986 on DNA damage and CA frequency, and ERCC4 rs1800067 on MN and CA frequencies. Our results show that the metal(loid) contamination in the Panasqueira mine area induced genotoxic damage both in individuals working in the mine or living in the area. The observed effects are closely associated to the internal exposure dose, and are more evident in susceptible genotypes. The urgent intervention of authorities is required to protect exposed populations.

Molecular Characterization of Circulating Respiratory Syncytial Virus (RSV) Genotypes in Gilgit Baltistan Province of Pakistan during 2011-2012 Winter Season

Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory tract infections in young children, but very little is known about its epidemiology and circulating genotypes in Pakistan. This study analyzed the epidemiological and molecular characteristics of RSV genotypes detected in Pakistani children less than 2 years of age with acute respiratory tract infections (ARIs) in a tertiary care hospital in Gilgit Baltistan (GB) province during 2011-12 winter season. RSV was detected in 75 out of 105 children presenting with acute respiratory infection. Male infants between 2-6 months age made up the highest percentage of RSV positive cases. Epidemiological factors such as pre-maturity, mean weight, clinical features and diagnosis when compared between RSV positive and negative groups were found to be statistically insignificant. Phylogenetic analysis classified all 75 of the RSV strains into 71 strains of subgroups A and 4 strains of subgroup B, respectively. Strains belonging to subgroups A and B were further subdivided into NA1/GA2 and BA, respectively. The nucleotide and deduced amino acid sequence identities were relatively high among these strains (>90%). Both RSV-A and RSV-B isolates had two potential N-glycosylation sites in HVR2 of G protein and with heavy O-glycosylation of serine and threonine residues (G scores of 0.5-0.7). This report highlights the significance of RSV as a dominant viral etiologic agent of pediatric ARIs, and need for continued molecular epidemiological surveys for early detection of prevalent strains and newly emerging genotypes to understand epidemiology of RSV infections in various regions of Pakistan.

Assessing calves as carriers ofCryptosporidiumandGiardiawith zoonotic potential on dairy and beef farms within a water catchment area by mutation scanning

In the present study, we undertook a molecular epidemiological survey of Cryptosporidium and Giardia in calves on three dairy and two beef farms within an open drinking water catchment area (Melbourne, Australia). Faecal samples (n = 474) were collected from calves at two time points (5 months apart) and tested using a PCR-based mutation scanning-targeted sequencing phylogenetic approach, employing regions within the genes of small subunit (SSU) of ribosomal RNA (designated partial SSU), 60 kDa glycoprotein (pgp60) and triose phosphate isomerase (ptpi) as genetic markers. Using partial SSU, the C. bovis, C. parvum, C. ryanae and a new genotype of Cryptosporidium were characterised from totals of 74 (15.6%), 35 (7.3%), 37 (7.8%) and 9 (1.9%) samples, respectively. Using pgp60, C. parvum genotype IIa subgenotype A18G3R1 was detected in 29 samples. Using ptpi, G. duodenalis assemblages A and E were detected in totals of 10 (2.1%) and 130 (27.4%) samples, respectively. The present study showed that a considerable proportion of dairy and beef calves in this open water catchment region excreted Cryptosporidium (i.e. subgenotype IIaA18G3R1) and Giardia (e.g. assemblage A) that are consistent with those infecting humans, inferring that they are of zoonotic importance. Future work should focus on exploring, in a temporal and spatial way, whether these parasites occur in the environment and water of the catchment reservoir.

Molecular epidemiological survey on seven viral pathogens in children with diarrhea disease in Urumqi area in 2011

Objective To study the infection status of seven viral pathogens in children under 5-years old with diarrhea in Urumqi in 2011 and their epidemiological characteristics. Methods A total of 315 fecal samples were collected from in-patient and out-patient children 5 years and younger with diarrhea from January to December of 2011 in Urumqi. Polymerase chain reaction （PCR） was used to detect adenovirus （AdV）, and multiplex reverse transcription-polymerase chain reaction （RT-PCR） was performed to determine rotavirus （RV）, aichi virus （AIV）, human parechovirus （HPeV）, human calicivirus（ HuCV）, enterovirus （EV） and astrovirus （AstV）. Results The positive rates of RV, AIV, HPeV, HuCV, EV, AdV and AstV in 315 fecal samples were 27.30% （86/315）, 18.41% （58/315）, 12.38% （39/315）, 12.06% （38/315）, 5.08% （ 16/315 ）, 3.81% （ 12/315 ） and 0.00% respectively, moreover, mixed infection was detected in 54 of 315 samples （ 17.14% ）. Children under 2-years old accounted for most of the patients with above mentioned infec- tion. Seasonal differences were found in infections of the six viruses, which were characterized by RV infection was mainly occurred in autumn and winter, AIV infection most in the latter half of the year, HPeV, HuCV and EV infection in summer, while AdV infection had no seasonal fluctuation. Conclusion Rotavirus was the pre- dominant pathogen causing infant diarrhea in Urmuqi, followed by AIV, HPeV and HuCV, moreover, EV and AdV infection also played an unignorable role in the infantile diarrhea. Key words: Children; Diarrhea; Diarrhea virus; Diarrhea-related virus