Proffered Papers 16.00–16.30 Monday 15 September 2003 6 Development of a multiplex PCR protocol for rapid screening of ThinPrep® cervical samples for HPV and Chlamydia trachomatis

Human papillomaviruses and Chlamydia trachomatis are two of the most commonly found sexually transmitted infections in cervical Pap smears. They are often asymptomatic and if left untreated can progress to cause serious complications such as pre‐cancerous lesions and tubal factor infertility, respectively. The aim of this study was to develop a rapid multiplex PCR for the simultaneous detection of HPV and C. trachomatis in ThinPrep^® liquid cytology samples. Two multiplexes were optimized. (A) For the detection of C. trachomatis using primers for the cryptic plasmid and for a chromosomal gene (Hsp60); (B) for the simultaneous detection of HPV and C. trachomatis using consensus primers for HPV and plasmid primers for C. trachomatis. Both multiplexes included a set of primers for a human housekeeping gene‐β‐globin. DNA from 34 ThinPrep^® cervical samples was extracted using the QiAmp DNA Mini Kit (Qiagen Ltd, UK). All 34 samples were previously confirmed positive for C. trachomatis using another nucleic‐acid based test. Using multiplex A.for the detection of C. trachomatis, 33 of 34 samples were positive for C. trachomatis by either the plasmid or chromosomal gene primer set. All samples were positive for β‐globin. Ten of the 34 C. trachomatis positive samples were known positives for HPV. Using the combined HPV and C. trachomatis multiplex 10 of 10 were positive for both HPV and C. trachomatis. These simple multiplexes are cost‐effective, rapid and have potential for rapid screening of cervical ThinPrep samples simultaneously for both HPV and C. trachomatis.     

Short-read reading-frame predictors are not created equal: sequence error causes loss of signal

ABSTRACT: BACKGROUND: Gene prediction algorithms (or gene callers) are an essential tool for analyzing shotgun nucleic acid sequence data. Gene prediction is a ubiquitous step in sequence analysis pipelines; it reduces the volume of data by identifying the most likely reading frame for a fragment, permitting the out-of-frame translations to be ignored. In this study we evaluate five widely used ab initio gene-calling algorithms--FragGeneScan, MetaGeneAnnotator, MetaGeneMark, Orphelia, and Prodigal--for accuracy on short (75-1000 bp) fragments containing sequence error from previously published artificial data and "real" metagenomic datasets. RESULTS: While gene prediction tools have similar accuracies predicting genes on error-free fragments, in the presence of sequencing errors considerable differences between tools become evident. For error-containing short reads, FragGeneScan finds more prokaryotic coding regions than does MetaGeneAnnotator, MetaGeneMark, Orphelia, or Prodigal. This improved detection of genes in error-containing fragments, however, comes at the cost of much lower (50%) specificity and overprediction of genes in noncoding regions. CONCLUSIONS: Ab initio gene callers offer a significant reduction in the computational burden of annotating individual nucleic acid reads and are used in many metagenomic annotation systems. For predicting reading frames on raw reads, we find the hidden Markov model approach in FragGeneScan is more sensitive than other gene prediction tools, while Prodigal, MGA, and MGM are better suited for higher-quality sequences such as assembled contigs.     

Seasonal Influenza Vaccination amongst Medical Students: A Social Network Analysis Based on a Cross-Sectional Study

Introduction

The Chief Medical Officer for England recommends that healthcare workers have a seasonal influenza vaccination in an attempt to protect both patients and NHS staff. Despite this, many healthcare workers do not have a seasonal influenza vaccination. Social network analysis is a well-established research approach that looks at individuals in the context of their social connections. We examine the effects of social networks on influenza vaccination decision and disease dynamics.

Methods

We used a social network analysis approach to look at vaccination distribution within the network of the Lancaster Medical School students and combined these data with the students’ beliefs about vaccination behaviours. We then developed a model which simulated influenza outbreaks to study the effects of preferentially vaccinating individuals within this network.

Results

Of the 253 eligible students, 217 (86%) provided relational data, and 65% of responders had received a seasonal influenza vaccination. Students who were vaccinated were more likely to think other medical students were vaccinated. However, there was no clustering of vaccinated individuals within the medical student social network. The influenza simulation model demonstrated that vaccination of well-connected individuals may have a disproportional effect on disease dynamics.

Conclusions

This medical student population exhibited vaccination coverage levels similar to those seen in other healthcare groups but below recommendations. However, in this population, a lack of vaccination clustering might provide natural protection from influenza outbreaks. An individual student’s perception of the vaccination coverage amongst their peers appears to correlate with their own decision to vaccinate, but the directionality of this relationship is not clear. When looking at the spread of disease within a population it is important to include social structures alongside vaccination data. Social networks influence disease epidemiology and vaccination campaigns designed with information from social networks could be a future target for policy makers.     

Comparison of DNA extraction from cervical cells collected in PreservCyt solution for the amplification of Chlamydia trachomatis

OBJECTIVE: The aim of this study was to compare and evaluate three methods of DNA extraction for the amplification of Chlamydia trachomatis in uterine cervical samples collected in PreservCyt solution. ThinPrep is the trade name for the slide preparation. METHODS: Thirty-eight samples collected in LCx buffer medium, which were identified as C. trachomatis infected by ligase chain reaction (LCR), were selected for this study. DNA from the PreservCyt samples was extracted by three methods: (i) QIAamp kit, (ii) boiling in Tris-EDTA buffer with Chelex purification, and (iii) Proteinase K digestion with Chelex purification. Sample DNA was tested for the presence of C. trachomatis by PCR using cryptic plasmid research (CTP) primers and major outer membrane protein research momp gene (MOMP) primers. Real-time (LightCycler) PCR for relative C. trachomatis quantification following DNA extraction was performed using primers (Hsp 60) for the 60 kDa heat-shock protein hsp60 gene. RESULTS: Amplification using CTP primers was the most successful with each of the extraction protocols. Boiling in buffer was the least successful extraction method. QIAamp was the best extraction method, yielding the most positives with both the CTP and MOMP primers. Proteinase K-Chelex extraction gave similar sensitivity to QIAamp extraction with CTP primers but lower for MOMP primers. CONCLUSIONS: The DNA extraction method must be carefully selected to ensure that larger PCR amplicons can be successfully produced by PCR and to ensure high sensitivity of detection of C. trachomatis. In this study it was found that the QIAamp extraction method followed by PCR with the CTP primers was the most successful for amplification of C. trachomatis DNA.     

Connexin 43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia

Gap junctions are assemblies of intercellular channels that regulate a variety of physiologic and developmental processes through the exchange of small ions and signaling molecules. These channels consist of connexin family proteins that allow for diversity of channel composition and conductance properties. The human connexin 43 gene, or GJA1, is located at human chromosome 6q22-q23 within the candidate region for the oculodentodigital dysplasia locus. This autosomal dominant syndrome presents with craniofacial (ocular, nasal, and dental) and limb dysmorphisms, spastic paraplegia, and neurodegeneration. Syndactyly type III and conductive deafness can occur in some cases, and cardiac abnormalities are observed in rare instances. We found mutations in the GJA1 gene in all 17 families with oculodentodigital dysplasia that we screened. Sixteen different missense mutations and one codon duplication were detected. These mutations may cause misassembly of channels or alter channel conduction properties. Expression patterns and phenotypic features of gja1 animal mutants, reported elsewhere, are compatible with the pleiotropic clinical presentation of oculodentodigital dysplasia.