A novel diagnostic gene region for distinguishing between two pest fruit flies: Bactrocera tryoni (Froggatt) and Bactrocera neohumeralis (Hardy) (Diptera: Tephritidae).

Bactrocera tryoni and Bactrocera neohumeralis are morphologically similar sibling pest fruit fly species that possess different biological attributes, geographic distributions, and host ranges. The need to differentiate between the two species is critical for accurate pest status assessment, management, biosecurity, and maintenance of reference colonies. While morphologically similar, adults may be separated based on subtle characters; however, some characters exhibit intraspecific variability, creating overlap between the two species. Additionally, there is currently no single molecular marker or rapid diagnostic assay that can reliably distinguish between B. neohumeralis and B. tryoni; therefore, ambiguous samples remain undiagnosed. Here we report the first molecular marker that can consistently distinguish between B. tryoni and B. neohumeralis. Our diagnostic region consists of two adjacent single nucleotide polymorphisms (SNPs) within the pangolin (pan) gene region. We confirmed the genotypes of each species are consistent across their distributional range, then developed a tetra-primer amplification refractory mutation system (ARMS) PCR assay for rapid diagnosis of the species. The assay utilizes four primers in multiplex, with two outer universal primers, and two internal primers: one designed to target two adjacent SNPs (AA) present in B. tryoni and the other targeting adjacent SNPs present in B. neohumeralis (GG). The assay accurately discriminates between the two species, but their SNP genotypes are shared with other nontarget tephritid fruit fly species. Therefore, this assay is most suited to adult diagnostics where species confirmation is necessary in determining ambiguous surveillance trap catches; maintaining pure colony lines; and in Sterile Insect Technique management responses.

Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar.

Insect identification and preservation of voucher specimens is integral to pest diagnostic and surveillance activities; yet bulk-trapped insects are a diagnostic challenge due to high catch numbers and the susceptibility of samples to environmental damage. Many insect trap catches rely on examination of morphological characters for species identifications, which is a time consuming and highly skilled task, hence there is a need for more efficient molecular approaches. Many bulk DNA extraction methods require destructive sampling of specimens, resulting in damaged, or fully destroyed, voucher specimens. We developed an inexpensive, rapid, bulk DNA isolation method that preserves specimens as pinned vouchers to a standard that allows for post-extraction morphological examination and inclusion in insect reference collections. Our protocol was validated using a group of insects that are time-consuming to identify when trapped in large numbers-the dacine fruit flies (Diptera: Tephritidae: Dacinae). In developing our method, we evaluated existing protocols against the following criteria: effect on morphology; suitability for large trap catches; cost; ease of handling; and application to downstream molecular diagnostic analyses such as real-time PCR and metabarcoding. We found that the optimum method for rapid isolation of DNA extraction was immersing flies in a NaOH:TE buffer at 75°C for 10 minutes, without the need for proteinase K or detergents. This HotSOAK method produced sufficient high-quality DNA whilst preserving morphological characters suitable for species-level identification with up to 20,000 flies in a sample. The lysates performed well in down-stream analyses such as loop-mediated isothermal amplification (LAMP) and real-time PCR applications, while for metabarcoding PCR the lysate required an additional column purification step. Development of this method is a key step required for upscaling our capacity to accurately detect insects captured in bulk traps, whether for biodiversity, biosecurity, or pest management objectives.

Loop-mediated isothermal amplification (LAMP) assays for detection of the New Guinea fruit fly Bactrocera trivialis (Drew) (Diptera: Tephritidae).

The cue-lure-responding New Guinea fruit fly, Bactrocera trivialis, poses a biosecurity risk to neighbouring countries, e.g., Australia. In trapping programs, lure caught flies are usually morphologically discriminated from non-target species; however, DNA barcoding can be used to confirm similar species where morphology is inconclusive, e.g., Bactrocera breviaculeus and B. rufofuscula. This can take days-and a laboratory-to resolve. A quicker, simpler, molecular diagnostic assay would facilitate a more rapid detection and potential incursion response. We developed LAMP assays targeting cytochrome c oxidase subunit I (COI) and Eukaryotic Translation Initiation Factor 3 Subunit L (EIF3L); both assays detected B. trivialis within 25 min. The BtrivCOI and BtrivEIF3L assay anneal derivatives were 82.7 ± 0.8 °C and 83.3 ± 1.3 °C, respectively, detecting down to 1 × 101 copies/µL and 1 × 103 copies/µL, respectively. Each assay amplified some non-targets from our test panel; however notably, BtrivCOI eliminated all morphologically similar non-targets, and combined, the assays eliminated all non-targets. Double-stranded DNA gBlocks were developed as positive controls; anneal derivatives for the COI and EIF3L gBlocks were 84.1 ± 0.7 °C and 85.8 ± 0.2 °C, respectively. We recommend the BtrivCOI assay for confirmation of suspect cue-lure-trapped B. trivialis, with BtrivEIF3L used for secondary confirmation when required.

In Vivo Characterisation of Five Strains of Bovine Viral Diarrhoea Virus 1 (Subgenotype 1c).

Bovine viral diarrhoea virus 1 (BVDV-1) is strongly associated with several important diseases of cattle, such as bovine respiratory disease, diarrhoea and haemoragic lesions. To date many subgenotypes have been reported for BVDV-1, currently ranging from subgenotype 1a to subgenotype 1u. While BVDV-1 has a world-wide distribution, the subgenotypes have a more restricted geographical distribution. As an example, BVDV-1 subgenotypes 1a and 1b are frequently detected in North America and Europe, while the subgenotype 1c is rarely detected. In contrast, BVDV-1 subgenotype 1c is by far the most commonly reported in Australia. Despite this, uneven distribution of the biological importance of the subgenotypes remains unclear. The aim of this study was to characterise the in vivo properties of five strains of BVDV-1 subgenotype 1c in cattle infection studies. No overt respiratory signs were reported in any of the infected cattle regardless of strain. Consistent with other subgenotypes, transient pyrexia and leukopenia were commonly identified, while thrombocytopenia was not. The quantity of virus detected in the nasal secretions of transiently infected animals suggested the likelihood of horizontal transmission was very low. Further studies are required to fully understand the variability and importance of the BVDV-1 subgenotype 1c.

Altered Expression of Angiotensinogen and Mediators of Angiogenesis in Ileal Crohn's Disease.

BACKGROUND AND AIMS: Angiotensin II (AII) is a powerful splanchnic vasoconstrictor with pro-inflammatory and pro-fibrotic properties. Angiotensin converting enzyme (ACE) inhibitors and AII Receptor Antagonists (ARBs) are therapeutic in animal models of colitis. The aim of this case-control study is to determine the expression of angiotensinogen and related genes in human ileal Crohn's disease. METHODS: Using quantitative real-time polymerase chain reaction (RT-PCR), we measured mRNA expression levels of angiotensinogen (AGT), hypoxia inducible factor (HIF)1α and melanoma cell adhesion molecule (MCAM; CD146) in 101 human samples (69 biopsy, 12 resection) from affected ileum (inflamed CD cases, n=36) and unaffected ileum (non-inflamed CD cases, n=45 and controls, n=20). Immunohistochemistry for angiotensinogen was also performed. The study was of case-control design in a tertiary care setting. RESULTS: Ileal expression of AGT was lower in CD cases compared to controls (p<0.0001), in inflamed CD samples (p=0.017) and current smokers (p=0.02). HIF1α expression was lower in non-inflamed CD biopsy samples than controls (p=0.006). The presence of disease-associated NOD2 variants was associated with increased expression of markers of angiogenesis (HIF1α p=0.009; MCAM p=0.007) in inflamed CD samples. Angiotensinogen immunohistochemical staining supported the quantitative RT-PCR expression findings. CONCLUSIONS: Angiotensinogen expression is down regulated in human ileal CD, particularly in the presence of inflammation and current cigarette smoking, implicating the mesenteric vasculature and mucosal hypoxia as co-factors in ileal CD pathogenesis. A novel reduction in HIF1α expression in non-inflamed ileal mucosa in CD patients was also demonstrated.

The first genome sequence of a metatherian herpesvirus: Macropodid herpesvirus 1.

BACKGROUND: While many placental herpesvirus genomes have been fully sequenced, the complete genome of a marsupial herpesvirus has not been described. Here we present the first genome sequence of a metatherian herpesvirus, Macropodid herpesvirus 1 (MaHV-1). RESULTS: The MaHV-1 viral genome was sequenced using an Illumina MiSeq sequencer, de novo assembly was performed and the genome was annotated. The MaHV-1 genome was 140 kbp in length and clustered phylogenetically with the primate simplexviruses, sharing 67% nucleotide sequence identity with Human herpesviruses 1 and 2. The MaHV-1 genome contained 66 predicted open reading frames (ORFs) homologous to those in other herpesvirus genomes, but lacked homologues of UL3, UL4, UL56 and glycoprotein J. This is the first alphaherpesvirus genome that has been found to lack the UL3 and UL4 homologues. We identified six novel ORFs and confirmed their transcription by RT-PCR. CONCLUSIONS: This is the first genome sequence of a herpesvirus that infects metatherians, a taxonomically unique mammalian clade. Members of the Simplexvirus genus are remarkably conserved, so the absence of ORFs otherwise retained in eutherian and avian alphaherpesviruses contributes to our understanding of the Alphaherpesvirinae. Further study of metatherian herpesvirus genetics and pathogenesis provides a unique approach to understanding herpesvirus-mammalian interactions.

Identification of non-essential loci within the Meleagrid herpesvirus 1 genome.

BACKGROUND: Meleagrid herpesvirus 1 (MeHV-1) infectious bacterial artificial chromosomes (iBACs) are ideal vectors for the development of recombinant vaccines for the poultry industry. However, the full potential of iBACS as vectors can only be realised after thorough genetic characterisation, including identification of those genetic locations that are non-essential for virus replication. Generally, transposition has proven to be a highly effective strategy for rapid and efficient mutagenesis of iBAC clones. The current study describes the characterisation of 34 MeHV-1 mutants containing transposon insertions within the pMeHV1-C18 iBAC genome. METHODS: Tn5 and MuA transposition methods were used to generate a library of 76 MeHV-1 insertion mutants. The capacity of each mutant to facilitate the recovery of infectious MeHV-1 was determined by the transfection of clone DNA into chicken embryo fibroblasts. RESULTS: Attempts to recover infectious virus from the modified clones identified 14 genetic locations that were essential for MeHV-1 replication in cell culture. Infectious MeHV-1 was recovered from the remaining 14 intragenic insertion mutants and six intergenic insertion mutants, suggesting that the respective insertion locations are non-essential for MeHV-1 replication in cell culture. CONCLUSIONS: The essential and non-essential designations for those MeHV-1 genes characterised in this study were generally in agreement with previous reports for other herpesviruses homologues. However, the requirement for the mardivirus-specific genes LORF4A and LORF5 are reported for the first time. These findings will help direct future work on the development of recombinant poultry vaccines using MeHV-1 as a vector by identifying potential transgene insertion sites within the viral genome.

Genomic deletions and mutations resulting in the loss of eight genes reduce the in vivo replication capacity of Meleagrid herpesvirus 1.

Meleagrid herpesvirus 1 (MeHV-1 or turkey herpesvirus) has been widely used as a vaccine in commercial poultry. Initially, these vaccine applications were for the prevention of Marek's disease resulting from Gallid herpesvirus 2 infections, while more recently MeHV-1 has been used as recombinant vector for other poultry infections. The construction of herpesvirus infectious clones that permit propagation and manipulation of the viral genome in bacterial hosts has advanced the studies of herpesviral genetics. The current study reports the construction of five MeHV-1 infectious clones. The in vitro properties of viruses recovered from these clones were indistinguishable from the parental MeHV-1. In contrast, the rescued MeHV-1 viruses were significantly attenuated when used in vivo. Complete sequencing of the infectious clones identified the absence of two regions of the MeHV-1 genome compared to the MeHV-1 reference sequence. These analyses determined the rescued viruses have seven genes, UL43, UL44, UL45, UL56, HVT071, sorf3 and US2 either partially or completely deleted. In addition, single nucleotide polymorphisms were identified in all clones compared with the MeHV-1 reference sequence. As a consequence of one of the polymorphisms identified in the UL13 gene, four of the rescued viruses were predicted to encode a serine/threonine protein kinase lacking two of three domains required for activity. Thus four of the recovered viruses have a total of eight missing or defective genes. The implications of these findings in the context of herpesvirus biology and infectious clone construction are discussed.

Smoking behaviour modifies IL23r-associated disease risk in patients with Crohn's disease.

BACKGROUND AND AIM: The etiology of Crohn's disease (CD) implicates both genetic and environmental factors. Smoking behavior is one environmental risk factor to play a role in the development of CD. The study aimed to assess the contribution of the interleukin 23 receptor (IL23R) in determining disease susceptibility in two independent cohorts of CD, and to investigate the interactions between IL23R variants, smoking behavior, and CD-associated genes, NOD2 and ATG16L1. METHODS: Ten IL23R single-nucleotide polymorphisms (SNPs) were genotyped in 675 CD cases, and 1255 controls from Brisbane, Australia (dataset 1). Six of these SNPs were genotyped in 318 CD cases and 533 controls from Canterbury, New Zealand (dataset 2). Case-control analysis of genotype and allele frequencies, and haplotype analysis for all SNPs was conducted. RESULTS: We demonstrate a strong increased CD risk for smokers in both datasets (odds ratio 3.77, 95% confidence interval 2.88-4.94), and an additive interaction between IL23R SNPs and cigarette smoking. Ileal involvement was a consistent marker of strong SNP-CD association (P ≤ 0.001), while the lowest minor allele frequencies for location were found in those with colonic CD (L2). Three haplotype blocks were identified across the 10 IL23R SNPs conferring different risk of CD. Haplotypes conferred no further risk of CD when compared with single SNP analyses. CONCLUSION: IL23R gene variants determine CD susceptibility in the Australian and New Zealand population, particularly ileal CD. A strong additive interaction exists between IL23R SNPs and smoking behavior resulting in a dramatic increase in disease risk depending upon specific genetic background.

The Meleagrid herpesvirus 1 genome is partially resistant to transposition.

The propagation of herpesvirus genomes as infectious bacterial artificial chromosomes (iBAC) has enabled the application of highly efficient strategies to investigate gene function across the genome. One of these strategies, transposition, has been used successfully on a number of herpesvirus iBACs to generate libraries of gene disruption mutants. Gene deletion studies aimed at determining the dispensable gene repertoire of the Meleagrid herpesvirus 1 (MeHV-1) genome to enhance the utility of this virus as a vaccine vector have been conducted in this report. A MeHV-1 iBAC was used in combination with the Tn5 and MuA transposition systems in an attempt to generate MeHV-1 gene interruption libraries. However, these studies demonstrated that Tn5 transposition events into the MeHV-1 genome occurred at unexpectedly low frequencies. Furthermore, characterization of genomic locations of the rare Tn5 transposon insertion events indicated a nonrandom distribution within the viral genome, with seven of the 24 insertions occurring within the gene encoding infected cell protein 4. Although insertion events with the MuA system occurred at higher frequency compared with the Tn5 system, fewer insertion events were generated than has previously been reported with this system. The characterization and distribution of these MeHV-1 iBAC transposed mutants is discussed at both the nucleotide and genomic level, and the properties of the MeHV-1 genome that could influence transposition frequency are discussed.

Common PPARγ variants C161T and Pro12Ala are not associated with inflammatory bowel disease in an Australian cohort.

BACKGROUND AND AIMS: Peroxisome proliferator-activated receptor (PPAR) γ is a transcription factor, highly expressed in colonic epithelial cells, adipose tissue and macrophages, with an important role in the regulation of inflammatory pathways. The common PPARγ variants C161T and Pro12Ala have recently been associated with Ulcerative Colitis (UC) and an extensive UC phenotype respectively, in a Chinese population. PPARγ Pro12Ala variant homozygotes appear to be protected from the development of Crohn's disease (CD) in European Caucasians. METHODS: A case-control study was performed for both variants (CD n=575, UC n=306, Controls n=360) using a polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis in an Australian IBD cohort. A transmission disequilibrium test was also performed using CD trios for the PPARγ C161T variant. Genotype-phenotype analyses were also undertaken. RESULTS: There was no significant difference in genotype distribution data or allele frequency between CD and UC patients and controls. There was no difference in allele transmission for the C161T variant. No significant relationship between the variants and disease location was observed. CONCLUSIONS: We were unable to replicate in a Caucasian cohort the recent association between PPARγ C161T and UC or between PPARγ Pro12Ala and an extensive UC phenotype in a Chinese population. There are significant ethnic differences in genetic susceptibility to IBD and its phenotypic expression.

KCNN4 gene variant is associated with ileal Crohn's Disease in the Australian and New Zealand population.

OBJECTIVES: Crohn's disease (CD; MIM 266600) is one of the most common forms of inflammatory bowel disease (IBD), and represents a significant burden to health care in developed countries. Our aim was to determine whether a gene in the IBD linkage region on chromosome 19q13, with a role in Paneth cell secretion and T-cell activation, conferred genetic susceptibility to the development of CD. METHODS: In total, 792 CD cases and 1,244 controls of Australian origin (Caucasian) were genotyped for seven single-nucleotide polymorphisms (SNPs) in the gene encoding the intermediate conductance calcium-activated potassium channel protein (KCNN4) at 19q13.2. CD cases were phenotyped using the Montreal classification. The replication set comprised an additional 326 CD cases and 951 population-based Caucasian controls. Analysis of the KCNN4 mRNA transcript was carried out using quantitative reverse transcriptase-PCR. RESULTS: KCNN4 SNP rs2306801 was associated with CD (primary P=0.0008, odds ratio (OR) (95% confidence interval (CI)): 0.76 (0.65-0.89); replication P=0.01, OR (95% CI): 0.77 (0.61-0.97). Stratification by disease location identified the association between SNP rs2306801 and ileal CD (P=0.01). Non-inflamed ileal mucosa from CD patients carrying any of the common disease-predisposing NOD2 variants (R702W, G908R, 1007fs) had significantly reduced levels of KCNN4 mRNA expression (P=0.001). KCNN4 protein expression was detected in Paneth cells, and in T cells in inflamed lamina propria. CONCLUSIONS: Our data implicate the role of KCNN4 in ileal CD. The dual roles of KCNN4 in Paneth cell secretion and T-cell activation and also its nature as a potassium channel make it an important and practical therapeutic target.

ATG16L1 T300A shows strong associations with disease subgroups in a large Australian IBD population: further support for significant disease heterogeneity.

OBJECTIVES: Crohn's disease (CD) and ulcerative colitis (UC) are the two most common forms of inflammatory bowel disease (IBD), representing a significant health-care burden. A variant in the autophagy gene ATG16L1 (T300A) has been newly identified as a CD susceptibility locus by genome-wide association. Our aim was to assess the contribution of T300A in determining disease susceptibility and phenotype in two independent Australian IBD cohorts and explore the relationship between T300A and known CD risk factors (NOD2[nucleotide-binding oligomerization domain containing 2] status and smoking). METHODS: In total, 669 CD and 543 UC cases, and 1,244 controls (study 1), 154 CD cases and 420 controls (study 2), and 702 unaffected parents from both groups were genotyped. We conducted case-control and family association analyses, and investigated relationships between T300A and disease subgroups and between NOD2 status and cigarette smoking (CD only). RESULTS: The strong association between CD and T300A was confirmed (P < 0.001), with a two-fold increase in disease risk associated with the GG genotype (odds ratio [OR] 1.96, 95% confidence interval [CI] 1.49-2.58), while ileal CD risk was almost three-fold (OR 2.73, CI 1.87-4.0). ATG16L1 and NOD2 were found to contribute independently to CD risk. A greater than seven-fold increased CD risk was observed for current smokers with a GG genotype (vs nonsmoking AA genotype; P < 0.001, OR 7.65, CI 4.21-13.91). A significant inverse association was found between T300A and UC (P= 0.002). This was strongest for patients with extensive, severe disease. CONCLUSIONS: We confirm the strong association between T300A and CD, specifically ileal subphenotype, and also report the first strong association of this variant with UC.

Novel NOD2 haplotype strengthens the association between TLR4 Asp299gly and Crohn's disease in an Australian population.

BACKGROUND: The first major Crohn's disease (CD) susceptibility gene, NOD2, implicates the innate intestinal immune system and other pattern recognition receptors in the pathogenesis of this chronic, debilitating disorder. These include the Toll-like receptors, specifically TLR4 and TLR5. A variant in the TLR4 gene (A299G) has demonstrated variable association with CD. We aimed to investigate the relationship between TLR4 A299G and TLR5 N392ST, and an Australian inflammatory bowel disease cohort, and to explore the strength of association between TLR4 A299G and CD using global meta-analysis. METHODS: Cases (CD = 619, ulcerative colitis = 300) and controls (n = 360) were genotyped for TLR4 A299G, TLR5 N392ST, and the 4 major NOD2 mutations. Data were interrogated for case-control analysis prior to and after stratification by NOD2 genotype. Genotype-phenotype relationships were also sought. Meta-analysis was conducted via RevMan. RESULTS: The TLR4 A299G variant allele showed a significant association with CD compared to controls (P = 0.04) and a novel NOD2 haplotype was identified which strengthened this (P = 0.003). Furthermore, we identified that TLR4 A299G was associated with CD limited to the colon (P = 0.02). In the presence of the novel NOD2 haplotype, TLR4 A299G was more strongly associated with colonic disease (P < 0.001) and nonstricturing disease (P = 0.009). A meta-analysis of 11 CD cohorts identified a 1.5-fold increase in risk for the variant TLR4 A299G allele (P < 0.00001). CONCLUSIONS: TLR 4 A299G appears to be a significant risk factor for CD, in particular colonic, nonstricturing disease. Furthermore, we identified a novel NOD2 haplotype that strengthens the relationship between TLR4 A299G and these phenotypes.

Prevalence of CARD15/NOD2 mutations in Caucasian healthy people.

BACKGROUND: Crohn's disease (CD) has been associated with CARD15/NOD2 mutations in Caucasians. The R702W, G908R, and 1007fs mutations represent 82% of the mutated chromosomes. The relative risk of developing CD in homozygous or compound heterozygous people has been estimated as between 10 and 40 times that of the general population. This high risk may support the opinion that CARD15/NOD2 variants are strong CD risk factors at the individual and population levels. SUBJECTS AND METHODS: The allele and genotype frequencies were calculated for the R702W, G908R, and 1007fs mutations in 3,575 Caucasian healthy controls recruited by 15 groups distributed on three continents. Geographic homogeneity was tested and the observed proportion of double mutants was compared with the expected value using chi2 tests. RESULTS: The allele frequencies of the R702W, G908R, and 1007fs mutations were 4.3% (3.6-4.9), 1.2% (0.8-1.6), and 2.3% (1.8-2.8), respectively, with large geographic fluctuations of the G908R, 1007fs, and wild-type alleles (P<0.0001). At the population level, no simple relationship was observed between mutation frequencies and the disease incidences in the studied populations. At the individual level, no significant deficit of double-dose mutation carriers among healthy controls was found, providing strong evidence that the penetrances of the most at-risk genotypes are low. CONCLUSION: Altogether, these data confirm that CARD15/NOD2 acts in interaction with other unknown risk cofactors.

Differential changes in Plasmodium falciparum var transcription during adaptation to culture.

Plasmodium falciparum erythrocyte membrane protein 1, which is encoded by the var multigene family, is expressed on the surface of P. falciparum-infected erythrocytes and has been implicated in many of the complications associated with falciparum malaria. Transcriptional switching of var is commonly investigated using in vitro cultured parasites, because parasite material from patients is limited. We investigated the affect of short-term in vitro cultivation on var gene transcription in patient samples. A significant reduction in the overall abundance of var transcripts was observed during the first approximately 10 days of culture. The rate of down-regulation was not constant among all var genes; genes with an upsA, -D, and -E 5' flanking region had a significantly faster rate than genes with an upsB or -C flanking region. These results have significant implications for the investigation of associations between var transcription and clinical manifestations using parasites that have been enriched by in vitro culture.

Physical linkage to drug resistance genes results in conservation of var genes among West Pacific Plasmodium falciparum isolates.

The multicopy var gene family encoding the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 is highly diverse, with little overlap between different P. falciparum isolates. We report 5 var genes (varS1-varS5) that are shared at relatively high frequency among 63 genetically diverse P. falciparum isolates collected from 5 islands in the West Pacific region. The varS1, varS2, and varS3 genes were localized to the internal region on chromosome 4, approximately 200 kb from pfdhfr-ts, whereas varS4 and varS5 were mapped to an internal region of chromosome 7, within 100 kb of pfcrt. The presence of varS2 and varS3 were significantly correlated with the pyrimethamine-resistant pfdhfr genotype, whereas varS4 was strongly correlated with the chloroquine-resistant pfcrt genotype. Thus, the conservation of these var genes is the result of their physical linkage with drug-resistant genes in combination with the antimalarial drug pressure in the region.

Detection sensitivity and quantitation of Plasmodium falciparum var gene transcripts by real-time RT-PCR in comparison with conventional RT-PCR.

Antigenic variation in Plasmodium falciparum erythrocyte membrane protein 1, caused by a switch in transcription of the encoding var gene, is an important feature of malaria. In this study, we quantified the relative abundance of var gene transcripts present in P. falciparum parasite clones using real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional RT-PCR combined with cloning and sequencing, with the aim of directly comparing the results obtained. When there was sufficient abundance of RNA for the real-time RT-PCR assay to be operating within the region of good reproducibility, RT-PCR and real-time RT-PCR tended to identify the same dominant transcript, although some transcript-specific issues were identified. When there were differences in the estimated relative amounts of minor transcripts, the RT-PCR assay tended to produce higher estimates than real-time RT-PCR. These results provide valuable information comparing RT-PCR and real-time RT-PCR analysis of samples with small quantities of RNA as might be expected in the analysis of field or clinical samples.

Clinically active Crohn's disease in the presence of a low C-reactive protein.

OBJECTIVE: Clinical interest in C-reactive protein (CRP) -- a component of the innate immune system -- has focused mainly on its worth as an indicator of disease activity. There has been a resurgence of interest in CRP in the Crohn's disease (CD) literature because several trials of new treatments for active CD have been characterized by both a large proportion of patients with low CRP (<10 mg/l) at entry to the trials and by a negative therapeutic outcome. It is therefore of interest to study the clinical characteristics of patients who are thought to have at the same time both active CD and a low CRP. MATERIAL AND METHODS: Patients were prospectively recruited as part of the Brisbane IBD clinical and research programme. Subjects were included in the low CRP group only if there were complete datasets for CRP on all occasions of active CD, and CRP was < 10 mg/l. Active disease was defined as CD activity index (CDAI)>200. The low CRP group was compared with patients in the raised CRP group for a range of clinical variables as well as the major NOD2 variants. RESULTS: There were data sets for 223 CD patients, with a mean disease duration of 12 years. Of these, 22 patients fulfilled the criteria for low CRP. The low CRP group (group 1) showed significant differences for disease site (p<0.01) and for BMI (p=0.006) compared to the raised CRP group (group 2). Specifically, group 1 had a predominance of pure ileal disease (95% versus 53%) and lack of pure colonic disease (0% versus 24%) compared to group 2, and their BMI was significantly lower (20.3 kg/m(2) versus 25.0 kg/m(2)). Groups 1 and 2 did not differ with respect to Vienna behaviour at diagnosis, smoking, appendicectomy, extra-intestinal manifestations of CD, or NOD2 SNP variants. There was a trend for low CRP patients with previous ileal resection to evolve to a stricturing phenotype. Fat wrapping was noted in 11/13 (85%) of low CRP patients undergoing ileal resections. CONCLUSIONS: Patients with CD and a persistently low CRP in the face of active disease were characterized by an almost exclusive ileal disease distribution and a low BMI, compared to those with a raised CRP. These patients had a similar frequency and distribution of NOD2/CARD15 variants. Stricturing (v inflammatory or penetrating) behaviour may explain some low CRP. Despite the abnormally low BMI, fat wrapping was noted in the majority of low CRP patients undergoing ileal resection.