Phase I, first-in-human study of MSC-1 (AZD0171), a humanized anti-leukemia inhibitory factor monoclonal antibody, for advanced solid tumors.

BACKGROUND: Activation of leukemia inhibitory factor (LIF) is linked to an immunosuppressive tumor microenvironment (TME), with a strong association between LIF expression and tumor-associated macrophages (TAMs). MSC-1 (AZD0171) is a humanized monoclonal antibody that binds with high affinity to LIF, promoting antitumor inflammation through TAM modulation and cancer stem cell inhibition, slowing tumor growth. In this phase I, first-in-human, open-label, dose-escalation study, MSC-1 monotherapy was assessed in patients with advanced, unresectable solid tumors. MATERIALS AND METHODS: Using accelerated-titration dose escalation followed by a 3 + 3 design, MSC-1 doses of 75-1500 mg were administered intravenously every 3 weeks (Q3W) until progression or unmanageable toxicity. Additional patients were enrolled in selected cohorts to further evaluate safety, pharmacokinetics (PK), and pharmacodynamics after escalation to the next dose had been approved. The primary objective was characterizing safety and determining the recommended phase II dose (RP2D). Evaluating antitumor activity and progression-free survival (PFS) by RECIST v1.1, PK and immunogenicity were secondary objectives. Exploratory objectives included pharmacodynamic effects on circulating LIF and TME immune markers. RESULTS: Forty-one patients received treatment. MSC-1 monotherapy was safe and well tolerated at all doses, with no dose-limiting toxicities. The maximum tolerated dose was not reached and the RP2D was determined to be 1500 mg Q3W. Almost half of the patients had treatment-related adverse events (TRAEs), with no apparent trends across doses; no patients withdrew due to TRAEs. There were no objective responses; 23.7% had stable disease for ≥2 consecutive tumor assessments. Median PFS was 5.9 weeks; 23.7% had PFS >16 weeks. On-treatment changes in circulating LIF and TME signal transducers and activators of transcription 3 signaling, M1:M2 macrophage populations, and CD8+ T-cell infiltration were consistent with the hypothesized mechanism of action. CONCLUSIONS: MSC-1 was very well tolerated across doses, with prolonged PFS in some patients. Biomarker and preclinical data suggest potential synergy with checkpoint inhibitors.

CD133+ brain tumor-initiating cells are dependent on STAT3 signaling to drive medulloblastoma recurrence.

Medulloblastoma (MB), the most common malignant paediatric brain tumor, is currently treated using a combination of surgery, craniospinal radiotherapy and chemotherapy. Owing to MB stem cells (MBSCs), a subset of MB patients remains untreatable despite standard therapy. CD133 is used to identify MBSCs although its functional role in tumorigenesis has yet to be determined. In this work, we showed enrichment of CD133 in Group 3 MB is associated with increased rate of metastasis and poor clinical outcome. The signal transducers and activators of transcription-3 (STAT3) pathway are selectively activated in CD133+ MBSCs and promote tumorigenesis through regulation of c-MYC, a key genetic driver of Group 3 MB. We screened compound libraries for STAT3 inhibitors and treatment with the selected STAT3 inhibitors resulted in tumor size reduction in vivo. We propose that inhibition of STAT3 signaling in MBSCs may represent a potential therapeutic strategy to treat patients with recurrent MB.

Quantifying intrinsic and extrinsic control of single-cell fates in cancer and stem/progenitor cell pedigrees with competing risks analysis.

The molecular control of cell fate and behaviour is a central theme in biology. Inherent heterogeneity within cell populations requires that control of cell fate is studied at the single-cell level. Time-lapse imaging and single-cell tracking are powerful technologies for acquiring cell lifetime data, allowing quantification of how cell-intrinsic and extrinsic factors control single-cell fates over time. However, cell lifetime data contain complex features. Competing cell fates, censoring, and the possible inter-dependence of competing fates, currently present challenges to modelling cell lifetime data. Thus far such features are largely ignored, resulting in loss of data and introducing a source of bias. Here we show that competing risks and concordance statistics, previously applied to clinical data and the study of genetic influences on life events in twins, respectively, can be used to quantify intrinsic and extrinsic control of single-cell fates. Using these statistics we demonstrate that 1) breast cancer cell fate after chemotherapy is dependent on p53 genotype; 2) granulocyte macrophage progenitors and their differentiated progeny have concordant fates; and 3) cytokines promote self-renewal of cardiac mesenchymal stem cells by symmetric divisions. Therefore, competing risks and concordance statistics provide a robust and unbiased approach for evaluating hypotheses at the single-cell level.

Kaiso depletion attenuates transforming growth factor-ß signaling and metastatic activity of triple-negative breast cancer cells.

Triple-negative breast cancers (TNBCs) represent a subset of breast tumors that are highly aggressive and metastatic, and are responsible for a disproportionate number of breast cancer-related deaths. Several studies have postulated a role for the epithelial-to-mesenchymal transition (EMT) program in the increased aggressiveness and metastatic propensity of TNBCs. Although EMT is essential for early vertebrate development and wound healing, it is frequently co-opted by cancer cells during tumorigenesis. One prominent signaling pathway involved in EMT is the transforming growth factor-ß (TGFß) pathway. In this study, we report that the novel POZ-ZF transcription factor Kaiso is highly expressed in TNBCs and correlates with a shorter metastasis-free survival. Notably, Kaiso expression is induced by the TGFß pathway and silencing Kaiso expression in the highly invasive breast cancer cell lines, MDA-MB-231 (hereafter MDA-231) and Hs578T, attenuated the expression of several EMT-associated proteins (Vimentin, Slug and ZEB1), abrogated TGFß signaling and TGFß-dependent EMT. Moreover, Kaiso depletion attenuated the metastasis of TNBC cells (MDA-231 and Hs578T) in a mouse model. Although high Kaiso and high TGFßR1 expression is associated with poor overall survival in breast cancer patients, overexpression of a kinase-active TGFßR1 in the Kaiso-depleted cells was insufficient to restore the metastatic potential of these cells, suggesting that Kaiso is a key downstream component of TGFß-mediated pro-metastatic responses. Collectively, these findings suggest a critical role for Kaiso in TGFß signaling and the metastasis of TNBCs.

Insecticide Efficacy and Timing for Control of Western Bean Cutworm (Lepidoptera: Noctuidae) in Dry and Snap Beans.

The western bean cutworm, Striacosta albicosta (Smith) (Lepidoptera: Noctuidae), is a recent pest of corn, dry,and snap beans, in the Great Lakes region, and best practices for its management in beans need to be established.Insecticide efficacy and application timing field studies, conducted in 2011­2013, determined that lambda-cyhalothrin and chlorantraniliprole were capable of reducing western bean cutworm feeding damage in dry beans from 2.3 to 0.4% in preharvest samples, and in snap beans from 4.8 to 0.1% of marketable pods, respectively. The best application timing in dry beans was determined to be 4­18 d after 50% egg hatch. No economic benefit was found when products were applied to dry beans, and despite high artificial inoculation rates, damage to marketable yield was relatively low. Thiamethoxam, methoxyfenozide, and spinetoram were also found to be effective at reducing western bean cutworm damage in dry bean to as low as 0.3% compared to an untreated control with 2.5% damaged pods. In snap beans, increased return on investment between CAD$400 and CAD$600 was seen with multiple applications of lambda-cyhalothrin, and with chlorantraniliprole applied 4 d after egg mass infestation.

Detection of single-nucleotide polymorphisms in Plasmodium falciparum by PCR primer extension and lateral flow immunoassay.

The resistance of Plasmodium falciparum to some antimalarial drugs is linked to single-nucleotide polymorphisms (SNPs). Currently, there are no methods for the identification of resistant parasites that are sufficiently simple, cheap, and fast enough to be performed at point-of-care, i.e., in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum positioned at amino acids 86, 184, and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1) and amino acid 76 of the chloroquine resistance transporter gene (pfcrt). The PEXT products were visualized by a nucleic acid lateral flow immunoassay (NALFIA) with carbon nanoparticles as the detection labels. PCR-PEXT-NALFIAs showed good correlation to the reference methods, quantitative PCR (qPCR) or direct amplicon sequence analysis, in an initial open-label evaluation with 17 field samples. The tests were further evaluated in a blind study design in a set of 150 patient isolates. High specificities of 98 to 100% were found for all 4 PCR-PEXT genotyping assays. The sensitivities ranged from 75% to 100% when all PEXT-positive tests were considered. A number of samples with a low parasite density were successfully characterized by the reference methods but failed to generate a result in the PCR-PEXT-NALFIA, particularly those samples with microscopy-negative subpatent infections. This proof-of principle study validates the use of PCR-PEXT-NALFIA for the detection of resistance-associated mutations in P. falciparum, particularly for microscopy-positive infections. Although it requires a standard thermal cycler, the procedure is cheap and rapid and thus a potentially valuable tool for point-of-care detection in developing countries.

Mammary glands exhibit molecular laterality and undergo left-right asymmetric ductal epithelial growth in MMTV-cNeu mice.

Significant left-right (L-R) differences in tumor incidence and disease outcome occur for cancers of paired organs, including the breasts; however, the basis for this laterality is unknown. Here, we show that despite their morphologic symmetry, left versus right mammary glands in wild-type mice have baseline differences in gene expression that are L-R independently regulated during pubertal development, including genes that regulate luminal progenitor cell renewal, luminal cell differentiation, mammary tumorigenesis, tamoxifen sensitivity and chemotherapeutic resistance. In MMTV-cNeu(Tg/Tg) mice, which model HER2/Neu-amplified breast cancer, baseline L-R differences in mammary gene expression are amplified, sustained or inverted in a gene-specific manner and the mammary ductal epithelium undergoes L-R asymmetric growth and patterning. Comparative genomic analysis of mouse L-R mammary gene expression profiles with gene expression profiles of human breast tumors revealed significant linkage between right-sided gene expression and decreased breast cancer patient survival. Collectively, these findings are the first to demonstrate that mammary glands are lateralized organs, and, moreover, that mammary glands have L-R differential susceptibility to HER2/Neu oncogene-mediated effects on ductal epithelial growth and differentiation. We propose that intrinsic molecular laterality may have a role in L-R asymmetric breast tumor incidence and, furthermore, that interplay between the L-R molecular landscape and oncogene activity may contribute to the differential disease progression and patient outcome that are associated with tumor situs.

Autoantibodies to MUC1 glycopeptides cannot be used as a screening assay for early detection of breast, ovarian, lung or pancreatic cancer.

BACKGROUND: Autoantibodies have been detected in sera before diagnosis of cancer leading to interest in their potential as screening/early detection biomarkers. As we have found autoantibodies to MUC1 glycopeptides to be elevated in early-stage breast cancer patients, in this study we analysed these autoantibodies in large population cohorts of sera taken before cancer diagnosis. METHODS: Serum samples from women who subsequently developed breast cancer, and aged-matched controls, were identified from UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) and Guernsey serum banks to formed discovery and validation sets. These were screened on a microarray platform of 60mer MUC1 glycopeptides and recombinant MUC1 containing 16 tandem repeats. Additional case-control sets comprised of women who subsequently developed ovarian, pancreatic and lung cancer were also screened on the arrays. RESULTS: In the discovery (273 cases, 273 controls) and the two validation sets (UKCTOCS 426 cases, 426 controls; Guernsey 303 cases and 606 controls), no differences were found in autoantibody reactivity to MUC1 tandem repeat peptide or glycoforms between cases and controls. Furthermore, no differences were observed between ovarian, pancreatic and lung cancer cases and controls. CONCLUSION: This robust, validated study shows autoantibodies to MUC1 peptide or glycopeptides cannot be used for breast, ovarian, lung or pancreatic cancer screening. This has significant implications for research on the use of MUC1 in cancer detection.

Gamma-secretase inhibitors target tumor-initiating cells in a mouse model of ERBB2 breast cancer.

Human breast tumors comprise a minor sub-population of tumor-initiating cells (TICs), commonly termed cancer stem cells. TICs are thought to sustain tumor growth and to confer resistance to current anticancer therapies. Hence, targeting TIC may be essential to achieving durable cancer cures. To identify molecular targets in breast TIC, we employed a transgenic mouse model of ERBB2 breast cancer; tumors arising in this model comprise a very high frequency of TIC, which is maintained in tumor cell populations propagated in vitro as non-adherent tumorspheres. The Notch pathway is dysregulated in human breast tumors and overexpression of constitutively active Notch proteins induces mammary tumors in mice. The Notch pathway has also been implicated in stem cell processes including those of mammary epithelial stem cells. Hence, we investigated the potential that the Notch pathway is required for TIC activity. We found that an antagonist of Notch signaling, a gamma (γ)-secretase inhibitor termed MRK-003, inhibited the survival of tumorsphere-derived cells in vitro and eliminated TIC as assessed by cell transplantation into syngeneic mice. Whereas MRK-003 also inhibited the self-renewal and/or proliferation of mammosphere-resident cells, this effect of the inhibitor was reversible thus suggesting that it did not compromise the survival of these cells. MRK-003 administration to tumor-bearing mice eliminated tumor-resident TIC and resulted in rapid and durable tumor regression. MRK-003 inhibited the proliferation of tumor cells, and induced their apoptosis and differentiation. These findings suggest that MRK-003 targets breast TIC and illustrate that eradicating these cells in breast tumors ensures long-term, recurrence-free survival.

MR imaging of parotid tumors: typical lesion characteristics in MR imaging improve discrimination between benign and malignant disease.

BACKGROUND AND PURPOSE: The surgical approach to parotid tumors is different for benign and malignant neoplasms, but the clinical symptoms do not correlate well with histology. Difficulties in tumor classification also arise in imaging modalities, in which sonography has the lowest and MR imaging, the highest accuracy. The purpose of this study was to review our experience using conventional MR imaging of the neck in the evaluation of parotid tumors and to evaluate which MR imaging findings are best able to predict malignant histology. MATERIALS AND METHODS: Eighty-four consecutive patients (43 males, 41 females; median age, 56 years; range, 9-85 years) with parotid gland tumors who underwent MR imaging before surgery were prospectively included in the present study and retrospectively analyzed. Histology was available for all tumors. We analyzed the following MR imaging parameters: signal intensity, contrast enhancement, lesion margins (well-defined versus ill-defined), lesion location (deep/superficial lobe), growth pattern (focal, multifocal, or diffuse), and extension into neighboring structures, perineural spread, and lymphadenopathy. RESULTS: The 57 (68%) benign and 27 (32%) malignant tumors consisted of 29 pleomorphic adenomas, 17 Warthin tumors, 11 various benign tumors, 5 mucoepidermoid carcinomas, 3 adenoid cystic carcinomas, 1 acinic cell carcinoma, 1 carcinoma ex pleomorphic adenoma, 9 metastases, and 8 various malignant neoplasms. Specific signs predictive of malignancy were the following: T2 hypointensity of the parotid tumor (P = .048), ill-defined margins (P = .001), diffuse growth (P = .012), infiltration of subcutaneous tissue (P = .0034), and lymphadenopathy (P = .012). CONCLUSIONS: Low signal intensity on T2-weighted images and postcontrast ill-defined margins of a parotid tumor are highly suggestive of malignancy.

Modeling distribution and abundance of soybean aphid in soybean fields using measurements from the surrounding landscape.

Soybean aphid (Aphis glycines Matsumura) is a severe pest of soybean in central North America. Outbreaks of the aphid in Ontario are often spotty in distribution, with some geographical areas affected severely and others with few or no aphid populations occurring in soybean for the duration of the season. A. glycines spend summers on soybean and overwinter on buckthorn, a shrub that is widespread in southern Ontario and is commonly found in agricultural hedgerows and at the margins of woodlots. A. glycines likely use both short distance migratory flights from buckthorn and longer distance dispersal flights in the search for acceptable summer hosts. This study aims to model colonization of soybean fields by A. glycines engaged in early-season migration from overwintering hosts. Akaike's information criterion (AIC) was used to rank numerous competing linear and probit models using field parameters to predict aphid presence, colonization, and density. The variable that best modeled aphid density in soybean fields in the early season was the ratio of buckthorn density to field area, although dramatic differences in relationships between the parameters were observed between study years. This study has important applications in predicting areas that are at elevated risk of developing economically damaging populations of soybean aphid and which may act as sources for further infestation.

Predation by Coccinella septempunctata and Harmonia axyridis (Coleoptera: Coccinellidae) on Aphis glycines (Homoptera: Aphididae).

Coccinella septempunctata L. and Harmonia axyridis Pallas are key natural enemies of soybean aphid, Aphis glycines Matsumura, in North America. Third instars, adult females, and adult males of both C. septempunctata and H. axyridis exhibited a type II functional response for predation toward adult soybean aphids at 26 +/- 1 degrees C. In C. septempunctata, the functional response curve of adult males differed from those of third instars and adult females, but there was no difference between third instars and adult females. In H. axyridis, the functional response curves of larvae, adult females, and adult males all differed significantly. Third instars and adult females consumed significantly more soybean aphids than did adult males at prey densities of 150 and 180 aphids per arena for C. septempunctata and at prey densities of 60, 90, 120, 150, and 180 aphids per arena for H. axyridis. The theoretical maximum daily predation rate of adult aphids by C. septempunctata was predicted to be 204 per third instar, 277 per adult female, and 166 per adult male, and 244, 156, and 73, respectively, for H. axyridis. Third instars and adult females of both species consumed significantly more aphids than did adult males on soybean plants with the recommended action threshold of 250 soybean aphids per plant. Both C. septempunctata and H. axyridis have high predation capacities and are important in suppressing soybean aphid populations.

Role of visual and olfactory cues from agricultural hedgerows in the orientation behavior of multicolored Asian lady beetle (Coleoptera: Coccinellidae).

Harmonia axyridis Pallas is an introduced lady beetle common in eastern North American agroecosystems. Two-choice behavioral bioassays were performed to determine whether visual and olfactory stimuli from prey and host habitats could elicit taxis in wild-collected H. axyridis adults and whether beetles exhibit a preference among stimuli. Soybean aphid (Aphis glycines Matsumura) spends much of the year in agricultural hedgerows residing on buckthorn (Rhamnus cathartica L), and H. axyridis is frequently observed feeding on aphids in this habitat. Olfactory bioassays were performed in a Y-tube olfactometer and tested the response of beetles to the odor of buckthorn leaves, apple leaves (Malus domestica Borkh.), and buckthorn leaves both naturally and artificially infested with A. glycines. No differences were observed between the numbers of beetles moving toward the odor of buckthorn artificially infested with A. glycines and uninfested buckthorn, but more beetles preferred naturally infested buckthorn over uninfested buckthorn. Visual bioassays were performed in an acrylic tube arena,and tested beetle response to silhouettes and to apple and buckhorn leaves. Beetles were significantly more likely to choose silhouettes over blank space in visual trials. Significantly more beetles moved toward buckthorn leaves than blank space, but beetles did not discern between apple and buckthorn until olfactory cues were also included. This study lays the foundation for future work examining the response of H. axyridis to visual and olfactory cues in Ontario agroecosystems, which could help enhance effectiveness of H. axyridis as a biological control and mitigate its impacts as a pest species.

Amodiaquine and artemether-lumefantrine select distinct alleles of the Plasmodium falciparum mdr1 gene in Tanzanian children treated for uncomplicated malaria.

The artemisinin-based combination therapies artemether-lumefantrine (AL) and amodiaquine (AQ) plus artesunate have been adopted for treatment of Plasmodium falciparum malaria in many African countries. Molecular markers of parasite resistance suitable for surveillance have not been established for any of the component drugs in either of these combinations. We assessed P. falciparum mdr1 (Pfmdr1) alleles present in 300 Tanzanian children presenting with uncomplicated falciparum malaria, who were enrolled in a clinical trial of antimalarial therapy. Pfmdr1 genotype analysis was also performed with isolates from 182 children who failed AQ monotherapy and 54 children who failed AL treatment. Pfmdr1 alleles 86Y, 184Y, and 1246Y were more common among treatment failures in the AQ group than among pretreatment infections. The converse was found in the AL-treated group. Children presenting with the 86Y/184Y/1246Y Pfmdr1 haplotype and treated with AQ were significantly more likely to retain this haplotype if they were parasite positive during posttreatment follow-up than were children treated with AL (odds ratio, 33.25; 95% confidence interval, 4.17 to 1441; P, <0.001). We conclude that AL and AQ exert opposite within-host selective effects on the Pfmdr1 gene of P. falciparum.

Parental reporting of smelly urine and urinary tract infection.

BACKGROUND: Parents often report that young children have "smelly urine" or a particular urinary odour. There is little evidence that these observations are relevant to the diagnosis of urinary tract infection (UTI). AIMS: To determine whether parental reporting of smelly urine is of any relevance to the diagnosis of UTI in children less than 6 years of age. METHODS: Parents whose children were having urine collected as part of their admission to a large district hospital were given a simple questionnaire to complete regarding the current smell of their child's urine. Parents were asked whether their child's urine smelled different from usual or had a particular smell. Microscopy and culture results of the child's urine were compared to their parent's questionnaire answers to see if there was a association between parental reporting of a different or particular urine smell and a diagnosis of UTI. RESULTS: One hundred and ten questionnaires and urine samples were obtained. Fifty two per cent of parents thought that their child's urine smelled different from usual or had a particular smell. Only 6.4% of children were diagnosed as having a UTI. There was no statistically significant association between parental reporting of abnormal urine smell and diagnosis of UTI. CONCLUSION: In determining whether a young child has a UTI, asking parents about urine smell is unlikely to be of benefit.

Polymerase chain reaction-restriction fragment-length polymorphism method to distinguish Liriomyza huidobrensis from L. Langei (Diptera: Agromyzidae) applied to three recent leafminer invasions.

A molecular method is presented for differentiating the morphologically cryptic leafminers Liriomyza langei Frick and L. huidobrensis (Blanchard). This method requires polymerase chain reaction (PCR) amplification of a 1031-bp region of mitochondrial cytochrome oxidase DNA followed by restriction fragment analysis using the restriction enzymes SpeI and EcoRV. Spel cuts the mitochondrial fragment of L. langei into two fragments, but does not cut the L. huidobrensis fragment. EcoRV cuts the L. huidobrensis fragment into two fragments, but does not cut the L. langei fragment. This PCR-restriction fragment-length polymorphism (RFLP) method is faster and less costly than DNA sequencing,which is currently the only other way to differentiate these two species. We apply the method to samples from recently introduced leafminer populations in Sri Lanka, Canada, and South Africa and find that the invasive leafminer in all three locations is L. huidobrensis.

Lack of association between hepatotropic transfusion transmitted virus infection and oral lichen planus in British and Italian populations.

BACKGROUND: A possible association between oral lichen planus (OLP) and chronic hepatic disease has been found in some populations, although this is probably geographically influenced. In 1997 a new hepatotropic virus, transfusion transmitted virus (TTV), was identified but has not been studied in relation to OLP. OBJECTIVE: The present investigation evaluated the genoprevalence of TTV DNA in the sera of British and Italian patients suffering from OLP using two different sets of primers to identify TTV subgenomic DNA. METHODS: Study groups comprised 40 adult subjects (21 British, 19 Italian) with OLP. For each country, two control groups, a disease-control group and a healthy-control group, were included. The presence of TTV DNA in the sera of patients and control subjects was assessed using two different polymerase chain reactions (PCR) and DNA sequence analysis. RESULTS: Statistical analysis did not reveal evidence of any association between TTV infection and OLP or country of residence. CONCLUSION: An association between TTV and OLP is unlikely.

The epidemiology of adenovirus infections in Greater Manchester, UK 1982-96.

Data relating to 3313 adenovirus isolates from patients in Greater Manchester, UK between 1982 and 1996 were analysed using chi2 tests and 95% confidence intervals. Of the 3098 isolates that were typed, 18.6% were serotype 2, 14.9% serotype 3, 12.1% serotype 1 and 10.9% serotype 41. There was evidence of a seasonal occurrence of serotype 7 (March-August), serotype 2 (January-April), serotype 4 (June-August) and subgenus F (September-November). Children less than 5 years old were the most common group of patients with adenovirus infection (61.3%) compared to 24.2% for adults and only 5.6% for school children (5-15 years). Gastric symptoms were the most common amongst infants (47.6%) followed by respiratory (27.5%) and general symptoms (12.9%). In adults, the overwhelming clinical condition was conjunctivitis (88.9%). Despite the traditional association with adenoviruses, remarkably few cases of pharyngoconjunctival fever were recorded (1.7%).

Molecular epidemiology of a large outbreak of hepatitis B linked to autohaemotherapy.

BACKGROUND: Unregulated skin-piercing procedures potentially facilitate the transmission of bloodborne pathogens. In February, 1998, a patient who had recently received autohaemotherapy at an alternative medicine clinic in the UK was diagnosed with acute hepatitis B. The autohaemotherapy procedure involved the drawing of 1 mL of the patient's blood, mixing with saline, and reinjection of the autologous blood mixture. We investigated the extent of hepatitis B virus (HBV) infection in patients and staff of the clinic. METHODS: Patients who had attended the clinic between January, 1997, and February, 1998, were tested for serological markers of HBV, and for HBV DNA by PCR. HBV DNA was sequenced to assess the relatedness of the virus identified in the cases. We analysed the number and dates of visits with regard to HBV status. FINDINGS: Serum samples were received from 352 patients and four staff members. Serological evidence of exposure to HBV was found in 57 (16%). Of the 33 patients and staff who were positive for hepatitis B surface antigen, 30 (91%) showed complete nucleotide identity in the DNA segments derived from the surface and core genes. Five patients with linked infection had markers of chronic hepatitis B, and one of these was regarded as the likely source of the outbreak. The attack rate was associated with the number of visits (p<0.0001) and the week of visit (p=0.011). Contaminated saline in a repeatedly used bottle was the probable vehicle of transmission. INTERPRETATION: We have described a large community-based outbreak of hepatitis B due to transmission by a single HBV variant. Our findings emphasise the continuing risk of transmission of bloodborne viruses in all health-care settings where skin-piercing procedures are used.

Characterization of a highly divergent TT virus genome.

A novel TT virus (TTV)-like DNA sequence was detected in the serum of a patient (PM) with acute non-A-E hepatitis. The full-length genome sequence, referred to here as PM virus (PMV), was obtained and its relationship to other full or near full-length TTV sequences examined. Although it shares a common genomic arrangement and short conserved regions, the majority of the genome is extremely divergent, displaying an average genetic distance of 0.60 from all other TTV sequences. By comparing PMV with TTV genomes representing the most divergent types so far described, six major groups can be distinguished. The level of genetic diversity seen between these genomes is higher than would be expected within a single virus species. Indeed, PMV could be considered the prototype of an independent taxonomic group within the Circoviridae: family. A genoprevalence study of sera from blood donors and patients with acute hepatitis suggests that PMV is rare.