Competitive inhibition flow analysis assay for the non-culture-based detection and serotyping of pneumococcal capsular polysaccharide.

Traditional confirmation procedures for the identification of a pneumococcal serotype require an isolate. Non-culture-based confirmation protocols are available. Some of these confirm only the presence of pneumococci, and others are capable of identifying a limited number of serotypes. The increased use of pneumococcal polysaccharide and conjugate vaccines, especially in high-risk patient groups, and the likely increase in the number of serotypes included in future versions of the conjugate vaccines have necessitated the need for improved enhanced surveillance in order to assess their impact on public health. Since 2006, a multiplexed assay has been used at the Health Protection Agency of the United Kingdom for the detection of 14 pneumococcal serotypes which requires pneumococcal serotype-specific monoclonal antibodies (MAbs). We have developed a microsphere competitive inhibition method capable of detecting 23 pneumococcal capsular polysaccharide serotypes in cerebrospinal fluid (CSF) and urine and serotyping pneumococcal suspensions, utilizing an international reference serum, 89-SF. The assay was shown to be reproducible and specific for homologous polysaccharide. Validation of the assay was performed with a selection of MAbs specific for pneumococcal capsular polysaccharide serotypes, which confirmed the specificity of the assay. Analysis of pneumolysin PCR-positive CSF samples in the competitive inhibition assay determined a serotype for 89% of the samples. The assay developed here is well suited to large-scale epidemiologic studies because the assay is simple, robust, and rapid and utilizes readily available resources.

Mental illness and disability among elders in developing countries: the case of Nepal.

OBJECTIVE: This article attempts to document the prevalence of psychiatric disorders among elders in a rural village in Nepal. In addition, we investigate the relationship between psychiatric illness and functional disability to assess the impact of disorder on social functioning. METHOD: A semistructured interview checklist to diagnose six disorders was used (N = 182). In addition, elders older than age 60 were examined to assess the functional impact of mental health conditions by measuring functional disability. RESULTS: Eighteen percent of elders seem to have a diagnosable psychiatric disorder. Furthermore, in general these elders were also less likely to receive assistance with the disabilities they report, compared with those who do not experience a psychiatric disorder. DISCUSSION: Documenting the extent of psychiatric disorder among elders in developing societies sensitizes health planners to the growing reality of aging in their societies and the need for expanded physical and psychiatric health care services.

Surveillance of individuals at intermediate risk of colorectal cancer--the impact of new guidelines.

OBJECTIVE: Individuals with two first degree relatives, or one diagnosed at age < 45 years, with colorectal cancer are at sufficient risk to merit surveillance. Most undergo colonoscopy four to five yearly, starting 10 years before the youngest case. The aim of this study was to assess the impact of a proposed new surveillance protocol. SUBJECTS AND METHODS: We identified individuals with these risk criteria seen in our clinic from 1989 to 2001 and reviewed their notes with respect to colonoscopy. RESULTS: Colonoscopy (n = 295) was performed on 186 patients in accordance with current recommendations. Cancer was detected in three and adenoma in 21 individuals. Applying the proposed protocol, 123 (42%) fewer colonoscopies would have been performed. No cancers would have been missed, but in five cases a small adenoma would not have been detected. CONCLUSIONS: Proposed new guidelines for surveillance of those at intermediate risk reduce the burden of colonoscopy without compromising identification of significant neoplasia.

Measuring community mental health in developing societies: evaluation of a checklist format in Nepal.

BACKGROUND: There is growing recognition of the importance of mental health problems in developing countries. In large part, however, we have very limited epidemiological data at national and/or community levels about the prevalence of mental illnesses. AIMS: The purpose of this paper is to describe the reliability and validity characteristics of an assessment tool that may be useful for conducting community-level surveys (particularly in rural communities of developing countries) to obtain prevalence rates of mental illnesses. METHODS: We used a sample of adults residing in a rural village in Nepal to assess disorders with a modified version of the DSM-III-R Checklist. We evaluated construct validity, scale reliability, convergent validity and discriminant validity. RESULTS: There is strong evidence for the construct validity of generalized anxiety and depression in our sample. By contrast, the symptoms associated with mania and schizophrenia were not empirically distinct. Convergent validity is acceptable. As a test of validity characteristics, the pattern of sociodemographic correlations suggests that the specific social origins of disorder in Nepal will require further investigation. CONCLUSION: The first step in obtaining high quality information on the distribution of mental illness in developing countries is to establish some reliable and valid indicators of disorder. The checklist format for assessing disorder appears to meet this objective and offers the possibility that community-level prevalence studies can be reasonably conducted.

Expression of c-myc is not critical for cell proliferation in established human leukemia lines.

BACKGROUND: A study was undertaken to resolve preliminary conflicting results on the proliferation of leukemia cells observed with different c-myc antisense oligonucleotides. RESULTS: RNase H-active, chimeric methylphosphonodiester / phosphodiester antisense oligodeoxynucleotides targeting bases 1147-1166 of c-myc mRNA downregulated c-Myc protein and induced apoptosis and cell cycle arrest respectively in cultures of MOLT-4 and KYO1 human leukemia cells. In contrast, an RNase H-inactive, morpholino antisense oligonucleotide analogue 28-mer, simultaneously targeting the exon 2 splice acceptor site and initiation codon, reduced c-Myc protein to barely detectable levels but did not affect cell proliferation in these or other leukemia lines. The RNase H-active oligodeoxynucleotide 20-mers contained the phosphodiester linked motif CGTTG, which as an apoptosis inducing CpG oligodeoxynucleotide 5-mer of sequence type CGNNN (N = A, G, C, or T) had potent activity against MOLT-4 cells. The 5-mer mimicked the antiproliferative effects of the 20-mer in the absence of any antisense activity against c-myc mRNA, while the latter still reduced expression of c-myc in a subline of MOLT-4 cells that had been selected for resistance to CGTTA, but in this case the oligodeoxynucleotide failed to induce apoptosis or cell cycle arrest. CONCLUSIONS: We conclude that the biological activity of the chimeric c-myc antisense 20-mers resulted from a non-antisense mechanism related to the CGTTG motif contained within the sequence, and not through downregulation of c-myc. Although the oncogene may have been implicated in the etiology of the original leukemias, expression of c-myc is apparently no longer required to sustain continuous cell proliferation in these culture lines.

Oligodeoxynucleotide 5mers containing a 5'-CpG induce apoptosis through a mitochondrial mechanism in T lymphocytic leukaemia cells.

A chimeric methylphosphonodiester/phosphodiester 15mer oligodeoxynucleotide of randomly selected sequence was observed to rapidly induce apoptosis in MOLT-4 and Jurkat E6 T lymphocytic leukaemia cells following intracytoplasmic delivery. A series of further methylphosphonate substitutions and mutations and truncations of the oligodeoxynucleotide served to establish that the phosphodiester-linked sequence CGGTA present in the 15mer was responsible for this biological activity. End-protected CpG oligodeoxy-nucleotide 5mers of sequence type CGNNN exhibited a range of apoptosis-inducing potencies, with CGTTA being the most active. The latter was shown to significantly reduce the rate of RNA synthesis in MOLT-4 cells within 1 h; DNA laddering and redistribution of phosphatidylserine to the outer surface of the plasma membrane were marked by 160 min and mitochondrial transmembrane potential collapsed over roughly the same time scale. Pro-caspase 8 was reduced within 130 min and the proteolytically activated caspase 8 substrate Bid was also down by this time, implicating release of cytochrome c from mitochondria by the active 15 kDa fragment of Bid. Substantial proteolytic activation of pro-caspase 3 was relatively delayed. These findings support a mitochondrial amplification mechanism for apoptosis triggered by CpG 5mers.

Advising patients with genital warts--a consensus approach.

This article reviews the evidence available to guide practitioners when advising patients with genital warts, by seeking to answer the questions most commonly asked in medical practice. It highlights where evidence is lacking and is intended to inform patients about their condition, and to identify areas where further research is warranted.

Clinical use of streptolysin-O to facilitate antisense oligodeoxyribonucleotide delivery for purging autografts in chronic myeloid leukaemia.

Antisense oligodeoxyribonucleotides (ODN) targeted against the breakpoint in BCR-ABL mRNA will specifically decrease BCR-ABL mRNA, provided cells are first permeabilised with streptolysin-O (SL-O). We used 18-mer chimeric methylphosphonodiester: phosphodiester linked (4-9-4) ODN complementary to 9 bases either side of the BCR-ABL junction to purge harvests ex vivo in three CML patients who remained completely Ph positive after multiple chemotherapy courses. After CD34+ cell selection and SL-O permeabilisation, harvests were purged with 20 microM ODN. After purging, all individual CFU-GM colonies grown from the two b3a2 breakpoint cases remained positive for BCR-ABL mRNA. In contrast, all 24 colonies grown from the b2a2 breakpoint case were BCR-ABL mRNA negative. Patients were conditioned with busulphan 16 mg/kg. The initial post-transplant course was uneventful, although the time to return to 0.5 x 10(9)/l neutrophils was slow at 25-51 days. Both chronic phase patients remain in haematological remission at +724 and +610 days, although each has cytogenetic evidence of relapse. The b2a2 accelerated phase patient died of myeloid blast transformation at day +91. The present SL-O-facilitated ODN purging strategy appears to be without significant toxicity, and offers considerable improvements in ODN delivery to the cytosol.

Evaluation of self-taken samples for the presence of genital Chlamydia trachomatis infection in women using the ligase chain reaction assay.

The aim of this study was to evaluate the sensitivity and acceptability of self-taken vulval-introital (VI) samples, first-catch urine (FCU) samples and clinician-obtained cervical samples for the presence of genital Chlamydia trachomatis infections in women using the ligase chain reaction (LCR) assay. One hundred and four patients were enrolled, of whom 54 patients had chlamydial DNA in at least one of the samples tested. The sensitivity of the cervical sample was 96.3%, vulval-introital sample in LCR buffer 92.6%, vulval-introital swab collected dry 88.9%, FCU stored at +2-8 degrees C 81.5%, FCU stored at room temperature 77.8% and FCU stored with 2% w/v boric acid at room temperature 87.0%. Self-taken vulval-introital LCR samples were shown to be an acceptable alternative to a clinician-obtained LCR sample. The addition of boric acid may overcome the need for a continuous cold chain for FCU samples.

The influence of target protein half-life on the effectiveness of antisense oligonucleotide analog-mediated biologic responses.

During the course of a study aimed at improving antisense oligodeoxynucleotide-mediated ex vivo bone marrow purging of patients suffering from chronic myeloid leukemia (CML), the properties of a number of antisense structures intended to reduce the expression of c-myc, mutant p53, and bcr-abl mRNAs and proteins were examined. The majority of the antisense oligodeoxynucleotides were designed to be capable of directing ribonuclease H (RNase H) cleavage of their target mRNAs. Streptolysin O (SLO) reversible permeabilization was used to deliver the oligodeoxynucleotides into the CML line KYO-1. We found that the efficiency and specificity of antisense oligonucleotide-induced reductions of target protein expression depended on target protein half-life, the oligonucleotide structure, and the specific sequence within the target mRNA. Transient reductions of c-myc mRNA and protein were achieved with a chimeric methylphosphonate-phosphodiester oligodeoxynucleotide antisense to the initiation codon, but cell proliferation was unaffected. In contrast, a chimeric oligodeoxynucleotide of similar structure targeted to an alternative site in the coding region of c-myc mRNA reduced target mRNA and protein levels for over 24 hours and halted cell proliferation. Chimeric methylphosphonate-phosphodiester oligodeoxynucleotide antisense to a point mutation in KYO-1 p53 mRNA efficiently reduced target mRNA expression, but only small, transient reductions in p53 protein expression were observed. However, a chimeric methylphosphonate-phosphorothioate oligodeoxynucleotide targeted to the same site reduced p53 protein to 30% of control levels over a 48-hour period. BCR-ABL protein expression was unaffected by chimeric oligodeoxynucleotides targeted to the breakpoint in bcr-abl mRNA, even when mRNA levels at early times were substantially reduced.

Outcome after multiple colorectal tumours.

BACKGROUND: Patients with primary colorectal cancers have a higher risk of development of second tumours synchronously or metachronously. This special group of patients raise a particular interest in their characteristics and outcome. METHODS: The records of 1009 patients with colorectal cancer were scrutinized. A group with multiple cancers was identified. Perioperative investigations, patterns of follow-up, pathological variables and outcome were noted. RESULTS: There were 22 patients with metachronous tumours and 39 with synchronous tumours following 'curative' operations in 20 and 28 respectively. There was no difference in Dukes classification between the two groups: Polyps were associated with metachronous lesions in ten of 22 patients and synchronous lesions in 17 of 39 patients. Five-year survival was 75 per cent for patients with metachronous tumours and only 18 per cent for those with synchronous tumours. CONCLUSION: In this study patients with metachronous tumours seemed to do very well while those with synchronous lesions did very badly. There were no identifiable demographic or clinical characteristics to account for this. There is a need to study this group of patients and identify factors like tumour biology or host resistance which prevent spread of tumour.

Preclinical studies of streptolysin-O in enhancing antisense oligonucleotide uptake in harvests from chronic myeloid leukaemia patients.

Antisense oligodeoxyribonucleotides (ODN) have been shown to produce a sequence-specific cleavage of BCR-ABL mRNA. They may therefore have clinical potential for purging harvests from chronic myeloid leukaemia (CML) patients, prior to autografting. Whilst ODN are highly effective in cell-free systems, their uptake into intact cells is very poor. We have previously reported that reversible permeabilisation of CML cell lines with Streptolysin-O (SL-O) can dramatically increase intracytoplasmic and nuclear ODN uptake. In this study, we examined whether SL-O permeabilisation could be used to enhance ODN uptake into bone marrow (BM) and peripheral blood stem cell (PBSC) harvests from CML patients, without undue toxicity. All 19 harvests studied were from patients in stable chronic phase of CML. Samples studied were either fresh BM harvests following leucoconcentration, fresh PBSC collections, or from previously cryopreserved harvests. Cells were permeabilised by SL-O to load them with fluorescein-labelled ODN. The proportion of permeabilised and viable cells was assessed by fluorescein uptake and propidium iodide exclusion, respectively, by flow cytometry. The effect of SL-O on ODN uptake and cell toxicity was unpredictable on simple mononuclear fractions of harvests. In contrast, SL-O consistently significantly enhanced ODN uptake in samples which were first selected for CD34-positive cells, and this was achieved without either direct toxicity or inhibition of CFU-GM growth. The SL-O concentration required for optimal permeabilisation varied considerably from case to case, in line with previous data on cell lines. PBSC harvests positively selected for CD34-positive cells tended to achieve superior permeabilisation to CD34 positively selected BM harvests. SL-O can be used to safely enhance the intracellular uptake of antisense ODN. This is best achieved on harvests which are first selected for CD34-positive cells.

Molecular status of individual CFU-GM colonies derived from chemotherapy-mobilised peripheral blood stem cells in chronic myeloid leukaemia.

Following chemotherapy in chronic myeloid leukaemia (CML), some peripheral blood (PB) cells may be Philadelphia (Ph) chromosome negative. The BCR-ABL mRNA status of residual Ph+ progenitors is not known. We examined the BCR-ABL mRNA status of individual colony-forming-unit granulocyte-macrophage (CFU-GM) colonies derived from PB harvested following chemotherapy. Seven patients were treated with 200 mg/m2/day cytarabine and 20 mg/m2/day Idarubicin and followed by Lenograstim. PB collections commenced daily when the white blood cell count reached 0.6 x 10(9)/l and continued until at least 5 x 10(8)/kg nucleated cells were obtained. CD34+ cells, Ph status, and CFU-GM were estimated at each harvest. For each patient, up to 24 individual CFU-GM colonies were analysed for BCR-ABL status. Two cases were BCR-ABL negative on all colonies and completely Ph-, and another case was BCR-ABL positive in all colonies and completely Ph+. In contrast, in two patients all colonies were BCR-ABL negative, despite virtually complete Ph+ metaphases. The final assessible case had five of nine BCR-ABL negative colonies, despite 94% Ph+ metaphases. After chemotherapy priming, the PB may contain Ph+ CFU-GM that do not express BCR-ABL.

Faecal occult blood screening for colorectal neoplasia in a targeted high-risk population.

A general practice-based programme was initiated in 1987 to identify individuals at high risk of developing large bowel cancer and offer them screening of faecal occult blood. In all, 5298 people from 21 general practices in the Guildford area were offered 7510 screening tests. In total, 5934 tests were completed (compliance rate 79.0 per cent) with 287 positive results (4.8 per cent). Of the patients with positive results, 44 had cancer and 38 polyps. The sensitivity of the test for cancer was 63 per cent, the specificity 96 per cent and the positive predictive value for all neoplasia 29 per cent. The detection rate of 44 cancers per 5934 people screened compares favourably with data from the Nottingham-based screening of an unselected population (0.74 versus 0.23 per cent).

Extent of mesorectal spread and involvement of lateral resection margin as prognostic factors after surgery for rectal cancer.

The extent of tumour growth beyond the muscularis propria (mesorectal spread) was measured in specimens from 167 consecutive patients with rectal cancer. The 5-year survival was significantly greater in patients with slight mesorectal spread (4 mm or less) than in those with more extensive mesorectal spread (55% [95% confidence interval 42-66%] vs 25% [13-38%]). The prognostic value for survival of mesorectal spread was independent of the presence of lymphnode metastases. There were also significant differences in survival between patients with slight and extensive mesorectal spread among patients with Dukes' stage B tumours (66% [41-82%] vs 37% [14-60%]) and those with Dukes' stage C tumours (30% [12-52%] vs 18% [6-34%]). Thus mesorectal spread of rectal cancer is an important determinant of survival, and its accurate measurement may serve to subdivide Dukes' B and C cases. In this study tumour involvement of the lateral resection margin was not a useful predictor of local recurrence, but it did correlate with poor prognosis.