Automated telephone reminder messages can assist electronic diabetes care

Telephone reminder systems have been used to assist in the treatment of many chronic diseases. However, it is unclear if these systems can increase medication and appointment adherence in patients with diabetes without direct patient-provider telephone contact. We tested the feasibility of using an automated telephone reminder system (ATRS) to deliver reminder messages to 253 adults with diabetes enrolled in a randomized controlled trial. Eighty-four percent of the patients were able to register using voice recognition and at least one reminder was delivered to 95% of registered patients over a period of 7.5 months. None of the demographic features studied predicted a patient's ability to enroll or to receive reminder calls. At the end of the study, 63% of patients indicated that they wished to continue to receive ATRS calls. The level of system use as determined by the number of received reminder calls was not associated with a change in the number of physician visits or diabetes-related laboratory tests during follow-up. The clinical benefits and sustainability of ATRS remain unproven, but our results indicate that an automated reminder system can be effective for providing messages to a large group of older patients with diabetes.     

A Two-Tube Combined TaqMan/SYBR Green Assay to Identify Mycobacteria and Detect Single Global Lineage-Defining Polymorphisms in Mycobacterium tuberculosis

We have developed a novel real-time PCR assay to identify and perform preliminary genotyping of mycobacteria in a manner tailored to our local service. Within a single thermocycler run, mycobacterial 16S rDNA and the Mycobacterium tuberculosis global lineage-defining RD750 polymorphism are targeted in separate reaction tubes, each of which includes both TaqMan and SYBR Green chemistries. The results of this 16S-RD assay differentiate M. tuberculosis complex (MTBC) from nontuberculous mycobacteria (NTM) and recognize whether or not MTBC isolates belong to the East African-Indian lineage, the single most frequently isolated global MTBC lineage in our service. If required, NTM amplicons may be sequenced to provide more specific identities. We report the technical performance of this assay on 88 mycobacteria-positive cultures and discuss its use in the initial management of mycobacterial infections. The 16S-RD assay correctly identified all 70 MTBC-positive cultures and 17 NTM-positive cultures while contemporaneously recognizing 26 MTBC isolates as within and 44 outside the East African-Indian lineage. In artificial samples, the combined assay also showed limited potential to detect mixed mycobacterial infections (MTBC/NTM) and tuberculosis infections involving more than one global MTBC lineage. The approach we have established can be readily tailored to targets of particular value for any mycobacterial diagnostic service, thereby optimizing the value of the results for local clinical and public health management of mycobacterial infections.When a possible case of mycobacterial infection is detected, both the clinical and public health management depend critically on whether the causative agent is Mycobacterium tuberculosis or a nontuberculous mycobacterium (NTM). In the former case, treatment and prevention of further person-to-person transmission are urgent concerns while, in the latter, infections are predominantly sporadic, derive from environmental sources of infection, and often reflect pre-existing conditions in the patient.^1,2,3 Although there are several promising direct molecular assays for M. tuberculosis,^4,5,6,7,8,9 culture is the most sensitive means to detect specific mycobacterial infections and remains the mainstay of diagnostic services in well-resourced laboratories. However, when cultures are detected positive and the presence of mycobacteria confirmed by acid fast staining, it is not known whether the isolated mycobacteria belong to the Mycobacterium tuberculosis complex (MTBC) or the NTM group. Furthermore, when the possibility of recent transmission of tuberculosis (TB) arises, no information is available regarding the relatedness of the isolate to other contemporary isolates with potential epidemiological connections to the new case.Cultures positive for mycobacteria are referred for further testing to address these points and, in the UK, this generally involves transmission to a reference laboratory where the caseload enables economies of scale in deploying established molecular identification and MTBC genotyping procedures. Thus, definitive recognition of infections as MTBC or NTM and the detection of potential epidemiological links may be delayed by days in the former case and weeks in the latter. In our service, where TB and NTM infections affect between 200 and 400 patients per annum, these delays may affect the management of a significant number of patients. Until identity and genotyping information is available, a precautionary response to a particular case in which mycobacteria have been isolated may lead to inappropriate isolation and treatment of patients for TB and unnecessary public health investigations.To address these issues, we have developed a real-time PCR assay for analysis of mycobacteria-positive cultures. The assay targets the mycobacterial 16S rDNA and the global lineage-defining RD750 polymorphism in separate reactions within a single PCR run; it also combines TaqMan and SYBR Green technologies to simultaneously differentiate two products in each reaction tube. The 16S assay distinguishes between MTBC and NTM by combining newly designed universal mycobacterial 16S primers and a MTBC-specific 16S-targeted TaqMan probe. The RD assay enables differentiation of MTBC strains with intact (RD750⁺) and deleted RD750 (RD750⁻) regions by use of hemi-nested primers and an RD750⁺-specific TaqMan probe. SYBR Green chemistry is used in both assays to obtain a total PCR amplicon readout. The principles of the combined 16S-RD assay are illustrated in Figures 1 and ​and22.Open in a separate windowFigure 1Schematic illustration of the combined 16S-RD assay. A: For the 16S assay (tube 1), TaqMan (T) signals show the presence or absence of MTBC-specific 16S rDNA sequence, whereas the SYBR Green (S) signals indicate the presence or absence of Mycobacterium genus-specific 16S rDNA sequence. B: For the RD assay (tube 2), T signals show the presence or absence of RD750, whereas the S signals act as further confirmation of MTBC-specific sequence and a positive assay control when the T signal is negative. T+ S− signals would be anomalous in both assays, indicating amplicons that do not interact with SYBR Green; no results were obtained in this category.Open in a separate windowFigure 2Diagrammatic representation of the RD assay targets.RD750 was first described as a deletion in Rv1519 present in M. tuberculosis strain CH, a local isolate responsible for a large school-associated TB outbreak in Leicester in 2001.¹⁰ This large sequence polymorphism was subsequently designated RD750 and recognized as one of six phylogeographical lineage-defining deletions in a global collection of M. tuberculosis strains.¹¹ It defines the East African-Indian (EA-I) lineage,¹¹ the differentiation of which is also supported by genomic and multilocus sequencing data^11,12 as well as spoligotyping. By the latter method, it is also designated as the Central Asian lineage.¹³ Intriguingly, the RD750 deletion has been associated with an immunosubversive phenotype¹⁴; it also occurs in approximately half of our local M. tuberculosis isolates (J. Malkin and H. Perera, unpublished data). The predominance of this lineage among Leicester M. tuberculosis isolates likely reflects our diverse ethnic population. In the 2001 census, 31% of Leicester city was reported to have an Asian ethnic background with the majority being Indian from either East Africa or Gujarat.¹⁵ In accordance with previous studies in the UK, our regional genotyping service reports a preponderance of the five-locus variable-number tandem repeat (VNTR) profile x2234/5 in this ethnic group.¹⁶ In this context, it will be appreciated that, in our service, early recognition of whether new M. tuberculosis isolates belong to the EA-I group through a single rapid assay offers the highest probability of obtaining strain information that might be discriminatory in a local context. For example, where two potentially linked cases yield RD750⁺and RD750⁻ isolates, transmission can be excluded. Also, in the longer term, specific identification of M. tuberculosis lineages will facilitate detection of associations between clinical phenotypes and bacterial genotypes.^17,18,19 This approach can be adapted to target any lineage-defining polymorphisms that are appropriate to a particular local service.In the present study, we report evaluation of the 16S-RD assay on positive cultures. A by-product of the assay design (Figure 1A and 1B) is that is has the potential to detect mixed infections (MTBC + NTM or RD750⁺ + RD750⁻) in clinical specimens, a phenomenon previously recognized by molecular analysis.²⁰ We therefore also report on the performance of our combined assay to detect mixed mycobacterial infections.     

V(D)J germline gene repertoire analysis of monoclonal D antibodies and the implications for D epitope specificity

BACKGROUND: The D antigen is a highly immunogenic human RBC antigen. Alloimmunization against the D antigen produces high-affinity antibodies that cause hemolytic transfusion reactions and HDN. STUDY DESIGN AND METHODS: Cloning and subsequent sequence analysis of 11 new samples of monoclonal anti-D was performed in an attempt to identify V(D)J germline gene usage. Sequences were compared and analyzed with 37 previously published samples of anti-D for identification of V(H) and V(L) pairings, canonical structures, and conformation of restricted germline gene usage. RESULTS: The V(H) and V(L) pairings used by the new D MoAbs resulted in seven canonical combinations, three of which had not been described previously. Preferential usage of gene segments from the VH3 and VH4 families and of D3, D6, JH6, and DPK9 germline gene segments was also determined. Three samples of anti-D from different donors were found to use similar V(H) and V(kappa) germline genes, despite the fact that two of the antibodies recognized epD6/7 and the third recognized epD1. From the cumulative analysis of the anti-D IgG, 24 V(H) and V(L) gene pairings were identified, resulting in only 10 canonical structures. CONCLUSIONS: Despite the potential for diversity, only a minority of V(H) and V(L) germline genes are used by anti-D. Consequently, V(H) and V(L) pairings and the resulting canonical structures are similarly restricted.