The challenges of detecting and responding to a Lassa fever outbreak in an Ebola-affected setting.

OBJECTIVES: Lassa fever (LF), a priority emerging pathogen likely to cause major epidemics, is endemic in much of West Africa and is difficult to distinguish from other viral hemorrhagic fevers, including Ebola virus disease (EVD). Definitive diagnosis requires laboratory confirmation, which is not widely available in affected settings. The public health action to contain a LF outbreak and the challenges encountered in an EVD-affected setting are reported herein. METHODS: In February 2016, a rapid response team was deployed in Liberia in response to a cluster of LF cases. Active case finding, case investigation, contact tracing, laboratory testing, environmental investigation, risk communication, and community awareness raising were undertaken. RESULTS: From January to June 2016, 53 suspected LF cases were reported through the Integrated Disease Surveillance and Response system (IDSR). Fourteen cases (26%) were confirmed for LF, 14 (26%) did not have a sample tested, and 25 (47%) were classified as not a case following laboratory analysis. The case fatality rate in the confirmed cases was 29%. One case of international exportation was reported from Sweden. Difficulties were identified in timely specimen collection, packaging, and transportation (in confirmed cases, the time from sample collection to sample result ranged from 2 to 64 days) and a lack of response interventions for early cases. CONCLUSIONS: The delay in response to this outbreak could have been related to a number of challenges in this EVD-affected setting: a need to strengthen the IDSR system, develop preparedness plans, train rapid response teams, and build laboratory capacity. Prioritizing these actions will aid in the timely response to future outbreaks.

The Carmarthenshire Glaucoma Referral Refinement Scheme, a safe and efficient screening service.

AIM: It has been previously shown that community refinement of glaucoma referrals is an efficient way to investigate and treat glaucoma suspects. The potential for false negatives has not been explored previously and we describe a scheme in which effort has been made to both assess and control for this, and report on its success. METHODS: Trained optometrists were recruited to examine and investigate the patients referred with suspected glaucoma, with a view to decreasing false-positive rates in accordance with an agreed protocol. The randomly selected notes of 100 patients referred onward to the Hospital Eye Service (HES) by trained, accredited optometrists, and the notes and optic disc images of 100 randomly selected patients retained in the community were examined in order to determine the efficiency and safety of the scheme. RESULTS: The scheme resulted in a 53% reduction in the total number of referrals to HES with a cost saving of £117 per patient. Analysis of patients referred resulted in a diagnosis of glaucoma or retention of patients in HES with suspected glaucoma in 83% and a good correlation between the hospital and optometric measurements. Analysis of notes and optic nerve images of patients not referred indicated no compromise on patient safety. CONCLUSION: This study suggests that suspected glaucoma can be successfully refined in the community with benefits to both the patient and the hospital. We also suggest that such a scheme may be safe as well as cost-effective, a conclusion that has not as yet been reached by any other study.

Management of post-infectious olfactory dysfunction: a nationwide survey of UK ENT consultants.

OBJECTIVE: To determine the current practice of UK ENT consultants in investigating cases of suspected post-infectious olfactory dysfunction. METHOD: We prepared questionnaires and posted these to every consultant ENT surgeon registered with the British Association of Otolaryngologists Head and Neck Surgeons (BAO-HNS). Questions included preferred radiological investigation, smelling test and follow up. RESULTS: A total of 510 questionnaires were sent, with a response rate of 51%. Most UK consultant ENT surgeons investigated suspected post-infectious olfactory dysfunction by performing either a CT (44%) or an MRI (29%) scan, but only 37% performed formal smell tests. Most consultant ENT surgeons (76%) decide not to follow up post-infectious olfactory dysfunction. CONCLUSION: This study gives an indication that suspected post-infectious olfactory dysfunction is not investigated thoroughly in the United Kingdom, with only a minority of clinicians performing formal smell tests and a lack of consensus as to radiological investigation. Standardisation of investigations is key to adequately managing suspected post-infectious olfactory dysfunction.

Testicular venous sampling in determining the possible origin of tumour markers and its influence on management.

Testicular vein sampling is a well-known technique in the investigation of testicular function, or in determining the origin of certain plasma protein markers. We describe the application of this technique in the case of a patient who had previously undergone right radical orchidectomy for testicular teratoma, to discover the source of persistently raised tumour markers following the operation. We found this diagnostic tool useful in avoiding unnecessary procedures of the remaining testis and in guiding further treatment.

The impact of H2-DM on humoral immune responses.

H2-DM (DM, previously H2-M) facilitates the exchange of peptides bound to MHC class II molecules. In this study, we have used H2-DM-deficient (DM(-/-)) mice to analyze the influence of DM in the priming of B cell responses in vivo and for Ag presentation by B cells in vitro. After immunization, IgG Abs could be raised to a T-dependent Ag, 4-hydroxy-5-nitrophenylacetyl-OVA, in DM(-/-) mice, but closer analysis revealed the IgG response to be slower, diminished in titer, and composed of low-affinity Abs. The Ab response correlated with a vast reduction in the number of germinal centers in the spleen. The presentation of multiple epitopes by H2-A(b) from distinct Ags was found to be almost exclusively DM-dependent whether B cells internalized Ags via fluid phase uptake or using membrane Ig receptors. The poor B cell response in vivo could be largely, but not completely restored by expression of a H2-Ea(d) transgene, despite the fact that Ag presentation by H2-E(d/b) molecules was found to be highly DM dependent. Hence, while substantial Ab responses can be raised in the absence of DM, this molecule is a crucial factor both for Ag processing and for the normal maturation of T-dependent humoral immune responses in vivo.

Unequal VH gene rearrangement frequency within the large VH7183 gene family is not due to recombination signal sequence variation, and mapping of the genes shows a bias of rearrangement based on chromosomal location.

Much of the nonrandom usage of V, D, and J genes in the Ab repertoire is due to different frequencies with which gene segments undergo V(D)J rearrangement. The recombination signal sequences flanking each segment are seldom identical with consensus sequences, and this natural variation in recombination signal sequence (RSS) accounts for some differences in rearrangement frequencies in vivo. Here, we have sequenced the RSS of 19 individual V(H)7183 genes, revealing that the majority have one of two closely related RSS. One group has a consensus heptamer, and the other has a nonconsensus heptamer. In vitro recombination substrate studies show that the RSS with the nonconsensus heptamer, which include the frequently rearranging 81X, rearrange less well than the RSS with the consensus heptamer. Although 81X differs from the other 7183-I genes at three positions in the spacer, this does not significantly increase its recombination potency in vitro. The rearrangement frequency of all members of the family was determined in microMT mice, and there was no correlation between the in vitro recombination potential and V(H) gene rearrangement frequency in vivo. Furthermore, genes with identical RSS rearrange at different frequencies in vivo. This demonstrates that other factors can override differences in RSS potency in vivo. We have also determined the gene order of all V(H)7183 genes in a bacterial artificial chromosome contig and show that most of the frequently rearranging genes are in the 3' half of the region. This suggests that chromosomal location plays an important role in nonrandom rearrangement of the V(H)7183 genes.

The development of a post-baccalaureate certificate program in molecular diagnostics.

A post-baccalaureate certificate program in diagnostic molecular sciences was created in 1995 by the Diagnostic Genetic Sciences Program in the School of Allied Health at the University of Connecticut. The required on-campus lecture and laboratory courses include basic laboratory techniques, health care issues, cell biology, immunology, human genetics, research, management, and molecular diagnostic techniques and laboratory in molecular diagnostics. These courses precede a 6-month, full-time practicum at an affiliated full-service molecular laboratory. The practicum includes amplification and blotting methods, a research project, and a choice of specialized electives including DNA sequencing, mutagenesis, in situ hybridization methods, or molecular diagnostic applications in microbiology. Graduates of the program are immediately eligible to sit for the National Credentialing Agency examination in molecular biology to obtain the credential Clinical Laboratory Specialist in Molecular Biology (CLSp(MB). This description of the University of Connecticut program may assist other laboratory science programs in creating similar curricula.

Wound complications of abdominoplasty in obese patients.

The records of 90 patients who underwent an abdominoplasty at the University of Virginia Health Sciences Center were analyzed to determine the effect of obesity on the incidence of complications after this surgery. The study patients were divided into three groups-obese, borderline, and nonobese-based on the degree to which their preoperative weights varied from their ideal body weight. A history of previous bariatric surgery was also analyzed to determine what impact that might have on subsequent abdominoplasty. Results showed that 80% of obese patients had complications compared with the borderline and nonobese patients, who had complication rates of 33% and 32.5% respectively (p = 0.001). Previous gastric bypass surgery had no significant effect on the incidence of postabdominoplasty complications. Based on these findings the authors conclude that obesity at the time of abdominoplasty has a profound influence on the wound complication rate following surgery, regardless of any previous weight reduction surgery.

DNA fragmentation during exposure of rat cerebella to ethanol under hypoxia imposed in vitro.

To gain a better understanding into the mechanisms of damage incurred by neurons in periods following heavy alcohol exposure during development, we used an in vitro system to monitor the effects of alcohol and hypoxia on cell survival and DNA integrity. Samples representing the first few hours of exposure to alcohol and hypoxia were compared to those resulting from hypoxia alone. Measurements were taken from cell counts using Trypan blue exclusion and TUNEL assays as well as digital scans of the ethidium bromide fluorescence of genomic DNA isolated from the treated tissue. We found that DNA degradation from hypoxia was accelerated by several hours in the presence of 100 mM ethanol. This result depended on age, with adult animals (>8 months) having a similar response to 4-day postnatal animals, while the effect on 10-day postnatal animals and those of intermediate age (45 days postnatal) was increasingly delayed. Different methods of inducing the processive degradation of DNA produced laddering typical of apoptosis, a biphasic degradative process, or patterns usually associated with necrosis.

CD4+ T cell responses in mice lacking MHC class II molecules specifically on B cells.

The role of B lymphocytes in initiating and maintaining a CD4+ T cell response has been examined using a variety of strategies, but remains controversial because of weaknesses inherent to each of the approaches. Here, we address this issue by measuring CD4+ T cell priming both in mutant mice devoid of B cells and in chimeric animals lacking major histocompatibility complex class II molecules specifically on B cells. We find that peptide and some protein antigens do not require B cells expressing class II molecules, nor B cells themselves, to efficiently prime. This could be demonstrated by the usual lymph node proliferation assay, a rather indirect in vitro measure of priming, and by a direct ex vivo assay of population expansion and activation marker expression. Interestingly, one protein antigen, conalbumin, could not prime in the absence of B cells, but could in the presence of B cells devoid of class II molecules. This finding constrains the possible mechanisms whereby B lymphocytes contribute to the initiation of a CD4+ T cell response, arguing against the importance of surface immunoglobulin-mediated antigen presentation by B cells.

Mice lacking the transcription factor CIITA--a second look.

We have generated a second line of mice lacking a transcription factor thought to be a critical regulator of MHC class II gene expression, CIITA (for class II transactivator). Our and the previously published lines differ in the deletion that was engineered and by the fact that we removed the neomycin-resistance promoter and structural gene via the cre-loxP recombination system. Characterization of our line led to two new findings. First, a substantial number of cells can express class II molecules in the absence of CIITA, albeit at 5-fold reduced levels, most notably dendritic cells in s.c. lymph nodes; therefore, the CIITA gene cannot be an absolute 'master gene' controlling the expression of class II molecules, as had been thought. Second, in contrast to recent results on human cell lines, CIITA is not critically involved in the IFN-gamma-induced up-regulation of MHC class I genes.

Renal transplantation in anticardiolipin antibody-positive lupus erythematosus patients.

Up to 44% of patients with systemic lupus erythematosus have antiphospholipid antibodies, which in some patients have been associated with thrombocytopenia, in vitro coagulation abnormalities, and a thrombotic tendency. Patients with lupus nephritis who reach end-stage renal disease may continue to have anticardiolipin antibodies (ACL-Ab) at the time of renal transplantation despite a lack of clinical or serologic activity of systemic lupus. The risk of thrombotic and other complications has not been previously reported in these patients. To evaluate the effect of ACL-Ab on the course of renal transplantation, the clinical course of eight ACL-Ab-positive lupus patients transplanted in the period from January 1990 to June 1992 were compared with five ACL-Ab-negative lupus patients transplanted during the same period. All patients had lupus nephritis as the cause of renal failure. There were four thrombotic episodes in the patients who were ACL-Ab-positive but none in the control group. Neither of the two groups differed in the number of rejection episodes, the rate of graft loss, or renal function at last follow-up. Patients with systemic lupus erythematosus who are ACL-Ab-positive can be successfully transplanted. However, these patients require attention to possible thrombotic events with a potential role for prophylactic anticoagulation.

Early B cell development requires mu signaling.

In vitro studies with Abelson murine leukemia virus (AMuLV)-transformed murine pre-B cell lines demonstrated that wild-type mu but not mutant mu chains lacking the first constant domain (mu delta 1) can efficiently induce Ig light (L) chain gene rearrangement. Using antibodies against the cytoplasmic tail of the immunoglobulin co-receptor beta (Ig beta) chain we find mu, but not mu delta 1 chains associated with Ig beta. Since a heterodimer of surface-labeled proteins was co-precipitated with mu we conclude that only wild-type mu is associated with the Ig alpha/Ig beta co-receptor on the surface of pre-B cell lines. Mutant mu delta 1 chains achieve their surface expression by utilizing a glycophospholipid anchor. In vivo analysis of transgenic mice expressing either mu or mu delta 1 transgenes revealed the expected "normal" B cell development in the case of wild-type mu transgenic lymphocytes, but a block in differentiation of mu delta 1 transgenic lymphocytes. The maturation block occurs at the developmental transition of pre-B lymphocytes from the CD43/S7+, CD45R/B220low stage to the CD43/S7-, B220low/high stage in which the majority of L chain gene rearrangements occur. These results, together with the observed inability of the mu delta 1 chains to signal activation of L chain gene joining and to associate Ig alpha/Ig beta in pre-B cell lines suggests that signals mediated by the protein complex composed to mu/Ig alpha/Ig beta are crucial during differentiation of pre-B lymphocytes.

A prospective comparison of two endogenous creatinine clearance testing methods in hospitalized hypertensive gravid women.

OBJECTIVE: Although 24-hour endogenous creatinine clearance testing is common in pregnancies complicated by hypertension, inaccuracies limit its usefulness. We controlled the conditions under which 4-hour endogenous creatinine clearance testing was performed and compared the results with outcomes of 24-hour tests from the same patients. STUDY DESIGN: In 83 women hospitalized with mild hypertension in the third trimester, we measured endogenous creatinine clearance with a 4-hour urine collection during lateral recumbency and supervised oral hydration. This test was paired with a 24-hour test performed immediately thereafter. No restrictions or recommendations regarding ambulation or oral intake were imposed for the 24-hour test. RESULTS: The 4-hour endogenous creatinine clearance value exceeded the 24-hour value in 133 of the 136 paired comparisons (p < 0.0001). Results of the tests from only the 29 patients with multiple paired tests showed more similarity (p < 0.005) among the 4-hour than among the 24-hour clearances. CONCLUSION: The 4-hour endogenous creatinine clearance test, as described, provides a higher and less variable estimate of renal function in hypertensive pregnant women than does the 24-hour test.

Molecular requirements for the mu-induced light chain gene rearrangement in pre-B cells.

During B cell differentiation rearrangement of immunoglobulin (Ig) genes is partially regulated by the Ig proteins. Rearrangement of heavy (H) chain genes is inhibited, whilst that of light (L) chain genes is induced by the membrane form of the mu H chain. In order to analyse additional structural requirements of mu induced L chain gene rearrangement we transfected wild-type mu and mutant mu constructs lacking functional exons encoding the first or second constant domains into Abelson murine leukemia virus (AMuLV) transformed pre-B cells. All mu chains are expressed on the surface of the pre-B cell and all associate with omega and iota, two proteins forming a surrogate light chain, necessary for mu membrane expression. Nevertheless, only wild-type mu and not the mutant mu proteins promote L gene rearrangement. A heterodimer of proteins with Mr of 33 kd and 36 kd was found associated with wild-type but not with the mutant mu proteins. Continuous presence of mu is required for L chain gene recombination since loss of mu stopped and readdition of mu started L gene rearrangement. We propose that the protein complex composed of mu and the 33 kd/36 kd protein heterodimer is responsible for the activation of the L chain gene locus and its rearrangement.

Class II antigen expression in peripheral neuropathies.

The expression of class II antigen was studied in sural nerve biopsies from patients with peripheral neuropathies. These included patients with chronic demyelinating polyradiculoneuropathy (CIDP), non-immune mediated neuropathies of diverse etiologies and controls without evidence of neuropathy. The major finding in CIDP was a marked increase in class II expression on Schwann cells. Endoneurial Schwann cell staining to the same degree as in CIDP was seen in diabetic symmetric proximal motor neuropathy, neuropathies associated with monoclonal gammopathies and hereditary sensory and autonomic neuropathy type 1. In the control nerves and the other non-immune mediated neuropathies class II expression was mainly restricted to endothelial and perineurial cells. Increased endoneurial expression of class II antigen was found to correlate with elevated cerebrospinal fluid (CSF) protein levels but not with other clinical variables or demyelination as defined by electrophysiologic criteria or teased fiber analysis. The increased expression of class II antigen on Schwann cells may be indicative of a breakdown in immunological tolerance but should not be used as a diagnostic marker for dysimmune neuropathies due to overlap with non-immune mediated neuropathies.