Evaluation of three newly developed enzyme-linked immunosorbent assays and two agglutination tests for detecting Salmonella enterica subsp. enterica serovar dublin infections in dairy cattle

In this study test characteristics of three newly developed enzyme-linked immunosorbent assays (ELISAs) for Salmonella enterica subsp. enterica serovar Dublin were evaluated and compared with two agglutination tests. The ELISAs involved were an indirect ELISA with serovar Dublin lipopolysaccharide (LPS ELISA), an indirect ELISA with serovar Dublin flagellar antigen (GP ELISA), and a double-antibody sandwich blocking ELISA that uses monoclonal antibodies against S. enterica subsp. enterica serovar Enteritidis flagellin (GM-DAS ELISA). The agglutination tests involved were two routine serum agglutination tests with either somatic (O) or flagellar (H) antigen. Diagnostic specificity of the three ELISAs was determined using 840 serum samples from seven dairy herds without any history of serovar Dublin infection. Cutoff values at a titer of 100, 100, and 10, respectively, for the LPS ELISA, GP ELISA, and GM-DAS blocking ELISA resulted in a specificity of 99.3, 100, and 100%, respectively. Using these cutoff values the LPS ELISA, GP ELISA, and GM-DAS ELISA were able to detect, respectively, 30, 46, and 38% of 50 fecal culture-positive animals from 13 herds with a recent serovar Dublin infection. With the same cutoff values, active carriers (n = 18) were detected for 94.4% with the LPS ELISA and for 100% with the GP and GM-DAS ELISAs. Kappa values determined on the results of all tests from 8 of the 13 serovar Dublin-infected herds and the 7 control herds demonstrated a good correlation between the results of all ELISAs and the H-agglutination test. The results of the O-agglutination test failed to correlate with those of the other tests. Using a set of sera from 170 aborting cows (with 25 abortions due to serovar Dublin), test results of the ELISAs and the H-agglutination test were comparable. The H-agglutination test may be used successfully for single sample testing, especially to diagnose abortion due to serovar Dublin. It is concluded that the ELISAs are useful diagnostic tools in serovar Dublin control programs and that they are preferred to agglutination tests for reasons of automation and costs.     

Ovarian stimulation by clomiphene citrate and hMG in combination with cetrorelix acetate for ICSI cycles

BACKGROUND: The introduction of GnRH antagonists such as cetrorelix acetate has made possible the simplification of ovarian stimulation. However, the most effective protocol for their administration has not yet been clearly defined. METHODS: Forty women with male-factor infertility undergoing 40 ICSI cycles were included in the study. Clomiphene citrate at 100 mg a day was given from cycle day 3 through day 7. hMG at 150 IU was given on cycle days 4, 6 and 8, and was adjusted from day 9 according to the follicular and hormone responses. Cetrorelix acetate at 2.5 mg was administered when the leading follicle reached 14 mm. The remaining 0.5 mg was divided into two 0.25 mg injections for possible later use. Serum FSH, LH, estradiol and progesterone levels were measured daily from the day of cetrorelix acetate injection until hCG was given. RESULTS: Serum LH level was suppressed effectively for 4 days. Four patients (10%) needed one or two additional injections of 0.25 mg cetrorelix acetate. No premature LH surge was detected in any of the women treated. Sixteen women became pregnant (40%), of which 14 pregnancies (35%) were ongoing at the time of writing. CONCLUSIONS: This study demonstrates that this new protocol is feasible for couples with male-factor infertility undergoing ICSI.     

Bilateral simultaneous tubal sextuplets: pregnancy after in-vitro fertilization--embryo transfer following salpingectomy

The presence of a damaged tube has been suggested in recent studies to have a negative effect on in-vitro fertilization (IVF) outcome. Performing bilateral salpingectomy prior to IVF to maximize pregnancy rates may also result in unnecessary surgery. This case is also an example of the occurrence of interstitial pregnancy after salpingectomy. This unusual type of ectopic pregnancy must be kept in mind when evaluating a patient suspected of a possible early abnormal gestation after assisted reproductive technolologies.      

Human anti-JC virus serum reacts with native but not denatured JC virus major capsid protein VP1

The immunoreactivity of human anti-JC virus (JCV) serum against the major capsid protein VP1 of JCV was analyzed by Western blot, dot blot, and hemagglutination inhibition (HAI) assays. JCV-positive human serum reacted with native but not denatured JCV major capsid protein VP1, as demonstrated by dot blot and Western blot. Rabbit antiserum raised against native JCV capsid had immunoreactivities similar to those of human anti-JCV serum. These results indicate that the antigenecity of native and denatured JCV VP1 is different. In addition, both JCV-positive human serum and rabbit antiserum raised against native JCV capsid protein inhibited the hemagglutination activity of JCV capsid particles. In contrast, rabbit antiserum raised against denatured JCV VP1 did not inhibit hemagglutination. These findings reveal that denaturation may alter the antigenic epitopes of JCV VP1. Therefore, keeping the JCV capsid protein native appears to be essential for serological or other immunological analyses of the virus.     

Amino acid substitutions within the heptad repeat domain 1 of murine coronavirus spike protein restrict viral antigen spread in the central nervous system

Targeted recombination was carried out to select mouse hepatitis viruses (MHVs) in a defined genetic background, containing an MHV-JHM spike gene encoding either three heptad repeat 1 (HR1) substitutions (Q1067H, Q1094H, and L1114R) or L1114R alone. The recombinant virus, which expresses spike with the three substitutions, was nonfusogenic at neutral pH. Its replication was significantly inhibited by lysosomotropic agents, and it was highly neuroattenuated in vivo. In contrast, the recombinant expressing spike with L1114R alone mediated cell-to-cell fusion at neutral pH and replicated efficiently despite the presence of lysosomotropic agents; however, it still caused only subclinical morbidity and no mortality in animals. Thus, both recombinant viruses were highly attenuated and expressed viral antigen which was restricted to the olfactory bulbs and was markedly absent from other regions of the brains at 5 days postinfection. These data demonstrate that amino acid substitutions, in particular L1114R, within HR1 of the JHM spike reduced the ability of MHV to spread in the central nervous system. Furthermore, the requirements for low pH for fusion and viral entry are not prerequisites for the highly attenuated phenotype.     

Clinical features and risk factors for mortality in Aeromonas bacteremic adults with hematologic malignancies.

BACKGROUND AND PURPOSE: Aeromonas spp. often cause infections in immunocompromised patients. To specifically understand the clinical features of Aeromonas bacteremic adults with hematologic malignancies, we investigated the demographic, clinical and microbiologic characteristics of Aeromonas bacteremia in this patient population. METHODS: Retrospective study performed in a tertiary medical center in southern Taiwan, in which adults with hematologic malignancies suffered from Aeromonas bacteremia admitted between 1995 and 2003 were included for study. RESULTS: There were 45 episodes of Aeromonas bacteremia in 41 adults with hematologic malignancies. Episodes of Aeromonas bacteremia which occurred at least 2 months apart were counted as separate cases in the analysis. A total of 30 men and 15 women (mean age: 53.2 years), with 4 patients experiencing 2 episodes, was included. The 3 leading underlying hematologic malignancies were acute myelogenous leukemia (37.8%), myelodysplastic syndrome (26.7%) and non-Hodgkin's lymphoma (17.8%). No cluster of Aeromonas bacteremia was found during the study period. Twenty nine (64.4%) of the 31 patients with nosocomial Aeromonas bacteremia had received recent antineoplastic chemotherapy. The 3 leading clinical manifestations were fever (88.9%), septic shock (40%), and altered consciousness (26.7%). Eleven (24.4%) episodes of bacteremia were polymicrobial. Sixteen (35.6%) patients died within 14 days of onset of bacteremia. The mean duration from sampling blood for culture to death was 3.81 days. Altered consciousness (odds ratio, 8.999; 95% confidence interval, 1.787-45.33; p=0.008) was the only independent prognostic factor for mortality. High resistance rates (11.1% to piperacillin and 35.6% to imipenem) among Aeromonas isolates were also noted. CONCLUSION: In febrile patients with hematologic malignancies and suspected Aeromonas infections, particular attention to the development of alteration of consciousness is needed as it is an independent risk factor for mortality.     

Identification and characterization of a structural protein of hepatitis B virus: a polymerase and surface fusion protein encoded by a spliced RNA

The hepatitis B virus (HBV) genome is known to contain four conserved and overlapped open reading frames (ORFs) encoding the viral core, polymerase (P), surface (S), and X proteins. Whether HBV encodes other proteins has long been a major interest in the field. Using (32)P-labeling of an introduced protein kinase A site attached to the N- or C-terminus of the HBV polymerase gene, a 43-kDa P-S fusion protein was detected in cell lysate, secreted virions, and 22-nm subviral particles. Immunobiochemical studies showed that the 43-kDa protein contains the epitopes of the N-terminus of polymerase and most parts of the surface proteins. This 43-kDa protein was shown to be a glycoprotein, similar to the surface protein. RT-PCR and sequence analyses identified a spliced mRNA which was derived from pregenomic RNA with a deletion of 454 nucleotides (nt) from nt 2447 to 2902. This splice event creates a P-S fusion ORF. This finding is consistent with the result obtained from an immunobiochemical study. Mutations at the splice donor or acceptor site on the HBV genome abrogated the production of the 43-kDa protein. These mutants had no effect on viral replication in transfected HuH-7 cells. However, this P-S fusion protein is able to substitute for the LS protein in virion maturation. On the basis of these results, we conclude that the 43-kDa protein is a polymerase-surface fusion protein encoded by a spliced RNA. Similar to the LS protein, the 43-kDa P-S fusion protein is a structural protein of HBV and might play a role in the HBV life cycle.     

The possible role of protein degradation in altered membrane structure and function

Possible changes in membrane lipid assemetry may result in altered function with aging. Membrane proteolysis is an additional factor which must be considered, both with respect to modulation of membrane function and also as a methodological problem in analyses of membrane dynamics.     

Estimating the relative frequency of leukodystrophies and recommendations for carrier screening in the era of next‐generation sequencing

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A‐related leukodystrophy, Peroxisomal biogenesis disorders, POLR3‐related Leukodystrophy, Vanishing White Matter, and Pelizaeus‐Merzbacher Disease. Despite the relative frequency of these conditions, carrier‐screening laboratories regularly test only 20 of the 55 leukodystrophy‐related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening.     

Comparison of different PCR approaches for characterization of Burkholderia (Pseudomonas) cepacia isolates.

In this study, we evaluated three PCR methods for epidemiological typing of Burkholderia (Pseudomonas) cepacia--PCR-ribotyping, arbitrarily primed PCR (AP-PCR) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR)--and compared them with pulsed-field gel electrophoresis. The analysis was performed with 31 isolates of B. cepacia, comprising 23 epidemiologically unrelated isolates and 8 isolates collected from the same patient during two episodes of bacteremia. Pulsed-field gel electrophoresis, ERIC-PCR, and AP-PCR identified 23 distinct types among the 23 unrelated isolates, while PCR-ribotyping only identified 12 strain types, even after AluI digestion of the amplification products. Among the eight isolates collected from the same patient, all typing techniques revealed two clones of strains. The day-to-day reproducibilities of PCR-ribotyping and ERIC-PCR were good, while greater day-to-day variations were noted in the fingerprints obtained by AP-PCR. We conclude that all three PCR techniques are useful for rapid epidemiological typing of B. cepacia, but ERIC-PCR seems to be more reproducible and discriminative.