Identification of two genetic markers that distinguish pathogenic and nonpathogenic strains of Acanthamoeba spp.

Species-level identification of Acanthamoeba isolates is difficult and gives little or no indication of the isolate's pathogenicity. We identified two amplification-based genetic markers that were highly correlated with pathogenicity in Acanthamoeba spp. One marker, designed to amplify a 485-bp fragment of the small-subunit ribosomal RNA gene (ssrDNA), was preferentially amplified from the nonpathogenic strains; amplifications from the pathogenic strains yielded anomalous fragments of 650 and 900?bp. A second marker was developed on the basis of the anomalous 650-bp fragment. Primers to this sequence preferentially amplified a noncoding locus (called Ac6) only from the pathogenic strains. These two genetic markers may be useful for identification of pathogenic Acanthamoeba spp. strains.     

Differentiation of Aedes triseriatus (Say) from Aedes hendersoni Cockerell (Diptera: Culicidae) by restriction fragment length polymorphisms of amplified ribosomal DNA

Aedes triseriatus is the primary vector of LaCrosse (LAC) virus, which can cause encephalitis, especially in young children. Aedes hendersoni, a sibling species of Ae. triseriatus, has a salivary gland barrier to LAC virus and, therefore, is not considered a vector of this virus. Adults of Ae. triseriatus are morphologically indistinguishable from those of Ae. hendersoni, and the two species are sympatric in the eastern United States. A definitive method of identifying field specimens is an important part of any disease surveillance program, particularly in the case of LAC virus. This study identifies restriction enzymes that produce species-specific restriction fragment length polymorphisms (RFLPs) from amplified ribosomal (r) DNA. In addition, sequences of the internal transcribed spacers 1 and 2 and the 5.8S regions of the rDNA were used to confirm the RFLP patterns. This study is the first to compare nucleotide sequences from Ae. triseriatus and Ae. hendersoni.     

Elimination of malaria in country Georgia

Malaria is well known in Georgia since ancient times, causing national disasters with associated significant mortality and economic losses. By 1970 Georgia managed to reach complete and sustained elimination of the disease as a result of comprehensive anti-malaria measures undertaken in the country. However from the mid-1990s, economic collapse following disintegration of Soviet Union causing breakdown of important public health networks including anti-malaria preventive and control infrastructure resulted in gradual increase of malaria cases in the country with a peak of 437 and 474 cases in 2001 and 2002, respectively. From 2000 two major anti-malaria efforts were carried out by National Center for Disease Control and Public Health, WHO and Global Fund to Fight AIDS, tuberculosis and malaria and as result of comprehensive and collaborative work in 2010 the level of zero cases of local mosquito-borne malaria transmission was achieved and the country entered the elimination phase.     

Detection of St. Louis encephalitis virus in mosquitoes by use of the polymerase chain reaction.

We recently developed an assay using the polymerase chain reaction (PCR) for the specific detection of St. Louis encephalitis (SLE) virus RNA. This assay was tested in a blind study on 7 samples of pooled mosquitoes (50 mosquitoes/pool) which were also characterized for SLE virus by plaque assay in Vero cell culture. One sample was positive for the SLE virus as determined by both the PCR assay and a combination of the plaque assay and the indirect fluorescent antibody assay. The remaining 6 samples were negative for the presence of SLE virus as determined by both methods. These data indicate that this PCR assay can be used to monitor for the presence of SLE virus in pools of homogenized mosquitoes. This approach could provide early data on which to base disease control decisions.     

Evaluation of Fontan liver disease: Correlation of transjugular liver biopsy with magnetic resonance and hemodynamics

Introduction: Liver fibrosis and cirrhosis are late complications in Fontan palliation. Liver biopsy is the gold standard. The goal of this study is to correlate transjugular liver biopsy (TJLB) in the setting of Fontan palliation with noninvasive testing and hemodynamics.
Methods: Between August 2014 and July 2017, 49 Fontan patients underwent TJLB. All the patients had hemodynamic evaluation, 28 patients had MRE (magnetic reso‐ nance elastography) and 40 patients had cardiopulmonary exercise test. Histologic liver fibrosis was quantitated using traditional histologic scoring systems and a modi‐ fied Ishak congestive hepatic fibrosis score.
Results: Median age 17.8 years, median time since Fontan 15.2 years. Primary diagnosis and Fontan type were variables, but predominantly LV morphology (30/49), lateral tun‐ nel Fontan (29/49), originally fenestrated (37/49), and 11/49 had a pacemaker. Histologic fibrosis correlated with MRE (R = 0.62, P ≤ .001). Histologic fibrosis and MRE correlated with Fontan pressure (R = 0.38, P = .008 & R = 0.59, P ≤ .001). Morphology of the single ventricle did not correlate with liver fibrosis. The presence of a fenestration resulted in a higher cardiac index (P = .026) but did not resulted in lower liver fibrosis (P = .64).
Conclusion: Noninvasive tests, such as MRE, may be suitable for longitudinal follow‐up in patients with single ventricle physiology. Our data suggest that there is reasonable cor‐ relation of MRE liver stiffness with biopsy scoring systems and Fontan pressures. We demonstrated the feasibility of TJLB in the setting of Fontan palliation and demonstrated its correlation with noninvasive measures particularly MRE. We recommend selective use of TJLB when MRE score is >5 KPa or when there are other clinical signs of cirrhosis.     

IMMEDIATE FREE FLAP MANDIBULAR RECONSTRUCTION IN OSTEOSARCOMA OF THE MANDIBLE IN CHILDHOOD

Mandibular osteogenic sarcoma (OS) is a very rare entity in childhood. Adequate surgical rejection with a wide margin of normal tissue is the mainstay of treatment of this site, while the role of adjuvant chemotherapy remains uncertain. A case is presented of a 15 1/2-year-old male with a huge OS of the mandible. The boy underwent surgical resection of the mandible with immediate fibula free flap reconstruction and is alive and free of disease 6 1/2 years following unitial diagnosis. This case suggests that immediate bone reconstitution with vascularized grafts have good functional and morphological results for osteosarcoma of the lower jaw.     

Impact of stimulation duration and gonadotropin type on the incidence of premature progesterone elevation – a retrospective analysis of the Ensure data

Abstract

Elevated progesterone levels on the day of trigger negatively impact the outcome of assisted reproductive technique (ART) treatment and forced ovarian stimulation might be a cause of progesterone elevation during ovarian stimulation. To analyze the impact of forced and prolonged stimulation on the progesterone elevation, this data analysis from the Ensure study compared hormonal stimulation with corifollitropin alpha (CFA)-only with CFA plus recombinant (rec) follicle-stimulating hormone (FSH) after day 8 (CFA-plus group) of ovarian stimulation. In the Ensure study, 268 patients underwent ovarian stimulation with 100?µg CFA and 128 patients with recombinant FSH. A total of 35 patients (13.1%) from the CFA-arm received the hCG trigger after stimulation with CFA-only, 233 patients (86.9%) needed additional rec FSH from day 8 onwards to meet the criteria for trigger. Progesterone levels >0.8?ng/ml on the trigger day occurred in 90 patients (38.6%) from the CFA plus FSH group and only in one patient (2.8%) in the CFA-only group (p?<?.001). The ongoing pregnancy rate (OPR) was 31.4% (11/35) for patients in the CFA-only group and 24.5% (57/233) for patients CFA-plus group with additional recFSH after day 8 (p?=?.378). This set of data demonstrates that prolongation of stimulation in combination with intense stimulation leads to a statistically significant increased incidence of progesterone elevation on the day of trigger.     

Highly structured sequence homology between an insertion element and the gene in which it resides

The recessive allele for soybean seed lectin results from the insertion of a DNA segment (designated Tgm1) into the coding region of the gene. The termini of Tgm1 display structural features characteristic of a transposable element. The complete sequence of Tgm1 contains 3550 base pairs (bp) and can be divided into three regions (left arm, mid-section, and right arm). No large open reading frames were found, but an extensive, highly structured border with homology to the lectin gene was revealed. The left border (726 bp) comprising most of the left arm and extreme right border (144 bp) of the right arm consist of various forms of a basic 54-bp repeating unit. This 54-bp unit is comprised of a stem-loop structure and interhairpin sequence that occurs 13 times in the left arm and 2 times in the right arm of Tgm1. Progressively degenerate forms of this repeating unit appear toward the termini of Tgm1, but the dyad symmetry remains highly conserved. Seven nucleotides (A-C-A-T-C-G-G and its complement) maintained within the stem also appear as a subset of inverted repeats found at nearly equal distances from the target site in the lectin gene. Together with the inverted repeat termini and a duplication in the left arm, this 7-bp sequence occurs a total of 33 times in Tgm1. We infer that the dyad symmetries containing this sequence are involved in target gene selection. The repeating unit format of Tgm1 describes a distinct class of eukaryotic elements that includes representatives known to be mobile in snapdragon and maize.     

Differential polymerase chain reaction for detection of wild-type and a vaccine strain of Aujeszky's disease (pseudorabies) virus.

A polymerase chain reaction (PCR) strategy for differentiating between a vaccine mutant strain and wild-type (WT) strains of Aujesky's disease (pseudorabies) virus (ADV) was evaluated. With this approach, a single virus or a concurrent WT and vaccine virus infection could be distinguished by targeting the genomic alteration within the vaccine strain. PCR primers were designed for a recombinant vaccine virus that has almost all of the WT gX gene replaced by the lacZ gene. One primer, corresponding to a conserved sequence upstream of the altered region, was selected for common use. The differentiating primers were chosen from the unique WT gX and vaccine lacZ gene sequences. The sensitivity of the differential PCR was analyzed using extracted viral DNA and in vitro infected cell lysates. Approximately 10 and between 10 to 100 molecules of WT and vaccine viral DNAs, respectively, could be detected, regardless of the presence or absence of uninfected cell lysates. Detection of viral DNA from in vitro infected cell cultures approximated this level of sensitivity. The specificity of the amplifications was verified by restriction endonuclease analysis and Southern hybridization. Although the vaccine primer pair target was amplified to a lesser degree as compared to the WT primer pair product, utility of the differential PCR was demonstrated using trigeminal nerve ganglia from swine infected with vaccine virus and WT virus. Both viral targets were detected only by their specific primer pair, in either the single or dual infection.