Diamond burr superficial keratectomy for recurrent corneal erosions.

AIMS: To evaluate the efficacy and safety of diamond burr superficial keratectomy in the treatment of recurrent corneal erosions. METHODS: A retrospective review of 54 eyes (47 patients) with recurrent corneal erosions treated with diamond burr superficial keratectomy. Preoperative and postoperative visual acuities and refractions, slit lamp examination findings, and the incidence of recurrent erosion after keratectomy were studied. Specular microscopy was also performed in six patients before and after surgery. RESULTS: 30 eyes had underlying map dot fingerprint anterior basement membrane corneal dystrophy, while 24 eyes did not. Postoperative follow up time ranged from 3 to 53 months (mean 12.3 months). Corneal erosion recurred in three eyes (6%) after diamond burr superficial keratectomy. This procedure improved the best corrected visual acuity from 20/26 to 20/22 by logMAR statistical evaluation (p=0.002) and caused very little change in the refractive spherical equivalent. No endothelial cell loss or changes in morphology were noted on specular microscopy. CONCLUSION: Diamond burr superficial keratectomy appears to be an effective and safe method of treating recurrent erosions and is a good alternative therapy to needle stromal micropuncture, Nd:YAG induced epithelial adhesion, and excimer laser surface ablation.

Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States.

From October 4 to November 2, 2001, the first 10 confirmed cases of inhalational anthrax caused by intentional release of Bacillus anthracis were identified in the United States. Epidemiologic investigation indicated that the outbreak, in the District of Columbia, Florida, New Jersey, and New York, resulted from intentional delivery of B. anthracis spores through mailed letters or packages. We describe the clinical presentation and course of these cases of bioterrorism-related inhalational anthrax. The median age of patients was 56 years (range 43 to 73 years), 70% were male, and except for one, all were known or believed to have processed, handled, or received letters containing B. anthracis spores. The median incubation period from the time of exposure to onset of symptoms, when known (n=6), was 4 days (range 4 to 6 days). Symptoms at initial presentation included fever or chills (n=10), sweats (n=7), fatigue or malaise (n=10), minimal or nonproductive cough (n=9), dyspnea (n=8), and nausea or vomiting (n=9). The median white blood cell count was 9.8 X 10(3)/mm(3) (range 7.5 to 13.3), often with increased neutrophils and band forms. Nine patients had elevated serum transaminase levels, and six were hypoxic. All 10 patients had abnormal chest X-rays; abnormalities included infiltrates (n=7), pleural effusion (n=8), and mediastinal widening (seven patients). Computed tomography of the chest was performed on eight patients, and mediastinal lymphadenopathy was present in seven. With multidrug antibiotic regimens and supportive care, survival of patients (60%) was markedly higher (<15%) than previously reported.

Enzyme-linked immunosorbent assays for detection of antibodies to Ebola and Marburg viruses using recombinant nucleoproteins.

The full-length nucleoprotein (NP) of Ebola virus (EBO) was expressed as a His-tagged recombinant protein (His-EBO-NP) by a baculovirus system. Carboxy-terminal halves of NPs of EBO and Marburg virus (MBG) were expressed as glutathione S-transferase-tagged recombinant proteins in an Escherichia coli system. The antigenic regions on the NPs of EBO and MBG were determined by both Western blotting and enzyme-linked immunosorbent assay (ELISA) to be located on the C-terminal halves. The C-terminal 110 and 102 amino acids of the NPs of EBO and MBG, respectively, possess strong antigenicity. The full-length NP of EBO was strongly expressed in insect cells upon infection with the recombinant baculovirus, while expression of the full-length NP of MBG was weak. We developed an immunoglobulin G (IgG) ELISA using His-EBO-NP and the C-terminal halves of the NPs of EBO and MBG as antigens. We evaluated the IgG ELISA for the ability to detect IgG antibodies to EBO and MBG, using human sera collected from EBO and MBG patients. The IgG ELISA with the recombinant NPs showed high sensitivity and specificity in detecting EBO and MBG antibodies. The results indicate that ELISA systems prepared with the recombinant NPs of EBO and MBG are valuable tools for the diagnosis of EBO and MBG infections and for seroepidemiological field studies.

A reassortant bunyavirus isolated from acute hemorrhagic fever cases in Kenya and Somalia.

In late 1997 and early 1998, a large outbreak of hemorrhagic fever occurred in East Africa. Clinical samples were collected in Kenya and southern Somalia, and 27 of 115 (23%) hemorrhagic fever patients tested showed evidence of acute infection with Rift Valley fever (RVF) virus as determined by IgM detection, virus isolation, detection of virus RNA by reverse transcription-polymerase chain reaction (RT-PCR), or immunohistochemistry. However, two patients (one from Kenya and the other from Somalia) whose illness met the hemorrhagic fever case definition yielded virus isolates that were not RVF. Electron microscopy suggested these two virus isolates were members of the family Bunyaviridae. RT-PCR primers were designed to detect bunyavirus RNA in these samples. Regions of the S and L segments of the two isolates were successfully amplified, and their nucleotide sequences exhibited nearly complete identity with Bunyamwera virus, a mosquito-borne virus not previously associated with severe human disease. Unexpectedly, the virus M segment appeared to be reassorted, as the sequences detected exhibited 32-33% nucleotide and 28% amino acid differences relative to the corresponding M segment sequence of Bunyamwera virus. The association of this reassortant bunyavirus, proposed name Garissa virus, with severe disease is supported by the detection of the virus RNA in acute-phase sera taken from 12 additional hemorrhagic fever cases in the region.

Foot-and-mouth disease: a review of the virus and the symptoms.

Foot-and-mouth disease virus (FMDV) is the etiologic agent of foot-and-mouth disease (FMD), which is a disease of cattle, swine, and other cloven-footed animals. FMD is characterized by the formation of vesicles on the tongue, nose, muzzle, and coronary bands of infected animals. The virus has several unique characteristics that enable it to cause one of the most economically devastating diseases in today's world. The ease with which it may be transmitted by contact and aerosol, combined with its enhanced ability to initiate infections, virtually ensures that most, if not all, animals in a herd will contract FMD. The long-term survival of FMDV in infected animals' tissues and organs, especially when refrigerated, offers an opportunity for its national and international transmission through the food chain. Multiple serotypes and numerous subtypes reduce the effectiveness and reliability of vaccines. The possible development of carriers in vaccinated animals and those that have recovered from FMD provides additional potential sources of new outbreaks. These features create a disease that can have a major economic impact on farmers and entire nations.

Small, overlapping tectonic keratoplasty involving graft-host junction of penetrating keratoplasty.

PURPOSE: To report the indications for and postoperative course of small tectonic keratoplasties overlapping (and involving) the graft-host junction of preexisting penetrating keratoplasties. METHODS: A retrospective study of 15 consecutive eyes (15 patients) with small tectonic keratoplasties overlapping the graft-host junction of preexisting penetrating keratoplasties. RESULTS: After tectonic keratoplasty, follow-up times ranged from 5 months to 20 years (mean, 69 months). Clinical indications included sterile corneal ulceration (seven cases), bacterial keratitis (six cases), and fungal keratitis (two cases). In the six cases with bacterial keratitis, five were suture abscesses, with four resulting in wound dehiscence. Ten tectonic grafts were lamellar keratoplasties, and five were penetrating keratoplasties. Postoperative best-corrected visual acuities were unchanged from preoperative levels in every patient. After tectonic grafting, the mean +/- SD change in keratometric astigmatism in the parent penetrating keratoplasty was 1.75 +/- 1.50 diopters. The astigmatism increased in 10 cases, decreased in three, and remained unchanged in two. There was no case of recurrent ulceration or wound dehiscence in or around the tectonic grafts. The surgery did not result in new glaucoma or worsening of preexisting glaucoma. CONCLUSIONS: In the treatment of infectious or ulcerative foci at or near the graft-host junction of penetrating keratoplasties, a small extirpative, tectonic graft over the diseased junction appears to be a safe and effective alternative to either repeating the original penetrating keratoplasty or performing an oversize transplant.

Lamellar corneal patch grafts in the management of corneal melting.

PURPOSE: To evaluate the clinical indications and results of reconstructive (tectonic) lamellar keratoplasty in corneal melting. METHODS: A nonrandomized, uncontrolled retrospective case series of 64 consecutive patients (80 eyes) who underwent lamellar keratoplasty for corneal melting at our institution over a 17-year period. We reviewed the (a) clinical indications, (b) visual acuities, (c) postoperative corneal clarity, and (d) post-operative complications. Comparisons in visual acuity were made between central and peripheral corneal melts. The statistical influence of patient age, diagnosis, and corneal graft size on pre- and postoperative visual acuity values also was studied. RESULTS: Although reconstructive lamellar keratoplasty for active corneal melting was effective in saving the integrity of the globes in all but four patients, the postoperative visual acuity remained poor in the majority of cases because of the often devastating nature of the underlying ocular diseases. Only 14 patients had best postoperative visual acuities of 20/100 or better. Repeated lamellar keratoplasties were necessitated by corneal opacification, infection, or progressive postoperative corneal dissolution in 14 cases. Subsequent vision-restoring surgeries, consisting of penetrating keratoplasties or cataract extractions, were done in 11 eyes with modest improvement of visual acuity. Postoperative visual acuity was significantly better in peripheral corneal melts than in central melts (p = 0.004). CONCLUSION: Lamellar keratoplasty is an effective method of restoring the integrity of the eye ravaged by corneal melting. It is less invasive and consequently safer than penetrating keratoplasty in actively inflamed and unstable eyes. The primary purpose for this surgery is to salvage the integrity of the globe during the acute phase of disease and not so much to achieve visual improvement per se. It allows time for systemic immunosuppression to take effect and for the eye to quiet down before possible future vision-restoring surgery.

A universal virus inactivant for decontaminating blood and biopharmaceutical products.

Removal of virus infectivity from blood and biopharmaceutical products prepared from blood is an issue of considerable importance. Irrespective of the methods that are chosen it is vital that the biological activity of the product is not impaired. For blood and unfractionated plasma or serum, the problem is even more challenging. Selective inactivation of the genome is the key step in the preparation of killed virus vaccines. Imines have been used for more than 30 years for the preparation of inactivated foot-and-mouth disease virus vaccines without any evidence of survival of virus infectivity. Moreover, the immunogenicity of the virus is unimpaired. Viruses belonging to all the recognised families can be inactivated by imines. The biological properties of several proteins, including the cell growth-promoting factors in calf serum, are not impaired using conditions which ensure the inactivation of > 10(15) infectious units of poliovirus and foot-and-mouth disease virus (FMDV). Moreover, both viruses can be inactivated by imines at 4 degrees C, thus providing a method for removing infectivity from protein preparations which are unstable at higher temperatures. The mechanism by which FMDV is inactivated has been studied. We found that the RNA extracted from the virus after inactivation at 4 degrees C was not degraded and contained no hidden breaks but nevertheless was non-infectious. However, it could be amplified by PCR using primers corresponding to the gene coding for a portion of the viral RNA polymerase, but not from that coding for VP1, one of the structural proteins, showing that alteration of a base or bases had occurred in that region.

Central lamellar keratoplasty for optical indications.

PURPOSE: This study aims to evaluate the results of lamellar keratoplasty (LKP) for optical (nontectonic) indications over the past 19 years at our institution, noting the advantages and pitfalls of the procedure. METHODS: The study is a retrospective review of 52 central LKPs in 37 patients. Snellen visual acuity, preoperative clinical indications, and postoperative status of the cornea (donor graft, graft-host interface, and recipient cornea) were assessed. RESULTS: Postoperative follow-up ranged from 3 months to 18 years (median, 3 years). In descending order of frequency, corneal dystrophies, aniridic keratopathy, corneal scars, and keratoconus were the most common indications for surgery. After surgery, 38% of the eyes were able to achieve 20/50 or better visual acuity. The two most common causes of poor visual acuity were (1) opacification and/or blood vessel growth in the graft-host interface or on the graft surface and (2) high astigmatism. Persistent epithelial defects occurred in 21% of the eyes after LKP. CONCLUSION: Although LKP provides a safer alternative to penetrating keratoplasty, it is limited by vision-reducing graft-host interface problems, astigmatism, and difficult surgical technique. We postulate that the current results of LKP may be improved by (1) removing as much recipient corneal stroma as possible (e.g., dissecting down to Descemet's membrane) or, alternatively, using an automated microkeratome and (2) raising the currently used qualitative eyebank standards for accepting LKP donor tissue.

Differentiating foot-and-mouth disease virus-infected from vaccinated animals with baculovirus-expressed specific proteins.

We had shown in preliminary studies with a small number of animals that antibodies against 2C could be detected in cattle and pigs which had been infected with FMDV but not in animals which had been vaccinated against the disease. To determine whether this test was generally applicable, sera from several hundred animals which had been vaccinated with different products in many countries have been tested in an ELISA using baculovirus expressed 2C. Our results show that only 1-2% of the sera gave a positive reaction by this method. In contrast, 100% of sera from convalescent animals gave a positive reaction. To be useful in differentiating between convalescent and vaccinated animals it is necessary to know how long these antibodies can be detected by our ELISA. We have determined the levels of antibodies against 2C and also other virus-specific proteins which are present in cattle and pigs following infection with FMDV. Our results show that levels of anti-3ABC antibodies could be detected by ELISA with baculovirus-expressed protein up to one year after infection. In contrast, the levels of anti-2C antibodies fell more rapidly than those against 3ABC indicating that the latter protein may be preferable for detecting convalescent animals. Nevertheless, we envisage that the final test format should include several virus-specific proteins to determine accurately the immune status of an animal.

Cattle response to foot-and-mouth disease virus nonstructural proteins as antigens within vaccines produced using different concentrations.

Four groups of ten nine-month-old Nelore heifers were used for this study. Each group received one of four foot-and-mouth disease (FMD) trivalent vaccines for the duration of the experiment. The four vaccine formulations (Normal, 2X, 4X and 8X) differed in 140S content to determine the serological reactivities to FMD virus (FMDV) nonstructural proteins 2C, 3ABC and 3D. Vaccination was by the intramuscular administration of vaccine on day 0, 180 and 360. Bleedings were done at 30 days post vaccination (dpv), 90 dpv, 30 days post revaccination (dpr), 90 dpr, and 30 days post third administration (dprr). There was a general tendency to have higher mean 3D responses with increased vaccine application but not with increased concentration of antigen. With 2C and 3ABC this tendency was not seen, neither with repeated application of vaccine nor with increased antigen concentration. All individual animal observations to 2C and 3ABC remained within three standard deviations of the average observed for naive bovids. Percent of positive (PP) reactions was determined using an ELISA for nonstructural proteins 2C, 3ABC and 3D expressed in baculovirus as previously described. A value of > 25 PP to 2C or 3ABC could be considered as an indication of previous infection or of the presence of viral activity. PP results between 18 and 25 PP suggest viral activity and animals should be retested. Those responses below 15 PP are suggestive of vaccination or naive status. As diagnosis in the laboratory is not divorced from the field epidemiological scene, the intermediate zone between 10 and 20 PP should be considered and acted upon according to the overall zoosanitary situation of that country or region and the purposes of the ongoing FMD control efforts.

The capsid protein of vesicular exanthema of swine virus serotype A48: relationship to the capsid protein of other animal caliciviruses.

Vesicular exanthema of swine virus (VESV), the prototype calicivirus, is the etiologic agent of the porcine disease vesicular exanthema of swine (VES). VES is characterized by vesicle formation on the extremities, mouth and snout and causes abortions and stillbirths if infection occurs during pregnancy. VESV is considered an exotic agent in the US, following its eradication in 1956. The single capsid protein gene of VESV serotype A48 was cloned and sequenced. The capsid amino acid sequence was 69% similar to the San Miguel sea lion virus serotype 1 (SMSV 1) and 89% similar to the SMSV serotype 4 (SMSV 4) capsid proteins. The six functional regions (A-F) previously identified in SMSV 1, SMSV 4, feline calicivirus and rabbit hemorrhagic disease virus capsid proteins were present in VESV A48. Two sets of PCR primers were designed which directed amplification of the 5' end (A region) and the hypervariable (E region) sequences of the capsid protein precursor gene of these viruses, as well as seven additional SMSV serotypes. Alignment and phylogenetic analysis of the N-terminal sequences demonstrated the close relationship of these viruses. Alignment of the hypervariable region amino acid sequences of the ten viruses confirmed that a great variety of sequence exists in this region; however, a consensus sequence (NxT(N/H)F(K/R)GxYI(C/M)GxLx(T/R)) was derived which is also present in the feline calicivirus capsid protein. Comparison of the E region sequences provides further evidence that this area of animal calicivirus capsid protein may contain the major antigenic determinants.

A universal virus inactivant for decontaminating blood and biopharmaceutical products.

Removal of virus infectivity from blood and biopharmaceutical products prepared from blood is an issue of considerable importance. For biopharmaceutical products, removal can usually be achieved by a series of fractionation steps or by inactivation with a suitable reagent. Irrespective of the methods that are chosen it is vital that the biological activity of the product is not impaired. For blood and unfractionated plasma or serum, the problem is even more challenging. Selective inactivation of the genome is the key step in the preparation of killed virus vaccines. Viruses belonging to all the recognised families can be inactivated by imines. In this paper it is shown that the biological properties of several proteins, including the cell growth-promoting factors in calf serum, are not impaired using conditions which ensure the inactivation of > 10(15) infectious units of poliovirus and foot-and-mouth disease virus (FMDV). Also shown is that both viruses can be inactivated by imines at 4 degrees C, thus providing a method for removing infectivity from protein preparations which are unstable at higher temperatures. The RNA extracted from FMDV inactivated at 4 degrees C was not degraded and contained no hidden breaks but nevertheless was non-infectious. However, it could be amplified by PCR using primers corresponding to the gene coding for a portion of the viral RNA polymerase, but not from that coding for VP1, one of the structural proteins, showing that alteration of a base or bases had occurred in that region. Surprisingly, it could be translated in the rabbit reticulocyte system although some of the products were different from those obtained with unmodified RNA.

The utility of culturing corneal ulcers in a tertiary referral center versus a general ophthalmology clinic.

OBJECTIVE: The purpose of the study is to compare the utility of culturing corneal ulcers in a tertiary referral clinic and a general ophthalmology clinic. DESIGN: A retrospective review of medical and microbiologic records was performed. PARTICIPANTS: One hundred fifty-seven patients with corneal ulcers were included in the study. Eighty-two ulcers were treated in the referral clinic and 75 ulcers were treated in the general ophthalmology clinic. MAIN OUTCOME MEASURES: The authors determined the percentage of corneal ulcers in each clinical setting that failed to respond to empiric therapy and required a culture-directed change in treatment. RESULTS: One hundred fifty-seven ulcers were included. Eight (10%) of the 82 patients treated in the Cornea Clinic had treatment altered based on culture and sensitivity results. All 75 patients in the general clinic responded to empiric antibiotics, such that culture data never were required for modification of therapy (0%, P = 0.007). In contrast to patients treated in the Cornea Clinic, patients treated in the general clinic had smaller, more peripheral ulcers, shorter duration of symptoms, and fewer risk factors for corneal ulceration other than contact lens wear. CONCLUSIONS: Cornea specialists, who are referred the most severe cases, should consider culturing most corneal ulcers. However, it appears reasonable for general ophthalmologists to use culturing more judiciously. Patients with significant corneal ulcers should be cultured regardless of the clinic to which they present. However, small, peripheral ulcers respond extremely well to current, broad-spectrum antibiotics. Close follow-up is mandatory to discover the rare patient who will not respond to empiric therapy.

Assessment of health-related quality of life after corneal transplantation.

PURPOSE: To determine the extent to which commonly used clinical measures of corneal transplantation outcome are related to aspects of visual function and health-related quality of life. METHODS: In a cross-sectional validation study, ophthalmic examination information was collected by chart review of, and health-related quality of life information was collected by telephone contact with, patients (n = 77) undergoing routine follow-up examinations at least 1 year after corneal transplantation. A questionnaire that included the VF-14 and SF-36 instruments was completed for each participant. Associations between clinical and questionnaire outcomes were evaluated using analysis of variance and regression analyses. RESULTS: When the best-corrected visual acuity of both eyes was evaluated, there was a positive association (P < .0001) of visual acuity with the VF-14 score and with the following SF-36 scales: role limitations because of emotional problems (P = .04), emotional well-being (P = .08), and social functioning (P = .02). Those with a high degree of keratometric astigmatism showed an impact on social functioning (P = .005). Upon regression analysis, the single most important factor associated with the patients' reported visual function was their visual acuity in the better eye, followed by the extent of keratometric astigmatism. CONCLUSIONS: These findings demonstrate a high degree of criterion validity in using the VF-14 instrument to assess the outcome of corneal transplantation. Application of the more generic SF-36 measure shows effects of visual disability on other aspects of corneal transplant patients' health status, including their emotional and social functioning.

Baculovirus expressed 2C of foot-and-mouth disease virus has the potential for differentiating convalescent from vaccinated animals.

Determining whether animals have been infected with foot-and-mouth disease virus or vaccinated is important because infected animals frequently become carriers of the virus, shed it intermittently and thus may be the source of new outbreaks of the disease. We had shown previously that the sera of convalescent animals contain antibodies to 2C, a highly conserved non-structural protein, whereas the sera of vaccinated animals do not. This is explained by observation that 2C is retained on the membranes of cells used for growing the virus for vaccine production. In contrast, the non-structural protein 3D, which is released into the medium, is not removed by centrifugation or filtration during vaccine production and therefore stimulates an immune response in both vaccinated and convalescent cattle. In this study we produced 2C and 3D in insect cells infected with recombinant baculoviruses. As demonstrated by serology and electron microscopy, 2C is also retained on the membranes of the insect cells. Both expressed proteins react with sera of convalescent animals, indicating that they are conformationally similar, but the 2C does not react with sera from vaccinated animals. The baculovirus expressed 2C appears to be a suitable antigen for the development of a reliable diagnostic test.

Sequence identification of antigenic variants in plaque isolates of foot-and-mouth disease virus.

Foot-and-mouth disease virus isolates frequently contain mixtures of antigenic variants. Using synthetic mixtures of two 'pure' viruses which differed at one amino acid of the major epitope, it was found that a minor component present as 10% or less of the mixture would be undetected by nucleic acid sequencing.

Localization of foot and mouth disease virus RNA in tissue culture infected cells via in situ polymerase chain reaction.

Foot and mouth disease virus RNA was visualized in infected primary tissue culture cells by in situ PCR incorporating digoxigenin-labeled dUTP. The viral RNA polymerase gene was used as a target for amplification. Infected cells revealed cytoplasmic staining, predominantly perinuclear. The intensity of staining was in proportion to the degree of cytopathology observed and similar to the results obtained using immunoperoxidase staining. The in situ PCR technique for FMDV detection could be applied to formalin-fixed samples and be useful for the study of persistent infections.

Genetic relatedness of the caliciviruses: San Miguel sea lion and vesicular exanthema of swine viruses constitute a single genotype within the Caliciviridae.

The San Miguel sea lion viruses (SMSV) and vesicular exanthema of swine viruses (VESV) are related morphologically and antigenically, but little has been done to determine their genotypic relationship to each other and to other caliciviruses. To examine this relationship, reverse transcriptase PCRs were performed by using oligonucleotide primer sets designed to amplify portions of the 2C RNA helicase-like and RNA-dependent RNA polymerase regions with total cellular RNA purified from virus-infected cell cultures as a template. The 2C RNA helicase primers directed the amplification of this region from eight SMSV serotypes, five VESV serotypes, and four related viruses. The RNA polymerase primer sets amplified products from all these viruses except one. Phylogenetic comparison of the caliciviruses demonstrated that SMSV, VESV, and four related viruses are closely related while being distinct from feline calicivirus, the human caliciviruses (small, round-structured viruses), and rabbit hemorrhagic disease virus and that they should be classified as a single genotype within the Caliciviridae.

Pathogenesis of foot-and-mouth disease in swine, studied by in-situ hybridization.

Eight 7-month-old pigs were inoculated intradermally with 10(3) plaque-forming units of foot-and-mouth disease virus, type O, and killed 24, 48, 72, or 96 h later. Numerous tissues from each animal were collected and examined histopathologically and by in-situ hybridization to determine the presence of virus and its correlation with lesion development. The probe for in-situ hybridization was a biotinylated 500-base negative-sense transcription product corresponding to a portion of the gene encoding polymerase. With this technique, virus was shown to be widely disseminated in all epidermal tissues, regardless of histologically apparent cellular disruption.