Diagnostic applications of molecular and serological assays for bluetongue and African horse sickness.

The availability of rapid, highly sensitive and specific molecular and serologic diagnostic assays, such as competitive enzyme-linked immunosorbent assay (cELISA), has expedited the diagnosis of emerging transboundary animal diseases, including bluetongue (BT) and African horse sickness (AHS), and facilitated more thorough characterisation of their epidemiology. The development of assays based on real-time, reverse-transcription polymerase chain reaction (RT-PCR) to detect and identify the numerous serotypes of BT virus (BTV) and AHS virus (AHSV) has aided in-depth studies of the epidemiology of BTV infection in California and AHSV infection in South Africa. The subsequent evaluation of pan-serotype, real-time, RT-PCR-positive samples through the use of serotype-specific RT-PCR assays allows the rapid identification of virus serotypes, reducing the need for expensive and time-consuming conventional methods, such as virus isolation and serotype-specific virus neutralisation assays. These molecular assays and cELISA platforms provide tools that have enhanced epidemiologic surveillance strategies and improved our understanding of potentially altered Culicoides midge behaviour when infected with BTV. They have also supported the detection of subclinical AHSV infection of vaccinated horses in South Africa. Moreover, in conjunction with whole genome sequence analysis, these tests have clarified that the mechanism behind recent outbreaks of AHS in the AHS-controlled area of South Africa was the result of the reversion to virulence and/or genome reassortment of live attenuated vaccine viruses. This review focuses on the use of contemporary molecular diagnostic assays in the context of recent epidemiologic studies and explores their advantages over historic virus isolation and serologic techniques.

La disponibilité d'essais diagnostiques moléculaires et sérologiques rapides, hautement sensibles et spécifiques tels que l'épreuve immuno-enzymatique de compétition (ELISAc), a accéléré le diagnostic des maladies animales transfrontalières émergentes, dont la fièvre catarrhale ovine (FCO) et la peste équine, et contribué à dresser un tableau épidémiologique plus complet de ces maladies. Grâce à la mise au point d'essais basés sur l'amplification en chaîne par polymérase en temps réel couplée à une transcription inverse (RT­PCR) qui permettent de détecter et d'identifier les nombreux sérotypes du virus de la fièvre catarrhale du mouton et du virus de la peste équine, des études approfondies ont pu être conduites sur l'épidémiologie de l'infection par le virus de la fièvre catarrhale du mouton en Californie et de l'infection par le virus de la peste équine en Afrique du Sud. L'évaluation postérieure des échantillons positifs à une RT­PCR en temps réel de groupe (détectant le virus quel que soit le sérotype) au moyen de RT­PCR spécifiques de chaque sérotype permet d'identifier rapidement le sérotype causal et de limiter le recours à des méthodes classiques onéreuses et chronophages comme l'isolement viral ou les essais de neutralisation virale spécifiques de chaque sérotype. Les outils fournis par ces essais moléculaires et par les plateformes ELISAc ont renforcé les stratégies de surveillance épidémiologique et permis de mieux connaître les altérations potentielles de comportement chez les tiques Culicoides infectées par le virus de la fièvre catarrhale du mouton. Ils ont également contribué à détecter les cas d'infection asymptomatique par le virus de la peste équine chez des chevaux vaccinés en Afrique du Sud. En outre, associés avec l'analyse de séquences du génome entier, ces tests ont révélé que le mécanisme sous-jacent aux récents foyers de peste équine dans la zone de contrôle en Afrique du Sud correspondait à une réversion vers la virulence et/ou à un réassortiment du génome des souches de vaccin à virus vivant atténué. Les auteurs passent en revue l'utilisation des essais de diagnostic moléculaire de nouvelle génération dans le contexte de récentes études épidémiologiques et cherchent à établir leurs avantages par rapport aux techniques classiques d'isolement viral et de recherche sérologique.

La existencia de ensayos moleculares y serológicos de diagnóstico rápidos y de gran sensibilidad y especificidad, como el ensayo inmunoenzimático de competición (ELISAc), ha acelerado el diagnóstico de enfermedades animales transfronterizas emergentes, como la lengua azul o la peste equina, y facilitado una caracterización más exhaustiva de su epidemiología. La creación de ensayos basados en la reacción en cadena de la polimerasa acoplada a transcripción inversa (RT?PCR) en tiempo real para detectar y caracterizar los numerosos serotipos de los virus de la lengua azul y la peste equina ha ayudado a estudiar a fondo la epidemiología de sendos episodios infecciosos causados por el virus de la lengua azul en California y por el virus de la peste equina en Sudáfrica. El subsiguiente análisis de las muestras positivas a la prueba de RT?PC en tiempo real de cualquier serotipo con empleo de ensayos RT?PCR dirigidos específicamente contra uno u otro serotipo permite identificar rápidamente los serotipos víricos, lo que hace menos necesario el uso de métodos convencionales más caros y largos, como el aislamiento del virus o técnicas de neutralización vírica adaptadas específicamente a un serotipo. Estos dispositivos de ensayo molecular o de ELISAc ponen a nuestra disposición herramientas que potencian las estrategias de vigilancia epidemiológica y ayudan a conocer mejor las eventuales alteraciones del comportamiento de los jejenes Culicoides al ser infectados por el virus de la lengua azul. Estas técnicas han ayudado también a detectar en Sudáfrica casos de infección asintomática por el virus de la peste equina en caballos vacunados. Estas pruebas, además, empleadas en combinación con el análisis de secuencias genómicas completas, han servido para aclarar que el mecanismo subyacente a los recientes brotes de peste equina surgidos en la zona de Sudáfrica donde la enfermedad estaba bajo control fue fruto de la reversión a la virulencia y/o el reordenamiento genómico de virus vacunales atenuados. Los autores, centrándose en el uso de modernos ensayos moleculares de diagnóstico como parte de recientes estudios epidemiológicos, examinan las ventajas que ofrecen en comparación con las tradicionales técnicas serológicas y de aislamiento vírico.

Habitat preferences and phenology of Ochlerotatus triseriatus and Aedes albopictus (Diptera: Culicidae) in southwestern Virginia.

Recently, the number of reported human cases of La Crosse encephalitis, an illness caused by mosquito-borne La Crosse virus (LAC), has increased in southwestern Virginia, resulting in a need for better understanding of the virus cycle and the biology of its vectors in the region. This study examined the spatial and temporal distributions of the primary vector of LAC, Ochlerotatus triseriatus (Say), and a potential secondary vector, Aedes albopictus (Skuse). Ovitrapping surveys were conducted in 1998 and 1999 to determine distributions and oviposition habitat preferences of the two species in southwestern Virginia. Mosquitoes also were collected for virus assay from a tire dump and a human La Crosse encephalitis case site between 1998 and 2000. Oc. triseriatus and Ae. albopictus were collected from all ovitrap sites surveyed, and numbers of Oc. triseriatus eggs generally were higher than those of Ae. albopictus. Numbers of Oc. triseriatus remained high during most of the summer, while Ae. albopictus numbers increased gradually, reaching a peak in late August and declining thereafter. In Wise County, relative Ae. albopictus abundance was highest in sites with traps placed in open residential areas. Lowest numbers of both species were found in densely forested areas. Ovitrapping during consecutive years revealed that Ae. albopictus was well established and overwintering in the area. An oviposition comparison between the yard and adjacent forest at a human La Crosse encephalitis case site in 1999 showed that Ae. albopictus preferentially oviposited in the yard surrounding the home, but Oc. triseriatus showed no preference. LAC isolations from larval and adult collections of Oc. triseriatus females from the same case site indicated the occurrence of transovarial transmission.

Polychlorinated biphenyls (PCBs) selectively disrupt serotonergic cell growth in the developing Spisula embryo.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that exert neurotoxic effects during embryonic development. The present study demonstrates that early embryonic exposure to a mixture of PCBs (Aroclor 1254) results in a decrease in serotonergic cell growth. Using a novel, marine invertebrate embryo model, Spisula solidissima, immunocytochemistry, and confocal microscopy techniques, a dose-dependent decrease in serotonergic cell number was quantified within 24 h of exposure. This effect was seen with doses as low as 1 ppm Aroclor 1254. These findings demonstrate that environmentally relevant doses of Aroclor 1254 impair development of the serotonergic nervous system.

Detection of mutant p53 in clam leukemia cells.

Leukemia in the soft-shell clam, Mya arenaria, is characterized by tumor cells which are detected initially in the hemolymph. This disease is much more common in clams inhabiting polluted waters, suggesting an environmental component to its pathogenesis. In this study, leukemia cells were identified using a murine monoclonal antibody, 1E10, which recognizes a leukemia-specific protein expressed by tumor cells. Mutant p53 protein was detected using a murine monoclonal antibody (PAb 240) which reacts with mutant p53. Using immunofluorescence, the reactivity of clam cells to the 1E10 antibody was evaluated along with mutant p53 protein reactivity. Reverse transcriptase-polymerase chain reactions followed by sequence analyses were utilized to examine clams with hemocytes reacting with the p53 antibody for possible p53 gene mutations. Mutant p53 protein was expressed by tumor cells from five animals with advanced disease (in which greater than 90% of cells reacted with 1E10). A C-->G transversion was detected at the end of exon 6 from two of the five animals that reacted with both the mutant p53 antibody and 1E10. This substitution changes the amino acid of this codon from proline to alanine. Overall, our results suggest that environmentally induced alterations in p53 can contribute to the pathogenesis of leukemia in soft-shell clams inhabiting polluted water and/or sediment.

Maximizing efficiency in the management of the physician practice: survival under managed care.

The managed care environment is forcing physicians to maximize the efficiency of the management of their practices. Physicians can no longer afford to delegate billing and office management to inexperienced or untrained personnel. All physician practices must ensure they have resources available to maximize revenue, control expenses, and avoid costly billing errors and omissions. Use of outside resources such as practice management firms and delegation of all billing and collections to professional medical billing companies are the best alternatives available to the physician.

Prognostic variables in hairy cell leukemia after splenectomy as initial therapy.

Splenectomy has been used as initial therapy in hairy cell leukemia (HCL) for many years and usually causes rapid improvement in peripheral blood counts. However, most patients eventually require further therapy. We have analyzed the case histories of 194 patients with HCL who had splenectomy as initial therapy. The median time to failure (second therapy or death) was 18.8 months. Univariate analysis of prognostic variables demonstrated that age, hemoglobin level, platelet count, bone marrow cellularity, bone marrow hairy cells, and hairy cell index (HCI) were statistically significant predictors of failure-free survival (FFS). However, only the bone marrow cellularity and platelet count were significant independent prognostic variables by Cox analysis. The patients were divided into the following three subsets by prognosis after splenectomy: (1) low risk of failure (cellularity less than 85% and platelet count greater than or equal to 60,000/microliters), (2) intermediate risk (cellularity less than 85% and platelet count less than 60,000/microliters), and (3) high risk (cellularity greater than or equal to 85%). The median FFS for each of these subsets was 56.5 months, 11.7 months, and 5.4 months, respectively. The median overall survival after splenectomy has not been reached with follow-up of up to 22 years. Patients diagnosed since 1982 have a better overall prognosis than those diagnosed earlier, with 4-year survivals of 88% and 68%, respectively (P less than 0.01). We conclude that splenectomy should continue to be the standard initial therapy in HCL. However, patients with bone marrow cellularity of 85% or greater have a short duration of response to splenectomy, and thus, may be considered for initial systemic therapy.

Specific chromosomal abnormalities in acute nonlymphocytic leukemia correlate with drug susceptibility in vivo.

Specific chromosomal abnormalities are independent predictors of response to therapy in acute nonlymphocytic leukemia (ANLL) de novo. In a series of 149 patients with ANLL, we sought to determine whether the t(8;21), t(15;17), t(9;11) or other abnormalities of the long arm of chromosome 11, inv(16) or t(16;16), inv(3) or t(3;3), trisomy 8, and abnormalities of chromosome 5 (-5/5q-) or of chromosome 7 (-7/7q-) identify differences in susceptibility to chemotherapy drugs in vivo. The immediate outcome of the first cycle of remission induction chemotherapy was analyzed for patients in each cytogenetic subgroup as an index of the drug susceptibility of the leukemia cells in vivo. Patients with t(8;21), inv(16), t(16;16), or 11q abnormalities had high rates of complete remission after initial therapy (60-100%), whereas patients with -7/7q- or -5/5q- had low initial response rates (0-36%), suggestive of drug resistance in vivo. In general, cytogenetic groups with high initial complete remission rates ("drug sensitive") also had long disease-free survivals; those groups with low initial remission rates ("drug resistant") had short remission durations even if these patients eventually achieved complete remission with further therapy. Patients with acute promyelocytic leukemia (APL), all of whom had the t(15;17), were the exception; despite low initial remission rates, they had long disease-free survivals, possibly due to a more rapid cytotoxic effect of chemotherapy on the clonogenic APL cells than on the more numerous malignant promyelocytes. We conclude that the prognostic importance of specific chromosomal abnormalities in ANLL resides in part in differing susceptibilities to chemotherapy.

Chlamydial pneumonia in the low birthweight neonate.

Five low birthweight neonates who developed a severe pneumonia are described. Chlamydia trachomatis was isolated from the respiratory tract of all infants. They had protracted respiratory problems despite eradication of the organism by chemotherapy.

Olfactory mucosa in herpes simplex encephalitis.

The olfactory mucosa was examined in three patients dying from herpes simplex encephalitis. It showed changes attributed to infection by the herpes simplex virus. It is suggested that in some patients encephalitis may be a complication of infection of the olfactory mucosa.

Infantile gastroenteritis: a clinical study of reovirus-like agent infection.

In a clinical study of 32 infants with symptoms from infections with the human reovirus-like agent (R.I.A.) identified by electron microscopy (E.M.) of faecal extracts, a fairly consistent clinical pattern was found in 30 who had a gastroenteritis-like illness. The disease was usually mild, affecting mainly infants less than 2 years and males more commonly than females. The incubation period appeared to be 48-72 hours; and the onset was sudden, often with vomiting in the first 1-2 days of the illness. Loose yellow-green offensive stools without blood or mucus developed after a variable time, and there was often accompanying fever. Severe dehydration and electrolyte inbalance were uncommon; and with standard treatment the illness was uncomplicated, usually lasting 5-8 days. These features resemble those of previously reported winter epidemics of infantile non-bacterial gastroenteritis, and it is suggested that these epidemics were due to R.L.A. 2 infants in whom R.L.A. was identified in the stool did not have a gastroenteritis-like illness although both had protracted diarrhoea.