Monkeypox virus isolation from longitudinal samples from four patients to infer risk of onwards transmission: an interim analysis.

Monkeypox virus (mpxv) is a DNA virus in the Orthopoxvirus genus which causes Mpox (previously monkeypox). Symptoms include fever, lymphadenopathy and vesicular lesions. There is limited evidence for the duration of mpxv infectivity. This study used cell culture as a proxy for infectivity. Clinical samples from four patients with Mpox were inoculated into African green monkey kidney (Vero E6) cells and monitored for cytopathic effects (CPE). From one patient, infectious mpxv was recovered 25 days after illness onset. Infectious virus was not isolated from samples with an Orthopoxvirus polymerase chain reaction (PCR) Ct value over 31.0, nor from urine.

Detection of SARS-CoV-2 within the healthcare environment: a multi-centre study conducted during the first wave of the COVID-19 outbreak in England.

BACKGROUND: Understanding how severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is spread within the hospital setting is essential in order to protect staff, implement effective infection control measures, and prevent nosocomial transmission. METHODS: The presence of SARS-CoV-2 in the air and on environmental surfaces around hospitalized patients, with and without respiratory symptoms, was investigated. Environmental sampling was undertaken within eight hospitals in England during the first wave of the coronavirus disease 2019 outbreak. Samples were analysed using reverse transcription polymerase chain reaction (PCR) and virus isolation assays. FINDINGS: SARS-CoV-2 RNA was detected on 30 (8.9%) of 336 environmental surfaces. Cycle threshold values ranged from 28.8 to 39.1, equating to 2.2 x 105 to 59 genomic copies/swab. Concomitant bacterial counts were low, suggesting that the cleaning performed by nursing and domestic staff across all eight hospitals was effective. SARS-CoV-2 RNA was detected in four of 55 air samples taken <1 m from four different patients. In all cases, the concentration of viral RNA was low and ranged from <10 to 460 genomic copies/m3 air. Infectious virus was not recovered from any of the PCR-positive samples analysed. CONCLUSIONS: Effective cleaning can reduce the risk of fomite (contact) transmission, but some surface types may facilitate the survival, persistence and/or dispersal of SARS-CoV-2. The presence of low or undetectable concentrations of viral RNA in the air supports current guidance on the use of specific personal protective equipment for aerosol-generating and non-aerosol-generating procedures.

Development of an indirect ELISA method for the parallel measurement of IgG and IgM antibodies against Crimean-Congo haemorrhagic fever (CCHF) virus using recombinant nucleoprotein as antigen.

Recombinant nucleoprotein from Crimean-Congo Haemorrhagic Fever (CCHF) virus was successfully derived from a baculovirus expression system and purified for use in a novel enzyme-linked immunosorbent assay (ELISA) diagnostic test. Comparable tests were used for detection of IgG and IgM antibodies, thus allowing efficient detection of both antibodies in parallel. The major benefits of the assay also included removing any requirement for polyclonal sera, thus eliminating variation in preparations and allowing standardisation between laboratories. The assay was successfully tested using a panel of positive sera supplied from samples identified as being positive in Turkey, Tajikistan and Kosovo and shown to be sensitive and specific. It is envisaged that this simple diagnostic ELISA for CCHF virus infection which removes the reliance on polyclonal antibody preparations, will be accessible to a wider range of laboratories enabling them to carry out routine diagnosis. This will improve the efficiency of diagnosis and subsequent management of infected patients.

The actions of crustacean cardioactive peptide on adult and developing stomatogastric ganglion motor patterns.

The motor patterns produced by the stomatogastric ganglion (STG) are strongly influenced by descending modulatory inputs from anterior ganglia. With these inputs intact, in control saline, the motor patterns produced by the stomatogastric nervous system of embryonic and larval lobsters are slower and less regular than those of adult lobsters. We studied the effects of the hormonal modulator, crustacean cardioactive peptide (CCAP) on the discharge patterns of STG motor patterns in embryos, larvae, and adult Maine lobsters, Homarus americanus, with the anterior inputs present and absent. In adults, CCAP initiated robust pyloric rhythms from STGs isolated from their descending control and modulatory inputs. Likewise, CCAP initiated robust activity in isolated embryonic and larval STGs. Nonetheless, quantitative analyses revealed that the frequency and regularity of the STG motor neuron discharge seen in the presence of CCAP in isolated STGs from embryos were significantly lower than those seen late in larval life and in adults under the same conditions. In contrast, when the descending control and modulatory pathways to the STG were left intact, the embryonic and larval burst frequency seen in the presence of CCAP was increased by CCAP, whereas the burst frequency in adults was decreased by CCAP, so that in CCAP the frequencies at all stages were statistically indistinguishable. These data argue that immature embryonic motor patterns seen in the absence of CCAP are a function of immaturity in both the STG and in the descending and modulatory pathways.

Development of the peptidergic modulation of a rhythmic pattern generating network.

The stomatogastric ganglion (STG) of adult lobsters and crabs receives dense aminergic and peptidergic projections. The neuropeptides are found in sensory neurons and in descending interneurons that modulate the output of the rhythmic central pattern generating networks in the STG. We describe the presence of these peptidergic projections in the adult Homarus americanus, and the effects of some of these neuropeptides on the motor patterns of the adult STG. We describe the developmental acquisition of these neuropeptides during embryonic and larval times and demonstrate that the immature STG networks are already sensitive to a variety of neuromodulators.

Maturation of lobster stomatogastric ganglion rhythmic activity.

The stomatogastric ganglion of the adult lobster, Homarus americanus generates extremely regular pyloric rhythms with a characteristic period of 0.5-1.5 Hz. To study the changes in the pyloric rhythm during embryonic and larval development, we recorded excitatory junctional potentials evoked by lateral pyloric (LP) neuron activity. Early in development the motor discharge of the LP neuron was often irregular, preventing use of conventional analysis methods that rely on extracting burst times to calculate cycle frequency and its variability. Instead, cycle frequency was determined for the LP neuron from the peak of the power spectrum obtained from the occurrence times of excitatory junctional potentials in the p1 muscle. The ratio of the power in the peak to the power from 0 to 3 Hz was used as a relative measure of the regularity of the rhythm. Throughout embryonic and the first larval stage, LP neuron activity is slow, irregular, and only weakly periodic. The regularity of the rhythm increased during midlarval stages, and both the frequency and regularity increased considerably by the postlarval stage LIV.

Sequential developmental acquisition of cotransmitters in identified sensory neurons of the stomatogastric nervous system of the lobsters, Homarus americanus and Homarus gammarus.

We studied the developmental acquisition of three of the cotransmitters found in the gastropyloric receptor (GPR) neurons of the stomatogastric nervous systems of the lobsters Homarus americanus and Homarus gammarus. By using wholemount immunocytochemistry and confocal microscopy, we examined the distribution of serotonin-like, allatostatin-like, and FLRF(NH2)-like immunoreactivities within the stomatogastric nervous system of embryonic, larval, juvenile, and adult animals. The GPR neurons are peripheral sensory neurons that send proprioceptive information to the stomatogastric and commissural ganglia. In H. americanus, GPR neurons of the adult contain serotonin-like, allatostatin-like, and Phe-Leu-Arg-Phe-amide (FLRF(NH2))-like immunoreactivities. In the stomatogastric ganglion (STG) of the adult H. americanus and H. gammarus, all of the serotonin-like and allatostatin-like immunoreactivity colocalizes in neuropil processes that are derived exclusively from ramifications of the GPR neurons. In both species, FLRF(NH2)-like immunoreactivity was detected in the STG neuropil by 50% of embryonic development (E50). Allatostatin-like immunoreactivity was visible first in the STG at approximately E70-E80. In contrast, serotonin staining was not clearly visible until larval stage I (LI) in H. gammarus and until LII or LIII in H. americanus. These data indicate that there is a sequential acquisition of the cotransmitters of the GPR neurons.

Studies of soil-water transport by MRI.

Sequential spin-echo spin-warp MRI pulse sequences have been used to study soil-water transport processes including infiltration, redistribution, and drainage of water in soil columns. Those images provide a means for monitoring and quantifying spatial and temporal changes of soil-water distributions and the movement of wetting fronts. In addition, temporal-geometric changes of unstable wetting fronts during water redistribution were estimated from 2D images and the temporal development of the longest length of finger was described by a fractal relation t approximately L1.38. Bulk dispersion-time-dependent displacement and velocity spectra, as well as 2D maps of flow velocities and dispersion coefficients in soil macropores during saturated steady-state flow, were reconstructed from data obtained using the alternating-pulsed-field-gradient (APFG) pulse sequences.

Magnetic resonance imaging of soil-water phenomena.

2D and 3D images of static and dynamic water phenomena in packed clay soil columns were obtained by MRI. A spin-echo spin-warp Fourier imaging sequence, together with prefocused 90 degrees selective pulse for 2D imaging, was selected. The images demonstrate the potential of using MRI for studies of static and dynamic water phenomena in soil with modest iron content and adequate water content.

Ultrastructure and microanalyses of the calcareous corpuscles of the protoscoleces of Echinococcus granulosus.

The calcareous corpuscles of the protoscolex stage of Echinococcus granulosus are irregularly spherical or ovoid in shape and have a diameter ranging between 2 and 16 microns. The central region of immature corpuscles is composed of an electron-lucent matrix containing granular deposits and, in more mature corpuscles, paired membrane lamellae. Energy-dispersive X-ray microanalysis of sectioned immature corpuscles demonstrated calcium, magnesium and phosphorus, whilst a quantitative analysis indicated the presence of calcium and magnesium at 142.7 and 41.3 mg/g dry weight, respectively, and inorganic phosphate at 18.0 mg/g. Assuming that the anion is predominantly carbonate, the molar ratio of Ca:Mg:HPO4(2-):CO3(2-) is 1:0.48:0.08:1.41. X-ray diffraction patterns obtained from preparations of whole corpuscles indicated a poorly crystalline material including the mineral calcite. X-ray absorption near-edge spectra of corpuscles, taken over the phosphorus K edge, resembled those of brushite (CaHPO4.2H2O) and suggest that the phosphate, within the corpuscles, is present in an amorphous, hydrated form that could be readily solubilised and mobilised for the metabolic processes of the organism.

Ultrastructure and microanalyses of the protoscolex hooks of Echinococcus granulosus.

The rostellar distal cytoplasm of Echinococcus granulosus protoscoleces is characterized by extensive basal membrane infolding, prominent hemidesmosomes and is subtended by a lamina reticularis with microfibrils of approximately 10 nm diameter that occasionally show a 55 nm banding periodicity. The rostellar hooks, in 2 rows, each have a blade, guard and handle region and possess a central amorphous pulp, a middle microfibrillar medulla with microfibrils of approximately 4 nm diameter, and a complex outer cortex in all but the proximal region of the guard and the base of the handle. In these regions additional material, of similar electron density to the medulla, but lacking the fibrillar substructure, occurs and gives the areas a lobed appearance. Energy-dispersive X-ray microanalysis of whole hooks demonstrated the presence of sulphur and trace quantities of phosphorus. X-ray near-edge absorption spectra resembled those of cystine, feather and hair and showed the sulphur to be predominantly in the form of disulphide linkages. X-ray diffraction patterns of whole hook preparations revealed 2 diffuse rings with equatorial spacings of 7.99 A and 15.22 A, thus differing from vertebrate keratins.

Perioperative prophylactic chemotherapy of Echinococcus granulosus: determination of minimum effective length of albendazole therapy in in vitro protoscolex culture.

Protoscoleces of Echinococcus granulosus were cultured in vitro in 500, 250 or 100 micrograms/l albendazole sulphoxide for 1,3,7,10,14d and then 'rescued' (R) into drug-free medium for the remainder of the culture period. Successful minimum lengths of therapy were much longer than for praziquantel, and only at 500 micrograms/l was the 10dR treatment as effective as continuous therapy for 28d. Treatment with 100 micrograms/l both in continuous culture and in the 'R' experiments was ineffective over a 35d period. The results are compared with those from similar experiments using praziquantel.

Comparison of Albendazole and Praziquantel therapy of Echinococcus granulosus in naturally infected sheep.

The effects of albendazole (10 mg kg-1 day-1) and praziquantel (50 mg kg-1 day-1) for 6 weeks on naturally infected sheep with pulmonary cysts of Echinococcus granulosus of proven viability were studied. Immediately following therapy, one of three sheep treated with praziquantel had viable cysts and 7 months later one of two sheep had viable cysts. One sheep died during albendazole therapy, but 7 months following therapy only one of five sheep had viable cysts. Electron microscopy demonstrated necrotic germinal layer tissue in most albendazole-treated cysts and praziquantel also had an effect on cyst ultrastructure. These data suggest that recurrence in humans treated with albendazole may be small. Whilst praziquantel was not particularly effective in this animal model, its clear effect on the ultrastructure suggests that an increased dose and combination therapy with albendazole may be more effective.

Effect of albendazole on human hydatid cysts: an ultrastructural study.

Five patients with hepatic (3), pelvic (1) or spinal (1) hydatid cysts received 10 mg/kg/d albendazole for 1-3 months prior to surgery. Daughter cysts were present in the spinal hydatid and in one patient with hepatic disease. Electron microscope examination of the cyst tissue of the pelvic and the 2 hepatic cysts lacking daughter cysts showed no evidence of germinal layer, and the protoscoleces were dead. The primary cyst of the hepatic hydatid with daughter cysts (1 month therapy) was also judged dead but some pieces of the daughter cyst germinal layer appeared normal and had unaffected protoscoleces. The daughter cyst tissue of the spinal hydatid (3 month therapy) appeared normal and the protoscoleces viable. In view of the undetermined viability of human hydatids before chemotherapy, treatment of longer than 1 month is advocated for hepatic cysts, particularly if daughter cysts are present, and longer therapy is indicated for spinal disease.

Rapid recovery of Echinococcus granulosus following 'successful' albendazole therapy in a gerbil model.

Peritoneal Echinococcus granulosus in gerbils was treated with albendazole. Both early and late infections were studied; response to albendazole therapy and the ability of the parasite to recover after treatment was found to depend on dose and length of therapy. Even after treatment at 50 mg/kg for 2 months late infections retained the ability to recover over 3 months.

Comparison of albendazole, mebendazole and praziquantel chemotherapy of Echinococcus multilocularis in a gerbil model.

The efficacy of albendazole (50 mg/kg/d), mebendazole (50 mg/kg/d) and praziquantel (500 mg/kg/d) against established intraperitoneal infections of Echinococcus multilocularis in gerbils was compared by monitoring parasite weight and making ultrastructural observations on treated and untreated material. Praziquantel was the most active protoscolicidal agent, reducing protoscolex viability to less than 2%, although it did not inhibit cyst growth. Albendazole was the most effective agent in reducing cyst growth and was, when compared with other regimes significantly more effective than mebendazole (p less than 0.05), praziquantel (p less than 0.01) or untreated controls (p less than 0.01).

Echinococcus multilocularis: ultrastructural effect of in vivo albendazole and praziquantel therapy, singly and in combination.

Cotton rats with Echinococcus multilocularis infections of one month's standing were treated for six months with albendazole (50 mg kg-1 day-1), or praziquantel (500 mg kg-1 day-1), or both drugs simultaneously. The germinal layer of albendazole-treated cysts differed little from control tissue apart from marginal increase in cyton vesiculation and the presence of small lamellated residual bodies. Protoscolex tissue appeared unaffected. The germinal layer of praziquantel-treated cysts differed markedly from control tissue, with increased vesiculation and mitochondrial number in those with intact germinal layers, and disintegration and detachment of the layer in other cysts. Protoscoleces were necrotic. After combined therapy, the germinal layer was either intact but with increased vesiculation and residual bodies present, or was disintegrating and detaching from the laminated layer.

Albendazole is effective against established Echinococcus granulosus in gerbils: comparison of serum concentrations achieved by gavage and feed administration.

Serum levels of albendazole sulphoxide in gerbils infected with Echinococcus granulosus and treated with albendazole (20 or 50 mg kg-1 day-1) by gavage were dose dependent, whereas albendazole administered in feed at a rate equivalent to 80 mg kg-1 day-1 achieved serum levels calculated to be equivalent to those produced by administration of 40 mg kg-1 day-1 by gavage. This regimen, over a three-month period, was effective and reduced parasite weight to 4.6% of that in control gerbils, and the treated parasite tissue showed ultrastructural changes ranging from severe accumulation of residual bodies to total disintegration of the germinal layer.

Echinococcus granulosus: in vitro maintenance of whole cysts and the assessment of the effects of albendazole sulphoxide and praziquantel on the germinal layer.

Small entire cysts of Echinococcus granulosus of human and animal origin were cultured in vitro in the presence or absence of albendazole sulphoxide (1000 micrograms/litre) or praziquantel (500 micrograms/litre) for 10 or 11 d, and subsequently passaged into the peritoneal cavity of gerbils to assess viability by continued cyst growth. Viability was reduced in the presence of albendazole sulphoxide, and disintegration of the germinal layer immediately after culture was demonstrated at the ultrastructural level. Praziquantel had no apparent effect on cyst growth.