Investigation of removal and inactivation efficiencies of human sapovirus in drinking water treatment processes by applying an in vitro cell-culture system.

Here, we examined the efficiencies of drinking water treatment processes for the removal and inactivation of human sapovirus (HuSaV). We applied a recently developed in vitro cell-culture system to produce purified solutions of HuSaV containing virus concentrations high enough to conduct virus-spiking experiments, to develop an integrated cell culture-polymerase chain reaction (ICC-PCR) assay to quantify the infectivity of HuSaV, and to conduct virus-spiking experiments. In virus-spiking coagulation-sedimentation-rapid sand filtration (CS-RSF) and coagulation-microfiltration (C-MF) experiments, HuSaV removals of 1.6-3.7-log10 and 1.2->4.3-log10, respectively, were observed. The removal ratios observed with CS-RSF were comparable and correlated with those of murine norovirus (MNV, a widely used surrogate for human noroviruses) and pepper mild mottle virus (PMMoV, a potential surrogate for human enteric viruses in physical and physicochemical drinking water treatment processes), and those observed with C-MF were higher than but still correlated with those of MNV and PMMoV, indicating that MNV and PMMoV are both potential surrogates for HuSaV in CS-RSF and C-MF. For astrovirus (AstV, a representative human enteric virus), removal ratios of 1.8-3.3-log10 and 1.1->4.0-log10 were observed with CS-RSF and C-MF, respectively. The removal ratios of AstV observed with CS-RSF were comparable and correlated with those of PMMoV, and those observed with C-MF were higher than but still correlated with those of PMMoV, indicating that PMMoV is a potential surrogate for AstV in CS-RSF and C-MF. When the efficacy of chlorine treatment was examined by using the developed ICC-PCR assay, 3.8-4.0-log10 inactivation of HuSaV was observed at a CT value (free-chlorine concentration [C] multiplied by contact time [T]) of 0.02 mg-Cl2·min/L. The infectivity reduction ratios of HuSaV were comparable with those of MNV. For AstV, 1.3-1.7-log10 and >3.4-log10 inactivation, as evaluated by ICC-PCR, was observed at CT values of 0.02 and 0.09 mg-Cl2·min/L, respectively. These results indicate that HuSaV and AstV are both highly sensitive to chlorine treatment and more sensitive than a chlorine-resistant virus, coxsackievirus B5 (1.3-log10 inactivation at a CT value of 0.4 mg-Cl2·min/L, as evaluated by the ICC-PCR assay).

Evaluation of reduction efficiencies of pepper mild mottle virus and human enteric viruses in full-scale drinking water treatment plants employing coagulation-sedimentation-rapid sand filtration or coagulation-microfiltration.

Here, we evaluated the reduction efficiencies of indigenous pepper mild mottle virus (PMMoV, a potential surrogate for human enteric viruses to assess virus removal by coagulation-sedimentation-rapid sand filtration [CS-RSF] and coagulation-microfiltration [C-MF]) and representative human enteric viruses in four full-scale drinking water treatment plants that use CS-RSF (Plants A and B) or C-MF (Plants C and D). First, we developed a virus concentration method by using an electropositive filter and a tangential-flow ultrafiltration membrane to effectively concentrate and recover PMMoV from large volumes of water: the recovery rates of PMMoV were 100% when 100-L samples of PMMoV-spiked dechlorinated tap water were concentrated to 20 mL; even when spiked water volume was 2000 L, recovery rates of >30% were maintained. The concentrations of indigenous PMMoV in raw and treated water samples determined by using this method were always above the quantification limit of the real-time polymerase chain reaction assay. We therefore were able to determine its reduction ratios: 0.9-2.7-log10 in full-scale CS-RSF and 0.7-2.9-log10 in full-scale C-MF. The PMMoV reduction ratios in C-MF at Plant C (1.0 ± 0.3-log10) were lower than those in CS-RSF at Plants A (1.7 ± 0.5-log10) and B (1.4 ± 0.7-log10), despite the higher ability of MF for particle separation in comparison with RSF owing to the small pore size in MF. Lab-scale virus-spiking C-MF experiments that mimicked full-scale C-MF revealed that a low dosage of coagulant (polyaluminum chloride [PACl]) applied in C-MF, which is determined mainly from the viewpoint of preventing membrane fouling, probably led to the low reduction ratios of PMMoV in C-MF. This implies that high virus reduction ratios (>4-log10) achieved in previous lab-scale virus-spiking C-MF studies are not necessarily achieved in full-scale C-MF. The PMMoV reduction ratios in C-MF at Plant D (2.2 ± 0.6-log10) were higher than those at Plant C, despite similar coagulant dosages. In lab-scale C-MF, the PMMoV reduction ratios increased from 1-log10 (with PACl [basicity 1.5], as at Plant C) to 2-4-log10 (with high-basicity PACl [basicity 2.1], as at Plant D), suggesting that the use of high-basicity PACl probably resulted in higher reduction ratios of PMMoV at Plant D than at Plant C. Finally, we compared the reduction ratios of indigenous PMMoV and representative human enteric viruses in full-scale CS-RSF and C-MF. At Plant D, the concentrations of human norovirus genogroup II (HuNoV GII) in raw water were sometimes above the quantification limit; however, whether its reduction ratios in C-MF were higher than those of PMMoV could not be judged since reduction ratios were >1.4-log10 for HuNoV GII and 2.3-2.9-log10 for PMMoV. At Plant B, the concentrations of enteroviruses (EVs) and HuNoV GII in raw water were above the quantification limit on one occasion, and the reduction ratios of EVs (>1.2-log10) and HuNoV GII (>1.5-log10) in CS-RSF were higher than that of PMMoV (0.9-log10). This finding supports the usefulness of PMMoV as a potential surrogate for human enteric viruses to assess virus removal by CS-RSF.

Suitability of pepper mild mottle virus as a human enteric virus surrogate for assessing the efficacy of thermal or free-chlorine disinfection processes by using infectivity assays and enhanced viability PCR.

Evaluating the efficacy of disinfection processes to inactivate human enteric viruses is important for the prevention and control of waterborne diseases caused by exposure to those viruses via drinking water. Here, we evaluated the inactivation of two representative human enteric viruses (adenovirus type 40 [AdV] and coxsackievirus B5 [CV]) by thermal or free-chlorine disinfection. In addition, we compared the infectivity reduction ratio of a plant virus (pepper mild mottle virus [PMMoV], a recently proposed novel surrogate for human enteric viruses for the assessment of virus removal by coagulation‒rapid sand filtration and membrane filtration) with that of the two human enteric viruses to assess the suitability of PMMoV as a human enteric virus surrogate for use in thermal and free-chlorine disinfection processes. Finally, we examined whether conventional or enhanced viability polymerase chain reaction (PCR) analysis using propidium monoazide (PMA) or improved PMA (PMAxx) with or without an enhancer could be used as alternatives to infectivity assays (i.e., plaque-forming unit method for AdV and CV; local lesion count assay for PMMoV) for evaluating virus inactivation by disinfection processes. We found that PMMoV was more resistant to heat treatment than AdV and CV, suggesting that PMMoV is a potential surrogate for these two enteric viruses with regard to thermal disinfection processes. However, PMMoV was much more resistant to chlorine treatment compared with AdV and CV (which is chlorine-resistant) (CT value for 4-log10 inactivation: PMMoV, 84.5 mg-Cl2·min/L; CV, 1.15-1.19 mg-Cl2·min/L), suggesting that PMMoV is not useful as a surrogate for these enteric viruses with regard to free-chlorine disinfection processes. For thermal disinfection, the magnitude of the signal reduction observed with PMAxx-Enhancer-PCR was comparable with the magnitude of reduction in infectivity, indicating that PMAxx-Enhancer-PCR is a potential alternative to infectivity assay. However, for free-chlorine disinfection, the magnitude of the signal reduction observed with PMAxx-Enhancer-PCR was smaller than the magnitude of the reduction in infectivity, indicating that PMAxx-Enhancer-PCR underestimated the efficacy of virus inactivation (i.e., overestimated the infectious virus concentration) by chlorine treatment. Nevertheless, among the PCR approaches examined in the present study (PCR alone, PMA-PCR or PMAxx-PCR either with or without enhancer), PMAxx-Enhancer-PCR provided the most accurate assessment of the efficacy of virus inactivation by thermal or free chlorine disinfection processes.

Evaluation of the suitability of a plant virus, pepper mild mottle virus, as a surrogate of human enteric viruses for assessment of the efficacy of coagulation-rapid sand filtration to remove those viruses.

Here, we evaluated the removal of three representative human enteric viruses - adenovirus (AdV) type 40, coxsackievirus (CV) B5, and hepatitis A virus (HAV) IB - and one surrogate of human caliciviruses - murine norovirus (MNV) type 1 - by coagulation-rapid sand filtration, using water samples from eight water sources for drinking water treatment plants in Japan. The removal ratios of a plant virus (pepper mild mottle virus; PMMoV) and two bacteriophages (MS2 and φX174) were compared with the removal ratios of human enteric viruses to assess the suitability of PMMoV, MS2, and φX174 as surrogates for human enteric viruses. The removal ratios of AdV, CV, HAV, and MNV, evaluated via the real-time polymerase chain reaction (PCR) method, were 0.8-2.5-log10 when commercially available polyaluminum chloride (PACl, basicity 1.5) and virgin silica sand were used as the coagulant and filter medium, respectively. The type of coagulant affected the virus removal efficiency, but the age of silica sand used in the rapid sand filtration did not. Coagulation-rapid sand filtration with non-sulfated, high-basicity PACls (basicity 2.1 or 2.5) removed viruses more efficiently than the other aluminum-based coagulants. The removal ratios of MS2 were sometimes higher than those of the three human enteric viruses and MNV, whereas the removal ratios of φX174 tended to be smaller than those of the three human enteric viruses and MNV. In contrast, the removal ratios of PMMoV were similar to and strongly correlated with those of the three human enteric viruses and MNV. Thus, PMMoV appears to be a suitable surrogate for human enteric viruses for the assessment of the efficacy of coagulation-rapid sand filtration to remove viruses.

Assessment of the efficacy of membrane filtration processes to remove human enteric viruses and the suitability of bacteriophages and a plant virus as surrogates for those viruses.

Here, we evaluated the efficacy of direct microfiltration (MF) and ultrafiltration (UF) to remove three representative human enteric viruses (i.e., adenovirus [AdV] type 40, coxsackievirus [CV] B5, and hepatitis A virus [HAV] IB), and one surrogate of human caliciviruses (i.e., murine norovirus [MNV] type 1). Eight different MF membranes and three different UF membranes were used. We also examined the ability of coagulation pretreatment with high-basicity polyaluminum chloride (PACl) to enhance virus removal by MF. The removal ratios of two bacteriophages (MS2 and φX174) and a plant virus (pepper mild mottle virus; PMMoV) were compared with the removal ratios of the human enteric viruses to assess the suitability of these viruses to be used as surrogates for human enteric viruses. The virus removal ratios obtained with direct MF with membranes with nominal pore sizes of 0.1-0.22 µm differed, depending on the membrane used; adsorptive interactions, particularly hydrophobic interactions between virus particles and the membrane surface, were dominant factors for virus removal. In contrast, direct UF with membranes with nominal molecular weight cutoffs of 1-100 kDa effectively removed viruses through size exclusion, and >4-log10 removal was achieved when a membrane with a nominal molecular weight cutoff of 1 kDa was used. At pH 7 and 8, in-line coagulation-MF with nonsulfated high-basicity PACls containing Al30 species had generally a better virus removal (i.e., >4-log10 virus removal) than the other aluminum-based coagulants, except for φX174. For all of the filtration processes, the removal ratios of AdV, CV, HAV, and MNV were comparable and strongly correlated with each other. The removal ratios of MS2 and PMMoV were comparable or smaller than those of the three human enteric viruses and MNV, and were strongly correlated with those of the three human enteric viruses and MNV. The removal ratios obtained with coagulation-MF for φX174 were markedly smaller than those obtained for the three human enteric viruses and MNV. However, because MS2 was inactivated after contact with PACl during coagulation pretreatment, unlike AdV, CV, MNV, and PMMoV, the removal ratios of infectious MS2 were probably an overestimation of the ability of coagulation-MF to remove infectious AdV, CV, and caliciviruses. Thus, PMMoV appears to be a suitable surrogate for human enteric viruses, whereas MS2 and φX174 do not, for the assessment of the efficacy of membrane filtration processes to remove viruses.

Elimination of representative contaminant candidate list viruses, coxsackievirus, echovirus, hepatitis A virus, and norovirus, from water by coagulation processes.

We examined the removal of representative contaminant candidate list (CCL) viruses (coxsackievirus [CV] B5, echovirus type [EV] 11, and hepatitis A virus [HAV] IB), recombinant norovirus virus-like particles (rNV-VLPs), and murine norovirus (MNV) type 1 by coagulation. Water samples were subjected to coagulation with polyaluminum chloride (PACl, basicity 1.5) followed by either settling or settling and filtration. Together with our previously published results, the removal ratio order, as evaluated by a plaque-forming-unit method or an enzyme-linked immunosorbent assay after settling, was HAV>EV=rNV-VLPs≥CV=poliovirus type 1=MNV>adenovirus type 40 (range, 0.1-2.7-log10). Infectious HAV was likely inactivated by the PACl and therefore was removed to a greater extent than the other viruses. A nonsulfated high-basicity PACl (basicity 2.1), removed the CCL viruses more efficiently than did two other sulfated PACls (basicity 1.5 or 2.1), alum, or ferric chloride. We also examined the removal ratio of two bacteriophages. The removal ratios for MS2 tended to be larger than those of the CCL viruses, whereas those for φX174 were comparable with or smaller than those of the CCL viruses. Therefore, φX174 may be a useful conservative surrogate for CCL viruses during coagulation.

Investigation of enteric adenovirus and poliovirus removal by coagulation processes and suitability of bacteriophages MS2 and φX174 as surrogates for those viruses.

We evaluated the removal of enteric adenovirus (AdV) type 40 and poliovirus (PV) type 1 by coagulation, using water samples from 13 water sources for drinking water treatment plants in Japan. The behaviors of two widely accepted enteric virus surrogates, bacteriophages MS2 and φX174, were compared with the behaviors of AdV and PV. Coagulation with polyaluminum chloride (PACl, basicity 1.5) removed AdV and PV from virus-spiked source waters: the infectious AdV and PV removal ratios evaluated by means of a plaque-forming-unit method were 0.1-1.4-log10 and 0.5-2.4-log10, respectively. A nonsulfated high-basicity PACl (basicity 2.1) removed infectious AdV and PV more efficiently than did other commercially available PACls (basicity 1.5-2.1), alum, and ferric chloride. The MS2 removal ratios tended to be larger than those of AdV and PV, partly because of differences in the hydrophobicities of the virus particles and the sensitivity of the virus to the virucidal activity of PACl; the differences in removal ratios were not due to differences in the surface charges of the virus particles. MS2, which was more hydrophobic than the other viruses, was inactivated during coagulation with PACl. Therefore, MS2 does not appear to be an appropriate surrogate for AdV and PV during coagulation. In contrast, because φX174, like AdV and PV, was not inactivated during coagulation, and because the hydrophobicity of φX174 was similar to or somewhat lower than the hydrophobicities of AdV and PV, the φX174 removal ratios tended to be similar to or somewhat smaller than those of the enteric viruses. Therefore, φX174 is a potential conservative surrogate for AdV and PV during coagulation. In summary, the surface hydrophobicity of virus particles and the sensitivity of the virus to the virucidal activity of the coagulant are probably important determinants of the efficiency of virus removal during coagulation.

Improved virus removal by high-basicity polyaluminum coagulants compared to commercially available aluminum-based coagulants.

We investigated the effects of basicity, sulfate content, and aluminum hydrolyte species on the ability of polyaluminum chloride (PACl) coagulants to remove F-specific RNA bacteriophages from river water at a pH range of 6-8. An increase in PACl basicity from 1.5 to 2.1 and the absence of sulfate led to a reduction of the amount of monomeric aluminum species (i.e., an increase of the total amount of polymeric aluminum and colloidal aluminum species) in the PACl, to an increase in the colloid charge density of the PACl, or to both and, as a result, to high virus removal efficiency. The efficiency of virus removal at around pH 8 observed with PACl-2.1c, a nonsulfated high-basicity PACl (basicity 2.1-2.2) with a high colloidal aluminum content, was larger than that observed with PACl-2.1b, a nonsulfated high-basicity PACl (basicity 2.1-2.2) with a high polymeric aluminum content. In contrast, although extremely high basicity PACls (e.g., PACl-2.7ns, basicity 2.7) effectively removed turbidity and UV260-absorbing natural organic matter and resulted in a very low residual aluminum concentration, the virus removal ratio with PACl-2.7ns was smaller than the ratio with PACl-2.1c at around pH 8, possibly as a result of a reduction of the colloid charge density of the PACl as the basicity was increased from 2.1 to 2.7. Liquid (27)Al NMR analysis revealed that PACl-2.1c contained Al30 species, which was not the case for PACl-2.1b or PACl-2.7ns. This result suggests that Al30 species probably played a major role in virus removal during the coagulation process. In summary, PACl-2.1c, which has high colloidal aluminum content, contains Al30 species, and has a high colloid charge density, removed viruses more efficiently (>4 log10 for infectious viruses) than the other aluminum-based coagulants-including commercially available PACls (basicity 1.5-1.8), alum, and PACl-2.7ns-over the entire tested pH (6-8) and coagulant dosage (0.54-5.4 mg-Al/L) ranges.

Evaluation of norovirus removal performance in a coagulation-ceramic microfiltration process by using recombinant norovirus virus-like particles.

Norovirus (NV) is a prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis worldwide. Because of the lack of a cell culture system or an animal model for this virus, studies on drinking water treatment such as separation and disinfection processes are still hampered. In the present study, we investigated NV removal performance as particles during a coagulation-ceramic microfiltration (MF) process by using recombinant NV virus-like particles (rNV-VLPs), which are morphologically and antigenically similar to native NV. We also experimentally investigated the behaviors of two widely accepted surrogates for pathogenic waterborne viruses, bacteriophages Qbeta and MS2, for comparison with the behavior of rNV-VLPs. More than 4-log removal was observed for rNV-VLPs with a 1.08 mg-Al/L dose of polyaluminium chloride in the coagulation-ceramic MF process. This high removal ratio of rNV-VLPs satisfies the U.S. Environmental Protection Agency requirement of 4-log removal or inactivation. In addition, the removal ratios of Qbeta and MS2 were approximately 2-log and 1-log, smaller than the ratio of rNV-VLPs. Accordingly, both bacteriophages have the potential to become appropriate surrogates for native NV in the coagulation-ceramic MF process, and, of the two, Qbeta is the more conservative surrogate.

Estimation of norovirus removal performance in a coagulation-rapid sand filtration process by using recombinant norovirus VLPs.

Norovirus (NV) is an important human pathogen that causes epidemic acute nonbacterial gastroenteritis worldwide. Because of the lack of a cell culture system or an animal model for this virus, studies of drinking water treatment such as separation and disinfection processes are still hampered. We successfully estimated NV removal performance during a coagulation-rapid sand filtration process by using recombinant NV virus-like particles (rNV-VLPs) morphologically and antigenically similar to native NV. The behaviors of two widely accepted surrogates for pathogenic waterborne viruses, bacteriophages Qbeta and MS2, were also investigated for comparison with that of rNV-VLPs. Approximately 3-log(10) removals were observed for rNV-VLPs with a dose of 40 muM-Al or -Fe, as polyaluminum chloride at pH 6.8 or ferric chloride at pH 5.8, respectively. Smaller removal ratios were obtained with alum and ferric chloride at pH 6.8. The removal performance for MS2 was somewhat larger than that for rNV-VLPs, meaning that MS2 is not recommended as an appropriate surrogate for native NV. By comparison, the removal performance for Qbeta was similar to, or smaller than, that for rNV-VLPs. However, the removal performances for rNV-VLPs and Qbeta differed between the coagulation process and the following rapid sand filtration process. Therefore, Qbeta also is not recommended as an appropriate surrogate for native NV.

Comparison of behaviors of two surrogates for pathogenic waterborne viruses, bacteriophages Qbeta and MS2, during the aluminum coagulation process.

Differences in the behaviors of two surrogates for pathogenic waterborne viruses, F-specific RNA bacteriophages Qbeta and MS2, were investigated during the coagulation process by using river water spiked with these bacteriophages. The particle size and electrophoretic mobility of Qbeta and MS2 were similar, but the removal performances of infectious Qbeta and MS2, as measured by a plaque forming unit (PFU) method, differed markedly during the coagulation process. The removal ratio of the infectious Qbeta concentration was approximately 2log higher than that of the infectious MS2 concentration at all coagulant doses tested. The total Qbeta and MS2 bacteriophage concentrations, which were measured by a real-time reverse transcription-polymerase chain reaction (RT-PCR) method and represented the total number of bacteriophages regardless of their infectivity, were similar after the coagulation process, suggesting that the behaviors of Qbeta and MS2 as particles were similar during the coagulation process. The difference between total concentration and infectious concentration indicated that some of the bacteriophages were probably inactivated during the coagulation process. This difference was larger for Qbeta than MS2, meaning that Qbeta was more sensitive to the virucidal activity of the aluminum coagulant. Analysis of the PFU and real-time RT-PCR findings together suggested that the difference in removal performances of Qbeta and MS2 during the coagulation process was probably caused by differences not in the extent of bacteriophage entrapment in the aluminum floc particles but in the sensitivity to virucidal activity of the aluminum coagulant.

Analysing mass balance of viruses in a coagulation-ceramic microfiltration hybrid system by a combination of the polymerase chain reaction (PCR) method and the plaque forming units (PFU) method.

Virus removal experiments using river water spiked with bacteriophages were conducted by an in-line coagulation-ceramic microfiltration hybrid system to investigate the effects of filtration flux (62.5 and 125 L/(m2 x h)) and type of virus (Qbeta and MS2) on virus removal. In addition, the mass balance of viruses through the hybrid system was analysed by quantifying the infectious and inactive viruses by a combination of the polymerase chain reaction (PCR) method and the plaque forming units (PFU) method. Even when the system was operated at high filtration flux (125 L/(m2 x h)), high virus removal (> 6 log) with short coagulation time (2.4 s) was successfully achieved by dosing polyaluminium chloride (PACI) at more than 1.08 mg-Al/L. Removal performances were different between Qbeta and MS2, although their diameters are almost the same: greater virus removal was achieved for MS2 at PACI dosing of 0.54 mg-Al/L, and for Qbeta at PACI dosing of more than 1.08 mg-Al/L. The combination of the PCR and PFU methods revealed that two phenomena, adsorption to/entrapment in aluminium floc and virucidal activity of PACI, partially account for the high virus removal in the coagulation-MF hybrid system.

Dural Arteriovenous Fistula within the Hypoglossal Canal Successfully Treated by Transvenous Embolization. Case Report.

SUMMARY: We report a case of dural arteriovenous fistula (DAVF) within the left hypoglossal canal in a 64-year-old man who presented with tinnitus and ocular symptoms. Angiography revealed DAVF with the fistulous pouch medial to the left jugular bulb. The fistula was feeded by meningeal branches of the bilateral ascending pharyngeal arteries and the branches from the left vertebral artery. The fistula shunted into the left jugular bulb, with reflux into the left inferior petrosal (IPS) and cavernous sinuses (Cses), left superior ophthalmic vein (SOV) and cortical veins over the cerebral convexity. We performed transvenous coil embolization to occlude the fistula resulting in complete resolution of symptoms and signs.

Binding characteristics of bovine lactoferrin to the cell surface of Clostridium species and identification of the lactoferrin-binding protein.

The binding characteristics of bovine lactoferrin (bLf) to cells of the Clostridium species were observed by using a horseradish peroxidase-bLf conjugate. A bLf-binding protein (BP) having a relative molecular mass of about 33 kDa was confirmed in the surface layer components from 7 strains of the Clostridium species. The binding of the conjugate to bLf-BP or C. perfringens was strongly blocked by intact Lfs, lysine or arginine residues modified bLf, and deglycosylated bLf, but was not by other milk proteins or by the constituent sugars of glycan. Bacterial growth was inhibited by bLf, but was slightly inhibited by lysine residues modified bLf or deglycosylated bLf. Lactoferricin B did not block the binding of the conjugate, but strongly inhibited the bacterial growth. This suggests that the lysine or arginine residues and glycan of bLf hardly participated in binding bLf to the bacterial cells, but that the amino acid residues and glycan played an important role in inhibiting the growth of bacteria.

Cervical spondylotic radiculopathy involving two adjacent nerve roots. Anterior decompression through a single level intervertebral approach.

We describe a single level intervertebral approach to decompress two adjacent involved nerve roots in cases of cervical spondylosis. The operation was undertaken in 4 patients. We carried out discectomy, partial excision of the vertebral body with removal of the anteromedial part of the pedicles, removal of osteophytes and excision of the posterior longitudinal ligament, followed by an anterior interbody fusion. Fusion was achieved with the spine in normal lordosis and without complications. Pain and motor weakness was relieved in every case. This procedure can maintain movement at one additional disc level and has a better fusion rate than multilevel inter-body fusion.

Cervical myelopathy in patients with congenital cervical block vertebrae.

Thirty-two patients with congenital cervical block vertebrae are reviewed. Twenty-nine patients had single level fusion, one had two-level fusion, and the remaining two had multilevel fusion. Eighteen patients had cervical myelopathy; five of these had related trauma and 13 had no history of trauma. The five patients who had cervical myelopathy following trauma underwent magnetic resonance imaging (MRI); three of them had abnormalities in the spinal cord at the segment adjacent to fusion. In all five patients the symptoms and signs were attributed to the segment adjacent to fusion. Myelography, computed tomographic myelography and MRI were performed in 11 of the 13 patients with cervical myelopathy without trauma. In 9 of them maximum compression of the spinal cord was not seen at the segment adjacent to fusion. The major factor contributing to cervical myelopathy was associated spinal canal stenosis. Seven patients with cervical myelopathy without history of trauma were treated surgically, six of whom had spinal canal stenosis treated by enlargement of the spinal canal: subtotal corpectomy and arthrodesis was performed in three, and open-door expansive laminoplasty in three. Anterior interbody arthrodesis was performed in one patient without spinal canal stenosis. All recovered from the myelopathy postopera-tively. When a trauma occurs, it concentrates stress at the segment adjacent to fusion, resulting in possible spinal cord injury. On the other hand, when there is no trauma, spinal canal stenosis is the principal factor contributing to cervical myelopathy.

Corticotroph cell hyperplasia in a patient with Addison disease: case report.

A woman with Addison disease developed hyperpigmentation, headache, and nausea despite conventional replacement therapy with cortisone. Excessively elevated plasma adrenocorticotropic hormone (ACTH) with absence of response to administration of corticotropin-releasing factor (CRF), and roentgenological evidence of enlargement of the sella turcica, as well as detection of enlarged pituitary gland on magnetic resonance images, led to a diagnosis of ACTH-producing microadenoma, which was removed by transsphenoidal microsurgery. The specimen obtained at surgery evidenced corticotroph hyperplasia, as demonstrated by immunohistochemical staining for ACTH. Fine structure exhibited densely granulated cells with a few bundles of microfilaments and an abundance of large lysosomal bodies. Surgical removal of the hyperplasia alleviated the patient's symptoms, and hyperpigmentation faded remarkably. Her plasma ACTH level returned to normal, has remained normal for more than 3 years, and responds adequately to CRF administration.

Os odontoideum with posterior atlantoaxial instability.

Nine patients who had os odontoideum with posterior atlantoaxial instability are reviewed. Three parameters were measured on the lateral radiographs: the distance from the os odontoideum to the spinous process of the axis in extension (Dext), the distance from the os odontoideum to the posterior arch of the atlas (Datl), and the degree of instability (Inst). Patients were classified into four groups: Group I, local symptoms (N = 3); Group II, transient myelopathy (N = 0); Group III, progressive myelopathy (N = 6); and Group IV, cerebral symptoms (N = 0). The development of cervical myelopathy was not related to degree of instability but to distance from the os to the spinous process of the axis (Dext). Dext was more than 16 mm in Group I and less than or equal to 16 mm in Group III. Five of six patients in Group III underwent myelography. Based on myelographic findings, Group III was further subdivided into two groups, Group IIIA (N = 2) and Group IIIB (N = 3), according to the following characteristics: In Group IIIA, the distance from the os to the posterior arch of the atlas was more than 13 mm, and the spinal cord was impinged between the os odontoideum and the lamina of the axis in extension and reduced in flexion. In Group IIIB, Datl was less than or equal to 13 mm, and the spinal cord was compressed at the level of the atlas during flexion and extension. Stenotic Datl of 13 mm or less specifically defined severe cervical myelopathy. Surgical treatment for cervical myelopathy in os odontoideum with posterior instability is suggested as follows: in the absence of canal stenosis of the atlas (Group IIIA), atlantoaxial fusion in a reduced position is indicated; when associated with canal stenosis of the atlas (Group IIIB), laminectomy of the atlas followed by occiput-to-C2 arthrodesis is indispensable.

Structural abnormalities in freeze-fractured sciatic nerve fibres of diabetic mice.

Nodal and paranodal regions of myelinated sciatic nerve fibres from diabetic (db/db) mice were examined in freeze fracture replicas. In some fibres, the axolemma was found to display abnormalities in the paranodal region. These include shallow, undifferentiated junctional indentations, thinning of the indentations with widening of the non-junctional grooves between them, particle clusters within the non-junctional grooves, and patches in which axolemmal E-face particles are distributed randomly rather than in the form of linear strings within grooves. Nodal structure, in contrast, is hardly affected. Nodal E-face and P-face particle densities in db/db axons are not significantly different from those in age-matched controls, although we found a few examples in which the E-face density fell slightly below the normal range. Occasional fibres showing evidence of paranodal or segmental demyelination were also seen. The results support paranodal pathology as a potential basis for reduced nerve conduction velocity in diabetic nerves but provide no evidence for significant changes in nodal structure or in nodal Na channel density in sciatic nerve fibres of the db/db mouse.