Can fifth nerve mapping guide rhizotomy for recurrent trigeminal neuralgia? Case report.

INTRODUCTION: Microvascular decompression (MVD) is usually considered the first-line treatment for trigeminal neuralgia (TN) when medical treatments fail. Recurrence is rare and best treatment option is controversial. MVD was proposed as a feasible and effective technique for recurrent TN by many authors. Nevertheless, in a substantial number of cases, not any impingement or deterioration are found intraoperatively and partial selective rhizotomy is then advised. The rhizotomy site is mostly guided by anatomical landmarks, but variations due to scarring and adhesions are common pitfalls in these second surgeries. Intraoperative monitoring is infrequently used during MVD for trigeminal neuralgia. We describe the use of nerve mapping in a case of recurrence, revealing an unexpected rootlet distribution and thus safely guiding partial rhizotomy. CLINICAL PRESENTATION: A 53-year-old woman had suffered from bilateral trigeminal neuralgia for 10 years. Symptoms began on the right side. MVD resolved her symptoms but, after a few months, she developed left TN which persisted after left MVD, radiofrequency and radiosurgery. She was referred to our center for a second MVD on the left side. Intraoperative inspection detected no relevant findings, and nerve mapping followed by partial selective rhizotomy was performed. Complete pain relief was achieved. There were no complications. CONCLUSION: Rhizotomy is seldom employed for refractory trigeminal neuralgia. The effects of previous treatments can jeopardize anatomical landmarks. Nerve mapping seems a promising tool to improve results.

Comparative characterisation of human and ovine non-aureus staphylococci isolated in Sardinia (Italy) for antimicrobial susceptibility profiles and resistance genes.

We present the comparative characterisation of 195 non-aureus staphylococci (NAS) isolates obtained from sheep (n = 125) and humans (n = 70) in Sardinia, Italy, identified at the species level by gap gene polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis with AluI. Isolates were tested phenotypically with a disc diffusion method and genotypically by PCR, for resistance to 11 antimicrobial agents including cationic antiseptic agents. Among the ovine isolates, Staphylococcus epidermidis (n = 57), S. chromogenes (n = 29), S. haemolyticus (n = 17), S. simulans (n = 8) and S. caprae (n = 6) were the most prevalent species, while among human isolates, S. haemolyticus (n = 28) and S. epidermidis (n = 26) were predominant, followed by S. lugdunensis and S. hominis (n = 4). Of the 125 ovine isolates, 79 (63.2%) did not carry any of the resistance genes tested, while the remainder carried resistance genes for at least one antibiotic. The highest resistance rates among ovine isolates were recorded against tetracycline (20.8%), and penicillin (15.2%); none was resistant to methicillin and two exhibited multidrug resistance (MDR); one of which was positive for the antiseptic resistance smr gene. By contrast, most human isolates (59/70, 84.3%) were resistant to ⩾1 antimicrobials, and 41 (58.6%) were MDR. All 52 (74.3%) penicillin-resistant isolates possessed the blaZ gene, and 33 of 70 (47.1%) harboured the mec gene; of these, seven were characterised by the Staphylococcal Chromosomal Cassette (SCCmec) type IV, 6 the type V, 5 of type III and one representative each of type I and type II. The majority (57.1%) was erythromycin-resistant and 17 isolates carried only the efflux msrA gene, 11 the methylase ermC gene and an equal number harboured both of the latter genes. Moreover, 23 (32.8%) were tetracycline-resistant and all but one possessed only the efflux tetK gene. qacA/B and smr genes were detected in 27 (38.6%) and 18 (25.7%) human NAS, respectively. These results underline a marked difference in species distribution and antimicrobial resistance between ovine and human-derived NAS.

Biofilm formation and virulence factor analysis of Staphylococcus aureus isolates collected from ovine mastitis.

AIMS: To perform a phenotypic and genotypic characterization of 258 Staphylococcus aureus isolates from clinical ovine mastitis and used for the preparation of inactivated autogenous vaccines. METHODS AND RESULTS: The potential for biofilm production was determined by phenotypic test of Congo Red Agar (CRA) and by PCR for the detection of icaA/D genes. Isolates were also screened by PCR for the presence of enterotoxins (sea, seb, sec, sed and see), toxic shock syndrome toxin (tsst), leukotoxins (lukD-E, lukM and lukPV83), haemolysins (hly-ß and hly-γ), autolysin (atlA) genes and encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs: clfA, clfB, fnbA, fnbB, bbp, cna, eno, fib, epbs, sdrC, sdrD and SdrE). None of the 258 isolates showed biofilm-forming ability on CRA and harboured icaA/D genes. The most frequent pyrogenic toxin superantigen genes amplified were sec plus tsst-1, which were found strictly in combination with 71·3% of the Staph. aureus isolates tested. None of the isolates harboured the genes encoding sea and see. Of the 258 isolates tested, 159 (61·6%) possessed all lukD-E/lukM/lukPV83 genes, 123 (47·7%) harboured both hly-ß/hly-γ genes, whereas almost all (97·3%) were PCR positive for atlA gene. With respect to adhesion determinants, 179 (69·4%) isolates presented simultaneously four genes (fnbA, fib, clfA and clfB) for fibronectin- and fibrinogen-binding proteins. CONCLUSIONS: In this search, several putative virulence determinants have been identified in ovine Staph. aureus isolates collected in Sardinia. SIGNIFICANCE AND IMPACT OF THE STUDY: Some of the putative virulence determinants could be considered as components of a vaccine because of their role in ovine mastitis pathogenesis.

Hemilaminectomy for spinal meningioma: A case series of 20 patients with a focus on ventral- and ventrolateral lesions.

OBJECTIVE: To demonstrate the effectiveness and safety of hemilaminectomy in spinal-meningioma surgery, with special attention to ventral lesions. We also describe technical tips to enhance surgical-corridor width. PATIENTS AND METHODS: A total of 20 patients (14 female and 6 male) underwent hemilaminectomy for resection of a spinal meningioma between January 2005 and December 2015. Preoperative magnetic resonance imaging defined the tumor location (16 thoracic, 3 cervical, 1 lumbar) and the dural-attachment site (4 ventral, 11 ventrolateral, 3 lateral, 2 posterior). Pre- and postprocedural functional status was evaluated according to McCormick's classification. Intraoperative neurophysiological monitoring was employed in all patients. RESULTS: The unilateral approach allowed for complete resection (Simpson grade I-II resection) in 18 patients (90%), including tumors with a ventral dural attachment. In most patients (n=13), monosegmental hemilaminectomy was performed, a single patient required hemilaminectomy of 3 levels, while the remaining 6 patients underwent hemilaminectomy of 2 levels. No patients experienced either worsening of neurological status or procedure-related complications. All patients who had preoperative pain reported postoperative improvement. CONCLUSIONS: The goal of surgery for spinal tumors is to achieve gross tumor removal while minimizing morbidity. In our experience, hemilaminectomy is an effective surgical approach, even in patients with ventral- and ventrolateral spinal meningiomas. The procedure offers several advantages in terms of early patient mobilization and rehabilitation, management of postoperative pain, and preservation of spinal stability while achieving positive functional outcomes.

Development of a specific immunomagnetic capture-PCR for rapid detection of viable Mycoplasma agalactiae in sheep milk samples.

AIMS: To develop an immunomagnetic capture (IMC) to detect viable Mycoplasma agalactiae in routine ovine milk samples. METHODS AND RESULTS: Polyclonal antibodies against two M. agalactiae membrane surface proteins (P80 and P55) were covalently conjugated to magnetic beads (MBs) to form MB-Ab80 and MB-Ab55. Mycoplasma agalactiae cells were captured by a specific antigen-antibody reaction and magnetic separation. Immunomagnetic capture (IMC) was used to isolate and concentrate M. agalactiae in serial decimal dilutions and in artificially contaminated milk to facilitate subsequent detection by PCR. A 375-bp fragment of M. agalactiae was amplified using a pair of M. agalactiae-specific primers in PCR. The limit of detection of IMC-PCR method ranged from 10 to 10(2)  CCU ml(-1) when mycoplasmas were resuspended in PBS and from 10(2) to 10(3)  CCU ml(-1) when mycoplasmas were resuspended in uncontaminated ovine milk. This study also describes the application of IMC-PCR method to test for M. agalactiae in 516 milk samples collected from sheep with suspected contagious agalactia. Its performance was evaluated relative to culture. CONCLUSIONS: This report has demonstrated for the first time, the effective use of rapid and reliable IMC combined with PCR assay for the detection of viable M. agalactiae. SIGNIFICANCE AND IMPACT OF THE STUDY: The method IMC-PCR provides an alternative to conventional microbiological detection, method and it could be applied to quick detection of M. agalactiae in routine sheep milk samples.

Proteomic approach for identification of immunogenic proteins of Mycoplasma mycoides subsp. capri.

In this study, an immunoproteomic approach was used to identify immunodominant proteins from Mycoplasma mycoides subsp. capri isolates. Membrane proteins, extracted through TX-114 phase partitioning, were separated using mono- and two-dimensional electrophoresis and detected by Western blotting with pooled sera from naturally infected goats. A total of 27 immunoreactive spots, corresponding to 13 different proteins, were identified using nanoLC-ESI-MSMS. Function annotation revealed that most of these proteins were metabolic enzymes involved in carbohydrate and energy metabolism. The immunogenic proteins identified in this study: pyruvate dehydrogenase, dihydrolipoamide acetyltransferase, dihydrolipoyl dehydrogenase, phosphate acetyltransferase, phosphopyruvate hydratase, adenine phopshoribosyltransferase, transketolase, translation elongation factor G, translation elongation factor Ts, FMN-dependent NADH-azoreductase, peptide methionine sulfoxide reductase, inorganic diphosphatase and trigger factor may be used as biomarkers for the serological diagnosis of contagious agalactia caused by M. mycoides subsp. capri.

Isolation and characterization of Bartonella strains in cats in Italy.

Bartonella henselae and Bartonella clarridgeiae were isolated in two cats in Sardinia, Italy. Infection by B. clarridgeiae was characterized by fever and submandibular lymph nodes enlargement while B. henselae infection was asymptomatic. This is the first report of B. clarridgeiae in a cat in Italy and the first isolation of B. henselae and B. clarridgeiae in Sardinia.

Identification of coagulase-negative staphylococci isolated from ovine milk samples by PCR-RFLP of 16S rRNA and gap genes.

The identification of coagulase-negative staphylococci (CNS) causing ovine infections remains problematic, although these bacteria are considered the main etiologic agents of subclinical mastitis in sheep and goats. In this study, 226 CNS isolates were collected from 2201 milking sarda sheep belonging to 15 flocks with high somatic cell count scores. All isolates were subjected to identification with the API Staph ID test, and then to the amplification of staphylococcal 16S rRNA and gap genes by PCR assays. The gap gene was subjected to restriction fragment length polymorphism analysis with the restriction endonuclease AluI, whereas the 16S rRNA gene was subjected to ribosomal fingerprinting with the restriction endonucleases RsaI, PstI and AluI. When PCR-RFLP patterns of CNS isolates were different from those of their reference strains, gap gene amplicons were sequenced for definitive identification. The API Staph ID test, in alternative to the genotypic identification method, produced considerably different results in terms of species identified within each group. Using the PCR-RFLP assay, most of the isolates clustered together with the Staphylococcus epidermidis type strain (131, corresponding to 57.9%), followed by S. caprae (34, corresponding to 15%) and S. chromogenes (30, corresponding to 13.2%). In conclusion, the PCR-RFLP assay of 16S rRNA and gap genes is a more reliable and reproducible method than the API Staph ID test for the identification of CNS causing sheep mastitis.

First direct detection of rickettsial pathogens and a new rickettsia, 'Candidatus Rickettsia barbariae', in ticks from Sardinia, Italy.

The present study evaluated the molecular detection and identification of Rickettsia species in 83 ticks collected in Sardinia, Italy. Fifteen ticks were PCR-positive using gltA-specific and ompA-specific primers, leading to the identification of Rickettsia aeschlimannii in Hyalomma marginatum marginatum, R. massiliae in Rhipicephalus turanicus and in Rhipicephalus sanguineus, and a new rickettsia, previously referred to as PoTiRb169 in Portugal, in four Rhipicephalus turanicus. This new species was further characterized by amplification and sequencing of three additional genes (ompB, sca4 and rrs). Using the current criteria to name a rickettsia, this uncultivated rickettsia can be given a Candidatus status, and we propose to call it 'Candidatus Rickettsia barbariae'. The detection of three tick-borne rickettsiae in Sardinia raises the possibility that many cases of spotted fever considered by clinicians and health authorities as Mediterranean spotted fever due to R. conorii could, in fact, be due to other rickettsiae, including those found in this study. Analysing skin biopsies of inoculation eschars in patients with spotted fever would be, together with continuing entomological surveys, the best way to increase our knowledge of tick-borne rickettsioses in Sardinia and more generally in the Mediterranean basin.

Two cases of human granulocytic ehrlichiosis in Sardinia, Italy confirmed by PCR.

In this work we report the first two cases of human granulocytic ehrlichiosis (HGE) in Sardinia. In early September 2004, a 69-year-old woman (patient 1) was admitted to the Infectious Diseases Institute of Sassari for rickettsiosis like-syndrome: high fever (39.5-40 degrees C), dyspnea, reduced consciousness, vomiting, and cutaneous rash. In late September 2004, a 30-year-old man (patient 2) with high fever was admitted for an evident palmar and oral erythema, edema of the labium, very intense arthralgia, myalgia, and dyspnea. In these two hospitalized patients, the diagnosis was made through indirect IgM and IgG immunofluorescent technique and confirmed by the presence of the specific DNA in the leukocytes. The two patients were A. phagocytophilum-PCR positive.

Role of Chlamydophila abortus in ovine and caprine abortion in Sardinia, Italy.

Between 1999-2003, 14321 sera and 646 abortion samples (498 foetuses and 148 placentae) were analysed from 807 sheep and goat farms distributed all over the island of Sardinia. After notification of abortion in a flock, sera collected at random from adult animals were examined to detect antibodies specific to Chlamydophila (C.) abortus by ELISA, whereas foetuses and placenta were analysed by PCR assay. Specific IgG antibodies were detected in 611 (4.8%) sheep and 106 (5.8%) goats. From a total of 2050 ovine and 151 caprine fetal samples including muscle, liver, abomasum, spleen, brain and placenta, 29 (1.4%) ovine and 1 (0.6%) caprine samples were C. abortus PCR-positive. Placenta was the tissue with the highest detection rate. These results indicate that the seroprevalence of C. abortus infection in sheep and goats is very low in Sardinia, and PCR results demonstrate that C. abortus has no significant role in abortion, especially in goats.

Rapid differential diagnosis of Mycoplasma agalactiae and Mycoplasma bovis based on a multiplex-PCR and a PCR-RFLP.

The membrane-protein 81 gene (mb-mp81) of Mycoplasma bovis was cloned, sequenced and compared to membrane-protein 81 gene (ma-mp81) of Mycoplasma agalactiae. After alignment of both sequences, specific primers pairs were designed from variable or unchanging nucleotide segments. In this study, we describe the development and optimization of a multiplex-PCR (MPCR) for the rapid detection of M. agalactiae and M. bovis strains. In addition, a simple and rapid PCR-restriction fragment length polymorphism (RFLP) assay, using the restriction enzymes AluI, DraI, RsaI and XbaI, is described to distinguish between both species. The results suggest that MPCR and PCR-RFLP assays could be used as an alternative method in routine diagnosis for rapid and specific simultaneous detection of M. agalactiae and M. bovis.

Survey of ovine and caprine toxoplasmosis by IFAT and PCR assays in Sardinia, Italy.

During the period 1999-2002, we have analyzed 9639 serum samples and 815 aborted samples (670 fetuses and 145 placenta) from 964 ovine and caprine farms distributed over all Sardinia island. After abortion notification, sera collected at random from adult animals were examined to detect simultaneously IgG and IgM antibodies specific to Toxoplasma gondii by indirect immunofluorescence assay, whereas fetuses and placenta were analyzed by a single tube nested PCR assay. Specific IgG antibodies were detected in 2048 (28.4%) sheep and 302 (12.3%) goats, specific IgM antibodies were found in 652 (9%) sheep and 139 (5.6%) goats. From a total of 2471 ovine and 362 caprine fetal samples including muscle, liver, abomasum, spleen, brain and placenta, 271 (11.1%) ovine and 23 (6.4%) caprine samples were T. gondii PCR-positive. Although T. gondii DNA was amplified from different types of tissues, placenta was the tissue with the highest detection rate. On the one hand, these results indicate that the seroprevalence of T. gondii infection in sheep and goats is relatively high, on the other PCR results demonstrate that T. gondii has a significant role in ovine and caprine abortion. Adequate management might be useful and essential to control the toxoplasmosis in the sheep and goats herds of Sardinia.

Ehrlichiosis and rickettsiosis in a canine population of Northern Sardinia.

Ehrlichiosis and rickettsiosis are very common, widespread diseases. These diseases are present in Sardinia year round because its temperate weather permits the survival of many kinds of tick vectors. A thousand dogs were subjected to physical, hematological, biochemical examinations and serological tests. All 1,000 sera were analyzed by indirect immunofluorescence antibody test to detect antibodies against E. canis and R. rickettsii. A high seroprevalence (about 50%) was detected for both etiological agents, without differences in relation to sex, breed, or usage. A high seroprevalence, corresponding to 62.5% for ehrlichiosis and 64% for rickettsiosis, was observed in the age group of 13-60 months. The mortality was greatest in the males in the age group, which manifested the disease in the chronic phase.

Sequence, cloning, expression and characterisation of the 81-kDa surface membrane protein (P80) of Mycoplasma agalactiae.

Mycoplasma agalactiae, the causative agent of contagious agalactia in small ruminants, produces a protein, named P80, that is detectable in all wild-type isolates examined to date and that appears expressed during the early phase of infection. We describe here the identification, cloning and expression of the gene encoding P80 (ma-mp81). The deduced amino acid sequence is consistent with a hydrophobic and basic protein that possesses a lipoprotein signal peptide. Sequence analysis of gene ma-mp81 suggests that P80 is a membrane lipoprotein that shows significant homology with other putative lipoproteins of M. pneumoniae. An internal 1-kb fragment of ma-mp81 was expressed in Escherichia coli as a 6xHis-tagged protein. The purified recombinant protein greatly reacted with polyclonal anti-P80 sera raised in lamb.

A physical map of the Mycoplasma agalactiae strain PG2 genome.

We have constructed a physical map of the Mycoplasma agalactiae strain PG2 chromosome analyzing it by pulsed field gel electrophoresis in a contour-clamped homogeneous electric-field system. We mapped 33 cleavage sites generated with SmaI, XhoI, SalI, EclXI and BsiWI restriction endonucleases using double digestions, one- and two-dimensional pulsed electrophoresis, cross-hybridization and linking clones. We have also mapped the loci of some genes by Southern hybridization.

Experimental vaccination against Mycoplasma agalactiae using different inactivated vaccines.

Five sets of vaccines were prepared using Mycoplasma agalactiae washed cultures inactivated with phenol (1), formalin (2), heat-treatment (3), sodium hypochlorite (4) and saponin (5). All sets, except for saponin-vaccine, were adjuvated with aluminium hydroxide. Five groups of six sarda ewes were inoculated twice before pregnancy, once during pregnancy and challenged during the lactation period. Monthly blood samples were taken from the vaccinated sheep and from 12 controls: sera were assayed by immunoblotting, ELISA and growth inhibition tests. Four control sheep were infected by intracanalicular route with pooled mycoplasmas at concentrations of 10(4), 10(5), 10(6) and 10(7) CCU. The challenge involved using infected milk to contaminate the remaining sheep. All the controls and some ewes from groups 2, 3 and 4 developed contagious agalactia. Ewes vaccinated with phenol- and saponin-inactivated mycoplasmas resisted experimental challenge. These results suggest that these two vaccines are effective and that their use could limit the diffusion of M. agalactiae infection.

Comparison of restriction pattern polymorphism of Mycoplasma agalactiae and Mycoplasma bovis by pulsed field gel electrophoresis.

Mycoplasma agalactiae and Mycoplasma bovis are closely related species in phylogenetic terms. Pulsed Field Gel Electrophoresis (PFGE) of SmaI, EclXI, Bsi WI, MluI, BssHII, SalI, XhoI, NruI and ApaI digested DNAs were used to analyse and to compare restriction fragment length polymorphism between M. agalactiae and M. bovis and to estimate their genome sizes. SmaI, EclXI and Bsi WI enzymes cleaved DNAs of both microrganisms. MluI, BssHII, SalI, XhoI and NruI digested only M. agalactiae DNA whereas ApaI cut only M. bovis DNA. The total DNA length was established to be 945 +/- 8.4 Kb for M. agalactiae and 961 +/- 18.9 Kb for M. bovis.

Characterization of membrane surface proteins of Mycoplasma agalactiae during natural infection.

We have analyzed antigenic variation of seven M. agalactiae wild strains using different sera from naturally infected sheep. Only 30 day sera recognized all surface proteins and inhibited the growth of mycoplasmas. Furthermore, we have observed that two strongly immunogenic proteins: 55 and 35 kDa were digested using 500 micrograms/ml of trypsin. These two bands are immunoprecipitated together with four other proteins but only the 35 kDa protein is recognized by eluted antibodies.