Improvement of diagnostic approach to Lyme neuroborreliosis in children by using recombinant antigens in detection of intrathecally produced IgM/IgG.

BACKGROUND: The purpose of the study was to evaluate new 3rd-generation test kits, EIA Borrelia recombinant IgM and IgG (TestLine, Brno, Czech Republic), in serum and cerebrospinal fluid (CSF) of children with Lyme neuroborreliosis. METHODS: Comparison of three tests was used: the whole-cell EIA from Borrelia garinii (EIA 1) was compared with the EIA using recombinant antigens (EIA 2) and immunoblot. In total, 364 samples of serum and CSF were examined. Eighty-six paired sera and CSF samples were evaluated in the first group of children with Lyme neuroborreliosis. The second group consisted of 30 children with probable Lyme neuroborreliosis. Sixty-six samples from children with neuroinfections other than borrelial etiology were used as controls. RESULTS: In the first group of children with proven LNB, EIA 2 gave significantly more positive results for IgG in serum (P = 0.006; OR = 7.5) as in CSF (P < 0.001; OR = 4.5). There was no statistically significant difference in the IgM positivity of serum (P = 0.54; OR = 0.71). EIA 2 determined significantly (P = 0.001; OR = 0.06) less positive results of IgM in CSF in the LNB patients. IgG antibody index (AI) assessed by both methods revealed similar results (P = 0.646; OR = 1.38). Both methods are comparable, but IgM AI assessed by EIA 2 showed significantly less positive results (P < 0.001; OR = 0.04).The differences in the detection of positive IgM/IgG antibodies in serum and CSF did not reach statistical significance either in the groups of children with excluded LNB or in controls. CONCLUSIONS: EIA 2 showed better results than EIA 1 and western blot for the detection of positive IgG antibodies in serum and CSF. The difference in the calculation of AI IgG by EIA 1 and EIA 2 was not noticeable in the group of LNB patients. Comparing IgG and IgM AIs calculated from both tests, the sensitivity for EIA 2 was 68% for IgG and 26% for IgM. The specificity is 100% for both tests.

Assessment of antibodies against surface and outer membrane proteins of Anaplasma phagocytophilum in Lyme borreliosis and tick-borne encephalitis paediatric patients.

To examine evidence of positive antibodies against immunogenic proteins of Anaplasma phagocytophilum in patients with other tick-borne infections and to diagnose possible co-infections, 412 serum specimens were tested by immunoblotting using three specific Anaplasma antigens: surface proteins p44 and Asp62 and outer membrane protein A (OmpA). In total, 284 serum samples from children with Lyme borreliosis and 12 serum samples from children with tick-borne encephalitis were tested. Sera from patients with viral aseptic meningitis (n = 47) and from blood donors (n = 69) were used as controls. Among all serum specimens from patients with tick-borne infections submitted for this study, six samples (2·0%) showed positive IgM reactions and seven samples (2·4%) were IgG positive for A. phagocytophilum by immunoblot. Borderline reactivity was found in 30 samples (10·14%) for IgM and 36 samples (12·2%) for IgG. The difference between patients and blood donors was statistically significant for IgM (P = 0·006) and for IgG (P = 0·0007) antibodies. A statistically significant result was obtained for IgG (P = 0·02) but not for IgM between patients and children with aseptic meningitis. Immunoblot using three specific antigens provides novel information about the positivity of antibodies to A. phagocytophilum in children with other tick-borne infections. Taking into account clinical and laboratory findings of children despite antibody positivity, no case of human granulocytic anaplasmosis was demonstrated.

[Benign acute childhood myositis as a complication of influenza B and its differential diagnosis]. / Benigní akutní myozitidy u detí jako komplikace chripky B a jejich diferenciální diagnostika.

STUDY AIM: Analysis of the course of benign acute childhood myosistis during the influenza epidemic of 2012/2013, with a focus on the clinical specificities, laboratory findings, diagnostic and therapeutic approaches, and prognosis. METHODS: A retrospective study was conducted of 10 patients with myositis during influenza infection. The haematological and biochemistry parameters were analysed. A direct real time-PCR assay was used to detect the virus from nasopharyngeal swabs. RESULTS: Eight of 10 patients were males of an average age of 7.1 years. Muscle disorders emerged during recovery from influenza. They were characterized by a sudden onset of severe pain in the calf muscles with the consequent inability to walk in nine patients. Toe walking was reported in six children and wide-base gait in one patient. The full blood count showed leukopenia (eight cases) and thrombocytopenia (five cases). Blood biochemistry typically detected elevated muscle enzymes (creatine kinase and aspartate aminotransferase) and elevated myoglobin in all patients. Influenza B was confirmed in all study patients. They all recovered spontaneously within two to four days. CONCLUSIONS: Benign acute childhood myositis presents with the clinical picture of transient muscle disorders resulting in difficulty walking and very rarely can be complicated by rhabdomyolysis and kidney failure. It is most often associated with influenza B and affects school age children, mostly males. Good awareness of the condition facilitates the differential diagnosis and enables to rule out other more serious causes of difficulty walking. Symptomatic therapy is an adequate option and the patient recovers spontaneously within a couple of days. Early recognition of the disease avoids unnecessary diagnostic and therapeutic interventions.

Cerebrospinal-fluid profile in neuroborreliosis and its diagnostic significance.

Selected cerebrospinal-fluid (CSF) parameters (intrathecal synthesis of Borrelia-specific antibodies, oligoclonal IgG bands, CSF-to-serum quotient of albumin as a marker of blood-CSF barrier function and cytology) and typical CSF profile in neuroborreliosis were evaluated with the aim of elucidating possible clinical and laboratory similarities of neuroborreliosis (NB) and other neurological diseases (OND). From the cohort of 58 patients (38 diagnosed for NB, 20 with OND) NB patients had positive Borrelia-specific IgG antibodies in 97 % and positive Borrelia-specific IgM antibodies in 55 %; oligoclonal IgG bands were detected in 55%. The blood-CSF barrier was impaired in 89%, positive cytology was detected in 97% of the NB patients. Evaluation of specific intrathecal synthesis improves CSF diagnosis of NB, therefore, a combined CSF analysis has to be considered along with the clinical picture and medical history when formulating the diagnosis of NB.

[Distal protection during primary coronary angioplasty in patients with acute myocardial infarction]. / Distální protekce behem primární koronární angioplastiky u nemocných s akutním infarktem myokardu.

BACKGROUND: The aim of the study was to assess the safety, feasibility and efficacy of mechanical distal protection during primary angioplasty using FilterWire EZ (FW). METHOD AND RESULTS: Thirty-one patients with acute myocardial infarction (AMI) were treated by primary angioplasty with distal protection using FW. The results were compared with a matched control group consisting of 33 patients with AMI treated by primary angioplasty alone. Successful FW positioning was obtained in 30 patients (97%). In these patients a lower rate of distal embolisation (3 vs. 18%, p=0.04) was found and a more effective reperfusion was assessed by ST elevation's resolution >50% immediately after the procedure (83 vs. 61%, p=0.05). A higher number of patients with corrected TIMI frame count <27 in FW group supported more effective reperfusion to but this difference did not reached statistical significance (87 vs. 73%, p=0.09). There were no differences between groups regarding average peak CK and CK-MB. However, there is a trend to lower release in FW group (32.1+/-24.5 vs. 35.3+/-31.0, p=0.33; 4.2+/-3 vs. 4.4+3.7, p=0.44). CONCLUSIONS: The presented study confirmed that distal embolisation during primary angioplasty is a frequent phenomenon. In this setting, adjunctive use of the FW is feasible and save, and it may improve myocardial reperfusion by reducing the embolic events.

Relevance of immunological variables in neuroborreliosis and multiple sclerosis.

OBJECTIVES: The aims were to investigate the frequency of intrathecal synthesis of specific antibodies against measles (M), rubella (R) and varicella zoster (Z) viruses (MRZ reaction) as a diagnostic marker between multiple sclerosis (MS) and neuroborreliosis (NB) groups and to postulate the most typical cerebrospinal fluid (CSF) variables profile of these entities. METHODS: Three cohorts of patients were investigated: MS (n = 42), NB (n = 27) and other neurological diseases (OND) (n = 15). Measles, rubella, varicella zoster and borrelia-specific IgG antibodies were measured by ELISA, Q(alb) (CSF/serum albumin ratio) as a marker of blood-CSF barrier function and specific antibody indices (AI) were calculated according to relevant formulae. IgG oligoclonal bands (OB) were detected by isoelectric focusing and immunoenzymatic staining. RESULTS: Eighty-eight percent of MS patients had positive MRZ reaction and 26.2% had positive anti-borrelia AI. Eighty-nine percent of NB patients had positive anti-borrelia AI and two patients had individually anti-measles and rubella positive AI. MS-CSF variables profile included the presence of IgG OB in 81%, elevated Q(alb) in 31% and normal cell count in 66.7%. Of NB patients IgG OB were positive in 74%, elevated Q(alb) in 81.5% and normal cell count in 7.4%. CONCLUSION: MRZ reaction was proved as statistically significant marker in differential diagnosis between MS and NB. Typical CSF variables profile of these two entities is highly supportive, especially when MRZ is included.

The extracellular matrix of rat pacinian corpuscles: an analysis of its fine structure.

The Pacinian corpuscle consists of a sensory axon terminal that is enveloped by two different structures, the inner core and the capsule. Since proteoglycans are extremely water soluble and are extracted by conventional methods for electron microscopy, the current picture of the structural composition of the extracellular matrix in the inner core and the capsule of the Pacinian corpuscle is incomplete. To study the structural composition of the extracellular matrix of the Pacinian corpuscles, cationic dyes (ruthenium red, alcian blue, acridine orange) and tannic acid were applied simultaneously with the aldehyde fixation. The interosseal Pacinian corpuscles of the rat were fixed either in 2% formaldehyde and 1.5% glutaraldehyde, with the addition of one of these cationic dyes or, in Zamboni's fixative, with tannic acid added. The cationic dyes and tannic acid revealed a different structural pattern of proteoglycans in the extracellular matrix in the inner core and in the capsule of the rat Pacinian corpuscles. The inner core surrounding the sensory axon terminal is a compartment containing proteoglycans that were distributed not only in the extracellular matrix but also in the cytoplasm of the lamellae. In addition, this excitable domain was separated from the capsular fluid by a thick layer of proteoglycans on its surface. An enlarged interlamellar space of the capsule contained large amounts of proteoglycans that were removed by digestion with chondroitinase-ABC. Ruthenium red and alcian blue provided only electron dense granules, probably corresponding to collapsed monomeric proteoglycan molecules. Acridine orange and tannic acid preserved proteoglycans very well and made it possible to visualize them as "bottlebrush" structures in the electron microscope. These results show that the inner core and the capsule of rat Pacinian corpuscles have different structural patterns of proteoglycans, which are probably involved in different functions.

An immunocytochemical analysis of growing axons in a silicone chamber prefilled with artificial sponge matrix.

We have used antibodies against growth associated protein (GAP-43), phosphorylated neurofilament protein of 200 kDa molecular weight (RT-97) and substance P (SP) to analyze regrowing axons and their features in a silicone chamber filled with resorbable sponge matrix within the first two weeks after sciatic nerve transection in the rat. Growing axons identified with the GAP-43 antibody extended over a distance of about 7 mm from the proximal stump at 7 days and grew over a 10 mm gap within, 14 days. This is a markedly longer distance than in the case of the standard chamber model without artificial sponge matrix. The regrowing axons were labelled with RT-97 already on the 7th day up to a distance of 5 mm and they made up about 75% of all axons in the first segments. The number of RT-97-positive axons did not increase significantly over the next 7 days, although they could be identified over a longer distance. Some of the growing axons expressed SP-like immunoreactivity (LI) 14 days, but not 7 days after chamber application and constituted about 30% of all growing axons in the first segment. The SP-LI fibres also appeared to grow from the distal stump since they were found in larger numbers in the distal segments than in central ones. Those fibres accompanying blood vessels are probably sympathetic. Our findings demonstrate that axons are able to bridge a 10 mm gap within 14 days under appropriate substrate conditions, which are provided by the resorbable fibrin sponge.