Two cases of acute severe flaccid myelitis associated with enterovirus D68 infection in children, Norway, autumn 2014.

Enterovirus D68 (EV-D68), phylogenetic clade B was identified in nasopharyngeal specimens of two cases of severe acute flaccid myelitis. The cases were six and five years-old and occurred in September and November 2014. EV-D68 is increasingly associated with acute flaccid myelitis in children, most cases being reported in the United States. Awareness of this possible neurological complication of enterovirus D68 infection is needed.

spa typing alone is not sufficient to demonstrate endemic establishment of meticillin-resistant Staphylococcus aureus in a low-prevalence country.

BACKGROUND: The prevalence of meticillin-resistant Staphylococcus aureus (MRSA) in Norway is low but increasing. Over the last decade, numerous nursing homes have experienced MRSA outbreaks. One genetic lineage, spa type t304, has been identified at multiple nursing homes and has caused large outbreaks lasting for several years. AIM: To evaluate whether spa typing is sufficient for the detection of MRSA spread and endemic establishment in a low-prevalence area, using spa type t304 as the test organism. METHODS: All spa type t304 isolates detected in 1991-2010 in the most densely populated area of Norway were included. Time and place of bacterial sampling were recorded. The isolates were analysed using multi-locus sequence typing, staphylococcal cassette chromosome mec typing, detection of lukS/F-PV and pulsed-field gel electrophoresis (PFGE). FINDINGS: In total, 181 spa type t304 isolates were identified in three of 23 municipalities. Most (91%) of the isolates could be linked to 13 nursing homes, eight of which experienced outbreaks. PFGE analysis revealed three PFGE types, consisting of 19 PFGE patterns; 95% of the isolates were PFGE type 2. In total, PFGE types 2 and 3 accounted for 99% of all nursing home isolates, and included isolates from different nursing homes, different outbreaks and different time periods. Additional genetic analyses did not further differentiate between the spa type t304 isolates. CONCLUSION: MRSA spa type t304 appears to have established itself as an endemic genetic lineage in the study area. spa typing does not provide sufficient resolution when investigating the spread of an endemic-like genetic lineage in a low-prevalence area, and should be supplemented by additional typing techniques.

Depletion of mitochondrial DNA copies/cell in peripheral blood mononuclear cells in HIV-1-infected treatment-naïve patients.

OBJECTIVES: Mitochondrial toxicity is believed to be the main reason for adverse effects related to nucleoside reverse transcriptase inhibitors (NRTIs). The aim of the present study was to compare mitochondrial toxicity in NRTI-treated HIV-positive patients, HIV-positive treatment-naïve patients and HIV-negative controls by comparing mitochondrial DNA (mtDNA) copies/cell in peripheral blood mononuclear cells (PBMCs) and lactate/pyruvate (L/P) ratios in the different groups. METHODS: We enrolled 60 participants in the study: 31 patients on combined antiretroviral therapy (CART), 14 HIV-positive treatment-naive patients and 15 HIV-negative controls. mtDNA (copies/cell) in peripheral blood was analysed using quantitative real-time polymerase chain reaction (PCR). Standard curves and serial dilutions of plasmid-cloned mitochondrion and retinoblastoma (RB1) PCR products with known concentrations were generated to estimate the mtDNA and nuclear DNA (nDNA) copy numbers in each sample. The L/P ratio was enzymatically and spectrophotometrically analysed in samples from individuals in a fasted, non-exercise state. Results The median mtDNA copy number was 63 copies/cell (interquartile range 33-94) in HIV-positive patients and 153 (132-283) in HIV-negative controls (P<0.001). No significant difference was seen between the HIV-positive NRTI-exposed patients and the HIV-positive treatment-naive patients. Current use of didanosine was negatively correlated with depletion of mtDNA (r=-0.36, P=0.046). HIV-positive patients also had a higher L/P ratio compared with HIV-negative controls (P=0.004). CONCLUSIONS: The number of mtDNA copies/cell in PBMCs was depleted in HIV-positive treatment-naive patients as well as in HIV-positive NRTI-exposed patients. HIV-positive patients also had a higher L/P ratio compared with HIV-negative controls, which supports this conclusion. The study suggests that neither mtDNA in PBMCs nor L/P ratio is a good marker of NRTI-associated mitochondrial toxicity.

An algorithm-based genotypic resistance score is associated with clinical outcome in HIV-1-infected adults on antiretroviral therapy.

OBJECTIVES: The aim of this study was to evaluate the association between genotypic drug resistance and the occurrence of HIV-related diseases and death in HIV-1-infected adults on antiretroviral therapy. METHODS: We performed an observational study on patients from an out-patient clinic in a university hospital. Genotypic drug resistance analysis after virological treatment failure was performed in 141 patients receiving two or more antiretroviral drugs. All patients had follow up of at least 6 months after the resistance test. An algorithm was developed to estimate the level of genotypic drug resistance and to assign an actual resistance score (ARS) for the drugs prescribed to each patient. The patient population was divided into quartiles according to patients' ARS values. Our endpoint was the risk of developing an HIV-related disease [Centers for Disease Control and Prevention (CDC) category B or C] during the period starting 6 months prior to and ending 6 months after the genotypic resistance test, or death during the 6 months following the resistance test. RESULTS: There was a significant association between the level of resistance to the drugs prescribed (ARS) and our clinical endpoint: the odds ratio for an endpoint (with 95% confidence interval) was 3.20 (1.28-7.99), adjusted for CD4 cell count and HIV RNA, in patients in the highest ARS quartile compared with patients in the other three quartiles. CONCLUSIONS: Our study indicates that patients with high-level genotypic drug resistance are at increased risk of developing an HIV-related disease. This association could not be explained by differences in CD4 cell count or HIV RNA levels.

Mycobacterium interjectum: a new pathogen in humans?

Molecular genetic techniques have increased the number of species recognized within the genus Mycobacterium. The clinical significance of these is uncertain and their pathogenic potential has still to be proven. We describe here a case of mycobacterial lymphadenitis in a Swedish boy, which supports the role of M. interjectum as a human pathogen.

Identification of clinical isolates of Mycobacterium spp. by sequence analysis of the 16S ribosomal RNA gene. Experience from a clinical laboratory.

Twenty-one mycobacterial type strains and 334 clinical isolates of mycobacteria were identified by standardized sequence analysis using part of the gene encoding 16S rRNA. Apart from two clinical isolates, the resulting sequences corresponded to previously published sequences. The results of the molecular determinations of the type strains completely overlapped the identities obtained using conventional techniques (cultural characteristics, biochemical tests, commercial DNA probes, and gas chromatographic lipid profiles). Of 323 isolates conventionally identified as slow-growing mycobacteria, 318 (98.5%) were identified to the same species or group level by 16S rDNA sequence analysis, while 6 of the 11 strains of rapid growers obtained a corresponding identity with the two approaches. The sequencing protocol combined with a few cultural characteristics (i.e. growth rate, pigmentation and susceptibility testing) offers a rapid, reliable and usually definite identification of mycobacterial isolates.

A second European collaborative study on polymerase chain reaction for Toxoplasma gondii, involving 15 teams.

In order to investigate the accuracy and practicability of the polymerase chain reaction (PCR) in the antenatal diagnosis of congenital toxoplasmosis, a collaborative study involving 15 European laboratories was performed under the auspices of the Biomed 2 Programme of the European Community. Each team received 12 aliquots (four negative, eight positive) of 'artificial samples' made of amniotic fluid spiked with tachyzoites of the RH strain of Toxoplasma gondii. Each team performed its own PCR protocol (all were different). Nine of the 15 laboratories were able to detect a single parasite, but two of the 15 found all samples negative. Four of the 15 laboratories found one or more control samples to be falsely positive. This study highlights the lack of homogeneity between PCR protocols and performance and underlines the need for an external quality assurance scheme which could provide 'reference' samples that could be used by any laboratory wanting to establish and maintain an accurate diagnostic test based on PCR.

Diagnosis of congenital Toxoplasma gondii infection by polymerase chain reaction (PCR) on amniotic fluid samples. The Norwegian experience.

As part of a screening project for detection of Toxoplasma gondii infection among pregnant women in Norway, nested polymerase chain reaction (PCR) aimed at the detection of T. gondii in amniotic fluid samples was included in the diagnostic routine. The results were compared with the routine criteria for congenital infection: i) T. gondii detected in amniotic fluid or cord blood by mouse inoculation, ii) specific IgM or IgA in serum collected after birth, and/or iii) specific IgG persisting beyond one year of age. The PCR was based on the B1 gene with an internal control gene amplified together with the B1 gene. One hundred and two amniotic fluid samples collected during pregnancy and/or at delivery from 67 pregnant women with serological evidence of primary T. gondii infection were available for examination by both B1-PCR and mouse inoculation. Six samples were positive and 86 samples were negative by both methods (90% concordance). One sample was mouse inoculation positive and B1-PCR negative while nine samples were B1-PCR positive and mouse inoculation negative, of which five were associated with four infants without proven infection. 59%, and 41% of samples associated with infected infants were positive by B1-PCR and mouse inoculation, respectively. The difference was mainly due to a lower detection rate by mouse inoculation after antiparasitic treatment. The specificity of B1-PCR was 94%. Even though B1-PCR performed on amniotic fluid samples did not detect all infected infants, it represented a valuable tool in addition to conventional methods in the diagnosis of congenital T. gondii infection.

The effect of human cytomegalovirus on selected functions of peripheral blood monocytes.

The effect of in vitro infection of human cytomegalovirus (HCMV) on various monocyte functions relevant to antimicrobial defence mechanisms has been investigated: the phagocytic activity of monocytes, the release of lysozyme and intracellular concentration of acid phosphatase, and the release of the cytokines interleukin-1 (IL-1), IL-6, and tumour necrosis factor-alpha (TNF-alpha). HCMV significantly inhibited the release of lysozyme and intracellular concentration of acid phosphatase. Regarding the phagocytic activity and the release of cytokines, there was considerable variation in the HCMV effect among the different blood donors tested. There was no clear tendency in the observed results; both stimulation and inhibition were seen. The HCMV-specific pp65 was detected in the nucleus of about 1% of the monocytes 3 h after infection and HCMV-specific IE antigens were found in about 0.1% of the monocytes 2 days postinfection. No E- or L-gene expression was observed and no infectious virus was produced in the monocytes. Our results indicate that HCMV infection may influence monocyte functions in spite of no productive infection of these cells.

Infection with human cytomegalovirus (HCMV) stimulates monocyte production of complement factor 3.

Complement biosynthesis in monocytes is stimulated by different microorganisms including Gram negative bacteria and yeasts. We have tested the effect of human cytomegalovirus (HCMV) on complement factor 3 (C3) production by cultured human monocytes. The monocytes were challenged with either a crude or a purified HCMV preparation obtained from the supernatant of HCMV-infected fibroblasts. When the monocytes were infected with 2 pfu/cell of virus and cultured for 2 days, the increase in C3 production compared to control ranged from 3% to 162%, median 62% (p < 0.01). However, crude HCMV was even more potent in stimulating C3 production, as the increase in C3 values ranged from 104% to 507%, median 247% (p = 0.001). This indicates the presence in the crude HCMV preparation of a substance which acts synergistically with HCMV on the C3 production. When monocytes were stimulated by lipopolysaccharide (LPS), a well known inducer of C3, infection with crude or purified HCMV did not further increase C3 production. Both HCMV and substances produced during the propagation of HCMV in fibroblasts are able to stimulate C3 production in monocytes. Complement production by inflammatory cells may be of importance in host resistance against viral infections.

Direct growth suppression of myeloid bone marrow progenitor cells but not cord blood progenitors by human cytomegalovirus in vitro.

Recently, considerable interest has arisen as to use cord blood (CB) as a source of hematopoietic stem cells for allogenic transplantation when bone marrow (BM) from a familial HLA-matched donor is not available. Because human cytomegalovirus (HCMV) has been shown to inhibit the proliferation of BM progenitors in vitro, it was important to examine whether similar effect could be observed in HCMV-infected CB cells. Therefore, the effect of HCMV challenge on the proliferation of myeloid progenitors from BM and CB was compared using both mononuclear cells (MNC) and purified CD34+ cells. A clinical isolate of HCMV inhibited the colony formation of myeloid BM progenitors responsive to granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, macrophage-CSF, interleukin-3 (IL-3) and the combination of IL-3 and stem cell factor (SCF). In contrast, colony growth of CB progenitors was not affected. In addition, HCMV inhibited directly the growth of purified BM CD34+ cells responsive to IL-3 and SCF in single cell assay by 40%, wheras the growth of CD34+ progenitors obtained from CB was not suppressed. The HCMV lower matrix structural protein pp65 and HCMV DNA were detected in both CB and BM CD34+ cells after in vitro challenge. However, neither immediate early (IE)-mRNA nor IE proteins were observed in infected cells. Cell cyclus examination of BM and CB CD34+ cells revealed that 25.7% of BM progenitors were in S + G2/ M phase wheras only 10.7% of the CB progenitors. Thus, a clinical isolate of HCMV directly inhibited the proliferation of myeloid BM progenitors in vitro wheras CB progenitors were not affected. This difference in the susceptibility of CB and BM cells to HCMV may partly be caused by the slow cycling rate of naive CB progenitors compared to BM progenitors at the time of infection.

Human interferon reduces surface expression but not total production of herpes simplex virus type 1 glycoproteins gC and gE in heterologous hamster cells.

The effect of interferon (IFN) on herpes simplex virus type 1 (HSV-1)-induced glycoproteins gC and gE was investigated in a heterologous IFN/cell model. In this model, the effect on surface expression of the glycoproteins could be studied separately from the effect on virus multiplication. Pretreatment of baby hamster kidney cells (BHK) with heterologous human leukocyte IFN suppressed surface expression of HSV-1-encoded gC and gE but had no influence on total production of the glycoproteins. This was in contrast to the effect on human embryonic fibroblast cells (HE) (homologous IFN and cells), where surface expression as well as total production of glycoproteins were reduced. The surface expression was demonstrated by antibody-sensitized monodisperse polystyrene beads, and immunoblotting and two-dimensional electrophoretic analysis of radioisotope-labeled proteins were used to study the total production.

Shigella flexneri adherence to and multiplication in coxsackie B1 virus-infected HEp-2 cells.

Coxsackie B1 virus infection enhances the susceptibility of in vitro cultured HEp-2 cells to invasiveness by Shigella flexneri. We have studied the effect of viral infection on two phases of the invasiveness. Only a minor part was mediated by enhanced bacterial adherence to the cells, and the intracellular multiplication was unaffected by the virus. Enhanced adherence was not dependent on the presence of the gene product of the 140 Md virulence associated plasmid. Our data indicate that enhanced invasiveness induced by viral infection is mediated by an effect on other phases of the invasiveness.

Infection with human cytomegalovirus enhances bacterial adhesiveness and invasiveness in permissive and semipermissive cells.

The effect of human cytomegalovirus (HCMV) infection on adhesiveness and invasiveness of Salmonella typhimurium was examined in cells permissive (human embryo fibroblasts (HE)), semipermissive (A549) and nonpermissive (HEp-2) for the virus. Preinfection of the cells with HCMV induced enhanced adhesiveness and invasiveness of bacteria in the permissive HE cells. In the semipermissive A549 cells, where HCMV immediate-early (IE) mRNA transcripts and IE proteins were detected, a significant effect on the initial phase of invasiveness, the adherence phase, was demonstrated. HCMV had no effect on invasiveness of S. typhimurium in nonpermissive HEp-2 cells. Neither HCMV IE transcripts nor IE proteins could be detected in these cells.

Nonspecific oral immunity in individuals with HIV infection.

Lactoferrin, lysozyme, interferon, and neopterin levels were determined in parotid saliva from 44 individuals with different clinical stages of human immunodeficiency virus (HIV) infection and 19 HIV-seronegative controls. The secretory output of individual components was calculated according to the fluid flow rate. No parotid interferon activity was found in any of the HIV-infected subjects or controls, and no significant differences in parotid lysozyme or neopterin outputs were observed. The lactoferrin output was significantly decreased in HIV-seropositive subjects in parallel with their markedly reduced parotid secretory IgA output. This combined deficiency of parotid lactoferrin and secretory IgA may well contribute to the frequent oral infections seen in subjects with HIV infection.

Effect of interferon treatment on expression of gC and gE glycoproteins in herpes simplex virus-infected cells.

The effect of interferon treatment on the herpes simplex virus type 1 (HSV-1)-specific glycoproteins gC and gE in homologous and heterologous cells has been investigated. In human embryonic fibroblastic cells, human leukocyte interferon inhibited virus multiplication and expression of the HSV-1-specific glycoproteins gC and gE on the cell surface in a dose-dependent manner. In heterologous baby hamster kidney cells, the human interferon had no effect on virus multiplication. However, the surface expression of the HSV-1-specific glycoproteins was reduced, as shown by erythrocyte rosette formation, by attachment of monodisperse polystyrene particles coated with antibodies and by immunogold scanning electron microscopy.

Adhesiveness and invasiveness of staphylococcal species in a cell culture model.

A model was established for the study of adhesiveness and invasiveness of staphylococcal species. Five collection strains from each of the species Staphylococcus aureus, S. epidermidis, and S. saprophyticus and 26 fresh isolates from patients with urinary tract infections were tested for adhesiveness and invasiveness in HEp-2 cell cultures. All the strains of S. saprophyticus were able to invade the cells and localize intracellularly in the cultures, whereas the invasive potential among the strains of S. aureus and S. epidermidis was lower. The number of adhesive bacteria was also highest among the S. saprophyticus strains, whereas S. epidermidis was the least adhesive. The model may be suitable for further study of urinary tract infection strains.

Influence of a carcinogenic agent, 7.12 dimethylbenz (alpha) anthracene (DMBA), on production of interferon alpha/beta in murine and human cells.

Interferon production in mouse and human cells induced by paramyxovirus was inhibited by pretreatment of cells with 7.12 dimethylbenz (alpha) anthracene. The inhibition was moderate but reproducible. It was most pronounced in mouse fibroblast cells, somewhat less in the mouse L-929 cell line and in human embryo fibroblast cells. Addition of the microsomal activator systems was necessary in the human system. A possible employment of this phenomenon in testing carcinogenic potential is discussed.

Invasiveness of Salmonella typhimurium in HEp-2 cell cultures pretreated with UV-inactivated coxsackie virus.

The invasiveness of Salmonella typhimurium was significantly enhanced in cell cultures pretreated with UV-inactivated virus. During the first 3 hours of virus infection there was no difference between the enhancement achieved with non-inactivated and that achieved with UV-inactivated virus. After 4 and 5 hours pretreatment the effect of non-inactivated virus was more pronounced than that of UV-inactivated virus. The results indicate that during the early period of virus infection the enhancement of bacterial invasiveness by pretreatment with virus is the result of a direct interaction between the virus and the cell membrane. During the later phase of viral reproduction, viral RNA induced alteration of the cell metabolism, and these altered products might be involved in the interaction.