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Background/aims To compare outcomes of acute endophthalmitis (EO) managed with either primary vitrectomy (PV) or primary intravitreal antibiotics (vancomycin and ceftazidime) followed by early vitrectomy (PIAEV) combined with polymerase chain reaction (PCR)-based diagnostics. Methods This was a prospective, comparative observational study of acute EO cases admitted to a regional vitreoretinal service over 18 months. Depending on whether immediate vitrectomy (within 6 h) was achievable, the EO cases were treated with either (1) PV or (2) PIAEV. Microbiology samples were collected either (A) before or (B) after administration of intravitreal antibiotics. The samples were analysed with broad-range 16S PCR and culture. Results The study included 41 EO cases. There were 19 post-injection EO, 18 post-cataract EO, three post-vitrectomy EO, and one blebitis-related EO. Fifteen of 19 PV cases and 15 of 21 PIAEV had a clinically meaningful improvement in best-corrected visual acuity (BCVA) of at least 15 letters at 3 months (p = 0.58). One patient was lost to follow-up. Twenty-three cases were culture- and PCR-positive, and seven additional cases were culture-negative but PCR-positive (p = 0.02). PCR increased the diagnostic yield for samples collected both before and after administration of intravitreal antibiotics. Conclusion Primary vitrectomy or PIAEV allowed for vitrectomy for all cases of acute EO in a large region. Most eyes in both groups achieved a clinically meaningful improvement in BCVA. By combining culture with PCR in connection with the vitrectomy procedure, intravitreal antibiotics could be injected before microbiological sampling, thereby improving the door-to-treatment time without sacrificing microbial identification.

Objectives The emerging SARS-CoV-2 variants of concern (VoC), B.1.1.7, B.1.351 and P.1, with increased transmission and/or immune evasion, emphasise the need for broad and rapid variant monitoring. Our high-volume laboratory introduced a PCR variant assay (Variant PCR) in January 2021 based on the protocol by Vogels et al. Study design To assess whether Variant PCR could be used for rapid B.1.1.7, B.1.351 and P.1 screening, all positive SARS-CoV-2 airway samples were prospectively tested in parallel using both the Variant PCR and whole genome sequencing (WGS). Results In total 1,642 SARS-CoV-2 positive samples from individual patients were tested within a time span of 4 weeks. For all samples with valid results from both Variant PCR and WGS, no VoC was missed by Variant PCR (totalling 399 VoC detected). Conversely, all of the samples identified as “other lineages” (i.e., “non-VoC lineages”) by the Variant PCR, were confirmed by WGS. Conclusions The Variant PCR based on the protocol by Vogels et al., is an effective method for rapid screening for VoC, applicable for most diagnostic laboratories within a pandemic setting. WGS is still required to confirm the identity of certain variants and for continuous surveillance of emerging VoC.

Significance The new SARS-CoV-2 pandemic leads to COVID-19 with respiratory failure, substantial morbidity, and significant mortality. Overactivation of the innate immune response is postulated to trigger this detrimental process. The complement system is a key player in innate immunity. Despite a few reports of local complement activation, there is a lack of evidence that the degree of systemic complement activation occurs early in COVID-19 patients, and whether this is associated with respiratory failure. This study shows that a number of complement activation products are systemically, consistently, and long-lastingly increased from admission and during the hospital stay. Notably, the terminal sC5b-9 complement complex was associated with respiratory failure. Thus, complement inhibition is an attractive therapeutic approach for treatment of COVD-19., Respiratory failure in the acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is hypothesized to be driven by an overreacting innate immune response, where the complement system is a key player. In this prospective cohort study of 39 hospitalized coronavirus disease COVID-19 patients, we describe systemic complement activation and its association with development of respiratory failure. Clinical data and biological samples were obtained at admission, days 3 to 5, and days 7 to 10. Respiratory failure was defined as PO2/FiO2 ratio of ≤40 kPa. Complement activation products covering the classical/lectin (C4d), alternative (C3bBbP) and common pathway (C3bc, C5a, and sC5b-9), the lectin pathway recognition molecule MBL, and antibody serology were analyzed by enzyme-immunoassays; viral load by PCR. Controls comprised healthy blood donors. Consistently increased systemic complement activation was observed in the majority of COVID-19 patients during hospital stay. At admission, sC5b-9 and C4d were significantly higher in patients with than without respiratory failure (P = 0.008 and P = 0.034). Logistic regression showed increasing odds of respiratory failure with sC5b-9 (odds ratio 31.9, 95% CI 1.4 to 746, P = 0.03) and need for oxygen therapy with C4d (11.7, 1.1 to 130, P = 0.045). Admission sC5b-9 and C4d correlated significantly to ferritin (r = 0.64, P < 0.001; r = 0.69, P < 0.001). C4d, sC5b-9, and C5a correlated with antiviral antibodies, but not with viral load. Systemic complement activation is associated with respiratory failure in COVID-19 patients and provides a rationale for investigating complement inhibitors in future clinical trials.

Introduction: Primary amoebic meningoencephalitis (PAM) is a rare disease caused by the free-living amoeba Naegleria fowleri. Infection occurs by insufflation of water containing amoebae into the nasal cavity, and is usually associated with bathing in freshwater. Nasal irrigation is a more rarely reported route of infection. Case presentation: A fatal case of PAM in a previously healthy Norwegian woman, acquired during a holiday trip to Thailand, is described. Clinical findings were consistent with rapidly progressing meningoencephalitis. The cause of infection was discovered by chance, owing to the unexpected detection of N. fowleri DNA by a PCR assay targeting fungi. A conclusive diagnosis was established based on sequencing of N. fowleri DNA from brain biopsies, supported by histopathological findings. Nasal irrigation using contaminated tap water is suspected as the source of infection. Conclusion: The clinical presentation of PAM is very similar to severe bacterial meningitis. This case is a reminder that when standard investigations fail to identify a cause of infection in severe meningoencephalitis, it is of crucial importance to continue a broad search for a conclusive diagnosis. PAM should be considered as a diagnosis in patients with symptoms of severe meningoencephalitis returning from endemic areas.

Background HIV type-1 (HIV-1) drug resistance testing is rarely available in resource-limited settings because of high costs and stringent requirements for storage and transport of plasma. Dried blood spots (DBS) can be a convenient alternative to plasma, but the use of DBS needs validation under field conditions. We assessed the performance of DBS in genotypic resistance testing of patients who failed first-line antiretroviral therapy (ART) in rural Tanzania. Methods A total of 36 ART-experienced patients with viral loads >1,000 copies/ml (median 15,180 copies/ml [range 1,350–3,683,000]) and with various HIV-1 subtypes were selected for resistance testing. DBS were stored with desiccant at ambient temperature for a median of 29 days (range 8–89). Samples were amplified using an in-house reverse transcriptase-nested PCR method and sequenced using the ViroSeq™ assay (Abbott Molecular, Des Plaines, IL, USA). DBS-derived genotypes were compared with genotypes from plasma. Results Overall, 34 of 36 (94%) DBS specimens were successfully genotyped. In the protease region, of 142 polymorphisms found in plasma, 132 (93%) were also detected in DBS. In the reverse transcriptase region, of 57 clinically relevant mutations present in plasma, 51 (89%) were also detected in DBS. A total of 30 of 34 (88%) patients had identical resistance profiles to antiretroviral drugs in plasma and DBS. Conclusions Genotyping was successful in the vast majority of DBS specimens stored at ambient temperature for up to 3 months, and there was high concordance between mutations found in DBS and plasma. Our study suggests that DBS can be a feasible and reliable tool to monitor HIV-1 drug resistance in patients on ART in resource-limited settings.

Published version of an article from the journal: Journal of Antimicrobial Chemotherapy. Also available from the publisher: htttp://dx.doi.org/10.1093/jac/dkq234 Objectives: To assess long-term virological efficacy and the emergence of drug resistance in children who receive antiretroviral treatment (ART) in rural Tanzania. Patients and methods: Haydom Lutheran Hospital has provided ART to HIV-infected individuals since 2003. From February through May 2009, a cross-sectional virological efficacy survey was conducted among children (, 15 years) who had completed >= 6 months of first-line non-nucleoside reverse transcriptase inhibitor (NNRTI)-based ART. Genotypic resistance was determined in those with a viral load of >200 copies/mL. Results: Virological response was measured in 19 of 23 eligible children; 8 of 19 were girls and median age at ART initiation was 5 years (range 2-14 years). Median duration of ART at the time of the survey was 40 months (range 11-61 months). Only 8 children were virologically suppressed (50% harboured drug resistance. Results for children were markedly poorer than for adults attending the same programme, underscoring the need for improved treatment strategies for children in resource-limited settings.

Over a 6-month period in 2008, approximately 15% of all Staphylococcus aureus isolates from our neonatal intensive care unit were resistant to penicillin, gentamicin, erythromycin and clindamycin. Extended antibiotic susceptibility testing and molecular profiling revealed an outbreak of an S. aureus strain with a rare susceptibility pattern for a Scandinavian setting.

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.

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.

Objectives Most data on mitochondrial toxicity have been derived from peripheral blood mononuclear cells (PBMCs). However, whether mitochondrial DNA (mtDNA) content in PBMCs reflects the mitochondrial state in tissues remains elusive. We report herein on mitochondrial toxicity in skeletal muscle in HIV-infected patients naive to antiretroviral treatment (ART [HIV+ART-naive]; n=10) patients exposed to nucleoside reverse transcriptase inhibitors (NRTIs [HIV+NRTI+]; n=24) and healthy controls ( n=11), and compare these tissue data with mtDNA in PBMCs. Methods Muscle biopsies were examined for (i) mtDNA and nuclear DNA (nDNA) content using TaqMan realtime PCR system, (ii) mtDNA deletions using long expand PCR with subsequent gel electrophoresis, and (iii) mitochondrial myopathy expressed as cytochrome c oxidase (COX)-deficient muscle fibres. Results The mt/n DNA ratio in muscle from HIV+NRTI+patients was reduced compared with HIV-negative controls ( P=0.028). Moreover, mtDNA deletions were more frequent in HIV+NRTI+ patients than in both HIV-negative controls ( P=0.009) and HIV+ART-naive patients ( P=0.005). HIV+NRTI+ also tended to have more COX-deficient fibres than HIV-negative controls ( P=0.076). COX-deficient fibres were positively correlated with mtDNA deletions in HIV+NRTI+ patients (r=0.83, PConclusions In skeletal muscle, HIV+NRTI+ had a reduced mt/n DNA ratio, more frequent mtDNA deletions and possibly more COX-deficient muscle fibres than HIV-negative controls. However, the mtDNA/cell in peripheral blood was decreased in both HIV+NRTI+ and HIV+ART-naive patients. Thus, mtDNA in peripheral blood may not be a relevant marker of mitochondrial toxicity in organ-specific tissue.

The study objective was to identify optimal starting criteria regarding levels of CD4 cells and human immunodeficiency virus (HIV) RNA at initiation of highly active antiretroviral therapy (HAART) in chronically HIV-infected people. All 162 treatment-naive patients in the centre who were treated for at least 180 d with 2 nucleoside reverse transcriptase inhibitors plus at least 1 protease inhibitor or 1 non-nucleoside reverse transcriptase inhibitor were included. The patients were stratified according to their levels of CD4 cells and HIV RNA at initiation of therapy. Baseline CD4 groups were: group 1: CD40.1 x 10(9)/l; group 2: CD4or = 0.1 and0.2 x 10(9)/l; group 3: CD4or = 0.2 and0.35 x 10(9)/l; and group 4: CD4or = 0.35 x 10(9)/l. Two patients died and 38 developed an HIV-related disease (Centers for Disease Control category B or C) during the study. The prevalence of HIV-related disease before HAART was significantly increased in groups 1 and 2 compared with groups 3 and 4. The level of HIV RNA was not associated with HIV-related disease either before or after treatment initiation. Subjects in group 1 had an increased risk of HIV-related disease after treatment initiation both in univariate Cox analysis and after adjustment for HIV RNA, gender, mode of transmission and age, compared with group 2 [adjusted risk ratio with 95% confidence interval: 3.76 (1.48-9.61)], group 3 [5.90 (2.07-16.95)] and group 4 [5.05 (1.96-12.90)]. The association between CD4 count and morbidity appeared to be particularly strong for older subjects. In conclusion, this study suggests that in chronically HIV-infected individuals, in most cases HAART can be withheld until the CD4 cell count falls towards 0.2 x 10(9)/l.

Objectives An unexpectedly high proportion of children were admitted for severe respiratory infections at the Oslo University Hospital, Ulleval, Norway, during September and October, 2014. In light of the ongoing outbreak of enterovirus-D68 (EV-D68) in North America a real-time RT-PCR for screening of enterovirus and enterovirus D68 was established. Design We developed a duplex real-time RT-PCR for rapid screening of enterovirus D68. The method target the 5′ non-translated region (NTR) of the HEV genome at a location generally used for enterovirus detection. Sample Nasopharyngeal samples (n = 354), from children

Streptococcus agalactiae (GBS) is a leading cause of invasive neonatal infection. Serotyping of GBS is important in following epidemiological trends and vaccine development. Capsular serotyping of GBS by latex agglutination has been the predominant typing method, but more recently capsular genotyping has been introduced as an alternative method. The purpose of this study was to compare the relative performance of these methods in a contemporary population of pregnant women. We typed isolates from an unselected population of 426 colonized women at delivery using latex agglutination and a combination of four PCR methods. Antibiotic resistance was tested in 449 isolates. Capsular genotyping gave a result in all except three of 426 isolates. Fifty-nine of 426 isolates could not be typed by latex agglutination. Agreement between serotyping and genotyping was shown in 303 (71.1%) of the isolates. 10.2% of the isolates were resistant to erythromycin, 9.6% to clindamycin, 76.6% to tetracycline and none to penicillin. In conclusion, a substantial proportion of the colonizing strains were non-typeable by serotyping, but typeable by genotyping. This suggests that a diagnostic genotyping strategy is preferable to serotyping of the GBS polysaccharide capsule in colonized, pregnant women.

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.

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.

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.

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.

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.

Summary Objectives The objectives of the study were to assess the utility of dried blood spots (DBS) for the detection of cytomegalovirus (CMV) antibody and viremia in a resource-poor setting, to study the prevalence of CMV antibody and viremia in HIV-infected patients with access to antiretroviral therapy (ART) in Tanzania, and to relate CMV viremia to outcome. Methods DBS were prepared from 168 ART-naive patients at baseline. Demographic, clinical, and laboratory data were obtained from patient records. CMV antibody was analyzed by chemiluminescent microparticle immunoassay and viremia by quantitative PCR. Results All patients were CMV-seropositive. At baseline 38 (22.6%) had detectable CMV viremia and 14 (8.3%) had a CMV viral load ≥200 copies/ml. In 135 patients available for follow-up, CMV ≥200 copies/ml was an independent risk factor for death with a hazard ratio of 5.0 (95% confidence interval 2.1–11.9) after adjusting for confounders. Symptoms compatible with CMV disease were common with viremia ≥200 copies/ml and CD4+ T cell counts 3 , but confirmatory diagnostic procedures were unavailable. Conclusions DBS are suitable for the detection of CMV antibody and viremia in HIV patients in resource-poor areas. CMV viremia was frequent and associated with an increased risk of death. Improved diagnosis and treatment of CMV may improve the prognosis for HIV-infected patients in developing countries and should be addressed in future studies.

An in-house nested polymerase chain reaction (PCR) was prospectively compared with culture for Bordetella pertussis detection in 435 nasopharyngeal and/or throat swabs from 304 patients. One hundred specimens - 21% of nasopharyngeal swabs and 25% of throat swabs - were PCR- and/or culture-positive. Seventy percent of positive nasopharyngeal samples and 44% of positive throat samples were culture-positive.

The human immunodeficiency virus type 1 (HIV-1) load is an important marker of disease progression and treatment efficacy in patients with HIV-1 infection. In recent years, an increase in the number of samples with detectable HIV-1 RNA has been reported among patients with previously suppressed viral loads, affecting clinical patient care and leading to repeat measurements of viral load and drug resistance. This rise seems to have coincided with the increased use of plasma preparation tubes (PPTs) for sample collection, and we have aimed to explain why PPTs might yield elevated HIV-1 RNA levels. The impacts of different sample-processing procedures on HIV-1 RNA levels were compared retrospectively. Prospectively, the presence of different cells and cell-associated HIV-1 nucleic acids in paired plasma samples from PPTs centrifuged before (PPT1) and after (PPT2) transportation to the laboratory was compared. A retrospective analysis of 4,049 patient samples with

Background Virological response to antiretroviral treatment (ART) in rural Africa is poorly described. We examined virological efficacy and emergence of drug resistance in adults receiving first-line ART for up to 4 years in rural Tanzania. Methods Haydom Lutheran Hospital has provided ART to HIV-infected patients since October 2003. A combination of stavudine or zidovudine with lamivudine and either nevirapine or efavirenz is the standard first-line regimen. Nested in a longitudinal cohort study of patients consecutively starting ART, we carried out a cross-sectional virological efficacy survey between November 2007 and June 2008. HIV viral load was measured in all adults who had completed at least 6 months first-line ART, and genotypic resistance was determined in patients with viral load >1000 copies/mL. Results Virological response was measured in 212 patients, of whom 158 (74.5%) were women, and median age was 35 years (interquartile range [IQR] 29–43). Median follow-up time was 22.3 months (IQR 14.0–29.9). Virological suppression, defined as

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.

OBJECTIVE Mitochondrial DNA (mtDNA) loss in peripheral blood mononuclear cells (PBMCs) has been found in both nucleoside reverse-transcriptase inhibitor (NRTI)-exposed and antiretroviral therapy (ART)-naive patients with human immunodeficiency virus (HIV) infection. Persistent immune activation might play a role in this phenomenon in HIV-infected, ART-naive patients. PBMC subsets with differential growth kinetics were therefore purified to study this similarity. METHODS CD4(+) and CD8(+) T cells, CD19(+) B cells, and CD14(+) monocytes were purified from PBMCs. mtDNA levels were quantified using real-time polymerase chain reaction and compared among the 2 groups of HIV-infected patients and a group of HIV-negative control subjects. mtDNA levels in a separate group of ART-naive patients stratified by the rate of disease progression were also evaluated with respect to their relationship to immune-activation markers (i.e., CD38 and programmed cell death-1 [PD-1]) on CD8(+) T cells and the rate of CD4(+) T cell loss. RESULTS mtDNA levels in CD8(+) T cells and B cells from 15 ART-naive patients were approximately 50% less than those observed for 14 control subjects (P < or = .01). mtDNA levels in all lymphocyte subsets correlated negatively with CD38(+)PD-1(+) expression (r= -0.66 P < -0.9; P < or = .03), and mtDNA levels in B cells correlated with the rate of CD4(+) T cell loss (r =0.66; P< .3). In 17 HIV-infected, NRTI-exposed patients, mtDNA loss was observed in both T cell subsets (P < or = .02) and was most pronounced in patients who received didanosine (P < or = .002). CONCLUSIONS In HIV-infected, ART-naive patients, mtDNA loss was found in CD8(+) T cells and B cells. These losses correlated with immune activation and, in B cells, with the rate of CD4(+) T cell loss. In patients receiving ART, only T lymphocytes had reduced mtDNA levels. This finding was probably associated with NRTI use, because it was most pronounced in patients with a history of didanosine exposure.

A recent nonrandomized pilot trial showed that hepatitis C virus (HCV) patients with genotype 2/3 and rapid virological response (RVR) had a 90% sustained virological response (SVR) rate after 14 weeks of treatment. We aimed to assess this concept in a randomized controlled trial. In the trial, 428 treatment-naive HCV RNA–positive patients with genotype 2 or 3 were enrolled. Patients with RVR were randomized to 14 (group A) or 24 (group B) weeks of treatment. Patients were treated with pegylated interferon α-2b (1.5 μg/kg) subcutaneously weekly and ribavirin (800-1400 mg) orally daily. The noninferiority margin was set to be 10% between the two groups with a one-sided 2.5% significance level. RVR was obtained in 302 of 428 (71%), and 298 of these were randomized to group A (n = 148) or group B (n = 150). In the intention-to-treat analysis, SVR rates were 120 of 148 (81.1%) in group A and 136 of 150 (90.7%) in group B (difference, 9.6%; 95% confidence interval, 1.7-17.7). Among patients with an HCV RNA test 24 weeks after the end of treatment, 120 of 139 (86.3%) patients in group A achieved SVR compared with 136 of 146 (93.2%) in group B (difference, 6.9%; 95% confidence interval, −0.1 to +13.9). Conclusion: We cannot formally claim that 14 weeks of treatment is noninferior to 24 weeks of treatment. However, the SVR rate after 14 weeks of treatment is high, and although longer treatment may give slightly better SVR, we believe economical savings and fewer side effects make it rational to treat patients with genotype 2 or 3 and RVR for only 14 weeks.

Background Patients with advanced HIV infection at the time of diagnosis and patients not responding to antiretroviral therapy are at risk of cytomegalovirus (CMV) disease. Earlier studies of patients with HIV infection have demonstrated that the diagnosis is often first made post-mortem. In recent years new molecular biological tests have become available for diagnosis of CMV disease. Although clinical evaluation of tests for diagnosis of CMV disease in HIV-infected individuals is suboptimal without autopsy, no results from such studies have been published. The aim of this study was to explore the diagnostic utility of CMV quantitative polymerase chain reaction (PCR) in plasma from HIV and CMV seropositive patients who died during the period 1991–2002 and in whom autopsy was performed. Methods Autopsy was performed in all cases, as part of routine evaluation of HIV-infected cases followed at Ullevaal University Hospital. Of 125 patients included, 53 had CMV disease, 37 of whom were first diagnosed at autopsy. CMV disease was diagnosed either by ophthalmoscopic findings typical of CMV retinitis, biopsy or autopsy. One or two plasma samples taken prior to the first diagnosis of CMV disease (alive or at autopsy) or death without CMV disease were analysed by CMV quantitative PCR. Sensitivity, specificity, positive and negative predictive values were calculated for different CMV viral load cut-offs and according to detection of viraemia in one versus two samples. Results Twenty-seven of 53 patients with CMV disease (51%) and 10 of 72 patients without CMV disease (14%) had detectable viraemia in at least one sample. Sensitivity and negative predictive value (NPV) of the test, maximised with a cut-off at the test's limit of detection of CMV viraemia (400 copies/mL), were 47% and 70%, respectively. With cut-off at 10 000 copies/mL, specificity and positive predictive value (PPV) were 100%. With a requirement for CMV viraemia in two samples, specificity and PPV were 100% in patients with CMV viraemia above the limit of detection. Conclusion Our results indicate that quantitative CMV PCR is best used to rule in, rather than to rule out CMV disease in HIV-infected individuals at high risk.

Purpose: To explore the relations between insulin resistance, plasma lactate, and mitochondrial (mt) DNA alterations in skeletal muscle in HIV-infected patients treated with nucleoside reverse transcriptase inhibitors (HIV+NRTI+). Method: Insulin resistance was estimated using the homeostatic model assessment (HOMA-IR). Mitochondrial dysfunction was determined by plasma lactate at rest and after subanaerobic exercise, mitochondrial/nuclear DNA (mt/nDNA) ratio, and mtDNA deletions in skeletal muscle. Results: HIV+NRTI+ patients (n = 27) had higher levels of HOMA-IR, higher lactate at rest as well as after exercise, and more frequent mtDNA deletions and decreased mt/nDNA ratios compared with controls (n = 15). Only in HIV+NRTI+ patients, HOMA-IR correlated with resting lactate (r = 0.5, p = .02) and probably also lactate 3, 5, and 8 minutes after exercise (r = 0.4; p = .075, p = .048, and p = .056, respectively). In contrast, neither HOMA-IR nor the lactate levels correlated with mt/nDNA ratio and mtDNA deletions in skeletal muscle in HIV+NRTI+ patients (r .6), whereas resting lactate correlated with mt/nDNA ratio in HIV seronegative controls (r = –0.7, p = .02). Conclusion: In HIV+NRTI+ patients, both resting and postexercise levels of lactate were related to insulin resistance rather than mtDNA alterations in skeletal muscle. Key words: HIV-1 infection, insulin resistance, lactate, mitochondrial dysfunction

Background Bloodstream infection is a common cause of hospitalization, morbidity and death in children. The impact of antimicrobial resistance and HIV infection on outcome is not firmly established. Methods We assessed the incidence of bloodstream infection and risk factors for fatal outcome in a prospective cohort study of 1828 consecutive admissions of children aged zero to seven years with signs of systemic infection. Blood was obtained for culture, malaria microscopy, HIV antibody test and, when necessary, HIV PCR. We recorded data on clinical features, underlying diseases, antimicrobial drug use and patients' outcome. Results The incidence of laboratory-confirmed bloodstream infection was 13.9% (255/1828) of admissions, despite two thirds of the study population having received antimicrobial therapy prior to blood culture. The most frequent isolates were klebsiella, salmonellae, Escherichia coli, enterococci and Staphylococcus aureus. Furthermore, 21.6% had malaria and 16.8% HIV infection. One third (34.9%) of the children with laboratory-confirmed bloodstream infection died. The mortality rate from Gram-negative bloodstream infection (43.5%) was more than double that of malaria (20.2%) and Gram-positive bloodstream infection (16.7%). Significant risk factors for death by logistic regression modeling were inappropriate treatment due to antimicrobial resistance, HIV infection, other underlying infectious diseases, malnutrition and bloodstream infection caused by Enterobacteriaceae, other Gram-negatives and candida. Conclusion Bloodstream infection was less common than malaria, but caused more deaths. The frequent use of antimicrobials prior to blood culture may have hampered the detection of organisms susceptible to commonly used antimicrobials, including pneumococci, and thus the study probably underestimates the incidence of bloodstream infection. The finding that antimicrobial resistance, HIV-infection and malnutrition predict fatal outcome calls for renewed efforts to curb the further emergence of resistance, improve HIV care and nutrition for children.

To monitor the evolving molecular epidemiology and genetic diversity of HIV in a country where many distinct strains cocirculate, we performed genetic analyses on sequences from 75 HIV-1-infected Cameroonians: 74 were group M and 1 was group O. Of the group M sequences, 74 were classified into the following env gp41 subtypes or recombinant forms: CRF02 (n = 54), CRF09 (n = 2), CRF13 (n = 2), A (n = 5), CRF11 (n = 4), CRF06 (n = 1), G (n = 2), F2 (n = 2), and E (n = 1, CRF01), and 1 was a JG recombinant. Comparison of phylogenies for 70 matched gp41 and protease sequences showed inconsistent classifications for 18 (26%) strains. Our data show that recombination is rampant in Cameroon with recombinant viruses continuing to recombine, adding to the complexity of circulating HIV strains. This expanding genetic diversity raises public health concerns for the ability of diagnostic assays to detect these unique HIV mosaic variants and for the development of broadly effective HIV vaccines.

The aim of this study was to evaluate the prognostic value of changes in CD4 counts and human immunodeficiency virus (HIV) RNA following 6 months of highly active antiretroviral therapy (HAART) in chronic HIV-1 infection. 148 treatment-naive patients treated with 2 nucleoside analogue reverse transcriptase inhibitors (NRTIs) + at least 1 protease inhibitor or non-NRTI for at least 180 d were included. Mean follow-up time after 6 months on HAART was 758 d. The patients were divided into 2 groups based on the increase in CD4 count (deltaCD4) from therapy initiation: groups A (n = 37, deltaCD40.052 x 10(9)/l) and B (n = 111, deltaCD4or = 0.052 x 10(9)/l). Patients were also stratified according to achievement of HIV RNA400 copies/ml (n = 122) oror = 400 copies/ml (n = 26). Endpoints were the occurrence of subsequent HIV-related disease (CDC category B or C) or death after 6 months on HAART. Subjects in group A had an increased risk of HIV-related disease compared with group B when adjusted for CD4 count at initiation of therapy [adjusted risk ratio (RR) 2.62, 95% confidence interval (95% CI) 1.07-6.40]. Viral loador = copies/ml versus reaching viral suppression400 copies/ml was associated with an increased risk of HIV-related disease only in patients with deltaCD40.052 x 10(9)/l (RR 4.20, 95% CI 1.05-16.9). Thus, this study indicates that patients with no or a small increase in CD4 counts after 6 months of HAART and low CD4 levels at initiation of therapy have an increased risk of HIV-related disease.

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.

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.

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.

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.

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.