Mona Holberg-Petersen

Antimicrobial resistance predicts death in Tanzanian children with bloodstream infections: a prospective cohort study

BackgroundBloodstream 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.MethodsWe 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.ResultsThe 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.ConclusionBloodstream 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.

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.

Virological efficacy and emergence of drug resistance in adults on antiretroviral treatment in rural Tanzania

BackgroundVirological 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.MethodsHaydom 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.ResultsVirological 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 <400 copies/mL, was observed in 187 patients (88.2%). Overall, prevalence of ≥1 clinically significant resistance mutation was 3.9, 8.4, 16.7 and 12.5% in patients receiving ART for 1, 2, 3 and 4 years, respectively. Among those successfully genotyped, the most frequent mutations were M184I/V (64%), conferring resistance to lamivudine, and K103N (27%), Y181C (27%) and G190A (27%), conferring resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs), whereas 23% had thymidine analogue mutations (TAMs), associated with cross-resistance to all nucleoside reverse transcriptase inhibitors (NRTIs). Dual-class resistance, i.e. resistance to both NRTIs and NNRTIs, was found in 64%.ConclusionVirological suppression rates were good up to 4 years after initiating ART in a rural Tanzanian hospital. However, drug resistance increased with time, and dual-class resistance was common, raising concerns about exhaustion of future antiretroviral drug options. This study might provide a useful forecast of drug resistance and demand for second-line antiretroviral drugs in rural Africa in the coming years.

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.

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

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.

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.

Drug resistance is widespread among children who receive long-term antiretroviral treatment at a rural Tanzanian hospital

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 (≤40 copies/mL), whereas 11 children had clinically relevant resistance mutations in the reverse transcriptase gene. The most frequent mutations were M184V (nu200a=u200a11), conferring resistance to lamivudine and emtricitabine, and Y181C (nu200a=u200a4), G190A/S (nu200a=u200a4) and K103N (nu200a=u200a4), conferring resistance to NNRTIs. Of concern, three children had thymidine analogue mutations, associated with cross-resistance to all nucleoside reverse transcriptase inhibitors. Despite widespread resistance, however, only one child experienced a new WHO stage 4 event and none had a CD4 cell count of <200 cells/mm3. Conclusions Among children on long-term ART in rural Tanzania, >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.

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.

HIV type-1 drug resistance testing on dried blood spots is feasible and reliable in patients who fail antiretroviral therapy in rural Tanzania.

BACKGROUNDnHIV 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.nnnMETHODSnA 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.nnnRESULTSnOverall, 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.nnnCONCLUSIONSnGenotyping 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.

Distinct Mechanisms for Mitochondrial DNA Loss in T and B Lymphocytes from HIV-Infected Patients Exposed to Nucleoside Reverse-Transcriptase Inhibitors and Those Naive to Antiretroviral Treatment

OBJECTIVEnMitochondrial 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.nnnMETHODSnCD4(+) 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.nnnRESULTSnmtDNA 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).nnnCONCLUSIONSnIn 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.