Ten years of surveillance for invasive Streptococcus pneumoniae during the era of antiretroviral scale-up and cotrimoxazole prophylaxis in Malawi

Objective

To document trends in invasive pneumococcal disease (IPD) in a central hospital in Malawi during the period of national scale-up of antiretroviral therapy (ART) and cotrimoxazole prophylaxis.

Methods

Between 1 January 2000 and 31 December 2009 almost 100,000 blood cultures and 40,000 cerebrospinal fluid (CSF) cultures were obtained from adults and children admitted to the Queen Elizabeth Central Hospital, Blantyre, Malawi with suspected severe bacterial infection.

Results

4,445 pneumococcal isolates were obtained over the 10 year period. 1,837 were from children: 885 (19.9%) from blood and 952 (21.4%) from CSF. 2,608 were from adults: 1,813 (40.8%) from blood and 795 (17.9%) from CSF. At the start of the surveillance period cotrimoxazole resistance was 73.8% and at the end was 92.6%. Multidrug resistance (MDR) was present in almost one third of isolates and was constant over time. Free ART was introduced in Malawi in 2004. From 2005 onwards there was a decline in invasive pneumococcal infections with a negative correlation between ART scale-up and the decline in IPD (Pearson''s correlation r = −0.91; p<0.001).

Conclusion

During 2004–2009, national ART scale-up in Malawi was associated with a downward trend in IPD at QECH. The introduction of cotrimoxazole prophylaxis in HIV-infected groups has not coincided with a further increase in pneumococcal cotrimoxazole or multidrug resistance. These data highlight the importance of surveillance for high disease burden infections such as IPD in the region, which will be vital for monitoring pneumococcal conjugate vaccine introduction into national immunisation programmes.     

Neuron navigator 3a regulates liver organogenesis during zebrafish embryogenesis

Endodermal organogenesis requires a precise orchestration of cell fate specification and cell movements, collectively coordinating organ size and shape. In Caenorhabditis elegans, uncoordinated-53 (unc-53) encodes a neural guidance molecule that directs axonal growth. One of the vertebrate homologs of unc-53 is neuron navigator 3 (Nav3). Here, we identified a novel vertebrate neuron navigator 3 isoform in zebrafish, nav3a, and we provide genetic evidence in loss- and gain-of-function experiments showing its functional role in endodermal organogenesis during zebrafish embryogenesis. In zebrafish embryos, nav3a expression was initiated at 22 hpf in the gut endoderm and at 40 hpf expanded to the newly formed liver bud. Endodermal nav3a expression was controlled by Wnt2bb signaling and was independent of FGF and BMP signaling. Morpholino-mediated knockdown of nav3a resulted in a significantly reduced liver size, and impaired development of pancreas and swim bladder. In vivo time-lapse imaging of liver development in nav3a morphants revealed a failure of hepatoblast movement out from the gut endoderm during the liver budding stage, with hepatoblasts being retained in the intestinal endoderm. In hepatocytes in vitro, nav3a acts as a positive modulator of actin assembly in lamellipodia and filipodia extensions, allowing cellular movement. Knockdown of nav3a in vitro impeded hepatocyte movement. Endodermal-specific overexpression of nav3a in vivo resulted in additional ectopic endodermal budding beyond the normal liver and pancreatic budding sites. We conclude that nav3a is required for directing endodermal organogenesis involving coordination of endodermal cell behavior.     

Safety and Effectiveness of Combination Antiretroviral Therapy during the First Year of Treatment in HIV-1 Infected Rwandan Children: A Prospective Study

Background

With increased availability of paediatric combination antiretroviral therapy (cART) in resource limited settings, cART outcomes and factors associated with outcomes should be assessed.

Methods

HIV-infected children <15 years of age, initiating cART in Kigali, Rwanda, were followed for 18 months. Prospective clinical and laboratory assessments included weight-for-age (WAZ) and height-for-age (HAZ) z-scores, complete blood cell count, liver transaminases, creatinine and lipid profiles, CD4 T-cell count/percent, and plasma HIV-1 RNA concentration. Clinical success was defined as WAZ and WAZ >−2, immunological success as CD4 cells ≥500/mm³ and ≥25% for respectively children over 5 years and under 5 years, and virological success as a plasma HIV-1 RNA concentration <40 copies/mL.

Results

Between March 2008 and December 2009, 123 HIV-infected children were included. The median (interquartile (IQR) age at cART initiation was 7.4 (3.2, 11.5) years; 40% were <5 years and 54% were female. Mean (95% confidence interval (95%CI)) HAZ and WAZ at baseline were −2.01 (−2.23, −1.80) and −1.73 (−1.95, −1.50) respectively and rose to −1.75 (−1.98, −1.51) and −1.17 (−1.38, −0.96) after 12 months of cART. The median (IQR) CD4 T-cell values for children <5 and ≥5 years of age were 20% (13, 28) and 337 (236, 484) cells/mm³respectively, and increased to 36% (28, 41) and 620 (375, 880) cells/mm³. After 12 months of cART, 24% of children had a detectable viral load, including 16% with virological failure (HIV-RNA>1000 c/mL). Older age at cART initiation, poor adherence, and exposure to antiretrovirals around birth were associated with virological failure. A third (33%) of children had side effects (by self-report or clinical assessment), but only 9% experienced a severe side effect requiring a cART regimen change.

Conclusions

cART in Rwandan HIV-infected children was successful but success might be improved further by initiating cART as early as possible, optimizing adherence and optimizing management of side effects.     

Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation

The Na,K-ATPase is an ion-translocating transmembrane protein that actively maintains the electrochemical gradients for Na⁺ and K⁺ across the plasma membrane. The functional protein is a heterodimer comprising a catalytic α-subunit (four isoforms) and an ancillary β-subunit (three isoforms). Mutations in the α₂-subunit have recently been implicated in familial hemiplegic migraine type 2, but almost no thorough studies of the functional consequences of these mutations have been provided. We investigated the functional properties of the mutations L764P and W887R in the human Na,K-ATPase α₂-subunit upon heterologous expression in Xenopus oocytes. No Na,K-ATPase-specific pump currents could be detected in cells expressing these mutants. The binding of radiolabelled [³H]ouabain to intact cells suggested that this could be due to a lack of plasma membrane expression. However, plasma membrane isolation showed that the mutated pumps are well expressed at the plasma membrane. ⁸⁶Rb⁺-flux and ATPase activity measurements demonstrated that the mutants are inactive. Therefore, the primary disease-causing mechanism is loss-of-function of the Na,K-ATPase α₂-isoform.     

Impaired routing of wild type FXYD2 after oligomerisation with FXYD2-G41R might explain the dominant nature of renal hypomagnesemia

Autosomal dominant renal hypomagnesemia, associated with hypocalciurea, has been linked to a G to A mutation at nucleotide position 121 in the FXYD2 gene, resulting in the substitution of Gly with Arg at residue 41 of the protein. FXYD2, also called the Na,K-ATPase gamma-subunit, binds to Na,K-ATPase and influences its cation affinities. In this paper, we provide evidence for the molecular mechanism underlying the dominant character of the disorder. Co-immunoprecipitation experiments using tagged FXYD2 proteins demonstrated that wild type FXYD2 proteins oligomerise. Moreover, FXYD2-G41R also shows oligomerisation with itself and with the wild type protein. In the case of FXYD2-G41R, however, formation of homo-oligomers was prevented by addition of DTT or introduction of the C52A mutation. Finally, we demonstrated that artificial glycosylation of the wild type FXYD2 is reduced when co-expressed with FXYD2-G41R. These data indicate that binding of FXYD2-G41R to wild type FXYD2 subunit might abrogate the routing of wild type FXYD2 to the plasma membrane thus causing the dominant nature of this mutation.     

Clinical course of homozygous familial hypercholesterolemia during childhood: report on 4 unrelated patients with homozygous or compound heterozygous mutations in theLDLR gene

Natural history of the disease in 4 unrelated Polish children with homozygous familial hypercholesterolemia (FH) is described. Their phenotypic homozygosity was established by identification of known LDLR gene mutations on both alleles, respectively: p.G592E & p.G592E in Patient 1; p.G592E & p.C667Y in Patient 2; p.S177L & p.R350X in Patient 3; and p.G592E & deletion in the promoter region, exons 1 and 2 in Patient 4. Heterozygosity of the mutations was revealed in all patients' mothers and fathers (obligatory heterozygotes) and in 1 out of 4 siblings studied. FH was diagnosed at the age of 4 months to 9 years by cholesterol screening among family members of patients with early cardiovascular disease episodes. At the time of FH detection, the children were asymptomatic. Only in 2, some skin eruptions were found. Antihyperlipidemic therapy was started, including a lipid-lowering diet, cholestyramine, and HMG-CoA inhibitors if necessary. No cardiovascular symptoms appeared during the observation up to the age of 18, 20, 19, and 17 years, respectively. An increase in external carotid artery diameter was found in a patient at the age of 9 years, and LDL-apheresis was introduced in his therapy. We conclude that the analysis of LDLR gene mutations in the studied FH children made it possible to identify 4 presymptomatic FH homozygotes and to introduce early appropriate treatment. Multicenter analysis of such persons would finally determine if the early lipid-lowering procedures can significantly reduce the risk of premature cardiovascular disease in homozygous FH.     

Rapid reversal of human intestinal ischemia-reperfusion induced damage by shedding of injured enterocytes and reepithelialisation

Background

Intestinal ischemia-reperfusion (IR) is a phenomenon related to physiological conditions (e.g. exercise, stress) and to pathophysiological events (e.g. acute mesenteric ischemia, aortic surgery). Although intestinal IR has been studied extensively in animals, results remain inconclusive and data on human intestinal IR are scarce. Therefore, an experimental harmless model for human intestinal IR was developed, enabling us to clarify the sequelae of human intestinal IR for the first time.

Methods and Findings

In 30 patients undergoing pancreatico-duodenectomy we took advantage of the fact that in this procedure a variable length of jejunum is removed. Isolated jejunum (5 cm) was subjected to 30 minutes ischemia followed by reperfusion. Intestinal Fatty Acid Binding Protein (I-FABP) arteriovenous concentration differences across the bowel segment were measured before and after ischemia to assess epithelial cell damage. Tissue sections were collected after ischemia and at 25, 60 and 120 minutes reperfusion and stained with H&E, and for I-FABP and the apoptosis marker M30. Bonferroni''s test was used to compare I-FABP differences. Mean (SEM) arteriovenous concentration gradients of I-FABP across the jejunum revealed rapidly developing epithelial cell damage. I-FABP release significantly increased from 290 (46) pg/ml before ischemia towards 3,997 (554) pg/ml immediately after ischemia (p<0.001) and declined gradually to 1,143 (237) pg/ml within 1 hour reperfusion (p<0.001). Directly after ischemia the intestinal epithelial lining was microscopically normal, while subepithelial spaces appeared at the villus tip. However, after 25 minutes reperfusion, enterocyte M30 immunostaining was observed at the villus tip accompanied by shedding of mature enterocytes into the lumen and loss of I-FABP staining. Interestingly, within 60 minutes reperfusion the epithelial barrier resealed, while debris of apoptotic, shedded epithelial cells was observed in the lumen. At the same time, M30 immunoreactivity was absent in intact epithelial lining.

Conclusions

This is the first human study to clarify intestinal IR induced cell damage and repair and its direct consequences. It reveals a unique, endogenous clearing mechanism for injured enterocytes: rapid detachment of damaged apoptotic enterocytes into the lumen. This process is followed by repair of the epithelial continuity within an hour, resulting in a normal epithelial lining.     

Late entry to HIV care limits the impact of anti-retroviral therapy in The Netherlands

Objective

To explain differences in survival in the first three years of combination anti-retroviral therapy (cART) between HIV treatment centres in the Netherlands.

Methodology/Principal Findings

We developed a mathematical simulation model, parameterised using data from the ATHENA cohort that describes patients entering care, being monitored and starting cART. Three scenarios were used to represent three treatment centres with widely varying mortality rates on cART that were differentiated by: (i) the distribution of CD4 counts of patients entering care; (ii) the age distribution of patients entering care; (iii) the average rate of monitoring the patients not on cART. At the level of the treatment centre, the fraction of Dutch MSM dying in the first three years of treatment ranged from 0% to 8%. The mathematical model captured the large variation in observed mortality between the three treatment centres. Manipulating the age-distribution of patients or the frequency of monitoring did not affect the model predictions. In contrast, when the same national average distribution of CD4 count at entry was used in all the scenarios, the variation in predicted mortality between all centres was diminished.

Conclusions/Significance

Patients entering care with low CD4 counts appears to be the main source of variation in the mortality rates between Dutch treatment centres. Recruiting HIV-infected individuals to care earlier could lead to substantial improvements in cART outcomes. For example, if patients were to present with at least 400 CD4 cells/mm³, as they do already in some centres, then our model predicts that the mortality in the first three years of cART could be reduced by approximately 20%.     

Intracellular Salmonella metabolism

Growth of Salmonella inside infected host cells is a key aspect of their ability to cause local enteritis or systemic disease. This growth depends on exploitation of host nutrients through a large Salmonella metabolism network with hundreds of metabolites and enzymes. Studies in cell culture infection models are unravelling more and more of the underlying molecular and cellular mechanisms but also show striking Salmonella metabolic plasticity depending on host cell line and experimental conditions. In vivo studies have revealed a qualitatively diverse, but quantitatively poor, host‐Salmonella nutritional interface, which on one side makes Salmonella fitness largely resilient against metabolic perturbations, but on the other side severely limits Salmonella biomass generation and growth rates. This review discusses goals and techniques for studying Salmonella intracellular metabolism, summarises main results and implications, and proposes key issues that could be addressed in future studies.     

The non-gastric H,K-ATPase is oligomycin-sensitive and can function as an H+,NH4(+)-ATPase

We used the baculovirus/Sf9 expression system to gain new information on the mechanistic properties of the rat non-gastric H,K-ATPase, an enzyme that is implicated in potassium homeostasis. The alpha2-subunit of this enzyme (HKalpha2) required a beta-subunit for ATPase activity thereby showing a clear preference for NaKbeta1 over NaKbeta3 and gastric HKbeta. NH4(+), K+, and Na+ maximally increased the activity of HKalpha2-NaKbeta1 to 24.0, 14.2, and 5.0 micromol P(i) x mg(-1) protein x h(-1), respectively. The enzyme was inhibited by relatively high concentrations of ouabain and SCH 28080, whereas it was potently inhibited by oligomycin. From the phosphorylation level in the presence of oligomycin and the maximal NH4(+)-stimulated ATPase activity, a turnover number of 20,000 min(-1) was determined. All three cations decreased the steady-state phosphorylation level and enhanced the dephosphorylation rate, disfavoring the hypothesis that Na+ can replace H+ as the activating cation. The potency with which vanadate inhibited the cation-activated enzyme decreased in the order K+ > NH4(+) > Na+, indicating that K+ is a stronger E2 promoter than NH4(+), whereas in the presence of Na+ the enzyme is in the E1 form. For K+ and NH4(+), the E2 to E1 conformational equilibrium correlated with their efficacy in the ATPase reaction, indicating that here the transition from E2 to E1 is rate-limiting. Conversely, the low maximal ATPase activity with Na+ is explained by a poor stimulatory effect on the dephosphorylation rate. These data show that NH4(+) can replace K+ with similar affinity but higher efficacy as an extracellular activating cation in rat nongastric H,K-ATPase.