Additional file 1: of Exploring neurodevelopmental outcome measures used in children with cerebral malaria: the perspectives of caregivers and health workers in Malawi

Topic guide for Interviews for health professionals. (DOCX 12 kb

Recognition of severity of illness.

<p>Recognition of severity of illness.</p

Summary of proven and probable ABM paediatric and adult cases interviewed by outcome and HIV status (where known).

<p>Summary of proven and probable ABM paediatric and adult cases interviewed by outcome and HIV status (where known).</p

Delays in action: need to validate severity and social position.

<p>Delays in action: need to validate severity and social position.</p

Subject population virological and phylogenetic characteristics.

a,b<p>VL, viral load; HIV-1 RNA (log₁₀ copies/ml).</p>c<p>CSF/blood VL ratio.</p>d<p>The percent of CSF <i>env</i> sequences that were compartmentalized (comp.) for each subject. CSF <i>env</i> sequences were considered compartmentalized when ≥4 sequences were found within the same clade and bootstrap values ≥40 were observed.</p>e<p><i>P</i> values used to measure genetic compartmentalization between the blood plasma and CSF HIV-1 populations were obtained using the Slatkin-Maddison test for gene flow between populations <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003094#ppat.1003094-Slatkin1" target="_blank">[31]</a>. A <i>P</i> value<0.05 indicated statistically significant genetic compartmentalization.</p>f<p><i>P</i> value<0.05 was obtained for subject 3040, but visual assessment of the neighbor-joining tree structure indicated the presence of an additional HIV-1 population within the blood plasma that was largely absent from the CSF.</p>g<p><i>P</i> value<0.05 was obtained for subject 4013, but visual assessment of neighbor-joining tree structure indicated significant compartmentalization was not present.</p>h<p>HIV-1 population characteristics in the CSF compartment (compart). Eq, equilibrated blood plasma and CSF populations; Inter (Intermediate), a minor subpopulation of the CSF was compartmentalized; Comp, significant compartmentalization in the CSF; Amp, clonal amplification of ≥3 variants detected in the CSF; N/A, not applicable when the CSF viral load was too low to obtain enough CSF <i>env</i> sequences.</p>i<p>The time to most recent common ancestor (TMRCA) analyzed by Bayesian Evolutionary Analysis by Sampling Trees (BEAST) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003094#ppat.1003094-Drummond1" target="_blank">[33]</a>.</p>j<p>For patient 4004, ≥3 transmitted variants observed: the first variant evolved at 52 months; the second and third variants evolved at 47 months.</p>k<p>Number of variants transmitted.</p>l<p>TMRCA for compartmentalized (Comp) population for intermediate and compartmentalized subjects. Equilibrated subjects were not analyzed (N/A). When recombination occurred between transmitted variants, BEAST was unable to date TMRCA (*).</p

Central Nervous System Compartmentalization of HIV-1 Subtype C Variants Early and Late in Infection in Young Children

<div><p>HIV-1 subtype B replication in the CNS can occur in CD4+ T cells or macrophages/microglia in adults. However, little is known about CNS infection in children or the ability of subtype C HIV-1 to evolve macrophage-tropic variants. In this study, we examined HIV-1 variants in ART-naïve children aged three years or younger to determine viral genotypes and phenotypes associated with HIV-1 subtype C pediatric CNS infection. We examined HIV-1 subtype C populations in blood and CSF of 43 Malawian children with neurodevelopmental delay or acute neurological symptoms. Using single genome amplification (SGA) and phylogenetic analysis of the full-length <em>env</em> gene, we defined four states: equilibrated virus in blood and CSF (n = 20, 47%), intermediate compartmentalization (n = 11, 25%), and two distinct types of compartmentalized CSF virus (n = 12, 28%). Older age and a higher CSF/blood viral load ratio were associated with compartmentalization, consistent with independent replication in the CNS. Cell tropism was assessed using pseudotyped reporter viruses to enter a cell line on which CD4 and CCR5 receptor expression can be differentially induced. In a subset of compartmentalized cases (n = 2, 17%), the CNS virus was able to infect cells with low CD4 surface expression, a hallmark of macrophage-tropic viruses, and intermediate compartmentalization early was associated with an intermediate CD4 entry phenotype. Transmission of multiple variants was observed for 5 children; in several cases, one variant was sequestered within the CNS, consistent with early stochastic colonization of the CNS by virus. Thus we hypothesize two pathways to compartmentalization: early stochastic sequestration in the CNS of one of multiple variants transmitted from mother to child, and emergence of compartmentalized variants later in infection, on average at age 13.5 months, and becoming fully apparent in the CSF by age 18 months. Overall, compartmentalized viral replication in the CNS occurred in half of children by year three.</p> </div

Compartmentalized HIV-1 CSF populations in children can have a low CD4 entry phenotype.

<p>Single-cycle infection of HIV-1 Env-pseudotyped reporter viruses on CD4^lowCCR5^high 293-Affinofile cells <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003094#ppat.1003094-Johnston1" target="_blank">[34]</a>. Receptor expression was measured as follows: CD4^low = 1,702 receptors/cell, CD4^high = 63,387 receptors/cell, CCR5^high = 26,117 receptors/cell. The data are averaged from triplicate wells for each of 2 to 3 env clones that were generated per indicated amplicon. Amplicons were selected for cloning to represent different portions of the phylogenetic tree. Panels show infection results for: (a) equilibrated subjects; (b) intermediate; and (c) compartmentalized subjects. Selected HIV-1 <i>env</i> sequences are indicated by a black square followed by the <i>env</i> name on neighbor joining trees for several subjects (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003094#ppat-1003094-g001" target="_blank">Figure 1</a>).</p

Older age and a higher CSF/blood viral load ratio are strong determining factors for compartmentalization.

<p>Relationships between clinical characteristics and genetic compartmentalization were assessed using the Mann-Whitney test in GraphPad Prism 4. Horizontal bars represent median values. A <i>P</i> value<0.05 was considered statistically significant. (a) Comparisons between age of the subjects and compartmentalization. (b) Comparisons between the CSF/blood viral load ratio of the subjects and compartmentalization.</p

Evidence of multiple transmission events during pediatric HIV-1 subtype C infection.

<p>Phylogenetic and sequence analysis of plasma and CSF HIV-1 populations for subject 3002. (a) Neighbor-joining tree. Sequences from the CSF are labeled with solid blue circles, and plasma sequences (PL) are labeled with solid red triangles. Bootstrap values ≥40 are indicated (<b>*</b>) at the appropriate nodes. Genetic distance is scaled at the bottom of the figure (0.001) and indicates the number of nucleotide substitutions per site between <i>env</i> sequences. The subject's age is noted, as well as the overall TMRCA and the TMRCA of the two transmitted viruses. The CNS sequestered population is represented by an open black circle. (b) Highlighter plot of aligned <i>env</i> plasma and CSF sequences, generated at <a href="http://www.hiv.lanl.gov" target="_blank">www.hiv.lanl.gov</a>. The HXB2 nucleotide position number is indicated on the <i>x</i> axis, and the sequence identifier is indicated on the <i>y</i> axis. Nucleotide changes are indicated by the following ticks on the highlighter plot: A, green; T, red; G, orange; and C, blue. The two transmitted populations are separated by a heavy black line.</p

Neurologic Manifestations Associated with an Outbreak of Typhoid Fever, Malawi - Mozambique, 2009: An Epidemiologic Investigation

<div><p>Background</p><p>The bacterium <i>Salmonella enterica</i> serovar Typhi causes typhoid fever, which is typically associated with fever and abdominal pain. An outbreak of typhoid fever in Malawi-Mozambique in 2009 was notable for a high proportion of neurologic illness.</p><p>Objective</p><p>Describe neurologic features complicating typhoid fever during an outbreak in Malawi-Mozambique</p><p>Methods</p><p>Persons meeting a clinical case definition were identified through surveillance, with laboratory confirmation of typhoid by antibody testing or blood/stool culture. We gathered demographic and clinical information, examined patients, and evaluated a subset of patients 11 months after onset. A sample of persons with and without neurologic signs was tested for vitamin B6 and B12 levels and urinary thiocyanate.</p><p>Results</p><p>Between March – November 2009, 303 cases of typhoid fever were identified. Forty (13%) persons had objective neurologic findings, including 14 confirmed by culture/serology; 27 (68%) were hospitalized, and 5 (13%) died. Seventeen (43%) had a constellation of upper motor neuron findings, including hyperreflexia, spasticity, or sustained ankle clonus. Other neurologic features included ataxia (22, 55%), parkinsonism (8, 20%), and tremors (4, 10%). Brain MRI of 3 (ages 5, 7, and 18 years) demonstrated cerebral atrophy but no other abnormalities. Of 13 patients re-evaluated 11 months later, 11 recovered completely, and 2 had persistent hyperreflexia and ataxia. Vitamin B6 levels were markedly low in typhoid fever patients both with and without neurologic signs.</p><p>Conclusions</p><p>Neurologic signs may complicate typhoid fever, and the diagnosis should be considered in persons with acute febrile neurologic illness in endemic areas.</p></div