Phenotypic consequences of LYS4 gene disruption in Candida albicans.

A BLAST search of the Candida Genome Database with the Saccharomyces cerevisiae LYS4 sequence known to encode homoaconitase (HA) revealed ORFs 19.3846 and 19.11327. Both alleles of the LYS4 gene were sequentially disrupted in Candida albicans BWP17 cells using PCR-based methodology. The null lys4Δ mutant exhibited lysine auxotrophy in minimal medium but was able to grow in the presence of l-Lys and α-aminoadipate, an intermediate of the α-aminoadipate pathway, at millimolar concentrations. The presence of d-Lys and pipecolic acid did not trigger lys4Δ growth. The C. albicans lys4Δ mutant cells demonstrated diminished germination ability. However, their virulence in vivo in a murine model of disseminated neonatal candidiasis appeared identical to that of the wild-type strain. Moreover, there was no statistically significant difference in fungal burden of infected tissues between the strains.

The seroepidemiology of Haemophilus influenzae type b prior to introduction of an immunization programme in Kathmandu, Nepal.

Haemophilus influenzae type b (Hib) is now recognized as an important pathogen in Asia. To evaluate disease susceptibility, and as a marker of Hib transmission before routine immunization was introduced in Kathmandu, 71 participants aged 7 months-77 years were recruited and 15 cord blood samples were collected for analysis of anti-polyribosylribitol phosphate antibody levels by enzyme-linked immunosorbent assay. Only 20% of children under 5 years old had levels considered protective (>0.15 µg/ml), rising to 83% of 15-54 year-olds. Prior to introduction of Hib vaccine in Kathmandu, the majority of young children were susceptible to disease.

Prevalence and genetic analysis of phenotypically Vi- negative Salmonella typhi isolates in children from Kathmandu, Nepal.

The Vi capsular polysaccharide (ViPS) protects Salmonella enterica subspecies enterica serotype Typhi (S.Typhi) in vivo by multiple mechanisms. Recent microbiological reports from typhoid endemic countries suggest that acapsulate S.Typhi may occur in nature and contribute to clinical typhoid fever that is indistinguishable from disease caused by capsulate strains. The prevalence and genetic basis of ViPS-negative S.Typhi isolates in children from Kathmandu, Nepal, were tested in 68 isolates. Although 5.9% of isolates tested negative for capsular expression by slide agglutination tests, a novel multiplex PCR assay and individual PCR analyses demonstrated the presence of all 14 genes responsible for the synthesis, transportation and regulation of the ViPS. These data suggest that phenotypically acapsulate S.Typhi may not have a genetic basis for the same.

Chorea and developmental regression associated with human herpes virus-6 encephalitis.

We report a 14-month old child with multiple episodes of febrile status epilepticus, followed by chorea and developmental regression, caused by human herpes virus-6 encephalitis. Chorea has been described as a complication of relapsing herpes simplex virus I infection, but not as a manifestation of human herpes virus-6 infection. It is uncertain whether the chorea was an autoimmune phenomenon or a direct effect of the virus. The child was treated with levetiracetam, intravenous immunoglobulin, and foscarnet. The seizures and chorea resolved with treatment, but developmental regression, with loss of language skills, persisted 6 months after the illness. This child illustrates a new clinical presentation of human herpes virus-6 encephalitis, adds to the spectrum of disorders caused by this virus, and strengthens the case for routine identification of specific viral agents in all cases of childhood viral infections with central nervous system symptoms to determine optimal treatment and prognosis.

High-throughput bacterial SNP typing identifies distinct clusters of Salmonella Typhi causing typhoid in Nepalese children.

BACKGROUND: Salmonella Typhi (S. Typhi) causes typhoid fever, which remains an important public health issue in many developing countries. Kathmandu, the capital of Nepal, is an area of high incidence and the pediatric population appears to be at high risk of exposure and infection. METHODS: We recently defined the population structure of S. Typhi, using new sequencing technologies to identify nearly 2,000 single nucleotide polymorphisms (SNPs) that can be used as unequivocal phylogenetic markers. Here we have used the GoldenGate (Illumina) platform to simultaneously type 1,500 of these SNPs in 62 S. Typhi isolates causing severe typhoid in children admitted to Patan Hospital in Kathmandu. RESULTS: Eight distinct S. Typhi haplotypes were identified during the 20-month study period, with 68% of isolates belonging to a subclone of the previously defined H58 S. Typhi. This subclone was closely associated with resistance to nalidixic acid, with all isolates from this group demonstrating a resistant phenotype and harbouring the same resistance-associated SNP in GyrA (Phe83). A secondary clone, comprising 19% of isolates, was observed only during the second half of the study. CONCLUSIONS: Our data demonstrate the utility of SNP typing for monitoring bacterial populations over a defined period in a single endemic setting. We provide evidence for genotype introduction and define a nalidixic acid resistant subclone of S. Typhi, which appears to be the dominant cause of severe pediatric typhoid in Kathmandu during the study period.

Appearance of peripheral blood plasma cells and memory B cells in a primary and secondary immune response in humans.

In humans, the kinetics of the appearance of memory B cells and plasma cells during primary immunization are not well defined. In this study, we assessed the primary B-cell response of rabies-antigen naive volunteers during a 3-dose course of rabies vaccine compared with the B-cell response to a booster dose of rabies vaccine given to previously immunized volunteers. After a single dose of vaccine, in the naive group plasma and memory B cells appeared later (peak at day 10) than in the primed group (peak at day 7) and were at lower frequency. The most rapid responses (day 4) were detected after a third immunization in the naive group. This is the first study to document the detailed kinetics of the plasma cell and memory B-cell responses to immunization in adult humans and to demonstrate differences in the responses that relate to the preexisting immune status of the persons.

Kinetics of the natural, humoral immune response to Salmonella enterica serovar Typhi in Kathmandu, Nepal.

Typhoid fever is a major public health problem in developing countries, conservatively estimated to occur in 17 million cases and be responsible for 200,000 deaths annually. We investigated the acquisition of natural immunity to Salmonella enterica serovar Typhi in a region where typhoid is endemic by testing sera from an age-stratified sample of 210 healthy participants in Kathmandu, Nepal, for bactericidal activity toward S. Typhi and for anti-Vi capsular polysaccharide antibodies. Bactericidal titers in children were significantly lower than those in newborns and adults (P < 0.0001). Anti-S. Typhi bactericidal geometric mean titers were age dependent, increasing 10-fold during childhood. Anti-Vi polysaccharide antibody geometric mean concentrations were also lower in children than in adults. Data presented here indicate the possibility of a relationship between low levels of bactericidal activity toward S. Typhi in serum and susceptibility to disease, as observed for other polysaccharide-encapsulated bacteria. Bactericidal antibody may be a marker of protective immunity against S. Typhi.

Biliary cirrhosis in a child with inherited interleukin-12 deficiency.

Interleukin-12 (IL-12) is a key cytokine in the defense against intracellular bacteria notably Mycobacteria and Salmonella species. We report a case of disseminated mycobacterial infection, following BCG vaccination, in a child who later developed tuberculosis. Functional tests and a novel diagnostic polymerase chain reaction (PCR) assay, revealed a loss-of-function deletion in the IL12 gene. Analysis of samples from the parents and siblings of the patient indicated an autosomal recessive inheritance pattern with varying degrees of phenotypic expression in identical genotypes. Interferon-gamma (IFN-gamma) therapy was associated with marked clinical improvement. Biliary cirrhosis, a hitherto unreported complication of IL-12 deficiency, developed later and required liver transplantation. A defect in the IL-12-IFN-gamma pathway should be suspected in patients presenting with multiple, repeated or persistent infection with intracellular bacteria. The diagnostic work-up and the immuno-genetic assay described here can aid in the quick and reliable diagnosis of IL-12 deficiency resulting from genetic defects and its subsequent management.