Defective natural killer cell anti-viral capacity in paediatric HBV infection.

Natural killer (NK) cells exhibit dysregulated effector function in adult chronic hepatitis B virus (HBV) infection (CHB), which may contribute to virus persistence. The role of NK cells in children infected perinatally with HBV is less studied. Access to a unique cohort enabled the cross-sectional evaluation of NK cell frequency, phenotype and function in HBV-infected children relative to uninfected children. We observed a selective defect in NK cell interferon (IFN)-γ production, with conserved cytolytic function, mirroring the functional dichotomy observed in adult infection. Reduced expression of NKp30 on NK cells suggests a role of impaired NK-dendritic cell (DC) cellular interactions as a potential mechanism leading to reduced IFN-γ production. The finding that NK cells are already defective in paediatric CHB, albeit less extensively than in adult CHB, has potential implications for the timing of anti-viral therapy aiming to restore immune control.

Nosocomial measles cluster in Denmark following an imported case, December 2008-January 2009.

A cluster of six confirmed cases with identical measles virus genotype was reported in Denmark between December 2008 and January 2009. The findings highlight the importance of vaccination before travelling and adherence to the routine vaccination schedule.

Attaching and effacing Escherichia coli isolates from Danish children: clinical significance and microbiological characteristics.

This study describes the prevalence, clinical manifestations and microbiological characteristics of attaching and effacing Escherichia coli isolates, i.e., enteropathogenic E. coli (EPEC) belonging to the classical EPEC serotypes, non-EPEC attaching and effacing E. coli (A/EEC) and verocytotoxin-producing E. coli (VTEC), isolated in a case-control study of Danish children aged <5 years. Among 424 children with diarrhoea and 866 healthy controls, EPEC and VTEC were more prevalent in cases (2.4% and 2.6%, respectively) than in controls (0.7% and 0.7%, respectively). There was a high frequency of A/EEC isolates (n = 121), but these were equally prevalent in cases (11.3%) and controls (12.5%), and comprised a heterogeneous distribution of O:H serotypes. The intimin (eae) subtypes in A/EEC isolates showed an even distribution; the eae-gamma subtype predominated in classical EPEC cases. The virulence genes encoding the bundle-forming pilus (bfpA) and enteroaggregative heat-stable enterotoxin (astA) were rare among all isolates, and seemed to be of limited pathogenic importance in this population. Virulence characterisation of A/EEC isolates did not reveal any significant differences between cases and controls. Colonisation of children with A/EEC was associated with contact with sheep or goats (OR 2.2). The role of A/EEC, not being VTEC or belonging to the classical EPEC serotypes, requires further clarification, but serotyping is useful in discriminating between EPEC and A/EEC strains.

Excretion patterns of human metapneumovirus and respiratory syncytial virus among young children.

BACKGROUND: As respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause serious respiratory tract infections, the routes of transmission of these viruses are important to elucidate. We examined the modes of virus shedding and shedding duration of RSV and hMPV in young children. METHODS: From each child in a group of 44 children (37 RSV-positive, 6 hMPV-positive, and 1 co-infected child), aged between 0.5-38 months, hospitalised at Hvidovre Hospital, Copenhagen, Denmark, one nasopharyngeal aspirate (NPA), saliva, urine, and faeces sample were collected at inclusion and weekly in a three-week period. Sweat and blood samples were obtained at inclusion. The presence of RSV and hMPV RNA was detected using real-time RT-PCR. RESULTS: We detected RSV RNA in 28 saliva specimens, 5 stool samples, and 3 sweat samples. hMPV RNA was detected in one saliva specimen and two sweat samples. Four of the five children shedding RNA in faeces had diarrhoea and children shedding RNA in sweat were either less than five weeks of age or had a chronic lung disease. RSV and hMPV RNA was shed in nasal secretions for a median of 11.5 and 5.0 days respectively (p = 0.001). More than 75% of the family members of the infected children showed to have an upper respiratory tract infection when following up. CONCLUSION: Viral RNA was present in nasal secretions, saliva, sweat, and faeces, but whether or not the virions were infectious and constitute a potential mode of transmission remains to be shown in future studies.

The national neonatal screening programme for congenital toxoplasmosis in Denmark: results from the initial four years, 1999-2002.

AIMS: To describe the outcome of four years' nationwide neonatal screening for congenital toxoplasmosis in liveborn newborns. METHODS: Congenital toxoplasmosis was diagnosed if specific Toxoplasma gondii IgM antibodies were detected in eluate from the PKU Guthrie filter paper card from a child. Infants diagnosed with congenital toxoplasmosis were examined for intracranial and retinal lesions and treated for three months with sulphadiazine, pyrimethamine, and folinic acid continuously. RESULTS: Eluates from PKU-cards from 262 912 newborns were analysed. The birth prevalence of congenital toxoplasma infection was 2.1 per 10 000 liveborns. Congenital toxoplasmosis was suspected in 96 infants and confirmed in 55. Forty seven children were examined for intracranial and retinal lesions soon after birth; 12 had clinical signs at this first examination. Of these, 5 had intracranial calcifications, 2 had retinochoroidal lesions, 4 had intracranial calcifications and retinochoroidal lesions, and 1 had hydrocephalus, intracranial calcifications, and retinochoroidal lesions. Ninety four eyes were examined soon after birth; there were central retinochoroidal lesions in 9. Two children had macular lesion of both eyes, five had macular lesions of one eye. At 1 year of age, 10/68 eyes had central lesions, and at 3 years of age, 5/32 had central lesions. Thus new retinochoroidal lesions developed in three eyes in the observation period. CONCLUSIONS: Neonatal screening is feasible for diagnosing children with congenital toxoplasmosis at birth in low endemic areas. Retinochoroiditis with macular lesion was diagnosed in 9.6% of the eyes at birth and in 15.6% of the eyes examined at 3 years of age.

The C-terminal part of the surface-associated protein MopE of the methanotroph Methylococcus capsulatus (Bath) is secreted into the growth medium.

A protein with an apparent molecular mass of 46 kDa was detected as the major polypeptide in the culture medium of the biotechnologically important methanotrophic bacterium Methylococcus capsulatus (Bath). The protein cross-reacted with polyclonal antibodies raised against the outer-membrane-associated protein MopE. The antiserum was used to identify a positive clone from a lambda gt11 library. The nucleotide sequence determined for the clone demonstrated that MopE and the secreted protein are encoded by the same gene, and that the secreted protein represents an N-terminally truncated form of MopE. By using monospecific antibodies against MopE in immunogold electron microscopy, the protein was localized at the cell surface and cell periphery. The mopE gene was expressed in Escherichia coli. The MopE protein synthesized was found in the periplasmic space of E. coli. No protein with sequence similarity over the entire length of MopE was detected in the databases, but some sequence similarity to the copper-repressible CorA protein of the methanotroph Methylomicrobium albus (Berson and Lidstrom 1997) was observed for the C-terminal region of MopE.

[Hepatitis B screening of pregnant women and follow-up vaccination of infants with HBsAG positive mothers. A survey from the Hvidovre hospital in 1999]. / Hepatitis B-screening af gravide kvinder og opfølgende vaccination of børn af HBsAg-positive mødre. En undersøgelse fra Hvidovre Hospital 1999.

The aim of this study was to assess the implementation of the recommendations of the Danish National Health Service concerning screening of women from hepatitis B (HB) endemic areas and follow-up vaccination of infants of carrier mothers. We examined all birth notification (n = 836) at Hvidovre Hospital during the first three months of 1999. Among 188 women from HB endemic areas only 134 (71%) women had been screened for HBsAg before delivery. Six women were HBsAg positive one of whom was examined after the delivery. Four out of six infants (67%) received Aunativ and Engerix-B within 12 hours of birth, one infant received Aunativ and Engerix-B at four days of age, the infant born of the mother who was screened after delivery didn't receive either Aunativ or Engerix-B. Three out of the six infants (50%) had received all the scheduled Engerix-B vaccinations. Our results suggest it may be necessary to reconsider the current screening and vaccination procedures to prevent an increase in the number of children becoming chronic HB carriers in Denmark.

Atovaquone-proguanil versus chloroquine-proguanil for malaria prophylaxis in non-immune travellers: a randomised, double-blind study. Malarone International Study Team.

BACKGROUND: Chloroquine plus proguanil is widely used for malaria chemoprophylaxis despite low effectiveness in areas where multidrug-resistant malaria occurs. Studies have shown that atovaquone and proguanil hydrochloride is safe and effective for prevention of falciparum malaria in lifelong residents of malaria-endemic countries, but little is known about non-immune travellers. METHODS: In a double-blind equivalence trial, 1083 participants travelling to a malaria-endemic area were randomly assigned to two treatment groups: atovaquone-proguanil plus placebos for chloroquine and proguanil, or chloroquine, proguanil, and placebo for atovaquone-proguanil. Follow-up was by telephone 7 and 60 days after travel and at a clinic at 28 days. Serum samples were tested for antibodies to a malaria circumsporozoite protein. Blood and serum samples of participants with a potential malaria diagnosis were tested in a reference laboratory. FINDINGS: 7 days after travel, at least one adverse event was reported by 311 (61%) of 511 participants who received atovaquone-proguanil and 329 (64%) of 511 who received chloroquine-proguanil. People receiving atovaquone-proguanil had a lower frequency of treatment-related gastrointestinal adverse events (59 [12%] vs 100 [20%], p=0.001), and of treatment-related adverse events of moderate or severe intensity (37 [7%] vs 56 [11%], p=0.05). There were fewer treatment-related adverse events that caused prophylaxis to be discontinued in the atovaquone-proguanil group than in the chloroquine-proguanil group (one [0.2%] vs ten [2%], p=0.015). INTERPRETATION: Overall the two preparations were similarly tolerated. However, significantly fewer adverse gastrointestinal events were observed in the atovaquone-proguanil group in than in the chloroquine-proguanil group.

Anopheles arabiensis and An. funestus are equally important vectors of malaria in Matola coastal suburb of Maputo, southern Mozambique.

Transmission characteristics of malaria were studied in Matola, a coastal suburb of Maputo, the capital City, in southern Mozambique, from November 1994 to April 1996. The local climate alternates between cool dry season (May-October) and hot rainy season (November-April) with mean annual rainfall 650-850 mm. Saltmarsh and freshwater pools provide mosquito breeding sites in Matola. Malaria prevalence reached approximately 60% among people living nearest to the main breeding sites of the vectors. Plasmodium falciparum caused 97% of malaria cases, others being P. malariae and P. ovale. Potential malaria vector mosquitoes (Diptera: Culicidae) collected at Matola during daytime indoor-resting (n = 1021) and on human bait at night (n = 5893) comprised 12% Anopheles coustani Laveran (93% biting outdoors), 46% An. funestus Giles (68% biting indoors) and 42% An. gambiae Giles sensu lato (60% biting outdoors). All 215 specimens of An. gambiae s.l. identified genetically were An. arabiensis Patton. Anopheles funestus populations remained stable throughout the year, whereas densities of the An. gambiae complex fluctuated considerably, with An. arabiensis peaking during the rainy season. No concomitant rise in malaria incidence was observed. Human landing indices of An. funestus and An. arabiensis averaged 1.8 and 3.8 per man-night, respectively. Overall Plasmodium sporozoite rates were 2.42+/-1.24% in 2181 An. funestus and 1.11+/-1.25% in 1689 An. arabiensis dissected and examined microscopically. Mean daily survival rates were 0.79 for both vector species. Estimated infective bites/person/year were 15 An. funestus and 12 An. arabiensis. Biting rates were greatest at 2100-24.00 hours for An. funestus (68% endophagic) and 21.00-03.00 hours for An. arabiensis (40% endophagic). The entomological inoculation rate (EIR) declined sharply over very short distances (50% per 90m) away from breeding-sites of the vectors. Consequently, P. falciparum prevalence among Matola residents was halved 350 m within the town. Implications for the protective effectiveness of a 'cordon sanitaire' by residual house-spraying and/or the use of insecticide-treated bednets are discussed.

The mosquito transmission of malaria: the effects of atovaquone-proguanil (Malarone) and chloroquine.

Despite its recognized importance, the prevention of patients with malaria from continuing to infect mosquitoes after treatment is not always achieved in practice. An inevitable consequence of the prolonged life-span and relative metabolic stasis of the mature gametocytes of Plasmodium falciparum is that they are not cleared by most antimalarials, and few antimalarials block infection in the mosquito vector. Previous research on the constituents of Malarone, a new 'combined antimalarial', suggested that the active components, atovaquone and proguanil, might inhibit infectivity of gametocytes to mosquitoes. We contrast here the impact of atovaquone-proguanil and chloroquine on the transmission of P. falciparum and P. berghei. While chloroquine enhanced infectivity of P. falciparum, atovaquone-proguanil caused a significant reduction. Surprisingly, sporontocidal activity against the rodent parasite persisted long after the levels of the constituent drugs would have been expected to have fallen below effective plasma concentrations on the basis of the established pharmacokinetics of atovaquone and proguanil. The P. berghei model may thus have provided a sensitive bioassay, detecting drug(s) at levels below that normally found with the usual chemical assays.

Different molecular events result in low protein levels of mannan-binding lectin in populations from southeast Africa and South America.

Previous studies have shown that three point mutations in exon 1 and a particular promoter haplotype of the mannan-binding lectin (MBL) gene lead to a dramatic decrease in the serum concentration of MBL. In this study, MBL genotypes and serum concentrations were determined in unrelated individuals in a population from Mozambique (n = 154) and in two native Indian tribes from Argentina (i.e., the Chiriguanos (n = 43) and the Mapuches (n = 25)). In both populations, the MBL concentrations were low compared with those found in Eskimo, Asian, and European populations. In Africans, the low serum concentrations were due to a high allele frequency (0.24) of the codon 57 (C) variant, which resulted in a high frequency of individuals with MBL deficiency (0.06), and were also due to the effect of a relatively high frequency (0.13) of low-producing promoter haplotypes. The low concentrations in the South American populations were primarily due to an extremely high allele frequency of the codon 54 (B) variant in both the Chiriguanos (0.42) and the Mapuches (0.46), resulting in high frequencies of individuals with MBL deficiency (0.14 and 0.16, respectively). In the search for additional genetic variants, we found five new promoter mutations that might help to elucidate the evolution of the MBL gene. Taken together, the results of this study show that different molecular mechanisms are the basis for low MBL levels on the two continents.

The differing impact of chloroquine and pyrimethamine/sulfadoxine upon the infectivity of malaria species to the mosquito vector.

Using serum or infected blood from Danish volunteers and Plasmodium falciparum-infected Mozambican patients, respectively, the impact of curative doses of chloroquine and pyrimethamine/sulfadoxine upon infectivity of P. falciparum to Anopheles arabiensis and An. gambiae or of P. berghei to An. stephensi was studied. Both treatments cleared circulating P. falciparum gametocytes within 28 days. Before this clearance, chloroquine enhanced infectivity to An. arabiensis, whereas pyrimethamine/sulfadoxine decreased infectivity. Patients harboring chloroquine-resistant parasites as opposed to -sensitive ones were 4.4 times more likely to have gametocytes following treatment. In contrast, pyrimethamine/sulfadoxine-resistant parasites were 1.9 times less likely to produce gametocytes. In laboratory infections using replicated P. berghei or P. falciparum preparations, serum from chloroquine-treated, uninfected, nonimmune volunteers enhanced gametocyte infectivity with increasing efficiency for 21 days following treatment, whereas pyrimethamine/sulfadoxine significantly suppressed infectivity. The observed enhancement in infectivity induced by the use of chloroquine combined with increased gametocytemias in chloroquine-resistant strains may in part explain the rapid spread of chloroquine resistance in endemic populations.

The Matola malaria project: a temporal and spatial study of malaria transmission and disease in a suburban area of Maputo, Mozambique.

A temporal and spatial study of malaria transmission in a suburban area of Maputo, Mozambique with a mean population density of 2,737/km2 was made from December 1992 to June 1995. A steep but continuous gradient was observed in the Plasmodium falciparum prevalence from 59.0% adjacent to the breeding sites to 5.4% only a few hundred meters distant. The entomologic inoculation rate ranged from a number too low to be determined in some districts to 20 infectious bites per person per year in the others. The risk of malaria was 6.2 times higher for individuals living less than 200 meters from the breeding sites than for individuals living 500 meters or more away from the breeding sites. In areas of high human density, mosquito and parasite dispersion is very limited, and therefore malaria control strategies could be more specifically targeted.

Population studies of the human V kappa A18 gene polymorphism in Caucasians, blacks and Eskimos. New functional alleles and evidence for evolutionary selection of a more restricted antibody repertoire.

Immunoglobulin gene polymorphisms are interesting because they reflect differences in the available antibody repertoire which may affect the susceptibility to specific infections. Until recently, the human V kappa gene, A18, was known as a nonfunctional gene only. In this study, we cloned and sequenced four apparently functional alleles and determined the gene frequencies in three well-defined populations: Danish Caucasians, eastern Greenland Eskimos and Mozambican blacks. The A18b allele that was recently described in Native American Navajos by Atkinson et al. was found in all three populations with gene frequencies of 8%, 45% and 23% in Caucasians, Eskimos and blacks, respectively. Conversely, the frequencies of the nonfunctional A18a allele were 92%, 55% and 57%. Further, three new A18 alleles, c, d, and e were found exclusively in blacks, among whom they had an total frequency of 19%. These data indicate that both the A18a and A18b alleles originated before the diversification of Africans and non-Africans 90,000 years ago, whereas the A18c, A18d and A18e alleles may have a more recent origin. The functionality of the A18b allele was documented by the demonstration of properly rearranged and somatically hypermutated A18b messenger RNA present in the blood lymphocytes of individuals carrying this allele. The expression clearly exceeded that of a known functional V gene, A2, indicating that functional A18 alleles contribute significantly to the available antibody repertoire. In this context, it is surprising that the functional A18b allele apparently has been negatively selected in the Caucasian population, among whom 85% completely lack a functional gene.

Cloning of the fomA gene, encoding the major outer membrane porin of Fusobacterium nucleatum ATCC10953.

The major outer membrane protein, FomA, of the Gram-negative human oral pathogen Fusobacterium nucleatum functions as a porin and is assumed to act as a receptor protein in coaggregation with other oral pathogenic bacteria such as Streptococcus sanguis and Porphyromonas gingivalis. We describe here the cloning of fomA from F. nucleatum in E. coli. Using pGEM3Zf(+), three recombinant plasmids were carrying parts of the fomA gene, but none of these contained regions upstream of the coding sequence. From these plasmids a clone was constructed which contained the whole fomA gene. The ATCC 10953 fomA gene was cloned under the phosphate limitation-inducible phoE promoter, using a vector derived from pACYC184. The protein was found to be incorporated into the outer membrane of the host in an apparently normal manner, as judged by heat-modifiability, trypsin-accessibility, and accessibility to antibodies to the protein in a whole cell enzyme-linked immunosorbent assay. The cloned FomA was found to exhibit pore-forming activity.

Clinical and parasitological studies on immunity to Plasmodium falciparum malaria in children.

Malaria remains one of the major health problems in many tropical countries. Plasmodium falciparum is the most common malaria parasite in Africa, and it causes much more severe and progressive illness than any of the other types of malaria parasite. Children living in sub-Saharan Africa are bearing the major burden of the disease and the mortality. Whatever parameter is used to measure the mortality or the morbidity from malaria, the true problem is likely to be underestimated. The pattern of morbidity and mortality depends on the transmission intensity; the more intensity of malaria transmission is increased, the earlier and more confined the age range of symptomatic malaria. The asymptomatic carrier status is common, and 60-80% of the children in highly endemic areas have P. falciparum parasitaemia at any given time. Consequently a case definition based on the mere presence of parasites in the blood is non-informative in terms of measuring morbidity. Recognizing that there are no specific diagnostic clinical parameters for malaria, but that fever is very common, and that morbidity is to some extent dependent on the parasite density, we described using a logistic regression model the probability of being sick from malaria in relation to body temperature and parasite density. Acquired clinical and parasitological immunity develop progressively over several years after repeated exposure to infection. Protection is acquired first against death or severe clinical disease, then against milder clinical attacks, but protection against infection is never complete. Clinical and parasitological immunity develop concomitantly, as demonstrated by relating the parasite densities to measured body temperature. However, the ability to control the disease and parasite density develops earlier than the ability to prevent the parasite infection. The individual immune mechanisms that are responsible for the acquired immunity remain uncertain, but classical transfer experiments with polyvalent gamma globulin from immune donors to non-immune individuals showed that antibodies play an important role. Potential targets for malarial vaccines include antigens on the surface of the sporozoites and the merozoites. Several protein antigens from P. falciparum have been characterized at the molecular level, and most of the characterized antigens have the common characteristic that they are recognized by immune sera from individuals living in malaria endemic areas. Working on the approach that potentially useful targets for protective vaccine development can be identified by correlating the naturally acquired immune responses with defined P. falciparum antigens, we examined antigens from both the sporozoite stage (CS-protein) and the blood stages (Pf155/RESA, GLURP, and MSP1), as well as P. falciparum induced neoantigens on the red blood cell (band-3 neoantigens). The relationship between the immune response to these defined P. falciparum antigens and clinical and parasitological protection was analysed in the individual age groups. The contribution of the antigen-specific immune response was evaluated, and a positive correlation of parasite density or probability of an episode of clinical malaria with antibody response to the individual antigens was identified in defined age groups. This correlation, however, did not span all age groups, and thus overall responses to defined antigens are not considered to be reliable indicators of protection. The findings may contribute to the understanding of immunological and clinical host responses to parasitaemia and to defined P. falciparum antigens. The studies on the impact of asexual stage infection and the human immune response led to studies on specific and non-specific responses to P. falciparum blood-stage parasites and observations on gametocytaemia. We demonstrated that pyrimethamine/sulfadoxine and chloroquine did not induce gametocytogenesis as suggested previously, but preformed gametocytes persisted after

Interval censored survival data and multistate compartmental models in the analysis of first appearance of Plasmodium falciparum parasites in infants.

A statistical model for interval censored data is described. Assuming a piecewise constant incidence enables us to analyse very unbalanced data in a generalized linear model. The distribution of age at first appearance of P. falciparum parasites in infants in Liberia has been estimated. A new graphical method for presentation of test results on all children was developed. In an illness--death model it is described how the proportion of undetected and detected malaria parasitemias depends on parasite rates and testing frequency. The incidence of detectable malaria parasitemia was 0.14 per month in infants under 4 months of age, and 0.60 per month in children over 4 months (p < 0.001). The congenital resistance to malaria in African infants living in a highly endemic area had largely disappeared by the age of 4 months; before this age children were partly protected.

Relationship between maternally derived anti-Plasmodium falciparum antibodies and risk of infection and disease in infants living in an area of Liberia, west Africa, in which malaria is highly endemic.

In areas where Plasmodium falciparum is endemic, immunoglobulin G is acquired by the fetus in utero, mainly during the third trimester of pregnancy. The potential protective effect of transferred anti-P. falciparum maternal antibodies was examined in a longitudinal study of 100 infants from birth to 1 year of age. The probability of acquiring a P. falciparum infection and developing an episode of clinical malaria was determined in relation to the P. falciparum-specific antibody level of the infant at birth against P. falciparum schizont antigen or recombinant merozoite surface protein MSP1(19) antigen. The risk of acquiring an episode of clinical malaria increased from birth to 6 months of age, after which it decreased. The overall prevalence of P. falciparum parasitemia was highest (48.9%) in the 6-month-old infants. The age-specific hematocrit value showed the lowest mean value (30.2) from 6 to 9 months, and the spleen rate was the highest (69.8%) at the same age. There was a lower risk of developing an episode of clinical malaria during the first year of life in the infants with high levels of anti-MSP1(19) antibodies at birth. The level of maternally derived overall anti-schizont antigen antibodies did not seem to play a role in the relative risk of developing malaria infection or disease during the first year of life, though the level of specific anti-MSP1(19) antibodies may be associated with protection.