Two-year analysis of Clostridium difficile ribotypes associated with increased severity.

BACKGROUND: Certain Clostridium difficile ribotypes have been associated with complex disease phenotypes including recurrence and increased severity, especially the well-described hypervirulent RT027. This study aimed to determine the pattern of ribotypes causing infection and the association, if any, with severity. METHODS: All faecal samples submitted to a large diagnostic laboratory for C. difficile testing between 2011 and 2013 were subject to routine testing and culture. All C. difficile isolates were ribotyped, and associated clinical and demographic patient data were retrieved and linked to ribotyping data. RESULTS: In total, 86 distinct ribotypes were identified from 705 isolates of C. difficile. RT002 and RT015 were the most prevalent (22.5%, N=159). Only five isolates (0.7%) were hypervirulent RT027. Ninety of 450 (20%) patients with clinical information available died within 30 days of C. difficile isolation. RT220, one of the 10 most common ribotypes, was associated with elevated median C-reactive protein and significantly increased 30-day all-cause mortality compared with RT002 and RT015, and with all other ribotypes found in the study. CONCLUSIONS: A wide range of C. difficile ribotypes were responsible for C. difficile infection presentations. Although C. difficile-associated mortality has reduced in recent years, expansion of lineages associated with increased severity could herald increases in future mortality. Enhanced surveillance for emerging lineages such as RT220 that are associated with more severe disease is required, with genomic approaches to dissect pathogenicity.

The diagnostic efficacy of fine-needle aspiration using cytology and culture in tuberculous lymphadenitis.

OBJECTIVE: To retrospectively assess the diagnostic efficacy of fine-needle aspiration (FNA) using cytological and microbiological examinations in tuberculous lymphadenitis. METHODS: Patients with tuberculous lymphadenitis treated at St Mary's Hospital, London, between January 2001 and June 2007 were identified. The cytological and microbiological reports of 97 patients were found. The criteria for a definite diagnosis of tuberculous lymphadenitis were based on a compatible clinical history, tuberculin positivity and either an indicative cytological result or positive culture. RESULTS: In 77 of the 97 (79%) cases, FNA cytology showed evidence of a tuberculous process. In 65 cases, Mycobacterium tuberculosis was cultured from the aspirates, and 54 of these 65 cases showed corresponding cytological evidence of a tuberculous process; 23 cases were diagnosed by cytology but not microbiology, while 11 cases were diagnosed by microbiology but not cytology. CONCLUSION: Cytological and microbiological results appeared to correlate well, but each also gives an exclusive diagnosis. When combining both modalities, the diagnostic efficacy of FNA rises to 91%. A definitive microbiological diagnosis was achieved in 67% of cases and provided information on drug susceptibility. We conclude that samples should be provided for both cytological and microbiological examination when using FNA to diagnose possible tuberculous lymphadenitis.

Host genetic contribution to the cellular immune response to Chlamydia trachomatis: Heritability estimate from a Gambian twin study.

If the cellular immune response to Chlamydia trachomatis is subject to genetic influences, the degree and mechanisms of such genetic control may have important implications for vaccine development. We estimated the relative contribution of host genetics to the total variation in lymphoproliferative responses to C. trachomatis antigen by analyzing these responses in 64 Gambian twin pairs from trachoma endemic areas. Zygosity was determined by restriction fragment length polymorphism analysis of minisatellite probes and microsatellite typing. Proliferative responses to serovar A elementary body antigen were estimated in monozygotic (MZ) and dizygotic (DZ) twin pairs. We found a stronger correlation and lower within-pair variability in these responses in MZ than in DZ twin pairs. The heritability estimate was 0.39 (P = 0.07) suggesting that host genetic factors contributed 39% of the variation. A better understanding of these genetic influences will contribute to the elucidation of preventive therapies for ocular C. trachomatis infection and may identify important mechanisms in protection for rational vaccine construction.

Genetic regulation of acquired immune responses to antigens of Mycobacterium tuberculosis: a study of twins in West Africa.

The role of genetic factors in clinical tuberculosis is increasingly recognized; how such factors regulate the immune response to Mycobacterium tuberculosis in healthy individuals is unclear. In this study of 255 adult twin pairs residing in The Gambia, West Africa, it is apparent that memory T-cell responses to secreted mycobacterial antigens (85-kDa antigen complex, "short-term culture filtrate," and peptides from the ESAT-6 protein), as well as to the 65-kDa heat shock protein, are subject to effective genetic regulation. The delayed hypersensitivity response to intradermal tuberculin also demonstrates significant genetic variance, while quantitative T-cell and antibody responses to the 38-kDa cell membrane protein appear to be determined largely by environmental factors. Such findings have implications for vaccine development.

Linkage disequilibrium between two chromosomally distinct loci associated with increased resistance to chloroquine in Plasmodium falciparum.

Chloroquine-resistance in Plasmodium falciparum is associated with polymorphisms in a locus on or near the cg2 gene on chromosome 7, and in the pfmdr1 gene on chromosome 5. In this study we typed P. falciparum DNA from uncomplicated malaria cases in The Gambia in 1990, 1995 and 1996 for size polymorphism in the omega repeat of cg2, for sequence polymorphisms in pfmdr1 at codons 86 and 184, in dhfr at codon 108 and in the msp2 gene. Chloroquine sensitivity tests were conducted in vitro. A significant but incomplete association was found between the presence of the cg2 Dd2-like omega repeat size polymorphism and in vitro resistance, and between the tyr-86 allele of pfmdr1 and in vitro resistance. Furthermore there was strong linkage disequilibrium between the pfmdr1 asn-86 allele and the cg2 not Dd2-like omega repeat allele located on different chromosomes. In contrast, no linkage disequilibrium was found between these alleles and either the dhfr ser-108 allele or the msp2 IC sequence polymorphism. No significant linkage was measured between pfmdr1 asn-86 and phe-184 although these loci are separated only by 296 base pairs. Our results suggest that genetic elements linked to the cg2 and the pfmdr1 genes are important determinants of chloroquine resistance. It can be concluded that the observed linkage disequilibrium is maintained epistatically through selection by chloroquine.

Male breast cancer.

Male breast cancer is a rare disease, often with a late presentation and poor prognosis. The mainstay of treatment is modified radical mastectomy, with axillary node dissection to assess stage, prognosis and the need for adjuvant treatment. When matched for age, tumour size, grade and axillary nodal status, the prognosis is similar for males and females. Concerted efforts must be made to educate both the public and health professionals, in order to make earlier diagnoses and thereby improve prognosis.

Causes of mortality in twins in a rural region of The Gambia, West Africa.

Little is known about birth or mortality rates of twins in The Gambia. There are no reports of the causes of death among twins in sub-Saharan Africa. We identified twin births and deaths from a community-based study which had been carried out in a large rural region of The Gambia over a 5-year period from 1989 to 1993. The overall twinning rate excluding stillbirths was 14.3 (95% CI 12.9, 15.8) per 1000 live deliveries. This was significantly lower among the Mandinka ethnic group (8.8 per 1000) than among the Serahulis (15.1 per 1000) or Fulas (18.3 per 1000). The early-neonatal, late-neonatal and post-neonatal twin mortality rates were 114.0, 45.9 and 64.2 per 1000 live twin births, respectively. In comparison, the early-neonatal, late-neonatal and post-neonatal mortality rates of singletons were 18.6, 16.0 and 41.1 per 1000, respectively. In the post-neonatal period, malnutrition was more frequently a cause of death among twins than among singletons (7.8 per 1000 twin births vs 2.0 per 1000 singleton births; p = 0.0008). Appropriate strategies for preventing malnutrition are required for this high-risk group.

PIP: A community-based study was conducted over a 5-year period (1989-93) in the rural Upper River Division of The Gambia to investigate causes of mortality among children under 5 years old focusing on the differences between singleton and twin mortality. During 1989-93, 26,496 deliveries were recorded. These include 396 twin pairs, 1 triplet, and 1 quadruplet delivery. Of the 396 twin deliveries, 7 pairs were stillborn and 11 pairs consisted of 1 stillbirth and 1 live birth, giving an overall twinning rate of 14.3/1000 live deliveries excluding stillbirths. The Mandinka ethnic group twinning rate excluding stillbirths was significantly lower than that among Fulas and Serahulis, which were recorded from June 1992 until the end of the study. On the other hand, there were 193 deaths of twins and 3583 singleton deaths recorded. The early neonatal, late-neonatal, and post-neonatal mortality rates of twins were 114.0, 45.9, and 64.2 per 1000 twins born alive, respectively. In comparison, the early-neonatal, late-neonatal, and post-neonatal mortality rates of singletons were 18.6, 16.0, and 41.1 per 1000 live births, respectively. Mortality rates were higher among males than in females. This held true among twins and singletons. The survey showed that malaria was frequently diagnosed in both twins and singletons while malnutrition was the etiologic factor of death among twins more often than singletons. In summary, it showed that mortality among twins, particularly among boys, was extremely high, which was likely due to the consequence of very low birth weight. However, malnutrition was a major cause of death among twins in the post-neonatal period.

Genetic analysis of host-parasite coevolution in human malaria.

Recent twin studies of clinical malaria and immune responses to malaria antigens have underscored the importance of both major histocompatability complex (MHC) and non-MHC genes in determining variable susceptibility and immune responsiveness. By using a combination of whole genome genetic linkage studies of families and candidate genes analysis, non-MHC genes are being mapped and identified. Human leucocyte antigen (HLA) genotype was found to affect susceptibility to severe malaria in a large study of West African children. T lymphocytes that may mediate such resistance have been identified and their target antigens and epitopes characterized. Some of these epitopes show substantial polymorphism, which appears to result from immune selection pressure. Natural variant epitopes have been found to escape T-cell recognition in cytolytic and other T-cell assays. More recently a novel immune escape mechanism has been described in viral infections, altered peptide ligand antagonism, whereby variants of a T-cell epitope can downregulate or ablate a T cell response to the index peptide. The likely implications of such immune escape mechanisms for the population structure of malaria parasites, for HLA associations with malaria infection and disease, and for the design of new malaria vaccines, are discussed. The evolutionary consequences of such molecular interactions can be assessed by using mathematical models that capture the dynamic of variable host and parasite molecules. Combined genetic, immunological and mathematical analysis of host and parasite variants in natural populations can identify some mechanisms driving host-parasite coevolution.

Genetic linkage of mild malaria to the major histocompatibility complex in Gambian children: study of affected sibling pairs.

PIP: Case-control studies have indicated that genes for the major histocompatibility complex influence the presentation and outcome of severe Plasmodium falciparum disease. To assess the role of genetic factors in mild malaria, an analysis was conducted in 217 pairs of Gambian twins (mean age, 5.3 years) concordant for this phenotype. The twins were monitored weekly during three rainy seasons (1991-93) for fever and P. falciparum infection. This surveillance produced a total of 40 pairs of twins who were concordant for clinical malaria; none had severe disease. In the 22 of these 40 families with complete information, 11 had two shared alleles (expected value, 5.5), 10 shared one allele (expected value, 11.0), and 1 shared no allele (expected value, 5.5). If a locus is genetically linked to disease, affected siblings will share a higher than expected number of alleles identical by descent at that locus. Sharing of major histocompatibility complex alleles was not increased among the 13 pairs of dizygous twins who were discordant for malaria. These findings confirm the importance of genetic factors to the risk of uncomplicated malaria.^ieng

Analysis of the T-cell receptor V beta usage in monozygotic and dizygotic twins living in a Plasmodium falciparum endemic area in west Africa.

To investigate the influence of genetic and/or environmental factors in the development and shaping of the human peripheral T cell repertoire the authors studied the T-cell receptor (TCR) V beta usage in 10 adult monozygous (Mz) and nine dizygous (Dz) twin pairs living in a Plasmodium falciparum endemic area in West Africa. The TCR repertoire was determined using a small panel of anti-V beta specific monoclonal antibodies (MoAbs) using conventional immunofluorescence assays. The results revealed that the V beta repertoire was similar to that recently described for a Caucasian population using a similar panel of antibodies. The frequencies of particular V beta genes tested were influenced neither by anti-malarial antibody titres nor by parasite densities, indicating that the P. falciparum parasite is not a dominating factor in determining the peripheral T cell repertoire. All donors were human leucocyte antigen (HLA) class I and II typed; no association was found between the expression of any V beta genes and MHC haplotype. The V beta usage was more concordant within the Mz than within the Dz pairs. For a group comprising four HLA class II identical individuals, the average within-pair difference was significantly greater than for the whole Mz group, but similar to that seen for the total Dz group. Thus, the data suggest that genetic, rather than environmental, factors have a profound effect on the shaping of the human circulating T cell repertoire and that the major genetic factors are encoded by non-HLA class II genes.

Quantification of the relative contribution of major histocompatibility complex (MHC) and non-MHC genes to human immune responses to foreign antigens.

Understanding the extent to which genetic factors influence the immune response is important in the development of subunit vaccines. Associations with HLA gene polymorphisms appear insufficient to explain the range of variation in immune responses to vaccines and to infections by major pathogens. In this study of Gambian twins we report that regulation of the immune response to a variety of antigens from Plasmodium falciparum and Mycobacterium tuberculosis is controlled by factors which are encoded by genes that lie both within and outside the major histocompatibility complex (MHC). We define the relative contribution of these genes, which varies for different antigens. The cumulative genetic contribution of non-MHC genes to the total phenotypic variance exceeds that of the MHC-encoded genes.

Ankylosing spondylitis in West Africans--evidence for a non-HLA-B27 protective effect.

OBJECTIVE: To determine the prevalence of ankylosing spondylitis in the Fula ethnic group in The Gambia, and relate the disease prevalence to the B27 frequency and subtype distribution of that population. METHODS: 215 first degree relatives of 48 B27 positive Fula twin pairs, and 900 adult Fula males were screened for ankylosing spondylitis by clinical and, where appropriate, radiographic means. The B27 prevalence was determined by PCR/sequence specific oligonucleotides on finger prick samples from 100 unrelated Fula, and B27 subtype distribution by SSCP on unrelated B27 positive individuals. This data were then compared with the prevalence of ankylosing spondylitis among B27 positive Caucasians. RESULTS: No case of ankylosing spondylitis was seen. Six per cent of Fula are B27 positive, of which 32% are B*2703 and 68% B*2705. Assuming the penetrance of ankylosing spondylitis in B27 positive Fula is the same as in B27 positive Caucasians, the probability of not observing any cases of ankylosing spondylitis among the Fula examined is remote (P = 6.7 x 10(-6)). Similarly, the chance of not seeing any cases among those expected to be either B*2705 or B*2703 was small (P = 3.2 x 10(-4) for B*2705, and P = 0.02 for B*2703). CONCLUSIONS: The risk of developing ankylosing spondylitis in B27 positive Fula is lower than in B27 positive Caucasians. This is not explained by the B27 subtype distribution among Fula, and suggests the presence of some non-B27 protective factor reducing the prevalence of ankylosing spondylitis in this population.

Changes in the pattern of infant and childhood mortality in upper river division, The Gambia, from 1989 to 1993.

A surveillance system was used to detect births and deaths in children in a large, rural, West African population from 1989 to 1993. Cause of death was investigated using post-mortem questionnaires. Overall infant (age 0-11 months) and child (age 1-4 years) mortality rates of 80.1 and 18.8 per 1000 per year were recorded. These were reasonably consistent over the period of surveillance. The most frequent cause of death in infants was acute respiratory infection (ARI), whereas in children it was malaria: these two conditions accounted for 41% of the deaths in children under 5 years old. Other leading causes of death were acute gastroenteritis, malnutrition, and septicaemia. Deaths attributed to ARI decreased over the 5-year period, but mortality rates from other causes were either unchanged or increased slightly. Mortality from all causes peaked in the rainy season and was slightly higher in villages which were part of a primary health care programme than in those which were not. There were also no differences between male and female mortality rates beyond one year of age. Despite the introduction of a number of health interventions, there has been no major change in the overall pattern of mortality in children in a rural area of The Gambia. Malaria and ARI remain the main causes of death.

Selective recognition of malaria antigens by human serum antibodies is not genetically determined but demonstrates some features of clonal imprinting.

Malaria infection induces the production of serum antibodies to a variety of malaria antigens but the prevalence of antibodies to any particular antigen is typically much less than 100%. It has been assumed that non-responsiveness to defined antigens in malaria immune subjects is due to HLS-mediated restriction of the immune response. In this study we have investigated the role of HLA and non-HLA genes in the antibody response to two merozoite surface antigens (MSP1 and MSP2) and a sexual stage antigen (Ps260/230) of Plasmodium falciparum, and conclude that host genotype is not a major determinant of responsiveness. Although antibody levels vary in accordance with seasonal variations in malaria transmission in semi-immune children, antibody levels remain stable in clinically immune adults. Antigen recognition is selective with individual donors showing consistent high titre responses to some antigens/epitopes whilst consistently failing to recognize adjacent regions/epitopes of the same protein. An alternative explanation, consistent with the data presented here, is that selective antibody responses to malaria antigens in immune individuals result from a process akin to clonal imprinting (original antigenic sin).

Genetic regulation of fever in Plasmodium falciparum malaria in Gambian twin children.

The role of genetic factors in determining the clinical response of children to Plasmodium falciparum infection is not fully understood. A longitudinal survey of malaria morbidity in a cohort of 258 pairs of twin children was conducted in a rural area of Gambia to assess the extent to which genetic factors determine the host's susceptibility and clinical response to infection. The marginal correlation (which measures the excess probability of both twins being affected above that expected assuming independence) for malaria was higher in dizygous (DZ) than in monozygous (MZ) twin pairs, indicating that infection per se is largely determined by environmental factors. Once infected however, both members of an MZ pair were more likely to develop fever than were twins of a DZ pair, suggesting that genetic factors influence the presentation of clinical disease.

Relationships between maternal malaria and malarial immune responses in mothers and neonates.

Immune responses of 97 Gambian women and their neonates were studied. New methods distinguished between active and previous placental malaria, were used to examine relationships between maternal malaria and neonatal immune responses. Many placentas (61%) had active or previous malarial infection. Maternal and cord malarial IgG levels correlated (P < 0.001). Malarial IgG was raised in cord blood in active placental malaria; IgM was not detected. Mean lymphoproliferation and the proportion of responders to soluble P. falciparum antigens (F32) and conserved regions of p190 expressed on trophozoites and schizonts (190L and 190N) were higher in neonates than mothers. There was no clear relationship between maternal malaria and neonatal mean lymphoproliferation to malarial antigens, although fewer neonates responded when mothers were actively infected. Matched maternal and neonatal lymphoproliferation responses did not correlate. However, first born neonatal lymphoproliferation to PPD and malarial antigens appeared lower than other neonates, in agreement with lower lymphoproliferation in primigravidae compared with multigravidae. Also in common with mothers, autologous plasma suppressed neonatal lymphoproliferation to PPD and malarial antigens, suggesting common immunoregulation. Higher cortisol or other circulating factors in first pregnancies may be implicated. The relevance of cell-mediated malarial immune responses detected at birth remains to be established.

Human antibody responses to Pfs 230, a sexual stage-specific surface antigen of Plasmodium falciparum: non-responsiveness is a stable phenotype but does not appear to be genetically regulated.

The 230 kD gamete surface protein of the malaria parasite Plasmodium falciparum (Pfs 230) is a target of transmission blocking antibodies. Anti-Pfs 230 antibodies are induced following natural infection with malaria but are not found in all P. falciparum-exposed individuals. In this study we have shown that approximately 40% of malaria-exposed Gambians do not make antibodies to the native Pfs 230 molecule. This phenotype is remarkably stable over time and does not appear to be related to age, malaria exposure or major histocompatibility complex genotype. Comparison of antibody responses in twins indicates that the anti-Pfs 230 response is not strictly genetically controlled, but a high degree of concordance within both dizygous and monozygous twin pairs suggests that factors associated with exposure to malaria in childhood may be important in determining the subsequent immune response.

Twin children in The Gambia: evidence for genetic regulation of physical characteristics in the presence of sub-optimal nutrition.

It has been demonstrated that physical growth characteristics are subject to genetic regulation. However, in developing countries, environmental factors such as food availability and frequent infections are associated with growth faltering which is particularly marked in infancy. We have conducted anthropometric measurements of a cohort of twin children aged less than 14 years living in a rural area of The Gambia to ascertain the extent to which genetic factors influence physical growth in the presence of a sub-optimal diet. Almost 25% of the children were more than 2SD below the median of the reference population in terms of their height-for-age Z score, indicating a marked level of undernutrition. Nevertheless, the within-pair variances were significantly less for monozygous than for dizygous twin pairs for the following variables: height, head circumference and body mass index (p < 0.01); weight (p < 0.02) and mid upper arm circumference (p < 0.1), indicating that there is a strong genetic influence on growth regulation despite the sub-optimal nutrition.

PIP: At the Medical Research Council (MRC) field station serving villages in the Upper River Division (URD) of The Gambia in April 1991, health workers took anthropometric measurements and a blood sample from 39 pairs of monozygous (MZ) twins and 115 pairs of same-sexed dizygous (DZ) twins who had been living together and lived together at the time of the survey. All the twins were less than 14 years old. Researchers wanted to determine whether genetic factors influence physical growth in the presence of a suboptimal diet and infectious diseases, especially diarrhea and malaria. The study was conducted at the end of the dry season when food availability was limited. The frequency distribution curves of height-for-age (HAZ) and weight-for-height (WHZ) for the study population were left of the distribution curve for the US National Center for Health Statistics' reference population. This leftward shift suggests that the twins were undernourished at the time of the survey and for a prolonged time, resulting in growth stunting. 17.8% of the children had a WHZ score that was less than or equal to two standard deviations below the median of the reference population. 24.6% had an HAZ score less the median of the reference population. Stunting was most common in children younger than 2 years old. No significant difference between the 2 total variances of the 2 twin types existed. On the other hand, except for skinfold thickness, the within-pair variances were significantly less for MZ than for DZ twin pairs (height, head circumference, and body mass index [p 0.01); weight [p 0.02], and mid-upper arm circumference [p 0.1]). The environmental constraints (i.e., suboptimal diet and presence of infections) may have concealed the genetic influences of skinfold thickness. These findings suggest that genetic factors influence height, head circumference, body mass index, weight, and mid-upper arm circumference.