Commonality and biosynthesis of the O-methyl phosphoramidate capsule modification in Campylobacter jejuni

In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the beta-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-alpha-L-gluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.     

Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents

We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently.     

Ivermectin Treatment and Sanitation Effectively Reduce Strongyloides stercoralis Infection Risk in Rural Communities in Cambodia

BackgroundStrongyloides stercoralis is the only soil-transmitted helminth with the ability to replicate within its host, leading to long-lasting and potentially fatal infections. It is ubiquitous and its worldwide prevalence has recently been estimated to be at least half that of hookworm. Information on the epidemiology of S. stercoralis remains scarce and modalities for its large-scale control are yet to be determined.Conclusions/SignificanceChemotherapy-based control of S. stercoralis is feasible and highly beneficial, particularly in combination with improved sanitation. The impact of community-based ivermectin treatment on S. stercoralis was high, with over 85% of villagers remaining negative one year after treatment. The integration of S. stercoralis into existing STH control programs should be considered without further delay.     

Dengue incidence in urban and rural Cambodia: results from population-based active fever surveillance, 2006-2008

Background

Dengue vaccines are now in late-stage development, and evaluation and robust estimates of dengue disease burden are needed to facilitate further development and introduction. In Cambodia, the national dengue case-definition only allows reporting of children less than 16 years of age, and little is known about dengue burden in rural areas and among older persons. To estimate the true burden of dengue in the largest province of Cambodia, Kampong Cham, we conducted community-based active dengue fever surveillance among the 0-to-19–year age group in rural villages and urban areas during 2006–2008.

Methods and Findings

Active surveillance for febrile illness was conducted in 32 villages and 10 urban areas by mothers trained to use digital thermometers combined with weekly home visits to identify persons with fever. An investigation team visited families with febrile persons to obtain informed consent for participation in the follow-up study, which included collection of personal data and blood specimens. Dengue-related febrile illness was defined using molecular and serological testing of paired acute and convalescent blood samples. Over the three years of surveillance, 6,121 fever episodes were identified with 736 laboratory-confirmed dengue virus (DENV) infections for incidences of 13.4–57.8/1,000 person-seasons. Average incidence was highest among children less than 7 years of age (41.1/1,000 person-seasons) and lowest among the 16-to-19–year age group (11.3/1,000 person-seasons). The distribution of dengue was highly focal, with incidence rates in villages and urban areas ranging from 1.5–211.5/1,000 person-seasons (median 36.5). During a DENV-3 outbreak in 2007, rural areas were affected more than urban areas (incidence 71 vs. 17/1,000 person-seasons, p<0.001).

Conclusion

The large-scale active surveillance study for dengue fever in Cambodia found a higher disease incidence than reported to the national surveillance system, particularly in preschool children and that disease incidence was high in both rural and urban areas. It also confirmed the previously observed focal nature of dengue virus transmission.     

The structures of the lipooligosaccharide and capsule polysaccharide of Campylobacter jejuni genome sequenced strain NCTC 11168.

Campylobacter jejuni infections are one of the leading causes of human gastroenteritis and are suspected of being a precursor to Guillain-Barré and Miller-Fisher syndromes. Recently, the complete genome sequence of C. jejuni NCTC 11168 was described. In this study, the molecular structure of the lipooligosaccharide and capsular polysaccharide of C. jejuni NCTC 11168 was investigated. The lipooligosaccharide was shown to exhibit carbohydrate structures analogous to the GM1a and GM2 carbohydrate epitopes of human gangliosides (shown below): The high Mr capsule polysaccharide was composed of beta-d-Ribp, beta-d-GalfNAc, alpha-d-GlcpA6(NGro), a uronic acid amidated with 2-amino-2-deoxyglycerol at C-6, and 6-O-methyl-d-glycero-alpha-l-gluco-heptopyranose as a side-branch (shown below): The structural information presented here will aid in the identification and characterization of specific enzymes that are involved in the biosynthesis of these structures and may lead to the discovery of potential therapeutic targets. In addition, the correlation of carbohydrate structure with gene complement will aid in the elucidation of the role of these surface carbohydrates in C. jejuni pathogenesis.     

Occurrence of a Hybrid Between Taenia saginata and Taenia asiatica Tapeworms in Cambodia

Human infection with Taenia asiatica or a hybrid between Taenia saginata and T. asiatica has not been reported in Cambodia. We detected for the first time a hybrid form between T. saginata and T. asiatica in Preah Vihear Province, Cambodia. An adult tapeworm specimen, i.e., 75 cm long strobila without scolex, was expelled from a 27-year-old man after praziquantel medication and purging. It was morphologically indistinguishable between T. saginata and T. asiatica. Several proglottids were molecularly analyzed to confirm the tapeworm species. The mitochondrial gene encoding cytochrome c oxidase subunit 1 (cox1) and nuclear genes encoding elongation factor-1α (ef1) and ezrin-radixin-moesin (ERM)-like protein (elp) were sequenced, and a single-allele analysis was performed to confirm the haploid genotype. The results revealed that our sample showed a discrepancy between the mitochondrial and 2 nuclear genes. It possessed homozygous sequences typical of T. saginata at cox1 and ef1 loci. However, it was heterozygous at the elp locus, with 1 allele in T. asiatica (elpA) and 1 in T. saginata (elpC), which indicates that it is a hybrid between T. saginata and T. asiatica. The present results confirmed the presence of a hybrid between T. saginata and T. asiatica in Cambodia and strongly suggest the existence of also ‘pure’ T. asiatica in Cambodia.     

Morphological and Molecular Diagnosis of Necator americanus and Ancylostoma ceylanicum Recovered from Villagers in Northern Cambodia

Human hookworm infections caused by adult Ancylostoma spp. and Necator americanus are one of the most important tropical diseases. We performed a survey of intestinal helminths using the Kato-Katz fecal examination technique targeting 1,156 villagers residing in 2 northern provinces (Preah Vihear and Stung Treng) of Cambodia in 2018. The results revealed a high overall egg positive rate of intestinal helminths (61.9%), and the egg positive rate of hookworms was 11.6%. Nine of the hookworm egg positive cases in Preah Vihear Province were treated with 5–10 mg/kg pyrantel pamoate followed by purging with magnesium salts, and a total of 65 adult hookworms were expelled in diarrheic stools. The adult hookworms were analyzed morphologically and molecularly to confirm the species. The morphologies of the buccal cavity and dorsal rays on the costa were observed with a light microscope, and the nucleotide sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were analyzed. The majority of the hookworm adults (90.7%) were N. americanus, whereas the remaining 9.3% were Ancylostoma ceylanicum, a rare hookworm species infecting humans. The results revealed a high prevalence of hookworm infections among people in a northern part of Cambodia, suggesting the necessity of a sustained survey combined with control measures against hookworm infections.     

Strongyloides stercoralis: Global Distribution and Risk Factors

Background

The soil-transmitted threadworm, Strongyloides stercoralis, is one of the most neglected among the so-called neglected tropical diseases (NTDs). We reviewed studies of the last 20 years on S. stercoralis''s global prevalence in general populations and risk groups.

Methods/Principal Findings

A literature search was performed in PubMed for articles published between January 1989 and October 2011. Articles presenting information on infection prevalence were included. A Bayesian meta-analysis was carried out to obtain country-specific prevalence estimates and to compare disease odds ratios in different risk groups taking into account the sensitivities of the diagnostic methods applied. A total of 354 studies from 78 countries were included for the prevalence calculations, 194 (62.4%) were community-based studies, 121 (34.2%) were hospital-based studies and 39 (11.0%) were studies on refugees and immigrants. World maps with country data are provided. In numerous African, Asian and South-American resource-poor countries, information on S. stercoralis is lacking. The meta-analysis showed an association between HIV-infection/alcoholism and S. stercoralis infection (OR: 2.17 BCI: 1.18–4.01; OR: 6.69; BCI: 1.47–33.8), respectively.

Conclusions

Our findings show high infection prevalence rates in the general population in selected countries and geographical regions. S. stercoralis infection is prominent in several risk groups. Adequate information on the prevalence is still lacking from many countries. However, current information underscore that S. stercoralis must not be neglected. Further assessments in socio-economic and ecological settings are needed and integration into global helminth control is warranted.