Detection of Diabetes by Macrovascular Tortuosity of Superior Bulbar Conjunctiva.

More than 8% of world population have diabetes which causes long term complications such as retinopathy, neuropathy, nephropathy and foot ulcers. Growing patient numbers has prompted large scale screening methods to detect early symptoms of diabetes (rather than elevated blood glucose levels which is a late symptom). Vascular tortuosity (twisted and curved nature of blood vessels) in retinal fundus images has proven to reflect the effect of diabetes on macrovasculature. However, large scale patient screening using retinal fundus images has limitations due to the requirement of a retinal camera. Therefore, we hypothesize that the vasculature of superior bulbar conjunctiva which could be captured using a regular camera could be used to measure tortuosity instead of retinal fundus images enabling mass screening.To test this hypothesis, a total of 168 scleral images were acquired from 50 healthy subjects and 34 diabetic patients using a digital camera. The sclera region was segmented using Chan-Vese algorithm and macrovasculature of superior bulbar conjunctiva was segmented using B-COSFIRE filters. Results revealed that the superior bulbar conjunctival macrovascular tortuosity of diabetic patients was significantly less than that of non-diabetic group (p-value =0.015). A similar result was yielded (p-value =0.049) from a group of participants who were less than 40 years old which excluded the age related variation of tortuosity.

Eighth International Meeting of the European Laboratory Working Group on Diphtheria and the Diphtheria Surveillance Network--June 2004: progress is needed to sustain control of diphtheria in European Region.

The Eighth International Meeting of the European Laboratory Working Group on Diphtheria (ELWGD) and the Diphtheria Surveillance Network (DIPNET) was held and co-organised with the WHO Regional Office for Europe, Copenhagen, Denmark, in June 2004. This article provides an international updated review of progress in clinical, epidemiological and microbiological aspects of diphtheria in the European region as presented at the meeting. It highlights the need for improved immunisation coverage, surveillance and epidemiological studies to sustain control of diphtheria in European Region.

Eighth International Meeting of the European Laboratory Working Group on Diphtheria and the Diphtheria Surveillance Network - June 2004: Progress is needed to sustain control of diphtheria in European Region.

The Eighth International Meeting of the European Laboratory Working Group on Diphtheria (ELWGD) and the Diphtheria Surveillance Network (DIPNET) was held and co-organised with the WHO Regional Office for Europe, Copenhagen, Denmark, in June 2004. This article provides an international updated review of progress in clinical, epidemiological and microbiological aspects of diphtheria in the European region as presented at the meeting. It highlights the need for improved immunisation coverage, surveillance and epidemiological studies to sustain control of diphtheria in European Region.

The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129.

Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms. The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria. This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme. However, the disease has made a dramatic return in recent years, in particular within the Eastern European region. The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s. We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak. The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids. It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins. This is in contrast to Corynebacterium's nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition. The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.

Nontoxigenic corynebacterium diphtheriae: an emerging pathogen in England and Wales?

Confirmed isolates of nontoxigenic Corynebacterium diphtheriae in England and Wales increased substantially from 1986 to 1994. Ribotyping of 121 isolates confirmed in 1995 showed that 90 were of a single strain isolated exclusively from the throat; none had previously been identified in toxigenic strains from U.K. or non-U.K. residents. The upward trend in nontoxigenic C. diphtheriae probably represented increased ascertainment, although dissemination of a particular strain or clone may have been a factor.

Use of amplified fragment length polymorphisms for typing Corynebacterium diphtheriae.

Amplified fragment length polymorphism (AFLP) was investigated for the differentiation of Corynebacterium diphtheriae isolates. Analysis using Taxotron revealed 10 distinct AFLP profiles among 57 isolates. Strains with ribotype patterns D1, D4, and D12 could not be distinguished; however, the technique discriminated isolates of ribotype patterns D3, D6, and D7 further. AFLP was rapid, fairly inexpensive, and reproducible and could be used as an alternative to ribotyping.

Molecular epidemiology of diphtheria.

Molecular subtyping of Corynebacterium diphtheriae identified significant genetic diversity within the species and led to the identification of a unique clonal group that emerged in Russia in 1990 at the beginning of the current epidemic. Strains of this group belong to a distinct electrophoretic type complex and are of ribotypes D1 and D4. Identification of the group allowed for precise monitoring of the epidemic's progression and for rapid detection of cases imported to other countries. The evolution of this clonal group was monitored, and changes were identified. Molecular analysis revealed that no amino acid substitutions have occurred in the diphtheria toxin gene of the epidemic clone strains, reaffirming the use of the current vaccine as the single most effective preventive measure. Application of molecular subtyping methods and continuous monitoring of the spread of these clones has made it possible to distinguish rapidly between epidemic, endemic, and imported cases, allowing for implementation of timely and adequate preventive measures and providing reassurance that no secondary spread resulted from importations.

PCR typing of Corynebacterium diphtheriae by random amplification of polymorphic DNA.

The usefulness of random amplification of polymorphic DNA (RAPD) analysis with Ready-To-Go RAPD beads was investigated for the rapid differentiation of Corynebacterium diphtheriae isolates from Eastern Europe and neighbouring countries. A selection of 45 C. diphtheriae isolates of known origin, biotype, toxigenicity status and ribotype were examined by RAPD. Twenty RAPD profiles (designated Rp1-Rp20) were revealed among the 45 isolates. There was 100% correlation between RAPD profiles and ribotypes. Preliminary studies showed that the use of crude DNA preparations resulted in poor amplification and the patterns were not reproducible. Different thermal cycler models produced different RAPD profiles from the same DNA sample. Reproducibility of the technique was good when the same thermal cycler was used throughout. RAPD proved to be a simple and a rapid method for analysing C. diphtheriae and it is a method which can be used as a potential alternative to ribotyping or as a screening technique during outbreak investigations.

Diphtheria in the Republic of Georgia: use of molecular typing techniques for characterization of Corynebacterium diphtheriae strains.

Sixty-six Corynebacterium diphtheriae strains (62 of the gravis biotype and 4 of the mitis biotype) isolated during the Georgian diphtheria epidemic of 1993 to 1998 and 13 non-Georgian C. diphtheriae strains (10 Russian and 3 reference isolates) were characterized by (i) biotyping, (ii) toxigenicity testing with the Elek assay and PCR, (iii) the randomly amplified polymorphic DNA (RAPD) technique, and (iv) pulsed-field gel electrophoresis (PFGE). Fifteen selected strains were ribotyped. Six RAPD types and 15 PFGE patterns were identified among all strains examined, and 12 ribotypes were found among the 15 strains that were ribotyped. The Georgian epidemic apparently was caused by one major clonal group of C. diphtheriae (PFGE type A, ribotype R1), which was identical to the predominant epidemic strain(s) isolated during the concurrent diphtheria epidemic in Russia. A dendrogram based on the PFGE patterns revealed profound differences between the minor (nonpredominant) epidemic strains found in Georgia and Russia. The methodologies for RAPD typing, ribotyping, and PFGE typing of C. diphtheriae strains were improved to enable rapid and convenient molecular typing of the strains. The RAPD technique was adequate for biotype differentiation; however, PFGE and ribotyping were better (and equal to each other) at discriminating between epidemiologically related and unrelated isolates.

Molecular typing of Legionella pneumophila serogroup 1 by pulsed-field gel electrophoresis with SfiI and comparison of this method with restriction fragment-length polymorphism analysis.

A panel of 131 well-characterised Legionella pneumophila serogroup 1 strains, previously used to evaluate a restriction fragment-length polymorphisms (RFLP) typing scheme, was examined by pulsed-field gel electrophoresis (PFGE) with the restriction endonuclease SfiI. The data obtained show that PFGE with SfiI is a highly discriminatory method yielding an index of discrimination (IOD) of 0.992 and 0.975, with 100% and 90% similarity thresholds respectively, compared with an IOD of 0.909 for the RFLP typing method. Reproducibility of PFGE profiles within gels was excellent and it was possible to compare the profiles visually. However, the reproducibility of the technique between gels was poor and visual comparison of the patterns was extremely difficult. Computer-aided analysis assisted the assessment of inter-gel reproducibility. Of 11 duplicates examined only four pairs showed 100% similarity, although 9 of 11 showed > or =90% similarity. In an attempt to determine if the PFGE banding patterns were sufficiently unambiguous to allow the method to be used as a definitive typing method, 20 coded strains were examined. At a 90% similarity level, 16 of these were placed in the correct PFGE type and four were not allocated to a type. Partial digestion of DNA by SfiI was noted despite careful control of DNA and enzyme concentrations, suggesting that an alternative enzyme might give more reproducible results.

Diagnosis and epidemiology of diphtheria.

Prior to the late 1980s diphtheria was regarded in many countries as one of "those rare and forgotten" diseases associated with the preimmunization era of the 1940s. Despite the success of many immunization programs, there is still much to be learned about this disease that has made a dramatic "return" in the 1990s, particularly to countries of the former Soviet Union (1) Since 1989, there has been a rapidly expanding epidemic of the disease in these countries, and thus has severe implications even for developed countries that have successfully controlled the disease for several decades (2). Diphtheria is also endemic in other countries of the world (3). The increase m international travel, migration from Eastern Europe, and also the emergence of "new strams" of the causative organism, Corynebacterlum diphtheria, causing disease have emphasized the importance of both clinical and laboratory-awareness. In addition, current immunization programs within each country should be reviewed, particularly for adults, to ensure that population immunity is adequate to prevent the re-emergence of epidemic disease in the Western world.

Corynebacterium imitans sp. nov. isolated from patients with suspected diphtheria.

A 5-month-old boy of a Romanian family traveling via Ukraine to Poland developed a respiratory disease that resembled and that was initially diagnosed as pharyngeal diphtheria. The child recovered after treatment with antidiphtheria antitoxin. A coryneform bacterium had been isolated from a nasopharyngeal specimen from the child and was initially identified as an atypical Corynebacterium diphtheriae strain. Seven adults who had contact with either the child or an adult contact person also developed symptoms of pharyngeal diphtheria, were also treated with antitoxin, and recovered uneventfully. Coryneform bacteria similar to that originating from the index patient were also isolated from the throat swabs of three adults. Detailed biochemical and chemotaxonomic investigations revealed that the coryneform bacteria belonged to the genus Corynebacterium and could be differentiated from all other defined species of this genus. Ribotyping and pulsed-field gel electrophoresis demonstrated that all four patients' isolates were of clonal origin. The diphtheria toxin gene and its product were not detected either by PCR assays or by the Elek test, making a possible disease association of the Corynebacterium more unlikely. Comparative 16S rRNA gene sequence analysis revealed that the coryneform bacterium represented a new subline within the genus Corynebacterium, for which the name Corynebacterium imitans sp. nov. is proposed. The type strain is NCTC 13015 (DSM 44264; CCUG 36877).

Molecular epidemiology of Corynebacterium diphtheriae from northwestern Russia and surrounding countries studied by using ribotyping and pulsed-field gel electrophoresis.

A selection of 100 Corynebacterium diphtheriae isolates from asymptomatic carriers and clinical cases from five regions in northwestern Russia were examined. Six additional isolates from patients in Finland and Estonia with epidemiological links to Russia were also examined. All isolates were characterized by biotyping, toxigenicity testing, ribotyping, and pulsed-field gel electrophoresis (PFGE). Hybridization of genomic DNA digested with BstEII revealed five ribotype patterns among the biotype gravis isolates (G1 through G5) and two patterns among the biotype mitis isolates (M1 and M2). PFGE using SfiI was not able to distinguish between ribotypes G1, G2, and G4. The predominant ribotype pattern, G1, found in cases of disease in all the areas studied, appears to be disseminating, in view of the isolates received from imported cases in Finland and Estonia. Among the 106 isolates examined, 68 produced pattern G1 and 24 produced pattern M1. Most of the M1 isolates were from the Leningrad Oblast region. Distinct ribotypes such as G2, G3, G4, G5, and M2 could represent endemic disease.

Method to estimate relative transmission efficiencies of Anopheles species (Diptera: Culicidae) in human malaria transmission.

A mathematical expression was derived to estimate the relative malaria transmission efficiency of an anopheline species with respect to a standard well-characterized species for which all vector parameters can be sufficiently determined. The method is particularly useful in situations where multiple anopheline species contribute to human malaria transmission and requires the estimation of the man-biting rate, the sporozoite rate, and the human malaria incidence. Under stable conditions of vector abundance, the average sporozoite rate in a species during a transmission season would by itself reflect its relative transmission efficiency. This "efficiency" then was used to calculate the "effective human-biting rate"; i.e., the human-biting rate of that species if it were to have ecological properties identical to those of the standard species. The standard well-characterized species then could be used with the effective human-biting rate of all species to quantify transmission, thus overcoming the need to measure vector parameters for all anopheline species contributing to transmission. An expression also was derived to calculate the relative contribution made by each species to malaria transmission. The usefulness of this method was illustrated using entomological and epidemiological data from Kataragama, Sri Lanka.

Clustering of malaria infections within an endemic population: risk of malaria associated with the type of housing construction.

The occurrence of malaria infections due to Plasmodium vivax and P. falciparum was monitored in a population of 3,023 people living in six contiguous villages in Kataragama, an area of endemic malaria in southern Sri Lanka, over a period of 17 months. The annual incidence of malaria in this population during the study period was 25.8%. Malaria attacks were clustered, occurring more frequently than expected in certain individuals and housing groups and less frequently than expected in others. In one of these villages, the distribution of cases was examined in relation to locality and to the type of house construction. There was a strong association between the malaria incidence and house construction, independent of location. The risk of getting malaria was greater for inhabitants of the poorest type of house construction (incomplete, mud, or cadjan (palm) walls, and cadjan thatched roofs) compared to houses with complete brick and plaster walls and tiled roofs. Houses that were better constructed had a significantly lower malaria incidence rate (10.5%) than those that were poorly constructed (21.2%; P less than 0.01, by Student's t-test). There was also a significantly higher number of indoor resting mosquitoes collected from the poorly constructed houses than from those better constructed; the average (geometric mean) of mosquito densities found in houses of better versus poor construction were 0.97 and 1.89 per collection in the dry season, and 1.95 and 3.42 per collection in the wet season, respectively (P less than 0.05 in both seasons). This indicated that the higher malaria risk associated with poorly constructed houses was at least partly due to higher human-mosquito contact among their inhabitants.

A mathematical model for Plasmodium vivax malaria transmission: estimation of the impact of transmission-blocking immunity in an endemic area.

We have developed a multi-state mathematical model to describe the transmission of Plasmodium vivax malaria; the model accommodates variable transmission-blocking/enhancing immunity during the course of a blood infection, a short memory for boosting immunity, and relapses. Using the model, we simulated the incidence of human malaria, sporozoite rates in the vector population, and the level of transmission-blocking immunity for the infected population over a period of time. Field data from an epidemiological study conducted in Kataragama in the south of Sri Lanka were used to test the results obtained. The incidence of malaria during the study period was simulated satisfactorily. The impact of naturally-acquired transmission-blocking immunity on malaria transmission under different vectorial capacities was also simulated. The results show that at low vectorial capacities, e.g., just above the threshold for transmission, the effect of transmission-blocking immunity is very significant; however, the effect is lower at higher vectorial capacities.