Epidemiology and cost of hospital care for Lyme borreliosis in Germany: lessons from a health care utilization database analysis.

To date, relatively little is known about the economic and medical impact of Lyme borreliosis (LB) on European health care systems, especially for the inpatient sector. This retrospective analysis is based on data provided for the years 2007-2011 by a German statutory health insurance company (DAK-Gesundheit) covering approximately 6 million insured. Total cost was calculated for a 1-year period both from the third-party payers and from the societal perspective, respectively. In our cohort the incident diagnosis of LB was coded for 2163 inpatient cases during the years 2008-2011. The median inpatient time was 9 days resulting in a median direct medical cost per hospital stay of 3917€ for adolescents and 2843€ for adults. Based on extrapolation of our findings to the German population, we would expect an average hospital admission of 5200 adults and 2300 adolescents (<18 years) for LB treatment incurring direct medical costs of more than 23 million Euro annually. The annual indirect costs due to loss of productivity would add up to more than 7 million Euro as assessed by the human capital method. Cases tended to accumulate between June and September with remarkable changes in disease manifestations in the course of the year documented in the coded secondary diagnoses. Also specific differences in the disease pattern of adolescents and adults became obvious. Age-specific incidence showed male predominance and a bimodal distribution. Incidence was highest in children aged between 3 and 17 (highest mean incidence of 29 cases/100,000 inhabitants in 6-9 year olds) with a second peak in 60-79 year old individuals. During the study period the nationwide inpatient incidence was 9/100,000 with marked regional variability. In summary, our study is one of the first European investigations on hospital care for LB inpatients and identifies LB as a possibly underestimated socioeconomic factor for health care in Germany.

Evaluating frequency, diagnostic quality, and cost of Lyme borreliosis testing in Germany: a retrospective model analysis.

BACKGROUND: Data on the economic impact of Lyme borreliosis (LB) on European health care systems is scarce. This project focused on the epidemiology and costs for laboratory testing in LB patients in Germany. MATERIALS AND METHODS: We performed a sentinel analysis of epidemiological and medicoeconomic data for 2007 and 2008. Data was provided by a German statutory health insurance (DAK) company covering approx. 6.04 million members. In addition, the quality of diagnostic testing for LB in Germany was studied. RESULTS: In 2007 and 2008, the incident diagnosis LB was coded on average for 15,742 out of 6.04 million insured members (0.26%). 20,986 EIAs and 12,558 immunoblots were ordered annually for these patients. For all insured members in the outpatient sector, a total of 174,820 EIAs and 52,280 immunoblots were reimbursed annually to health care providers (cost: 2,600,850€). For Germany, the overall expected cost is estimated at 51,215,105€. However, proficiency testing data questioned test quality and standardization of diagnostic assays used. CONCLUSION: Findings from this study suggest ongoing issues related to care for LB and may help to improve future LB disease management.

Commensal ecology, urban landscapes, and their influence on the genetic characteristics of city-dwelling Norway rats (Rattus norvegicus).

Movement of individuals promotes colonization of new areas, gene flow among local populations, and has implications for the spread of infectious agents and the control of pest species. Wild Norway rats (Rattus norvegicus) are common in highly urbanized areas but surprisingly little is known of their population structure. We sampled individuals from 11 locations within Baltimore, Maryland, to characterize the genetic structure and extent of gene flow between areas within the city. Clustering methods and a neighbour-joining tree based on pairwise genetic distances supported an east-west division in the inner city, and a third cluster comprised of historically more recent sites. Most individuals (approximately 95%) were assigned to their area of capture, indicating strong site fidelity. Moreover, the axial dispersal distance of rats (62 m) fell within typical alley length. Several rats were assigned to areas 2-11.5 km away, indicating some, albeit infrequent, long-distance movement within the city. Although individual movement appears to be limited (30-150 m), locations up to 1.7 km are comprised of relatives. Moderate F(ST), differentiation between identified clusters, and high allelic diversity indicate that regular gene flow, either via recruitment or migration, has prevented isolation. Therefore, ecology of commensal rodents in urban areas and life-history characteristics of Norway rats likely counteract many expected effects of isolation or founder events. An understanding of levels of connectivity of rat populations inhabiting urban areas provides information about the spatial scale at which populations of rats may spread disease, invade new areas, or be eradicated from an existing area without reinvasion.

Cryptic species within Anopheles longipalpis from southern Africa and phylogenetic comparison with members of the An. funestus group.

House-resting Anopheles mosquitoes are targeted for vector control interventions; however, without proper species identification, the importance of these Anopheles to malaria transmission is unknown. Anopheles longipalpis, a non-vector species, has been found in significant numbers resting indoors in houses in southern Zambia, potentially impacting on the utilization of scarce resources for vector control. The identification of An. longipalpis is currently based on classical morphology using minor characteristics in the adult stage and major ones in the larval stage. The close similarity to the major malaria vector An. funestus led to investigations into the development of a molecular assay for identification of An. longipalpis. Molecular analysis of An. longipalpis from South Africa and Zambia revealed marked differences in size and nucleotide sequence in the second internal transcribed spacer (ITS2) region of ribosomal DNA between these two populations, leading to the conclusion that more than one species was being analysed. Phylogenetic analysis showed the Zambian samples aligned with An. funestus, An. vaneedeni and An. parensis, whereas the South African sample aligned with An. leesoni, a species that is considered to be more closely related to the Asian An. minimus subgroup than to the African An. funestus subgroup. Species-specific primers were designed to be used in a multiplex PCR assay to distinguish between these two cryptic species and members of the An. funestus subgroup for which there is already a multiplex PCR assay.

Feeding and indoor resting behaviour of the mosquito Anopheles longipalpis in an area of hyperendemic malaria transmission in southern Zambia.

Anopheles longipalpis (Theobald) (Diptera: Culicidae) is a predominantly zoophilic mosquito that has not been implicated in malaria transmission. However, this species was collected indoors with An. funestus s.l. in southern Zambia, where transmission of Plasmodium falciparum is hyperendemic, and we initially misidentified it morphologically and molecularly as An. funestus s.l. The indoor resting density and blood-feeding behaviour of An. longipalpis were investigated during the 2004-05 and 2005-06 transmission seasons in Mufwafwi village in southern Zambia. Numbers of endophilic An. longipalpis increased towards the end of the rainy season. Although specimens were collected during human landing catches, the feeding behaviour of An. longipalpis was significantly biased towards cattle (88.7%), with other bloodmeals originating from dogs, goats and chickens. None of the 177 specimens of An. longipalpis were infected with P. falciparum. These data are consistent with existing reports that An. longipalpis is not involved in malaria transmission. However, more extensive sampling is necessary. Importantly, the correct identification of An. longipalpis is crucial for malaria control programmes in areas where An. funestus s.l and An. longipalpis exist sympatrically so that scarce resources are not wasted on the control of a non-vector.

Genetic differentiation of Anopheles gambiae s.s. populations in Mali, West Africa, using microsatellite loci.

Anopheles gambiae sensu stricto is a principal vector of malaria through much of sub-Saharan Africa, where this disease is a major cause of morbidity and mortality in human populations. Accordingly, population sizes and gene flow in this species have received special attention, as these parameters are important in attempts to control malaria by impacting its mosquito vector. Past measures of genetic differentiation have sometimes yielded conflicting results, in some cases suggesting that gene flow is extensive over vast distances (6000 km) and is disrupted only by major geological disturbances and/or barriers. Using microsatellite DNA loci from populations in Mali, West Africa, we measured genetic differentiation over uniform habitats favorable to the species across distances ranging from 62 to 536 km. Gene flow was strongly correlated with distance (r(2) = 0.77), with no major differences among chromosomes. We conclude that in this part of Africa, at least, genetic differentiation for microsatellite DNA loci is consistent with traditional models of isolation by distance.

Phylogeography of the neotropical sand fly Lutzomyia longipalpis inferred from mitochondrial DNA sequences.

Sand flies in the Lutzomyia longipalpis species complex include the primary vector of Leishmania chagasi, the etiologic agent of visceral leishmaniasis in the Neotropics. Twelve L. longipalpis populations from South and Central America were compared using the cytochrome c oxidase I (COI) gene from the mitochondrial genome. The haplotype profiles for each population revealed that the majority of sequence variation was inter-population (98%) rather than intra-population, suggesting that sequence polymorphisms at the COI locus should provide excellent characters for the study of phylogenetic relationships among populations. Phylogenetic reconstruction using distance (neighbor-joining) and maximum parsimony analysis revealed the existence of four clades among the L. longipalpis populations studied: (1) Laran, (2) Brazilian, (3) cis-Andean and (4) trans-Andean. We suggest that these clades represent species. A biogeographical interpretation of the molecular phylogeny suggests that the process of speciation in the L. longipalpis complex began in the Pliocene, from a sub-Andean-Amazonian gene pool resulting from the Andean orogeny (formation of the East Andean Cordillera). The four clades probably diverged as a result of vicariance events that occurred throughout the late Pliocene and Pleistocene. We propose and discuss several historical scenarios, based on the biogeography and historical geology of Central and South America.

DNA analysis of transferred sperm reveals significant levels of gene flow between molecular forms of Anopheles gambiae.

Anopheles gambiae populations in west Africa are complex, being composed of multiple, sympatric subpopulations. Recent studies have failed to reveal significant genetic differences among subpopulations, stimulating a debate regarding the levels of gene flow among them. The observed homogeneity may be the consequence of substantial contemporary gene flow or it may be that reproductive isolation is complete, but too recent for the accumulation of significant levels of genic divergence. Here, we report the results of a study estimating contemporary levels of gene flow between An. gambiae subpopulations by analysing females and transferred sperm removed from their reproductive systems. A total of 251 female and associated sperm extracts was analysed from a single site in Mali. Two molecular forms of An. gambiae, the M- and S-forms, occurred in sympatry at this site. Overall, we found very strong positive assortative mating within forms, however, we did observe significant hybridization between forms. In the M subpopulation 2/195 females (1.03%) contained sperm from S-form males and in 55 S-form females we found one female containing M-form sperm (1.82%). We also identified a mated M xS hybrid adult female. From mating frequencies, we estimate the Nem between the M- and S-form at 16.8, and from the adult hybrid frequency at 5.6. These values are consistent with our earlier estimate, based on FST for 21 microsatellite loci in which Nem = 5.8. We conclude that the general lack of genetic divergence between the M and S subpopulations of An. gambiae can be explained entirely by contemporary gene flow.

Atmospheric scattering for varying degrees of saturation and turbulent intermittency.

Atmospheric turbulence is inherently inhomogeneous and intermittent. Short periods of high activity are embedded in longer periods of relative calm. Local spatial and temporal changes in sound speed associated with this intermittency increase the likelihood of measuring large values of scattered acoustic signals. Previous work successfully predicted the probability density functions (pdf's) of fully saturated, scattered signals measured within an acoustic shadow zone [Wilson et al., J. Acoust. Soc. Am. 99, 3393-3400 (1996)]. The more general case of incompletely saturated scattering is considered in this paper; using the Rice-Nagakami distribution a theory is developed. The predicted intensity pdf has two free parameters: one to describe the degree of intermittency and a second for the degree of saturation. For validation purposes, outdoor propagation measurements were made over a flat, hard ground at ranges of 146-283 m and at frequencies of 50-540 Hz. The saturation parameter was determined from the acoustic data and also estimated from the turbulence conditions. The degree of saturation increased with frequency, and measured intensity pdf's were found to be in excellent agreement with the theory.

Microsatellite DNA polymorphism and heterozygosity among field and laboratory populations of Anopheles gambiae ss (Diptera: Culicidae).

We compared microsatellite polymorphism at nine loci located on chromosome 3 among two colonies and a field population of Anopheles gambiae sensu stricto Giles mosquitoes. Numbers of microsatellite alleles observed at each locus and mean heterozygosities were drastically reduced among laboratory colonies. Genetic analysis of the field population used in this study revealed an unprecedented frequency of rare alleles (<0.05). In contrast, colony samples revealed large numbers of alleles with frequencies >0.50. Partitioning of field data to assess the impact of rare alleles, null alleles, and sample size on estimates of mean heterozygosity revealed the plasticity of this measurement and suggests that heterozygosity may be reliably estimated from relatively small collections using microsatellites.

Gene flow among populations of the malaria vector, Anopheles gambiae, in Mali, West Africa.

The population structure of the Anopheles gambiae complex is unusual, with several sibling species often occupying a single area and, in one of these species, An. gambiae sensu stricto, as many as three "chromosomal forms" occurring together. The chromosomal forms are thought to be intermediate between populations and species, distinguishable by patterns of chromosome gene arrangements. The extent of reproductive isolation among these forms has been debated. To better characterize this structure we measured effective population size, N(e), and migration rates, m, or their product by both direct and indirect means. Gene flow among villages within each chromosomal form was found to be large (N(e)m > 40), was intermediate between chromosomal forms (N(e)m approximately 3-30), and was low between species (N(e)m approximately 0.17-1.3). A recently developed means for distinguishing among certain of the forms using PCR indicated rates of gene flow consistent with those observed using the other genetic markers.

Sibling species in the Llutzomyia longipalpis complex differ in levels of mRNA expression for the salivary peptide, maxadilan.

Maxadilan is a small ( approximately 7 kDa) protein found in the saliva of sand fly species in the Lutzomyia longipalpis complex, vectors of the parasite causing visceral leishmaniasis, Leishmania chagasi. It is a potent vasodilator and also has immunomodulatory affects. Maxadilan recovered from different sibling species of the Lu. longipalpis complex differ in amino acid content by as much as 23%, however all variants possess equivalent vasodilatory activity. Therefore, the dramatic differences in vasodilatory activity of the saliva from different sibling species is probably due to differences in the amounts of maxadilan in their saliva. This is significant because it has been suggested that maxadilan may influence the pathogenesis of leishmanial infections. In this study we measured the amount of maxadilan messenger RNA (mRNA) per pair of salivary glands from individual sand flies by quantitative reverse transcription polymerase chain reaction (RT-PCR) using a competitive method. We report a method using the gene of interest, in this case maxadilan, amplified by the PCR from genomic DNA, as a competitor in the quantitative RT-PCR, taking advantage of differences in the size of these products due to the presence of an intron. Significant differences in amounts of maxadilan mRNA among colonies from Central and South America are described. We found a strong correlation between the amount of maxadilan mRNA detected in salivary glands of different Lu. longipalpis sibling species and previously described differences in the size of erythemas produced by the bite of these species. Therefore, variation in the amount of mRNA suggests that differences in the vasodilatory properties of saliva among the different sibling species are the result of differences in the amount of maxadilan present in the saliva and not differences in the potency of maxadilan peptide variants. The geographical distribution of species with high or low levels of maxadilan gene expression are concordant with the distribution of atypical cutaneous leishmaniasis resulting from infection with Le. chagasi, lending credence to earlier suggestions that maxadilan may be involved with visceralization of this parasite.

Population genetics and phylogenetic analysis of Colorado Borrelia burgdorferi.

Borrelia burgdorferi is transmitted in an enzootic cycle in Colorado between the tick Ixodes spinipalpis and the woodrat Neotoma mexicana. The genetic relationship of Colorado isolates to other B. burgdorferi isolates is unknown nor have relationships among various Colorado isolates been determined. Portions of the flagellin (fla), 66-kD protein, and outer surface protein A (ospA) genes were amplified from 71 Colorado isolates, screened for genetic variability using single strand conformation polymorphism analysis, and unique alleles were sequenced. Colorado isolates were most similar to tick isolates from California and New York isolate 25015. Genetic distances among Colorado ospA sequences were the same or higher than distances among other isolates whereas distances among fla sequences tended to be the same or lower. The index of association (I(A)) was calculated among all loci as a measure of clonality. The I(A) among Colorado isolates was similar to I(A) previously estimated among other United States isolates.

Culturing selects for specific genotypes of Borrelia burgdorferi in an enzootic cycle in Colorado.

In Colorado, Borrelia burgdorferi sensu stricto, the etiologic agent of Lyme disease, is maintained in an enzootic cycle between Ixodes spinipalpis ticks and Neotoma mexicana rats (27). The frequencies of flagellin (fla), 66-kDa protein (p66), and outer surface protein A (ospA) alleles were examined in 71 B. burgdorferi isolates from samples from Colorado. Approximately two-thirds of these samples were isolates from I. spinipalpis ticks that had been cultured in BSK-H medium prior to DNA extraction. The remaining samples were from total DNA extracted directly from infected I. spinipalpis ticks. A portion of each gene was amplified by PCR and screened for genetic variability by single-strand conformation polymorphism (SSCP) analysis. We identified three alleles in the fla gene, seven in the p66 gene, and seven in the ospA gene. Sequencing verified that the amplified products originated from B. burgdorferi template DNA and indicated 100% sensitivity and specificity of the SSCP analysis. The frequencies of the p66 and ospA alleles were significantly different between cultured and uncultured spirochetes. The number of three-locus genotypes and the genetic diversity of alleles at all loci were consistently lower in cultured spirochetes, suggesting that culturing of B. burgdorferi in BSK-H medium may select for specific genotypes.

Taxonomic status of Ixodes neotomae and I. spinipalpis (Acari: Ixodidae) based on mitochondrial DNA evidence.

Ixodes spinipalpis Hadwen & Nuttall and I. neotomae Cooley are enzootic vectors of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner in western North America. The taxa overlap in host preference, habitat, and morphology. Mitochondrial DNA was compared between the taxa to test for reproductive isolation. A 300-bp region of the mitochondrial 16S ribosomal DNA gene was amplified in 28 specimens of I. neotomae and 149 specimens of I. spinipalpis. These products were screened for sequence variation using single-strand conformation polymorphism analysis, and 9 haplotypes were detected. Haplotype frequencies varied between taxa; however, Shannon diversity analysis indicated that most variation arose among collections within each taxon, and no unique haplotypes characterized either one. Phylogenetic analysis of 18 sequences, representing a replicate of each of the 9 haplotypes, was performed with Ixodes pacificus Cooley & Kohls and Ixodes jellisoni Cooley & Kohls as outgroups. Strong monophyletic support was found for a clade containing I. neotomae and I spinipalpis and within this clade no lineages comprised exclusively either taxon. These results argue against treatment of I. neotomae and I. spinipalpis as distinct species.

Acquired resistance and antibody response of raccoons (Procyon lotor) to sequential feedings of Ixodes scapularis (Acari: Ixodidae).

Captive-bred raccoons (Procyon lotor) developed immune resistance to infestation by the larval stage of the ixodid tick, Ixodes scapularis, the vector of Borrelia burgdorferi, following repeated applications of both nymphs and larvae. Resistance was expressed as a significant decrease in the proportion of engorged larvae recovered from each cohort. Resistance to nymphs was not noted, but, only two such cohorts were applied. Utilizing an enzyme-linked immunosor-bent assay (ELISA) developed to detect raccoon serum antibodies to tick salivary gland antigens, raccoons evidenced a two to ten-fold increase in anti-tick salivary extract antibody titer following the application of two cohorts of nymphs and eights cohorts of larvae. The tick saliva antigens recognized by both pre- and post-exposure raccoon sera were evaluated by Western blotting. The production of antibodies correlated with the development of resistance to infestation, suggesting that the resistance was immune-mediated and could be measured by anti-tick salivary extract antibody titers. Resistance in exposed raccoons prevents nearly 90% of larvae from prolonged feeding. Prolonged feeding is required for engorgement and the transmission of various infectious agents, such as B. burgdorferi.

Experimental infection of the raccoon (Procyon lotor) with Borrelia burgdorferi.

The reservoir competence of the raccoon (Procyon lotor) for the Lyme disease spirochete (Borrelia burgdorferi) was evaluated in the laboratory during September 1991 to April 1993. Five raccoons were exposed to spirochete-infected (JD1 and Wisconsin 210 Wise strains) Ixodes scapularis nymphs (20/raccoon). A second feeding of spirochete-infected (Wisconsin 210 Wise strain) nymphs (20/raccoon) was performed with four of the original raccoons. Xenodiagnosis with cohorts of I. scapularis larvae (300/cohort) or nymphs (150/cohort) that were periodically placed on each animal was used to detect infection. We examined 1943 engorged ticks by a indirect immunofluorescence monoclonal antibody assay, but no spirochetes were detected. After exposure to spirochete-infected ticks, blood samples were collected at approximately weekly intervals and ear-skin biopsy samples were taken from each animal every third week. These tissues were placed in Barbour-Stoenner-Kelly media. Spirochetes were isolated in cultures of skin (wk 3, 5, 9, 81, and 83) and blood (wk 5, 8, 9, 11, and 12) of one raccoon and the skin (wk 28 and 31) of another raccoon. Antibody response of each animal was monitored through enzyme-linked immunosorbent assays and immunoblotting of blood serum against B. burgdorferi proteins. Except for one animal, raccoons did not have an antibody response until they were fed upon by a second cohort of infected I. scapularis nymphs. Based on Western blot analyses, raccoons exposed to B. burgdorferi via tick bite responded to the 31- (OspA) and 34-KDa (OspB) antigens. Response to other antigens varied among raccoons. Based on our results raccoons may be inefficient reservoirs for B. burgdorferi. Although some raccoons can become infected with B. burgdorferi, they may not transfer the infection to attached ticks.

Population genetics of Ixodes scapularis (Acari: Ixodidae) based on mitochondrial 16S and 12S genes.

Blacklegged tick, Ixodes scapularis Say, is the principal vector of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner in the eastern half of the United States. Populations exhibit extreme variation in morphology, host usage, development time, and behavior. We examined sequence variation in the 16S and 12S mitochondrial ribosomal DNA genes to determine genetic relationships among I. scapularis collections from throughout its range. Single strand conformation polymorphism analysis of 300 bp of the 16S molecule was used to identify different haplotypes and estimate their relative frequencies among 198 ticks. Eleven different haplotypes were detected. Haplotype diversity was least in northeastern collections and greatest in the southeast. The 11 haplotypes were sequenced in 24 specimens. In total, 462 bp in the 16S gene and 420 bp in the 12S gene were sequenced to reveal 66 informative sites. Phylogenetic analysis, using I. ricinus L. and I. pacificus Cooley & Kohls as outgroups, revealed 2 clades within I. scapularis. One clade was limited to the South and the other was distributed throughout the range of I. scapularis. Specimens from the Southern United States were basal in the broadly distributed clade. Random amplified polymorphic DNA by polymerase chain reaction patterns examined between members of the 2 clades provided no evidence for reproductive isolation. These patterns suggest that I. scapularis arose in the South but that a large geographic split gave rise to 2 distinct lineages. These lineages now interbreed and are partially sympatric.