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Background West Nile virus (WNV) is the leading cause of mosquito-borne illness in the continental USA. WNV occurrence has high spatiotemporal variation, and current approaches to targeted control of the virus are limited, making forecasting a public health priority. However, little research has been done to compare strengths and weaknesses of WNV disease forecasting approaches on the national scale. We used forecasts submitted to the 2020 WNV Forecasting Challenge, an open challenge organized by the Centers for Disease Control and Prevention, to assess the status of WNV neuroinvasive disease (WNND) prediction and identify avenues for improvement. Methods We performed a multi-model comparative assessment of probabilistic forecasts submitted by 15 teams for annual WNND cases in US counties for 2020 and assessed forecast accuracy, calibration, and discriminatory power. In the evaluation, we included forecasts produced by comparison models of varying complexity as benchmarks of forecast performance. We also used regression analysis to identify modeling approaches and contextual factors that were associated with forecast skill. Results Simple models based on historical WNND cases generally scored better than more complex models and combined higher discriminatory power with better calibration of uncertainty. Forecast skill improved across updated forecast submissions submitted during the 2020 season. Among models using additional data, inclusion of climate or human demographic data was associated with higher skill, while inclusion of mosquito or land use data was associated with lower skill. We also identified population size, extreme minimum winter temperature, and interannual variation in WNND cases as county-level characteristics associated with variation in forecast skill. Conclusions Historical WNND cases were strong predictors of future cases with minimal increase in skill achieved by models that included other factors. Although opportunities might exist to specifically improve predictions for areas with large populations and low or high winter temperatures, areas with high case-count variability are intrinsically more difficult to predict. Also, the prediction of outbreaks, which are outliers relative to typical case numbers, remains difficult. Further improvements to prediction could be obtained with improved calibration of forecast uncertainty and access to real-time data streams (e.g. current weather and preliminary human cases). Graphical Abstract

The disaccharide galactose-α-1,3-galactose (alpha-gal) is expressed in mammals other than humans, apes, and old-world monkeys. In humans, elevated immunoglobulin-E (IgE) antibodies specific for alpha-gal can result in allergic hypersensitivity known as alpha-gal syndrome (AGS). Case reports and series suggest that tick bites can induce alpha-gal specific IgE antibodies.Evaluate tick exposure as a risk factor for AGS and elevated alpha-gal specific IgE (sIgE).We conducted a case-control study comparing AGS patients from a North Carolina allergy clinic with controls who were patients at a nearby internal medicine clinic. Cases and controls were administered a questionnaire to obtain information about demographics, home environment, outdoor activities, and recollection of tick bite. Serum samples taken at the time of enrollment were tested for total IgE, alpha-gal sIgE, and antibodies to other tickborne pathogens.AGS patients were more likely to recall finding a tick on themselves (OR=11.20, 95% CI 4.97-25.15), live near wooded forest (OR=2.27, 95% CI 0.92-5.55), and spend 17 or more hours per week outdoors in wooded areas (OR=5.58, 95% CI 2.56-12.19). AGS patients were also more likely to report 4 or more tick bites (OR=33.05, 95% CI 9.92-155.12) and reactions at the site of tick bites (OR=7.93, 95% CI 3.74-16.80). Elevated alpha-gal sIgE was also observed in 33% of controls and was also associated with tick exposure in the controls (OR=4.25, 95% CI 2.21-8.18).The results define tick bite as a risk factor for AGS and elevated alpha-gal sIgE.

Alpha-gal syndrome (AGS) is an IgE-mediated allergy to galactose-alpha-1,3-galactose. Clinical presentation ranges from hives to anaphylaxis; episodes typically occur 2-6 h after exposure to alpha-gal-containing products. In the United States, lone star tick bites are associated with the development of AGS. To characterize features of AGS, we evaluated a cohort of patients presenting for care at the University of North Carolina, focusing on symptoms, severity, and identifying features unique to specific alpha-gal-containing product exposures.We performed a chart review and descriptive analysis of 100 randomly selected patients with AGS during 2010-2019.Median age at onset was 53 years, 56% were female, 95% reported White race, 86% reported a history of tick bite, and 75% met the criteria for anaphylaxis based on the involvement of ≥2 organ systems. Those reporting dairy reactions were significantly less likely to report isolated mucocutaneous symptoms (3% vs. 24%; ratio [95% CI]: 0.1 [0.1, 0.3]) than those who tolerated dairy, and were more likely to report gastrointestinal symptoms (79% vs. 59%; ratio [95% CI]: 1.3 [0.7, 2.6]), although this difference was not statistically significant. Dairy-tolerant patients demonstrated higher alpha-gal sIgE titers (as a percentage of total IgE) than dairy-reactive patients (GM 4.1 [95% CI: 2.7, 6.1] vs. GM 2.5 [95% CI: 1.3, 4.8], respectively; ratio -1.6 [95% CI: -1.0, 3.9]).While tick exposure is common in the southern United States, nearly all AGS patients reported a tick bite. Gastrointestinal symptoms were prominent among those reporting reactions to dairy. Anaphylaxis was common, underscoring the severity and need to raise awareness of AGS among patients and providers.

Plague, a fleaborne rodent-associated zoonosis, is a neglected disease with most recent cases reported from east and central Africa and Madagascar. Because of its low incidence and sporadic occurrence, most of our knowledge of plague ecology, prevention, and control derives from investigations conducted in response to human cases. Long-term studies (which are uncommon) are required to generate data to support plague surveillance, prevention, and control recommendations. Here we describe a 15-year, multidisciplinary commitment to plague in the West Nile region of Uganda that led to significant advances in our understanding of where and when persons are at risk for plague infection and how to reduce morbidity and mortality. These findings provide data-driven support for several existing recommendations on plague surveillance and prevention and may be generalizable to other plague foci.

Background Vector-borne diseases are an important cause of morbidity and mortality in the USA. Effective, convenient prevention methods are needed. Long-lasting permethrin-impregnated (LLPI) clothing can prevent tick bites, however, additional information is needed on the real-world effectiveness and safety of this preventative measure. Methods In this pilot study, we recruited state and county park employees from North Carolina to wear LLPI uniforms for three months during the summer of 2016. We collected spot urine samples for biomonitoring of permethrin metabolites at one week, one month and three months after first use of the LLPI uniform. Following three months of wear, we collected pants and socks and analyzed them for permethrin content and mortality to ticks and mosquitoes. Results Thirteen park employees were included in the analysis. Bioactive amounts of permethrin remained in all clothing swatches tested, although there was great variability. Tick mortality was high, with 78% of pant and 88% of sock swatches having mean knockdown percentages ≥ 85%. In contrast, mosquito mortality was low. Over the study period, the absorbed dosage of permethrin averaged < 4 μg/kg/d of body weight based on measurements of three metabolites. Conclusions LLPI clothing retained permethrin and bioactivity against ticks after three months of use in real-world conditions. The estimated absorbed dosage of permethrin was well below the U.S. EPA level of concern, suggesting that LLPI clothing can be used safely by outdoor workers for tick bite prevention. Electronic supplementary material The online version of this article (10.1186/s13071-019-3314-1) contains supplementary material, which is available to authorized users.

Background Alpha-gal syndrome (AGS) is an emerging immunoglobulin E (IgE)–mediated allergy to galactose-alpha-1,3-galactose (alpha-gal). The geographic distribution and burden of AGS in the United States are unknown. Objective To characterize alpha-gal IgE testing patterns and describe the trends and distribution from 2010 to 2018 in the United States. Methods This retrospective analysis included all persons tested for alpha-gal IgE antibodies by Viracor-IBT Laboratories (Lee’s Summit, Missouri), the primary site of testing in the United States. Data included age and sex of person tested, specimen state of origin, collection date, and result value; persons with at least 1 positive test result (≥0.1 kU/L) were compared with negatives. Proportions tested and with positive test results were calculated using the US Census population estimates. Results Overall, 122,068 specimens from 105,674 persons were tested for alpha-gal IgE during July 1, 2010, to December 31, 2018. Nearly one-third (34,256, 32.4%) had at least 1 positive result. The number of persons receiving positive test results increased 6-fold from 1110 in 2011 to 7798 in 2018. Of those receiving positive test results, mean [SD] age was 46.9 (19.8) years; men were more likely to test positive than women (43.3% vs 26.0%). Arkansas, Virginia, Kentucky, Oklahoma, and Missouri had the highest number of persons who were tested and had a positive result per 100,000 population. Conclusion More than 34,000 persons, most presumably symptomatic, have received positive test results for IgE antibodies to alpha-gal, suggesting AGS is an increasingly recognized public health problem. The geographic distribution of persons who tested positive is consistent with exposure to Amblyomma americanum ticks.

CDC has updated its interim guidance for U.S. health care providers caring for pregnant women with possible Zika virus exposure, to include the emerging data indicating that Zika virus RNA can be detected for prolonged periods in some pregnant women. To increase the proportion of pregnant women with Zika virus infection who receive a definitive diagnosis, CDC recommends expanding real-time reverse transcription-polymerase chain reaction (rRT-PCR) testing. Possible exposures to Zika virus include travel to or residence in an area with active Zika virus transmission, or sex* with a partner who has traveled to or resides in an area with active Zika virus transmission without using condoms or other barrier methods to prevent infection.(†) Testing recommendations for pregnant women with possible Zika virus exposure who report clinical illness consistent with Zika virus disease(§) (symptomatic pregnant women) are the same, regardless of their level of exposure (i.e., women with ongoing risk for possible exposure, including residence in or frequent travel to an area with active Zika virus transmission, as well as women living in areas without Zika virus transmission who travel to an area with active Zika virus transmission, or have unprotected sex with a partner who traveled to or resides in an area with active Zika virus transmission). Symptomatic pregnant women who are evaluated

The blacklegged tick, Ixodes scapularis Say, is the primary vector to humans in the eastern United States of the Lyme disease spirochete Borrelia burgdorferi, as well as causative agents of anaplasmosis and babesiosis. Its close relative in the far western United States, the western blacklegged tick Ixodes pacificus Cooley and Kohls, is the primary vector to humans in that region of the Lyme disease and anaplasmosis agents. Since 1991, when standardized surveillance and reporting began, Lyme disease case counts have increased steadily in number and in geographical distribution in the eastern United States. Similar trends have been observed for anaplasmosis and babesiosis. To better understand the changing landscape of risk of human exposure to disease agents transmitted by I. scapularis and I. pacificus, and to document changes in their recorded distribution over the past two decades, we updated the distribution of these species from a map published in 1998. The presence of I. scapularis has now been documented from 1,420 (45.7%) of the 3,110 continental United States counties, as compared with 111 (3.6%) counties for I. pacificus. Combined, these vectors of B. burgdorferi and other disease agents now have been identified in a total of 1,531 (49.2%) counties spread across 43 states. This marks a 44.7% increase in the number of counties that have recorded the presence of these ticks since the previous map was presented in 1998, when 1,058 counties in 41 states reported the ticks to be present. Notably, the number of counties in which I. scapularis is considered established (six or more individuals or one or more life stages identified in a single year) has more than doubled since the previous national distribution map was published nearly two decades ago. The majority of county status changes occurred in the North-Central and Northeastern states, whereas the distribution in the South remained fairly stable. Two previously distinct foci for I. scapularis in the Northeast and North-Central states appear to be merging in the Ohio River Valley to form a single contiguous focus. Here we document a shifting landscape of risk for human exposure to medically important ticks and point to areas of re-emergence where enhanced vector surveillance and control may be warranted.

Lyme disease has increased both in incidence and geographic extent in the United States and Canada over the past two decades. One of the underlying causes is changes during the same time period in the distribution and abundance of the primary vectors: Ixodes scapularis Say and Ixodes pacificus Cooley and Kohls in eastern and western North America, respectively. Aside from short periods of time when they are feeding on hosts, these ticks exist in the environment where temperature and relative humidity directly affect their development, survival, and host-seeking behavior. Other important factors that strongly influence tick abundance as well as the proportion of ticks infected with the Lyme disease spirochete, Borrelia burgdorferi, include the abundance of hosts for the ticks and the capacity of tick hosts to serve as B. burgdorferi reservoirs. Here, we explore the linkages between climate variation and: 1) duration of the seasonal period and the timing of peak activity; 2) geographic tick distributions and local abundance; 3) enzootic B. burgdorferi transmission cycles; and 4) Lyme disease cases. We conclude that meteorological variables are most influential in determining host-seeking phenology and development, but, while remaining important cofactors, additional variables become critical when exploring geographic distribution and local abundance of ticks, enzootic transmission of B. burgdorferi, and Lyme disease case occurrence. Finally, we review climate change-driven projections for future impact on vector ticks and Lyme disease and discuss knowledge gaps and research needs.

Serological assays and a two-tiered test algorithm are recommended for laboratory confirmation of Lyme disease. In the United States, the sensitivity of two-tiered testing using commercially available serology-based assays is dependent on the stage of infection and ranges from 30% in the early localized disease stage to near 100% in late-stage disease. Other variables, including subjectivity in reading Western blots, compliance with two-tiered recommendations, use of different first- and second-tier test combinations, and use of different test samples, all contribute to variation in two-tiered test performance. The availability and use of sample sets from well-characterized Lyme disease patients and controls are needed to better assess the performance of existing tests and for development of improved assays. To address this need, the Centers for Disease Control and Prevention and the National Institutes of Health prospectively collected sera from patients at all stages of Lyme disease, as well as healthy donors and patients with look-alike diseases. Patients and healthy controls were recruited using strict inclusion and exclusion criteria. Samples from all included patients were retrospectively characterized by two-tiered testing. The results from two-tiered testing corroborated the need for novel and improved diagnostics, particularly for laboratory diagnosis of earlier stages of infection. Furthermore, the two-tiered results provide a baseline with samples from well-characterized patients that can be used in comparing the sensitivity and specificity of novel diagnostics. Panels of sera and accompanying clinical and laboratory testing results are now available to Lyme disease serological test users and researchers developing novel tests.

Local knowledge of when humans are at elevated risk for exposure to tick vectors of human disease agents is required both for the effective use of personal protection measures to avoid tick bites and for implementation of control measures to suppress host-seeking ticks. Here, we used previously published data on the seasonal density of host-seeking Ixodes pacificus Cooley and Kohls nymphs, the primary vectors of Lyme disease spirochetes in the far western USA, collected across a broad habitat and climate gradient in northwestern California to identify predictors of periods of time within the year when questing nymphal density is elevated. Models based on calendar week alone performed similarly to models based on calendar week and woodland type, or meteorological variables. The most suitable model for a given application will depend on user objectives, timescale of interest, and the geographic extent of predictions. Our models sought not only to identify when seasonal host-seeking activity commences, but also when it diminishes to low levels. Overall, we report a roughly 5–7 month period in Mendocino County during which host-seeking nymphal densities exceed a low threshold value.

Lyme disease, which is caused by the spirochetal agent Borrelia burgdoferi, is the most common vector-borne illness in the United States. In 1998, the US Food and Drug Administration approved a recombinant Lyme disease vaccine that was later voluntarily withdrawn from the market by the manufacturer. Current Lyme disease prevention efforts focus on a combination of methods and approaches, including area acaricides, landscape management, host-targeted interventions, management of deer populations, and personal protective measures, such as the use of insect repellant and tick checks. Although these methods are generally safe and relatively inexpensive, the primary limitations of these methods are that their effectiveness has been difficult to demonstrate conclusively and that rates of compliance are generally poor. An effective human Lyme disease vaccine that has been adequately evaluated in the highest-risk population groups could be very beneficial in preventing Lyme disease; however, it would need to meet high standards regarding safety, efficacy, cost, and public acceptance.

Background: When Pneumocystis DNA is recovered from respiratory specimens of patients without Pneumocystis pneumonia (PCP), patients are said to be colonised with Pneumocystis , although the significance of this state is unknown. Understanding risk factors for and outcomes of colonisation may provide insights into the life cycle and transmission dynamics of Pneumocystis jirovecii . Methods: We performed a cross sectional study of the prevalence and clinical predictors of Pneumocystis colonisation in 172 HIV infected, PCP negative inpatients undergoing diagnostic evaluation of 183 episodes of pneumonia at either the Medical Center of Louisiana at New Orleans between 2003 and 2005 or San Francisco General Hospital between 2000 and 2005. DNA was extracted from sputum and bronchoalveolar lavage specimens and amplified using a nested PCR assay at the mitochondrial large subunit (18S) ribosomal RNA locus. Colonisation was deemed present if Pneumocystis DNA was identified by both gel electrophoresis and direct DNA sequencing. Results: 68% (117/172) of all patients were colonised with Pneumocystis . No strong associations with colonisation were identified for any demographic factors. Among clinical factors, having a CD4+ T cell count ⩽50 cells/μl (unadjusted OR 2.4, 95% CI 1.09 to 5.48; p = 0.031) and using PCP prophylaxis (unadjusted OR 0.55, 95% CI 0.29 to 1.07; p = 0.077) were associated with Pneumocystis colonisation, although the latter association may have been due to chance. After adjustment for CD4+ T cell count, use of PCP prophylaxis was associated with a decreased odds of colonisation (adjusted OR 0.45, 95% CI 0.21 to 0.98; p = 0.045). 11 patients who were colonised were subsequently readmitted for evaluation of a second episode of pneumonia; three were found to be colonised again, but none had PCP. Conclusions: The majority of hospitalised HIV infected patients with non-PCP pneumonia are colonised with Pneumocystis . Failure to use co-trimoxazole prophylaxis and severe immunosuppression are associated with an increase in the odds of colonisation. Pneumocystis colonisation among hospitalised patients does not commonly lead to PCP.

Pneumocystis pneumonia (PCP), caused by infection with Pneumocystis jirovecii, remains an important opportunistic infection in humans. A reverse transcriptase polymerase chain reaction assay has been shown to specifically detect viable P. jirovecii organisms. In the current study, we evaluated this assay on different types of respiratory samples. The assay had a diagnostic sensitivity of 100% and a specificity of 86% when applied to bronchoalveolar lavage samples. The assay's performance declined when applied to less invasive induced sputum and oropharyngeal wash (OPW) samples. The sensitivity, when applied to OPWs, was improved by examining multiple sequential OPW samples and was affected by clinical sampling parameters that could increase or decrease the number of potential organisms in the oropharynx. When used in conjunction with an optimized clinical sampling protocol, this assay may become a useful tool for detecting and monitoring P. jirovecii in minimally invasive clinical samples.

Polymerase chain reaction analysis, direct DNA sequencing, and histological staining were used to determine whether Pneumocystis jirovecii was present in lung tissue specimens obtained, at autopsy, from 58 infants without identifiable immunodeficiency. The results of genotyping of these specimens were compared with the results of genotyping of specimens obtained from 384 human immunodeficiency virus (HIV)-infected adults with Pneumocystis pneumonia. P. jirovecii DNA was detected at the mitochondrial large subunit rRNA and dihydropteroate synthase loci in 100% and 53%, respectively, of the specimens obtained from infants. All specimens obtained from adults tested positive for P. jirovecii at both loci. Genotype distributions at both loci were significantly different in the 2 populations (P < .0001). The observation of different strains circulating in immunocompetent infants and HIV-infected adults suggests independent transmission cycles that warrant further study.

Ixodes scapularis transmits several pathogens including Borrelia burgdorferi. Bioactive compounds in tick saliva support tick feeding and influence pathogen transmission to the mammalian host. These studies utilized oral delivery of dsRNA to silence an anticomplement gene (isac) in I. scapularis nymphs. Silencing of isac significantly reduced fed-tick weight compared to delivery of control lacZ dsRNA, and immunoblots specific for FlaB protein indicated a reduction in spirochete load in isac-silenced infected nymphs. SDS-PAGE demonstrated that isac gene silencing affected expression of a number of salivary and non-salivary gland proteins in ticks. Finally, multiple isac cDNA homologues were cloned, and these may represent a new gene family coexpressed during tick feeding. This work presents a novel oral delivery approach for specific gene silencing in I. scapularis nymphs and characterizes the effect of isac on blood-feeding in an attempt to block transmission of B. burgdorferi.

Several typing methods, with different strengths and weaknesses, are available for studies of Pneumocystis pneumonia., Pneumocystis pneumonia (PCP) caused by the opportunistic fungal agent Pneumocystis jirovecii (formerly P. carinii) continues to cause illness and death in HIV-infected patients. In the absence of a culture system to isolate and maintain live organisms, efforts to type and characterize the organism have relied on polymerase chain reaction–based approaches. Studies using these methods have improved understanding of PCP epidemiology, shedding light on sources of infection, transmission patterns, and potential emergence of antimicrobial resistance. One concern, however, is the lack of guidance regarding the appropriateness of different methods and standardization of these methods, which would facilitate comparing results reported by different laboratories.

Triatoma brasiliensis (Hemiptera: Reduviidae: Triatominae) is the most important Chagas disease vector in the semiarid areas of Northeast Brazil. We analyzed mitochondrial cytochrome b sequence variation among 136 individuals representing 16 populations from across the species' distribution. Neighbor-joining and parsimony tree-building methods were used in conjunction with nested clade analysis to describe the systematics and phylogeography of this species. Our results indicate that T brasiliensis is composed of four genetically distinct chromatic forms (referred to as brasiliensis, macromelasoma, juazeiro, and melanica) that present inter-population divergence values (0.027-0.119, corrected K2-p) and a pattern of haplotype geographic distribution compatible with the existence of a species complex. As a consequence, such forms can be treated as isolated targets in vector control programs. We were unable to infer what is shaping the population structure of the brasiliensis form as we obtained mutually exclusive causes of structure, namely a barrier to gene flow caused by past population fragmentation, and isolation by distance between populations (which would permit gene flow). We found indication of mitochondrial DNA introgression occurring among forms in putative hybrid zones. (C) 2004 Elsevier Inc. All rights reserved.

Oral-wash samples obtained during 113 episodes of suspected Pneumocystis pneumonia (PCP) in human immunodeficiency virus-infected patients were tested by use of a quantitative touch-down PCR (QTD PCR) assay. QTD PCR had a sensitivity of 88% and a specificity of 85%. Treatment for PCP prior to oral wash collection had an impact on the sensitivity, and PCR-positive oral-wash samples obtained within < or =1 day of treatment from patients without PCP had significantly fewer copies per tube than did those from patients with PCP; thus, application of a post hoc cut-off value of 50 copies/tube increased the specificity to 100%. QTD PCR of oral-wash samples can be an accurate and noninvasive method for diagnosis of PCP.

Background: The epidemiology and transmission of Pneumocystis are poorly understood. The incidence of colonization, or detection of organisms without signs of disease, has been debated, and risk factors for colonization are largely unknown. Objective: To determine the rate of Pneumocystis colonization among HIV-infected patients at autopsy and analyze associated clinical variables. Methods: Subjects were selected from the Multicenter AIDS Cohort Study. Subjects who died from causes other than Pneumocystis pneumonia and consented to autopsy were included in analysis. DNA was extracted from lung tissue, and nested PCR was performed to detect the presence of Pneumocystis. Clinical data were obtained from the Multicenter AIDS Cohort database. Univariate and multivariate analyses were performed to determine predictors of Pneumocystis colonization. Results: Pneumocystis DNA was detected in 42 of 91 (46%) subjects by nested PCR. Clinical variables such as CD4 cell count, use of Pneumocystis prophylaxis or antiretroviral drugs, and history of previous Pneumocystis pneumonia were not related to risk of colonization. Multivariate analysis demonstrated that cigarette smoking was related to an increased risk of colonization [odds ratio (OR), 4.5; 95% confidence interval (Cl), 1.27-15.6; P = 0.02] and risk also varied by city of residence (OR, 0.12; 95% Cl, 0.03-0.45; P= 0.002 for living in Los Angeles). Conclusions: This study found a high rate of Pneumocystis colonization among HIV-infected patients. We also identified cigarette smoking and city of residence as novel, independent risk factors for colonization. The role of subclinical colonization in disease transmission and the effects of Pneumocystis colonization on the lung require further study.

To clarify the epidemiologic importance of Triatoma brasiliensis, the most important Chagas disease vector in the Northeastern of Brazil, capture data related to this species, its distribution, capture index, and percentages of natural infection by Trypanosoma cruzi were examined in 12 different Brazilian states. The Brazilian National Health Foundation collected these data from 1993 to 1999, a period during which a total of 1,591,280 triatomines (21 species) were captured in domiciles within the geographic range of T. brasiliensis. Of this total, 422,965 (26.6%) were T. brasiliensis, 99.8% of which were collected in six states, and 54% in only one state (Ceará). The percentage of bugs infected with T. cruzi varied significantly among states, ranging from 0% (Goiás, Maranhão, Sergipe, and Tocantins) to more than 3% (Alagoas, Minas Gerais, and Rio Grande do Norte) with an average of 1.3%. This latter value represents a dramatic reduction in the natural infection percentages since 1983 (6.7%) suggesting that, despite the impossibility of eradicating this native species, the control measures have significantly reduced the risk of transmission. However, the wide geographic distribution of T. brasiliensis, its high incidence observed in some states, and its variable percentages of natural infection by T. cruzi indicate the need for sustained entomological surveillance and continuous control measures against this vector.

The phylogeographical structure of the closely related species Rhodnius prolixus and R. robustus is presented based on a 663-base pair (bp) fragment of the mitochondrial cytochrome b gene. Twenty haplotypes were recovered from 84 samples examined, representing 26 populations from seven Latin American countries. The resulting phylogenetic tree is composed of five major reciprocally monophyletic clades, one representing R. prolixus and four representing R. robustus. While R. prolixus is a very homogeneous assemblage, R. robustus has deeper clades and is paraphyletic, with the clade comprising R. robustus from Venezuela (Orinoco region) more closely related to the R. prolixus clade than to the other R. robustus populations from the Amazon region. The R. robustus paraphyly was supported further by the analysis of a nuclear gene (D2 region of the 28S RNA) for a subset of specimens. The data support the view that R. robustus represents a species complex. Levels of sequence divergence between clades within each region are compatible with a Pleistocene origin. Nucleotide diversity (pi) for all R. prolixus populations was extremely low (0.0008), suggesting that this species went through a recent bottleneck, and was subsequently dispersed by man.

The disease known as Pneumocystis carinii pneumonia (PCP) is a major cause of illness and death in persons with impaired immune systems. While the genus Pneumocystis has been known to science for nearly a century, understanding of its members remained rudimentary until DNA analysis showed its extensive diversity. Pneumocystis organisms from different host species have very different DNA sequences, indicating multiple species. In recognition of its genetic and functional distinctness, the organism that causes human PCP is now named Pneumocystis jiroveci Frenkel 1999. Changing the organism's name does not preclude the use of the acronym PCP because it can be read "Pneumocystis pneumonia." DNA sequence variation exists among samples of P. jiroveci, a feature that allows reexamination of the relationships between host and pathogen. Instead of lifelong latency, transient colonization may be the rule.

In the context of emerging infectious diseases, opportunistic infections (OIs) have assumed importance because of immunocompromised populations that are either new or have increased over the past two decades. Such populations may be immunocompromised as the result of a pathologic process that occurs in human immunodeficiency virus (HIV) infection, or because of medical treatments that induce immunosuppression, such as radiation or chemotherapy for cancer or immunosuppressive therapy for organ transplantation. The challenges posed by Ols in HIV-infected persons are numerous and go beyond simple measurements of incidence of disease and costs of medical care. They are perhaps best illustrated by a discussion of two Ols that present a different array of challenges- Pneumocystis carinii pneumonia (PCP) and cryptosporidiosis. A variety of other conditions are associated with immunosuppression and increased susceptibility to OIs. Vaccinations pose challenges in terms of efficacy in inununocompromised persons and possible stimulation of HIV replication and acceleration of HIV disease. OIs pose significant challenges as emerging infectious diseases in increasing populations of immunocompromised persons, including persons receiving bone marrow or solid organ transplants, and the elderly.

Summary Background Investigators have reported that patients infected with Pneumocystis carinii containing mutations in the DHPS (dihydropteroate synthase) gene have a worse outcome than those infected with P carinii containing wild-type DHPS . We investigated patients with HIV-1 infection and P carinii pneumonia to determine if DHPS mutations were associated with poor outcomes in these patients. Methods We compared presence of mutations at the DHPS locus with survival and response of patients to co-trimoxazole or other drugs. Findings For patients initially given co-trimoxazole, nine (14%) of 66 with DHPS mutant died, compared with nine (25%) of 36 with wild type (risk ratio=0·55 [95% CI=0·24–1·25]; p=0·15). Ten (15%) of 66 patients with a DHPS mutant did not respond to treatment, compared with 13 (36%) of 36 patients with the wild type (0·42 [0·20–0·86]; p=0·02). For patients aged 40 years or older, four (14%) of 29 with the mutant and nine (56%) of 16 with the wild type died (0·25 [0·09–0·67]; p=0·005). Interpretation These results, by contrast with those of previous studies, suggest that patients with wild-type P carinii do not have a better outcome than patients with the mutant when given co-trimoxazole. Our results suggest that presence of a DHPS mutation should be only one of several criteria guiding the choice of initial drug treatment of P carinii pneumonia in patients with HIV-1 infection.

The 17 019 bp mitochondrial genome of Triatoma dimidiata is composed of thirteen protein coding sequences, twenty-two tRNAs, small and large ribosomal units, and a control region. The gene order and orientation are identical to that of Drosophila yakuba. The nucleotide composition is biased toward adenine and thymine (69.5% A + T). The 2.1 kb putative control region, known as the A + T rich region in most insects, has an A + T bias of 66%, but contains a 400 bp sequence that is 77.5% A + T and two other distinct regions: (1) one with a lower A + T bias (60.1%) and (2) a region of eight tandem repeat units. The identified 1.4 kb nuclear copy of mitochondrial sequences encompasses the string of Gs and the beginning of the cytochrome c oxidase 1 gene but lacks the 1.8 kb region spanning the eight tandem repeats and the 5' end of the NADH dehydrogenase subunit II gene.

Paratransgenesis, a strategy for control of certain arthropod-borne infectious diseases, involves expression of disease transmission-blocking molecules by genetically altered symbiotic bacteria of insect vectors. Field use of this approach relies on a method for dispersal of genetically transformed bacteria to their host insects. Rhodococcus rhodnii , a symbiotic bacterium of the Chagas disease vector, Rhodnius prolixus (Stal), has been transformed genetically to express several molecules. Here we report a strategy for dispersal of genetically altered R. rhodnii to target populations of R. prolixus that uses a synthetic substrate termed CRUZIGARD. This preparation mimics natural feces of R. prolixus and, when impregnated with genetically altered symbiotic bacteria, simulates the natural coprophagic method of symbiont dispersal used by the reduviid bug. CRUZIGARD laden with genetically altered symbionts was applied monthly to closed cages of R. prolixus nymphs. Uptake and retention of the recombinant bacteria was demonstrated under laboratory conditions and simulated field conditions in which competing environmental microbes were present. In the simulated field study conducted in Guatemala, nearly 50% of R. prolixus nymphs exposed to bacteria-laden CRUZIGARD retained the transgenic microbes throughout their development. In these paratransgenic insects, the genetically altered symbionts comprised nearly 95% of the bacterial colony forming units. No adverse effects of CRUZIGARD on insect development and fecundity were noted. CRUZIGARD represents a potentially powerful method for dispersal of recombinant symbiotic bacteria among field populations of the Chagas disease vector, R. prolixus .

Isolates from 25 (13 sporadic and 12 outbreak) cryptosporidiosis cases, 24 of which were from British Columbia, Canada, were characterized using nested polymerase chain reaction amplification of the polymorphic internal transcribed spacer 1 locus. Two predominant Cryptosporidium parvum genotypes were found. Twelve (8 sporadic and 4 outbreak) isolates amplified with the cry7/cry21 primer pair and 12 (5 sporadic and 7 outbreak) isolates amplified with the cry7/cryITS1 primer pair. Multi-locus gene analysis using sequence polymorphisms on 3 other loci, i.e., the thrombospondin-related adhesion protein gene, the dihydrofolate reductase gene, and the 18S rRNA gene on 8 (4 outbreak and 4 sporadic) isolates showed non-random association among the human and animal alleles of the 4 different C. parvum gene loci. Associations between these 2 parasite genotypes and different routes of cryptosporidiosis transmission such as zoonotic, anthroponotic, and waterborne transmission were studied using municipal population and agricultural information, as well as detection of C. parvum oocysts in municipal drinking water specimens of the residential communities of sporadic and outbreak cases.

Expression within insects of foreign antiparasitic gene products via microbial symbionts could be used to prevent transmission of vector-borne pathogens to vertebrate hosts. Genetically transformed symbiotic bacteria Rhodococcus rhodnii expressed functional antibody fragments (rDB3 encoding murine V(H)/K which binds progesterone) that were exported into the gut lumen of the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae), a vector of Chagas disease. Transgenic symbionts were maintained in successive nymphal instars and adults of Rhodnius prolixus despite competition with native untransformed Rhodococcus rhodnii. This is the first description of a functional mammalian antibody fragment expressed in an insect. Our system is a model for constructing paratransgenic insects (insects carrying transformed symbionts) with compromised ability to transmit pathogens.

Bacterial symbionts may be used as vehicles for expressing foreign genes in arthropods. Expression of selected genes can render an arthropod incapable of transmitting a second microorganism that is pathogenic for humans and is an alternative approach to the control of arthropod-borne diseases. We discuss the rationale for this alternative approach, its potential applications and limitations, and the regulatory concerns that may arise from its use in interrupting disease transmission in humans and animals.

We report the results of molecular analysis of 39 isolates of Cryptosporidium parvum from human and bovine sources in nine human outbreaks and from bovine sources from a wide geographic distribution. All 39 isolates could be divided into either of two genotypes, on the basis of genetic polymorphism observed at the thrombospondin-related adhesion protein (TRAP-C2) locus. Genotype 1 was observed only in isolates from humans. Genotype 2, however, was seen in calf isolates and in isolates from a subset of human patients who reported direct exposure to infected cattle or consumed items thought to be contaminated with cattle feces. Furthermore, experimental infection studies showed that genotype 2 isolates were infective to mice or calves under routine laboratory conditions, whereas genotype 1 isolates were not. These results support the occurrence of two distinct transmission cycles of C. parvum in humans.

Pneumocystis jirovecii dihydropteroate synthase (DHPS) gene mutations are well-reported. Although sulfa prophylaxis generally is associated with DHPS mutant infection, whether mutant infection is associated with poorer clinical outcomes is less clear. The differing definitions of sulfa prophylaxis and the different mortality endpoints used in these studies may be one explanation for the conflicting study results. Applying different definitions of prophylaxis, mortality endpoints and DHPS mutant to 301 HIV-infected patients with Pneumocystis pneumonia, we demonstrate that prophylaxis, irrespective of definition, increased the risk of infection with pure mutant (any prophylaxis: AOR 4.00, 95% CI: 1.83–8.76, p0.05). Future studies should standardize key variables associated with DHPS mutant infection as well as examine DHPS mutant subtypes (pure mutant vs. mixed infections) – perhaps even individual DHPS mutant genotypes – so that data can be pooled to better address this issue.

The polyubiquitin gene from the mosquito Anopheles gambiae has been cloned and sequenced, and its structure is reported along with sequence analysis results. The gene consists of approximately seven tandem head-to-tail repeat units of the seventy-six amino acid-coding ubiquitin monomer. It is expressed constitutively in larvae, pupae and adults of An. gambiae, as well as in a cell line derived from this mosquito species. A probe made from a DNA fragment containing the coding region of the gene recognizes transcripts of approximately 3.6 kb and 4.4 kb in RNA isolated from all mosquito developmental stages and a unique transcript of approximately 3.0 kb in RNA from the cell line. Single monomeric units of the An. gambiae polyubiquitin gene shared from 75.9% to 85.5% identity at the DNA level with homologous sequences from other organisms ranging from yeast to man. A comparison of individual repeat units of the An. gambiae gene revealed that, in general, the 5' ends of the individual monomers are more highly conserved than the 3' ends. The gene mapped by in situ hybridization on ovarian nurse cell polytene chromosomes to a primary site at division 12C on chromosome 2R and to a secondary site at division 9C on the same chromosome.

Vectorial transmission, first cases, diagnostic methods, treatment, epidemiology, serological surveys, cardiac disease, CF test, ECG, seropositive patients, transfusion transmission, accidental cases, immunosuppression, seropositive donors, Latin Americans, immigrants, Latin America, endemic areas, achalasia, megaesophagus, accidental infections, acute myocarditis, blood cultures, serological reactions, treatment

After critically reviewing old and new literature, a general picture of the situation of Chagas disease in the United States is presented by the authors. In the first two chapters, besides an historical background, several aspects of the local transmission potential by the vectors are presented; their distribution, present relationship with human dwellings, infection rates with T. cruzi , ethology and bionomics, and the severe allergic reactions they may cause are discussed. In another chapter, the zoonotic angle of the disease is considered; both wild and domestic reservoirs are described, and the epidemiological importance of some animals such as wood rats, opossums, raccoon, and dogs is stressed. There is a chapter dedicated to the different characteristics of the locally isolated parasites, including experimental infections and molecular genotype analysis. The final chapter discusses the present situation concerning the disease in human beings; specifically, indigenous cases, serological prevalence, blood transfusion, congenital and organ transplant transmission, the disease in immigrants, and laboratory-acquired infections. An overview is presented that considers the disease as a new endemic disease that is transmitted in certain areas of the country by a vector; the burden to the United States caused by Latin America immigrants who carry the disease with them is also considered. This book will help to create greater awareness about the status of Chagas disease in the United States and will help in the evaluation of certain parameters from the standpoints of ecoepidemiological and public health, facilitating future control actions.

Some of the world's most devastating diseases are transmitted by arthropod vectors. Attempts to control these arthropods are currently being challenged by the widespread appearance of insecticide resistance. It is therefore desirable to develop alternative strategies to complement existing methods of vector control. In this review, Charles Beard, Scott O'Neill, Robert Tesh, Frank Richards and Serap Aksoy present an approach for introducing foreign genes into insects in order to confer refractoriness to vector populations, ie. the inability to transmit disease-causing agents. This approach aims to express foreign anti-parasitic or anti-viral gene products in symbiotic bacteria harbored by insects. The potential use of naturally occurring symbiont-based mechanisms in the spread of such refractory phenotypes is also discussed.

Two isolates of bacterial endosymbionts, GP01 and GM02, were established in cell free medium from haemolymph of the tsetse, Glossina pallidipes and G. morsitans. These microorganisms appear similar to rickettsia-like organisms reported previously from various tsetse species. The 16S rRNA sequence analysis, however, placed them within the gamma subdivision of the Proteobacteria, phylogenetically distinct from most members of the Rickettsiaceae which align with the alpha subdivision. Distinct multiple endogenous plasmids are harboured by GP01 and GM02, suggesting that the two isolates are different. Restriction mapping analysis showed that one of the conserved plasmids is present in high copy number and is at least 80 kb in size. A heterologous plasmid pSUP204, which contains the broad host range oriV replication origin, was used to transfect bacterial cultures. The symbiont GM02 was transformed, and it expressed plasmid encoded resistance to the antibiotics ampicillin, tetracycline and chloramphenicol. Transformation of these symbionts may provide a novel means for expressing anti-parasitic genes within tsetse populations.

Plague, a life-threatening flea-borne zoonosis caused by Yersinia pestis, has most commonly been reported from eastern Africa and Madagascar in recent decades. In these regions and elsewhere, prevention and control efforts are typically targeted at fine spatial scales, yet risk maps for the disease are often presented at coarse spatial resolutions that are of limited value in allocating scarce prevention and control resources. In our study, we sought to identify sub-village level remotely sensed correlates of elevated risk of human exposure to plague bacteria and to project the model across the plague-endemic West Nile region of Uganda and into neighboring regions of the Democratic Republic of Congo. Our model yielded an overall accuracy of 81%, with sensitivities and specificities of 89% and 71%, respectively. Risk was higher above 1,300 meters than below, and the remotely sensed covariates that were included in the model implied that localities that are wetter, with less vegetative growth and more bare soil during the dry month of January (when agricultural plots are typically fallow) pose an increased risk of plague case occurrence. Our results suggest that environmental and landscape features play a large part in classifying an area as ecologically conducive to plague activity. However, it is clear that future studies aimed at identifying behavioral and fine-scale ecological risk factors in the West Nile region are required to fully assess the risk of human exposure to Y. pestis.