Blanca I. Restrepo

Antigenic variation by Borrelia hermsii occurs through recombination between extragenic repetitive elements on linear plasmids

The relapsing fever agent Borrelia hermsii undergoes multiphasic antigenic variation through gene conversion of a unique expression site on a linear plasmid by an archived variable antigen gene. To further characterize this mechanism we assessed the repertoire and organization of archived variable antigen genes by sequencing ∼85% of plasmids bearing these genes. Most archived genes shared with the expressed gene a ≤ 62 nucleotide (nt) region, the upstream homology sequence (UHS), that surrounded the start codon. The 59 archived variable antigen genes were arrayed in clusters with 13 repetitive, 214 nt long downstream homology sequence (DHS) elements distributed among them. A fourteenth DHS element was downstream of the expression locus. Informative nucleotide polymorphisms in UHS regions and DHS elements were applied to the analysis of the expression site of relapse serotypes from 60 infected mice in a prospective study. For most recombinations, the upstream crossover occurred in the UHS’s second half, and the downstream crossover was in the DHS’s second half. Usually the closest archival DHS element was used, but occasionally a more distant DHS was employed. The downstream extragenic crossover site in B. hermsii contrasts with the downstream extragenic crossover site for antigenic variation in African trypanosomes.

Brain Granulomas in Neurocysticercosis Patients Are Associated with a Th1 and Th2 Profile

ABSTRACT Neurocysticercosis (NCC) is a common central nervous system (CNS) infection caused by Taenia solium metacestodes. Despite the well-documented importance of the granulomatous response in the pathogenesis of this infection, there is limited information about the types of cells and cytokines involved. In fact, there has been limited characterization of human brain granulomas with any infectious agent. In the present study a detailed histological and immunohistochemical analysis of the immune response was performed on eight craniotomy specimens where a granuloma surrounded each T. soliummetacestode. The results indicated that in all the specimens there was a dying parasite surrounded by a mature granuloma with associated fibrosis, angiogenesis, and an inflammatory infiltrate. The most abundant cell types were plasma cells, B and T lymphocytes, macrophages, and mast cells. Th1 cytokines were prevalent and included gamma interferon, interleukin-18 (IL-18), and the immunosuppressive, fibrosis-promoting cytokine transforming growth factor β. The Th2 cytokines IL-4, IL-13, and IL-10 were also present. These observations indicate that a chronic immune response is elicited in the CNS environment with multiple cell types that together secrete inflammatory and anti-inflammatory cytokines. In addition, both collagen type I and type III deposits were evident and could contribute to irreversible nervous tissue damage in NCC patients.

Type 2 diabetes and tuberculosis in a dynamic bi-national border population

The epidemic of type 2 diabetes in the United States prompted us to explore the association between diabetes and tuberculosis (TB) on the South Texas-Mexico border, in a large population of mostly non-hospitalized TB patients. We examined 6 years of retrospective data from all TB patients (n=5049) in South Texas and northeastern Mexico and found diabetes self-reported by 27.8% of Texan and 17.8% of Mexican TB patients, significantly exceeding national self-reported diabetes rates for both countries. Diabetes comorbidity substantially exceeded that of HIV/AIDS. Patients with TB and diabetes were older, more likely to have haemoptysis, pulmonary cavitations, be smear positive at diagnosis, and remain positive at the end of the first (Texas) or second (Mexico) month of treatment. The impact of type 2 diabetes on TB is underappreciated, and in the light of its epidemic status in many countries, it should be actively considered by TB control programmes, particularly in older patients.

Tuberculosis in poorly controlled type 2 diabetes: altered cytokine expression in peripheral white blood cells.

BACKGROUND Although the biological basis for the increased susceptibility of diabetic patients to tuberculosis remains unclear, the world is undergoing a type 2 diabetes pandemic. We hypothesize that chronic hyperglycemia leads to immunocompromise that facilitates progression to active tuberculosis. To assess this possibility, we determined whether patients with tuberculosis and diabetes (particularly those with chronic hyperglycemia), compared with patients with tuberculosis who did not have diabetes, presented altered cytokine responses to a mycobacterial antigen. METHODS Samples of whole blood from patients with tuberculosis and diabetes and from patients with tuberculosis who did not have diabetes was stimulated in vitro with purified protein derivative from Mycobacterium tuberculosis. We then determined whether there was an association between the levels of innate and adaptive cytokines secreted in response to the antigen and diabetes status, or diabetes with chronic hyperglycemia (measured by glycosylated hemoglobin level), after controlling for possible confounders. RESULTS Innate and type 1 cytokine responses were significantly higher in patients with tuberculosis who had diabetes than in nondiabetic control subjects. The effect was consistently and significantly more marked in diabetic patients with chronic hyperglycemia. CONCLUSIONS These data provide preliminary evidence that type 2 diabetes, especially type 2 diabetes involving chronic hyperglycemia, is associated with an altered immune response to M. tuberculosis. More-detailed knowledge of the underlying mechanisms should focus on the effect of chronic hyperglycemia on the immune response to help in understanding the enhanced susceptibility of diabetic patients to tuberculosis.

Antigenic variation and strain heterogeneity in Borrelia spp.

Antigenic variation and strain heterogeneity have been demonstrated for the pathogenic Borrelia species, i.e. B. burgdorferi and the relapsing fever borreliae. In relapsing fever, new borrelia serotypes emerge at a high rate spontaneously, a mechanism that is caused by DNA rearrangements on linear plasmid translocating genes coding for variable major proteins from previous silent to expression sites (i.e. from inner sites to telomeric sites of the plasmid). As a result of this variation, the borreliae escape the immune response of the host, thus leading to the relapse phenomenon. In B. burgdorferi, which is the causative agent of the multisystem disorder Lyme borreliosis, there is also a growing body of findings that antigenic variation is involved in pathogenesis of the disease. Phenotypic variation of strains in vitro concerns the size and the amount of surface-associated proteins (OspA, OspB and pC). There are indications that OspA and OspB truncations are due to deletions within the ospAB operon caused by recombination events, and that OspA/OspB-less mutants lack the 49-kb plasmid that bears the ospAB operon. With the increasing number of isolates obtained from various geographic and biological sources, it became apparent that B. burgdorferi is immunologically and genetically more heterogeneous, as previously believed. The major outer surface proteins OspA and OspB (which have been efficient antigens in vaccine studies) are heterogeneous at a genetic level. The same degree of genetic non-identity was observed for the pC protein. Other proteins like flagellin and the highly specific immunodominant p100 range protein show a lower degree of non-identity. Recombinant OspA, pC, p100 range protein and flagellin have been hyperexpressed in E. coli and these proteins are immunologically reactive. This allows further research for development of vaccines and diagnostic tools. B. burgdorferi isolates have been investigated with genotyping (DNA hybridization, PCR and 16S rRNA analysis) as well as serotyping by various authors. Comparison of the different methods has shown good agreement when the same strains have been investigated. No correlation could be found between different phenotypic and genotypic groups with respect to the ability to cause arthritis in SCID mice. A serotyping system based on immunological differences in OspA detected by a panel of monoclonal antibodies has been proposed. Serotyping a large number of B. burgdorferi isolates has shown a striking predominance of the OspA serotype 2 among European isolates from human skin, in contrast to isolates from ticks or CSF.(ABSTRACT TRUNCATED AT 400 WORDS)

Pathogen escape from host immunity by a genome program for antigenic variation

The vector-borne bacterium Borrelia hermsii, a relapsing fever agent, switches gene expression of a surface protein between different antigenic variants, thereby causing sequential waves of immune escape within hosts and increasing the likelihood of transmission. Analogous programmed systems of antigenic variation occur in African trypanosomes and Plasmodium falciparum. In these examples, switch rates to individual variants differ over a wide range. We studied how B. hermsii determines switch rates in two experimental infections: one where variants were identified by specific antisera and one based on identification by DNA sequence. Unexpressed loci of variant antigens copy into a single expression site at rates determined by extragenic features of silent loci rather than similarity between coding sequences of variants at silent sites and the single expression site. Two elements, in particular, determine switch rates. One set of elements overlaps the 5′ ends of the expressed gene and the silent loci; greater sequence identity between elements was associated with a higher switch rate. The second set of elements flanks the expression site on the 3′ side and occurs at variable distances downstream from silent loci; the nearer an element to a silent locus, the greater the switch rate of that locus into the expression site. In combination, these two features of the genome provide a simple mechanism to modulate switch rate whereby silent loci form a hierarchy of switch rates into the expression site. Although the switching hierarchy causes changes in individual cells that are stochastic, ordering of variants within hosts is semipredictable.

Cross-sectional assessment reveals high diabetes prevalence among newly-diagnosed tuberculosis cases

Introduction Tuberculosis (TB) continues to be the leading killer among bacterial diseases worldwide. In 2009, more than 9 million new cases were diagnosed and 1.7 million people died from the disease. (1) The World Health Organization (WHO) suspects that TB control is being undermined by the growing number of patients with diabetes mellitus in the world, which currently stands at an estimated 285 million but is anticipated to reach 438 million by 2030. (2,3) Prior to the 1950s reports of an association between diabetes (primarily type 1) and TB were frequent in the literature, but they waned as insulin and drugs against TB became available. (4,5) This association (now with type 2 diabetes) was rccognized again in the 1990s (6-9) and is currently supported by a growing body of literature. (6-17) According to a recent meta-analysis, diabetes patients have three times the risk of contracting TB as non-diabetics (95% confidence interval, CI: 2.3-4.3) (18) and studies report the fraction of TB cases attributable to diabetes to be between 15% and 25%. (9,13,16) The biological basis for the association between both diseases is not fully understood but studies suggest that diabetes depresses the immune response, which in turn facilitates infection with Mycobacterium tuberculosis and/or progression to symptomatic disease. This is corroborated by the fact that diabetes is generally diagnosed before TB develops. (4,19-22) Despite the suggested importance of diabetes as a risk factor for TB, most contemporary studies ate based on secondary data or self-reported diabetes status. (6-17) The contribution of diabetes to the burden of TB may be more conspicuous in countries where both diseases are highly prevalent: Bangladesh, Brazil, China, India, Indonesia, Pakistan, and the Russian Federation ate high-burden countries and rank among the 10 countries with the highest numbers of diabetes patients (2,23) and also classified as high-burden for TB. However, the risk of TB attributable to diabetes has only been reported for India and it was estimated from secondary data. (13) We need to gain a deeper understanding of the differences between TB patients with and without diabetes to assist in the development of guidelines to prevent co-morbidity. Our research team is strategically located on the Texas-Mexico border, where TB is endemic. In 2007 the overall incidence of TB was 10.5 cases per 100 000 in south Texas and 38 per 100 000 in north-eastern Mexico (personal communications, JL Robles, Secretaria de Salud de Tamaulipas [SSA-Tamaulipas]; Brian Smith and Nita Ngo, Texas Department of State and Health Services [DSHS]). Diabetes prevalence among adults over the age of 20 years is 19.4% in South Texas and 15.1% in northeastern Mexico. (24,25) Our previous studies were conducted with data extracted from existing surveillance databases for TB control and were based on self-reported diabetes. (17) In this study our objective was to estimate the risk of TB attributable to diabetes in this population along the Mexican border using primary data from patients newly diagnosed with TB and tested for diabetes. Methods Patient enrolment Referral patients with probable TB were enrolled between March 2006 and September 2008 at the TB clinics in Hidalgo and Cameron County Health Departments, which are the TB reference centres for South Texas counties (South Texas), and the SSA-Tamaulipas in Matamoros, Mexico (north-eastern Mexico). Jail inmates and individuals For TB diagnosis we used standard WHO definitions: culture positive for Mycobacterium tuberculosis (confirmed case), sputum smear positive for acid-fast bacilli when culture data were not available (smear-positive case), of clinical diagnosis only when microbiological test results were negative or not available (clinical case: symptoms compatible with TB and documentation of anti-TB treatment for at least 6 months). …OBJECTIVE To estimate the contribution of clinically-confirmed diabetes mellitus to tuberculosis (TB) rates in communities where both diseases are prevalent as a way to identify opportunities for TB prevention among diabetic patients. METHODS This is a prospective study in which TB patients ≥ 20 years old at TB clinics in the Texas-Mexico border were tested for diabetes. The risk of tuberculosis attributable to diabetes was estimated from statistics for the corresponding adult population. FINDINGS The prevalence of diabetes among TB patients was 39% in Texas and 36% in Mexico. Diabetes contributed 25% of the TB cases studied, whereas human immunodeficiency virus (HIV) infection contributed 5% or fewer. Among TB patients, fewer Mexicans than Texans were aware that they had diabetes before this study (4% and 19%, respectively). Men were also less frequently aware than women that they had diabetes (P = 0.03). Patients who knew that they had diabetes before the study had an 8-year history of the disease, on average, before being diagnosed with TB. CONCLUSION Patients with diabetes are at higher risk of contracting TB than non-diabetic patients. Integrating TB and diabetes control programmes worldwide would facilitate TB prevention among diabetes patients and increase the number of diabetics who learn of their condition, particularly among males. Such a strategy would lead to earlier case detection and improve the management of both TB and diabetes.

Convergence of the Tuberculosis and Diabetes Epidemics: Renewal of Old Acquaintances

The current pandemic of type 2 diabetes mellitus is accelerating[1, 2] in a world where approximately one third of the population is latently infected with Mycobacterium tuberculosis [3]. Diabetes affects 230 million worldwide, anticipated to reach 366 million by 2030 at which time 80% of those affected will be living in low- and middle-income countries where active tuberculosis is widespread [4, 5]. Eight of the ten countries with the highest incidence of diabetes worldwide[5] are also classified as high burden countries for tuberculosis by the World Heath Organization [6]. The consequences of these converging epidemics are likely to be substantial. The association between diabetes and tuberculosis was documented by Avicenna (980–1027). In the early 20th century it was said that the patient who did not die in a diabetic coma was likely to do so from tuberculosis, particularly if they were poor[7, 8]. The discovery of insulin in the 1920s and the later discovery of effective antibiotics led to the eclipse of the combination of these life-threatening diseases. Nevertheless, large surveys prior to the 1960s indicated tuberculosis was 2–4 times more prevalent in individuals with diabetes than those without diabetes [7–10]. The patients’ characteristics (onset at young age, insulin use) suggested at least half had type 1 diabetes [7, 9]. Today with type 2 now accounting for 90 to 95% of all diabetes we are “re-discovering” this association [11–21]. The current literature on tuberculosis and diabetes is sometimes contradictory and difficult to interpret. Most reports have limitations inherent in retrospective studies, lack confirmation of diabetes, or provide no measures of blood glucose control. Many contain data only on hospitalized patients, with bias towards the most seriously ill. Nevertheless, in the aggregate they point to a significant impact of diabetes on tuberculosis and these reports cannot be ignored. For example, data consistently shows that the odds ratio of having type 2 diabetes in patients with active tuberculosis ranges from 1.3 to 7.8-fold [14, 16, 18, 22–24], indicating that diabetes clearly increases the risk for tuberculosis. Though the risk of tuberculosis is less at the individual level for diabetes compared with that of AIDS (113 to 170-fold)[25, 26], the sheer numbers of patients with diabetes are likely to have an equal or greater effect at the population and public health level. In at least one region of the United States the number of excess cases of tuberculosis attributable to diabetes has already reached that attributable to HIV infection [14]. What is more difficult to interpret from the current literature is the effect of type 2 diabetes on the clinical presentation and treatment response during tuberculosis. Though results are sometimes conflicting, several studies, including our own, indicate that tuberculosis patients with diabetes present higher bacillary load in sputum[17, 27], delayed mycobacterial clearance [27, 28] and higher rates of multi-drug resistance[12]. These results imply that tuberculosis patients with diabetes may be more seriously ill and pose higher risk for spread of (drug-resistant) mycobacteria in the community. Thus, these issues require urgent attention. On page XX of this journal[29], Alisjahbana and collaborators present prospective data from a cohort of tuberculosis patients in Indonesia where prevalence of confirmed diabetes among tuberculosis patients is 14.8%, compared with 3.2% in the general population[18]. Their study contrasts with the mostly retrospective reports cited above by providing detailed prospective data (including HIV documentation and exclusion), clinical manifestations, diabetes classification and microbiological findings at diagnosis, two and six months of DOT treatment on at least 543 patients. Their most significant conclusion was that after six months of treatment, diabetes patients were 7.65-fold more likely to have positive cultures in a multivariate model which controlled for age, gender, study site, body-mass index, chest x-ray and culture conversion at eight weeks. This careful and complete prospective study provides strong evidence of the deleterious effect of diabetes on tuberculosis treatment, and its potential impact on control. Will similar findings be observed in other areas where diabetes and tuberculosis are prevalent? We can only know this when we have more prospective studies. In the meantime we can speculate on why we might see variations between reports. For instance, in contrast to other publications [17, 27], Alisjahbana and collaborators were not able to detect differences between the prevalence of positive smears at the start of treatment between tuberculosis patients with and without diabetes. Sputum smears are highly insensitive, and since a positive smear is the defining criteria for tuberculosis diagnosis in Indonesia, as in many developing countries, differences in smear positivity can only be reported in programs where diagnosis is based on clinical criteria, cultures and smears. The detectable impact of diabetes on tuberculosis in a given population will depend on the characteristics of those with tuberculosis but not diabetes. In places where AIDS is highly prevalent, the immunosuppression induced by this infection is likely to be so strong that it will probably “mask” the impact of diabetes. A similar scenario but to a lesser degree may be encountered with other risk factors for tuberculosis, such as incarceration, alcohol or drug abuse. Thus, a critical component of data analysis is proper modeling based on understanding the role of risk factors for tuberculosis as possible confounders or effect modifiers. In the study by Alisjahbana et al, the effect of diabetes on tuberculosis was detected in a population where less than 1% of the tuberculosis patients were HIV-positive (and were excluded) and none reported a history of incarceration, alcohol or substance-abuse. Notably, by the end of DOTS there were still 38/322 tuberculosis patients with diabetes who remained culture positive in the study from Indonesia. This delayed clearance may be related to drug resistance. The authors report no association between multi-drug resistant tuberculosis and diabetes at diagnosis, but no further evaluation of drug resistance was conducted during the course of treatment. This is an important consideration since multi-drug resistant tuberculosis may be more frequent among tuberculosis-diabetes patients from certain populations, including ours [12], but whether this is due to primary or acquired resistance remains unknown. Studies in Indonesia by the same research team have in fact indicated that plasma levels of rifampin were 53% lower in tuberculosis-diabetes patients in contrast to tuberculosis patients without diabetes [19]. It may well be that the metabolism of rifampin is affected by diabetes rendering it less effective and predisposing patients to acquisition of resistance to rifampin during treatment. In summary, the findings by Alisjahbana and collaborators highlight the need for further research aimed at understanding how the current global epidemic of type 2 diabetes is affecting tuberculosis control and prevention. Many questions are yet unanswered: What is the magnitude and impact of these converging epidemics worldwide? Should every patient with diabetes be screened for latent tuberculosis infection, and if positive, how should tuberculosis progression be monitored or prevented? Should patients with tuberculosis and diabetes be treated with a different drug regimen compared to those with tuberculosis alone? What is the biological basis of the association between tuberculosis and diabetes? What is the most cost-effective measure to assess diabetes among tuberculosis patients? In the meantime, simple and economically-realistic approaches can be immediately implemented at every tuberculosis clinic worldwide. This includes documenting self-reported diabetes in every new tuberculosis patient, and where feasible, performing a finger stick glucometer assay for a random blood sugar. In any patient with diabetes and active tuberculosis, readings will likely be very high. These patients can then be flagged for potential treatment failure, and be accorded special attention.

Type 2 diabetes and multidrug-resistant tuberculosis.

The association between tuberculosis (TB) and diabetes is re-emerging with the epidemic of type 2 diabetes (T2DM). We analyzed retrospective data from 2878 TB patients across the Texas/Mexico border. Overall, 161/2878 (5.6%) patients had MDR TB (resistance to rifampin and isoniazid): Texas 49/1442 (3.4%) and Mexico 112/1436 (7.8%). In Texas, MDR TB was significantly associated with T2DM (OR 2.1, 95% CI 1.1–4.2) when adjusted for age, gender, drug and alcohol abuse, HIV infection and history of previous episode of TB; and in Mexico (OR 1.80, 95% CI 1.1–2.9) when adjusted for age and gender. Patients with T2DM in both countries were more likely to be compliant with DOTS therapy (Texas: OR 2.4, 95% CI 1.1–5.4) than patients without T2DM. In Texas, all but 3 of the T2DM patients with MDR TB were resistant at their first culture at the time of diagnosis. It is possible that impaired immunity in T2DM increases susceptibility to infection with resistant strains.

Activation of a vmp pseudogene in Borrella hermsii: an alternate mechanism of antigenic variation during relapsing fever

The relapsing fever agent, Borrella hermsii, undergoes multiphasic antigenic variation to evade its hosts immune response. A frequently observed switch is serotype 7 to 26. Unlike silent vmp genes previously characterized, the transcriptionally silent vmp26 sequence was a pseudogene in lacking a start codon. In serotype 7 the location of the silent vmp26 sequence just downstream of vmp7 on the expression plasmid, as well as on the silent plasmid, was also unique. The demonstration of a predicted circular recombination product in serotype 7 but not serotype 21 populations indicates that the pseudogene was activated by an intramolecular recombination producing a deletion of DNA between 20‐nucleotide direct repeats in vmp7 and Ψvmp26.