Molecular Epidemiological Techniques Research Articles

Pulmonary fungal infections

This review details some of the advances that have been made in the recent decade in the diagnosis, treatment and epidemiology of pulmonary fungal infections. These advances have occurred because of increasing knowledge regarding the fungal genome, better understanding of the structures of the fungal cell wall and cell membrane and the use of molecular epidemiological techniques. The clinical implications of these advances are more rapid diagnosis and more effective and less toxic antifungal agents. For example, the diagnosis of invasive pulmonary aspergillosis, as well as histoplasmosis and blastomycosis, has improved with the use of easily performed antigen detection systems in serum and bronchoalveolar lavage fluid. Treatment of angioinvasive moulds has improved with the introduction of the new azoles, voriconazole and posaconazole that have broad antifungal activity. Amphotericin B is less frequently used, and when used is often given as lipid formulation to decrease toxicity. The newest agents, the echinocandins, are especially safe as they interfere with the metabolism of the fungal cell wall, a structure not shared with humans cells. Epidemiological advances include the description of the emergence of Cryptococcus gattii in North America and the increase in pulmonary mucormycosis and pneumonia due to Fusarium and Scedosporium species in transplant recipients and patients with haematological malignancies. The emergence of azole resistance among Aspergillus species is especially worrisome and is likely related to increased azole use for treatment of patients, but also to agricultural use of azoles as fungicides in certain countries.

Salmonella paratyphi B and Salmonella litchfield outbreaks associated with pet turtle exposure in Spain

IntroductionSalmonellosis is an important public health problem. Turtles are increasingly involved in the role of transmitters of this infection to humans. MethodsSalmonella cases are reported to the local Surveillance Agency where interviews are carried out to address possible exposures. Molecular epidemiology techniques were used to identify species. ResultsIn this article we report two examples of this type of infection in two places, 300km apart in Spain. In Barcelona a turtle transmitted the disease to a small baby via her mother, and in Castellón 5 related cases of Salmonella infections were detected, and all were transmitted by imported turtles. Molecular epidemiology techniques confirmed the turtle-person transmissions and showed strong relationships between cases in Castellón and Barcelona. DiscussionThese examples represent the tip of the iceberg of what is happening with pet reptiles as regards transmission of this infection. We believe that it is important to assess the impact of this type of infection in each country, in order to subsequently promote prevention strategies such as: regulations for pet shops, and educating/informing families who buy reptiles as pets.

Detection of European Strain ofEchinococcus multilocularisin North America

Detection of European Strain of<i>Echinococcus multilocularis</i>in North America

Multicenter Evaluation of the LightCycler MRSA Advanced Test, the Xpert MRSA Assay, and MRSASelect Directly Plated Culture with Simulated Workflow Comparison for the Detection of Methicillin-Resistant Staphylococcus aureus in Nasal Swabs

Rapid detection of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) followed by appropriate infection control procedures reduces MRSA infection and transmission. We compared the performance and workflow of two Food and Drug Administration-approved nucleic acid amplification assays, the LightCycler MRSA Advanced Test and the Xpert MRSA test, with those of directly plated culture (MRSASelect) using 1202 nasal swabs collected at three U.S. sites. The sensitivity of the LightCycler test (95.2%; 95% CI, 89.1% to 98.4%) and Xpert assay (99%; 95% CI, 94.8% to 100%) did not differ compared with that of culture; the specificity of the two assays was identical (95.5%; 95% CI, 94.1% to 96.7%) compared with culture. However, sequencing performed on 71 samples with discordant results among the three methods confirmed the presence of MRSA in 40% of samples that were positive by both molecular methods but negative by culture. Workflow analysis from all sites including batch runs revealed average hands-on sample preparation times of 1.40, 2.35, and 1.44 minutes per sample for the LightCycler, Xpert, and MRSASelect methods, respectively. Discrete event simulation analysis of workflow efficiencies revealed that the LightCycler test used less hands-on time for the assay when greater than eight batched samples were run. The high sensitivity and specificity, low hands-on time, and efficiency gains using batching capabilities make the LightCycler test suitable for rapid batch screening of MRSA colonization.

The impact of globalisation on the distribution of Echinococcus multilocularis

In the past three decades, Echinococcus multilocularis, the cause of human alveolar echinococcosis, has been reported in several new countries both in definitive hosts (canids) as well as in people. Unless treated, infection with this cestode in people is fatal. In previously endemic countries throughout the Northern Hemisphere, geographic ranges and human and animal prevalence levels seem to be increasing. Anthropogenic influences, including increased globalisation of animals and animal products, and altered human/animal interfaces are thought to play a vital role in the global emergence of this pathogenic cestode. Molecular epidemiological techniques are a useful tool for detecting and tracing introductions, and differentiating these from range expansions.

Hospital Cluster of HBV Infection: Molecular Evidence of Patient-to-Patient Transmission through Lancing Device

IntroductionIn western countries the transmission of hepatitis B virus (HBV) transmission through multi-patients lancing devices has been inferred since early ‘90s, however no study has ever provided biological evidence which directly link these device with HBV cross-infection. Here we present results of an outbreak investigation which could associate, by molecular techniques, the use of lancing device on multiple patients with HBV transmission in an Italian oncohematology unit.MethodsThe outbreak investigation was designed as a retrospective cohort study to identify all potential cases. All cases identified were eventually confirmed through molecular epidemiology techniques. Audit of personnel including extensive review of infection control measures and reviewing personnel's tests for HBV was done identify transmission route.ResultsBetween 4 May 2006 and 21 February 2007, six incident cases of HBV infection were reported among 162 patients admitted in the oncohematology. The subsequent molecular instigation proved that 3 out 6 incident cases and one prevalent cases (already infected with HBV at the admission) represented a monophyletic cluster of infection. The eventual environmental investigation found that an identical HBV viral strain was present on a multi-patients lancing device in use in the unit and the inferential analysis showed a statistically significant association between undergoing lancing procedures and the infection.DiscussionThis investigation provide molecular evidence to link a HBV infection cluster to multi-patients lancing device and highlights that patients undergoing capillary blood sampling by non-disposable lancing device may face an unacceptable increased risk of HBV infection. Therefore we believe that multi-patients lancing devices should be banned from healthcare settings and replace with disposable safety lancets that permanently retract to prevent the use of the same device on multiple patients. The use of non-disposable lancing devices should be restricted to individual use at patients' home.

Real-Time Molecular Epidemiology of Tuberculosis by Direct Genotyping of Smear-Positive Clinical Specimens

We applied MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing) to directly analyze the bacilli present in 61 stain-positive specimens from tuberculosis patients. A complete MIRU type (24 loci) was obtained for all but one (no amplification in one locus) of the specimens (98.4%), and the allelic values fully correlated with those obtained from the corresponding cultures. Our study is the first to demonstrate that real-time genotyping of Mycobacterium tuberculosis can be achieved, fully transforming the way in which molecular epidemiology techniques can be integrated into control programs.

Overview of silkworm pathology in China

In this study, we elaborated the history and progress of studies on silkworm diseases in China through summarizing and reviewing the achievements on silkworm pathology, pathogenic molecular biology, epidemiology, pathogen detection and diagnostic techniques, damage from non-infectious silkworm diseases and control of non-infectious silkworm diseases. These summaries and reviews would provide good references for further studies in silkworm diseases. Key words : Silkworm, pathology, disease control, China.

Factors associated with recently transmitted Mycobacterium tuberculosis strain MS0006 in Hinds County, Mississippi.

The objective of this study was to investigate risk factors associated with tuberculosis (TB) transmission that was caused by Mycobacterium tuberculosis strain MS0006 from 2004 to 2009 in Hinds County, Mississippi. DNA fingerprinting using spoligotyping, mycobacterial interspersed repetitive unit, and IS6110-based restriction fragment length polymorphism of culture-confirmed cases of TB was performed. Clinical and demographic factors associated with strain MS0006 were analyzed by univariate and multivariate analysis. Of the 144 cases of TB diagnosed during the study period, 117 were culture positive with fingerprints available. There were 48 different strains, of which 6 clustered strains were distributed among 74 patients. The MS0006 strain accounted for 46.2% of all culture-confirmed cases. Risk factors for having the MS0006 strain in a univariate analysis included homelessness, HIV co-infection, sputum smear negativity, tuberculin skin test negativity, and noninjectable drug use. Multivariate analysis identified homelessness (odds ratio 7.88, 95% confidence interval 2.90-21.35) and African American race (odds ratio 5.80, 95% confidence interval 1.37-24.55) as independent predictors of having TB caused by the MS0006 strain of M. tuberculosis. Our findings suggest that a majority of recently transmitted TB in the studied county was caused by the MS0006 strain. African American race and homelessness were significant risk factors for inclusion in the cluster. Molecular epidemiology techniques continue to provide in-depth analysis of disease transmission and play a vital role in effective contact tracing and interruption of ongoing transmission.

Exploring the use of molecular epidemiology to track bovine tuberculosis in Nigeria: An overview from 2002 to 2004

Tuberculosis remains a major public health problem in Nigeria. While human to human transmission of Mycobacterium tuberculosis is clearly of major importance in driving the tuberculosis epidemic in Nigeria, the impact of Mycobacterium bovis transmission from infected cattle is largely unknown. Molecular epidemiology of M. bovis in Nigeria will increase our understanding of this endemic disease and provide tools to assess cattle-to-human transmission. Between 2002 and 2004, molecular techniques including spoligotyping, variable number of tandem repeats (VNTR) typing and deletion typing were used to track and analyze a sample of strains of the M. tuberculosis complex circulating in the cattle population in Ibadan, Southwestern Nigeria. In all, 180 isolates were typed with a view to elucidating epidemiological information on circulating strains, occurrence of transborder transmission and molecular diversity of the M. bovis strains. Results obtained showed that 99% (178/180) of the isolates were M. bovis, while the remaining were M. tuberculosis and M. africanum. In all, strains of M. bovis had 34 different spoligotypes: strains with spoligotype pattern SB0944 (as designated by www.mbovis.org) were the most common (46% of strains). This molecular type is also common in countries neighbouring Nigeria. Strains with this spoligotype pattern could be further divided into 40 different VNTR types. This analysis shows the value of simple molecular epidemiological techniques applied to strains of M. bovis and suggests that further epidemiological studies will shed more light on the transmission dynamics of bovine tuberculosis locally and across neighbouring African countries.

Personalized nutrigenomics: tailoring the diet to the aging diabesity population

Personalized nutrigenomics: tailoring the diet to the aging diabesity population Douglas M Ruden1, Xiangyi Lu21Wayne State University, Department of Obstetrics and Gynecology, C. S. Mott Center for Human Growth and Development, Institute of Environmental Health Sciences, Detroit, MI, USA; 2Institute of Environmental Health Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Detroit, MI, USAAbstract: According to the International Diabetes Federation (IDF), 190 million people worldwide suffer from diabetes, and this number is estimated to double by the year 2025. Diabetes is especially prominent in the elderly population because the IDF indicates age above 45 years as a major risk factor for diabetes. The most common trials for controlling diabetes focus on tighter glucose control as a means to reduce the long-term complications. However, whether tight blood sugar control or other dietary or pharmaceutical interventions in the elderly are more appropriate is not known. Major changes have taken place in our diet over the past 10,000 years since the beginning of the Agricultural Revolution, but our genes have not changed. Furthermore, the large numbers of diabetic elderly in the population are a recent phenomenon, because those with diabetes have historically died young. Genetically speaking, humans today live in a nutritional environment that differs from that for which our genetic constitution was selected. For example a high omega-6/omega-3 ratio, found in today&rsquo;s Western diets, promotes the pathogenesis of many chronic diseases, including cardiovascular disease and diabetes. Knowing who is at risk would be useful if it meant that one could avoid the environmental triggers that convert susceptibility to disease. The prospect of targeting specific dietary treatments at the elderly, who are predicted to gain the most therapeutic benefits, clearly has important clinical and economic consequences. In this review, we will discuss modern molecular genetic and epidemiological techniques which are now, or soon will be, made available by inexpensive whole-genome sequencing and other whole genome approaches to treat the elderly diabetic population.Keywords: diabetes, elderly diabetic population, DNA, genome sequencing&nbsp;

Molecular epidemiology of tuberculosis in the Tula area, Central Russia, before the introduction of the Directly Observed Therapy Strategy

Tuberculosis remains a major public health concern in Russia and worldwide. Given the great geographical, ethnic, and socio-economic heterogeneities between Russian regions, epidemiological data cannot be generalized from a regional to a country-wide level. We present data on the epidemiology of tuberculosis in Central Russia. We report a high level of resistance to major antitubercular drugs in both new and previously treated patients in the region. The level of drug resistance in new cases was almost twice as high as the estimated average national level. The Mycobacterium tuberculosis strains that circulated in the region were predominantly represented by LAM-RUS and Beijing genotypes. These two lineages were strongly associated with drug resistance and clustering. Using molecular epidemiology techniques, we showed a high interpenetration by M. tuberculosis strains between the prison and civilian populations. A limited number of identical strains were responsible for the majority of drug-resistant tuberculosis cases in both settings.

Molecular epidemiology of a hepatitis C virus epidemic in a haemodialysis unit: outbreak investigation and infection outcome

BackgroundHCV is a leading cause of liver chronic diseases all over the world. In developed countries the highest prevalence of infection is reported among intravenous drug users and haemodialysis (HD) patients. The present report is to identify the pathway of HCV transmission during an outbreak of HCV infection in a privately run haemodialysis (HD) unit in Italy in 2005.MethodsDynamics of the outbreak and infection clinical outcomes were defined through an ambi-directional cohort study. Molecular epidemiology techniques were used to define the relationships between the viral variants infecting the patients and confirm the outbreak. Risk analysis and auditing procedures were carried out to define the transmission pathway(s).ResultsOf the 50 patients treated in the HD unit 5 were already anti-HCV positive and 13 became positive during the study period (AR = 28.9%). Phylogenic analysis identified that, all the molecularly characterized incident cases (10 out of 13), were infected with the same viral variant of one of the prevalent cases. The multivariate analysis and the auditing procedure disclosed a single event of multi-dose vials heparin contamination as the cause of transmission of the infection in 11 out of the 13 incident cases; 2 additional incident cases occurred possibly as a result of inappropriate risk management.DiscussionMore than 30% of all HCV infections in developed countries results from poor application of standard precautions during percutaneous procedures. Comprehensive strategy which included: educational programmes, periodical auditing on standard precaution, use of single-dose vials whenever possible, prospective surveillance for blood-borne infections (including a system of prompt notification) and risk assessment/management dedicated staff are the cornerstone to contain and prevent outbreaks in HDConclusionsThe outbreak described should serve as a reminder to HD providers that patients undergoing dialysis are at risk for HCV infection and that HCV may be easily transmitted whenever standard precautions are not strictly applied.

Mycobacterium bovisandM.tuberculosisin Goats, Nigeria

To the Editor: Documentation of possible tuberculosis (TB) in goats in Nigeria was reported by Ojo (1) on the basis of gross lesions without culture confirmation. Livestock owners in Nigeria normally graze cattle and goats together, and this practice poses a high risk for transmission of bovine TB among these animals (1). This practice is especially a threat to goats in Nigeria because several reports have described bovine TB in cattle in Nigeria (2–5). However, reports of diagnosis of TB in goats in Nigeria are lacking. Molecular epidemiologic techniques such as deletion typing and spoligotyping have been used to characterize members of the Mycobacterium tuberculosis complex (MTC) and to provide information on transmission of mycobacterial diseases between animals and humans (6). However, because of limited resources and lack of expertise, these techniques are not commonly used in most developing nations such as Nigeria, where TB is endemic (3). Because slaughterhouses provide excellent opportunities for detecting diseases of economic and public health importance, we investigated the presence of mycobacteria in slaughtered goats with lesions suggestive of TB. The investigation was conducted at the Bodija Municipal Abattoir in Ibadan in southwestern Nigeria over a period of 6 months. Slaughtered goats were obtained from local herds and herds in northern Nigeria. A total of 10,389 male and female goats of 2 breeds (West African Dwarf and Red Sokoto) and 1–2 years of age were slaughtered; 1,387 were inspected for gross lesions of TB. Of 1,387 animals screened, 62 (4.47%) had lesions suggestive of TB in the liver, lungs, and mesenteric lymph nodes. Five (0.36%) goats were confirmed positive by culture as described (2). Deletion typing (6) with the RD9 deletion was used to distinguish M. tuberculosis from other members of the MTC. Those isolates with a deletion in this region were further investigated with primers specific for RD4. This reaction distinguishes between M. bovis, M. caprae, and other members of the MTC. Spoligotyping was performed as described (7) to type an M. tuberculosis isolate from a goat after identification of this bacterium by deletion typing. We isolated 4 strains of M. bovis and 1 strain of M. tuberculosis from the goats (Table). Spoligotyping identified the M. tuberculosis isolate as belonging to the East African Indian (EAI)–5 family in the SpolDB4 database. All M. bovis isolates were M. bovis bovis, not M. bovis caprae, according to their deletion typing profile (6). One M. bovis isolate was obtained from a male goat; the 3 remaining M. bovis isolates and the M. tuberculosis isolate were obtained from female goats. Table Results of deletion typing for Mycobacterium tuberculosis and M. bovis in goats, Nigeria* Epidemiologic inferences can be made from the results of our study. First, M. bovis, which is naturally found in cattle, was isolated from 4 slaughtered goats. Although M. bovis caprae was the M. bovis variant most frequently isolated from goats in some areas (8), in our study, only M. bovis bovis was isolated. This finding is consistent with results reported by Crawshaw et al. (9), and suggests transmission from cattle, rather than transmission from the goat reservoir. Second, because the infected goats were adult females, infection may be transmitted to their offspring. Third, M. tuberculosis was isolated from a goat. Its presence in this goat may have been caused by direct transmission from humans because this bacterium may be a natural pathogen of humans. Transmission caused by close cohabitation of goats and humans with advanced TB may occur, given the endemic nature of TB in humans in Nigeria (10). TB cases caused by EAI strains have been found in humans in southwestern Nigeria (4; S.I. Cadmus et al., unpub. data), a finding that supports zoonotic transmission of this organism from humans to goats. However, different lineages of M. tuberculosis may vary in host range, and EAI genotype strains may be adapted to human and animal hosts. Conversely, human-to-animal transmission of M. tuberculosis has been reported in Nigeria relative to infection in cattle (3,4). Thus, confirmation of TB in goats supports the possibility of risk for TB transmission between humans and animals in Nigeria. This study should be interpreted in the context of its limitations. Because the sources of the animals were unknown, we could not determine whether the organisms were imported from a neighboring country (3). In addition, we lacked information on the breed and condition of the animals. However, we have identified M. tuberculosis and TB in goats in Nigeria. Additional studies of other slaughterhouses in Nigeria are needed to confirm our results.

Inter-Continental Patterns of HIV-1 Control: HLA and the Quest for a T Cell Vaccine

toxins A and B and possibly binary toxins. Improvements in molecular epidemiological techniques are increasingly enhancing our understanding of C. difficile. The epidemiology of C. difficile is not entirely delineated, but it is clear that the organism can be carried asymptomatically in addition to causing disease. Studies from the 1980s and 1990s have suggested that asymptomatic carriage of C. difficile is common in infants, although research into the organism as a pathogen in the paediatric population at that time was inconclusive. More recent work from North America has, however, again focused on the apparent increase in CDAD amongst children, and a prevalence of up to 20% of the hypervirulent BI/NAP1/027 strain in paediatric patients with diarrhoea who are positive for C. difficile has been reported. This strain has been responsible for epidemic outbreaks in adults over the last six years and is associated with more severe disease most likely due to increased toxin production mediated via deletions in the tcdC toxin regulator gene. This study was designed as a pilot to assess methodology for detailed molecular epidemiological investigations of C. difficile in children.

Diabetes, insulin, and development of acute lung injury

Recently, many studies have investigated the immunomodulatory effects of insulin and glucose control in critical illness. This review examines evidence regarding the relationship between diabetes and the development of acute lung injury/acute respiratory distress syndrome (ALI/ARDS), reviews studies of lung injury related to glycemic and nonglycemic metabolic features of diabetes, and examines the effect of diabetic therapies. A MEDLINE/PubMed search from inception to August 1, 2008, was conducted using the search terms acute lung injury, acute respiratory distress syndrome, hyperglycemia, diabetes mellitus, insulin, hydroxymethylglutaryl-CoA reductase inhibitors (statins), angiotensin-converting enzyme inhibitor, and peroxisome proliferator-activated receptors, including combinations of these terms. Bibliographies of retrieved articles were manually reviewed. Available studies were critically reviewed, and data were extracted with special attention to the human and animal studies that explored a) diabetes and ALI; b) hyperglycemia and ALI; c) metabolic nonhyperglycemic features of diabetes and ALI; and d) diabetic therapies and ALI. Clinical and experimental data indicate that diabetes is protective against the development of ALI/ARDS. The pathways involved are complex and likely include effects of hyperglycemia on the inflammatory response, metabolic abnormalities in diabetes, and the interactions of therapeutic agents given to diabetic patients. Multidisciplinary, multifaceted studies, involving both animal models and clinical and molecular epidemiology techniques, are essential.

A random PCR screening system for the identification of type 1 human herpes simplex virus

Several viral diseases exhibit measles-like symptoms. Differentiation of suspected cases of measles with molecular epidemiological techniques in the laboratory is useful for measles surveillance. In this study, a random PCR screening system was undertaken for the identification of isolates from patients with measles-like symptoms who exhibited cytopathic effects, but who had negative results for measles virus-specific reverse transcription (RT)-PCR and indirect immunofluorescence assays. Sequence analysis of random amplified PCR products showed that they were highly homologous to type 1 human herpes simplex virus (HSV-1). The results were further confirmed by an HSV-1-specific TaqMan real-time PCR assay. The random PCR screening system described in this study provides an efficient procedure for the identification of unknown viral pathogens. Measles-like symptoms can also be caused by HSV-1, suggesting the need to include HSV-1 in differential diagnoses of measles-like diseases.

Molecular epidemiology of isolates of the Cryptococcus neoformans species complex from Spain

To study genetic diversity of Cryptococcus neoformans species complex in Spain, 97 isolates of the yeast recovered from human, animal and environmental samples have been analysed using three molecular epidemiological techniques. One of these, URA5 gene fragment length polymorphism (RFLP) analysis, has been previously described as a molecular epidemiology tool. Thus, standard profiles and reference strains have been defined for it. In addition, 5S rDNA/IGS RFLP and [GACA] 4 microsatellite PCR fingerprinting were also used. Our results show five of the previously defined URA5 genotypes with a high frequency (33%) of the VNI type, which is in concordance with other studies. The high presence of VNIII pattern (28.9%) among our strains is remarkable and could be a specific feature of the isolates from our country. 5S rDNA/IGS RFLP showed a low intra-species discriminative power. Three different molecular profiles (S1-3), which showed a good correlation with the different species, varieties and genotypes, were obtained. [GACA] 4 microsatellite PCR-fingerprinting analysis showed a high variability of patterns among the studied strains. Molecular profiles represented in a dendrogram clustered strains in four main groups related with the source of the yeast and also in concordance with some of the described genotypes (VNI-IV and VGI).

Tuberculosis associated with household crowding in a developed country

Background:Tuberculosis (TB) remains an important infectious disease in New Zealand (NZ) and globally, but risk factors for transmission are still poorly understood. This research aimed to identify whether household crowding...

Current trends in molecular epidemiology studies of Mycobacterium tuberculosis

Mycobacterium tuberculosis is one of the most harmful human pathogens worldwide, and there are mass efforts for controlling this pathogen. One of the powerful tools to find out and control this pathogen is molecular epidemiology techniques. Currently, wide ranges of techniques are available to type mycobacterium tuberculosis, and choosing the correct technique as a portable and standard method is difficult. IS6110 restriction fragment length polymorphism (RFLP) remains gold standard method on genotyping to date, but it is labor intensive and inefficient on samples which have fewer than six copy numbers of IS6110. In the recent years, some new methods have been introduced for genotyping of mycobacterium tuberculosis such as mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) and Spoligotyping. The present review tries to introduce new approaches in molecular techniques for epidemiological investigation of tuberculosis and to illustrate advantages and problems associated with them. Key words: Molecular epidemiology, Mycobacterium tuberculosis, fingerprinting, transmission, genotype.