MLVA Genotyping Research Articles

Genetic diversity of Francisella tularensis in Poland with comments on MLVA genotyping and a proposition of a novel rapid v4-genotyping

We investigated the genotypes of Francisella tularensis (F. tularensis) strains isolated in Poland during the period 1953–2013 and studied their genetic relationship to F. tularensis strains isolated in other countries using MLVA. We examined the mosquito and tick samples collected in Poland for the presence of F. tularensis DNA using PCR. Our results revealed a high genetic diversity among the strains of F. tularensis collected from Poland, suggesting that the bacterium is commonly found in the environment. However, we did not detect F. tularensis DNA in ticks and mosquitoes, showing that the arthropod bites might not be the main source of infection.We also propose the application of a practical assay called v4-genotyping that can be directly performed on the clinical and environmental samples.In addition, we discovered genetic variations among Schu S4 reference strains used in various laboratories and showed that MLVA analysis should not be based on amplicon sizes only because point mutations occurring within the MLVA loci might not always be manifested by a change in the amplicon size.

Epidemiological characteristics of Brucella species isolated from different regions of the world using the MLVA genotyping

Objective: To study the molecular-epidemiological characteristics of Brucella species isolated from different countries, using the multiple locus tandem-repeat (MLVA) analysis. Methods: Eleven variable-number tandem-repeat (VNTR) loci were selected. VNTR strains of Brucella isolated from 48 different countries in 1953-2013, were analyzed by using the BioNumerics software. Unweighted Paired Arithmetic Average method was used to cluster and draw phylogenetic tree as well as the minimum spannin. Results: The evolutionary relationship of Brucella phylogenetic tree was consistent with the classical biological typing method. However, the Brucella suis biovar 5 strains were different from the other Brucella suis biovars 1, 2, 3 and 4. Brucella ceti strains were divided into two parts and different from each other. Worldwide epidemics of brucellosis were emerged from 2005 to 2008 under the MLVA11 Orsay analysis. China has been a brucellosis-prone regions, with Brucella melitensis as the main epidemic Brucella species, followed by Brucella abortus. Brucella suis was mainly identified in the southern provinces, but Brucella canis was mainly found in dogs. No human cases were found. Conclusion: Molecular-epidemiological characteristics of the Brucella strains were related to factors as time, region and hosts of isolation, which are important to setting up prevention and control programs on brucellosis.

Homogeneity of Mycoplasma agalctiae vaccine strains in an agalaxy- high-burden environment

Interrogation of the genomic relations between Iranian Mycoplasma agalactiae vaccine strains of Taliqan, Lorestan and Shiraz. Two MLVA (covering VNTR loci of 5, 9, 17 and 19) and MLST (comprising dnaA, gltX, gyrB, C, tufA genes) genotyping systems plus nucleotide structure analysis of P80 gene, was conducted. The shared MLVA pattern represented by the three strains differed to that of the Mag PG2 laboratory strain, only at locus VNTR19 where the PG2 genome hold a 3 bp longer stretch. In MLST analysis, at dnaA, gltX, gyrB, metS and tufA loci, the three strains displayed alleles 1, 21, 2, 2 and 1, respectively. At, gltX locus a new allele (21) was detected where a new sequence type (ST33) was identified. Besides, the trio strains hold an identical nucleotide structure in their ma-mp81 gene. In explanation, lack of efficient disease control measures, has possibly contributed in evolution of a clone or a few clones that gradually overwhelmed the population over the time. Besides, the similarity between the Iranian and the PG2 strains, might be due to homoplasy or farming exercises such as animal importation. Inclusion of further local isolates in next studies will help to assess these assumptions.

Genetic Profiling of the Causative Agents of Natural-Focal infections, Circulating in the Stavropol Territory

Objective of the study was genetic typing of the strains and nucleic acid isolates of causative agents of natural focal infections, both bacterial and viral etiology, accumulation of data on genetic features of regional strains cir­culating in the Stavropol Territory . Material and methods. To study the genetic spectrum of causative agents of natural- focal infections, analysis of the strains and nucleic acids isolates detected in the samples of field and clinical material was carried out. Indication of causative agents of natural focal infections in the samples was carried out by PCR. For genetic typing of the strains and DNA/RNA isolates of natural focal infections agents, MLVA and sequencing of genome regions with subsequent phylogenetic analysis were used. The analysis of territorial distribution of the causative agent genetic variants and mapping was performed in ArcGIS 10.1. Results and conclusions. MLVA-25 typing of 20 strains of Francisella. tularensis, MLVA-10 typing of 4 Coxiella burnetii isolates, species identification of 20 isolates of Rickettsia sp., sub-species genetic typing of 40 RNA isolates of CCHF virus and 8 RNA isolates of hantaviruses circulating in the Stavropol Territory in 2016-2017 were performed. The studied strains of F. tularensis belong to eight MLVA genotypes. They are mainly confined to specific areas. The isolates of C. burnetii have the same MLVA type. Rickettsia, belonging to 5 species: R. massiliae R. raoultii, R. sibirica, R. aeschlimannii, R. slovaca, RNA-isolates of hantavirus of the «Tula» genotype and variants of the CCHF virus of the Europe-1 and Europe-3 genotypes were identified. The obtained data can be used in the epidemiological investigation of possible cases of infectious diseases to determine the source and pathways of infection.

Genotyping of Coxiella burnetii in sheep and goat abortion samples

BackgroundQ fever, caused by Coxiella burnetii, is a zoonosis that presents a worldwide distribution and affects both humans and animals. The route of dispersal of the pathogen by ruminants into the environment usually involves stages of abortion and parturition, nevertheless the agent can, also, be detected in other animal samples. Therefore it is considered as important in terms of proper diagnosis, as well as, for epidemiology and surveillance purposes, to genotype the pathogen. The aim of the current study was to investigate the presence of different genotypes of the agent in animals that had suffered from abortion during a two-year survey in Greece.ResultsSixty nine tissue samples (37 stomach contents, 11 liver samples, 21 cotyledons) were collected from 59 abortion cases in sheep (N = 45) and goats (N = 14) from 65 farms at eight different areas of Greece. Samples were screened by qPCR and positive ones were further genotyped using a 10-locus multiple loci (ms 1, 3, 7, 12, 20, 21, 22, 26, 30 and 36) variable number of tandem repeat analysis (MLVA) method.Three genotypes were identified in sheep (A, B, C). Samples representing each of the obtained MLVA profile were further used for MST genotyping. Ten spacers (Cox 2, 5, 6, 18, 20, 22, 37, 51, 56 and 57) were amplified. A close relatedness among the identified MLVA genotypes was confirmed since they all belonged to MST group 32.ConclusionsThe current study introduces into the aspect of genotyping of C. burnetii in Greece. Further studies are needed to explore the presence of more genotypes, to associate the genotypes circulating in the animal and tick population with those causing human disease in order to further expand on the epidemiological aspects of the pathogen.

Staphylococcus epidermidis, Clonality and Accessory Gene Regulator Diversity in Clinical Isolates

Background: Quorum-sensing systems are considered as important mechanisms for pathogenesis and bacterial communication. The accessory gene regulator (agr) is one of the quorum-sensing systems in staphylococci, which is generally conserved. It is believed that there is a correlation between agr groups and infection. Multiple-locus VNTR analysis is a method for bacterial typing, previously applied for several different species of bacteria. This study aimed at determining the diversity of Staphylococcus epidermidis accessory gene regulator and clonality in clinical isolates from intensive care unit patients, Tehran, Iran. Methods: A total of 59 Staphylococcus epidermidis isolates were obtained from intensive care unit patients. The MLVA was performed for S. epidermidis isolates, using seven VNTR loci, including SE2395, SE0331, SE 828, SE1632, SE0175, Se2, and Se4. Specific primers were used for agr diversity determination. Results: The agr type I was observed in 29 (49%), while each of the agr type II and agr type III were observed in 10 (17%). Furthermore, 10 (17%) isolates were untypeable with using primers. In total, 49 MLVA genotypes were discriminated. Isolates were classified to six clonal complexes. Of the 59 isolate, 33 were included in clonal complex 1 (CC1), the largest of which harbored 15 (45.4%) agr type I. Conclusions: Agr type I was observed in the majority of the isolates. The MLVA results of this study suggest that there was a clone with 33 samples, comprised of 56% of isolates; smaller clones each comprised of two to seven isolates, and four isolates in the form of singleton. It seems that big clone isolates were settled in the intensive care unit (ICU), and singleton isolates entered the ward by visitors or medical personnel and caused infection.

Genotypic Expansion Within the Population Structure of Classical Brucella Species Revealed by MLVA16 Typing of 1404 Brucella Isolates From Different Animal and Geographic Origins, 1974-2006.

Previous studies have shown the usefulness of MLVA16 as a rapid molecular identification and classification method for Brucella species and biovars including recently described novel Brucella species from wildlife. Most studies were conducted on a limited number of strains from limited geographic/host origins. The objective of this study was to assess genetic diversity of Brucella spp. by MLVA16 on a larger scale. Thus, 1404 animal or human isolates collected from all parts of the world over a period of 32 years (1974-2006) were investigated. Selection of the 1404 strains was done among the approximately 4000 strains collection of the BCCN (Brucella Culture Collection Nouzilly), based on classical biotyping and on the animal/human/geographic origin over the time period considered. MLVA16 was performed on extracted DNAs using high throughput capillary electrophoresis. The 16 loci were amplified in four multiplex PCR reactions. This large scale study firstly confirmed the accuracy of MLVA16 typing for Brucella species and biovar identification and its congruence with the recently described Extended Multilocus Sequence Analysis. In addition, it allowed identifying novel MLVA11 (based upon 11 slowly evolving VNTRs) genotypes representing an increase of 15% relative to the previously known Brucella MLVA11 genotypes. Cluster analysis showed that among the MLVA16 genotypes some were genetically more distant from the major classical clades. For example new major clusters of B. abortus biovar 3 isolated from cattle in Sub-Saharan Africa were identified. For other classical species and biovars this study indicated also genotypic expansion within the population structure of classical Brucella species. MLVA proves to be a powerful tool to rapidly assess genetic diversity of bacterial populations on a large scale, as here on a large collection of strains of the genomically homogeneous genus Brucella. The highly discriminatory power of MLVA appears of particular interest as a first step for selection of Brucella strains for whole-genome sequencing. The MLVA data of this study were added to the public Brucella MLVA database at http://microbesgenotyping.i2bc.paris-saclay.fr. Current version Brucella_4_3 comprises typing data from more than 5000 strains including in silico data analysis of public whole genome sequence datasets.

Association of virulence gene expression with colistin-resistance in Acinetobacter baumannii: analysis of genotype, antimicrobial susceptibility, and biofilm formation

BackgroundAcinetobacter baumannii causes difficult-to-treat nosocomial infections, which often lead to morbidity due to the development of antimicrobial drug resistance and expression of virulence genes. Data regarding the association of resistance to colistin, a last treatment option, and the virulence gene expression of A. baumannii is scarce.MethodsWe evaluated the MLVA genotype, antimicrobial resistance, and biofilm formation of 100 A. baumannii isolates from burn patients, and further compared the in vitro and in vivo expression of four virulence genes among five colistin-resistant A. baumannii (Cst-R-AB) isolates. Five Cst-R-AB isolates were tested; one from the present study, and four isolated previously.ResultsOur results showed that reduced expression of recA, along with increased in vivo expression of lpsB, dnaK, and blsA; are associated with colistin resistance among Cst-R-AB isolates. Differences in virulence gene expressions among Cst-R-AB isolates, may in part explain common discrepant in vitro vs. in vivo susceptibility data during treatment of infections caused by Cst-R-AB.ConclusionsOur findings highlight the intricate relationship between colistin-resistance and virulence among A. baumannii isolates, and underscore the importance of examining the interactions between virulence and antimicrobial resistance toward efforts to control the spread of multidrug-resistant A. baumannii (MDR-AB) isolates, and also to reduce disease severity in burn patients with MDR-AB infection.

Multiple Locus Variable-Number Tandem-Repeat and Single-Nucleotide Polymorphism-Based Brucella Typing Reveals Multiple Lineages in Brucella melitensis Currently Endemic in China.

Brucellosis is a worldwide zoonotic disease caused by Brucella spp. In China, brucellosis is recognized as a reemerging disease mainly caused by Brucella melitensis specie. To better understand the currently endemic B. melitensis strains in China, three Brucella genotyping methods were applied to 110 B. melitensis strains obtained in past several years. By MLVA genotyping, five MLVA-8 genotypes were identified, among which genotypes 42 (1-5-3-13-2-2-3-2) was recognized as the predominant genotype, while genotype 63 (1-5-3-13-2-3-3-2) and a novel genotype of 1-5-3-13-2-4-3-2 were second frequently observed. MLVA-16 discerned a total of 57 MLVA-16 genotypes among these Brucella strains, with 41 genotypes being firstly detected and the other 16 genotypes being previously reported. By BruMLSA21 typing, six sequence types (STs) were identified, among them ST8 is the most frequently seen in China while the other five STs were firstly detected and designated as ST137, ST138, ST139, ST140, and ST141 by international multilocus sequence typing database. Whole-genome sequence (WGS)-single-nucleotide polymorphism (SNP)-based typing and phylogenetic analysis resolved Chinese B. melitensis strains into five clusters, reflecting the existence of multiple lineages among these Chinese B. melitensis strains. In phylogeny, Chinese lineages are more closely related to strains collected from East Mediterranean and Middle East countries, such as Turkey, Kuwait, and Iraq. In the next few years, MLVA typing will certainly remain an important epidemiological tool for Brucella infection analysis, as it displays a high discriminatory ability and achieves result largely in agreement with WGS-SNP-based typing. However, WGS-SNP-based typing is found to be the most powerful and reliable method in discerning Brucella strains and will be popular used in the future.

Exploring the Diversity of Field Strains of Brucella abortus Biovar 3 Isolated in West Africa.

Brucellosis is one of the most widespread bacterial zoonotic diseases in the world, affecting both humans and domestic and wild animals. Identification and biotyping of field strains of Brucella are of key importance for a better knowledge of the epidemiology of brucellosis, for identifying appropriate antigens, for managing disease outbreaks and for setting up efficient preventive and control programmes. Such data are required both at national and regional level to assess potential threats for public health. Highly discriminative genotyping methods such as the multiple locus variable number of tandem repeats analysis (MLVA) allow the comparison and assessment of genetic relatedness between field strains of Brucella within the same geographical area. In this study, MLVA biotyping data retrieved from the literature using a systematic review were compared using a clustering analysis and the Hunter-Gaston diversity index (HGDI). Thus, the analysis of the 42 MLVA genotyping results found in the literature on West Africa [i.e., from Ivory Coast (1), Niger (1), Nigeria (34), The Gambia (3), and Togo (3)] did not allow a complete assessment of the actual diversity among field strains of Brucella. However, it provided some preliminary indications on the co-existence of 25 distinct genotypes of Brucella abortus biovar 3 in this region with 19 genotypes from Nigeria, three from Togo and one from Ivory Coast, The Gambia, and Niger. The strong and urgent need for more sustainable molecular data on prevailing strains of Brucella in this sub-region of Africa and also on all susceptible species including humans is therefore highlighted. This remains a necessary stage to allow a comprehensive understanding of the relatedness between field strains of Brucella and the epidemiology of brucellosis within West Africa countries.

MLVA Genotyping Characteristics of Human Brucella melitensis Isolated from Ulanqab of Inner Mongolia, China.

Brucellosis is a serious public health problem in Ulanqab, which is a region located in the middle of the Inner Mongolia Autonomous Region adjacent to Shanxi and Hebei provinces. The disease is prevalent in both the latter provinces and Ulanqab with the highest prevalence of brucellosis occurring in Inner Mongolia. The MLVA-16 scheme is a genotyping tool for assessing genetic diversity and relationships among isolates. Moreover, this genotyping tool can also be applied to epidemiological trace-back investigations. This study reports the occurrence of at least two B. melitensis biovars (1 and 3) in Ulanqab, encompassing 22 and 94 isolates, respectively. B. melitensis biovar 3 was the predominant biovar in the area examined. Panel 1 (MLVA-8) identified three genotypes (42, 63, and 114), with genotype 42 (n = 101) representing 87% of the tested strains. MLVA-11 identified eight genotypes (116, 111, 297, 291, and 342–345) from 116 of the analyzed isolates. All of these isolates were identified as belonging to the East Mediterranean group. Genotype 116 (n = 94) was the predominant genotype and represented 81% of the isolates. The isolates pertaining to this genotype were distributed throughout most of Ulanqab and neighboring regions. The MLVA-16 scheme showed the presence of 69 genotypes, with 46 genotypes being represented by single isolates. This analysis revealed that Ulanqab brucellosis cases had epidemiologically unrelated and sporadic characteristics. The remaining 23 genotypes were shared (between a total of 70 isolates) with each genotype being represented by two to eight isolates. These data indicate that these cases were epidemiologically related. MLVA genotyping confirmed the occurrence of a multipoint outbreak epidemic and intrafamilial brucellosis. Extensive genotype-sharing events were observed among isolates from different regions of Ulanqab and from other provinces of China. These findings suggest either a lack of control of animal movement between different regions or the circulation of contaminated animal products in the market. Our study is the first comprehensive genotyping and genetic analysis of B. melitensis in Ulanqab. We believe that this study will help to improve the effectiveness of brucellosis control programs.

Genotyping of human Brucella melitensis strains isolated in Shanxi province by MLVA-2B typing and rpoB typing

Objective To analyze the genotypes of Brucella melitensis strains isolated in Shanxi province by using multiple-locus variable-number tandem-repeat 2B typing (MLVA-2B) and rpoB typing. Methods Brucella strains were isolated by culturing the blood samples collected from patients with brucellosis in Shanxi province from 2014 to 2015 and then were identified with conventional methods. MLVA panel 2 (eight loci)was used to identify the genotypes of the Brucella melitensis strains, and the results were further analyzed by cluster analysis. The rpoB gene was molecularly characterized by PCR amplification and DNA sequencing in those strains. The rpoB nucleotide sequences of all Brucella strains were compared with the published rpoB gene sequence of the Brucella melitensis strain 16M to reveal specific nucleotide variations. Results A total of 41 Brucella strains were isolated in 2014 and 2015 and all of them belonged to Brucella melitensis biovar 3. Those strains were divided into five groups and thirty-one MLVA types based on MLVA panel 2B with a genetic similarity ranging from 64.78% to 100%. Four mutation sites in rpoB gene were investigated in the 41 Brucella strains and based on that analysis, those strains were grouped as rpoB-2 (629-GTG/985-GTC/1309-CTA, 95.12%) and rpoB-2 variant (629-GTG/1309-CTA, 4.88%). Conclusion MLVA genotyping was significant for analyzing the sources, epidemics and outbreaks of brucellosis. Analyzing the panel 2B loci was a simple and efficient way for genotyping the Brucella melitensis strains isolated in Shanxi province. The 41 strains belonged to two genotypes on the basis of rpoB typing, including 39 strains of rpoB-2 genotype and 2 strains of a new genotype rpoB-2 variant. There might be a correlation between MLVA and rpoB genotyping. Key words: Brucella; Multiple-locus variable-number tandem-repeat analysis; rpoB gene

Opportunistic fungal pathogen Candida glabrata circulates between humans and yellow-legged gulls.

The opportunistic pathogenic yeast Candida glabrata is a component of the mycobiota of both humans and yellow-legged gulls that is prone to develop fluconazole resistance. Whether gulls are a reservoir of the yeast and facilitate the dissemination of human C. glabrata strains remains an open question. In this study, MLVA genotyping highlighted the lack of genetic structure of 190 C. glabrata strains isolated from either patients in three hospitals or fecal samples collected from gull breeding colonies located in five distinct areas along the French Mediterranean littoral. Fluconazole-resistant isolates were evenly distributed between both gull and human populations. These findings demonstrate that gulls are a reservoir of this species and facilitate the diffusion of C. glabrata and indirect transmission to human or animal hosts via environmental contamination. This eco-epidemiological view, which can be applied to other vertebrate host species, broadens our perspective regarding the reservoirs and dissemination patterns of antifungal-resistant human pathogenic yeast.

Genotyping of Brucella melitensis and Brucella abortus strains currently circulating in Xinjiang, China

Brucellosis is a well-known zoonotic disease that can cause severe economic and healthcare losses. Xinjiang, one of the biggest livestock husbandry sectors in China, has gone through increasing incidence of brucellosis in cattle and small ruminants recently. In this paper, 50 B. melitensis strains and 9 B. abortus strains collected from across Xinjiang area (from 2010 to 2015) were genotyped using multiple locus variable-number tandem-repeat (VNTR) analysis (MLVA) and multi-locus sequence typing (MLST). Based on 8 loci (MLVA-8), 50 B. melitensis strains were classified into three genotypes. Genotypes 42 (n=38, 76%) and 63 (n=11, 22%) were part of the East Mediterranean group, and one genotype with pattern of 1-5-3-13-2-4-3-2 represents a single-locus variant from genotype 63. MLVA-16 resolved 50 B. melitensis strains into 28 genotypes, of which 15 are unique to Xinjiang and 10 are in common with those in adjacent country Kazakhstan and neighboring provinces of China. Minimum Spanning Tree (MST) analysis implies that B. melitensis strains collected from across Kazakhstan, Xinjiang and China areas may share a common origin. Nine B. abortus strains were sorted into three genotypes by MLVA-8, genotypes 36 (n=7, 77.8%), 86 (n=1, 11.1%) and a new genotype with pattern of 4-5-3-13-2-2-3-1. Each B. abortus strain showed distinct MLVA-16 genotypes, suggesting that B. abortus species may possess more genetic diversity than B. melitensis. Using MLST, most B. melitensis strains (n=49) were identified as sequence type ST8, and most B. abortus strains (n=8) were recognized as ST2. Two new sequence types, ST37 and ST38, represented by single strain from B. melitensis and B. abortus species respectively, were also detected in this study. These results could facilitate the pathogen surveillance in the forthcoming eradication programs and serve as a guide in source tracking in case of new outbreaks occur.

Multiple Locus Variable number of tandem repeat Analysis: A molecular genotyping tool for Paenibacillus larvae.

SummaryAmerican Foulbrood, caused by Paenibacillus larvae, is the most severe bacterial disease of honey bees (Apis mellifera). To perform genotyping of P. larvae in an epidemiological context, there is a need of a fast and cheap method with a high resolution. Here, we propose Multiple Locus Variable number of tandem repeat Analysis (MLVA). MLVA has been used for typing a collection of 209 P. larvae strains from which 23 different MLVA types could be identified. Moreover, the developed methodology not only permits the identification of the four Enterobacterial Repetitive Intergenic Consensus (ERIC) genotypes, but allows also a discriminatory subdivision of the most dominant ERIC type I and ERIC type II genotypes. A biogeographical study has been conducted showing a significant correlation between MLVA genotype and the geographical region where it was isolated.

Coxiella burnetii Genotypes in Iberian Wildlife.

To investigate if Coxiella burnetii, the causative agent of Q fever, genotypes circulating in wildlife are associated with those infecting livestock and humans, multiple-locus variable number tandem-repeat analysis (MLVA-6-marker) was carried out over C. burnetii obtained from red deer (Cervus elaphus), Eurasian wild boar (Sus scrofa), European wild rabbit (Oryctolagus cuniculus), black rat (Rattus rattus), and wood mouse (Apodemus sylvaticus). MLVA typing was performed by using six variable loci in C. burnetii: Ms23, Ms24, Ms27, Ms28, Ms33, and Ms34. The C. burnetii cooperative database from MLVABank 5.0 was employed to compare genotypes found in this study with 344 isolates of diverse origin. Twenty-two genotypes from wildlife and two genotypes from domestic goats were identified. Some MLVA genotypes identified in wildlife or in farmed game clustered with genotypes of human Q fever clinical cases, supporting the idea that humans and wildlife share C. burnetii genotypes. The major part of genotypes identified in coexisting red deer and rabbits clustered according to their host of origin, suggesting host specificity for particular C. burnetii genotypes. These findings provide important insights to understand the epidemiology of C. burnetii at the wildlife-livestock-human interface.

Cases of human brucellosis in Sweden linked to Middle East and Africa.

BackgroundHuman brucellosis cases are still reported each year in Sweden despite eradication of the disease in animals. Epidemiological investigation has never been conducted to trace back the source of human infection in the country. The purpose of the study was to identify the source of infection for 16 human brucellosis cases that occurred in Sweden, during the period 2008–2012.ResultsThe isolates were identified as Brucella melitensis and MLVA-16 genotyping revealed 14 different genotypes of East Mediterranean and Africa lineages. We also reported one case of laboratory-acquired brucellosis (LAB) that was shown to be epidemiological linked to one of the cases in the current study.ConclusionsBrucella melitensis was the only species diagnosed, confirming its highest zoonotic potential in the genus Brucella, and MLVA-16 results demonstrated that the cases of brucellosis in Sweden herein investigated, are imported and linked to travel in the Middle East and Africa. Due to its zoonotic concerns, any acute febrile illness linked to recent travel within those regions should be investigated for brucellosis and samples should be processed according to biosafety level 3 regulations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2074-7) contains supplementary material, which is available to authorized users.

Analyzing Deoxyribose Nucleic Acid from Malaria Rapid Diagnostic Tests to Study Plasmodium falciparum Genetic Diversity in Mali.

We evaluated the use of positive malaria rapid diagnostic tests (mRDTs) to determine genetic diversity of Plasmodium falciparum in Mali. Genetic diversity was assessed via multiple loci variable number of tandem repeats analysis (MLVA). We performed DNA extraction from 104 positive and 30 negative used mRDTs that had been stored at ambient temperature for up to 14 months. Extracted DNA was analyzed via quantitative polymerase chain reaction (qPCR), and MLVA genotyping was then assessed on positive qPCR samples. Eighty-three of the positive mRDTs (83/104, 79.8%) and none of the negative mRDTs were confirmed P. falciparum positive via qPCR. We achieved complete genotyping of 90.4% (75/83) of the qPCR-positive samples. Genotyping revealed high genetic diversity among P. falciparum populations in Mali and an absence of population clustering. We show that mRDTs are useful to monitor P. falciparum genetic diversity and thereby can provide essential data to guide malaria control programs.

Diagnosis of Human Brucellosis

Brucellosis is a worldwide zoonosis with a high degree of morbidity in humans. The disease may be overlooked and misdiagnosed because of the difficult diagnosis and the absence and lack of experience with laboratory testing. As the clinical picture of human brucellosis is fairly non-specific, a definitive diagnosis requires isolation of the causative organism, or the demonstration of the high levels of specific antibodies, or organism specific DNA in samples. Isolation of organism is time-consuming and hazardous, so must be performed by highly skilled personnel. So serological methods are preferred. Advanced serological methods like ELISA, Brucellacapt are more sensitive techniques. Combination of culture and serological test should be used to avoid misdiagnosis. Molecular assays come with high sensitivities and specificities and may reduce diagnostic delays in clinical laboratories. Several PCR based assays have been studied for standardizing them for brucellosis diagnosis. Another rapid, sensitive and inexpensive molecular technique, LAMP has been developed by designing primers specific for Brucella abortus genes. This technique can prove helpful in resource limited settings in developing countries. Highthroughput MLVA-16 genotyping techniques are also being studied and developed for brucella typing for tracing the source of brucellosis infection. This test can be useful for brucella diagnosis and surveillance studies. This review article describes procedures, advantages or limitations of some useful conventional diagnostic techniques to more sophisticated molecular techniques for brucellosis diagnosis.

Bacillus anthracis Diversity and Geographic Potential across Nigeria, Cameroon and Chad: Further Support of a Novel West African Lineage.

Zoonoses, diseases affecting both humans and animals, can exert tremendous pressures on human and veterinary health systems, particularly in resource limited countries. Anthrax is one such zoonosis of concern and is a disease requiring greater public health attention in Nigeria. Here we describe the genetic diversity of Bacillus anthracis in Nigeria and compare it to Chad, Cameroon and a broader global dataset based on the multiple locus variable number tandem repeat (MLVA-25) genetic typing system. Nigerian B. anthracis isolates had identical MLVA genotypes and could only be resolved by measuring highly mutable single nucleotide repeats (SNRs). The Nigerian MLVA genotype was identical or highly genetically similar to those in the neighboring countries, confirming the strains belong to this unique West African lineage. Interestingly, sequence data from a Nigerian isolate shares the anthrose deficient genotypes previously described for strains in this region, which may be associated with vaccine evasion. Strains in this study were isolated over six decades, indicating a high level of temporal strain stability regionally. Ecological niche models were used to predict the geographic distribution of the pathogen for all three countries. We describe a west-east habitat corridor through northern Nigeria extending into Chad and Cameroon. Ecological niche models and genetic results show B. anthracis to be ecologically established in Nigeria. These findings expand our understanding of the global B. anthracis population structure and can guide regional anthrax surveillance and control planning.