Molecular Epidemiology of Cryptosporidiosis in Children in Malawi

ABSTRACT: Few studies have examined the molecular epidemiology of cryptosporidiosis in developing countries. In this study, DNA of 69 microscopy-positive human fecal samples collected from Malawi were examined by multilocus genetic analyses. From 43, 27 and 28 of the samples, the SSU rRNA, 70 kDa heat shock protein (HSP70) and 60 kDa glycoprotein (GP60) genes, respectively, were successfully PCR-amplified. Restriction analysis of the SSU PCR products showed that 41 of the 43 PCR-positive samples had C. hominis and 2 had C. parvum. Sequence analysis of the HSP70 and GP60 gene contirmed the species identification by SSU rRNA PCR-RFLP analysis, but also revealed high intraspecific variations. Altogether, six HSP70 subtypes and six GP60 subtypes (belonging to lour subtype alleles) of C. hominis were found. Linkage diseyuilibrum analysis of the two genetic loci showed possible intraspecitic recombination. Thus, cryptosporidiosis in the study area was largely caused by anthroponotic transmission. The high intraspecitic variation and existence of genetic recombination were probably results of high transmission of cryptosporidiosis in this area.     

Extrahuman Epidemiology of Acinetobacter baumannii in Lebanon

The presence of Acinetobacter baumannii outside hospitals is still a controversial issue. The objective of our study was to explore the extrahospital epidemiology of A. baumannii in Lebanon. From February 2012 to October 2013, a total of 73 water samples, 51 soil samples, 37 raw cow milk samples, 50 cow meat samples, 7 raw cheese samples, and 379 animal samples were analyzed by cultural methods for the presence of A. baumannii. Species identification was performed by rpoB gene sequencing. Antibiotic susceptibility was investigated, and the A. baumannii population was studied by two genotyping approaches: multilocus sequence typing (MLST) and bla_OXA-51 sequence-based typing (SBT). A. baumannii was detected in 6.9% of water samples, 2.7% of milk samples, 8.0% of meat samples, 14.3% of cheese samples, and 7.7% of animal samples. All isolates showed a susceptible phenotype against most of the antibiotics tested and lacked carbapenemase-encoding genes, except one that harbored a bla_OXA-143 gene. MLST analysis revealed the presence of 36 sequence types (STs), among which 24 were novel STs reported for the first time in this study. bla_OXA-51 SBT showed the presence of 34 variants, among which 21 were novel and all were isolated from animal origins. Finally, 30 isolates had new partial rpoB sequences and were considered putative new Acinetobacter species. In conclusion, animals can be a potential reservoir for A. baumannii and the dissemination of new emerging carbapenemases. The roles of the novel animal clones identified in community-acquired infections should be investigated.     

肠道病毒71型分子流行病学研究进展

肠道病毒71型(Enterovirus type71,EV71),自1974年首次报道以来,在世界范围内引起多次爆发与流行[1].EV71感染主要引起患者手足口病(hand,foot and mouth disease,HFMD),在临床上与柯萨奇病毒A16(Coxsakie A16,CA16)感染所引起的手足口病难以区别,但EV71还能够引起多种与神经系统相关的疾病[2].近年来,EV71病毒的流行在亚太地区呈上升趋势[3～5],其中最令人关注的是在该地区的EV71感染引起越来越严重的中枢神经系统症状.     

Molecular Epidemiology of Carbapenem Non-Susceptible Acinetobacter baumannii in France

Carbapenem-resistant Acinetobacter baumannii have emerged globally. The objective of this study was to investigate the epidemiology, clonal diversity and resistance mechanisms of imipenem non-susceptible A. baumannii isolates in France. Between December 2010 and August 2011, 132 notifications were collected, including 37 outbreaks corresponding to 242 cases (2 to 55 per cluster). Multilocus sequence typing, pulsed-field gel electrophoresis (PFGE) and characterisation of carbapenemase-encoding genes were performed on 110 non-repetitive isolates. Gene bla_OXA-23 was the most frequently detected (82%), followed by bla_OXA-24 (11%) and bla_OXA-58 (7%). Eleven sequence types (ST) were distinguished, among which sequence types ST1, ST2 (64%), ST20, ST25, ST85 and ST107. Isolates from epidemiological clusters had the same ST and resistance genes, indicating probable transmission within centres. In contrast, PFGE types of isolates differed among centres, arguing against transmission among centers. This study provides the first epidemiological snapshot of the population of A. baumannii with reduced susceptibility to carbapenems from France, and further underlines the predominance of international clones.     

Molecular Epidemiology of Crimean-Congo Hemorrhagic Fever Virus in Kosovo

Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic agent that causes severe, life-threatening disease, with a case fatality rate of 10–50%. It is the most widespread tick-borne virus in the world, with cases reported in Africa, Asia and Eastern Europe. CCHFV is a genetically diverse virus. Its genetic diversity is often correlated to its geographical origin. Genetic variability of CCHFV was determined within few endemic areas, however limited data is available for Kosovo. Furthermore, there is little information about the spatiotemporal genetic changes of CCHFV in endemic areas. Kosovo is an important endemic area for CCHFV. Cases were reported each year and the case-fatality rate is significantly higher compared to nearby regions. In this study, we wanted to examine the genetic variability of CCHFV obtained directly from CCHF-confirmed patients, hospitalized in Kosovo from 1991 to 2013. We sequenced partial S segment CCHFV nucleotide sequences from 89 patients. Our results show that several viral variants are present in Kosovo and that the genetic diversity is high in relation to the studied area. We also show that variants are mostly uniformly distributed throughout Kosovo and that limited evolutionary changes have occurred in 22 years. Our results also suggest the presence of a new distinct lineage within the European CCHF phylogenetic clade. Our study provide the largest number of CCHFV nucleotide sequences from patients in 22 year span in one endemic area.     

Seroepidemiology and Molecular Epidemiology of Enterovirus 71 in Russia

Enterovirus 71 (EV71) is an emerging human pathogen causing massive epidemics of hand, foot and mouth disease with severe neurological complications in Asia. EV71 also circulates in Europe, however it does not cause large outbreaks. The reason for distinct epidemiological patterns of EV71 infection in Europe and Asia and the risk of EV71 epidemic in Europe and Russia remain unknown. Seroepidemiology of EV71 and molecular epidemiology of occasional EV71 isolates were studied to explore circulation of EV71 in Russia. In six regions of Russian Federation, seroprevalence of EV71 in sera collected in 2008 ranged from 5% to 20% in children aged 1–2 years and from 19% to 83% in children aged 3–5 years. The seroprevalence among elder children was significantly higher (41–83% vs. 19–27%) in Asian regions of Russia. EV71 strains identified in Russia in 2001–2011 belonged to subtypes C1 and C2, while genotype C4 that was causing epidemics in Asia since 1998 emerged in 2009 and became dominant in 2013.     

First Molecular Epidemiology Study of Mycobacterium tuberculosis in Kiribati

Tuberculosis incidence rates in Kiribati are among the highest in the Western Pacific Region, however the genetic diversity of circulating Mycobacterium tuberculosis complex strains (MTBC) and transmission dynamics are unknown. Here, we analysed MTBC strains isolated from culture positive pulmonary tuberculosis (TB) cases from the main TB referral centre between November 2007 and October 2009. Strain genotyping (IS6110 typing, spoligotyping, 24-loci MIRU-VNTR and SNP typing) was performed and demographic information collected. Among 73 MTBC strains analysed, we identified seven phylogenetic lineages, dominated by Beijing strains (49%). Beijing strains were further differentiated in two main branches, Beijing-A (n = 8) and -B (n = 28), that show distinct genotyping patterns and are characterized by specific deletion profiles (Beijing A: only RD105, RD207 deleted; Beijing B: RD150 and RD181 additionally deleted). Many Kiribati strains (59% based on IS6110 typing of all strains) occurred in clusters, suggesting ongoing local transmission. Beijing-B strains and over-crowded living conditions were associated with strain clustering (likely recent transmission), however little evidence of anti-tuberculous drug resistance was observed. We suggest enhanced case finding amongst close contacts and continued supervised treatment of all identified cases using standard first-line drugs to reduce TB burden in Kiribati. Beijing strains can be subdivided in different principle branches that might be associated with differential spreading patterns in the population.     

Molecular Analysis of Acinetobacter baumannii Strains Isolated in Lebanon Using Four Different Typing Methods

This study analyzed 42 Acinetobacter baumannii strains collected between 2009–2012 from different hospitals in Beyrouth and North Lebanon to better understand the epidemiology and carbapenem resistance mechanisms in our collection and to compare the robustness of pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), repetitive sequence-based PCR (rep-PCR) and bla _OXA-51 sequence-based typing (SBT). Among 31 carbapenem resistant strains, we have detected three carbapenem resistance genes: 28 carried the bla _OXA-23 gene, 1 the bla _OXA-24 gene and 2 strains the bla _OXA-58 gene. This is the first detection of bla _OXA-23 and bla _OXA-24 in Lebanon. PFGE identified 11 types and was the most discriminating technique followed by rep-PCR (9 types), bla _OXA-51 SBT (8 types) and MLST (7 types). The PFGE type A''/ST2 was the dominant genotype in our collection present in Beyrouth and North Lebanon. The clustering agreement between all techniques was measured by adjust Wallace coefficient. An overall agreement has been demonstrated. High values of adjust Wallace coefficient were found with followed combinations: PFGE to predict MLST types  = 100%, PFGE to predict bla _OXA-51 SBT = 100%, bla _OXA-51 SBT to predict MLST = 100%, MLST to predict bla _OXA-51 SBT = 84.7%, rep-PCR to predict MLST = 81.5%, PFGE to predict rep-PCR = 69% and rep-PCR to predict bla _OXA-51 SBT = 67.2%. PFGE and MLST are gold standard methods for outbreaks investigation and population structure studies respectively. Otherwise, these two techniques are technically, time and cost demanding. We recommend the use of bla _OXA-51 SBT as first typing method to screen isolates and assign them to their corresponding clonal lineages. Repetitive sequence-based PCR is a rapid tool to access outbreaks but careful interpretation of results must be always performed.     

Molecular Epidemiology of Cases of Mycoplasma californicum Infection in Japan

Bovine mastitis due to Mycoplasmacalifornicum is often accompanied by huge economic losses, and the disease spreads very quickly. An appropriate molecular epidemiological analysis is needed to prevent and control infectious disease, but molecular epidemiological analysis methods for M. californicum have not yet been reported. Here we developed a combination of multiple-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) methods, which are common genotyping methods for various bacteria, for M. californicum. The MLVA is based on four interspersed repeat units that were found in the M. californicum genome data. The MLVA using these repeat units showed sufficient discriminatory power for a molecular epidemiological analysis; i.e., a Hunter-Gaston diversity index (HGDI) of 0.949, against M. californicum strains in Japan and M. californicum strain ATCC 33461. The PFGE for M. californicum also showed sufficient discriminatory power, with an HGDI of 0.985. Strain ATCC 33461 showed MLVA profiles and pulsotypes that differed greatly from those of strains from Japan. These results indicate that MLVA and PFGE are good tools for identifying M. californicum transmission events more accurately. Our combined MLVA and PFGE analysis suggests the persistence of M. californicum infection among herds in a specific area for a long period of time, as well as the movement of cows and heifers accompanying the expansion of M. californicum infection. Failure to identify asymptomatic infected cows is suspected as one of the central causes of the present M. californicum infection scenario in Japan.     

Molecular Epidemiology of Human Oral Chagas Disease Outbreaks in Colombia

Background

Trypanosoma cruzi, the causative agent of Chagas disease, displays significant genetic variability revealed by six Discrete Typing Units (TcI-TcVI). In this pathology, oral transmission represents an emerging epidemiological scenario where different outbreaks associated to food/beverages consumption have been reported in Argentina, Bolivia, Brazil, Ecuador and Venezuela. In Colombia, six human oral outbreaks have been reported corroborating the importance of this transmission route. Molecular epidemiology of oral outbreaks is barely known observing the incrimination of TcI, TcII, TcIV and TcV genotypes.

Methodology and Principal Findings

High-throughput molecular characterization was conducted performing MLMT (Multilocus Microsatellite Typing) and mtMLST (mitochondrial Multilocus Sequence Typing) strategies on 50 clones from ten isolates. Results allowed observing the occurrence of TcI, TcIV and mixed infection of distinct TcI genotypes. Thus, a majority of specific mitochondrial haplotypes and allelic multilocus genotypes associated to the sylvatic cycle of transmission were detected in the dataset with the foreseen presence of mitochondrial haplotypes and allelic multilocus genotypes associated to the domestic cycle of transmission.

Conclusions

These findings suggest the incrimination of sylvatic genotypes in the oral outbreaks occurred in Colombia. We observed patterns of super-infection and/or co-infection with a tailored association with the severe forms of myocarditis in the acute phase of the disease. The transmission dynamics of this infection route based on molecular epidemiology evidence was unraveled and the clinical and biological implications are discussed.     

Molecular Ordering of the Initial Signaling Events of CD95

Binding of either ligand or agonistic antibodies to the death receptor CD95 (APO-1/Fas) induces the formation of the death-inducing signaling complex (DISC). We now show that signal initiation of CD95 in type I cells can be further separated into at least four distinct steps. (i) The first step is ligand-induced formation of CD95 microaggregates at the cell surface. (ii) The second step is recruitment of FADD to form a DISC. This step is dependent on actin filaments. (iii) The third step involves formation of large CD95 surface clusters. This event is positively regulated by DISC-generated caspase 8. (iv) The fourth step is internalization of activated CD95 through an endosomal pathway. The latter step is again dependent on the presence of actin filaments. The data indicate that the signal initiation by CD95 is a complex process actively regulated at various levels, providing a number of new drug targets to specifically modulate CD95 signaling.     

广东湛江听神经病分子流行病学调查

目的:研究听神经病在广东湛江耳聋人群中的分子流行病学。方法:收集耳聋患者静脉血样标本362例,选取WFS1、OTOF两个基因上的3个位点,利用Taqman实时荧光定量聚合酶链反应对其上热点突变进行基因分型分析。结果:共发现39例含有听神经病热点突变,检出率高达10.8%。结论:遗传性听神经病在湛江耳聋患者中占有很大比例,应将高检出率位点纳入遗传性耳聋分子病因学诊断,为听神经病的临床治疗提供参考依据。     

Molecular Epidemiology of Simian Immunodeficiency Virus Infection in Wild-Living Gorillas

Chimpanzees and gorillas are the only nonhuman primates known to harbor viruses closely related to HIV-1. Phylogenetic analyses showed that gorillas acquired the simian immunodeficiency virus SIVgor from chimpanzees, and viruses from the SIVcpz/SIVgor lineage have been transmitted to humans on at least four occasions, leading to HIV-1 groups M, N, O, and P. To determine the geographic distribution, prevalence, and species association of SIVgor, we conducted a comprehensive molecular epidemiological survey of wild gorillas in Central Africa. Gorilla fecal samples were collected in the range of western lowland gorillas (n = 2,367) and eastern Grauer gorillas (n = 183) and tested for SIVgor antibodies and nucleic acids. SIVgor antibody-positive samples were identified at 2 sites in Cameroon, with no evidence of infection at 19 other sites, including 3 in the range of the Eastern gorillas. In Cameroon, based on DNA and microsatellite analyses of a subset of samples, we estimated the prevalence of SIVgor to be 1.6% (range, 0% to 4.6%), which is significantly lower than the prevalence of SIVcpzPtt in chimpanzees (5.9%; range, 0% to 32%). All newly identified SIVgor strains formed a monophyletic lineage within the SIVcpz radiation, closely related to HIV-1 groups O and P, and clustered according to their field site of origin. At one site, there was evidence for intergroup transmission and a high intragroup prevalence. These isolated hot spots of SIVgor-infected gorilla communities could serve as a source for human infection. The overall low prevalence and sporadic distribution of SIVgor could suggest a decline of SIVgor in wild populations, but it cannot be excluded that SIVgor is still more prevalent in other parts of the geographical range of gorillas.Simian immunodeficiency viruses (SIVs) have been identified in approximately 40 African primate species, but chimpanzees and gorillas are the only nonhuman primates known to harbor viruses closely related to human immunodeficiency virus type 1 (HIV-1) (38). These viruses have been transmitted to humans on at least four occasions, leading to four different HIV-1 groups, M to P (14, 26). West central African chimpanzees (Pan troglodytes troglodytes) in southern Cameroon are recognized as the reservoir of the ancestors of HIV-1 group M, which resulted in the AIDS pandemic, and of HIV-1 group N, which has been identified in only a few individuals in Cameroon (15). Western lowland gorillas (Gorilla gorilla gorilla) are infected with SIVgor, which is closely related to the two other HIV-1 lineages, termed group O, which represents 1% of HIV-1 infections in west central Africa, and group P, recently described from a single Cameroonian patient residing in France (26, 36).The phylogenetic relationships between SIVcpz, SIVgor, and HIV-1 show that chimpanzees are the original reservoir of SIVs found in gorillas and humans (31, 36). Pan troglodytes troglodytes apes were most likely the original source of SIVgor, because SIVgor is significantly more closely related to SIVcpzPtt, from Pan troglodytes troglodytes in west central Africa, than to SIVcpzPts, from Pan troglodytes schweinfurthii in east Africa. In addition, an ancestral SIVcpzPtt lineage from which SIVgor and HIV-1 group O viruses are derived has been identified in the form of mosaic pol fragments in present-day SIVcpzPtt recombinants (2, 31). However, the ways of transmission and the exact origin of SIVgor infection in gorillas are not yet resolved. Because of the extensive overlap in habitat and diet (6, 23, 29, 33, 40), direct encounters between gorillas and chimpanzees seem inevitable, but they have rarely been observed and have been described as primarily nonaggressive (17, 28). The primate source of HIV-1 groups O and P also remains unclear, since current data do not allow one to differentiate between a chimpanzee and a gorilla reservoir, especially for HIV-1 group O (26, 31, 36).To determine the geographic distribution, prevalence, and species association of SIVgor, we performed a comprehensive survey of wild gorilla populations in west central (Gorilla gorilla gorilla) and east (Gorilla beringei graueri) Africa. We found an overall prevalence of SIVgor of 1.6%, with infection confirmed at only three field sites. At two of these sites, however, the prevalence of SIVgor was 4.6%, indicating efficient virus spread within and between different communities. The geographic distribution of SIVgor is thus far limited to only a few sites in Cameroon. However, isolated hot spots of infection do exist, which could serve as a source of human infection.     

Molecular Epidemiology of Tuberculosis in Finland, 2008-2011

In industrialized countries the majority of tuberculosis (TB) cases are linked to immigration. In Finland, most cases are still Finnish born but the number of foreign born cases is steadily increasing. In this 4-year population based study, the TB situation in Finland was characterized by a genotypic analysis of Mycobacterium tuberculosis isolates. A total of 1048 M. tuberculosis isolates (representing 99.4% of all culture positive cases) were analyzed by spoligotyping and MIRU. Spoligotype lineages belonging to the Euro-American family were predominant among the Finnish isolates, particularly T (n=346, 33.0%) and Haarlem (n=237, 22.6%) strains. The lineage signature was unknown for 130 (12.4%) isolates. Out of the 17 multi-drug resistant TB strains, 10 (58.8%) belonged to the Beijing lineage. In total, 23 new SIT designations were given and 51 orphan strains were found, of which 58 patterns were unique to Finland. Phylogeographical TB mapping as compared to neighboring countries showed that the population structure in Finland most closely resembled that observed in Sweden. By combining spoligotyping and MIRU results, 98 clusters comprising 355 isolates (33.9%) were found. Only 10 clusters contained both Finnish and foreign born cases. In conclusion, a large proportion of the M. tuberculosis isolates were from Finnish born elderly patients. Moreover, many previously unidentified spoligotype profiles and isolates belonging to unknown lineages were encountered.     

Exploring the Molecular Epidemiology and Evolutionary Dynamics of Influenza A Virus in Taiwan

The evolution and population dynamics of human influenza in Taiwan is a microcosm of the viruses circulating worldwide, which has not yet been studied in detail. We collected 343 representative full genome sequences of human influenza A viruses isolated in Taiwan between 1979 and 2009. Phylogenetic and antigenic data analysis revealed that H1N1 and H3N2 viruses consistently co-circulated in Taiwan, although they were characterized by different temporal dynamics and degrees of genetic diversity. Moreover, influenza A viruses of both subtypes underwent internal gene reassortment involving all eight segments of the viral genome, some of which also occurred during non-epidemic periods. The patterns of gene reassortment were different in the two subtypes. The internal genes of H1N1 viruses moved as a unit, separately from the co-evolving HA and NA genes. On the other hand, the HA and NA genes of H3N2 viruses tended to segregate consistently with different sets of internal gene segments. In particular, as reassortment occurred, H3HA always segregated as a group with the PB1, PA and M genes, while N2NA consistently segregated with PB2 and NP. Finally, the analysis showed that new phylogenetic lineages and antigenic variants emerging in summer were likely to be the progenitors of the epidemic strains in the following season. The synchronized seasonal patterns and high genetic diversity of influenza A viruses observed in Taiwan make possible to capture the evolutionary dynamic and epidemiological rules governing antigenic drift and reassortment and may serve as a “warning” system that recapitulates the global epidemic.     

2000年～2007年山东省风疹病毒分子流行病学研究

本研究对使用Vero细胞、RK13细胞或Vero/SLAM细胞从2000年～2007年山东省6个市风疹暴发和散发病例的咽拭子标本中所分离的风疹病毒,用RT-PCR方法扩增其E1基因的1107个核苷酸片段,并对其PCR产物进行序列测定和分析。结果提示,在基于WHO基因定型靶序列739个核苷酸片段构建的基因亲缘关系树上,16株风疹病毒株分属三个基因型:1E(12株)、1F(1株)和2A(3株)。1E基因型于2001年在山东省首次检测到,并逐年成为我省风疹病毒流行的优势基因型,其中,2006～2007年间流行的1E基因型风疹病毒与2001年和2002年流行的1E基因型风疹病毒存在差异,但无明显的时间和地理分布倾向;1F基因型和2A基因型分别于2000年、2001年分离到,至2007年间未再出现;3株2A基因型风疹病毒与我国风疹疫苗株(BRDII)密切相关。16株风疹病毒大部分核苷酸的突变为无义突变,氨基酸序列高度保守,均无重要抗原位点的改变;山东省2001～2007年间分离的所有1E基因型风疹病毒在E1蛋白的第338位氨基酸发生相同突变(Leu338→Phe338),以往报道相同。