Prevalence and carrier status of leptospirosis in smallholder dairy cattle and peridomestic rodents in Kandy, Sri Lanka

Leptospirosis is an important bacterial zoonotic disease globally and one of the notifiable diseases in Sri Lanka. Other than human leptospirosis, little information is available on leptospirosis in domestic and feral animals in Sri Lanka. Thus, this study attempted to determine the prevalence and carrier status of leptospirosis in smallholder dairy cattle and peridomestic rodents to understand the impact of the disease on public health in Kandy, Sri Lanka. Cattle and rodent samples were collected from the Yatinuwara and Udunuwara divisional secretaries in Kandy. Serum samples were analyzed for the presence of antileptospiral antibodies using microscopic agglutination test. DNA was extracted from cattle urine and rodent kidney tissue samples, in which polymerase chain reaction was carried out to detect the Leptospira flaB gene. The cattle in 19 (38.8%) of the 49 farms harbored antileptospiral antibodies. Out of 113 cattle serum samples, 23 (20.3%) were positive; 17 (73.9%) and 6 (26.1%) reacted with serogroups Sejroe and Hebdomadis, respectively. Out of the 74 rodent samples, 13 (17.5%) were positive; 8 (61.5%) and 4 (30.8%) had reactions to serogroups Javanica and Icterohaemorrhagiae, respectively. Leptospiral DNA was detected in one cattle urine sample and identified as Leptospira interrogans. This study revealed a high prevalence of leptospirosis in cattle and rodents in Kandy. These animals were infected with a wide array of leptospiral serogroups, which are consistent with the research findings observed in humans in Kandy. Overall, serological data indicate that relative to rodents, cattle may be a more significant reservoir for human transmission and a greater source of potential risk to local agricultural communities.     

The regional sero-epidemiology of rhinosporidiosis in Sri Lankan humans and animals

No data is available in the world literature on serum anti-rhinosporidial antibody levels in animals, and as far as we aware this is the first report. Although rhinosporidiosis in farm and domestic animals has been widely reported from other countries, rhinosporidiosis in animals has not been reported in Sri Lanka, though this country has the highest world-wide prevalence of human rhinosporidiosis on a unit-population basis. Serum IgG titres in 6 species of Sri Lankan animals (buffalo, cat, cattle, dog, goat, horse; total 291) were assayed by the Immuno blot (dot-ELISA) method on nitrocellulose paper and were compared with serum IgG titres in normal Sri Lankan human subjects (total 211) in different geographical areas, and in human Sri Lankan patients with rhinosporidiosis as reference values (total 36). Sensitization to rhinosporidial antigen(s) was detected in all 6 species of animals and the highest titres (1/3200) were found in cats, and free-grazing horses. Cattle showed higher levels of antibody than buffaloes. The titres in these animals are compared with world reports on overt rhinosporidiosis in these species, and with titres in normal Sri Lankan humans. Human, but not animal titres showed variations compatible with the regional prevalence of rhinosporidiosis. The variations in titres in animals especially horses, were probably more related to their mode of feeding, while in humans the titres in normal persons were probably related to the rhinosporidial-endemicity of their respective regions. No conclusions from sero-positivity in animals could be made regarding the absence of reports on rhinosporidiosis as an overt disease in these Sri Lankan animal species but the possibility of a genetically-determined insusceptibility to rhinosporidiosis in Sri Lanka, is considered. Rhinosporidium seeberi-specific PCR positive reactions were obtained with nasal scrapings from cattle that microscopically showed PAS+ bodies that were compatible with rhinosporidial sporangia. Sequence-analysis of the reactions products from five positive R. seeberi-specific PCR samples (four in this study and 1 in a previous study) gave results confirmatory of R. seeberi.     

Molecular epidemiology of Leptospira borgpetersenii serovar Arborea, Queensland, Australia, 1998-2005

Leptospira borgpetersenii serovar Arborea is an emerging cause of leptospirosis in Australia. It was not previously recognized as an endemic serovar before the 1990s, but at that point, human infections with the serovar increased significantly. Using fluorescent-amplified fragment-length polymorphism (FAFLP) molecular typing, human and rodent isolates were compared genetically. Typing revealed 11 unique profiles among the 23 isolates examined; however, there was no clonality revealed between the human and rodent isolates. There was clonality among rodent isolates from geographically related areas. This study highlights the utility of Leptospira culture combined with FAFLP for the examination of the epidemiology of this disease.     

Leptospirosis outbreak in Sri Lanka in 2008: lessons for assessing the global burden of disease

Global leptospirosis disease burden estimates are hampered by the lack of scientifically sound data from countries with probable high endemicity and limited diagnostic capacities. We describe the seroepidemiologic and clinical characteristics of the leptospirosis outbreak in 2008 in Sri Lanka. Definitive/presumptive case definitions proposed by the World Health Organization Leptospirosis Epidemiology Reference Group were used for case confirmation. Of the 404 possible cases, 155 were confirmed to have leptospirosis. Highest titers of patient seum samples reacted with serovars Pyrogenes (28.7%), Hardjo (18.8%), Javanica (11.5%), and Hebdomadis (11.5%). Sequencing of the 16S ribosomal DNA gene identified six infections: five with Leptospira interrogans and one with L. weilli. In this patient population, acute renal failure was the main complication (14.8%), followed by myocarditis (7.1%) and heart failure (3.9%). The case-fatality rate was 1.3%. This report strengthens the urgent need for increasing laboratory diagnostic capabilities to determine the causes of epidemic and endemic infectious diseases in Sri Lanka, a finding relevant to other tropical regions.     

Case Report: Saw-Scaled Viper Bites in Sri Lanka: Is It a Different Subspecies? Clinical Evidence from an Authenticated Case Series

The saw-scaled viper (SSV) (Echis carinatus) is considered to be a highly venomous snake in Sri Lanka despite any published clinical justification. Being a rarity, the clinical profile of SSV bites is not well established in Sri Lanka. We report a series of 48 (n-48) SSV bites from the Northern Province of Sri Lanka. The majority (65%) of victims had evidence of local envenoming at the site of the bite; however, 29% showed spontaneous bleeding and 71% had coagulopathy. There were no deaths in the series. The envenoming was mild in contrast to the mortality and significant morbidity associated with SSV bites in West Africa and some parts of India. These observations need to be further explored with laboratory studies to identify the venom components, study of morphological characteristics, and genetic profiling of the Sri Lankan SSV to see if it is different from the subspecies found elsewhere.     

Manifestations and outcomes of leptospirosis during local outbreaks in high endemic districts of Sri Lanka: A retrospective multi-center study

Objective: To determine the clinical presentations and disease outcomes of suspected and confirmed cases of leptospirosis from 3 high endemic districts of Sri Lanka, during outbreaks reported between 2013 and 2017. Methods: The retrospective multi-center study was carried out during 2013-2017 in 5 selected hospitals representing 3 high endemic districts in Sri Lanka. Clinically suspected leptospirosis patients were recruited according to the Communicable Disease Epidemiology Profile Sri Lanka, WHO. Leptospirosis was confirmed by either single microscopic agglutination test titre 1: 400 or by positive polymerase chain reaction(PCR) test result. Results: Out of 372 clinically suspected cases, 29.00% were confirmed as leptospirosis cases by either microscopic agglutination test(50.00%) or positive polymerase chain reaction(52.77%) and 12.90% were presumptively identified as leptospirosis. Clinical symptoms(headache, vomiting, jaundice and dyspnoea) and variations in haematological parameters(haemoglobin, platelet count) and biochemical parameters(serum creatinine, serum urea, serum bilirubin and C-reactive protein) were associated with confirmed leptospirosis(P0.05). Acute kidney injury, meningitis, myocarditis, pulmonary haemorrhage and acute liver failure was seen among 21.30%, 12.04%, 6.48%, 6.48%, 5.56%, respectively with 4.63% fatality among the leptospirosis confirmed patients. The sensitivity, specificity, positive predictive value and negative predictive value of the case definition of Ministry of Health, Sri Lanka were 96.29%, 9.09%, 31.13%and 85.71%, respectively, when benchmarked against either positive polymerase chain reaction or microscopic agglutination test as the gold standard. Conclusions: Acute kidney injury is the predominant complication observed among the leptospirosis confirmed patients. However, pulmonary haemorrhage is predominantly associated with mortality. The case definition of Ministry of Health, Sri Lanka is found to have higher sensitivity and enabled the screening of all probable cases of leptospirosis.     

Candidate gene study of susceptibility to cutaneous leishmaniasis in Sri Lanka

Objectives To investigate the association between selected single nucleotide polymorphisms (SNPs) in TNF, LTA and SLC11A1 genes and risk of endemic cutaneous leishmaniasis (CL) in Sri Lanka through a case–control disease association study. Methods An anonymized DNA resource representative of the Sri Lankan population was genotyped initially to establish baseline parameters. This was followed up by genotyping 200 patients and 200 matched controls. Published or modified PCR/RFLP methods were employed for genotyping. Results Comparison of the different ethnic groups showed the distribution of alleles of LTA +252 A>G to differ significantly in Tamils and Moors when compared with Sinhalese. The differences seen at allele level were also reflected in the haplotypes defined by these SNPs at the TNF locus. The case–control analysis did not show an association between the SNPs or the haplotypes investigated and CL. The distribution of these variant alleles in other populations, where they are positively associated with leishmaniasis, differed significantly from the Sri Lankan study cohort. Conclusions The selected polymorphisms do not predispose to CL in the Sri Lankan population. The study of extended haplotypes at these loci using a sufficiently powered sample collection would elaborate the findings of this study. In the face of an evolving disease pattern in the country with other forms of leishmaniasis now being reported, prevalence of polymorphisms predisposing to these forms calls for heightened surveillance and preparedness.     

Serological evidence of Thailand virus-related hantavirus infection among suspected leptospirosis patients in Kandy, Sri Lanka

A cross-sectional study was undertaken to determine the current prevalence of leptospirosis and hantaviral infections, and the socio-demographic characteristics and risk factors of infected patients, in Kandy, Sri Lanka. This report discusses the serological evidence of hantavirus infections among 105 suspected leptospirosis patients, 8 of whom had hantavirus antibodies. Serotyping ELISA showed that these 8 patients had high optical density values for Thailand virus. Most of the sera showed that the focus reduction neutralization test titer against Thailand virus was higher than that against Seoul virus, thereby suggesting that the hantaviral antibodies found in Sri Lanka are different from Seoul virus but closely related to Thailand virus. These findings imply that the hantaviral infection found in Kandy, Sri Lanka appears to be due to a virus similar to Thailand virus. Epidemiological analysis revealed that the association between hantavirus infection and socio-demographic characteristics was not statistically significant.     

Phylogeography and Molecular Epidemiology of an Epidemic Strain of Dengue Virus Type 1 in Sri Lanka

In 2009, a severe epidemic of dengue disease occurred in Sri Lanka, with higher mortality and morbidity than any previously recorded epidemic in the country. It corresponded to a shift to dengue virus 1 as the major disease-causing serotype in Sri Lanka. Dengue disease reached epidemic levels in the next 3 years. We report phylogenetic evidence that the 2009 epidemic DENV-1 strain continued to circulate within the population and caused severe disease in the epidemic of 2012. Bayesian phylogeographic analyses suggest that the 2009 Sri Lankan epidemic DENV-1 strain may have traveled directly or indirectly from Thailand through China to Sri Lanka, and after spreading within the Sri Lankan population, it traveled to Pakistan and Singapore. Our findings delineate the dissemination route of a virulent DENV-1 strain in Asia. Understanding such routes will be of particular importance to global control efforts.     

Thyroglobulin epitope recognition in a post iodine-supplemented Sri Lankan population

OBJECTIVE: We previously reported a high prevalence of raised thyroglobulin autoantibodies (TgAb) in apparently healthy Sri Lankan schoolgirls following salt iodination. To characterize these antibodies further we determined the epitopes on thyroglobulin (Tg) with which they react and compared these with serum obtained from both healthy subjects and established autoimmune thyroid disease (AITD) patients from the UK. To extend our study to a wider population within Sri Lanka, we in addition determined the epitopes recognized by a group of AITD patients selected from a thyroid clinic in Sri Lanka, as well as apparently healthy female Sri Lankan tea workers of distinct ethnicity from the schoolgirls and AITD patients. DESIGN: Sri Lankan schoolgirls (n = 282) and adult female tea estate workers (n = 208) were examined for thyroid autoimmune markers. Sera with high TgAb (> 98 kIU/l) were selected from these two groups (n = 36 and 45, respectively) to study epitope-binding patterns. We also examined the sera from 16 AITD patients attending a thyroid clinic in Colombo, 16 patients with AITD from the thyroid clinic at the University Hospital of Wales and 16 sera from healthy control UK women with no evidence of thyroid disease. To determine the epitopes on Tg recognized by the subjects' TgAb, we employed a panel of Tg mouse monoclonal antibodies labelled with alkaline phosphatase in a competitive enzyme-linked immunosorbent assay reaction with the subjects' serum. RESULTS AND CONCLUSIONS: A majority of the Sri Lankan schoolgirls did not react with the immunodominant epitopes and did not differ significantly from healthy subjects from the UK in their Tg epitope recognition pattern. On the other hand, tea estate workers and Sri Lankan AITD patients recognized typical autoimmune thyroid disease epitopes and, in addition, recognized a separate cluster not previously associated with either the autoimmune state or the healthy state. The significance of this cluster requires further clarification.     

Chronic kidney disease of uncertain aetiology: adding vital piece of information to the national project team report of Sri Lanka

In a recent study published by the National Project Team on chronic kidney diseases of unknown origin in Sri Lanka, identified cadmium as a major risk factor but strong conclusions were not made as the identified environmental toxins were within the permissible levels.Sri Lankan food consumption pattern is different so that approach of total exposure of cadmium by food and water been calculated. Such calculation point out that total exposure of cadmium exceed the provisional tolerable weekly intake determined by international agencies.     

贵州省3株钩端螺旋体分离株PFGE分型与基因种鉴定

目的应用脉冲场电泳（PFGE）分型技术和16S rRNA基因测序分析技术分别对贵州省3株动物宿主钩 端螺旋体分离菌株进行分子分型和基因种鉴定,了解贵州省钩端螺旋体的分子流行病学特征。方法应用 DNA限制性内切酶Not I对钩端螺旋体染色体DNA酶切后,用PFGE将DNA片段分离,采用BionumericsV4.0将3 株钩体菌株PFGE图谱与中国15群15型参考菌株进行聚类分析;同时,应用PCR扩增几乎全长的钩体16S rRNA基因片段,并将扩增产物进行双向序列测定,并与GenBank数据库已注册的核酸序列进行同源性比对 、确定基因种、分析亲缘进化关系。结果来自贵州省的3株动物宿主分离钩体菌株PFGE带型命名为LepNot I 002和LepNot I 003,经聚类分析,3株菌株与黄疸出血群黄疸出血型赖株的相似性大于95%。16S rRNA 基因测序和分析表明,3株贵州分离钩体菌株之间的同源性为100%,与致病性钩体问号钩端螺旋体种（L. interrogans）不同血清型参考菌株的同源性达100%。结论3株贵州动物分离钩体菌株经PFGE分型鉴定与黄 疸出血群赖型赖株的相似性大于95%,经16S rRNA基因测序分析鉴定为L. interrogans种,上述两种方法 对贵州省钩体分离菌株的鉴定结果一致,有助于贵州省钩端螺旋体病的主动监测、暴发调查和传染源追踪 。     

Sri Lankan National Melioidosis Surveillance Program Uncovers a Nationwide Distribution of Invasive Melioidosis

The epidemiologic status of melioidosis in Sri Lanka was unclear from the few previous case reports. We established laboratory support for a case definition and started a nationwide case-finding study. Suspected Burkholderia pseudomallei isolates were collated, identified by polymerase chain reaction assay, referred for Matrix Assisted Laser Desorption Ionization-Time of Flight analysis and multilocus sequence typing (MLST), and named according to the international MLST database. Between 2006 and early 2014, there were 32 patients with culture-confirmed melioidosis with an increasing annual total and a falling fatality rate. Patients were predominantly from rural communities, diabetic, and male. The major clinical presentations were sepsis, pneumonia, soft tissue and joint infections, and other focal infection. Burkholderia pseudomallei isolates came from all parts of Sri Lanka except the Sabaragamuwa Province, the south central hill country, and parts of northern Sri Lanka. Bacterial isolates belonged to 18 multilocus sequence types, one of which (ST 1137) was associated with septicemia and a single-organ focus (Fisher''s exact, P = 0.004). Melioidosis is an established endemic infection throughout Sri Lanka, and is caused by multiple genotypes of B. pseudomallei, which form a distinct geographic group based upon related sequence types (BURST) cluster at the junction of the southeast Asian and Australasian clades.     

A multi-center study in order to further define the molecular basis of beta-thalassemia in Thailand, Pakistan, Sri Lanka, Mauritius, Syria, and India, and to develop a simple molecular diagnostic strategy by amplification refractory mutation system-polymerase chain reaction.

The spectrum of the beta-thalassemia mutations of Thailand, Pakistan, India, Sri Lanka, Mauritius and Syria has been further characterized by a multi-center study of 1,235 transfusion-dependent patients, and the mutations discovered used to assess the fidelity of a simple diagnostic strategy. A total of 44 beta-thalassemia mutations were identified either by allele-specific oligonucleotide hybridization, amplification with allele-specific primers, or DNA sequencing of amplified product. The results confirm and extend earlier findings for Thailand, Pakistan, India, Mauritius and Syria. This is the first detailed report of the spectrum of mutations for Sri Lanka. Two novel mutations were identified, codon 55 (-A) and IVS-I-129 (A-->C), both found in Sri Lankan patients. Two beta-thalassemia mutations were found to coexist in one beta-globin gene: Sri Lankan patients homozygous for the beta0 codon 16 (-C) frameshift were also homozygous for the beta+ codon 10 (C-->A) mutation. Studies of Sri Lankan, Pakistani, and Indian carriers suggest the codon 10 (C-->A) mutation is just a rare polymorphism on an ancestral allele, on which the beta0 codon 16 (-C) mutation has arisen. Each country was found to have only a few common mutations accounting for 70% or more of the beta-thalassemia alleles. A panel of primers to diagnose the majority of the mutations by the amplification refractory mutation system was developed, enabling a simple molecular diagnostic strategy to be introduced for each country participating in the multi-center study.     

Locally acquired cutaneous leishmaniasis in Sri Lanka.

Cutaneous leishmaniasis in a Sri Lankan, who had not left the country, is reported. The diagnosis was confirmed by detecting the parasite in smears under microscopic examination. This is the first case report of locally acquired leishmaniasis in Sri Lanka.     

A MULTI-CENTER STUDY IN ORDER TO FURTHER DEFINE THE MOLECULAR BASIS OF β-THALASSEMIA IN THAILAND,PAKISTAN, SRI LANKA,MAURITIUS, SYRIA,AND INDIA,AND TO DEVELOP A SIMPLE MOLECULAR DIAGNOSTIC STRATEGY BY AMPLIFICATION REFRACTORY MUTATION SYSTEM-POLYMERASE CHAIN REACTION

The spectrum of the β-thalassemia mutations of Thailand, Pakistan, India, Sri Lanka, Mauritius and Syria has been further characterized by a multi-center study of 1,235 transfusion-dependent patients, and the mutations discovered used to assess the fidelity of a simple diagnostic strategy. A total of 44 β-thalassemia mutations were identified either by allele-specific oligonucleotide hybridization, amplification with allele-specific primers, or DNA sequencing of amplified product. The results confirm and extend earlier findings for Thailand, Pakistan, India, Mauritius and Syria. This is the first detailed report of the spectrum of mutations for Sri Lanka. Two novel mutations were identified, codon 55 (?A) and IVS-I-129 (A → C), both found in Sri Lankan patients. Two β-thalassemia mutations were found to coexist in one β-globin gene: Sri Lankan patients homozygous for the β⁰ codon 16 (?C) frameshift were also homozygous for the β⁺ codon 10 (C → A) mutation. Studies of Sri Lankan, Pakistani, and Indian carriers suggest the codon 10 (C → A) mutation is just a rare polymorphism on an ancestral allele, on which the β⁰ codon 16 (?C) mutation has arisen. Each country was found to have only a few common mutations accounting for 70% or more of the β-thalassemia alleles. A panel of primers to diagnose the majority of the mutations by the amplification refractory mutation system was developed, enabling a simple molecular diagnostic strategy to be introduced for each country participating in the multi-center study.     

Determinants of leptospirosis in Sri Lanka: Study Protocol

Background  

Leptospirosis is becoming a major public health threat in Sri Lanka as well as in other countries. We designed a case control study to determine the factors associated with local transmission of leptospirosis in Sri Lanka, in order to identify major modifiable determinants of leptospirosis. The purpose of this paper is to describe the study protocol in detail prior to the publishing of the study results, so that the readership will be able to understand and interpret the study results effectively.     

福建省鼠感染钩端螺旋体调查和分离株基因种的鉴定北大核心CSCD

目的了解福建省鼠感染钩端螺旋体(钩体)现状及分离株基因种的特征,为钩体病防控提供科学依据。方法2015-2019年根据疫情选取14个调查点,采用笼夜法捕鼠并采集心血或肾脏标本进行钩体培养,测定血清抗体;对鼠中分离保存的23株钩体菌株提取DNA,PCR扩增16S rRNA基因、PCR产物纯化测序,与GenBank数据库收录的钩体17种基因型进行比对确定基因种,并用Mega 6.0软件构建系统进化树并进行序列分析。结果福建省鼠种以黄毛鼠为主要优势鼠种,占40.87%(499/1221)、其次依次是黄胸鼠19.57%(239/1221)、针毛鼠16.79%(205/1221)和褐家鼠8.85%(108/1221),钩体血清抗体阳性率为29.32%(207/706),以黄毛鼠最高,为42.16%(121/287),不同地区鼠种构成及鼠钩体感染率不同。23株菌株均扩增出16S rRNA基因的目标条带1484 bp,测序及序列分析属于致病性的基因种有13株,其中L.borgpetersenii(伯氏)占8株,L.interrogans(问号)占5株,属于非致病的基因种10株。钩体菌株分离自福建省的优势鼠种(黄毛鼠、黄胸鼠、针毛鼠)及不同地区(闽东、闽南、闽西和闽北)。结论福建省鼠类感染钩体普遍,以致病性基因种(L.borgpetersenii和L.interrogans)为主,也携带非致病性基因种,需加强钩体病传染源啮齿动物的监测工作。     

2016-2018年江西省钩端螺旋体病鼠类动物流行病学调查与分离菌株鉴定

目的 初步了解江西省钩端螺旋体病主要宿主动物鼠类种类构成、带菌率及血清学和分子流行病学特征,为钩体病的防控提供科学依据。方法 2016-2018年选取江西省钩体病4个国家级监测点上饶、上高、上犹及浮梁县作为现场监测地点,开展鼠类种类调查、钩体分离培养工作。利用暗视野显微镜凝集试验和MLST技术对分离菌株进行血清学鉴定和MLST基因分型。结果 共计捕鼠14 387只,平均鼠密度为9.76%,分离菌株64株,鼠类钩体分离率为4.56%。鼠种构成包括黑线姬鼠、褐家鼠、黄毛鼠、黄胸鼠及社鼠5个种类。4个监测点优势鼠种均以黑线姬鼠和黄毛鼠为主。血清群鉴定结果显示:64株菌株隶属于3个血清群,其中黄疸出血群为主要优势血清群,占81.25%,其次是爪哇群和致热群。MLST研究显示:64株菌株隶属于6个ST型别,分别为ST1、ST17、ST92、ST143、ST216和ST224,其中ST1为主要基因型,占76.56%。BioNumerics软件分析显示:64株菌株隶属于6个Clusters,分别对应6个ST型,血清学、MLST基因型分布具有明显地域差异。结论 江西省以黑线姬鼠和黄毛鼠为优势鼠种,黄疸出血群和ST1为江西省钩体病主要流行型别,充分了解江西省钩体病可能的宿主动物以及血清学和分子流行病学特征,对钩体病的预防控制有一定的指导意义。