• 1.

    Levett PN, 2007. Sequence-based typing of Leptospira: epidemiology in the genomic era. PLoS Negl Trop Dis 1: e120.

  • 2.

    Ahmed N, Devi SM, Valverde M de L, Vijayachari P, Machang'u RS, Ellis WA, Hartskeerl RA, 2006. Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species. Ann Clin Microbiol Antimicrob 5: 28.

    • Search Google Scholar
    • Export Citation
  • 3.

    Thaipadungpanit J, Wuthiekanun V, Chierakul W, Smythe LD, Petkanchanapong W, Limpaiboon R, Apiwatanaporn A, Slack AT, Suputtamongkol Y, White NJ, Feil EJ, Day NP, Peacock SJ, 2007. A dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand. PLoS Negl Trop Dis 1: e56.

    • Search Google Scholar
    • Export Citation
  • 4.

    Agampodi SB, Peacock SJ, Thevanesam V, Nugegoda DB, Smythe L, Thaipadungpanit J, Craig SB, Burns MA, Dohnt M, Boonsilp S, Senaratne T, Kumara A, Palihawadana P, Perera S, Vinetz JM, 2011. Leptospirosis outbreak in Sri Lanka in 2008: lessons for assessing the global burden of disease. Am J Trop Med Hyg 85: 471478.

    • Search Google Scholar
    • Export Citation
  • 5.

    Drummond AJAB, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A, 2011. Geneious Pro 5.4.6. Available at: http://www.geneious.com/.

    • Search Google Scholar
    • Export Citation
  • 6.

    Ren SX, Fu G, Jiang XG, Zeng R, Miao YG, Xu H, Zhang YX, Xiong H, Lu G, Lu LF, Jiang HQ, Jia J, Tu YF, Jiang JX, Gu WY, Zhang YQ, Cai Z, Sheng HH, Yin HF, Zhang Y, Zhu GF, Wan M, Huang HL, Qian Z, Wang SY, Ma W, Yao ZJ, Shen Y, Qiang BQ, Xia QC, Guo XK, Danchin A, Saint Girons I, Somerville RL, Wen YM, Shi MH, Chen Z, Xu JG, Zhao GP, 2003. Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing. Nature 422: 888893.

    • Search Google Scholar
    • Export Citation
  • 7.

    Sehgal SC, Vijayachari P, Smythe LD, Norris M, Symonds M, Dohnt M, Korver H, v d Kemp H, Hartskeerl RA, Terpstra WJ, 2000. Lai-like Leptospira from the Andaman Islands. Indian J Med Res 112: 135139.

    • Search Google Scholar
    • Export Citation
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Utility and Limitations of Direct Multi-Locus Sequence Typing on qPCR-Positive Blood to Determine Infecting Leptospira Strain

Suneth B. AgampodiDepartment of Community Medicine, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Saliyapura, Sri Lanka; Division of Infectious Diseases, Department of Medicine, University of California at San Diego, La Jolla, California

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Angelo C. MorenoDepartment of Community Medicine, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Saliyapura, Sri Lanka; Division of Infectious Diseases, Department of Medicine, University of California at San Diego, La Jolla, California

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Joseph M. VinetzDepartment of Community Medicine, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Saliyapura, Sri Lanka; Division of Infectious Diseases, Department of Medicine, University of California at San Diego, La Jolla, California

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Michael A. MatthiasDepartment of Community Medicine, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Saliyapura, Sri Lanka; Division of Infectious Diseases, Department of Medicine, University of California at San Diego, La Jolla, California

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Culture-independent molecular characterization of infecting Leptospira human blood specimens from a 2008 outbreak of human leptospirosis in central Sri Lanka was carried out. Of 58 quantitative real-time polymerase chain reaction-positive samples analyzed for seven multi-locus sequence typing (MLST) housekeeping genes (mreA, pfkB, pntA, sucA, tpiA, fadD, and glmU), interpretable data was obtained from 12 samples. Mean bacterial load was 2.2 × 105 among specimens with complete MLST profiles compared with 1.3 × 104 among specimens without complete MLST profiles; all specimens with complete profiles had at least 4.9 × 104 Leptospira/mL (t = 5, P < 0.001). Most (11/12) identified sequence types were ST1 (L. interrogans serovar Lai) and ST44 (L. interrogans serovar Geyaweera). MLST can be used to directly identify infecting Leptospira strains in blood samples obtained during acute illness without the need for culture isolation, but it shows important limitations related to bacterial load.

Introduction

Leptospirosis is a globally emerging zoonotic disease. Epidemiological investigations of leptospirosis outbreaks are a major challenge in most settings because of varied clinical presentation, difficult diagnosis, and complex disease transmission cycle. These challenges are particularly apparent in areas where the disease is highly endemic and resources and technical capacity are lacking. Because culture isolation is insensitive and may take up to several months for a definitive result, characterization of the infecting strain, especially in outbreak investigations, is difficult. Recently, sequence-based typing techniques (including multiple-locus variable number tandem repeat analysis [MLVA], multi-locus sequence typing [MLST], etc.) have emerged as alternatives to serotyping.1 MLST has advantages in typing Leptospira because of reproducibility.2 Two MLST schemes for Leptospira have been published.2,3 Importantly, MLST has typically required isolation of Leptospira to provide sufficient DNA for analysis. Nonetheless, because of difficulties in obtaining isolates of Leptospira from clinical samples (expense, expertise, problems with contamination, and intrinsically poor isolation rates), being able to identify infecting Leptospira in clinical samples is a high priority for clinical, epidemiological, and basic science purposes. In this study, we used a previously published MLST approach to identify the infecting strain in serum and whole-blood specimens collected during a 2008 outbreak of leptospirosis in Sri Lanka.

Methods

A systematically collected sample of serum and whole blood from a 2008 outbreak of leptospirosis in Sri Lanka was used in the present study. Patient recruitment, sample collection, and processing have been published previously.4 Of the original 401 patients, we selected samples from 58 quantitative polymerase chain reaction (qPCR) -positive patients for the present study. For MLST, DNA was extracted from 100 μL serum or whole blood using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer's directions. For analysis, we used a previously published MLST scheme based on the amplification of seven housekeeping genes (mreA, pfkB, pntA, sucA, tpiA, fadD, and glmU).3 Reaction mixes were prepared using HotStar Taq master mix (Qiagen) with 5 pmol each primer and 5 μL DNA in 25 μL total volume. Amplification was carried out using DNA Engine Dyad thermal cycler (BioRad, Hercules, CA). PCR products were cleaned using ExoSAP-IT (Affymetrix, Santa Clara, CA) and Sanger-sequenced in both directions using Big Dye chemistry and an ABI automated sequencer. At least four reads were obtained for each sequence. Individual sequences were assembled using Genius Pro 5.4.6 software.5 Samples from six patients included sequences with ambiguous bases, which are potentially indicative of mixed infections, and these samples were excluded from analysis. Assembled sequences were trimmed and aligned with reference sequences downloaded from leptospira.mlst.net to assign allele numbers for all seven loci. For strain identification, allelic profiles were searched against the Leptospira MLST database.

Results and Discussion

Of the 58 samples tested, 12 samples provided interpretable data. Of these 12 patients, 5 patients were severe cases with myocarditis and/or renal failure, 6 patients were uncomplicated (no specific end organ involvement), and one patient lacked clinical data. Amplification and sequencing reactions were repeated to improve confidence in the underlying data. Of the seven loci, the glmU data were the least reliable. Three samples had complete MLST profiles (i.e., sequences from all seven loci) (Table 1), allowing confident serovar assignment. Of the specimens from which we obtained incomplete MLST profiles (and hence, tentative serovar identification), one shared two (of two) alleles with L. interrogans serovar Geyaweera strain Geyaweera, which is unique to Sri Lanka. All other strain types (STs) were similar to ST1, L. interrogans sv Lai, strains Lai and 55631. Because the MLST profiles were mostly incomplete, nodes were poorly supported after 500 bootstrap replicates (data not shown).

Table 1

Allelic profiles of 12 positive samples with useful sequence data obtained from MLST scheme

Sample Primers ST type Closest serovar
sucA tpiA fadD glmU pntA pfkB mreA
1 1 1 1 1 1 1 1 1 Lai
2 1 1 1 1 1 1 1 1 Lai
3 1 1 1 1 1 1 1 1 Lai
4 1 1 1 1 1 1 1 Lai
5 1 1 1 1 1 Lai
6 1 1 1 1 1 1 Lai
7 1 1 1 1 1 1 Lai
8 1 1 1 1 Lai
9 1 1 1 Lai
10 1 1 1 1 Lai
11 1 1 1 1 1 Lai
12 5 5 44 Geyaweera

No amplification is indicated (–).

To determine whether the completeness of MLST profiles correlated with bacterial load, we compared mean leptospiremia of specimens with and without complete MLST profiles, which also included PCR-negative specimens. Mean bacterial load was 2.2 × 105 among specimens with complete MLST profiles compared with 1.3 × 104 among specimens without, with a cutoff of 4.9 × 104 Leptospira/mL observed for specimens with complete profiles (t = 5, P < 0.001).

The data presented here suggest that ST1 was the predominant infecting ST among the study population during the 2008 outbreak of leptospirosis in Sri Lanka. Based on serology, ST1 seems to be L. interrogans serovar Lai, previously reported to be a highly virulent leptospire, and for the first time, it was reported to be present in Sri Lanka.6 In the Sri Lanka setting, serovar Lai seems to be associated with both severe and non-severe disease, which has been reported both from China6 and the Andaman Islands.7 Whether the Sri Lanka outbreak of leptospirosis in 2008 was predominantly caused by ST1 is difficult to answer considering the incomplete MLST profiles and consequent poor bootstrap support. One possible explanation would be that some of the infecting serovars could be from other novel STs not currently represented in the MLST database.

The present study is the first report of using a full MLST typing scheme to directly identify infecting Leptospira in human clinical specimens. Although MLST was found to be relatively insensitive in this study and the poor amplification results might indicate limited homology between the primers used and target gene sequence, this method has important potential for future clinical, epidemiological, and basic science studies.

ACKNOWLEDGMENTS

This work was supported by US Public Health Service Grants 1R01TW05860, 1U01AI075420, 1K24AI068903, 5R25GM083275, and 1D43TW007120. We would like to thank Prof. Sharon Peacock and Janjira Thaipudungpanit for their expert advice on the interpretation of the MLST results and Jason Lehmann for support for molecular work. The authors thank Paula Maguina (University of California at San Diego) for essential scientific and logistical contributions essential to carrying out this study.

  • 1.

    Levett PN, 2007. Sequence-based typing of Leptospira: epidemiology in the genomic era. PLoS Negl Trop Dis 1: e120.

  • 2.

    Ahmed N, Devi SM, Valverde M de L, Vijayachari P, Machang'u RS, Ellis WA, Hartskeerl RA, 2006. Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species. Ann Clin Microbiol Antimicrob 5: 28.

    • Search Google Scholar
    • Export Citation
  • 3.

    Thaipadungpanit J, Wuthiekanun V, Chierakul W, Smythe LD, Petkanchanapong W, Limpaiboon R, Apiwatanaporn A, Slack AT, Suputtamongkol Y, White NJ, Feil EJ, Day NP, Peacock SJ, 2007. A dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand. PLoS Negl Trop Dis 1: e56.

    • Search Google Scholar
    • Export Citation
  • 4.

    Agampodi SB, Peacock SJ, Thevanesam V, Nugegoda DB, Smythe L, Thaipadungpanit J, Craig SB, Burns MA, Dohnt M, Boonsilp S, Senaratne T, Kumara A, Palihawadana P, Perera S, Vinetz JM, 2011. Leptospirosis outbreak in Sri Lanka in 2008: lessons for assessing the global burden of disease. Am J Trop Med Hyg 85: 471478.

    • Search Google Scholar
    • Export Citation
  • 5.

    Drummond AJAB, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A, 2011. Geneious Pro 5.4.6. Available at: http://www.geneious.com/.

    • Search Google Scholar
    • Export Citation
  • 6.

    Ren SX, Fu G, Jiang XG, Zeng R, Miao YG, Xu H, Zhang YX, Xiong H, Lu G, Lu LF, Jiang HQ, Jia J, Tu YF, Jiang JX, Gu WY, Zhang YQ, Cai Z, Sheng HH, Yin HF, Zhang Y, Zhu GF, Wan M, Huang HL, Qian Z, Wang SY, Ma W, Yao ZJ, Shen Y, Qiang BQ, Xia QC, Guo XK, Danchin A, Saint Girons I, Somerville RL, Wen YM, Shi MH, Chen Z, Xu JG, Zhao GP, 2003. Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing. Nature 422: 888893.

    • Search Google Scholar
    • Export Citation
  • 7.

    Sehgal SC, Vijayachari P, Smythe LD, Norris M, Symonds M, Dohnt M, Korver H, v d Kemp H, Hartskeerl RA, Terpstra WJ, 2000. Lai-like Leptospira from the Andaman Islands. Indian J Med Res 112: 135139.

    • Search Google Scholar
    • Export Citation

Author Notes

* Address correspondence to Joseph M. Vinetz, University of California at San Diego, School of Medicine, Center for Tropical Medicine and Emerging Infectious Diseases, Division of Infectious Diseases, Department of Medicine, 9500 Gilman Drive 0741, Palade Laboratories, Room 125, La Jolla, CA 92093-0741. E-mail: jvinetz@ucsd.edu

Authors' addresses: Suneth B. Agampodi, Department of Community Medicine, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Saliyapura, Sri Lanka, E-mail: sunethagampodi@yahoo.com. Angelo C. Moreno, Graduate Program in Molecular Genetics and Microbiology, Duke University, Durham, NC, E-mail: angelo.moreno@duke.edu. Joseph M. Vinetz, Department of Medicine, School of Medicine, University of California at San Diego, La Jolla, CA, E-mail: jvinetz@ucsd.edu. Michael A. Matthias, Division of Infectious Diseases, Department of Medicine, University of California at San Diego, School of Medicine, La Jolla, CA, E-mail: mmatthias@ucsd.edu.

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