Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay

Stuart D Blacksell, Cherry Lim, Ampai Tanganuchitcharnchai, Suthatip Jintaworn, Pacharee Kantipong, Allen L Richards, Daniel H Paris, Direk Limmathurotsakul, Nicholas P J Day, Stuart D Blacksell, Cherry Lim, Ampai Tanganuchitcharnchai, Suthatip Jintaworn, Pacharee Kantipong, Allen L Richards, Daniel H Paris, Direk Limmathurotsakul, Nicholas P J Day

Abstract

The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus.

Copyright © 2016 Blacksell et al.

Figures

FIG 1
FIG 1
Distribution of IgM IFA titers in admission samples and convalescent/discharge samples.
FIG 2
FIG 2
Distribution of IgM ELISA ODs in admission samples (A) and convalescent/discharge samples (B) at sample dilutions from 1:100 to 1:102,400. The colored lines show the positions of the patients who had the OD at the 10th, 20th, and every 10th percentile up to the 90th percentile at a sample dilution of 1:100.

References

    1. Paris DH, Chattopadhyay S, Jiang J, Nawtaisong P, Lee JS, Tan E, Dela Cruz E, Burgos J, Abalos R, Blacksell SD, Lombardini E, Turner GD, Day NP, Richards AL. 2015. A nonhuman primate scrub typhus model: protective immune responses induced by pKarp47 DNA vaccination in cynomolgus macaques. J Immunol 194:1702–1716. doi:10.4049/jimmunol.1402244.
    1. Rodkvamtook W, Gaywee J, Kanjanavanit S, Ruangareerate T, Richards AL, Sangjun N, Jeamwattanalert P, Sirisopana N. 2013. Scrub typhus outbreak, northern Thailand, 2006-2007. Emerg Infect Dis 19:774–777. doi:10.3201/eid1905.121445.
    1. Dittrich S, Rattanavong S, Lee SJ, Panyanivong P, Craig SB, Tulsiani SM, Blacksell SD, Dance DA, Dubot-Peres A, Sengduangphachanh A, Phoumin P, Paris DH, Newton PN. 2015. Orientia, rickettsia, and leptospira pathogens as causes of CNS infections in Laos: a prospective study. Lancet Glob Health 3:e104–e112. doi:10.1016/S2214-109X(14)70289-X.
    1. McGready R, Prakash JA, Benjamin SJ, Watthanaworawit W, Anantatat T, Tanganuchitcharnchai A, Ling CL, Tan SO, Ashley EA, Pimanpanarak M, Blacksell SD, Day NP, Singhasivanon P, White NJ, Nosten F, Paris DH. 2014. Pregnancy outcome in relation to treatment of murine typhus and scrub typhus infection: a fever cohort and a case series analysis. PLoS Negl Trop Dis 8:e3327. doi:10.1371/journal.pntd.0003327.
    1. Paris DH, Shelite TR, Day NP, Walker DH. 2013. Unresolved problems related to scrub typhus: a seriously neglected life-threatening disease. Am J Trop Med Hyg 89:301–307. doi:10.4269/ajtmh.13-0064.
    1. Mayxay M, Castonguay-Vanier J, Chansamouth V, Dubot-Peres A, Paris DH, Phetsouvanh R, Tangkhabuanbutra J, Douangdala P, Inthalath S, Souvannasing P, Slesak G, Tongyoo N, Chanthongthip A, Panyanouvong P, Sibounheuang B, Phommasone K, Dohnt M, Phonekeo D, Hongvanthong B, Xayadeth S, Ketmayoon P, Blacksell SD, Moore CE, Craig SB, Burns MA, von Sonnenburg F, Corwin A, de Lamballerie X, Gonzalez IJ, Christophel EM, Cawthorne A, Bell D, Newton PN. 2013. Causes of non-malarial fever in Laos: a prospective study. Lancet Glob Health 1:e46–e54. doi:10.1016/S2214-109X(13)70008-1.
    1. Koh GC, Maude RJ, Paris DH, Newton PN, Blacksell SD. 2010. Diagnosis of scrub typhus. Am J Trop Med Hyg 82:368–370. doi:10.4269/ajtmh.2010.09-0233.
    1. Luce-Fedrow A, Mullins K, Kostik AP, St John HK, Jiang J, Richards AL. 2015. Strategies for detecting rickettsiae and diagnosing rickettsial diseases. Future Microbiol 10:537–564. doi:10.2217/fmb.14.141.
    1. Blacksell SD, Bryant NJ, Paris DH, Doust JA, Sakoda Y, Day NP. 2007. Scrub typhus serologic testing with the indirect immunofluorescence method as a diagnostic gold standard: a lack of consensus leads to a lot of confusion. Clin Infect Dis 44:391–401. doi:10.1086/510585.
    1. Phetsouvanh R, Thojaikong T, Phoumin P, Sibounheuang B, Phommasone K, Chansamouth V, Lee SJ, Newton PN, Blacksell SD. 2013. Inter- and intra-operator variability in the reading of indirect immunofluorescence assays for the serological diagnosis of scrub typhus and murine typhus. Am J Trop Med Hyg 88:932–936. doi:10.4269/ajtmh.12-0325.
    1. Suwanabun N, Chouriyagune C, Eamsila C, Watcharapichat P, Dasch GA, Howard RS, Kelly DJ. 1997. Evaluation of an enzyme-linked immunosorbent assay in Thai scrub typhus patients. Am J Trop Med Hyg 56:38–43.
    1. Coleman RE, Sangkasuwan V, Suwanabun N, Eamsila C, Mungviriya S, Devine P, Richards AL, Rowland D, Ching WM, Sattabongkot J, Lerdthusnee K. 2002. Comparative evaluation of selected diagnostic assays for the detection of IgG and IgM antibody to Orientia tsutsugamushi in Thailand. Am J Trop Med Hyg 67:497–503.
    1. Zhou XH, Castelluccio P, Zhou C. 2005. Nonparametric estimation of ROC curves in the absence of a gold standard. Biometrics 61:600–609. doi:10.1111/j.1541-0420.2005.00324.x.
    1. Limmathurotsakul D, Chantratita N, Teerawattanasook N, Piriyagitpaiboon K, Thanwisai A, Wuthiekanun V, Day NP, Cooper B, Peacock SJ. 2011. Enzyme-linked immunosorbent assay for the diagnosis of melioidosis: better than we thought. Clin Infect Dis 52:1024–1028. doi:10.1093/cid/cir080.
    1. Lim C, Wannapinij P, White L, Day NP, Cooper BS, Peacock SJ, Limmathurotsakul D. 2013. Using a web-based application to define the accuracy of diagnostic tests when the gold standard is imperfect. PLoS One 8:e79489. doi:10.1371/journal.pone.0079489.
    1. Choi YW, Collins M, Gardner I. 2006. Bayesian inferences for receiver operating characteristic curves in the absence of a gold standard. Agric Biol Environ Stat 11:19.
    1. Joseph L, Gyorkos TW, Coupal L. 1995. Bayesian estimation of disease prevalence and the parameters of diagnostic tests in the absence of a gold standard. Am J Epidemiol 141:263–272.
    1. Paris DH, Blacksell SD, Nawtaisong P, Jenjaroen K, Teeraratkul A, Chierakul W, Wuthiekanun V, Kantipong P, Day NP. 2011. Diagnostic accuracy of a loop-mediated isothermal PCR assay for detection of Orientia tsutsugamushi during acute Scrub Typhus infection. PLoS Negl Trop Dis 5:e1307. doi:10.1371/journal.pntd.0001307.
    1. Lim C, Paris DH, Blacksell SD, Laongnualpanich A, Kantipong P, Chierakul W, Wuthiekanun V, Day NP, Cooper BS, Limmathurotsakul D. 2015. How to determine the accuracy of an alternative diagnostic test when it is actually better than the reference tests: a re-evaluation of diagnostic tests for scrub typhus using Bayesian LCMs. PLoS One 10:e0114930. doi:10.1371/journal.pone.0114930.
    1. Lim C, Blacksell SD, Paris DH, Laongnualpanich A, Kantipong P, Day NP, Limmathurotsakul D. 2015. Optimal cut-off titres for scrub typhus indirect immunofluorescence assay in northern Thailand. J Clin Microbiol 53:3663–3666. doi:10.1128/JCM.01680-15.
    1. Luksameetanasan R, Blacksell SD, Kalambaheti T, Wuthiekanun V, Chierakul W, Chueasuwanchai S, Apiwattanaporn A, Stenos J, Graves S, Peacock SJ, Day NP. 2007. Patient and sample-related factors that effect the success of in vitro isolation of Orientia tsutsugamushi. Southeast Asian J Trop Med Public Health 38:91–96.
    1. Sonthayanon P, Chierakul W, Wuthiekanun V, Blacksell SD, Pimda K, Suputtamongkol Y, Pukrittayakamee S, White NJ, Day NP, Peacock SJ. 2006. Rapid diagnosis of scrub typhus in rural Thailand using polymerase chain reaction. Am J Trop Med Hyg 75:1099–1102.
    1. Paris DH, Blacksell SD, Stenos J, Graves SR, Unsworth NB, Phetsouvanh R, Newton PN, Day NP. 2008. Real-time multiplex PCR assay for detection and differentiation of rickettsiae and orientiae. Trans R Soc Trop Med Hyg 102:186–193. doi:10.1016/j.trstmh.2007.11.001.
    1. Paris DH, Aukkanit N, Jenjaroen K, Blacksell SD, Day NP. 2009. A highly sensitive quantitative real-time PCR assay based on the groEL gene of contemporary Thai strains of Orientia tsutsugamushi. Clin Microbiol Infect 15:488–495. doi:10.1111/j.1469-0691.2008.02671.x.
    1. Dendukuri N, Joseph L. 2001. Bayesian approaches to modeling the conditional dependence between multiple diagnostic tests. Biometrics 57:158–167. doi:10.1111/j.0006-341X.2001.00158.x.
    1. Lunn DJ, Thomas A, Best N, Spiegelhalter D. 2000. WinBUGS—a Bayesian modelling framework: concepts, structure, and extensibility. Stat Comput 10:325–337. doi:10.1023/A:1008929526011.
    1. Nielsen SS, Gronbaek C, Agger JF, Houe H. 2002. Maximum-likelihood estimation of sensitivity and specificity of ELISAs and faecal culture for diagnosis of paratuberculosis. Prev Vet Med 53:191–204. doi:10.1016/S0167-5877(01)00280-X.
    1. Wang C, Bruce WT, Yrjo TG, Soren SN. 2007. Nonparametric estimation of ROC curves based on Bayesian models when the true disease state is unknown. J Agric Biol Environ Stat 12:128–146. doi:10.1198/108571107X178095.
    1. Elisberg BL, Sangkasuvana V, Campbell JM, Bozeman FM, Bodhidatta P. 1967. Physiogeographic distribution of scrub typhus in Thailand. Acta Med Biol (Niigata) 15:61–67.
    1. Kollars TM Jr, Bodhidatta D, Phulsuksombati D, Tippayachai B, Coleman RE. 2003. Short report: variation in the 56-kD type-specific antigen gene of Orientia tsutsugamushi isolated from patients in Thailand. Am J Trop Med Hyg 68:299–300.
    1. Blacksell SD, Luksameetanasan R, Kalambaheti T, Aukkanit N, Paris DH, McGready R, Nosten F, Peacock SJ, Day NP. 2008. Genetic typing of the 56-kDa type-specific antigen gene of contemporary Orientia tsutsugamushi isolates causing human scrub typhus at two sites in north-eastern and western Thailand. FEMS Immunol Med Microbiol 52:335–342. doi:10.1111/j.1574-695X.2007.00375.x.
    1. Fournier PE, Siritantikorn S, Rolain JM, Suputtamongkol Y, Hoontrakul S, Charoenwat S, Losuwanaluk K, Parola P, Raoult D. 2008. Detection of new genotypes of Orientia tsutsugamushi infecting humans in Thailand. Clin Microbiol Infect 14:168–173. doi:10.1111/j.1469-0691.2007.01889.x.
    1. McGready R, Blacksell SD, Luksameetanasan R, Wuthiekanun V, Jedsadapanpong W, Day NP, Nosten F. 2010. First report of an Orientia tsutsugamushi type TA716-related scrub typhus infection in Thailand. Vector Borne Zoonotic Dis 10:191–193. doi:10.1089/vbz.2008.0199.
    1. Ruang-Areerate T, Jeamwattanalert P, Rodkvamtook W, Richards AL, Sunyakumthorn P, Gaywee J. 2011. Genotype diversity and distribution of Orientia tsutsugamushi causing scrub typhus in Thailand. J Clin Microbiol 49:2584–2589. doi:10.1128/JCM.00355-11.
    1. Coleman RE, Monkanna T, Linthicum KJ, Strickman DA, Frances SP, Tanskul P, Kollars TM Jr, Inlao I, Watcharapichat P, Khlaimanee N, Phulsuksombati D, Sangjun N, Lerdthusnee K. 2003. Occurrence of Orientia tsutsugamushi in small mammals from Thailand. Am J Trop Med Hyg 69:519–524.
    1. Lerdthusnee K, Nigro J, Monkanna T, Leepitakrat W, Leepitakrat S, Insuan S, Charoensongsermkit W, Khlaimanee N, Akkagraisee W, Chayapum K, Jones JW. 2008. Surveys of rodent-borne disease in Thailand with a focus on scrub typhus assessment. Integr Zool 3:267–273. doi:10.1111/j.1749-4877.2008.00100.x.
    1. Rodkvamtook W, Ruang-Areerate T, Gaywee J, Richards AL, Jeamwattanalert P, Bodhidatta D, Sangjun N, Prasartvit A, Jatisatienr A, Jatisatienr C. 2011. Isolation and characterization of Orientia tsutsugamushi from rodents captured following a scrub typhus outbreak at a military training base, Bothong district, Chonburi province, central Thailand. Am J Trop Med Hyg 84:599–607. doi:10.4269/ajtmh.2011.09-0768.
    1. Rodkvamtook W, Zhang Z, Chao CC, Huber E, Bodhidatta D, Gaywee J, Grieco J, Sirisopana N, Kityapan M, Lewis M, Ching WM. 2015. Dot-ELISA rapid test using recombinant 56-kDa protein antigens for serodiagnosis of scrub typhus. Am J Trop Med Hyg 92:967–971. doi:10.4269/ajtmh.14-0627.
    1. Chao CC, Huber ES, Porter TB, Zhang Z, Ching WM. 2011. Analysis of the cross-reactivity of various 56 kDa recombinant protein antigens with serum samples collected after Orientia tsutsugamushi infection by ELISA. Am J Trop Med Hyg 84:967–972. doi:10.4269/ajtmh.2011.10-0545.
    1. Leeflang MM, Bossuyt PM, Irwig L. 2009. Diagnostic test accuracy may vary with prevalence: implications for evidence-based diagnosis. J Clin Epidemiol 62:5–12. doi:10.1016/j.jclinepi.2008.04.007.

Source: PubMed

Sponsorer og samarbeidspartnere

Medisinsk tilstand

Legemiddelintervensjoner

3
Abonnere