Printer Friendly

Zika Virus IgM Detection and Neutralizing Antibody Profiles 12-19 Months after Illness Onset.

Zika virus is a flavivirus closely related to dengue, West Nile, Japanese encephalitis, and yellow fever viruses (1,2). Diagnostic testing for Zika virus infection is conducted using both molecular and serologic methods, which include testing for viral RNA and IgM and neutralizing antibodies (3-5). RNA detection is most sensitive during the acute phase of illness and confirms Zika virus infection, but sensitivity declines after the first week of illness and a negative result does not exclude infection. Zika virus IgM typically develops [less than or equal to] 4 days after symptom onset and remains detectable for at least 12 weeks (6-8). Data on the duration of IgM after Zika virus infection are lacking, but IgM against other flaviviruses can last for months to years following infection (9-13). Neutralizing antibodies develop shortly after IgM, persist for many years, and may confer lifelong immunity (13,14).

Cross-reactivity between Zika virus and other flaviviruses occurs both with IgM and neutralizing antibodies and makes distinguishing Zika virus from dengue virus infections especially challenging. Whereas primary Zika virus infections typically generate highly specific neutralizing antibodies, secondary flavivirus infections show a high degree of cross-reactivity (6,15,16). For secondary infections, it remains uncertain whether the infecting flavivirus neutralizing antibody response is significantly greater than the cross-reacting neutralizing response, allowing for differentiation, and whether cross-reactive neutralizing antibodies are maintained for months to years after infection (16-19).

In July 2016, the first Zika virus outbreak in the continental United States was identified in Florida, culminating in 300 locally acquired cases in 2016 (20,21). We collected serum specimens from patients with Zika virus infection confirmed by molecular testing to determine the proportion of patients with detectable Zika virus IgM and the ratio of Zika virus and dengue virus neutralizing antibodies at 12-19 months after their acute illness.

Methods

Eligible participants were residents of Miami-Dade County, Florida, USA, who had Zika virus disease confirmed by real-time reverse transcription PCR (rRT-PCR) and symptom onset during June-October 2016. Persons with asymptomatic infection, pregnant women, and infants with congenital infection were excluded from enrollment. We enrolled participants during October 16, 2017-February 1, 2018. We obtained written consent from study participants or their guardians.

Serum specimens were tested at the Centers for Disease Control and Prevention (Fort Collins, CO, USA) by IgM antibody capture ELISA (MAC-ELISA) for detection of Zika virus and dengue virus IgM and by plaque reduction neutralization test (PRNT) to detect Zika virus and dengue virus neutralizing antibodies (5,6,22). The PRNT endpoint titer was defined as the reciprocal of the dilution reducing the virus plaque count by 90%.

We obtained descriptive and clinical data for case-patients, including age, gender, race/ethnicity, reported symptoms, symptom onset, and origin of infection, from Merlin, the Florida Department of Health surveillance system. We used Pearson [chi square] and Fisher exact tests to examine associations between demographics, symptomology, and Zika virus IgM results. We performed all statistical analyses with SAS statistical software version 9.4 (https://www.sas.com/ en_us/software/sas9.html). This study was approved by the Florida Department of Health Institutional Review Board.

Results

Of 352 eligible PCR-confirmed Zika virus disease case patients, 62 (18%) were enrolled and provided follow-up serum specimens. The 62 enrolled participants and 290 eligible case-patients who were not enrolled were similar with regard to age, sex, race/ethnicity, and clinical manifestations; however, 55% of enrolled participants acquired their infections in Florida, compared with 45% of the unenrolled cases (Table 1).

Among the enrolled participants, 8 (13%) provided a specimen at 12 months after initial symptom onset, 1 (2%) at 13 months, 13 (21%) at 14 months, 21 (34%) at 15 months, 11 (18%) at 16 months, 3 (5%) at 17 months, 3 (5%) at 18 months, and 2 (3%) at 19 months. The median age of participants was 47 years (range 8-70 years); 60 (97%) were adults [greater than or equal to] 18 years of age (Table 1). Overall, 32 (52%) participants were male, and 42 (68%) were Hispanic. Two (3%) participants reported only 1 of the 4 main symptoms (fever, maculopapular rash, arthralgia, and conjunctivitis) at the time of their initial Zika virus diagnosis; 15 (24%) reported 2, 32 (52%) reported 3, and 13 (21%) reported all 4. From case investigations, we determined that 34 (55%) participants acquired their Zika virus infection locally in Miami-Dade County.

At follow-up, 45 (73%) patients had detectable Zika virus IgM, 12 (19%) had an equivocal result, and 5 (8%) were negative (all laboratory results provided in Appendix, http://wwwnc.cdc.gov/EID/article/25/2/18-1286-App1. pdf). Results by month since Zika virus symptom onset (Figure) showed that, overall, 39 (91%) of 43 specimens collected at 12-15 months postonset were IgM positive or equivocal, and 18 (95%) of 19 specimens collected at 16-19 months were positive or equivocal. No significant differences in IgM persistence were identified by age, gender, race/ethnicity, origin of infection, or time since illness onset (Table 2).

All participants had Zika virus neutralizing antibodies at 12-19 months after their acute illness, and 39 (63%) had dengue virus neutralizing antibody titers at follow-up. Using a definition of positive or equivocal Zika virus IgM with confirmatory Zika virus neutralizing antibodies, 57 (92%) would have had a diagnosis of recent Zika virus or flavivirus infection on the basis of results from their follow-up specimens (Table 3). Overall, regardless of Zika virus IgM results, 45 (73%) of the PCR-confirmed cases had Zika virus neutralizing antibody titers that were [greater than or equal to] 4- fold higher than dengue virus titers. However, substantial cross-reactivity in neutralizing antibodies was still observed; 12 (19%) patients had a <4-fold difference between Zika virus and dengue virus titers, and 5 (8%) had dengue virus titers that were [greater than or equal to] 4-fold higher than Zika virus titers.

Discussion

These findings demonstrate that 73% of persons with PCR-confirmed symptomatic Zika virus disease still had positive-IgM test results 12-19 months after their initial illness, and another 19% had equivocal results. Because all participants had Zika virus neutralizing antibodies, a high proportion (92%) would have had a recent Zika virus or flavivirus infection diagnosis on the basis of serologic testing performed at the follow-up time point. Current Zika virus testing guidance recommends Zika virus serologic diagnosis only for symptomatic patients with a clinically compatible Zika virus illness (3). However, given the limited specificity of the clinical symptoms associated with Zika virus disease, the prolonged detection of Zika virus IgM presents a particular challenge for serologic diagnosis in pregnant women, given the importance of determining if the infection occurred during the current pregnancy, and complicates the diagnosis of new Zika virus infections in locations with known previous outbreaks.

The prolonged detection of IgM after Zika virus infection is consistent with previous findings for related flaviviruses (9-13). In a study of patients with confirmed West Nile virus encephalitis, 9 (43%) of 21 patients had detectable IgM 300-400 days after onset of their acute illness, and 5 (42%) of 12 had IgM detected >500 days after onset (11). Among asymptomatic blood donors with West Nile virus viremia detected on routine screening, IgM was still detected an average of 156 days (95% CI 70-423 days) after the viremic donation (13). A similar finding was observed in a study looking at the immune response to yellow fever vaccine in adults, in which 29 (73%) of 40 persons had detectable levels of IgM 3-4 years postvaccination (12).

Serologic cross-reactivity has been demonstrated between Zika virus and related flaviviruses, including dengue virus, but the degree to which neutralizing antibody cross reactivity limits the ability to identify the specific virus responsible for the current infection is unclear (6,23). One published report suggests that relative levels of neutralizing antibody titers can distinguish Zika virus from dengue virus infections, especially in specimens collected months after infection (19). However, we found substantial neutralizing antibody cross-reactivity in >25% of specimens collected [greater than or equal to] 1 year after symptom onset.

These findings are subject to several limitations. This report presents data from a single follow-up specimen but how long IgM may persist after this timeframe remains unknown. We cannot exclude the possibility that some participants may have been reexposed to Zika virus or another flavivirus between their initial illness and follow-up testing. The timing of the follow-up specimen varied among the participants and was limited to 12-19 months following onset of Zika virus symptoms. This, coupled with the small number of specimens at some time points, prevented us from assessing possible trends in IgM persistence over time. The small sample size and lack of specimens from the acute illness also limited our ability to detect factors that may be associated with prolonged detection of IgM, including possible differences between primary and secondary infections. Our findings of prolonged IgM seropositivity are specific to the Centers for Disease Control and Prevention MAC-ELISA, which targets Zika virus premembrane and envelope glycoproteins; other IgM serologic assays targeting other proteins are currently available and may not produce comparable findings. Finally, because we enrolled only symptomatic disease case-patients, it is uncertain whether persons with asymptomatic infections would exhibit similar IgM persistence.

These findings support data for other flaviviruses and suggest that a substantial proportion of persons with Zika virus disease will still have detectable IgM 1-2 years after their initial infection. The results highlight the complexity of using serologic diagnosis to determine the specific timing of a recent infection, which is particularly important for pregnant women and challenging for residents of areas with previous or ongoing Zika virus activity. As such, the findings further support the current recommendations to use nucleic acid amplification for screening asymptomatic pregnant women with ongoing possible Zika virus exposure (3). Further study is needed to assess IgM persistence using other approved assays, determine the full duration of Zika virus IgM after infection, and evaluate possible differences in IgM duration following primary and secondary infections.

Acknowledgments

The research team thanks the Miami-Dade County residents who volunteered their time to participate in this study and gave their serum specimens to help further the understanding of Zika virus. The authors also acknowledge the work of the dedicated laboratorians, epidemiologists, and phlebotomists at the Centers for Disease Control and Prevention (Amanda J. Panella, Robert S. Lanciotti); Bureau of Public Health Laboratories; and the Florida Department of Health in Miami-Dade County (Lillian Rivera, Hannah Speaks, Anne Barrera).

Ms. Griffin is an outbreak epidemiologist at the Florida Department of Health in Miami, Florida, USA. Her primary research interest is emerging infectious diseases.

References

(1.) Hills SL, Fischer M, Petersen LR. Epidemiology of Zika virus infection. J Infect Dis. 2017;216(suppl_10):S868-74. http://dx.doi.org/10.1093/infdis/jix434

(2.) Petersen LR, Jamieson DJ, Honein MA. Zika virus. N Engl J Med. 2016;375:294-5. http://dx.doi.org/10.1056/NEJMc1606769

(3.) Oduyebo T, Polen KD, Walke HT, Reagan-Steiner S, Lathrop E, Rabe IB, et al. Update: Interim guidance for health care providers caring for pregnant women with possible Zika virus exposure-United States (including U.S. territories), July 2017. MMWR Morb Mortal Wkly Rep. 2017;66:781-93. http://dx.doi.org/10.15585/ mmwr.mm6629e1

(4.) Rabe IB, Staples JE, Villanueva J, Hummel KB, Johnson JA, Rose L, et al.; MTS. Interim guidance for interpretation of Zika virus antibody test results. MMWR Morb Mortal Wkly Rep. 2016;65:543-6. http://dx.doi.org/10.15585/mmwr.mm6521e1

(5.) Theel ES, Hata DJ. Diagnostic testing for Zika virus: a postoutbreak update. J Clin Microbiol. 2018;56:e01972-17. http://dx.doi.org/10.1128/JCM.01972-17

(6.) Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis. 2008;14:1232-9. http://dx.doi.org/10.3201/eid1408.080287

(7.) Bingham AM, Cone M, Mock V, Heberlein-Larson L, Stanek D, Blackmore C, et al. Comparison of test results for Zika virus RNA in urine, serum, and saliva specimens from persons with travel-associated Zika virus disease--Florida, 2016. MMWR Morb Mortal Wkly Rep. 2016;65:475-8. http://dx.doi.org/10.15585/ mmwr.mm6518e2

(8.) Paz-Bailey G, Rosenberg ES, Doyle K, Munoz-Jordan J, Santiago GA, Klein L, et al. Persistence of Zika virus in body fluids--final report. N Engl J Med. 2017;379:1234-43. http://dx/doi.org/10.1056/NEJMoa1613108

(9.) Centers for Disease Control and Prevention. Prolonged IgM antibody response in people infected with Zika virus: implications for interpreting serologic testing results for pregnant women. CDC Health Advisory. 2017 [cited 2018 May 4]. https://emergency.cdc.gov/ han/han00402.asp

(10.) Prince HE, Tobler LH, Yeh C, Gefter N, Custer B, Busch MP. Persistence of West Nile virus-specific antibodies in viremic blood donors. Clin Vaccine Immunol. 2007;14:1228-30. http://dx.doi.org/ 10.1128/CVI.00233-07

(11.) Roehrig JT, Nash D, Maldin B, Labowitz A, Martin DA, Lanciotti RS, et al. Persistence of virus-reactive serum immunoglobulin M antibody in confirmed West Nile virus encephalitis cases. Emerg Infect Dis. 2003;9:376-9. http://dx.doi.org/10.3201/eid0903.020531

(12.) Gibney KB, Edupuganti S, Panella AJ, Kosoy OI, Delorey MJ, Lanciotti RS, et al. Detection of anti-yellow fever virus immunoglobulin m antibodies at 3-4 years following yellow fever vaccination. Am J Trop Med Hyg. 2012;87:1112-5. http://dx.doi.org/10.4269/ajtmh.2012.12-0182

(13.) Busch MP, Kleinman SH, Tobler LH, Kamel HT, Norris PJ, Walsh I, et al. Virus and antibody dynamics in acute West Nile virus infection. J Infect Dis. 2008;198:984-93. http://dx.doi.org/ 10.1086/591467

(14.) Poland JD, Calisher CH, Monath TP, Downs WG, Murphy K. Persistence of neutralizing antibody 30-35 years after immunization with 17D yellow fever vaccine. Bull World Health Organ. 1981;59:895-900.

(15.) Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfield JS, Westaway EG, et al. Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J Gen Virol. 1989;70:37-43. http://dx.doi.org/10.1099/0022-1317-70-1-37

(16.) Johnson BW, Kosoy O, Martin DA, Noga AJ, Russell BJ, Johnson AA, et al. West Nile virus infection and serologic response among persons previously vaccinated against yellow fever and Japanese encephalitis viruses. Vector Borne Zoonotic Dis. 2005;5:137-45. http://dx.doi.org/10.1089/vbz.2005.5.137

(17.) Halstead SB, Rojanasuphot S, Sangkawibha N. Original antigenic sin in dengue. Am J Trop Med Hyg. 1983;32:154-6. http://dx.doi.org/10.4269/ajtmh.1983.32.154

(18.) Montoya M, Collins M, Dejnirattisai W, Katzelnick LC, Puerta-Guardo H, Jadi R, et al. Longitudinal analysis of antibody cross-neutralization following Zika virus and dengue virus infection in Asia and the Americas. J Infect Dis. 2018;218:536-45. http://dx.doi.org/10.1093/infdis/jiy164

(19.) Collins MH, McGowan E, Jadi R, Young E, Lopez CA, Baric RS, et al. Lack of durable cross-neutralizing antibodies against Zika virus from dengue virus infection. Emerg Infect Dis. 2017;23:77381. http://dx.doi.org/10.3201/eid2305.161630

(20.) Likos A, Griffin I, Bingham AM, Stanek D, Fischer M, White S, et al. Local mosquito-borne transmission of Zika virus-Miami-Dade and Broward counties, Florida, June-August 2016. MMWR Morb Mortal Wkly Rep. 2016;65:1032-8. http://dx.doi.org/10.15585/mmwr.mm6538e1

(21.) Florida Department of Health. 2016 Zika map. 2018 [cited 2018 Jul 19]. https://zikafreefl.org/map

(22.) Martin DA, Muth DA, Brown T, Johnson AJ, Karabatsos N, Roehrig JT. Standardization of immunoglobulin M capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections. J Clin Microbiol. 2000;38:1823-6.

(23.) Priyamvada L, Hudson W, Ahmed R, Wrammert J. Humoral cross-reactivity between Zika and dengue viruses: implications for protection and pathology. Emerg Microbes Infect. 2017;6:e33. http://dx.doi.org/10.1038/emi.2017.42

Address for correspondence: Isabel Griffin, Florida Department of Health in Miami-Dade County--Epidemiology, Disease Control, and Immunization Services, 8600 NW 17th St, Ste 200, Miami, FL 33126, USA; email: isabel.griffin@flhealth.gov

Author affiliations: Florida Department of Health in Miami-Dade County, Miami, Florida, USA (I. Griffin, A. Falise, O. Ponomareva, R. Jean); Centers for Disease Control and Prevention, Fort Collins, Colorado, USA (S.W. Martin, M. Fischer, T.V. Chambers, O. Kosoy); Bureau of Public Health Laboratories, Miami (L.D. Gillis); Florida Department of Health, Tallahassee, Florida, USA (C. Blackmore)

DOI: https://doi.org/10.3201/eid2502.181286

Caption: Figure. Zika virus IgM results for 62 participants in Miami-Dade County, Florida, USA, with PCR-confirmed Zika virus disease by follow-up specimen collection month.
Table 1. Demographic and clinical characteristics of confirmed Zika
virus disease case-patients in Miami-Dade County, Florida, USA

                                  No. (%) enrolled, *
Characteristic                          n = 62

Age group, y
  1-18                                   2 (3)
  19-64                                 54 (87)
  [greater than or equal to] 65         6 (10)
Sex
  M                                     32 (52)
  F                                     30 (48)
Race/ethnicity
  Non-Hispanic white                    13 (21)
  Non-Hispanic African American          3 (5)
  Hispanic                              42 (68)
  Unknown                                4 (6)
Main symptoms of Zika virus
disease ([double dagger])
  1 of 4                                 2 (3)
  2 of 4                                15 (24)
  3 of 4                                32 (52)
  4 of 4                                13 (21)
Origin of infection
  Florida                               34 (55)
  Outside Florida                       28 (45)

                                  No. (%) not enrolled, *    p value
Characteristic                            n = 290           ([dagger])

Age group, y                                                   0.19
  1-18                                    26 (9)
  19-64                                  247 (85)
  [greater than or equal to] 65           17 (6)
Sex                                                            0.85
  M                                      146 (50)
  F                                      144 (50)
Race/ethnicity                                                 0.55
  Non-Hispanic white                      41 (14)
  Non-Hispanic African American           14 (5)
  Hispanic                               209 (72)
  Unknown                                 26 (9)
Main symptoms of Zika virus                                    0.88
disease ([double dagger])
  1 of 4                                  12 (4)
  2 of 4                                  78 (27)
  3 of 4                                 130 (45)
  4 of 4                                  69 (24)
Origin of infection                                            0.19
  Florida                                133 (46)
  Outside Florida                        157 (54)

* All percentages are column percentages.

([dagger]) A p value [less than or equal to] 0.05 was considered
statistically significant.

([double dagger]) Main symptoms were defined as fever, maculopapular
rash, arthralgia, and conjunctivitis.

Table 2. Demographic and clinical characteristics of enrolled
participants in Miami-Dade County, Florida, USA, with PCR-confirmed
Zika virus disease by Zika virus IgM antibody result 12-19 months
after illness onset (n = 62)

                                       Zika virus IgM results 12-19 mo
                                       after symptom onset, no. (%) *

Characteristic                         Positive   Equivocal   Negative

Age group, y
  1-18, n = 2                          2 (100)        0          0
  19-64, n = 54                        38 (71)     12 (22)     4 (7)
  [greater than or equal to] 65,        5 (83)        0        1 (17)
    n = 6
Sex
  M, n = 32                            25 (78)     6 (19)      1 ( 3)
  F, n = 30                            20 (67)     6 (20)      4 (13)
Race/ethnicity
  Non-Hispanic white, n = 12           11 (92)      1 (8)        0
  Non-Hispanic African American,        1 (25)     2 (50)      1 (25)
    n = 4
  Hispanic, n = 42                     29 (69)     9 (21)      4 (10)
  Unknown, n = 4                       4 (100)        0          0
Main symptoms of Zika
virus ([double dagger])
  1 of 4, n = 2                         1 (50)     1 (50)        0
  2 of 4, n = 15                        8 (54)     5 (33)      2 (13)
  3 of 4, n = 32                       25 (78)     4 (13)      3 (9)
  4 of 4, n = 13                       11 (85)     2 (15)        0
Origin of infection
  Florida, n = 34                      24 (70)     6 (18)      4 (12)
  Outside Florida, n = 28              21 (75)     6 (21)      1 (4)

Characteristic                         p value ([dagger])

Age group, y                                  0.58
  1-18, n = 2
  19-64, n = 54
  [greater than or equal to] 65,
    n = 6
Sex                                           0.30
  M, n = 32
  F, n = 30
Race/ethnicity                                0.18
  Non-Hispanic white, n = 12
  Non-Hispanic African American,
    n = 4
  Hispanic, n = 42
  Unknown, n = 4
Main symptoms of Zika                         0.28
virus ([double dagger])
  1 of 4, n = 2
  2 of 4, n = 15
  3 of 4, n = 32
  4 of 4, n = 13
Origin of infection                           0.52
  Florida, n = 34
  Outside Florida, n = 28

* All percentages are row percentages.

([dagger]) A p-value [less than or equal to] 0.05 was considered
statistically significant.

[double dagger]) Main symptoms were defined as fever, maculopapular
rash, arthralgia, and conjunctivitis.

Table 3. Diagnostic test interpretations among participants with PCR-
confirmed Zika virus disease in Miami-Dade County, Florida, USA, based
on IgM and neutralizing antibody results 12-19 months after onset *

                              No. (%),
Test results                  n = 62     Interpretation ([dagger])

Zika virus IgM positive or
equivocal, n = 57
  Zika virus PRNT titer       21 (34)    Recent Zika virus infection
    [greater than or equal
    to] 10 and DENV PRNT
    titer <10
  Zika virus PRNT titer       21 (34)    Recent flavivirus infection
    [greater than or equal
    to] 4-fold higher than
    DENV PRNT titer
  <4-fold difference          11 (18)    Recent flavivirus infection
    between Zika virus and
    DENV PRNT titers
  DENV PRNT titer [greater
    than or equal to] 4-fold  4 (6)      Recent flavivirus infection
    higher than Zika virus
    PRNT titer
ZIKV IgM negative, n = 5
  Zika virus PRNT titer       2 ( 3)     Previous Zika virus infection
    [greater than or equal
    to] 10 and Zika virus
    PRNT titer <10
  Zika virus PRNT titer       1 (2)      Previous flavivirus infection
    [greater than or equal
    to] 4-fold higher than
    DENV PRNT titer
  <4-fold difference          1 (2)      Previous flavivirus infection
    between Zika virus and
    DENV PRNT titers
  DENV PRNT titer >4-fold     1 (2)      Previous flavivirus infection
    higher than Zika virus
    PRNT titer

* DENV, dengue virus; PRNT, plaque reduction neutralization test.

([dagger]) Recent Zika virus infection: Zika virus IgM positive or
equivocal with a Zika virus PRNT titer [greater than or equal to] 10
and dengue PRNT titer <10; recent flavivirus infection: Zika virus IgM
positive or equivocal with a Zika virus PRNT titer [greater than or
equal to] 10 or dengue PRNT titer  [greater than or equal to] 10;
previous Zika virus infection: Zika virus IgM negative with a
Zika virus PRNT titer [greater than or equal to] 10 and
dengue PRNT titer <10; previous flavivirus infection:
Zika virus IgM negative with a Zika virus PRNT titer [greater than or
equal to] 10 or dengue PRNT titer [greater than or equal to] 10.
COPYRIGHT 2019 U.S. National Center for Infectious Diseases
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2019 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:RESEARCH
Author:Griffin, Isabel; Martin, Stacey W.; Fischer, Marcs; Chambers, Trudy V.; Kosoy, Olga; Falise, Alyssa;
Publication:Emerging Infectious Diseases
Geographic Code:1U5FL
Date:Feb 1, 2019
Words:3635
Previous Article:Macrophage Activation Marker Soluble CD163 Associated with Fatal and Severe Ebola Virus Disease in Humans.
Next Article:Killing Clothes Lice by Holding Infested Clothes Away from Hosts for 10 Days to Control Louseborne Relapsing Fever, Bahir Dah, Ethiopia.
Topics:

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters