Cross-reactive IgM responses in patients with dengue or Japanese encephalitis.

BACKGROUND: Dengue and Japanese encephalitis viruses co-circulate in Thailand. IgM-capture enzyme-linked immunosorbent assay (ELISA) has been widely used for confirmation of dengue and Japanese encephalitis (JE). OBJECTIVES: To examine the cross-reactivity in IgM responses to dengue and JE viruses in serum and CSF samples from dengue and JE patients. STUDY DESIGN: Two hundred and fifty-eight serum samples from 177 confirmed dengue patients, and 99 serum samples and 37 cerebrospinal fluid (CSF) samples from confirmed JE patients were analyzed. RESULTS: Nine percent of serum samples from dengue patients were positive for anti-JE IgM. Thirteen percent of serum samples and 11% of CSF samples from JE patients were positive for anti-dengue IgM. Levels of cross-reactive IgM were lower than those of specific IgM in all the dengue and JE patients. CONCLUSIONS: Only specific IgM is detected in about 90% of dengue and JE patients, but cross-reactive IgM is also detected in the remainder. The presence of cross-reactive IgM responses should to be considered in the serodiagnosis of dengue and JE, especially in areas where dengue and JE viruses co-circulate.     

Evaluation of four methods for detection of immunoglobulin M antibodies to dengue virus.

Dengue has become hyperendemic in many islands of the Caribbean region. The performance in a diagnostic laboratory of four commercial assays for detection of immunoglobulin M (IgM) antibodies was evaluated. Sera from 62 patients with dengue virus infection were studied. These included 18 patients from whom dengue virus type 2 was isolated in a 1997 outbreak (specimens collected a mean of 14 days after onset of symptoms), 8 patients with dengue hemorrhagic fever (mean time after onset, 11 days), and 36 patients in whom dengue was previously confirmed by serology (mean time after onset, 10 days). Thirty serum specimens from blood donors in a country where dengue is not endemic were used as negative controls. The methods evaluated were two IgM-capture enzyme-linked immunosorbent assays (ELISA) (MRL Diagnostics, Cypress, Calif., and PanBio, Queensland, Australia), a dot ELISA dipstick assay (Integrated Diagnostics, Baltimore, Md.), and a rapid immunochromatographic assay for dengue IgG and IgM (PanBio IC). IgG antibodies were also detected by an ELISA method (MRL Diagnostics). The sensitivities of the four assays were as follows: MRL Diagnostics IgM ELISA, 98.4%; PanBio IgM ELISA, 85.5%; Integrated Diagnostics IgM dot ELISA, 96. 8%; and PanBio IC, 83.9%. The specificities of all tests were 100%. Evidence of secondary dengue was found in all patients with dengue hemorrhagic fever and in 83% of the remaining patients. The MRL Diagnostics IgM ELISA appears to be more sensitive than the PanBio IgM ELISA, and this may be significant when IgM titers are low, particularly in patients with secondary dengue infections. The dot ELISA dipstick assay is equally sensitive and may be more appropriate for use in laboratories with lower workloads.     

Analysis of specific IgM responses in secondary dengue virus infections: levels and positive rates in comparison with primary infections.

BACKGROUND: Dengue viruses are a serious cause of illness in tropical and subtropical areas of the world. Laboratory diagnosis is essential for confirmation of dengue virus infections. Detection of specific IgM by IgM-capture enzymed-linked immunoassay (ELISA) has been widely used as a main serological diagnostic technique. OBJECTIVES: The levels of specific IgM in secondary dengue virus infections were compared with those in primary infections. STUDY DESIGN: A total of 1780 samples collected from 924 confirmed dengue cases were tested for anti-dengue IgM by IgM-capture ELISA. RESULTS AND CONCLUSIONS: Specific IgM was detected in all the cases with primary dengue virus infection on disease day 9 or later. However, specific IgM cannot be detected in 28% (204/716) of the cases in secondary infections. The average titers of IgM were higher in primary infections than in secondary infections. The results confirmed that IgM detection is a reliable serological diagnostic test in primary dengue virus infections. Although IgM detection is also a useful test, other serological diagnostic tests or tests for dengue virus detection are necessary for confirmation of all the secondary dengue virus infections.     

Evaluation of Four Methods for Detection of Immunoglobulin M Antibodies to Dengue Virus

Dengue has become hyperendemic in many islands of the Caribbean region. The performance in a diagnostic laboratory of four commercial assays for detection of immunoglobulin M (IgM) antibodies was evaluated. Sera from 62 patients with dengue virus infection were studied. These included 18 patients from whom dengue virus type 2 was isolated in a 1997 outbreak (specimens collected a mean of 14 days after onset of symptoms), 8 patients with dengue hemorrhagic fever (mean time after onset, 11 days), and 36 patients in whom dengue was previously confirmed by serology (mean time after onset, 10 days). Thirty serum specimens from blood donors in a country where dengue is not endemic were used as negative controls. The methods evaluated were two IgM-capture enzyme-linked immunosorbent assays (ELISA) (MRL Diagnostics, Cypress, Calif., and PanBio, Queensland, Australia), a dot ELISA dipstick assay (Integrated Diagnostics, Baltimore, Md.), and a rapid immunochromatographic assay for dengue IgG and IgM (PanBio IC). IgG antibodies were also detected by an ELISA method (MRL Diagnostics). The sensitivities of the four assays were as follows: MRL Diagnostics IgM ELISA, 98.4%; PanBio IgM ELISA, 85.5%; Integrated Diagnostics IgM dot ELISA, 96.8%; and PanBio IC, 83.9%. The specificities of all tests were 100%. Evidence of secondary dengue was found in all patients with dengue hemorrhagic fever and in 83% of the remaining patients. The MRL Diagnostics IgM ELISA appears to be more sensitive than the PanBio IgM ELISA, and this may be significant when IgM titers are low, particularly in patients with secondary dengue infections. The dot ELISA dipstick assay is equally sensitive and may be more appropriate for use in laboratories with lower workloads.     

Detection of anti-West Nile virus immunoglobulin M in chicken serum by an enzyme-linked immunosorbent assay

The emergence of West Nile (WN) virus in New York and the surrounding area in 1999 prompted an increase in surveillance measures throughout the United States, including the screening of sentinel chicken flocks for antibodies. An enzyme-linked immunosorbent assay (ELISA) for the detection of chicken immunoglobulin M (IgM) to WN virus was developed, standardized, and characterized as a rapid and sensitive means to detect WN viral antibodies in sentinel flocks. Serum specimens from experimentally infected chickens were analyzed by using this assay, and IgM was detected as early as 3 to 7 days postinfection. Persistence of IgM varied from at least 19 to more than 61 days postinfection, which indicates the need to bleed sentinel flocks at least every 2 weeks for optimal results if this method is to be used as a screening tool. The ELISA was compared to hemagglutination-inhibition and plaque reduction neutralization tests and was found to be the method of choice when early detection of WN antibody is required. House sparrows and rock doves are potential free-ranging sentinel species for WN virus, and the chicken WN IgM-capture ELISA was capable of detecting anti-WN IgM in house sparrow serum samples from laboratory-infected birds but not from rock dove serum samples. The chicken WN IgM-capture ELISA detected anti-WN antibodies in serum samples from naturally infected chickens. It also detected IgM in serum samples from two species of geese and from experimentally infected ring-necked pheasants, American crows, common grackles, and redwinged blackbirds. However, the test was determined to be less appropriate than an IgG (IgY)-based assay for use with free-ranging birds. The positive-to-negative ratios in the ELISA were similar regardless of the strain of WN viral antigen used, and only minimal cross-reactivity was observed between the WN and St. Louis encephalitis (SLE) IgM-capture ELISAs. A blind-coded serum panel was tested, and the chicken WN IgM-capture ELISA produced consistent results, with the exception of one borderline result. A preliminary test was done to assess the feasibility of a combined SLE and WN IgM-capture ELISA, and results were promising.     

Development and evaluation of an IgM-capture ELISA for detection of recent infection with bluetongue viruses in cattle

An IgM-capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of recent infection of bluetongue virus (BTV) in cattle. The test is based on the use of biotinylated capture anti-bovine IgM antibodies bound to a streptavidin-coated ELISA plate. The captured IgM antibodies were detected by application of BTV VP7 antigen and a VP7 antigen-specific monoclonal antibody. The IgM-capture ELISA was compared with the competitive ELISA by testing serum samples from groups of calves infected experimentally with five USA and 19 South Africa serotypes of BTV. The IgM-capture ELISA was able to detect bovine anti-VP7 antibodies from all animals infected with the 24 BTV serotypes at 10 days post-infection, whereas the competitive ELISA was not. When the detectable IgM diminished after 40 days post-infection by the IgM-capture ELISA, the IgG anti-VP7 antibodies remained high. The IgM-capture ELISA is sensitive and can be applied for the detection of recent infection of BTV in cattle.     

Evaluation of immunoglobulin M and G capture enzyme-linked immunosorbent assay Panbio kits for diagnostic dengue infections.

BACKGROUND: Serological assays are widely used to confirm dengue virus infections and to differentiate between a primary and a secondary infection. OBJECTIVE: Two commercial dengue diagnostic kits, Panbio Dengue IgM Capture and Dengue IgG Capture ELISA (Brisbane, Australia) were evaluated. STUDY DESIGN: Three hundred and seventy-three serum samples were tested. Panel sera included samples from dengue confirmed cases (representing both primary and secondary infections), from non-dengue infectious diseases, and from healthy individuals. The MAC-ELISA/Dengue IPK was used for the detection of anti-dengue virus IgM antibody in the sera and the ELISA inhibition method (EIM/Dengue IPK) was used to differentiate between primary and secondary infections. Both these reference assays, which were previously developed in the Arbovirus Laboratory at the "Pedro Kouri" Tropical Medicine Institute, were employed as the gold standard. RESULTS: High sensitivity (96.8%) and specificity (99.4%) were found with the commercial diagnostics when compared to the reference methods. Furthermore, high concordance 95.5% in classifying dengue infection types (primary or secondary infections) was observed. CONCLUSIONS: The Panbio Dengue IgM and IgG assays offer a good alternative for dengue diagnosis. They are easy to perform and results can be obtained in less than 3h.     

Evaluation of the MRL diagnostics dengue fever virus IgM capture ELISA and the PanBio Rapid Immunochromatographic Test for diagnosis of dengue fever in Jamaica

We evaluated two new commercial dengue diagnostic tests, the MRL Diagnostics Dengue Fever Virus IgM Capture ELISA and the PanBio Rapid Immunochromatographic Test, on serum samples collected during a dengue epidemic in Jamaica. The MRL ELISA method correctly identified 98% (78 of 80) of the samples as dengue positive, while the PanBio test identified 100% (80 of 80). Both tests were 100% (20 samples of 20) specific.     

Comparison of two rapid diagnostic assays for detection of immunoglobulin M antibodies to dengue virus

Two easy-to-use commercial diagnostic assays, a dipstick enzyme-linked immunosorbent assay (ELISA) (Integrated Diagnostics, Baltimore, Md.) and an immunochromatographic card assay (PanBio, Brisbane, Australia) were evaluated for detection of immunoglobulin M (IgM) antibody to dengue virus with an in-house IgM antibody capture microplate ELISA as a reference assay. The dipstick ELISA was based on the indirect-ELISA format using dengue 2 virus as the only antigen and enzyme-labeled goat anti-human IgM antibody as the detector. The total assay time was 75 min. The immunochromatographic card assay was based on the antibody capture format and separately measured both anti-dengue virus IgM and IgG in the same test. Colloidal-gold-labeled anti-dengue virus monoclonal antibody bound with dengue virus 1 to 4 antigen cocktail was the detector, and anti-human IgM and IgG were the capture antibodies. The total assay time was <10 min. Sera from 164 individuals classified as either anti-dengue virus IgM positive (94) or anti-dengue virus IgM negative (70) in the reference microplate ELISA with a dengue virus 1 to 4 antigen cocktail were tested in the two commercial assays. The dipstick ELISA missed 7 of 94 positive samples, for a sensitivity of 92.6%, while the immunochromatographic card assay missed two positive samples, for a sensitivity of 97.9%. Of the 70 negative samples, four were false positive by the dipstick ELISA and two were false positive in the immunochromatographic card assay, resulting in specificities of 94.3 and 97.1%, respectively. Both commercial assays provide sensitive and specific detection of anti-dengue virus IgM antibody and could prove useful in settings where the microplate ELISA is impractical.     

Laboratory diagnosis of primary and secondary dengue infection.

BACKGROUND: Dengue fever is routinely detected in many laboratories using commercial tests for the specific detection of dengue IgM antibodies. OBJECTIVES: We have studied the sensitivity of IgM antibody detection in paired serum samples of 43 patients with either with primary dengue (PD) or secondary dengue (SD). STUDY DESIGN: Two consecutive samples were drawn from 23 Vietnamese and 20 German patients. All patients were selected for a positive PCR and for the fact that consecutive serum samples were available. The diagnosis of PD was based on seroconversion to dengue antigen and in SD on the detection of virus RNA in the presence of anti-dengue IgG antibodies. RESULTS: In samples of patients with PD fever taken during days 1-3 of the disease no IgM antibody could be detected. During days 4-7 and after day 7, IgM antibody was detected in 55% and 94%, respectively. In patients with SD fever, even less positive IgM samples were found in samples taken during days 4-7 (47%) and after day 7 (78%). IgG titers were significantly higher in SD compared to PD patients, although high (>1280) titers were also found in some PD patients. CONCLUSION: In numerous acute dengue fever patients an early diagnosis will be obtained only by combining IgM antibody detection with detection of virus or virus RNA using RT-PCR.     

Rapid diagnosis of human Crimean-Congo hemorrhagic fever and detection of the virus in naturally infected ticks.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect Crimean-Congo hemorrhagic fever (CCHF) virus-specific immunoglobulin M (IgM) in human serum samples. For this test, a heat-inactivated antigen was prepared from the brains of suckling mice infected with CCHF virus. The IgM-capture ELISA proved more sensitive than indirect fluorescence tests for IgM to this virus. A human serum containing high-titer IgM to CCHF virus was used for an antigen-capture ELISA to detect this virus in heat-inactivated suspensions of virus-infected ticks. The antigen-capture ELISA appeared to be as sensitive as virus isolation in suckling mice. The studies described suggest that the IgM-capture ELISA and the antigen-detection ELISA should provide a rapid and sensitive diagnosis of human CCHF virus infection and should be useful in ecologic studies of this virus.     

Value of serological testing for diagnosis of legionellosis in outbreak patients

Serum antibody detection tests and a urine antigen detection technique were compared in samples from 116 patients epidemiologically characterized as belonging to a legionellosis outbreak. Sera were tested by enzyme-linked immunosorbent assays (ELISAs) for immunoglobulin M (IgM) and IgG plus IgM and by immunofluorescent assays (IFAs) for IgG, IgM, IgA, and polyimmunoglobulin using commercial kits (Vircell); concentrated urines were tested with the Binax NOW Legionella test. ELISA for IgM, ELISA for IgG plus IgM, antigenuria detection, and IFA for IgM were able to diagnose 72.3%, 60.5%, 53.3%, and 51.4%, respectively, of patients. Antigenuria was present in 53.8% of first samples, ELISA detected IgM in 29.7%, ELISA detected IgG plus IgM in 7.9%, and IFA detected IgM in 3.9%. Ten antigenuria-negative first samples tested serologically positive, 9 of them to IgM by ELISA. Despite the single source of the samples included in the study, detection of IgM using a sensitive technique such as ELISA seems to be a suitable complement to antigenuria detection for the diagnosis of legionellosis.     

Comparison of different techniques for detection of anti-HLA antibodies in sera from patients awaiting kidney transplantation.

The aim of the present study was to compare different techniques of HLA antibody detection in patients waiting for a transplant. Two methods of HLA antibody screening were compared: the complement-dependent cytotoxicity (CDC) test and the enzyme-linked immunosorbent solubility assay (ELISA). Three different commercial kits for the latter assay were tested: the kits were produced by One Lambda (O.L.) and SANG STAT for prescreening and specificity determination, and GTI only for prescreening. Of the two techniques, only CDC can detect IgM. The study included 207 serum samples selected from 192 patients waiting for a first kidney transplant. The conclusions are that O.L. is more sensitive than CDC and the other ELISA kits and is much faster for analysing a serum (taking only a few hours), but costs about 16 times more than CDC and does not detect IgM antibodies, so can be used only in support of CDC.     

Comparison of different techniques for detection of anti‐HLA antibodies in sera from patients awaiting kidney transplantation

The aim of the present study was to compare different techniques of HLA antibody detection in patients waiting for a transplant. Two methods of HLA antibody screening were compared: the complement‐dependent cytotoxicity (CDC) test and the enzyme‐linked immunosorbent solubility assay (ELISA). Three different commercial kits for the latter assay were tested: the kits were produced by One Lambda (O.L.) and SANG STAT for prescreening and specificity determination, and GTI only for prescreening. Of the two techniques, only CDC can detect IgM. The study included 207 serum samples selected from 192 patients waiting for a first kidney transplant. The conclusions are that O.L. is more sensitive than CDC and the other ELISA kits and is much faster for analysing a serum (taking only a few hours), but costs about 16 times more than CDC and does not detect IgM antibodies, so can be used only in support of CDC.     

Development of dengue IgM-capture enzyme-linked immunosorbent assay with higher sensitivity using monoclonal detection antibody

An IgM-capture enzyme-linked immunosorbent assay (IgM-ELISA) is used widely for serodiagnosis of dengue. A dengue IgM-ELISA with higher sensitivity has been developed. In the new ELISA, anti-dengue IgM antibody, which had been captured on the solid phase, was reacted with tetravalent dengue viral antigens, and detected by a flavivirus group specific monoclonal antibody, D1-4G2-4-15 (4G2). Reaction of 4G2 to viral antigens was similar to that of dengue patients' IgG. Non-specific reaction of 4G2 to the control antigen, which was prepared from uninfected cell culture fluid of mosquito C6/36 cells, was much lower than that of patients' IgG. Thus, specificity of the ELISA with 4G2 was much higher than that with patients' IgG, and lower levels of specific IgM was detected in the serum samples. These results suggest that the modified dengue IgM-ELISA with monoclonal antibody 4G2 has many advantages over the original "in-house" ELISA.     

Comparative studies of commercially available particle agglutination assay and enzyme-linked immunosorbent assay for screening of human T-cell leukemia virus type I antibodies in blood donors.

Human T-cell leukemia virus type I (HTLV-I) transmission during blood transfusion can be prevented by screening for and eliminating blood containing anti-HTLV-I antibodies. For this purpose, we evaluated two commercial test kits for HTLV-I antibodies, Serodia-ATLA (particle agglutination assay [PA]) and Eitest-ATL (enzyme-linked immunosorbent assay [ELISA]), by using serum samples from Japanese blood donors. Of 2,316 serum samples, 39 (1.7%), 34 (1.5%), and 28 (1.2%) were positive for the antibody by PA, ELISA, and immunofluorescence (IF), respectively. The coincidence rate for antibody-positive and antibody-negative results was 99.5% between PA and IF, whereas it was 99.6% between ELISA and IF. The serum samples which were positive by IF were also positive by PA, whereas 2 of 26 IF-positive samples were negative by ELISA. All the samples positive by both PA and ELISA were shown to be positive by IF unequivocally. The samples positive by only one of the two methods (PA or ELISA) were different. Eleven and eight serum samples that were negative by IF but positive by PA and ELISA, respectively, were further studied for HTLV-I antibody by radioimmunoprecipitation. Three of the former but none of the latter were found to be positive for antibody. Moreover, a PA-positive but IF-negative serum sample was shown to have significantly decreased PA titers after 2-mercaptoethanol treatment. The PA was further shown to detect not only IgG antibody but also IgM antibody to HTLV-I after separation of this serum sample by high-performance liquid chromatography.     

Comparison of a dipstick dot-ELISA with commercial assays for anti-dengue virus IgM antibodies

Dengue virus infections have undergone dramatic expansion in range, affecting several tropical and subtropical regions of the world. Dengue virus causes life-threatening complications characterized by dengue hemorrhagic fever and dengue shock syndrome. No standard validated test systems are available for serological diagnosis of dengue virus infection. This creates problems in the diagnosis and proper management of patients. Evaluation of a Defense Research and Development Establishment (DRDE) dengue virus dipstick dot-ELISA test, developed in house, vis-à-vis commercially available immunodiagnostic kits was carried out for detection of IgM antibodies. The DRDE dengue dipstick dot-ELISA was performed on the basis of the dot-ELISA principle. Commercial tests, namely, the Panbio Dengue IgM Capture ELISA system (Panbio, Sinnamon Park, Australia) and Pathozyme Dengue M (Omega Diagnostics, Alva, UK), were performed according to the manufacturers' protocols. Ninety-one serum samples collected from the states of Kerala and Delhi, India during August and November of 2004 were used in the present study. The overall agreement among all three tests was found to be only 72.16% for IgM antibodies. Correlations between the DRDE dipstick dot-ELISA and the Panbio kit, between the DRDE dipstick dot-ELISA and the Pathozyme Dengue M kit, and between the Panbio kit and the Pathozyme Dengue M kit were found to be 96, 93, and 94%, respectively. Although the Panbio kit is widely used in various laboratories in India, the DRDE dipstick dot-ELISA promises to be a useful kit because of its field applicability and comparable sensitivity.     

Diagnosis of Mycoplasma pneumoniae Pneumonia in Children

We evaluated a commercial immunoglobulin M (IgM)-capture immunoassay for the detection of Mycoplasma pneumoniae infections in 278 pediatric patients with community-acquired, radiographically defined pneumonia. Acute- and convalescent-phase serum samples were collected from all patients and were tested for M. pneumoniae-specific IgM and IgG antibodies by Platelia enzyme immunoassays (Sanofi Diagnostica Pasteur, Marnes la Coquette, France). Nasopharyngeal aspirates (NPAs) were collected at the time of admission to the hospital. A total of 227 NPAs were subjected to the detection of M. pneumoniae DNA by PCR, and 191 NPAs were cultured by using the Pneumofast kit (International Mycoplasma, Signeswere, France). Southern hybridization of PCR products and the IgM test with solid-phase antigen (Serion Immunodiagnostica, Würzburg, Germany) were used for additional confirmation of a positive result, which required agreement of at least two different methods. A total of 24 (9%) confirmed diagnoses of mycoplasma infection were made, 5 (21%) of which were in children <5 years of age. Of the positive children, 24 of 24 (sensitivity, 100%) were positive by the IgM-capture test with convalescent-phase serum, 19 of 24 (79%) were positive by the IgM-capture test with acute-phase serum, 19 of 24 (79%) were positive by IgG serology, 10 of 20 (50%) were positive by PCR, and 8 of 17 (47%) were positive by culture. An additional 5 (of 254) children were positive by the Platelia IgM test alone (specificity, 98%). When the PCR with Southern hybridization result was combined with the IgM-capture test result with the acute-phase sera, the sensitivity of rapid laboratory diagnosis increased to 95%. In conclusion, the IgM serology test was the single most valuable tool for the diagnosis of M. pneumoniae pneumonia in children of any age.     

Comparison of Seven Commercial Antigen and Antibody Enzyme-Linked Immunosorbent Assays for Detection of Acute Dengue Infection

Seven commercial assays were evaluated to determine their suitability for the diagnosis of acute dengue infection: (i) the Panbio dengue virus Pan-E NS1 early enzyme-linked immunosorbent assay (ELISA), second generation (Alere, Australia); (ii) the Panbio dengue virus IgM capture ELISA (Alere, Australia); (iii) the Panbio dengue virus IgG capture ELISA (Alere, Australia); (iv) the Standard Diagnostics dengue virus NS1 antigen ELISA (Standard Diagnostics, South Korea); (v) the Standard Diagnostics dengue virus IgM ELISA (Standard Diagnostics, South Korea); (vi) the Standard Diagnostics dengue virus IgG ELISA (Standard Diagnostics, South Korea); and (vii) the Platelia NS1 antigen ELISA (Bio-Rad, France). Samples from 239 Thai patients confirmed to be dengue virus positive and 98 Sri Lankan patients negative for dengue virus infection were tested. The sensitivities and specificities of the NS1 antigen ELISAs ranged from 45 to 57% and 93 to 100% and those of the IgM antibody ELISAs ranged from 85 to 89% and 88 to 100%, respectively. Combining the NS1 antigen and IgM antibody results from the Standard Diagnostics ELISAs gave the best compromise between sensitivity and specificity (87 and 96%, respectively), as well as providing the best sensitivity for patients presenting at different times after fever onset. The Panbio IgG capture ELISA correctly classified 67% of secondary dengue infection cases. This study provides strong evidence of the value of combining dengue virus antigen- and antibody-based test results in the ELISA format for the diagnosis of acute dengue infection.