Enzyme immunoassay for separate detection of anti-HCV antibodies to individual HCV antigens as a confirmatory assay in diagnostics of HCV infection

Dijagnostika hepatitisa C temelji se na određivanju protutijela anti-HCV probirnim imunoenzimskim testovima (EIA). Svaki reaktivni nalaz anti-HCV probirnog testa prema recentnim smjernicama zahtijeva daljnje određivanje HCV RNK. U slučajevima negativnog nalaza HCV RNK, reaktivni nalaz EIA anti-HCV treba potvrditi imunoblot testom (IB). Komercijalno su dostupni i EIA testovi koji imaju mogućnost određivanja specifičnih protutijela na pojedinačne antigene HCV koji daju rezultate usporedive sa standardnim potvrdnim IB testovima. Cilj ovog rada bio je prikazati EIA potvrdni test, EIA-Anti-HCV-Spectrum (DSI, Italija) i usporediti rezultate testiranja sa standardnim IB testom. Analizirali smo 50 nasumično odabranih seruma koji su imali reaktivni odgovor protutijela na HCV u probirnom EIA testu (Monolisa HCV Ag-Ab ULTRA, Bio-Rad, Francuska). Za isključenje lažno pozitivnih rezultata serumi su dalje testirani potvrdnim IB testom (Deciscan HCV PLUS, Bio-Rad, Francuska). Odabranih 50 uzoraka testirano je testom EIA-Anti-HCV-Spectrum za određivanje pojedinačnih protutijela anti-HCV na antigene jezgre te antigene NS3, NS4 i NS5. Od 50 seruma s reaktivnim odgovorom protutijela na HCV u probirnom testu u oba potvrdna testa pozitivno je bilo 40, negativno 1 i granično 3 uzorka. Nepodudarnih rezultata potvrdnih testova bilo je 6: svi su bili granično pozitivni u IB testu, a negativni u alternativnom potvrdnom testu EIA-Anti-HCV-Spectrum. Predstavili smo imunoenzimski potvrdni test za protutijela anti-HCV koji je pokazao dobru korelaciju sa standardnim imunoblot testom premda je potrebno napraviti evaluaciju na ve}em broju uzoraka za sigurnije zaključke.Diagnostics of hepatitis C is based on the determination of anti-HCV antibodies by screening enzyme immunoassays (EIA). Each reactive anti-HCV screening result, according to recent guidelines, requires further determination of HCV RNA. In cases of negative HCV RNA findings, reactive anti-HCV results should be confirmed by immunoblotting (IB). Alternative confirmatory EIA with the ability to determine the antibodies against individual HCV antigens, which are comparable with the results of immunoblot tests, are commercially available. The aim of this study was to present an EIA test, the EIA-Anti-HCV-Spectrum (DSI, Italy) and compare the test results with standard IB test. We analyzed 50 randomly selected serum samples with reactive screening EIA (Monolisa HCV Ag-Ab ULTRA, Bio-Rad, France) results. In order to exclude false positives, serum samples were further tested with confirmatory IB test (Deciscan HCV PLUS, Bio-Rad, France). A total of 50 selected samples were tested with EIA-Anti-HCV-Spectrum for separate detection of anti-HCV antibodies against core, NS3, NS4 and NS5 antigens. Out of the 50 primary reactive samples, anti-HCV antibodies were positive in both confirmatory tests in 40, negative in 1 and borderline in 3 samples. Discordant results of confirmatory tests were recorded in 6 samples: all borderline in IB test and negative in the alternative EIA-Anti-HCV-Spectrum test. We present an EIA confirmatory test for anti-HCV antibodies which showed a good correlation with the standard immunoblot test, although it is ecessary to perform the evaluation on a large number of samples for safer conclusions

ARE BORRELIA MIYAMOTOI AND BORRELIA MAYONII POSSIBLE PATHOGENS IN CROATIA?

Borrelia miyamotoi pripada grupi borelija koje uzrokuju povratnu vrućicu, a prenosi se istim krpeljima (Ixodidae) kao i Borrelia burgdorferi, Anaplasma phagocytophilum, Babesia spp. te virus krpeljnog meningoencefalitisa. B. miyamotoi dokazana je u malih glodavaca u Hrvatskoj. U Europi bolest nije česta, a prezentira se vrućicom praćenom zimicom, znojenjem, glavoboljom, umorom, mialgijama i artralgijama. U imunokompromitiranih bolesnika bolest može biti teška s meningoencefalitisom. Infekcija B. miyamotoi dijagnosticira se molekularnim testovima nakon isključivanja drugih krpeljima prenosivih bolesti. Serološke studije rađene su testovima temeljenima na rekombinantnom GlpQ proteinu koji nisu komercijalno dostupni. Nakon detaljne kliničke i mikrobiološke evaluacije moguća infekcija B miyamotoi u Hrvatskoj može se dijagnosticirati molekularnim metodama in house. Borrelia mayonii izolirana je samo u SAD-u iz krpelja Ixodes scapularis i u malog broja bolesnika. Nema dokaza za postojanje B. mayonii u Europi. Bolesnici s B. mayonii imali su akutnu febrilnu bolest s osipom i mogućim neurološkim simptomima. Dijagnoza infekcije B. mayonii postavljena je nalazom spirohetemije koja je dokazana kultivacijom i mikroskopiranjem preparata krvnog razmata te metodom PCR. Serološki testovi za detekciju B. mayonii nisu komercijalno dostupni. Preporuke za liječenje infekcije B. miyamotoi i B. mayonii temelje se na objavljenim kliničkim prikazima bolesnika te preporukama za liječenje lajmske borelioze. Nakon provedene terapije bolesnici su se oporavili bez komplikacija.Borrelia (B.) miyamotoi belongs to the group of relapsing fever borrelia and is transmitted by the same ticks (Ixodidae) as Borrelia burgdorferi, Anaplasma phagocytophilum, Babesia species and tick-borne fl aviviruses. B. miyamotoi was detected in small rodents in Croatia. In Europe, B. miyamotoi infections are not common, and mainly present as febrile illness with chills, sweats, headache, fatigue, myalgias and arthralgias. In immunocompromised patients, the disease may be severe with meningoencephalitis. B. miyamotoi diseases have been diagnosed with molecular methods after excluding other tick-borne diseases. Serological assays based on recombinant B. miyamotoi GlpQ protein used in published serological studies are not commercially available. After detailed clinical and microbiological evaluation, the possible B. miyamotoi infection in Croatia can be diagnosed with in-house methods. Borrelia (B.) mayonii has only been detected among few patients and Ixodes scapularis ticks in the United States so far. There is no evidence for B. mayonii in Europe. Patients with B. mayonii infection had febrile illness with rash and probable neurological symptoms. The diagnosis of B. mayonii infection was defi ned by detection of spirochetes in peripheral blood with culture isolation, microscopic smears, and polymerase chain reaction method. Serological tests for B. mayonii are not commercially available. Treatment recommendations for B. miyamotoi and B. mayonii infections are based on the published case reports and recommendations for treatment of Lyme disease. After antimicrobial treatment, all patients recovered without complications

Enzyme immunoassay for separate detection of anti-HCV antibodies to individual HCV antigens as a confirmatory assay in diagnostics of HCV infection

Dijagnostika hepatitisa C temelji se na određivanju protutijela anti-HCV probirnim imunoenzimskim testovima (EIA). Svaki reaktivni nalaz anti-HCV probirnog testa prema recentnim smjernicama zahtijeva daljnje određivanje HCV RNK. U slučajevima negativnog nalaza HCV RNK, reaktivni nalaz EIA anti-HCV treba potvrditi imunoblot testom (IB). Komercijalno su dostupni i EIA testovi koji imaju mogućnost određivanja specifičnih protutijela na pojedinačne antigene HCV koji daju rezultate usporedive sa standardnim potvrdnim IB testovima. Cilj ovog rada bio je prikazati EIA potvrdni test, EIA-Anti-HCV-Spectrum (DSI, Italija) i usporediti rezultate testiranja sa standardnim IB testom. Analizirali smo 50 nasumično odabranih seruma koji su imali reaktivni odgovor protutijela na HCV u probirnom EIA testu (Monolisa HCV Ag-Ab ULTRA, Bio-Rad, Francuska). Za isključenje lažno pozitivnih rezultata serumi su dalje testirani potvrdnim IB testom (Deciscan HCV PLUS, Bio-Rad, Francuska). Odabranih 50 uzoraka testirano je testom EIA-Anti-HCV-Spectrum za određivanje pojedinačnih protutijela anti-HCV na antigene jezgre te antigene NS3, NS4 i NS5. Od 50 seruma s reaktivnim odgovorom protutijela na HCV u probirnom testu u oba potvrdna testa pozitivno je bilo 40, negativno 1 i granično 3 uzorka. Nepodudarnih rezultata potvrdnih testova bilo je 6: svi su bili granično pozitivni u IB testu, a negativni u alternativnom potvrdnom testu EIA-Anti-HCV-Spectrum. Predstavili smo imunoenzimski potvrdni test za protutijela anti-HCV koji je pokazao dobru korelaciju sa standardnim imunoblot testom premda je potrebno napraviti evaluaciju na ve}em broju uzoraka za sigurnije zaključke.Diagnostics of hepatitis C is based on the determination of anti-HCV antibodies by screening enzyme immunoassays (EIA). Each reactive anti-HCV screening result, according to recent guidelines, requires further determination of HCV RNA. In cases of negative HCV RNA findings, reactive anti-HCV results should be confirmed by immunoblotting (IB). Alternative confirmatory EIA with the ability to determine the antibodies against individual HCV antigens, which are comparable with the results of immunoblot tests, are commercially available. The aim of this study was to present an EIA test, the EIA-Anti-HCV-Spectrum (DSI, Italy) and compare the test results with standard IB test. We analyzed 50 randomly selected serum samples with reactive screening EIA (Monolisa HCV Ag-Ab ULTRA, Bio-Rad, France) results. In order to exclude false positives, serum samples were further tested with confirmatory IB test (Deciscan HCV PLUS, Bio-Rad, France). A total of 50 selected samples were tested with EIA-Anti-HCV-Spectrum for separate detection of anti-HCV antibodies against core, NS3, NS4 and NS5 antigens. Out of the 50 primary reactive samples, anti-HCV antibodies were positive in both confirmatory tests in 40, negative in 1 and borderline in 3 samples. Discordant results of confirmatory tests were recorded in 6 samples: all borderline in IB test and negative in the alternative EIA-Anti-HCV-Spectrum test. We present an EIA confirmatory test for anti-HCV antibodies which showed a good correlation with the standard immunoblot test, although it is ecessary to perform the evaluation on a large number of samples for safer conclusions

ARE BORRELIA MIYAMOTOI AND BORRELIA MAYONII POSSIBLE PATHOGENS IN CROATIA?

Borrelia miyamotoi pripada grupi borelija koje uzrokuju povratnu vrućicu, a prenosi se istim krpeljima (Ixodidae) kao i Borrelia burgdorferi, Anaplasma phagocytophilum, Babesia spp. te virus krpeljnog meningoencefalitisa. B. miyamotoi dokazana je u malih glodavaca u Hrvatskoj. U Europi bolest nije česta, a prezentira se vrućicom praćenom zimicom, znojenjem, glavoboljom, umorom, mialgijama i artralgijama. U imunokompromitiranih bolesnika bolest može biti teška s meningoencefalitisom. Infekcija B. miyamotoi dijagnosticira se molekularnim testovima nakon isključivanja drugih krpeljima prenosivih bolesti. Serološke studije rađene su testovima temeljenima na rekombinantnom GlpQ proteinu koji nisu komercijalno dostupni. Nakon detaljne kliničke i mikrobiološke evaluacije moguća infekcija B miyamotoi u Hrvatskoj može se dijagnosticirati molekularnim metodama in house. Borrelia mayonii izolirana je samo u SAD-u iz krpelja Ixodes scapularis i u malog broja bolesnika. Nema dokaza za postojanje B. mayonii u Europi. Bolesnici s B. mayonii imali su akutnu febrilnu bolest s osipom i mogućim neurološkim simptomima. Dijagnoza infekcije B. mayonii postavljena je nalazom spirohetemije koja je dokazana kultivacijom i mikroskopiranjem preparata krvnog razmata te metodom PCR. Serološki testovi za detekciju B. mayonii nisu komercijalno dostupni. Preporuke za liječenje infekcije B. miyamotoi i B. mayonii temelje se na objavljenim kliničkim prikazima bolesnika te preporukama za liječenje lajmske borelioze. Nakon provedene terapije bolesnici su se oporavili bez komplikacija.Borrelia (B.) miyamotoi belongs to the group of relapsing fever borrelia and is transmitted by the same ticks (Ixodidae) as Borrelia burgdorferi, Anaplasma phagocytophilum, Babesia species and tick-borne fl aviviruses. B. miyamotoi was detected in small rodents in Croatia. In Europe, B. miyamotoi infections are not common, and mainly present as febrile illness with chills, sweats, headache, fatigue, myalgias and arthralgias. In immunocompromised patients, the disease may be severe with meningoencephalitis. B. miyamotoi diseases have been diagnosed with molecular methods after excluding other tick-borne diseases. Serological assays based on recombinant B. miyamotoi GlpQ protein used in published serological studies are not commercially available. After detailed clinical and microbiological evaluation, the possible B. miyamotoi infection in Croatia can be diagnosed with in-house methods. Borrelia (B.) mayonii has only been detected among few patients and Ixodes scapularis ticks in the United States so far. There is no evidence for B. mayonii in Europe. Patients with B. mayonii infection had febrile illness with rash and probable neurological symptoms. The diagnosis of B. mayonii infection was defi ned by detection of spirochetes in peripheral blood with culture isolation, microscopic smears, and polymerase chain reaction method. Serological tests for B. mayonii are not commercially available. Treatment recommendations for B. miyamotoi and B. mayonii infections are based on the published case reports and recommendations for treatment of Lyme disease. After antimicrobial treatment, all patients recovered without complications

Hepatitis E in patients with hepatic disorders and HIV-infected patients in Croatia: is one diagnostic method enough for hepatitis E diagnosis?

We assessed hepatitis E virus (HEV) seroprevalence in patients with hepatic disorders as well as in human immunodeficiency virus (HIV)-infected patients and emphasised the issue of possible non-specific anti-HEV seroresponse and need for combining diagnostic methods for hepatitis E diagnosis. Over a two-year period, from March 2011 to February 2013, we determined anti-HEV immunoglobulin M (IgM) and IgG by enzyme immunoassays (EIA; Mikrogen, Germany) in 504 hepatitis patients negative for acute viral hepatitis A-C. Furthermore, 88 samples from randomly selected consecutive HIV-infected patients were also analysed. All EIA reactive samples were additionally tested by line immunoblot assays (LIA; Mikrogen, Germany). HEV nested reverse transcription polymerase chain reaction (RT-PCR) was carried out in 14 anti-HEV IgM LIA-positive patients. Anti-HEV IgM or IgG were detected in 16.9 % of patients by EIA and confirmed by LIA in 10.7 % [95 % confidence interval (CI) 8.3-13.7 %] of hepatitis patients. HEV RNA was detected in five patients. The agreement between EIA and LIA assessed by Cohen's kappa was 0.47 (95 % CI 0.55-0.75) for IgM and 0.83 (95 % CI 0.78-0.93) for IgG. Anti-HEV IgM and IgG seroprevalence in HIV-infected patients was 1.1 %, respectively. Our findings show a rather high HEV seroprevalence in patients with elevated liver enzymes in comparison to HIV-infected patients. Discordant findings by different methods stress the need to combine complementary methods and use a two-tier approach with prudent interpretation of reactive serological results for hepatitis E diagnosis

Slower Waning of Anti-SARS-CoV-2 IgG Levels Six Months after the Booster Dose Compared to Primary Vaccination

Anti-SARS-CoV-2 IgG titer decreases rapidly after primovaccination, leading to a mandatory booster vaccination. We analysed anti-SARS-CoV-2 Spike RBD IgG levels (positive ≥ 50 AU/mL) in 405 healthcare workers (3010 sera) who received a booster dose (BD) 9 months after two-dose BNT162b2 primovaccination. Median antibody titer at the time of BD (582.6 AU/mL) was 1.7-fold and 16.4-fold lower than the peak titer after the first (961.5 AU/mL) and the second vaccine dose (SVD) (10,232.6 AU/mL), respectively. One month after vaccination, IgG titer increased 40.6-fold after BD compared with a 10.8-fold increase after primovaccination. Three months after vaccination, post-booster antibodies decreased significantly slower (2.2-fold) than after primovaccination (3.3-fold). At six months, antibodies decreased slower after BD (4.5-fold; median 5556.0 AU/mL) than after primovaccination (9.6-fold; median 1038.5 AU/mL). Antibody titers before and one month after BD correlated weakly (r = 0.30) compared with a strong correlation (r = 0.65) between the corresponding post-primovaccination titers. Pre-vaccination COVID-19 had no effect on IgG levels after BD compared with a positive effect after primovaccination. Despite high post-booster IgG levels, 22.5% of participants contracted mild COVID-19. The trend of IgG decline indicates the need for further revaccination, but the vaccine type should be defined according to viral mutations

Decline of Anti-SARS-CoV-2 IgG Antibody Levels 6 Months after Complete BNT162b2 Vaccination in Healthcare Workers to Levels Observed Following the First Vaccine Dose

Research on post-vaccination antibody dynamics has become pivotal in estimating COVID-19 vaccine efficacy. We studied anti-SARS-CoV-2 Spike RBD IgG levels in 587 healthcare workers (2038 sera) who completed BNT162b2 vaccination. Average antibody titer 3 weeks after the first dose in COVID-19-naïve participants (median 873.5 AU/mL) was 18-fold higher than the test threshold, with a significant increase 1 month (median 9927.2 AU/mL) and an exponential decrease 3 (median 2976.7 AU/mL) and 6 (median 966.0 AU/mL) months after complete vaccination. Participants with a history of COVID-19 prior to vaccination showed significantly higher antibody levels, particularly after the first dose (median 14,280.2 AU/mL), with a slight decline 1 month (median 12,700.0 AU/mL) and an exponential decline in antibody titers 3 (median 4831.0 AU/mL) and 6 (median 1465.2 AU/mL) months after vaccination. Antibody levels of COVID-19-naïve subjects after the first dose were moderately correlated with age (r = −0.4). Multivariate analysis showed a strong independent correlation between IgG levels 6 months after vaccination and both IgG titers after the first dose and 1 month after vaccination (R2 = 0.709). Regardless of pre-vaccination COVID-19 history, IgG levels 6 months after vaccination were comparable to antibody levels reached by COVID-19-naïve participants after the first vaccine dose

Antibody response and the clinical presentation of patients with COVID-19 in Croatia: the importance of a two-step testing approach

According to anti-SARS-CoV-2 seroresponse in patients with COVID-19 from Croatia, we emphasised the issue of different serological tests and need for combining diagnostic methods for COVID-19 diagnosis. Anti-SARS-CoV-2 IgA and IgG ELISA and IgM/IgG immunochromatographic assay (ICA) were used for testing 60 sera from 21 patients (6 with severe, 10 moderate, and 5 with mild disease). The main clinical, demographic, and haemato-biochemical data were analysed. The most common symptoms were cough (95.2%), fever (90.5%), and fatigue and shortness of breath (42.9%). Pulmonary opacities showed 76.2% of patients. Within the first 7 days of illness, seropositivity for ELISA IgA and IgG was 42.9% and 7.1%, and for ICA IgM and IgG 25% and 10.7%, respectively. From day 8 after onset, ELISA IgA and IgG seropositivity was 90.6% and 68.8%, and for ICA IgM and IgG 84.4% and 75%, respectively. In general, sensitivity for ELISA IgA and IgG was 68.3% and 40%, and for ICA IgM and IgG 56.7% and 45.0%, respectively. The anti-SARS-CoV-2 antibody distributions by each method were statistically different (ICA IgM vs. IgG, p = 0.016; ELISA IgG vs. IgA, p < 0.001). Antibody response in COVID-19 varies and depends on the time the serum is taken, on the severity of disease, and on the type of test used. IgM and IgA antibodies as early-stage disease markers are comparable, although they cannot replace each other. Simultaneous IgM/IgG/IgA anti-SARS-CoV-2 antibody testing followed by the confirmation of positive findings with another test in a two-tier testing is recommended