Low-avidity Antibodies Research Articles

Modified phosphatidylserine-dependent antithrombin ELISA enables identification of patients negative for other antiphospholipid antibodies and also detects low avidity antibodies

Two approaches for detecting anti-prothrombin antibodies have been described. The first detects antibodies against prothrombin alone and the second, phos-phatidylserine-dependent antiprothrombin antibodies. The latter more often correlate with clinical manifestations of antiphospholipid syndrome and with lupus anticoagulant activity. In order to increase the capacity of antibody binding, we modified the previously described phosphatidylser-ine-dependent antiprothrombin ELISA and determined their avidity. We examined 203 patients with systemic autoimmune diseases and 222 blood donors. Our modification resulted in a greater intensity of antibody binding to prothrombin on phosphatidylserine-coated plate surfaces compared to the previously described method. By changing ELISA conditions, we were able to detect with one assay the two, presumably different, populations of antiprothrombin antibodies. Diagnostic specificities of both ELISAs for antiphospholipid syndrome were similar (92.5% vs. 93.1%), while the sensitivity of the modified phosphatidylserine-dependent antiprothrombin ELISA was significantly higher than the anti-prothrombin alone ELISA (59% vs. 25%). Low avidity antiprothrombin antibodies were only detected in the modified phosphatidylserine-dependent antiprothrombin ELISA. Four percent of patients with positive phosphatidylserine-dependent antiprothrombin antibodies, showing clinical manifestations of antiphospholipid syndrome, were negative for all other antiphospholipid antibodies. The risk for antiphospholipid syndrome increased with the number of antiphospholipid antibody positivity. We conclude that antibodies detected with a modified phosphatidylserine-dependent antiprothrombin ELISA could improve the diagnosis of antiphospholipid syndrome by offering additional information on the risk for thrombosis, especially in patients negative for other antiphospholipid antibodies.

Low‐avidity anti‐HPA‐1a alloantibodies are capable of antigen‐positive platelet destruction in the NOD/SCID mouse model of alloimmune thrombocytopenia

Neonatal alloimmune thrombocytopenia (NAIT) is mostly caused by maternal antibodies against human platelet antigen 1a (HPA-1a) expressed on glycoprotein (GP) IIb/IIIa. Accumulated evidence indicated that anti-HPA-1a could be overlooked by standard methods due to low avidity. Low-avidity HPA-1a antibodies were shown to be detectable by surface plasmon resonance (SPR). We sought to investigate the frequency and in vivo relevance of low-avidity anti-HPA-1a. A retrospective cohort consisting of 82 HPA-1bb mothers of HPA-1ab newborns with thrombocytopenia was analyzed using standard serologic methods. Maternal immunoglobulin (Ig)G fractions were investigated for low-avidity antibodies in SPR using purified GPIIb/IIIa (HPA-1a or -1b). The capability of HPA-1a antibodies to clear platelets (PLTs) in vivo was analyzed using the NOD/SCID mouse model of alloimmune thrombocytopenia. HPA antibodies were detectable in sera from 68 of 82 (83%) mothers using standard serologic methods and undetectable in 14 of 82 sera. In SPR, IgG fractions of sera reacting positive in monoclonal antibody immobilization of PLT antigen (MAIPA) assay showed specific binding to an HPA-1a flow cell (mean, 87 ± 21 resonance units [RU]). When MAIPA-negative sera were tested in SPR, binding with low avidity was observed in 7 of 14 to HPA-1a (mean, 31 ± 5 RU), but not to HPA-1b flow cell (mean, 5 ± 2 RU). In vivo, low-avidity antibodies were capable of clearing HPA-1ab PLTs but not HPA-1bb PLTs in a NOD/SCID mouse model. Elimination kinetics were slower than observed with MAIPA-positive antibodies. Low-avidity HPA-1a antibodies are present in a significant number of NAIT cases and, although they can escape detection by standard serology, they harbor the capability of PLT destruction in vivo.

Identification of Potential Serodiagnostic and Subunit Vaccine Antigens by Antibody Profiling of Toxoplasmosis Cases in Turkey

Toxoplasmosis, caused by infection of the protozoan parasite Toxoplasma gondii, is associated with mild disease in healthy individuals, whereas individuals with depressed immunity may develop encephalitis, neurologic disorders, and other organ diseases. Women who develop acute toxoplasmosis during pregnancy are at risk of transmitting the infection to the fetus, which may lead to fetal damage. A diagnosis is usually confirmed by measuring IgG, or IgM where it is important to determine the onset of infection. A negative IgM result essentially excludes acute infection, whereas a positive IgM test is largely uninterpretable because IgM can persist for up to 18 months after infection. To identify antigens for improved diagnosis of acute infection, we probed protein microarrays displaying the polypeptide products of 1357 Toxoplasma exons with well-characterized sera from Turkey. The sera were classified according to conventional assays into (1) seronegative individuals with no history of T. gondii infection; (2) acute infections defined by clinical symptoms, high IgM titers, and low avidity IgG; (3) chronic/convalescent cases with high avidity IgG but persisting IgM; (iv) true chronic infections, defined by high avidity IgG and no IgM. We have identified 38 IgG target antigens and 108 IgM target antigens that can discriminate infected patients from healthy controls, one or more of which could form the basis of a 'tier-1' test to determine current or previous exposure. Of these, three IgG antigens and five IgM antigens have the potential to discriminate chronic/IgM persisting or true chronics from recent acutely infected patients (a 'tier-2' test). Our analysis of the antigens revealed several enriched features relative to the whole proteome, which include transmembrane domains, signal peptides, or predicted localization at the outer membrane. This is the first protein microarray survey of the antibody response to T. gondii, and will help in the development of improved serodiagnostics and vaccines.

The Relationship between Cytomegalovirus Infection and Guillain–Barré Syndrome

The association between cytomegalovi- rus (CMV) and Guillain-Barresyn- drome (GBS) was first noted in 1967 (1) and was soon endorsed by other reports and case series. The largest previous case series showed that CMV infection was associated with GBS in 8% of 229 pa- tients from a European and North American trial (2). In this issue of Clinical Infectious Diseases, Orlikowski et al (2) confirmed this finding in a larger population of people with GBS by using the resources of the French reference centre in Garches, Paris, which has been a major source of useful research into the disease. Of 506 patients with GBS, 63 (12.5%) had serological evidence of primary CMV infection. It is difficult to distinguish between primary and reactivation of infection, an important problem, because 50% of the nonimmunosuppressed people in de- veloped countries have serological evi- dence of exposure to CMV. The serological methods used by Orlikowski and colleagues incorporated a test for high immunoglobulin (Ig) G avidity to exclude positive results due to re- activation. Low-avidity IgG antibody is found in primary infection before affinity maturation has occurred. The serological results were supported by the finding of CMV DNA in the plasma by poly- merase chain reaction (PCR) in 36 (62%) of 58 subjects who had positive serological test results. The sensitivity of CMV PCR nears 100% for acute in- fection, but it would be expected to be reduced in the late infectious phase, when GBS occurs because virus is being cleared. Unfortunately no conva- lescent-phase samples were available for analysis because of the intervening treat- ment and quantitative PCR was not performed for enough patients to publish the result. The authors used contemporary French epidemiological data to calculate, for the first time, that the risk of GBS following CMV is in the order of 0.6-2.2 cases per 1000 persons. There were no control subjects in the Orlikowski et al (2) study, but 2 earlier studies had shown that the frequency of recent CMV infection preceding GBS significantly exceeded that of control subjects: in the United Kingdom, 11% of 99 case patients and only 1% of control patients had evi- dence of recent CMV infection (3); in the Netherlamds, 13% of 154 case patients and only 2% of control subjects (4) had evidence of recent CMV infection. The study by Orlikowski et al (2)

West Nile virus neuroinvasive disease presenting with acute flaccid paralysis and bilateral sensorineural hearing loss

West Nile virus (WNV), is an emerging virus [1]. Most human infections are subclinical or manifest as a mild febrile illness, while fewer than 1% develop a neuroinvasive disease (WNND), including meningitis, encephalitis, and/or an acute flaccid paralysis (AFP) [2–4]. We describe a case of sensorineural hearing loss (SNHL) secondary to WNV infection. A 55 year-old previously healthy and normoacousic man, was admitted (in September 2010) for bilateral hearing loss and leg weakness. He complained, about 2 weeks before, of malaise, fever, generalized fatigue, and diffuse myalgias, initially attributed to a flu-like syndrome. After a few days he noted a mild weakness in both of his legs evolving over a week into a flaccid paraparesis. At the same time, he also developed a sudden bilateral hearing loss. He was afebrile, with stable vital signs. Routine and immunological laboratory data were within the normal range. Neurologic examination was remarkable for a flaccid areflexic paraparesis, weakness being more pronounced in the right leg and proximally (3/5 strength in the right leg, 4/5 strength in the left leg), with flexor plantar responses. No cranial nerve involvement, vestibular, cerebellar, brainstem or meningeal signs were noted. MRI of the brain and whole spine was normal without meningeal, parenchymal or ventral nerve root enhancement. Audiological tests, including the Weber and Rinne tuning fork tests, pure tone and speech audiometry, tympanic admittance, auditory evoked potentials (ABRs) and a screening test for acoustic emissions (OAEs), demonstrated a bilateral, down-sloping SNHL—mild on the left side, severe on the right—with normal acoustic admittance, and impaired OAEs, which were absent in the right ear. Weber test was lateralized to the left side, and the Rinne test was not significant. ABR with click at maximum output level (125 dB SPL) was not evocable by stimulating the right ear and normal for the left ear after latencies correction for the hearing threshold elevation. On the whole, the data were compatible with a bilateral asymmetric cochlear damage. Electrodiagnostic studies showed normal sensory and motor nerve conductions, and moderate decrease of compound motor action potential amplitudes. Late responses were normal. Concentric needle electromyography showed active denervation with a neurogenic recruitment pattern in all four limbs, with a clear predominance in legs. The findings fulfilled the diagnostic criteria for WNV AFP [4, 5]. Neurophysiological examination of cranial nerves and brainstem reflexes studies gave normal results. Since the symptoms began during a work-related stay in Romania where a WNV infection outbreak was occurring [6, 7], we considered the above infection in the differential diagnosis. Serum IgM and low-avidity IgG WNV-specific antibodies were detected (titre:1:1,600; 1:51,200, respectively). The presence of IgMand low avidity IgG WNV-specific I. Casetta (&) E. Cesnik Section of Neurology, Department of Medical and Neurological Sciences of Communication and Behaviour, University of Ferrara, Corso della Giovecca 203, 44121 Ferrara, Italy e-mail: cti@unife.it

Modified phosphatidylserine-dependent antiprothrombin ELISA enables identification of patients negative for other antiphospholipid antibodies and also detects low avidity antibodies

Article Modified phosphatidylserine-dependent antiprothrombin ELISA enables identification of patients negative for other antiphospholipid antibodies and also detects low avidity antibodies was published on September 1, 2011 in the journal Clinical Chemistry and Laboratory Medicine (CCLM) (volume 49, issue 9).

How Innate Immune Mechanisms Contribute to Antibody-Enhanced Viral Infections

Preexisting antibodies may enhance viral infections. In dengue, nonneutralizing antibodies raised by natural infection with one of four dengue viruses (DENVs) may enhance infection with a different virus by a process we term "intrinsic antibody-dependent enhancement" (iADE). In addition, nonprotective antibodies raised by formalin-inactivated respiratory syncytial virus (RSV) and measles virus vaccines have led to enhanced disease during breakthrough infections. Infections under iADE conditions not only facilitate the process of viral entry into monocytes and macrophages but also modify innate and adaptive intracellular antiviral mechanisms, suppressing type 1 interferon (IFN) production and resulting in enhanced DENV replication. The suppression observed in vitro has been documented in patients with severe (dengue hemorrhagic fever [DHF]) but not in patient with mild (dengue fever [DF]) secondary dengue virus infections. Important veterinary viral infections also may exhibit iADE. It is thought that use of formalin deconforms viral epitopes of RSV, resulting in poor Toll-like receptor (TLR) stimulation; suboptimal maturation of dendritic cells with reduced production of activation factors CD40, CD80, and CD86; decreased germinal center formation in lymph nodes; and the production of nonprotective antibodies. These antibodies fail to neutralize RSV, allowing replication with secondary stimulation of RSV-primed Th2 cells producing more low-avidity antibody, resulting in immune complexes deposited into affected tissue. However, when formalin-inactivated RSV was administered with a TLR agonist to mice, they were protected against wild-type virus challenge. Safe and effective vaccines against RSV/measles virus and dengue virus may benefit from a better understanding of how innate immune responses can promote production of protective antibodies.

Use of Dense Granule Antigen GRA6 in an Immunoglobulin G Avidity Test To Exclude AcuteToxoplasma gondiiInfection during Pregnancy

The usefulness of a specific immunoglobulin G (IgG) avidity enzyme-linked immunosorbent assay (ELISA) based on recombinant GRA6 antigen for distinguishing between acute and chronic Toxoplasma infection was investigated. Two sets of serum samples obtained from pregnant women with acute, chronic, or no Toxoplasma infection collected in France and Iran were used. Among the French subjects, 19 of 20 (95%) women who experienced seroconversion during the past 4 months before sampling displayed low-avidity IgG antibodies against GRA6, while all 17 (100%) women with chronic infection had high-avidity antibodies. When the Euroimmun IgG avidity ELISA was used, 15 of 19 (78.9%) recently infected women had low-avidity antibodies, and 20 of 22 (90.9%) women with chronic infection displayed high-avidity antibodies. The results suggested better performance of the GRA6 avidity ELISA than the Euroimmun avidity ELISA for exclusion of a recent infection occurring less than 4 months previously. Similarly, all 35 Iranian women with acute Toxoplasma infection had low-avidity antibodies against GRA6, whereas all 34 women with chronic infection displayed IgG antibodies of high avidity, indicating the value of GRA6 avidity testing for ruling out a recent infection. Avidity tests based on lysed whole-cell Toxoplasma gondii antigen are currently used to exclude recently acquired infections; however, the use of recombinant antigen(s) might improve the diagnostic performance of avidity tests and facilitate the development of more standardized assays.

Serological screening for antenatal toxoplasma infection in India

Purpose: Detection of infection caused by Toxoplasma gondii during pregnancy to prevent congenital infection. Materials and Methods: This study was carried out from January 2005 to 2006 in 300 pregnant women. Antitoxoplasma IgG, IgM, IgA antibody and IgG avidity were assessed using ELISA. Atleast two samples were taken atleast 3 weeks apart preferably one in each trimester. Result: Of these 300 pregnant women, anti toxoplasma IgG antibodies were detected in 46 (15.33%) cases, while 9 (3%) had positive anti toxoplasma IgM with IgA and /low IgG avidity antibodies suggestive of acute infection during or just before pregnancy. Conclusion: The results indicate that about 85% of female population of Chandigarh is susceptible to toxoplasma infection and thus should be specifically educated about prevention of this infection during pregnancy

High diagnostic efficiency of IgM-ELISA with the use of multiple antigen peptides (MAP1) from T. gondii ESA (SAG-1, GRA-1 and GRA-7), in acute toxoplasmosis

The main serological marker for the diagnosis of recent toxoplasmosis is the specific IgM antibody, along with IgG antibodies of low avidity. However, in some patients these antibodies may persist long after the acute/recent phase, contributing to misdiagnosis in suspected cases of toxoplasmosis. In the present study, the diagnostic efficiency of ELISA was evaluated, with the use of peptides derived from T. gondii ESA antigens, named SAG-1, GRA-1 and GRA-7. In the assay referred to, we studied each of these peptides individually, as well as in four different combinations, as Multiple Antigen Peptides (MAP), aiming to establish a reliable profile for the acute/recent toxoplasmosis with only one patient serum sample. The diagnostic performance of the assay using MAP1, with the combination of SAG-1, GRA-1 and GRA-7 peptides, demonstrated better discrimination of the acute/recent phase from non acute/recent phase of toxoplasmosis. Our results show that IgM antibodies to MAP1 may be useful as a serological marker, enhancing the diagnostic efficiency of the assay for acute/recent phase of toxoplasmosis.

Toxoplasma gondii: Usefulness of ROP 1 Recombinant Antigen in an Immunoglobulin G Avidity Assay for Diagnosis of Acute Toxoplasmosis in Humans

The results present in this study suggest that the Toxoplasma gondii recombinant ROP1 antigen in an IgG avidity test can be useful for detection of acute stage of infection. Specific antibodies of low avidity were detected in most of the sera from individuals with acute toxoplasmosis, while the absence or specific antibodies of high avidity were detected in sera from patients with chronic infection.

Development of Two Avidity-Based Assays to Detect Recent HIV Type 1 Seroconversion Using a Multisubtype gp41 Recombinant Protein

Current laboratory methods to detect recent HIV-1 infection for the estimation of incidence have various limitations, including varying performance in different subtypes or populations. Therefore, new methods are needed to detect recent infections with increased specificity. We developed a recombinant protein, rIDR-M, that covered divergent sequences from the immunodominant region (IDR) of gp41 from all major subtypes and recombinants of HIV-1 group M and expressed in Escherichia coli. The rIDR-M protein was highly reactive with HIV antibodies in sera from different subtypes and equivalently detected antibodies to divergent subtypes B and AE from Thailand, in contrast to individual gp41 peptides derived from respective subtypes, suggesting that it can be used for incidence assays. The protein was used in two different assay formats to measure antibody avidity: (1) a two-well avidity index assay (AI-EIA) and (2) a new one-well limiting antigen avidity assay (LAg-avidity EIA), both with a pH 3.0 buffer to dissociate low-avidity antibodies present during early infection. Limiting the amount of antigen allowed detection of recent HIV-1 infection, with or without dissociation buffer, but the detection was most efficient when the pH 3.0 dissociation buffer was included. When a well-characterized 41-member seroincidence panel (20 recent and 21 long-term) was used, both the two-well AI-EIA and one-well LAg-avidity EIA efficiently distinguished recent and long-term infections. The new avidity-based assays using rIDR-M antigen may improve the accuracy of detecting recent HIV-1 infection and allow a better estimation of incidence in diverse HIV-1 subtypes.

Poster Board III-457 Surface Plasmon Resonance (SPR) Analysis Detects Clinically Significant Low Avidity HPA-1a Antibodies Not Identified by Conventional Serology.

Abstract 3520Poster Board III-457Neonatal alloimmune thrombocytopenia (NATP) is caused by passively transmitted maternal antibodies specific for fetal platelet antigens inherited from the father. The diagnosis is made by demonstrating a maternal antibody specific for a paternal platelet alloantigen inherited by the affected infant. It is not unusual for mothers of suspected NATP cases to be antibody-negative using standard serologic methods. Assays routinely used to detect alloantibodies (flow cytometry, glycoprotein-specific solid phase assays) require repeated washing of the target cell or antigen to remove unbound antibody, followed by detection of bound antibody with a labeled secondary probe. Low avidity antibodies could dissociate from their targets during these washing steps and might, therefore, not be detected; however, the extent to which such antibodies cause human disease is controversial. Surface plasmon resonance (SPR) directly measures the accretion of ligand on an immobilized receptor in real time and could therefore lend itself to detection of low avidity, clinically significant human antibodies. In fact, Socher et al recently described use of SPR to detect maternal antibodies specific for the HPA-1a (PlA1) platelet antigen in two NATP cases not diagnosed by standard serology (Transf 49:943, 2009).We have also studied the utility of SPR for detection of clinically important human antibodies. HPA-1a/a-positive and -negative GPIIb/IIIa was isolated from platelets and was immobilized in the lanes of a CM5 Biacore chip. IgG used for testing was doubly purified from serum to remove vitronectin and other proteins that might react with the target integrin through RGD binding sites, thereby masking antibody-specific signals. The range for binding of IgG from normal subjects to these targets was defined with IgG from 30 healthy donors.In preliminary studies, we found that SPR analysis is about 5 times as sensitive as standard serology for detection of known HPA-1a-specific antibodies. We then studied maternal sera from 45 cases of suspected NATP in which the mother was HPA-1a-negative but no maternal antibody was detected. Seven of these sera were found to contain IgG that recognizes HPA-1a-positive, but not HPA-1a-negative GPIIb/IIIa. A comparison of the reaction kinetics of these antibodies with those of “conventional” HPA-1a-specific antibodies showed that the former have a significantly higher off rate, as would be expected of low avidity antibodies. We conclude that low avidity antibodies specific for HPA-1a are capable of causing NATP and may not be rare and that SPR may provide a unique tool for their detection. The extent to which SPR can be useful in studying other antibody-mediated human disorders deserves exploration. Disclosures:No relevant conflicts of interest to declare.

Spiramycin Treatment of Toxoplasma gondii Infection in Pregnant Women Impairs the Production and the Avidity Maturation of T. gondii -Specific Immunoglobulin G Antibodies

The aim of the study was to evaluate the influence of treatment with spiramycin on the increase of immunoglobulin G (IgG) titers and IgG avidity indexes (AI) in pregnant women with seroconversion from the beginning of therapy until delivery and after delivery. This group was compared with adult patients with recently acquired untreated toxoplasmosis. One hundred four samples from 32 pregnant women with seroconversion for toxoplasmosis and/or very low IgG AI were followed from the beginning of therapy with spiramycin until delivery. Twenty-nine women were further followed some months after delivery and interruption of therapy. Thirty-eight samples from 16 untreated, nonpregnant patients were evaluated as the control group. The Toxoplasma gondii-specific IgG antibody and the T. gondii-specific IgG AI were significantly delayed in pregnant women receiving therapy compared to nonpregnant, untreated controls, and the findings were consistent with the results of assays from two different manufacturers. The T. gondii-specific IgG AI increased in pregnant women after they gave birth. Avidity maturation is delayed during pregnancy and treatment, and low-avidity antibodies in pregnant women within 3 to 4 months cannot be taken as a sign of infection.

Low‐avidity HPA‐1a alloantibodies in severe neonatal alloimmune thrombocytopenia are detectable with surface plasmon resonance technology

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is mostly caused by maternal alloantibodies directed against the human platelet alloantigen (HPA)-1a. Currently, the serologic diagnosis of FNAIT is based on the characterization of the HPA alloantibodies in monoclonal antibody-based antigen-capture assays (e.g., MAIPA assay). Accumulated current evidence indicated that such assays may overlook some HPA-1a antibodies. This study employed surface plasmon resonance (SPR) technology using immunoaffinity-purified glycoprotein IIb/IIIa isoforms immobilized on biosensor chips to study the binding kinetics of HPA-1a alloantibodies from different FNAIT cases in real time. Analysis of HPA-1a alloantibodies from FNAIT cases (n = 9) in SPR showed a moderate relative response (22.2-69.7 resonance units [RU]) and slow antibody dissociation. After the dissociation phase, varying amounts of bound antibodies (41%-79%) remained on the chip. In contrast in HPA-1a alloantibodies from a patient suffering from posttransfusion purpura, a high relative response (approximately 490 RU) was observed at the end of the association phase and no dissociation of antibody binding was detectable. Of particular relevance, by the use of this SPR technique, HPA-1a alloantibodies were detected in two severe FNAIT cases that had determined as false negative by MAIPA assay. In SPR, these HPA-1a alloantibodies showed low-avidity nature characterized by gradual dissociation of antibody during the association phase and complete detachment of antibody binding after the dissociation phase. This high "off-rate" character of low-avidity HPA-1a alloantibodies indicates that such antibody binding is easily detachable by the extensive washing procedure of the MAIPA. Our results demonstrated that the SPR method can facilitate the diagnosis of clinically relevant low-avidity HPA-1a antibodies.

Lack of antibody affinity maturation due to poor Toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease

Respiratory syncytial virus (RSV) is a leading cause of hospitalization in infants. A formalin-inactivated RSV vaccine was used to immunize children in 1966 and elicited non-protective, pathogenic antibody. Two immunized infants died and 80% were hospitalized after subsequent RSV exposure. No vaccine was licensed since.A widely accepted hypothesis attributed vaccine failure to formalin disruption of protective antigens. Instead, we show that lack of protection was not due to alterations caused by formalin, but to low antibody avidity for protective epitopes. Lack of antibody affinity maturation followed poor Toll-like receptor stimulation. This study explains why the inactivated RSV vaccine failed to protect and consequently led to severe disease, hampering vaccine development for forty-two years. Also, it suggests that inactivated RSV vaccines may be rendered safe and effective by inclusion of TLR-agonists in their formulation. In addition, it identifies affinity maturation as a critical factor for the safe immunization of infants.

Detection of Acute Toxoplasma gondii infection in early pregnancy by IgG-avidity and PCR analysis

Acute Toxoplasma gondii infection in early pregnancy carries the risk of transmitting the infection to the fetus with serious sequelae. However, serological testing for IgG/IgM anti-Toxoplasma antibodies may fail to differentiate between a recent and past infection. Two hundred and twenty-four Kuwaiti women in their first trimester were screened for IgG/IgM antibodies by the Vitek Immuno Diagnostic Assay System (VIDAS) and VIDAS IgG-avidity tests. On serological screening, 119 (53.1 %) women were positive for IgG antibodies and 31 (13.8 %) for IgM antibodies. Nine of the IgM-positive and 7 IgM-negative women had low-avidity antibodies. However, the IgG-avidity test detected low-avidity antibodies only in 9 (29 %) of the 31 IgM-positive women, suggesting a recent infection; 19 (61.3 %) women had high-avidity antibodies, indicating that the infection was acquired in the distant past. Based on IgM serology alone, at least 31 IgM-positive women may have been wrongly labelled as having acute Toxoplasma infection thus warranting appropriate therapeutic intervention. All the 19 IgM-positive women with high-avidity antibodies were confirmed negative for Toxoplasma DNA on PCR analysis. Compared with PCR analysis, the VIDAS avidity test was a helpful tool for the diagnosis of recent Toxoplasma infection in IgM-negative women with low-avidity antibodies and IgM-positive women with high-avidity antibodies; the specificity was >85 -100 %. It is concluded that the VIDAS avidity test when used in combination with VIDAS IgG/IgM tests is a valuable assay for the exclusion of ongoing or recently acquired T. gondii infection in pregnant women in their first trimester and that it decreases significantly the necessity for follow-up testing and unnecessary therapeutic intervention.

Pattern of humoral immune response to Plasmodium falciparum blood stages in individuals presenting different clinical expressions of malaria

BackgroundThe development of protective immunity against malaria is slow and to be maintained, it requires exposure to multiple antigenic variants of malaria parasites and age-associated maturation of the immune system. Evidence that the protective immunity is associated with different classes and subclasses of antibodies reveals the importance of considering the quality of the response. In this study, we have evaluated the humoral immune response against Plasmodium falciparum blood stages of individuals naturally exposed to malaria who live in endemic areas of Brazil in order to assess the prevalence of different specific isotypes and their association with different malaria clinical expressions.MethodsDifferent isotypes against P. falciparum blood stages, IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA, were determined by ELISA. The results were based on the analysis of different clinical expressions of malaria (complicated, uncomplicated and asymptomatic) and factors related to prior malaria exposure such as age and the number of previous clinical malaria attacks. The occurrence of the H131 polymorphism of the FcγIIA receptor was also investigated in part of the studied population.ResultsThe highest levels of IgG, IgG1, IgG2 and IgG3 antibodies were observed in individuals with asymptomatic and uncomplicated malaria, while highest levels of IgG4, IgE and IgM antibodies were predominant among individuals with complicated malaria. Individuals reporting more than five previous clinical malaria attacks presented a predominance of IgG1, IgG2 and IgG3 antibodies, while IgM, IgA and IgE antibodies predominated among individuals reporting five or less previous clinical malaria attacks. Among individuals with uncomplicated and asymptomatic malaria, there was a predominance of high-avidity IgG, IgG1, IgG2 antibodies and low-avidity IgG3 antibodies. The H131 polymorphism was found in 44.4% of the individuals, and the highest IgG2 levels were observed among asymptomatic individuals with this allele, suggesting the protective role of IgG2 in this population.ConclusionTogether, the results suggest a differential regulation in the anti-P. falciparum antibody pattern in different clinical expressions of malaria and showed that even in unstable transmission areas, protective immunity against malaria can be observed, when the appropriated antibodies are produced.

Sin Nombre Virus Shedding Patterns in Naturally Infected Deer Mice (Peromyscus maniculatus) in Relation to Duration of Infection

A 2-year capture-mark-recapture study was conducted in southern Manitoba, Canada, to test for an association between the duration of Sin Nombre virus (SNV) infection in deer mice (Peromyscus maniculatus) and virus shedding. Hantavirus-specific IgG antibodies were detected in 22.2% of captured deer mice, and recently infected deer mice were identified based on the detection of low-avidity IgG antibodies. SNV RNA was detected in blood samples from the majority of seropositive deer mice with no significant difference in the association of SNV RNA between the low- and high-avidity groups (57.8% and 52.1%, respectively). A small subset of seropositive mice (11.6%) had detectable SNV RNA in oropharyngeal fluids (OPF) or urine. A greater proportion of deer mice with low-avidity antibodies had SNV RNA in OPF or urine compared with rodents with high-avidity antibodies (21% versus 6.8%, respectively). This is the first study of naturally infected deer mice to provide evidence that recently infected mice are more likely to shed SNV and thus might represent a greater risk of human infection.

Relationship of IgG avidity index and IgM levels for the differential diagnosis of primary from recurrent cytomegalovirus infections.

Since the incidence of symptomatic congenital cytomegalovirus infection is low (~0.05%) and risk factors are not well defined, it is difficult to develop strategies for prevention. The aim of this study was to recognise the utility of specific Immunoglobulin G avidity analysis for distinguishing primary infection from past/recurrent infection. Sera from 50 women with cytomegalovirus specific Immunoglobulin M antibodies without proven seroconversion and infants born to these women were tested for presence of Immunoglobulin M antibodies by commercial enzyme immunoassay. For cytomegalovirus specific immunoglobulin G avidity, sera were measured by commercial kit according to manufacturer's recommendations. Among 50 sera form mothers, 26 showed the presence of Immunoglobulin M antibodies out of which 15 had low avidity antibodies. Out of 50 sera from children, 18 showed the presence of Immunoglobulin M antibodies. Out of these 18 sera from children, 12 were symptomatic, which all showed the presence of low avidity antibodies. The results showed that an avidity index <40% and presence of Immunoglobulin M antibodies is highly suggestive of a recent primary infection.