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Autoimmune liver serology: Current diagnostic and clinical challenges
Dimitrios P Bogdanos, Pietro Invernizzi, Ian R Mackay, Diego Vergani
World Journal of Gastroenterology , 2008,
Abstract: Liver-related autoantibodies are crucial for the correct diagnosis and classification of autoimmune liver diseases (AiLD), namely autoimmune hepatitis types 1 and 2 (AIH-1 and 2), primary biliary cirrhosis (PBC), and the sclerosing cholangitis variants in adults and children. AIH-1 is specified by anti-nuclear antibody (ANA) and smooth muscle antibody (SMA). AIH-2 is specified by antibody to liver kidney microsomal antigen type-1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1). SMA, ANA and anti-LKM antibodies can be present in de-novo AIH following liver transplantation. PBC is specified by antimitochondrial antibodies (AMA) reacting with enzymes of the 2-oxo-acid dehydrogenase complexes (chiefly pyruvate dehydrogenase complex E2 subunit) and disease-specific ANA mainly reacting with nuclear pore gp210 and nuclear body sp100. Sclerosing cholangitis presents as at least two variants, first the classical primary sclerosing cholangitis (PSC) mostly affecting adult men wherein the only (and non-specific) reactivity is an atypical perinuclear antineutrophil cytoplasmic antibody (p-ANCA), also termed perinuclear anti-neutrophil nuclear antibodies (p-ANNA) and second the childhood disease called autoimmune sclerosing cholangitis (ASC) with serological features resembling those of type 1 AIH. Liver diagnostic serology is a fast-expanding area of investigation as new purified and recombinant autoantigens, and automated technologies such as ELISAs and bead assays, become available to complement (or even compete with) traditional immunofluorescence procedures. We survey for the first time global trends in quality assurance impacting as it does on (1) manufacturers/purveyors of kits and reagents, (2) diagnostic service laboratories that fulfill clinicians’ requirements, and (3) the end-user, the physician providing patient care, who must properly interpret test results in the overall clinical context.
A case of bilateral presumed chikungunya neuroretinitis  [cached]
Mahesh G,Giridhar A,Shedbele Archis,Kumar Ram
Indian Journal of Ophthalmology , 2009,
Abstract: Chikungunya fever is a relatively rare from of vector-borne viral fever caused by chikungunya virus and spread by bites of the Aedes aegypti and Aedes albopictus mosquito. Epidemics of chikungunya fever have been reported in the past from different parts of the world. Although the virus had been passive for quite some time, recent reports of outbreaks of chikungunya fever in several parts of Southern India have confirmed the re-emergence of this virus. Symptoms of this infection include abrupt onset of fever, chills, and headache, rash, severe joint pain, conjunctival injection and photophobia. Ocular manifestations have been recently reported with this infection. We report a case of a 48-year-old female patient, who presented with defective vision two weeks after a serology proven chikungunya infection. There was bilateral neuroretinitis with peripapillary cotton wool spots. These findings should be kept in mind as an ocular manifestation of chikungunya virus infection.
Serology using rROP2 antigen in the diagnostic of toxoplasmosis in pregnant women
Macre, Miriam de Souza;Pires, Márcio;Meireles, Luciana Regina;Angel, Sérgio O.;Andrade Jr., Heitor Franco de;
Revista do Instituto de Medicina Tropical de S?o Paulo , 2009, DOI: 10.1590/S0036-46652009000500009
Abstract: toxoplasma gondii causes severe fetal disease during acute infection in pregnant women, thus demanding early diagnosis for effective treatment and fetus preservation. fetal tests are inefficient and risky, and diagnosis is based on maternal igm serology, which had weak screening ability due to increased sensitivity, with alternative igg avidity tests. here, we performed elisa and avidity assays using a recombinant t. gondii antigen, rrop2, in samples from 160 pregnant women screened from a large public hospital who were referred due to positive igm assays. igg serology and avidity assays were compared using whole t. gondii extract or rrop2. elisa igg detection with rrop2 showed good agreement with assays performed with t. gondii extract, but rrop2 igg avidity assays were unrelated to whole extract antigen igg avidity, regardless of the chaotrope used. these data show that avidity maturation is specific to individual antigen prevalence and immune response during infection. elisa rrop2 igg assays may be an alternative serological test for the diagnosis of toxoplasmosis during pregnancy, although our data do not support their use in avidity assays.
Definition of a diagnostic routine in individuals with inconclusive serology for chagas disease
Picka, Mariele Cristina Modolo;Meira, Domingos Alves;Carvalho, Thaís Batista de;Peresi, Eliana;Marcondes-Machado, Jussara;
Brazilian Journal of Infectious Diseases , 2007, DOI: 10.1590/S1413-86702007000200012
Abstract: despite the existence of highly sensitive tests, inconclusive serological results are frequent in chronic chagasic infection. this study aimed to define a diagnostic conduct for 30 individuals with inconclusive serology (g3) for chagasic infection assisted at the outpatient unit for infectious and parasitic diseases of the botucatu school of medicine. twenty-one individuals with negative serology (g1) and 33 with positive serology (g2) were also studied. serological methods elisa, hai, ifi and immunoblotting tesa-cruzi were used for g1, g2 and g3, and parasitological methods xenodiagnosis, hemoculture and pcr-lit were used for g2 and g3 individuals. elisa, hai and ifi were performed in 5 different blood samples in g2 and g3. tesa-cruzi was carried out only once in g1, g2 and g3 and, since it is the most sensitive, it was utilized as standard. in g3, positivity for elisa reached 86% in the fifth blood sample; the elisa+hai+ifi combination showed a maximum of 44.8% in the second sample; and tesa-cruzi, 76% in one single sample. xenodiagnosis positivity was 9.4%; hemoculture showed 15.2%; and pcr-lit exhibited 22% positivity in g2. nevertheless, in g3, positivity percentage was 3.4% for xenodiagnosis, 6.7% for pcr-lit, and no positive result was found for hemoculture. in g3, pcr-lit resolved one case which was still inconclusive according to serology tests. in order to define inconclusive diagnoses, the results suggest the combined use of elisa+hai+ifi in 2 blood samples, decreasing the occurrence of false positive/negative results. if results remain inconclusive, the performance of tesa-cruzi and pcr-lit, if necessary, is recommended.
Inhibitors of Alphavirus Entry and Replication Identified with a Stable Chikungunya Replicon Cell Line and Virus-Based Assays  [PDF]
Leena Pohjala, Age Utt, Margus Varjak, Aleksei Lulla, Andres Merits, Tero Ahola, P?ivi Tammela
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0028923
Abstract: Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC50 values between 0.4 and 24 μM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.
A Polarized Cell Model for Chikungunya Virus Infection: Entry and Egress of Virus Occurs at the Apical Domain of Polarized Cells  [PDF]
Pei Jin Lim,Justin Jang Hann Chu
PLOS Neglected Tropical Diseases , 2014, DOI: 10.1371/journal.pntd.0002661
Abstract: Chikungunya virus (CHIKV) has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, and an increasing incidence of encephalitis. The re-emergence of CHIKV with more severe pathogenesis highlights its potential threat on our human health. In this study, polarized HBMEC, polarized Vero C1008 and non-polarized Vero cells grown on cell culture inserts were infected with CHIKV apically or basolaterally. Plaque assays, viral binding assays and immunofluorescence assays demonstrated apical entry and release of CHIKV in polarized HBMEC and Vero C1008. Drug treatment studies were performed to elucidate both host cell and viral factors involved in the sorting and release of CHIKV at the apical domain of polarized cells. Disruption of host cell myosin II, microtubule and microfilament networks did not disrupt the polarized release of CHIKV. However, treatment with tunicamycin resulted in a bi-directional release of CHIKV, suggesting that N-glycans of CHIKV envelope glycoproteins could serve as apical sorting signals.
Chikungunya Infection in India: Results of a Prospective Hospital Based Multi-Centric Study  [PDF]
Pratima Ray, Vinod H. Ratagiri, Sushil K. Kabra, Rakesh Lodha, Sumit Sharma, B. S. Sharma, Mani Kalaivani, Naveet Wig
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0030025
Abstract: Background Chikungunya (CHIKV) has recently seen a re-emergence in India with high morbidity. However, the epidemiology and disease burden remain largely undetermined. A prospective multi-centric study was conducted to evaluate clinical, epidemiological and virological features of chikugunya infection in patients with acute febrile illness from various geographical regions of India. Methods and Findings A total of 540 patients with fever of up to 7days duration were enrolled at Karnataka Institute of Medical Sciences (KIMS), Karnataka (South); Sawai Man Singh Medical College (SMS) Rajasthan (West), and All India Institute of Medical Sciences (AIIMS) New Delhi (North) from June 2008 to May 2009. Serum specimens were screened for chikungunya infection concurrently through RT-PCR and serology (IgM). Phylogenetic analysis was performed using Bioedit and Mega2 programs. Chikungunya infection was detected in 25.37% patients by RT-PCR and/or IgM-ELISA. Highest cases were detected in south (49.36%) followed by west (16.28%) and north (0.56%) India. A difference in proportion of positives by RT-PCR/ELISA with regard to duration of fever was observed (p<0.05). Rashes, joint pain/swelling, abdominal pain and vomiting was frequently observed among chikungunya confirmed cases (p<0.05). Adults were affected more than children. Anti-CHIK antibodies (IgM) were detected for more than 60days of fever onset. Phylogenetic analysis based on E1 gene from KIMS patients (n = 15) revealed ~99% homology clustering with Central/East African genotype. An amino acid change from lysine to glutamine at position 132 of E1 gene was frequently observed among strains infecting children. Conclusions The study documented re-emergence of chikungunya in high frequencies and severe morbidity in south and west India but rare in north. The study emphasizes the need for continuous surveillance for disease burden using multiple diagnostic tests and also warrants the need for an appropriate molecular diagnostic for early detection of chikungunya virus.
Utilization of an Eilat Virus-Based Chimera for Serological Detection of Chikungunya Infection  [PDF]
Jesse H. Erasmus?,James Needham?,Syamal Raychaudhuri?,Michael S. Diamond?,David W. C. Beasley?,Stan Morkowski?,Henrik Salje?,Ildefonso Fernandez Salas?,Dal Young Kim?,Ilya Frolov
PLOS Neglected Tropical Diseases , 2015, DOI: 10.1371/journal.pntd.0004119
Abstract: In December of 2013, chikungunya virus (CHIKV), an alphavirus in the family Togaviridae, was introduced to the island of Saint Martin in the Caribbean, resulting in the first autochthonous cases reported in the Americas. As of January 2015, local and imported CHIKV has been reported in 50 American countries with over 1.1 million suspected cases. CHIKV causes a severe arthralgic disease for which there are no approved vaccines or therapeutics. Furthermore, the lack of a commercially available, sensitive, and affordable diagnostic assay limits surveillance and control efforts. To address this issue, we utilized an insect-specific alphavirus, Eilat virus (EILV), to develop a diagnostic antigen that does not require biosafety containment facilities to produce. We demonstrated that EILV/CHIKV replicates to high titers in insect cells and can be applied directly in enzyme-linked immunosorbent assays without inactivation, resulting in highly sensitive detection of recent and past CHIKV infection, and outperforming traditional antigen preparations.
Persisting Mixed Cryoglobulinemia in Chikungunya Infection  [PDF]
Manuela Oliver,Marc Grandadam,Catherine Marimoutou,Christophe Rogier,Elisabeth Botelho-Nevers,Hugues Tolou,Jean-Luc Moalic,Philippe Kraemer,Marc Morillon,Jean-Jacques Morand,Pierre Jeandel,Philippe Parola,Fabrice Simon
PLOS Neglected Tropical Diseases , 2009, DOI: 10.1371/journal.pntd.0000374
Abstract: Background Chikungunya virus (CHIKV), an arbovirus, is responsible for a two-stage disabling disease, consisting of an acute febrile polyarthritis for the first 10 days, frequently followed by chronic rheumatisms, sometimes lasting for years. Up to now, the pathophysiology of the chronic stage has been elusive. Considering the existence of occasional peripheral vascular disorders and some unexpected seronegativity during the chronic stage of the disease, we hypothesized the role of cryoglobulins. Methods From April 2005 to May 2007, all travelers with suspected CHIKV infection were prospectively recorded in our hospital department. Demographic, clinical and laboratory findings (anti-CHIKV IgM and IgG, cryoglobulin) were registered at the first consultation or hospitalization and during follow-up. Results Among the 66 travelers with clinical suspicion of CHIKV infection, 51 presented anti-CHIKV IgM. There were 45 positive with the serological assay tested at room temperature, and six more, which first tested negative when sera were kept at 4°C until analysis, became positive after a 2-hour incubation of the sera at 37°C. Forty-eight of the 51 CHIKV-seropositive patients were screened for cryoglobulinemia; 94% were positive at least once during their follow-up. Over 90% of the CHIKV-infected patients had concomitant arthralgias and cryoglobulinemia. Cryoglobulin prevalence and level drop with time as patients recover, spontaneously or after short-term corticotherapy. In some patients cryoglobulins remained positive after 1 year. Conclusion Prevalence of mixed cryoglobulinemia was high in CHIKV-infected travelers with long-lasting symptoms. No significant association between cryoglobulinemia and clinical manifestations could be evidenced. The exact prognostic value of cryoglobulin levels has yet to be determined. Responsibility of cryoglobulinemia was suspected in unexpected false negativity of serological assays at room temperature, leading us to recommend performing serology on pre-warmed sera.
A Re-Examination of the History of Etiologic Confusion between Dengue and Chikungunya  [PDF]
Goro Kuno
PLOS Neglected Tropical Diseases , 2015, DOI: 10.1371/journal.pntd.0004101
Abstract: Contrary to the perception of many researchers that the recent invasion of chikungunya (CHIK) in the Western Hemisphere marked the first episode in history, a recent publication reminded them that CHIK had prevailed in the West Indies and southern regions of the United States from 1827–1828 under the guise of “dengue” (DEN), and that many old outbreaks of so-called “dengue” actually represented the CHIK cases erroneously identified as “dengue.” In hindsight, this confusion was unavoidable, given that the syndromes of the two diseases—transmitted by the same mosquito vector in urban areas—are very similar, and that specific laboratory-based diagnostic techniques for these diseases did not exist prior to 1940. While past reviewers reclassified problematic “dengue” outbreaks as CHIK, primarily based on manifestation of arthralgia as a marker of CHIK, they neither identified the root cause of the alleged misdiagnosis nor did they elaborate on the negative consequences derived from it. This article presents a reconstructed history of the genesis of the clinical definition of dengue by emphasizing problems with the definition, subsequent confusion with CHIK, and the ways in which physicians dealt with the variation in dengue-like (“dengue”) syndromes. Then, the article identifies in those records several factors complicating reclassification, based on current practice and standards. These factors include terms used for characterizing joint problems, style of documenting outbreak data, frequency of manifestation of arthralgia, possible involvement of more than one agent, and occurrence of the principal vector. The analysis of those factors reveals that while some of the old “dengue” outbreaks, including the 1827–1828 outbreaks in the Americas, are compatible with CHIK, similar reclassification of other “dengue” outbreaks to CHIK is difficult because of a combination of the absence of pathognomonic syndrome in these diseases and conflicting background information.
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