Microscopic haematuria as an occult filarial infection in bhubaneshwar an endemic area for bancroftian filariasis

Sera samples of 7 microscopic haematuria cases collected before and after treatment with Diethylcarbamazine citrate, (DEC), 9 microfilaraemic cases and 19 endemic normal individuals were analysed for filarial antigen and IgG antibody levels. Filarial antigen was detected in 5 of the 7 microscopic haematuria cases, of which 3 turned negative for antigen after treatment with DEC. While none of the 7 haematuria cases were positive for filarial IgG antibodies, before the DEC treatment, all of them turned positive after DEC treatment. The sensitivity and specificity values(to detect mf +ve cases) were 89% and 90% respectively for the detection of filarial antigen and 78% and 95% respectively for the detection of filarial IgG antibodies.     

Immunomonitoring followed by optimal dec therapy for successful management of clinical filariasis in an endemic area

Lymphatic filariasis continues to be the major cause of clinical morbidity in India and other developing tropical countries. One of the major lacunae in the effective management of clinical filarial cases is the non-availability of a suitable diagnostic test for confirming filaria aetiology in acute, chronic and occult clinical cases where microfilariae (mf) are not usually seen in peripheral circulation. Studies in our laboratory have shown the usefulness of filarial antibody and antigen assays using microfilarial excretory-secretory (mf ES) antigen in detecting microfilaraemic, acute and chronic filarial cases and in confirming filarial aetiology in occult infections. Diethylcarbamazine citrate (DEC) is the drug of choice for lymphatic filariasis. Different regimens of DEC have been explored in the treatment of microfilaraemic cases. Immunomonitoring has shown that the seroconversion of antigen and antibody positivity was found to be very helpful in determining appropriate period of DEC treatment for clinical relief and cure in clinical filarial patients and further they did not have recurrence in most of the cases. Optimal DEC (6mg/kg body wt/day for 21 days each month for 3–12 months) therapy was found to be very effective in acute and atypical clinical manifestations such as asthmatic bronchitis, pulmonary eosinophilia, monoarthritis, recurrent upper respiratory tract infections (URI), pneumonia (super imposed infections) in children and minimal hydrocele, epididymoorchitis, lymphangitis, lymphadenitis, acute abdomen, central serous retinopathy, tenosynovitis, pain and swelling in limbs and joints in adults living in filaria endemic areas.     

Filaria associated clinical manifestations in children in an endemic area and morbidity control by immunomonitoring and optimal DEC therapy: Sevagram experience

Lymphatic filariasis is a major public health problem in India with 412 million people living in bancroftian endemic areas and is a major cause of clinical morbidity. Twenty million people are reported to suffer from chronic disease manifestations such as lymphoedema, hydrocele or elephantiasis. At least twice the number have been shown to suffer from acute and occult filarial infections in an endemic area without diagnosis. Due to non-availability of suitable diagnostic test for confirming filaria aetiology other than parasitological examination, no significant study on filariasis in children has been reported earlier. Studies in our laboratory for more than a decade showed usefulness of microfilarial excretory-secretory antigen in confirming filarial aetiology in acute and occult infections in adults as well as in children. This study reports acute and atypical manifestations such as lymphadenopathy, asthmatic bronchitis, pulmonary eosinophilia, mono-arthritis, recurrent URI, pneumonia, nutritional anemia, pain in abdomen etc. in children living in filaria endemic area having no microfilaraemia but showing filaria aetiology by immunomonitoring for the presence of antibody or antigen and responding to optimal DEC therapy.     

<Emphasis Type="Italic">In vitro</Emphasis> released antigens in diagnosis and immunomonitoring of filaria and tuberculosis

In vitro released antigens by living parasites or bacteria underin vitro maintenance or short term culture showing specific humoral immune response have been explored in development of immunodiagnostics for infectious diseases such as filariasis and tuberculosis in our laboratory. ELISA usingB. malayi mf ES antigen has been explored for detecting IgG antibody by Indirect ELISA and antigen by Inhibition ELISA and in immunomonitoring of carriers as well as clinical filarial cases. A ten year follow up of mf carriers with DEC therapy showed disapperance of antigen and antibody followed by reappearance in few cases in an endemic area. None of the cases followed developed clinical symptoms suggesting the need for long term monitoring and treatment of microfilaraemic carriers. Further immunomonitoring was found to be useful in confirming filaria aetiology in the absence of microfilaremia and determining appropriate period of treatment of acute, early clinical and occult filarial infections for clinical relief and cure.Indirect Stick Penicillinase ELISA system using Mtb EST-6 antigen for detecting tuberculous IgG antibody and a Sandwich Penicillinase ELISA system using affinity purified antibody for detecting circulating antigen were explored in tuberculosis. A combination of both the assay systems with a sensitivity of 70% and specificity of 98% was found to be promising in the precise diagnosis of pulmonary tuberculosis. Further antigen detection was found to be useful in bone and joint tuberculosis.     

Modulation of cellular immune response by cytokines in bancroftian filariasis

Human lymphatic filariasis is a chronic helminth infection caused byWuchereria bancrofti andBrugia malayi. Wide spectrum of clinical manifestations are seen in different clinical groups of filariasis patients which comprises asymptomatic patients with circulating microfilaria (MF), individuals with chronic lymphatic obstruction (CP), Endemic normals (EN) who are asymptomatic and amicrofilaraemic or the relatively rare tropical pulmonary eosinophilia (TPE). The cellular immune response to this infection varies in different clinical groups of filarial patients. ranging from normal lymphocyte proliferative response in EN individuals to lymphocyte hyporesponsiveness in MF to total parasite antigen [Brugia malayi antigen (BMA)]. But in response to recombinant filarial antigen (pRJ51) the lymphocyte proliferation is restored in MF patients. Interestingly the lymphocytes from MFs responded normally to parasite antigen when EN serum was added in the culture whereas sera from MFs failed to revert the lymphocyte hyporesponsiveness. In order to study the molecular mechanisms responsible for parasite antigen specific lymphocyte hyporesponsiveness, we analysed both the Th1 and Th2 type cytokine gene expression profile in different clinical groups of filarasis patients. MF individuals expressed elevated Th2 type cytokines like IL-4, IL-5 and IL-10 and decreased levels of IL-2 and IFN-γ in response to parasite antigen. Chronic patients have elevated levels of both Th1 and Th2 type cytokines in response to parasite antigen. The EN individuals had a purely Th1 type pattern with absence of IL-4 and IL-5 expression. These studies clearly demonstrate the role of Th2 cytokine like IL-10 in antigen specific hyporesponsiveness seen in MF patients. Any methods to arrest the progression of this disease should concentrate on the means to revert the Th2 type into Th1 type response in the MF patients either by Th1 type cytokine therapy or by using recombinant filarial antigen which stimulates the Th1 response. Further the recombinant filarial antigen which induces Th1 type cytokine response could be used for immunoprophylactic studies.     

Filarial antibody and antigen detection in different clinical conditions in an endemic area

The study was conducted in filarial endemic area for various clinical presentations and diagnosis of occult filariasis. A total of 157 cases of various clinical presentations namely tropical pulmonary eosinophilia, monoarthritis, polyarthritis, glomerulonephropathy, tenosynovitis, inguinal lymphadenopathy, generalised lymphadenopathy, retroperitonial lymphadenopathy, endomyocardial fibrosis and acute conjunctivitis were screened for filarial antigen and antibody by enzyme linked immunosorbent assay (ELISA). Out of 157 cases, 107 cases were positive for antigen or antibody, suggesting the role of filarial infection in these clinical presentations. All the 107 cases were treated with diethylcarbamazine citrate (DEC) and some of the patients who were followed showed relief in signs and symptoms. Hence assay of filarial antigen and/or antibody may be useful for diagnosing occult filarial syndromes for better management and further appropriate treatment.     

Immunodiagnosis of human lymphatic filariasis: A review

A monoclonal antibody-based antigen detection system was used to detect the levels of circulating antigen in filarial patients before and after treatment with DEC and in normal individuals living in an area endemic forW. bancrofti infection in Chennai, India. The present study was to show the use of this assay as a means of efficient screening for filariásis in an endemic area where blood was absorbed onto the filter paper by finger prick during day time. The results of the antigen levels collected onto filter strips correlated with their corresponding plasma antigen levels (r=0.83). In microfilaraemics, DEC treatment did not alter the levels of circulating antigens upto a period of one month. We conclude that this monoclonal antibody based ELISA using filter strips may be used in daytime and can replace the existing routine night blood survey.     

Current status of the lateral flow immunoassay for the detection of SARS-CoV-2 in nasopharyngeal swabs

Early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and diagnosis of coronavirus disease 2019 (COVID-19) are priorities during the pandemic. Symptomatic and suspected asymptomatic individuals should be tested for COVID-19 to confirm infection and to be excluded from social interactions. As molecular testing capacity is overloaded during the pandemic, rapid antigen tests, such as lateral flow immunoassays (LFIAs), can be a useful tool as they allow greater test availability and obtain results in a very short time. This short review aims to present the analytical properties of LFIAs in the detection of SARS-CoV-2 in nasopharyngeal swabs. Lateral flow immunoassay is a method that combines thin-layer chromatography and indirect immunochemical sandwich method and allows the detection of a specific SARS-CoV-2 antigen in nasopharyngeal swabs. Swab specimens should be adequately collected and tested as soon as possible. Users should pay attention to quality control and possible interferences. Antigen tests for SARS-CoV-2 show high sensitivity and specificity in cases with high viral loads, and should be used up to five days after the onset of the first symptoms of COVID-19. False positive results may be obtained when screening large populations with a low prevalence of COVID-19 infection, while false negative results may happen due to improper specimen collection or insufficient amount of antigen in the specimen. So as to achieve reliable results, a diagnostic accuracy study of a specific rapid antigen test should be performed.     

Detection of tubercular antibody and antigen in sera of bone and joint tuberculosis

Trichloroacetic acid (TCA) solubilized and DEAE fractionatedMycobacterium tuberculosis H₃₇Ra excretory-secretory (ES) antigen viz., Mtb EST DE1 and affinity purified goat antibodies to the TCA solubilized ES antigen (Mtb EST) were explored in detecting tubercular antibody and antigen respectively in sera of bone and joint tuberculosis by indirect and sandwich ELISA. Out of total 36 bone & joint tuberculosis cases, tubercular antibody was detected by indirect ELISA in 30 patients (sensitivity 83%), while circulating tubercular antigen was detected by sandwich ELISA in 27 patients (sensitivity 75%). Out of 34 non tubercular disease control cases, 10 patients showed positive reaction for antibody while only 4 patients showed positive reaction for antigen. In another group of 34 healthy subjects who were screened, 4 individuals showed positive reaction for tubercular antibody and 2 cases for antigen. This study shows that antigen detection assay using affinity purified anti Mtb EST antigen antibody is superior with overall specificity of 91% as compared to antibody detection assay with 75% specificity in bone & joint tuberculosis.     

Interference of anti-T3 autoantibodies in the measurement of total and free T3 in serum

A case of anti-trilodothyronine autoantibodies is presented in this report. The thyroid hormone profile of a hypertensive patient, with multinodular goiter and history of exogenous thyroid hormone therapy, was found to be highly ambiguous. The total as well as free T3 levels were consistently high (out of range) whereas the T4 (total and free) values were always within normal limits. The thyrotropin was found to be partially suppressed. Very low T3-Uptake indicated some kind of interference in the immunoassays. We incubated the sera with the radio-iodine labelled T3 and observed that the patient's serum bound about ten times more radioactivity than a control run in parallel. On further resolving the serum proteins on cellulose acetate electrophoresis, the radioactivity was detected in the γ-globulin band. Therefore it was established that the patient's serum carried the antibodies reactive with T3 which were interfering in the immunoassays. Elevated anti-thyroid microsomal antibodies were also present in the patient's serum. The anti-T3 antibodies were highly specific for T3 and did not show any cross reactivity with the T4 or its analogues used in the estimation of free T4.     

Serodiagnosis of tuberculosis using two ELISA systems

Serodiagnosis by ELISA has been widely explored over the years, in the diagnosis of tuberculosis. Two ELISA systems were evaluated for detection of mycobacterial antibodies in pulmonary and extra pulmonary tuberculosis. The two test assays explored were ERBA LISA (TB IgG) test (Anda Biologicals) which uses A60 antigen complex found in the cytosol of typical and atypical mycobacteria, and SEVA TB (IgG) ELISA, which uses a 31 kDa, glycoprotein antigen purified fromM. tb H₃₇Ra culture filtrate. Sera from 98 proven tuberculosis [pulmonary TB (48), tuberculous lymphadenopathy (30), tuberculous meningitis (15) & genitourinary TB (5)] were studied along with 32 healthy controls. The overall positivity obtained using ERBA LISA (TB IgG) test and SEVA TB (IgG) ELISA test was 72.9% and 91.6% in pulmonary tuberculosis, 43.3% and 76.6% in tuberculous lymphadenopathy respectively. The sensitivity of ERBA LISA test in tuberculous meningitis and genito-urinary TB was significantly low (26.6% & 40% respectively) compared to sensitivity obtained using SEVA TB ELISA (86.6% & 60% respectively) with overall specificity of 60% and 87.5%. Thus SEVA TB IgG ELISA test was found to be more sensitive than ERBA LISA in detecting IgG antibodies in tuberculous sera, in particular in extra pulmonary tuberculosis cases.     

Diagnostic potential of fractionatedMycobacterium tuberculosis H37Ra excretory-secretory (EST-DE1) antigen in pulmonary tuberculosis

Tuberculosis is emerging as a major public health problem in developing and developed world. Early and precise diagnosis is of prime importance in successful control of infection. Indirect ELISA with penicillinase as marker was developed using purifiedM. tuberculosis excretory-secretory (EST-DE₁) antigen for detecting IgG antibodies in pulmonary tuberculosis. The assay System gave a overall sensitivity of 82% for both smear positive and smear negative pulmonary tuberculosis cases with a specificity of 84%. The positive and negative predictive values were 75% and 88% respectivaly. Further studies with EST-DE₁ antigen revealed that, it contains two of the active antigen fractions of Mtb EST antigen i.e. Mtb EST-4 (56–68 KDa) and Mtb EST-6 (37–45 KDa), as demonstrated by inhibition ELISA. Reactivity with monoclonal antibodies HGT 3a showed the presence of 38 KDa molecule in EST-DE₁ antigen.     

Host protective immunity and vaccine development studies in lymphatic filariasis

Lymphatic filariasis caused mainly by infection fromWuchereria bancrofti andBrugia malayi remains as the major cause of clinical morbidity in tropical and subtropical countries. Development of vaccine against filarial infection can act as additional measure to the existing therapeutic and vector control methods in the control of this disease. The main hurdles in the development of anti-filarial vaccine are the strict primate specificity ofWuchereria bancrofti, the paucity of parasite material, the diversity of clinical manifestations and their associated complex immune responses, lack of clear understanding on host-parasite interactions and the mechanisms involved in protective immunity. However in the past few years, the information generated in immuno-epidemiological studies, correlated with observations in experimental animals suggests that a filarial vaccine is feasible. Initially live irradiated infective larvae have been successfully used to induce high level of protective immunity in several animal models. Applying diverse strategies, variety of purified or recombinant filarial antigens have been explored for their ability to induce protection in different host-parasite systems. Some of these targeted filarial antigens induced high level of resistance in experimental animals against challenge infections. More focussed studies on thorough characterization of parasitological and immunological changes associated with resistance induced by such candidate protective antigens and on delivery mechanisms and safety aspects will be crucial in their selection for possible use in humans.     

Effect of nutritional supplements and protease inhibitor on the yield of es antigens ofBrugia malayi microfilariaein vitro

Brugia malayi microfilarial excretory-secretory (mf ES) antigens obtained byin vitro maintenance of mf are important tools in the immunodiagnosis of bancroftian filariasis. To increase the yield of mf ES products, the effect of nutritional supplements on the culture medium (RPMI 1640) and the maintenance temperature were studied. Supplementation of RPMI 1640 medium with organic acids and sugars of Grace's insect culture medium forin vitro maintenance of 10 lakhs of mf in 40 ml medium increased the yield of mf ES antigen from 152 μg to 364 μg of mean protein, content and the mean antigen titre from 200 to 400. Supplementation with phenyl methyl sulphonyl fluoride (PMSF) and shift in the culture temperature resulted in a further increase in the yield of mf ES antigen to 502 μg of mean total protein with an antigen titre of 800. The modification resulted in a net increase of 3 fold in the protein content and 4 fold in the antigen titre of ES products. The above modifications in thein vitro maintenance of mf did not affect the diagnostic quality of mf ES antigen, which gave a sensitivity and specificity of 80% and 90% respectively in detection of filarial IgG antibodies inWuchereria bancrofti infected cases.     

百色市山羊布鲁氏菌病及衣原体病血清流行病学调查报告

为掌握百色市山羊布鲁氏菌病及衣原体病感染状况。在2013年对百色市辖12个县区的养羊场进行调查并采集血清进行布病及衣原体检测。调查发现,1732个羊场中有159个发生流产、死胎的现象。431个存栏100只以上的羊场中,有87个发生过流产、死胎现象。对76个羊场采集924份血清样品使用虎红平板凝集试验及试管凝集试验进行检测,未检出布鲁氏菌阳性血清。送检520份血清,检出衣原体阳性血清52份,阳性率10%;其中检测种母羊血清139份,检出衣原体阳性血清14份,阳性率10.07%;检测种公羊血清72份,检出衣原体阳性血清9份,阳性率12.50%。经分析,百色市山羊的布病防控达到广西稳定控制标准要求;羊衣原体病在本场乃至整个百色地区有蔓延流行的风险。     

Microfluidic immunoassay for detection of serological antibodies: A potential tool for rapid evaluation of immunity against SARS-CoV-2

In December 2019, coronavirus disease 2019 became a pandemic affecting more than 200 countries and territories. Millions of lives are still affected because of mandatory quarantines, which hamstring economies and induce panic. Immunology plays a major role in the modern field of medicine, especially against virulent infectious diseases. In this field, neutralizing antibodies are heavily studied because they reflect the level of infection and individuals'' immune status, which are essential when considering resumption of work, flight travel, and border entry control. More importantly, it also allows evaluating the antiviral vaccine efficacy as vaccines are still known for being the ultimate intervention method to inhibit the rapid spread of virulent infectious diseases. In this Review, we first introduce the host immune response after the infection of SARS-CoV-2 and discuss the latest results using conventional immunoassays. Next, as an enabling platform for detection with sufficient sensitivity while saving analysis time and sample size, the progress of microfluidic-based immunoassays is discussed and compared based on surface modification, microfluidic kinetics, signal output, signal amplification, sample matrix, and the detection of anti-SARS-CoV-2 antibodies. Based on the overall comparison, this Review concludes by proposing the future integration of visual quantitative signals on microfluidic devices as a more suitable approach for general use and large-scale surveillance.     

Comparison of diagnostic accuracy for eight SARS-CoV-2 serological assays

IntroductionSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests have been suggested as an additional diagnostic tool in highly suspected cases with a negative molecular test and determination of seroprevalence in population. We compared the diagnostic performance of eight commercial serological assays for IgA, IgM, and IgG antibodies to the SARS-CoV-2 virus.Materials and methodsThe comparison study was performed on a total of 76 serum samples: 30 SARS-CoV-2 polymerase chain reaction (PCR)-negative and 46 SARS-CoV-2 PCR-positive patients with asymptomatic to severe disease and symptoms duration from 3-30 days. The study included: three rapid lateral flow immunochromatographic assays (LFIC), two enzyme-linked immunosorbent assays (ELISA), and three chemiluminescence immunoassays (CLIA).ResultsAgreement between IgM assays were minimal to moderate (kappa 0.26 to 0.63) and for IgG moderate to excellent (kappa 0.72 to 0.92). Sensitivities improved with > 10 days of symptoms and were: 30% to 89% for IgM; 89% to 100% for IgG; 96% for IgA; 100% for IgA/IgM combination; 96% for total antibodies. Overall specificities were: 90% to 100% for IgM; 85% to 100% for IgG; 90% for IgA; 70% for IgA/IgM combination; 100% for total antibodies. Diagnostic accuracy for IgG ELISA and CIA assays were excellent (AUC ≥ 0.90), without significant difference. IgA showed significantly better diagnostic accuracy than IgM (P < 0.001).ConclusionThere is high variability between IgM assays independently of the assay format, while IgG assays showed moderate to perfect agreement. The appropriate time for testing is crucial for the proper immunity investigation.     

Immunodiagnosis of tuberculosis: An update

Tuberculosis is still a major health problem in most developing countries and its incidence is rising in many developed countries. This resurgence has been attributed to the HIV epidemic and TB has been declared as a global health emergency by WHO in 1993. The diagnosis of tuberculosis mainly depends upon initial clinical suspicion and radiographic findings with subsequent bacteriological confirmation by sputum smear examination and culture. Lack of sensitivity in smear examination, non specificity of radiological findings, extended tum around time ofMycobacterium tuberculosis culture and difficulties in diagnosing paucibacillary, childhood and extrapulmonary tuberculosis has necessitated to explore the utility of immunodiagnosis of tuberculosis as a convenient and cost effective test to supplement clinical information for definite diagnosis. Many commercial tests are available in the market for diagnosis of TB. Most of these tests are based on the detection of IgG, IgA and IgM antibodies to specific mycobacterial antigen or mixture of antigens. Indigenous immunoassay systems have explored excretory-secretory ES-31 mycobacterial antigen for immunodiagnosis of TB. Many a time there is lack of consistent elevation in all the three Ig classes in active infection thus making it more important to determine the ideal antibody isotype assay for reliable diagnosis of tuberculosis and to save the costs of the patient for unnecessary investigations.     

Usage of Conventional PCR Technology for the Detection of HLA-B27 Allele: A Significant Molecular Marker of Ankylosing Spondylitis

Ankylosing spondylitis is a chronic inflammatory disease that has been linked to the human leukocyte antigen class I allele HLA-B27. More than 90 % of patients with ankylosing spondylitis possess the HLA-B27 allele, but only 1 % of people with HLA-B27 develop the disease. Ankylosing spondylitis predominately affects young males. The present study was planned to find out the involvement of HLA-B27 specific allele in relation to age and sex in symptomatic suspected patients of ankylosing spondylitis using conventional PCR technology. Forty symptomatic patients of ankylosing spondylitis were included in the present study. SSP–PCR technique was used to detect the HLA-B27 specific allele. This study showed (out of 40 symptomatic suspected cases of ankylosing spondylitis only 12 patients were detected positive with HLA-B27 allele, while remaining 28 were negative) that the positivity rate for ankylosing spondylitis with HLA-B27 allele is low. Furthermore, it was observed that the males above 50 years are more prone to develop AS with HLA-B27 specific allele. It could be concluded that the conventional PCR technology is a rapid, sensitive, and confirmatory method for the diagnosis of ankylosing spondylitis.     

Development and evaluation of realistic microbioassays in freely suspended droplets on a chip

A novel technique for biomolecular detection in microliter droplets floating on the surface of high density oil is presented. Each droplet was captured and manipulated dielectrophoretically and was used as a site for a microscopic bioassay based on agglutination of antibody-conjugated particles. The results were read out by the pattern of unagglomerated gold nanoparticles collected on the droplet surface. Two formats of bioassays, namely gold only agglutination and gold and latex agglutination, were investigated experimentally by varying analyte concentration, particle size and concentration, number of antigen binding sites per particle, time for incubation, and rate of particle collection on the droplet surface. The microbioassays performance was also evaluated with ricin antibodies and compared to the ricin assays in field use. It is estimated that the droplet based assays require 100× smaller sample volume and are ten times more sensitive, though they require longer times to complete. The experiments were interpreted by modeling the kinetics of particle agglutination and mass transfer processes inside the droplets. The incubation time and antigen concentration values calculated by the model correlate well with the experimental results. The results could allow for development of efficient immunoassays on a chip requiring even smaller sample volumes.