Convalescent Samples Research Articles

Persistently low plasma thioredoxin is associated with meningococcal septic shock in children

To compare plasma levels of thioredoxin (Trx), TNF-alpha and IL-1 beta in children during the acute phase of meningococcal septic shock (MSS) and in convalescence. Retrospective, observational study in the paediatric intensive care unit of a postgraduate teaching hospital. Thirty-five children requiring intensive care for meningococcal sepsis; paired convalescent samples from 30 survivors (median interval between samples 62 days); 25 healthy control children. Plasma Trx levels were significantly lower in the children with MSS, both during the acute illness (5.5 ng/ml, IQR 1.4-11.4) and in convalescence (2.5 ng/ml, IQR 0.4-6.9) than controls (18.8 ng/ml, IQR 7.9-25.0). Levels of IL-1 beta and TNF-alpha were higher in patients with acute MSS (30.3 pg/ml, IQR 3.6-63.6, and 145.9 pg/ml, IQR 31.8-278.1 respectively) than controls (3.7 pg/ml, IQR 0-36.9, and 23.8 pg/ml, IQR 0-124.3, respectively). Levels fell in convalescence (3.7 pg/ml, IQR 0-25.5, 3.7 pg/ml, IQR 0-304.8, respectively). Plasma Trx was higher in non-survivors, albeit a small group (n=5), than in survivors (n=30). Trx, IL-1 beta, and TNF-alpha levels were not correlated with predicted mortality as assessed by the paediatric risk of mortality (PRISM) score. Children with MSS exhibit persistently low plasma levels of Trx during acute illness and in convalescence.

Clinical and Immunologic Risk Factors for Meningococcal C Conjugate Vaccine Failure in the United Kingdom

The meningococcal serogroup C conjugate (MCC) vaccine was introduced into the United Kingdom with licensure based on immunogenicity data not efficacy data. All subjects with laboratory-confirmed meningococcal serogroup C (MenC) disease from January 2000 to December 2003 in England and Wales were followed up. A vaccine failure was defined as a laboratory-confirmed case of MenC disease occurring > or =10 days after the subject's last scheduled dose of MCC vaccine. Total immunoglobulins, serum bactericidal antibody (SBA) titers, MCC anticapsular antibody levels, and avidity indices (AIs) were measured in acute and convalescent serum samples from subjects with vaccine failure and unvaccinated subjects with MenC disease. Of 465 subjects with confirmed MenC disease identified among those eligible for vaccination, information on vaccination history was obtained for 462 (99.4%); of these, 53 were subjects with vaccine failure. SBA titers in convalescent serum samples and AIs in acute serum samples were significantly higher in subjects with vaccine failure than in unvaccinated subjects, (6.1-fold higher for SBA titers [P=.03] and 3.2-fold higher for AIs [P=.001]). The antibody response in the subjects with vaccine failure was consistent with an anamnestic response, suggesting that MenC disease occurred despite the MCC vaccine priming for immune memory. Persistence of antibodies may be a more appropriate correlate of long-term protection for MCC vaccines than the ability to generate a booster response on exposure.

Bovine Respiratory Syncytial Virus, Bovine Coronavirus, and Bovine Viral Diarrhea Virus Diagnosis by PCR Testing of Nasal Swabs

Diagnosis of viral infections has traditionally used viral isolation in cell culture and serologic testing (antibody levels) to detect active infection (rising antibody levels). These procedures require considerable time for the results, especially cell culture isolation (days to weeks for multiple passages). Serology requires the host response post infection (3-4 weeks for acute to convalescent samples), plus the antibody test performance. Use of cell culture systems for viral diagnostics is useful for selected viruses, yet some viruses may not be readily identified in cell culture or may require unique cell cultures not normally maintained in diagnostic laboratories. Also, some viruses may be quite labile and have reduced chance of recovery due to time and conditions of shipment. There are a limited number of rapid antigen detection systems available for diagnoses of viruses causing BRD. An antigen capture ELISA test for BVDV, used primarily as a test for persistently infected (Pl) BVDV cattle using fresh ear notches, is a newer and more rapid test.
 Molecular diagnostic testing, such as reverse transcriptase polymerase chain reaction (RT-PCR), offers a rapid means and greater sensitivity than the traditional tests like cell culture assay and serology. The nucleotide sequences for primer selection for the RT-PCR are available. The purpose of this study was two-fold: 1) implement RT-PCR tests for BRSV, BCV and BVDV using nasal swab samples from cattle; and 2) compare the RT-PCR results for BRSV, BCV and BVDV with cell culture assay results.

Transcriptional profiling of the peripheral blood response during tularemia

Tularemia is a febrile disease caused by the highly contagious bacterium Francisella tularensis. We undertook an analysis of the transcriptional response in peripheral blood during the course of ulceroglandular tularemia by use of Affymetrix microarrays comprising 14,500 genes. Samples were obtained from seven individuals at five occasions during 2 weeks after the first hospital visit and convalescent samples 3 months later. In total, 265 genes were differentially expressed, 95 of which at more than one time point. The differential expression was verified with real-time quantitative polymerase chain reaction for 36 genes (R(2)=0.590). The most prominent changes were noted in samples drawn on days 2-3 and a considerable proportion of the upregulated genes appeared to represent an interferon-gamma-induced response and also a proapoptotic response. Genes involved in the generation of innate and acquired immune responses were found to be downregulated, presumably a pathogen-induced event. A logistic regression analysis revealed that seven genes were good predictors of the early phase of tularemia. This is the first description of the transcriptional host response to ulceroglandular tularemia and the study has identified gene subsets relevant to the pathogenesis of the disease and subsets that may serve as early diagnostic biomarkers.

Bacterial coinfections in children with viral wheezing

Bacterial coinfections occur in respiratory viral infections, but the attack rates and the clinical profile are not clear. The aim of this study was to determine bacterial coinfections in children hospitalized for acute expiratory wheezing with defined viral etiology. A total of 220 children aged 3 months to 16 years were investigated. The viral etiology of wheezing was confirmed by viral culture, antigen detection, serologic investigation, and/or PCR. Specific antibodies to common respiratory bacteria were measured from acute and convalescent serum samples. All children were examined clinically for acute otitis media, and subgroups of children were examined radiologically for sinusitis and pneumonia. Rhinovirus (32%), respiratory syncytial virus (31%), and enteroviruses (31%) were the most common causative viruses. Serologic evidence of bacterial coinfection was found in 18% of the children. Streptococcus pneumoniae (8%) and Mycoplasma pneumoniae (5%) were the most common causative bacteria. Acute otitis media was diagnosed in 44% of the children. Chest radiographs showed alveolar infiltrates in 10%, and paranasal radiographs and clinical signs showed sinusitis in 17% of the older children studied. Leukocyte counts and serum C-reactive protein levels were low in a great majority of patients. Viral lower respiratory tract infection in children is often associated with bacterial-type upper respiratory tract infections. However, coexisting bacterial lower respiratory tract infections that induce systemic inflammatory response are seldom detected.

High seroprevalence of antibodies against dengue virus in a prospective study of schoolchildren in Managua, Nicaragua

To investigate the incidence of dengue virus (DENV) infection in Nicaragua, a 2-year prospective study was conducted in schoolchildren 4-16 years old in the capital city of Managua. Blood samples were collected before the rainy season in 2001, 2002 and 2003, and were assayed for DENV-specific antibodies. Participants were monitored for dengue-like illness, and acute and convalescent blood samples were collected from suspected dengue cases. In 2001 and 2002, 602 and 397 students were recruited, respectively, and paired annual serum samples were available from 467 and 719 participants in 2001-2002 and 2002-2003, respectively. The overall seroprevalence of anti-DENV antibodies was 91%, increasing from 75% at age 4 to 100% at age 16. The incidence of DENV infection was 12% in Year 1 and 6% in Year 2 (P < 0.001). During Year 1, four laboratory-confirmed dengue cases were detected, with one DENV2 isolate; during Year 2, there were six confirmed dengue cases, with one DENV1 isolate. These and additional circulating serotypes were confirmed by plaque reduction neutralisation test. This study demonstrates surprisingly high transmission of DENV in urban Nicaragua.

Detection of Chlamydophila pneumoniae IgG in paired serum samples: comparison of serological techniques in pneumonia cases

Serological diagnosis of Chamydophila pneumoniae is usually undertaken by complement fixation test (CFT) or by microimmunofluorescence (MIF). A number of commercial methods for detecting C. pneumoniae-specific IgG have been developed. The aim of this study was to compare the performance characteristics of six methods for the diagnosis of pneumonia due to C. pneumoniae, including CFT (in house), MIF (Vircell, Spain), and four ELISAs (Medac, Germany; Savyon, Israel; Serion, Germany; and DRG, Germany). ELISA-Medac, ELISA-Savyon, ELISA-DRG and MIF use C. pneumoniae antigens while ELISA-Serion and CFT use Chlamydophila genus-specific antigen. Acute and convalescent samples from 85 pneumonia patients were studied. Using CFT, cases were initially classified as due to Chlamydophila (43 cases); to other agents (23 cases) (influenza A and B, Mycoplasma pneumoniae, respiratory syncytial virus, adenovirus and Legionella pneumophila); or as negative (19 cases). Cases were considered positive if they showed seroconversion, a significant rise in titer or high titer; and were finally classified as positive if they gave a positive result in at least three assays. Sensitivity values ranged from 87% to 97.8%; and specificity from 84.6% to 97.4%. In conclusion, the assays compared appear to be useful tools for the diagnosis of pneumonia due to Chlamydophila.

The Comparative Accuracy of 8 Commercial Rapid Immunochromatographic Assays for the Diagnosis of Acute Dengue Virus Infection

The serological diagnosis of acute dengue virus infection relies on the detection of dengue-specific immunoglobulin M (IgM) antibodies. Immunochromatographic tests are rapid diagnostic tests (RDTs) that can be performed at the bedside, but they have not been fully validated for diagnosis of dengue infection. More than 20 RDTs for diagnosis of acute dengue infection are commercially available. Of these, 8 were selected for evaluation of performance by use of characterized dengue and nondengue serum specimens, and results were compared with those of a previously published dengue IgM/IgG enzyme-linked immunosorbent assay in conjunction with dengue virus serotyping by reverse-transcriptase polymerase chain reaction. Assay sensitivities were low, ranging from 6.4% (95% confidence interval [CI], 4.0%-9.7%) to 65.3% (95% CI, 59.9%-70.5%), and specificities ranged from 69.1% (95% CI, 61.4%-76.0%) to 100% (95% CI, 97.8%-100%). Of the 8 tests, only 2 had sensitivities of >50%, the level considered to be clinically useful, and, of these, 1 had relatively low specificity (69.1%). Samples collected early in the infection were less likely to test positive than those collected later. A thermal stability study demonstrated a loss in performance of some RDTs when they were stored at a high ambient temperature for 3 months. Users of RDTs for dengue should be aware that many of these tests have a diagnostic accuracy that falls well below the manufacturers' claims. If an acute specimen yields a negative result, a convalescent serum sample should be tested to confirm the result. No RDT adequately differentiated primary and secondary dengue infections, and the tests should not be used for this purpose.

Cytokines in the Nasal Washes of Children with Respiratory Syncytial Virus Bronchiolitis

Although respiratory syncytial (RS) virus is the major cause of bronchiolitis and pneumonia in young children, the factors that regulate the associated lung inflammation have not been defined. The levels of interleukin (IL)10, IL-12, and interferon (IFN) were determined in the nasal wash samples from 20 infants with a clinical diagnosis of bronchiolitis, seven with confirmed RS virus infections and 9 control children without respiratory illnesses. IL-10 levels were significantly higher in acute nasal wash samples (1-4 d post hospitalization) from RS virus-infected infants than in convalescent samples from these children (14-21 d post-hospitalization), from children with other forms of bronchiolitis and from control children. In contrast, only one RS virus-infected infant had detectable IL-12 in an acute nasal wash sample. IFN activity was not detected in any samples from RS virus-infected children. RS virus infection stimulates IL-10 expression but not IL-12 and IFN, possibly contributing to an ineffective cell-mediated immune response.

Human-to-human transmission of avian influenza A/H7N7, The Netherlands, 2003

An outbreak of highly pathogenic avian influenza A virus subtype H7N7 began in poultry farms in the Netherlands in 2003. Virus infection was detected by RT-PCR in 86 poultry workers and three household contacts of PCR-positive poultry workers, mainly associated with conjunctivitis. To determine the magnitude of and risk factors for human-to-human transmission of influenza A/H7N7 in the Netherlands, a retrospective cohort study among household members of infected poultry workers was undertaken. In total, 33 (58.9%) of 56 (among 62) participants who provided blood samples had positive H7 serology, using single convalescent serum samples obtained at least 3 weeks after onset of symptoms of the index case. Eight household members (12.9%) reported symptoms (conjunctivitis and/or ILI), of which four of five (80.0%) tested seropositive. On univariate analysis, significant risk factors for seropositivity included having at least two toilets, a pet bird, and using cloth handkerchiefs. It was not possible to obtain a stable model for binomial regression for the outcome of A/H7N7 infection. Further seroprevalence studies among contacts of asymptomatic H7 cases should be conducted.

Purification of severe acute respiratory syndrome hyperimmune globulins for intravenous injection from convalescent plasma

BACKGROUND: Severe acute respiratory syndrome (SARS) is a new infectious disease caused by the SARS virus. Current first‐line treatments are experimental, and their effectiveness remains open to question. For more effective treatment and prevention of SARS, human SARS hyperimmune globulins for intravenous (IV) injection were purified in this study. STUDY DESIGN AND METHODS: A combination of cold ethanol precipitation and ion‐exchange chromatography was used to process pooled SARS convalescent plasma samples. Virus inactivation and removal approaches were taken to ensure safety. RESULTS: The purified hyperimmune globulins were formulated as a 5 percent solution, with an antibody titer specifically against the SARS virus of 1:83, 1:1600, and 1:200, as determined by enzyme‐linked immunosorbent assay, immunofluorescence assay, and neutralizing antibody test, respectively. The purity of the SARS hyperimmune globulins was 99.0 percent, and the monomer and dimer content was 100 percent. Other variables analyzed met the Chinese Requirements of Biologics for IV immune globulin. The SARS hyperimmune globulins prepared were subsequently approved for clinical evaluation by the Chinese National Institute for the Control of Pharmaceutical & Biological Products. CONCLUSION: IV‐injectable, purified, and concentrated human SARS hyperimmune globulins were prepared from pooled convalescent plasma samples, which are ready to be further evaluated.

Comparison of antibody detection assays for the diagnosis of equine herpesvirus 1 and 4 infections in horses

To compare methods of detecting equine herpesvirus type 1 (EHV1)- and EHV4-specific antibodies in horse sera. 33 acute and convalescent serum samples from experimentally or naturally infected horses after confirmed EHV1 or EHV4 infection. For each sample, serum antibody titers against EHV1 and EHV4 were determined by use of virus neutralization (VN) and complement fixation (CF) assays. The ELISA absorbance values for each serum sample were determined against the EHV1 and EHV4 recombinant ELISA antigens. Values obtained for acute and convalescent sera in each assay were compared. Following experimental infection of foals, EHV1 or EHV4 antibodies that were specific for the inoculating virus were detected only by use of the ELISA. Results of VN and CF assays indicated that the foals seroconverted to EHV1 and EHV4 following infection with EHV4 only. After EHV1-induced abortion, myeloencephalitis, or respiratory tract disease, the VN and CF assay results revealed seroconversion to EHV1 and EHV4, whereas results of the ELISA revealed seroconversion to EHV1 only. Similarly, after confirmed EHV4-induced respiratory tract disease, increases in EHV4-specific antibodies were detected only by use of the ELISA with no indication of an increase in EHV1 antibodies. The CF and, to a lesser degree, VN assays revealed that seroconversion to EHV1 and EHV4 occurred between the time of obtaining acute and convalescent serum samples. The EHV1/EHV4 type-specific antibody ELISA clearly identifies horses that have been infected with EHV1 or EHV4 by use of acute and convalescent sera. Results of VN and CF assays indicate that cross-reactive antibodies greatly limit their use.

Bordetella pertussisin Adult Pneumonia Patients1

<i>Bordetella pertussis</i>in Adult Pneumonia Patients1

Hand-foot-and-mouth disease in calicut

Hand-Foot-and-Mouth Disease (HFMD) is a mild exanthematous illness seen worldwide, affecting mainly children under ten years of age. The causative agents were initially Coxsackie virus type A 16 and related serotypes. The situation changed drastically about thirty years ago with the advent of a new aetiological agent, Enterovirus type 71 (EV 71), which has caused very large outbreaks with severe complications and many deaths. The authors report an outbreak of papulovesicular lesions on the skin and oral mucosa compatible with the diagnosis of HFMD in children in and around Calicut in October 2003. Clinical and laboratory study in collaboration with the National Institute of Communicable Diseases, Delhi. Eighty one children with the syndrome were examined and followed up from October 2003 to February 2004, when the outbreak subsided. The outbreak was mild and all children recovered within 1 to 2 weeks. Acute and convalescent paired serum samples collected from 19 patients were examined at the NICD for IgM antibody against EV 71 by microneutralisation test in cell culture. All the paired samples tested showed significant rise in titre of antibodies, confirming the diagnosis of EV 71 infection in each of them.

A serological survey on neutralizing antibody titer of SARS convalescent sera.

A seroepidemiologic study was conducted in North China in 2003 to determine the neutralizing antibody titer of severe acute respiratory syndrome (SARS) convalescent sera. A total of 99 SARS convalescent serum samples were collected from patients from the Inner Mongolia Autonomous Region, Hebei Province, and Beijing 35–180 days after the onset of symptoms. The anti‐SARS antibodies were detected by enzyme‐linked immunosorbent assay (ELISA), neutralization assay, and Western blot. Eighty‐seven serum samples were confirmed to be positive for SARS antibodies. The neutralizing antibody titer of the 87 positive sera was analyzed quantitatively by neutralization assay. The geometric mean titer (GMT) of the 87 convalescent sera was 1:61. The Kolmogorov–Smirnov test showed that the neutralizing antibody titers conform to normal distribution, which suggests that the average anti‐SARS antibody level in this study was representative of the convalescent antibody level of the SARS population. This result could be useful for the development and quality control of SARS vaccines. J. Med. Virol. 77:147–150, 2005. © 2005 Wiley‐Liss, Inc.

Detection of pathogenic leptospires by real-time quantitative PCR

Definitive diagnosis of leptospirosis has traditionally depended upon the isolation of leptospires from clinical specimens or the demonstration of seroconversion in paired acute and convalescent serum samples. Both of these approaches require expertise not routinely available in clinical laboratories and usually result in delayed diagnosis. Conventional PCR assays have been developed, but all have limitations which have restricted their widespread use. In order to overcome these limitations, a real-time PCR assay was developed using a 423 bp target on the lipL32 gene, which is conserved among pathogenic serovars of LEPTOSPIRA: Reactions were monitored by SYBR green fluorescence and melting curve analysis. Representative serovars from 16 species of Leptospira and over 40 species of other bacteria and fungi were tested. Positive results were obtained with all pathogenic leptospiral serovars, with the exception of Leptospira fainei serovar Hurstbridge. The analytical sensitivity of this assay was 3 genome equivalents per reaction; approximately 10 genome equivalents were detectable in human urine. Leptospiral DNA was amplified from blood containing EDTA or citrate anticoagulants, but heparin, sodium polyanetholesulfonate and saponin were inhibitory. The assay successfully detected leptospiral DNA from serum and urine samples of patients with leptospirosis. This assay has the potential to facilitate rapid, sensitive diagnosis of acute leptospirosis.

Isolation of Escherichia coli Bacteriophages from the Stool of Pediatric Diarrhea Patients in Bangladesh

A 3-week coliphage survey was conducted in stool samples from 140 Bangladeshi children hospitalized with severe diarrhea. On the Escherichia coli indicator strain K803, all but one phage isolate had 170-kb genomes and the morphology of T4 phage. In spot tests, the individual T4-like phages infected up to 27 out of 40 diarrhea-associated E. coli, representing 22 O serotypes and various virulence factors; only five of them were not infected by any of these new phages. A combination of diagnostic PCR based on g32 (DNA binding) and g23 (major capsid protein) and Southern hybridization revealed that half were T-even phages sensu strictu, while the other half were pseudo-T-even or even more distantly related T4-like phages that failed to cross-hybridize with T4 or between each other. Nineteen percent of the acute stool samples yielded T4-like phages, and the prevalence was lower in convalescent stool samples. T4-like phages were also isolated from environmental and sewage water, but with low frequency and low titers. On the enteropathogenic E. coli strain O127:K63, 14% of the patients yielded phage, all of which were members of the phage family Siphoviridae with 50-kb genomes, showing the morphology of Jersey- and beta-4 like phages and narrow lytic patterns on E. coli O serotypes. Three siphovirus types could be differentiated by lack of cross-hybridization. Only a few stool samples were positive on both indicator strains. Phages with closely related restriction patterns and, in the case of T4-like phages, identical g23 gene sequences were isolated from different patients, suggesting epidemiological links between the patients.

Preliminary Findings from the Prospective Multicenter Surveillance, Epidemiology and Risk Factors for Thrombotic Thrombocytopenic Purpura (SERF-TTP) Study.

Background: Due to the rarity of TTP (approximately 4 cases per million), epidemiologic data and correlative laboratory samples from a prospectively identified incident cohort of TTP patients are needed.Methods: SERF TTP is a NHLBI-funded 15-site study targeted to identify 300 incident TTP patients and 600 age-/gender-matched community controls.Results: From nine IRB-approved collaborating sites, 22 incident TTP cases and 17 controls have been interviewed. Data and plasma (acute and convalescent samples) are available for 19 TTP patients (Table 1). Their mean age is 42.5 years (range, 20 – 80 years), and 90% are female. Using the Rose-Eldor TTP Scoring system based on platelets, creatinine, hemoglobin, and neurologic function, 37% would be classified as severely ill (score > 4 of a maximum 8 points). Mean time from symptom onset to diagnosis was 9.2 days, median 5.5 days (range, 0 to 33 days). Severity of TTP was poorer for the 5 patients whose treatment did not begin for at least 16 days (16–33 days) versus the other 14 patients (mean Rose-Eldor score of 4.6 versus 4). Medications which had been prescribed to the patients prior to TTP onset included herbal supplements (n= 5) and hormone replacement therapy or oral contraceptives (n= 6). All patients received at least daily therapeutic plasma exchange (TPE). A platelet count >150,000 was reached at a mean of 9.4 days following TPE initiation. At 30-days follow-up, all patients were alive, although 3 had an exacerbation requiring daily TPE reinstitution. Fourteen patients (78%) experienced an adverse event, including allergic reactions to plasma (n=9), citrate-related toxicity (n=9) and venous access complications (n=2) including one with major hemorrhage requiring transfer to the intensive care unit.Conclusion: The clinical characteristics of the SERF-TTP cohort are similar to those reported from large single-site studies, although the survival rate (100% versus 71% and 83%) is higher. Only a minority of incident TTP cases in the modern era present with renal insufficiency or neurologic findings - highlighting the importance of developing reliable diagnostic laboratory testing.Clinical Characteristics at PresentationStudyPlatelets <100,000Hgb <10 or Hct <.3Cr > 2mg/dlFeverNeurologic symptomsMortalitySERF-TTP n=1995%79%32%11%47%0%Washington University (1) (excluding HSCT/FK506 cases) n= 2896%82%29%32%50%29%Oklahoma TTP-HUS Registry (2) (ADAMTS13 < 5%) n=18100%100%22%NA (Not Available)56%17%(1)Zheng XL, Kaufman RM et al. Blood. 2004; 103: 4043; (2)Vesely SK, George JN et al. Blood. 2003; 102: 60. HSCT: Hematopoetic stem cell transplantation, FK-506: Tacrolimus.

Evaluation of a peptide-based enzyme immunoassay for anti-SARS coronavirus IgG antibody.

High throughput assays for anti‐SARS‐CoV IgG antibody detection are need for large‐scale epidemiologic studies. The performance of a microplate enzyme immunoassay, DETECT‐SARS™, was evaluated for the detection of anti‐SARS‐CoV IgG antibody. This assay is based on synthetic peptides derived from the nucleocapsid and spike proteins. The results showed that the assay provided a high degree of sensitivity (95.9%) for convalescent serum samples. The level of specificity was close to 90%, and did not show significant variation among different control groups. The high degree of sensitivity together with the high‐throughput nature makes it advantageous as a screening assay for studies where handling of a large number of specimens is required. J. Med. Virol. 74:517–520, 2004. © 2004 Wiley‐Liss, Inc.

Evidence of rotavirus AU32 like G9 strains from nontypeable fecal specimens of Indian children hospitalized during 1993-1994.

Serotyping of 432 rotavirus positive fecal specimens collected from hospitalized children during 1990-1997 was carried out at National Institute of Virology (NIV), Pune, India, using monoclonal antibodies (MAbs) directed against VP7 determinant of serotypes G1-G4, G6, G8, and G10. However, significant number of specimens, that is, 47.92% remained nontypeable. The aim of the present study was to culture adapt the nontypeable specimens and to characterize them further. Since the fecal specimens were not tested by MAb to G9 serotype, which has emerged as an important serotype infecting humans recently, presence of G9 serotype was expected in nontypeable specimens. Therefore, we selected specimens from those children, who showed higher neutralizing antibody (NAb) titer in their convalescent serum samples to G9 serotype than their mothers. Out of six isolates having long electropherotype, five isolates showed subgroup II, and one showed subgroup I, II. The isolates were confirmed as G9 by MAb based ELISA, neutralization assay, and PCR. The G9 specific nested PCR products of four isolates showed 96-99% identities to AU32 G9 strain reported from Japan. P type of four isolates was determined as P8. Besides isolates, four additional nontypeable fecal specimens were confirmed as G9 by MAb based ELISA. Thus, 10 (28.57%) out of 35 nontypeable specimens were identified as rotavirus serotype G9. The results indicate that serotype G9 may represent significant proportion of specimens, which were previously nontypeable.