Efficacy of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine in women aged 15-25 years with and without serological evidence of previous exposure to HPV-16/18.

In the Phase III PATRICIA study (NCT00122681), the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine (Cervarix(®), GlaxoSmithKline Biologicals) was highly efficacious against HPV-16/18 infections and precancerous lesions in women HPV-16/18 deoxyribose nucleic acid (DNA) negative and seronegative at baseline. We present further data on vaccine efficacy (VE) against HPV-16/18 in the total vaccinated cohort including women who may have been exposed to HPV-16/18 infection before vaccination. In women with no evidence of current or previous HPV-16/18 infection (DNA negative and seronegative), VE was 90.3% (96.1% confidence interval: 87.3-92.6) against 6-month persistent infection (PI), 91.9% (84.6-96.2) against cervical intraepithelial neoplasia (CIN)1+ and 94.6% (86.3-98.4) against CIN2+ [97.7% (91.1-99.8) when using the HPV type assignment algorithm (TAA)]. In women HPV-16/18 DNA negative but with serological evidence of previous HPV-16/18 infection (seropositive), VE was 72.3% (53.0-84.5) against 6-month PI, 67.2% (10.9-89.9) against CIN1+, and 68.8% (-28.3-95.0) against CIN2+ [88.5% (10.8-99.8) when using TAA]. In women with no evidence of current HPV-16/18 infection (DNA negative), regardless of their baseline HPV-16/18 serological status, VE was 88.7% (85.7-91.1) against 6-month PI, 89.1% (81.6-94.0) against CIN1+ and 92.4% (84.0-97.0) against CIN2+ [97.0% (90.6-99.5) when using TAA]. In women who were DNA positive for one vaccine type, the vaccine was efficacious against the other vaccine type. The vaccine did not impact the outcome of HPV-16/18 infections present at the time of vaccination. Vaccination was generally well tolerated regardless of the woman's HPV-16/18 DNA or serological status at entry.

The Kaposi's sarcoma-related herpesvirus (KSHV)-encoded chemokine vMIP-I is a specific agonist for the CC chemokine receptor (CCR)8.

The Kaposi's sarcoma-related herpesvirus (KSHV), also designated human herpesvirus 8, is the presumed etiologic agent of Kaposi's sarcoma and certain lymphomas. Although KSHV encodes several chemokine homologues (viral macrophage inflammatory protein [vMIP]-I, -II, and -III), only vMIP-II has been functionally characterized. We report here that vMIP-I is a specific agonist for the CC chemokine receptor (CCR)8 that is preferentially expressed on Th2 T cells. Y3 cells transfected with CCR8 produced a calcium flux in response to vMIP-I and responded vigorously in in vitro chemotaxis assays. In competition binding experiments, the interaction of vMIP-I with CCR8 was shown to be specific and of high affinity. In contrast to its agonist activity at CCR8, vMIP-I did not interact with CCR5 or any of 11 other receptors examined. Furthermore, vMIP-I was unable to inhibit CCR5-mediated HIV infection. These findings suggest that expression of vMIP-I by KSHV may influence the Th1/Th2 balance of the host immune response.

Infection of human thymocytes by Epstein-Barr virus.

The Epstein-Barr Virus (EBV) causes infectious mononucleosis, and has been strongly associated with certain human cancers. The virus is thought to exclusively bind to B lymphocytes and epithelial cells via receptors (CR2/CD21) that also interact with fragments of the third component of complement (C3). Recent evidence, however, has challenged this belief. We have used two-color immunofluorescence analysis using biotin-conjugated EBV and streptavidin-phycoerythrin along with fluorescein-conjugated anti-T cell antibodies and demonstrated that CD1-positive, CD3-dull (immature) human thymocytes express functional EBV receptors. In four replicate experiments, the binding of EBV to thymocytes ranged between 8 and 18%. This interaction is specific as evidenced by inhibition with nonconjugated virus, anti-CR2 antibodies, aggregated C3, and an antibody to the gp350 viral glycoprotein that the virus uses to bind to CR2. EBV can infect the thymocytes as evaluated by the presence of episomal EBV-DNA in thymocytes that had been incubated with the virus as short as 12 days or as long as 6 weeks. Episomal DNA analysis was performed by Southern blotting with a EBV-DNA probe that hybridizes to the first internal reiteration of the viral DNA. The presence of the EBV genome is also supported by the detection of EBV nuclear antigen 1 in infected thymocytes, assessed by Western blotting with EBV-immune sera. The EBV infection is specific as determined by blocking experiments using anti-CR2 and anti-gp350 antibodies. Finally, virus infection of thymocytes can act synergistically along with interleukin 2 and induce a lymphokine-dependent cellular proliferation. In view of previously reported cases of EBV-positive human T cell lymphomas, the possibility is raised that EBV may be involved in cancers of T lymphocytes that have not been previously appreciated.

The reduced expression of 6Ckine in the plt mouse results from the deletion of one of two 6Ckine genes.

6Ckine is an unusual chemokine capable of attracting naive T lymphocytes in vitro. It has been recently reported that lack of 6Ckine expression in lymphoid organs is a prominent characteristic of mice homozygous for the paucity of lymph node T cell (plt) mutation. These mice show reduced numbers of T cells in lymph nodes, Peyer's patches, and the white pulp of the spleen. The genetic reason for the lack of 6Ckine expression in the plt mouse, however, has remained unknown. Here we demonstrate that mouse 6Ckine is encoded by two genes, one of which is expressed in lymphoid organs and is deleted in plt mice. A second 6Ckine gene is intact and expressed in the plt mouse.

Aberrant in vivo T helper type 2 cell response and impaired eosinophil recruitment in CC chemokine receptor 8 knockout mice.

Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (-/-) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8(-/)- mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo.

Sustained efficacy and immunogenicity of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine: analysis of a randomised placebo-controlled trial up to 6.4 years.

BACKGROUND: Prophylactic human papillomavirus (HPV) vaccines have to provide sustained protection. We assessed efficacy, immunogenicity, and safety of the HPV-16/18 AS04-adjuvanted vaccine up to 6.4 years. METHODS: Women aged 15-25 years, with normal cervical cytology, who were HPV-16/18 seronegative and oncogenic HPV DNA-negative (14 types) at screening participated in a double-blind, randomised, placebo-controlled initial study (n=1113; 560 vaccine group vs 553 placebo group) and follow-up study (n=776; 393 vs 383). 27 sites in three countries participated in the follow-up study. Cervical samples were tested every 6 months for HPV DNA. Management of abnormal cytologies was prespecified, and HPV-16/18 antibody titres were assessed. The primary objective was to assess long-term vaccine efficacy in the prevention of incident cervical infection with HPV 16 or HPV 18, or both. We report the analyses up to 6.4 years of this follow-up study and combined with the initial study. For the primary endpoint, the efficacy analysis was done in the according-to-protocol (ATP) cohort; the analysis of cervical intraepithelial neoplasia grade 2 and above (CIN2+) was done in the total vaccinated cohort (TVC). The study is registered with ClinicalTrials.gov, number NCT00120848. FINDINGS: For the combined analysis of the initial and follow-up studies, the ATP efficacy cohort included 465 women in the vaccine group and 454 in the placebo group; the TVC included 560 women in the vaccine group and 553 in the placebo group. Vaccine efficacy against incident infection with HPV 16/18 was 95.3% (95% CI 87.4-98.7) and against 12-month persistent infection was 100% (81.8-100). Vaccine efficacy against CIN2+ was 100% (51.3-100) for lesions associated with HPV-16/18 and 71.9% (20.6-91.9) for lesions independent of HPV DNA. Antibody concentrations by ELISA remained 12-fold or more higher than after natural infection (both antigens). Safety outcomes were similar between groups: during the follow-up study, 30 (8%) participants reported a serious adverse event in the vaccine group versus 37 (10%) in the placebo group. None was judged related or possibly related to vaccination, and no deaths occurred. INTERPRETATION: Our findings show excellent long-term efficacy, high and sustained immunogenicity, and favourable safety of the HPV-16/18 AS04-adjuvanted vaccine up to 6.4 years. FUNDING: GlaxoSmithKline Biologicals (Belgium).

Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women.

BACKGROUND: The human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine was immunogenic, generally well tolerated, and effective against HPV-16 or HPV-18 infections, and associated precancerous lesions in an event-triggered interim analysis of the phase III randomised, double-blind, controlled PApilloma TRIal against Cancer In young Adults (PATRICIA). We now assess the vaccine efficacy in the final event-driven analysis. METHODS: Women (15-25 years) were vaccinated at months 0, 1, and 6. Analyses were done in the according-to-protocol cohort for efficacy (ATP-E; vaccine, n=8093; control, n=8069), total vaccinated cohort (TVC, included all women receiving at least one vaccine dose, regardless of their baseline HPV status; represents the general population, including those who are sexually active; vaccine, n=9319; control, n=9325), and TVC-naive (no evidence of oncogenic HPV infection at baseline; represents women before sexual debut; vaccine, n=5822; control, n=5819). The primary endpoint was to assess vaccine efficacy against cervical intraepithelial neoplasia 2+ (CIN2+) that was associated with HPV-16 or HPV-18 in women who were seronegative at baseline, and DNA negative at baseline and month 6 for the corresponding type (ATP-E). This trial is registered with ClinicalTrials.gov, number NCT00122681. FINDINGS: Mean follow-up was 34.9 months (SD 6.4) after the third dose. Vaccine efficacy against CIN2+ associated with HPV-16/18 was 92.9% (96.1% CI 79.9-98.3) in the primary analysis and 98.1% (88.4-100) in an analysis in which probable causality to HPV type was assigned in lesions infected with multiple oncogenic types (ATP-E cohort). Vaccine efficacy against CIN2+ irrespective of HPV DNA in lesions was 30.4% (16.4-42.1) in the TVC and 70.2% (54.7-80.9) in the TVC-naive. Corresponding values against CIN3+ were 33.4% (9.1-51.5) in the TVC and 87.0% (54.9-97.7) in the TVC-naive. Vaccine efficacy against CIN2+ associated with 12 non-vaccine oncogenic types was 54.0% (34.0-68.4; ATP-E). Individual cross-protection against CIN2+ associated with HPV-31, HPV-33, and HPV-45 was seen in the TVC. INTERPRETATION: The HPV-16/18 AS04-adjuvanted vaccine showed high efficacy against CIN2+ associated with HPV-16/18 and non-vaccine oncogenic HPV types and substantial overall effect in cohorts that are relevant to universal mass vaccination and catch-up programmes. FUNDING: GlaxoSmithKline Biologicals.

Chemokines and the arrest of lymphocytes rolling under flow conditions.

Circulating lymphocytes are recruited from the blood to the tissue by rolling along the endothelium until being stopped by a signaling event linked to the Gialpha subunit of a heterotrimeric GTP-binding protein; that event then triggers rapid integrin-dependent adhesion. Four chemokines are now shown to induce such adhesion to intercellular adhesion molecule-1 and to induce arrest of rolling cells within 1 second under flow conditions similar to those of blood. SDF-1 (also called PBSF), 6-C-kine (also called Exodus-2), and MIP-3beta (also called ELC or Exodus-3) induced adhesion of most circulating lymphocytes, including most CD4+ T cells; and MIP-3alpha (also called LARC or Exodus-1) triggered adhesion of memory, but not naïve, CD4+ T cells. Thus, chemokines can regulate the arrest of lymphocyte subsets under flowing conditions, which may allow them to control lymphocyte-endothelial cell recognition and lymphocyte recruitment in vivo.

Simulation case rehearsals for carotid artery stenting.

A case series of 5 patients is presented assessing the utility of simulation case rehearsals of individual patients for carotid artery stenting on an endovascular simulator. Simulated and operative device dimensions were similar. Results of subjective surveys indicated that face and content validity were excellent. The simulations predicted difficulty with vessel cannulation, however had difficulty predicting post-stent changes in bifurcation angulation. Our experience suggests that it may be feasible to use patient-specific CTA-derived data in the creation of a realistic case rehearsal simulation. The overall utility of this concept, including cost-benefit analysis, has yet to be determined.

Chemokines and lymphocyte biology.

The past few years have seen significant growth in the number of known chemokines. Along with expansion in this field has come an appreciation of a role for chemokines beyond the chemotaxis of leukocytes. In particular, it appears that these molecules may represent major products of activated lymphocytes and that, in addition to chemotaxis, some of them may also regulate the growth, activation, and differentiation of lymphocytes.

Subtelomere FISH analysis of 11 688 cases: an evaluation of the frequency and pattern of subtelomere rearrangements in individuals with developmental disabilities.

BACKGROUND: Subtelomere fluorescence in situ hybridisation (FISH) analysis has increasingly been used as an adjunct to routine cytogenetic testing in order to detect small rearrangements. Previous reports have estimated an overall abnormality rate of 6%, with a range of 2-29% because of different inclusion criteria. METHODS: This study presents data compiled from 11 688 cases referred for subtelomere FISH testing in three clinical cytogenetic laboratories. RESULTS: In this study population, the detection rate for clinically significant subtelomere abnormalities was approximately 2.5%, with an additional 0.5% detection of presumed familial variants. Approximately half of the clinically significant abnormalities identified were terminal deletions, the majority of which were de novo. Most of the remaining cases were unbalanced translocations between two chromosomes or two arms of the same chromosome. Approximately 60% of the unbalanced translocations were inherited from a parent carrying a balanced form of the rearrangement. Other abnormalities identified included tandem duplications, apparently balanced translocations, partial deletions, and insertions. Interestingly, 9 cases (0.08%) were found to have interstitial deletions of non-telomeric control loci, either BCR on 22q or PML on 15q. The most common clinically significant imbalances found were deletions of 1p, 22q, 4p, 9q, 8p, 2q and 20p. The most common familial variants were a deletion or duplication of 10q, deletion of 4q, deletion of Yq, and duplication of X/Yp onto Xq. CONCLUSIONS: This study of subtelomere rearrangements is a 20 fold increase in number over the previously reported largest study and represents an unbiased analysis of subtelomere rearrangements in a large, unselected patient population.

Transgenic overexpression of neuromedin U promotes leanness and hypophagia in mice.

Recent work has shown that neuromedin U (NmU), a peptide initially identified as a smooth muscle contractor, may play a role in regulating food intake and energy homeostasis. To further evaluate this putative function, we measured food intake, body weight, energy expenditure and glucose homeostasis in transgenic mice that ubiquitously overexpress murine proNmU. NmU transgenic mice were lighter and had less somatic and liver fat, were hypophagic, and had improved insulin sensitivity as judged by an intraperitoneal insulin tolerance test. Transgenic mice had higher levels of hypothalamic NPY, POMC and MCH mRNA. There was no difference in O2 consumption between genotypes; however, NmU transgenic mice displayed a modest increase in respiratory quotient during food deprivation and refeeding. There were no behavioral disturbances in the NmU transgenic mice that could account for the results (e.g. changes in locomotor activity). When placed on a high-fat diet, transgenic mice remained lighter than wild-type mice and ate less, but gained weight at a rate similar to wild-type mice. Despite the increased weight gain with high-fat feeding, glucose tolerance was significantly improved in the transgenic mice. These findings support the hypothesized role of NmU as an endogenous anorexigenic peptide.

Recent advances in chemokines and chemokine receptors.

Chemokines are a superfamily of small cytokine-like molecules which have been described primarily on the basis of their ability to mediate the migration of verious cell types, particularly those of lymphoid origin. The receptors for these molecules are all seven-transmembrane domain G protein-coupled receptors that have historically been excellent targets for small-molecule drugs. This fact, coupled with the advent of large-scale DNA database mining and the recognition that chemokine receptors are also coreceptors for HIV, has driven discovery in this field at a tremendous rate. This process has included not just an expansion of the number of known chemokines and chemokine receptors, but also a greater appreciation for the variety of functions that chemokines are involved in. We review here the molecules that have come from the most recent years of chemokine research as well as many of the new functions that have been ascribed to them.

Organisation of the pericentromeric region of chromosome 15: at least four partial gene copies are amplified in patients with a proximal duplication of 15q.

Clinical cytogenetic laboratories frequently identify an apparent duplication of proximal 15q that does not involve probes within the PWS/AS critical region and is not associated with any consistent phenotype. Previous mapping data placed several pseudogenes, NF1, IgH D/V, and GABRA5 in the pericentromeric region of proximal 15q. Recent studies have shown that these pseudogene sequences have increased copy numbers in subjects with apparent duplications of proximal 15q. To determine the extent of variation in a control population, we analysed NF1 and IgH D pseudogene copy number in interphase nuclei from 20 cytogenetically normal subjects by FISH. Both loci are polymorphic in controls, ranging from 1-4 signals for NF1 and 1-3 signals for IgH D. Eight subjects with apparent duplications, examined by the same method, showed significantly increased NF1 copy number (5-10 signals). IgH D copy number was also increased in 6/8 of these patients (4-9 signals). We identified a fourth pseudogene, BCL8A, which maps to the pericentromeric region and is coamplified along with the NF1 sequences. Interphase FISH ordering experiments show that IgH D lies closest to the centromere, while BCL8A is the most distal locus in this pseudogene array; the total size of the amplicon is estimated at approximately 1 Mb. The duplicated chromosome was inherited from either sex parent, indicating no parent of origin effect, and no consistent phenotype was present. FISH analysis with one or more of these probes is therefore useful in discriminating polymorphic amplification of proximal pseudogene sequences from clinically significant duplications of 15q.

"Molecular rulers" for calibrating phenotypic effects of telomere imbalance.

As a result of the increasing use of genome wide telomere screening, it has become evident that a significant proportion of people with idiopathic mental retardation have subtle abnormalities involving the telomeres of human chromosomes. However, during the course of these studies, there have also been telomeric imbalances identified in normal people that are not associated with any apparent phenotype. We have begun to scrutinize cases from both of these groups by determining the extent of the duplication or deletion associated with the imbalance. Five cases were examined where the telomere rearrangement resulted in trisomy for the 16p telomere. The size of the trisomic segment ranged from approximately 4-7 Mb and the phenotype included mental and growth retardation, brain malformations, heart defects, cleft palate, pancreatic insufficiency, genitourinary abnormalities, and dysmorphic features. Three cases with telomeric deletions without apparent phenotypic effects were also examined, one from 10q and two from 17p. All three deletions were inherited from a phenotypically normal parent carrying the same deletion, thus without apparent phenotypic effect. The largest deletion among these cases was approximately 600 kb on 17p. Similar studies are necessary for all telomeric regions to differentiate between those telomeric rearrangements that are pathogenic and those that are benign variants. Towards this goal, we are developing "molecular rulers" that incorporate multiple clones at each telomere that span the most distal 5 Mb region. While telomere screening has enabled the identification of telomere rearrangements, the use of molecular rulers will allow better phenotype prediction and prognosis related to these findings.

Subunit structure of a cortical granule lectin involved in the block to polyspermy in Xenopus laevis eggs.

The cortical granule lectin of Xenopus laevis eggs is a large molecular mass glycoprotein involved in the post-fertilization block to polyspermy. We have investigated the subunit structure of this lectin and found that the native molecule contains 10-12 monomers, each of which has considerable charge and size heterogeneity due to glycosylated side chains. In addition, significant amino acid sequence homology is indicated by peptide mapping of subunits separated by isoelectric focusing.

Centrally administered hemokinin-1 (HK-1), a neurokinin NK1 receptor agonist, produces substance P-like behavioral effects in mice and gerbils.

Hemokinin-1 (HK-1) is a recently described mouse tachykinin peptide whose biological functions are not fully understood. To date, a unique receptor for HK-1 has not been identified. Recent studies suggest HK-1 may have a role in immunological functions, but there has been little characterization of HK-1's effects in the central nervous system (CNS). In the present studies, we confirm that HK-1 is an endogenous agonist at all of the known tachykinin receptors, and is selective for the NK1 receptor over the NK2 and NK3 subtypes. CHO cells transfected with the human NK1 receptor released intracellular calcium in response to HK-1. In addition, HK-1 competed with substance P (SP) for binding to mouse NK1 and human NK1 receptors. In vivo central administration of HK-1 to gerbils and mice induced foot-tapping and scratching behaviors, respectively, similar to those observed following central administration of SP or the NK1 receptor agonist, GR-73632. Furthermore, these behavioral effects were blocked by the selective NK1 receptor antagonist, MK-869. Finally, a comprehensive expression analysis of HK-1 demonstrated that HK-1 mRNA is much more broadly expressed than previously reported with expression observed in many brain regions. Together these data demonstrate that HK-1 is a functional agonist at NK1 receptors and suggest that HK-1 may function both centrally and peripherally.

Targeted use of exoglycosidase digestion for the structural elucidation of neutral O-linked oligosaccharides.

Exoglycosidase digestion in combination with the catalog-library approach (CLA) is used with matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) to obtain the complete structure of oligosaccharides. The CLA is a collision-induced dissociation (CID)-based method used to determine the structure of O-linked neutral oligosaccharides. It provides both linkage and stereochemical information. Exoglycosidases are used to confirm independently the validity of the CLA. In some cases, the CLA provides structural information on all but a single residue. Exoglycosidase is used to refine these structures. In this way, exoglycosidase use is targeted employing only a small number of enzymes. Exoglycosidase arrays, which have been used with N-linked oligosaccharides, is avoided despite the larger variations in structures of O-linked species.

Comparative study of the effectiveness of cefixime and penicillin V for the treatment of streptococcal pharyngitis in children and adolescents.

An open label randomized trial conducted in rural Kentucky compared the efficacy and safety of cefixime (CFX), 8 mg/kg once daily, with those of penicillin V (PEN), 250 mg 3 times daily, in 110 pediatric patients with Group A beta-hemolytic streptococcal pharyngitis. Forty-eight CFX and 47 PEN patients were evaluable for efficacy. At the end of therapy bacteriologic eradication was 45 of 48 (94%) and 36 of 47 (77%) in the CFX and PEN V groups, respectively (P < 0.05). Up to 6 weeks posttherapy 10 (21%) CFX patients and 21 (45%) PEN patients had positive Group A beta-hemolytic Streptococcus cultures (P < 0.05). Concordant serotypes were identified from 4 of 7 CFX and 15 of 17 PEN patients with positive repeat cultures. All discordant serotypes (5 of 31) were identified at greater than 19 days posttherapy. Symptomatic treatment failures (concordant serotypes) occurred in 1 (2%) CFX and 8 (17%) PEN patients (P < 0.05). Drug-related adverse experiences consisted of 2 cases of mild diarrhea and loose stools in the CFX group and none in the PEN group. No clinically significant laboratory test abnormalities occurred in either group. CFX, once daily, was as safe as and significantly more effective than PEN given 3 times daily for the treatment of Group A beta-hemolytic streptococcal pharyngitis.

Relation of the outcome of conjunctivitis and the conjunctivitis-otitis syndrome to identifiable risk factors and oral antimicrobial therapy.

The epidemiology, microbiology and clinical outcome of the conjunctivitis-otitis syndrome (CJ-AOM) was investigated in a rural private practice concurrent to a double blind placebo-controlled study of orally administered amoxicillin for prevention of acute otitis media (AOM) secondary to conjunctivitis. Bacterial pathogens were isolated when greater than 15 polymorphonuclear leukocytes/high power field were observed on Gram-stained smear of conjunctival secretions. Nontypable Haemophilus influenzae biotype 2 predominated in CJ-AOM; however, Streptococcus pneumoniae was isolated nearly as frequently as H. influenzae in conjunctivitis without AOM. Younger age (P = 0.001) and more episodes of AOM in the previous year (P = 0.006) were risk factors for CJ-AOM. Persistence of AOM was frequently observed in CJ-AOM. The frequency of AOM secondary to conjunctivitis was reduced (P = .01) in amoxicillin recipients (2 of 41) compared with placebo (11 of 42), but amoxicillin failed to eradicate nasopharyngeal carriage of H. influenzae. More episodes of AOM per year (P less than 0.001) and day care (P less than 0.001) were found to be risk factors for AOM secondary to conjunctivitis.