Middle East respiratory syndrome coronavirus not detected in children hospitalized with acute respiratory illness in Amman, Jordan, March 2010 to September 2012.

Hospitalized children < 2 years of age in Amman, Jordan, admitted for fever and/or respiratory symptoms, were tested for Middle East respiratory syndrome coronavirus (MERS-CoV): MERS-CoV by real-time RT-PCR (rRT-PCR). This was a prospective year-round viral surveillance study in children <2 years of age admitted with acute respiratory symptoms and/or fever from March 2010 to September 2012 and enrolled from a government-run hospital, Al-Bashir in Amman, Jordan. Clinical and demographic data, including antibiotic use, were collected. Combined nasal/throat swabs were collected, aliquoted, and frozen at -80°C. Specimen aliquots were shipped to Vanderbilt University and the Centers for Disease Control and Prevention (CDC), and tested by rRT-PCR for MERS-CoV. Of the 2433 subjects enrolled from 16 March 2010 to 10 September 2012, 2427 subjects had viral testing and clinical data. Of 1898 specimens prospectively tested for other viruses between 16 March 2010 and 18 March 2012, 474 samples did not have other common respiratory viruses detected. These samples were tested at CDC for MERS-CoV and all were negative by rRT-PCR for MERS-CoV. Of the remaining 531 samples, collected from 19 March 2012 to 10 September 2012 and tested at Vanderbilt, none were positive for MERS-CoV. Our negative findings from a large sample of young Jordanian children hospitalized with fever and/or respiratory symptoms suggest that MERS-CoV was not widely circulating in Amman, Jordan, during the 30-month period of prospective, active surveillance occurring before and after the first documented MERS-CoV outbreak in the Middle East region.

Incidence of respiratory pathogens in persons hospitalized with pneumonia in two provinces in Thailand.

Although pneumonia is a leading cause of death from infectious disease worldwide, comprehensive information about its causes and incidence in low- and middle-income countries is lacking. Active surveillance of hospitalized patients with pneumonia is ongoing in Thailand. Consenting patients are tested for seven bacterial and 14 viral respiratory pathogens by PCR and viral culture on nasopharyngeal swab specimens, serology on acute/convalescent sera, sputum smears and antigen detection tests on urine. Between September 2003 and December 2005, there were 1730 episodes of radiographically confirmed pneumonia (34·6% in children aged <5 years); 66 patients (3·8%) died. A recognized pathogen was identified in 42·5% of episodes. Respiratory syncytial virus (RSV) infection was associated with 16·7% of all pneumonias, 41·2% in children. The viral pathogen with the highest incidence in children aged <5 years was RSV (417·1/100,000 per year) and in persons aged ≥50 years, influenza virus A (38·8/100,000 per year). These data can help guide health policy towards effective prevention strategies.

Adenovirus gastroenteritis in Hungary, 2003-2006.

The incidence and type distribution of enteric human adenoviruses (HAds) among diarrheic children in south-western Hungary was investigated from 2003 through 2006. Laboratory studies were conducted using commercial antigen detection tests (latex agglutination or immunochromatography), polymerase chain reaction (PCR) amplification, single-strand conformation polymorphism, and sequencing and phylogenetic analysis of a conservative region of the HAd hexon gene. The overall rate of HAd infection in childhood gastroenteritis cases during the 4-year study was 8.1%, with a gradual decrease in detection rates from 11.7% in 2003 to 5.7% in 2006. Molecular studies of a subset of HAd-positive samples found that enteric HAd type 40 strains were identified only in 2003 and 2004, while HAd type 41 strains were identified throughout the 4-year study. Higher detection rates of non-enteric HAds was documented during the first half of the study period when latex agglutination was used in our laboratory for detection. Our study suggests that the choice of diagnostic method may profoundly influence the epidemiologic picture and disease burden attributed to enteric HAd infections.

Molecular typing of human adenoviruses by PCR and sequencing of a partial region of the hexon gene.

Human adenoviruses (Ads) are responsible for a substantial disease burden. Type-specific identification of Ads can help guide therapeutic and disease prevention strategies and aid epidemiological investigations. Immunotyping of Ads by serum neutralization (SN) is laborious and time consuming and depends upon type-specific antisera that are in short supply. A rapid molecular typing assay based on polymerase chain reaction (PCR) amplification and sequencing of Ad hexon gene hyper-variable regions 1-6 (HVR(1-6)) known to contain type-specific epitopes was evaluated as an alternative to SN. Deduced amino acid sequences of HVR(1-6) obtained from all 51 currently recognized Ad prototype strains were well resolved, with the exception of types 15 and 29, which were identical. Of 192 temporally and geographically diverse Ad field isolates sequenced in this study, and 111 previously published sequences, all more closely matched their predicted prototype strains. Ads were also detected and correctly identified directly from 24 clinical specimens positive by culture or antigen detection. PCR and sequencing of HVR(1-6) offers a practical alternative to SN for typing most Ads and can be readily adapted for use in laboratories with molecular capabilities.

Sequence polymorphism in the E3 7.7K ORF of subspecies B1 human adenoviruses.

Sequences corresponding to the 7.7K open-reading frame (ORF) of the E3 region of subspecies B1 adenoviruses (Ads) were compared with prototype strains of Ad3, Ad7, Ad16, Ad21, and Ad50 and field isolates representing a variety of genome restriction types of Ad3 and Ad7 to better assess the extent of genetic variation in this intriguing region of the viral genome encoding a product whose function is still unknown. Alignment of 55 species B1 Ad sequences revealed a marked polymorphism in the 7.7K ORF and allowed the identification of eight distinct sequence profiles (SPs) characterized by (1) deletions that retain or change the reading frame, (2) single-base mutations (SBMs) that change the start codon (ATG to ATT or ATC), and (3) other SBMs. mRNAs of expected size for the observed sequence polymorphisms were identified by RT-PCR from DNAse I-treated total RNA extracts of infected cells. Predicted proteins ranged from 0 to 94 amino acids corresponding to molecular masses of 0-11 K. Together with the hypervariable regions of the hexon gene, the E3 7.7K ORF appears to be another area of the Ad genome in which genetic diversity may be generated by illegitimate recombination.

Active surveillance for respiratory virus infections in adults who have undergone bone marrow and peripheral blood stem cell transplantation.

Community-acquired respiratory virus (RV) infections are an important cause of disease in immunocompromised adults with cancer. To investigate the viral etiology, incidence, clinical presentation, and outcome of RV infections in an outpatient cohort of adult bone marrow or peripheral blood stem cell transplant (SCT) recipients, we monitored 62 outpatient volunteers from January 1 to April 30, 2001. A nasopharyngeal aspirate was collected from subjects when they reported new respiratory symptoms and tested for RV (influenza A, influenza B, human parainfluenza 1-3, adenovirus, and respiratory syncytial virus) by culture and reverse transcription polymerase chain reaction (RT-PCR) assay. Of 62 subjects enrolled, 27% had received allogeneic SCT and 45% were within 1 year of their transplant. In all, 35 participants (56%) reported 37 episodes of respiratory symptoms. Of the 37 specimens tested, five (14%) were positive for RV by culture and 20 (54%) were positive by RT-PCR. Only six patients with RV infections developed lower respiratory tract illnesses; these patients had received either autologous or allogeneic transplants and developed illnesses between 41 and 2666 days post transplant. Although RV infections were common in SCT outpatients during the RV season, most participants had upper respiratory tract infections, which resolved without the need for hospitalization.

Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes.

The common species C adenoviruses (serotypes Ad1, Ad2, Ad5, and Ad6) infect more than 80% of the human population early in life. Following primary infection, the virus can establish an asymptomatic persistent infection in which infectious virions are shed in feces for several years. The probable source of persistent virus is mucosa-associated lymphoid tissue, although the molecular details of persistence or latency of adenovirus are currently unknown. In this study, a sensitive real-time PCR assay was developed to quantitate species C adenovirus DNA in human tissues removed for routine tonsillectomy or adenoidectomy. Using this assay, species C DNA was detected in Ficoll-purified lymphocytes from 33 of 42 tissue specimens tested (79%). The levels varied from fewer than 10 to greater than 2 x 10(6) copies of the adenovirus genome/10(7) cells, depending on the donor. DNA from serotypes Ad1, Ad2, and Ad5 was detected, while the rarer serotype Ad6 was not. When analyzed as a function of donor age, the highest levels of adenovirus genomes were found among the youngest donors. Antibody-coated magnetic beads were used to purify lymphocytes into subpopulations and determine whether viral DNA could be enriched within any purified subpopulations. Separation of T cells (CD4/8- expressing and/or CD3-expressing cells) enriched viral DNA in each of nine donors tested. In contrast, B-cell purification (CD19-expressing cells) invariably depleted or eliminated viral DNA. Despite the frequent finding of significant quantities of adenovirus DNA in tonsil and adenoid tissues, infectious virus was rarely present, as measured by coculture with permissive cells. These findings suggest that human mucosal T lymphocytes may harbor species C adenoviruses in a quiescent, perhaps latent form.

Prospective study of etiologic agents of community-acquired pneumonia in patients with HIV infection.

OBJECTIVES: To study prospectively HIV-positive patients admitted to the hospital because of pneumonia by extensive laboratory tests to determine specific microbiologic diagnoses and to establish the best clinical diagnosis after review of all available data by expert clinicians. METHODS: Patients admitted to one of two hospitals had extensive questionnaires completed and defined diagnostic tests performed on blood, sputum, urine and bronchoalveolar lavage specimens, when available. RESULTS: A total of 230 patients had a diagnosis of pneumonia verified. A definite or probable etiologic diagnosis was made in 155 (67%) of these patients. Pneumocystis carinii caused 35% of all cases of pneumonia. Twenty-seven percent of cases of pneumonia with a single etiology had a definite or probable bacterial etiology. 'Atypical agents' were distinctly uncommon. Few clinical or laboratory parameters could differentiate specific etiologies. CONCLUSIONS: P. carinii continues to be a common cause of pneumonia in these patients. The rarity of 'atypical agents' could simplify the empiric approach to therapy. Despite the use of extensive testing we did not find a definite etiology in a large number of cases.

Outbreak of human parainfluenza virus 3 infections in a hematopoietic stem cell transplant population.

Clinical manifestations and epidemiological features are described for a cluster of 12 cases of human parainfluenza virus 3 (HPIV3) infection that occurred among 64 allogeneic hematopoietic stem cell transplant (SCT) recipients in an 11-week period during spring 2000. Upper respiratory symptoms predominated. Pneumonia occurred in 3 patients and was a contributing factor in the death of 1 patient. Exposure histories and molecular analysis of HPIV3 isolates suggested that both community acquired and nosocomially transmitted infections occurred during this outbreak. A chain of transmission within the outpatient clinic appeared to have occurred in 4 outpatients and to have extended to 2 hospitalized patients. Molecular epidemiology was useful in discerning routes of transmission in this outbreak.

Type-specific identification of human adenovirus 3, 7, and 21 by a multiplex PCR assay.

Human adenovirus (Ad) serotypes 3, 7, and 21 of DNA cluster B:1 are often associated with severe respiratory illness, particularly in infants and young children and, in addition to Ad4, are among the most important causes of acute respiratory disease syndrome in new military recruits. To address the inherent problems associated with classic typing methods, we developed a multiplex PCR assay for the rapid, specific identification of Ad3, Ad7, and Ad21 field isolates. To design type-specific primers for our assay, we sequenced the Ad21 hexon gene and compared this sequence with previously published sequences of Ad3, Ad7, and Ad16. The overall nucleotide (nt) and amino acid (aa) identities between Ad21 and Ad3, Ad7, and Ad16 were similar (ranges 78.3-80.8% nt; 84.1-86.2% aa), with significantly greater variability in the regions of the hexon that encode surface loops 1 and 2. Type-specific primers designed to the hypervariable regions correctly identified Ad3, Ad7, and Ad21 prototype strains and 53 previously typed Ad field isolates. No cross-reactions with other Ad serotypes were identified. Our multiplex PCR assay for type-specific identification of Ad3, Ad7, and Ad21 isolates will provide a rapid and convenient tool for the epidemiologic investigation of Ad-associated respiratory illness.

Basic fibroblast growth factor induced angiogenesis and prefabricated flap survival.

Prefabrication of a latissimus dorsi myocutaneous flap was performed in adult male Landrace pigs. Gelfoam sponges were used as a delivery system for basic fibroblast growth factor (bFGF) at the muscle-subcutaneous tissue interface. Skin survival and angiogenesis were augmented in the growth-factor-treated animals. These data support the use of basic fibroblast growth factor to enhance flap prefabrication.

Outbreak of adenovirus genome type 7d2 infection in a pediatric chronic-care facility and tertiary-care hospital.

An outbreak of adenovirus infection that involved residents of a pediatric chronic-care facility, staff of a tertiary-care hospital, and a nosocomial hospital case was studied. In the pediatric facility, 31 (33%) of 93 residents had adenovirus infection, and 8 died. Risk factors for illness were an age of < 7 years (P = .004), presence of a tracheostomy (P = .015), and residence on a particular floor (P < .001). In the tertiary-care hospital, 36 health care workers had adenovirus infection; 26 (72%) had failed to follow strict contact and droplet precautions, and 30 (83%) continued to care for patients while they had symptoms. A 5-month-old patient with underlying lung disease acquired severe adenovirus infection in this hospital. All isolates were adenovirus type 7 (Ad7). DNA restriction analysis revealed the band patterns of all isolates to be identical and characteristic of the genome type d2. Thus, Ad7d2 caused significant morbidity and mortality in persons in the pediatric chronic-care facility and tertiary-care hospital. This is the first published description of Ad7d2 strains in the United States.

Species-specific identification of human adenoviruses by a multiplex PCR assay.

A multiplex PCR assay was developed by using primers to the fiber gene that could differentiate human adenovirus (Ad) species A through F in a single amplification reaction. The assay correctly identified the species of all 49 recognized Ad prototype strains as well as 180 geographically and temporally diverse Ad field isolates. Ad serotype 6 (Ad6) (species C), Ad16 (species B), Ad31 (species A), and Ad40 and Ad41 (species F) could also be distinguished by amplicon size within each respective species. In comparison, a previously described Ad species-specific multiplex PCR assay that used primers to the Ad hexon gene gave equivocal results with several serotypes of species B, whereas our multiplex assay amplified all species B serotypes equally well. Our multiplex PCR assay will permit rapid, accurate, and cost-effective classification of Ad isolates.

Rapid molecular epidemiologic studies of human parainfluenza viruses based on direct sequencing of amplified DNA from a multiplex RT-PCR assay.

Sequencing studies of limited regions of the human parainfluenza viruses (HPIVs) genomes have helped describe patterns of virus circulation and characterize institutional outbreaks of HPIVs-associated respiratory illness. In this study, we sequenced reverse transcription polymerase chain reaction (RT-PCR)-amplified HPIVs RNA obtained from a multiplex RT-PCR assay described previously for simultaneous detection of HPIV-1, 2 and 3. Differences in the nucleotide sequences of limited regions of the HN gene allowed us to distinguish temporally and geographically diverse HPIV isolates (43 HPIV-1, 7 HPIV-2, 12 HPIV-3 isolates from this and previously published studies). In addition, an outbreak of HPIV-3-associated illness among infants on a pediatric ward was investigated by comparing sequences of three ward isolates with three matched community controls. Sequences of all ward isolates were identical and differed from those of the community controls, suggesting a single introduction and nosocomial transmission of the virus. Combining multiplex reverse transcription polymerase chain reaction (RT-PCR) assays with direct sequencing of the PCR products can provide an integrated system for rapid diagnosis and characterization of HPIVs.

Investigation of porcine parvovirus among persons with hemophilia receiving Hyate:C porcine factor VIII concentrate.

BACKGROUND: Porcine clotting factor has been used for more than 15 years to treat severe bleeding episodes in persons with hemophilia who have antibodies to human clotting factor. In 1996, QC procedures revealed for the first time the presence of porcine parvovirus (PPV) in the product. This report describes an investigation to determine the extent of product contamination and to evaluate past recipients of porcine clotting factor (Hyate:C, Speywood Biopharm) for evidence of PPV infection. STUDY DESIGN AND METHODS: Stored specimens from 22 lots of previously released Hyate:C were tested for the presence of PPV DNA by PCR and nested PCR assays. Serum specimens from 98 recipients of Hyate:C and 24 controls who did not receive Hyate:C were tested for PPV antibodies by an immunofluorescence assay. RESULTS: PPV DNA was detected in product from 21 of the 22 lots of Hyate:C, primarily by nested PCR testing. In contrast, none of the serum specimens from the 98 Hyate:C recipients tested positive for PPV IgG antibodies. CONCLUSION: The risk of human disease from percutaneous exposure to low levels of PPV seems to be low. Nevertheless, the theoretical risk of human infection with PPV has led to manufacturing changes, including PCR screening of all porcine plasma, which are designed to eliminate this risk.

The pattern of dyslipidemia among urban African-Americans with type 2 diabetes.

OBJECTIVE: To analyze lipid profiles from a large sample of African-American patients with type 2 diabetes who receive care at an urban outpatient diabetes clinic. RESEARCH DESIGN AND METHODS: Fasting serum lipid profiles of 4,014 African-Americans and 328 Caucasians with type 2 diabetes were retrieved from a computerized registry. American Diabetes Association criteria were applied to classify LDL cholesterol, HDL cholesterol, and triglyceride (TG) levels into risk categories. The proportion of patients who had none, one, two, and three lipoprotein concentrations outside of recommended clinical targets was examined. Multiple logistical regression analyses were performed to determine the influence of sex and race on the probability of having a lipid level outside of the recommended target. RESULTS: The percentages of African-Americans with high-, borderline-, and low-risk LDL cholesterol concentrations were 58, 26, and 16%, respectively, and the percentages for Caucasians were 54, 29, and 16%, respectively (P = 0.51). For HDL cholesterol, 41, 33, and 26% of African-Americans were in the high-, borderline-, and low-risk categories, respectively, compared with 73, 18, and 9% of Caucasians, respectively (P < 0.0001). Nearly 81% of African-Americans had TG concentrations that were in the low-risk category compared with only 50% of Caucasians. More women than men had high-risk LDL and HDL cholesterol profiles. The most common pattern of dyslipidemia was an LDL cholesterol level above target combined with an HDL cholesterol level below target, which was detected in nearly 50% of African-Americans and 42% of Caucasians. African-Americans had lower odds of having an HDL cholesterol or TG level outside of target. African-American women, compared to men, had greater probabilities of having abnormal levels of LDL and HDL, but a lower likelihood of having a TG level above goal. CONCLUSIONS: In a large sample of urban type 2 diabetic patients receiving care at a diabetes treatment program, race and sex differences in serum lipid profiles were present. Because hypertriglyceridemia was rare among African-American subjects, interventions will need to focus primarily on improving their LDL and HDL cholesterol levels. Further studies are required regarding how to best adapt these observed differences into more effective strategies to optimize lipid levels for this population of diabetic patients and to determine whether similar patterns of dyslipidemia occur in other clinical settings.

Non-enveloped viruses transmitted by blood and blood products.

Since the advent of solvent detergent (S-D) treatment for inactivation of enveloped viruses, there has been no transmission of human immunodeficiency virus, hepatitis B virus, or hepatitis C virus by treated blood products. However, shortly after the introduction of S-D treatment, transmission of hepatitis A with S-D treated factor concentrates was reported in Germany, Italy, Ireland, the United States and South Africa, and this raised awareness of the potential for blood transmission of non-enveloped viruses in general. This report summarizes the physical and epidemiological features of three non-enveloped viruses, hepatitis A virus, parvovirus B19, and the recently identified TT virus, and their transmission by blood and blood products.

Detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-PCR.

We describe a multiplex reverse transcription-PCR (m-RT-PCR) assay that is able to detect and differentiate all known human parainfluenza viruses (HPIVs). Serial dilution experiments with reference strains that compared cell culture isolation and m-RT-PCR showed sensitivities ranging from 0.0004 50% tissue culture infective dose (TCID(50)) for HPIV type 4B (HPIV-4B) to 32 TCID(50)s for HPIV-3. As few as 10 plasmids containing HPIV PCR products could be detected in all cases. When 201 nasopharyngeal aspirate specimens from pediatric patients hospitalized for lower respiratory illness were tested, m-RT-PCR assay detected 64 HPIVs (24 HPIV-3, 23 HPIV-1, 10 HPIV-4, and 7 HPIV-2), while only 42 of them (21 HPIV-1, 14 HPIV-3, 6 HPIV-2, and 1 HPIV-4 isolates) grew in cell culture. Our m-RT-PCR assay was more sensitive than either cell culture isolation or indirect immunofluorescence with monoclonal antibodies for the detection of HPIV infections. Also, HPIV-4 was more frequently detected than HPIV-2 in this study, suggesting that it may have been underestimated as a lower respiratory tract pathogen because of the insensitivity of cell culture.

An outbreak of acute respiratory disease caused by Mycoplasma pneumoniae and adenovirus at a federal service training academy: new implications from an old scenario.

Outbreaks of Mycoplasma pneumoniae and adenovirus have been reported in military institutions for several decades. During a recent outbreak in a federal service training academy, we performed an epidemiological and laboratory investigation to better characterize and control the outbreak. Of 586 students responding to a questionnaire, 317 (54%) reported having a respiratory illness during the outbreak period. Among 42 students who underwent complete laboratory testing, 24 (57%) had evidence of M. pneumoniae infection, 8 (19%) had evidence of adenovirus infection, and 4 (10%) had evidence of both. Polymerase chain reaction testing of oropharyngeal swabs revealed more acute M. pneumoniae infections (57% positive) than did serology or culture. Multivariate analysis revealed that visiting the campus health clinic >3 times for a nonrespiratory condition, such as injury, was a significant risk factor for illness among freshmen early in the course of the outbreak, whereas having an ill roommate was a risk factor throughout the duration of the outbreak.

Molecular characterization of a nosocomial outbreak of human respiratory syncytial virus on an adult leukemia/lymphoma ward.

Although nosocomial transmission of human respiratory syncytial virus (HRSV) and its effect on morbidity and mortality among immunocompromised adults are well recognized, few studies have applied molecular techniques to differentiate nosocomial from community-acquired infections. Between January and April 1997, an outbreak of HRSV occurred among adult patients in a leukemia/lymphoma ward. Among 45 hospitalized patients undergoing bronchoscopy for investigation of acute respiratory illness, 8 were identified with HRSV infection. One infected patient developed symptoms before admission and was thought to be the index case. However, subsequent sequencing of 7 HRSV isolates identified 2 distinct genotypes, GA5 (1 case) and GB3 (6 cases). The 6 GB3 isolates could be further differentiated into 2 strains with identical nucleotide sequences that differed from each other and from 14 community HRSV isolates. Instead of a single nosocomial outbreak of HRSV, multiple introductions of HRSV likely occurred with distinct lines of nosocomial transmission.