Antonietta Marchi

Correlation of Rhinovirus Load in the Respiratory Tract and Clinical Symptoms in Hospitalized Immunocompetent and Immunocompromised Patients

While human rhinoviruses (HRVs) are well accepted as a major cause of common cold syndromes (rhinitis), their role in the etiology of lower respiratory tract infections is still controversial, and their detection in asymptomatic patients is relatively common. The HRV pathogenic role in four groups of hospitalized patients (pediatric immunocompetent and immunocompromised patients, and adult immunocompetent and immunocompromised patients) was investigated by quantifying HRV load in nasopharyngeal aspirates or bronchoalveolar lavage samples by real‐time reverse transcription PCR (RT‐PCR). Real‐time RT‐PCR was performed in duplicate on all respiratory samples resulting positive by qualitative RT‐PCR. In addition, molecular typing allowed detection of all known HRV species (A, B, and C). In immunocompetent pediatric patients HRVs were mostly associated with lower respiratory tract infections (in the absence of other viral agents) and wheezing, when viral load was ≥106 RNA copies/ml. In young immunocompromised patients (stem cell transplantation recipients), an inverse correlation between HRV persistence over time and time at which the infection occurred after transplantation was observed, whereas in adult immunocompromised patients (lung transplant recipients) HRVs could be detected at a medium–low level (<105 RNA copies/ml) in bronchoalveolar lavage samples taken routinely from asymptomatic patients. In conclusion, when detected at high viral load, HRVs may cause severe upper and lower respiratory tract infections, whereas when detected at a medium–low viral load, an event more frequent in immunocompromised subjects, they may represent only bystander viruses. J. Med. Virol. 81:1498–1507, 2009.

Genetic variability of human coronavirus OC43-, 229E-, and NL63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients.

In the winter–spring seasons 2003–2004 and 2004–2005, 47 (5.7%) patients with acute respiratory infection associated with human coronavirus (hCoV) 229E‐, NL63‐, and OC43‐like strains were identified among 823 (597 immunocompetent and 226 immunocompromised) patients admitted to hospital with acute respiratory syndromes. Viral infections were diagnosed by either immunological (monoclonal antibodies) or molecular (RT‐PCR) methods. Each of two sets of primer pairs developed for detection of all CoVs (panCoV) failed to detect 15 of the 53 (28.3%) hCoV strains identified. On the other hand, all hCoV strains could be detected by using type‐specific primers targeting genes 1ab and N. The HuH‐7 cell line was found to be susceptible to isolation and identification of OC43‐ and 229E‐like strains. Overall, hCoV infection was caused by OC43‐like, 229E‐like, and NL63‐like strains in 25 (53.2%), 10 (21.3%), and 9 (19.1%) patients, respectively. In addition, three patients (6.4%) were infected by untypeable hCoV strains. NL63‐like strains were not found to circulate in 2003–2004, and 229E‐like strains did not circulate in 2004–2005, while OC43‐like strains were detected in both seasons. The monthly distribution reached a peak during January through March. Lower predominated over upper respiratory tract infections in each age group. In addition, hCoV infections interested only immunocompetent infants and young children during the first year of life, while all adults were immunocompromised patients. Coinfections of hCoVs and other respiratory viruses (mostly interesting the first year of life) were observed in 14 of the 47 (29.8%) patients and were associated with severe respiratory syndromes more frequently than hCoV single infections (P = 0.002). In conclusion, the use of multiple primer sets targeting different genes is recommended for diagnosis of all types of hCoV infection. In addition, the detection of still untypeable hCoV strains suggests that the number of hCoVs involved in human pathology might further increase. Finally, hCoVs should be screened routinely for in both infants and immunocompromised patients with acute respiratory infection. J. Med. Virol. 78:938–949, 2006.

Human respiratory coronavirus HKU1 versus other coronavirus infections in Italian hospitalised patients

Abstract Background Human respiratory coronavirus (hCoV) HKU1 infections were reported for the first time in 2005 in Hong Kong. Objective To investigate epidemiological, clinical, and diagnostic features of HKU1 infections. Study design Longitudinal, prospective study from November 2005 through May 2006 in a hospitalised patient population. Results Overall, 48/426 (11.3%) patients were found to be infected by hCoV acute respiratory tract infections (ARTI). Of these, 10 (19.2%) were caused by HKU1 (6 single infections and 4 coinfections) during the period January–May 2006. Diagnosis was made by using RT-PCR for all four hCoVs, and in parallel, in-house developed group-specific monoclonal antibodies (MAbs) for HKU1 and 229E. HKU1-specific MAb was able to retrospectively identify 8 of 10 HKU1 strains detected by RT-PCR. Phylogenetic analysis showed that four HKU1 strains were genotype A and six genotype B. In HKU1-infected patients, the predominant clinical symptom was rhinorrhea (nine patients). Within group II hCoV, HKU1-infected patients had a significantly lower rate of lower ARTI compared to OC43-infected patients. Conclusion HKU1 hCoV strains circulated in northern Italy during the winter–spring season 2005–2006. Both HKU1 genotypes were detected. HKU1-specific MAb may contribute to the rapid diagnosis of HKU1 infections currently performed by RT-PCR.

Detection and pathogenicity of human metapneumovirus respiratory infection in pediatric Italian patients during a winter–spring season

Abstract Background Some diagnostic, epidemiological and clinical features of the recently discovered human metapneumovirus remain to be investigated. Objectives To study the best approach for the diagnosis of human metapneumovirus infections by both conventional and molecular methods, along with the human metapneumovirus circulation rate in northern Italy and the severity of human metapneumovirus respiratory infections in a pediatric patient population. Study design Nasopharyngeal aspirates (NPA) were taken from 306 pediatric patients during the winter–spring season 2003–2004, and examined for conventional respiratory viruses by direct fluorescent staining and cell culture, while human coronavirus and human metapneumovirus were sought by RT-PCR. Results RT-PCR detected human metapneumovirus in 40/306 (13.1%) children positive for respiratory viruses, with an incidence intermediate between that of respiratory syncytial virus (58 patients, 18.9%) and that of influenzavirus infections (29 patients, 9.5%). Phylogenetic analysis showed cocirculation of both human metapneumovirus types (A and B) as well as their relevant subtypes (A1–A2 and B1–B2). Clinically, human metapneumovirus was found to be second to human respiratory syncytial virus alone, as a cause of respiratory tract infections, while duration of virus excretion appeared to correlate with severity of infection, and virus load in NPA with the stage of respiratory infection. Conclusion (i) Human metapneumovirus is a major viral pathogen in the Italian pediatric patient population; (ii) the severity of lower respiratory tract infections approaches that of human respiratory syncytial virus; (iii) there are preliminary indications that the duration of virus excretion may reach 2–3 weeks and that the level of viral load in NPA correlates with the clinical stage of human metapneumovirus infection.

Changing circulation rate of human metapneumovirus strains and types among hospitalized pediatric patients during three consecutive winter-spring seasons

Summary.From 2001 through 2004, 808 pediatric patients admitted to hospital because of acute respiratory infections were examined for presence of respiratory viruses by either direct fluorescent staining using monoclonal antibodies or RT-PCR during three consecutive winter-spring seasons. On the whole, 336 (42%) patients were detected as positive for one or more respiratory viruses. The most widely circulating virus was human respiratory syncytial virus (hRSV) infecting 50% of positive patients, followed by human metapneumovirus (hMPV) found in 13% of patients, and then by influenza virus type A, human parainfluenzaviruses and coinfections. Significant variations in the circulation rate of hRSV, hMPV and influenzavirus type A were observed during the individual seasons. In addition, the circulation rates of the different types of hMPV changed yearly. In 2001–2002 and 2002–2003 hMPV circulated at a significant lower proportion than hRSV, while in 2003–2004 the circulation rates of the two viruses were closer. In conclusion, the 4 hMPV subtypes circulated yearly in Northern Italy flanking hRSV as major respiratory pathogens in the infantile patient population.

Human rhinovirus and human respiratory enterovirus (EV68 and EV104) infections in hospitalized patients in Italy, 2008-2009.

The epidemiology of picornavirus infections along with associated risk factors for lower respiratory tract infections (LRTI) and duration of virus shedding were investigated in 985 hospitalized patients in the period October 2008-September 2009. One-third of patients were human rhinovirus (HRV)-positive. Of 336 HRV-associated episodes, 153 (45.5%) were sustained by HRV-A, 31 (9.2%) by HRV-B, and 93 (27.7%) by HRV-C, while 7 episodes showed multiple HRV types and 52 were sustained by undefined HRV species. Independent risk factors for LRTI included high viral load and age less than 5 years. Twenty (2.1%) patients were enterovirus (EV)-positive (12 had EV-68, 7 EV-104, and 1 E-13 infection). Half of the EV-positive patients had a LRTI and were younger with respect to patients with upper RTI (median 18 months versus 37 years; P < 0.001). HRVs are often the cause of LRTI in children less than 5 years, frequently in association with a high viral load.

Phylogenetic characterization of enterovirus 68 strains in patients with respiratory syndromes in Italy.

Enterovirus 68 (EV‐D68) was associated with mild to severe respiratory infections. In the last 4 years, circulation of different EV‐D68 strains has been documented worldwide. In this study, the phylogenetic characterization of nine EV‐D68 strains identified in patients in the 2010–2012 period and 12 additional EV‐D68 Italian strains previously identified in 2008 in Italy was described. From January 2010 to December 2012, a total of 889 respiratory specimens from 588 patients stayed or visited at the Fondazione IRCCS Policlinico San Matteo were positive for HRV or HEV. Extracted nucleic acids were amplified by one‐step RT‐PCR with primer specific for VP1 region of EV‐D68 and purified positive PCR products were directly sequenced. Overall, 9/3736 (0.24%) patients were EV‐D68 positive. Of these, 7/9 (77.8%) were pediatric and two (22.2%) were adults. Five out of seven (71.4%) pediatric patients had lower respiratory tract infection with oxygen saturation <94%. Four cases were detected from August through October 2010, while five other cases from September through December 2012. The Italian EV‐D68 strains in 2008 belonged to clade A (n = 5) and clade C (n = 7). In 2010 all the Italian strains belonged to clade A (n = 4) and in 2012, four Italian strains belonged to clade B and one to clade A. In conclusion, we provide additional evidence supporting a role of EV‐D68 in severe respiratory infection in pediatric patients. In addition, all the three EV‐D68 clades circulating worldwide were identified in Italy in a 5‐year period of time. J. Med. Virol. 86:1590–1593, 2014.

Human parechovirus infections in patients admitted to hospital in Northern Italy, 2008-2010

Human parechoviruses (HPeVs) infection is associated with a wide range of clinical syndromes such as respiratory, gastrointestinal, neurologic diseases, and neonatal sepsis‐like illness. The main objective of this study was to investigate the epidemiology of HPeVs infection in hospitalized patients in a period of 2 years. Respiratory samples from 3,525 patients with respiratory syndrome, cerebrospinal fluid (CSF) from 340 patients with neurologic syndrome as well as CSF and plasma samples from five neonatal patients with sepsis‐like illness collected from October 2008 to 2010 were tested retrospectively using HPeV‐specific real‐time RT‐PCR. Phylogenetic analysis of VP3/VP1 region was performed on the positive samples. Fourteen out of 3,525 (0.4%) patients with respiratory syndrome and five out of five patients with sepsis‐like illness were positive for HPeV. In 3/5 patients with sepsis‐like illness multiple samples (e.g., stool, plasma, CSF, or respiratory samples) were available, and HPeV was found in all specimens. In contrast, no positive CSF was detected among the 340 patients with neurologic syndromes. Eleven patients (57.9%) were infected with HPeV1 strain, 7 (36.8%) with HPeV3, and 1 (5.3%) with HPeV6 strains. Ten of the 14 HPeV patients with respiratory syndrome were co‐infected with other respiratory viruses (eight with rhinovirus and two with coronavirus OC43). All five patients with sepsis‐like illness were less than 1 month of age and were infected with HPeV3. Although not circulating at high frequency and unlikely to cause respiratory syndrome, HPeV was associated with severe clinical syndromes in a minority of newborns. J. Med. Virol. 84:686–690, 2012.

Human respiratory syncytial virus (hRSV) RNA quantification in nasopharyngeal secretions identifies the hRSV etiologic role in acute respiratory tract infections of hospitalized infants

Abstract Background Human respiratory syncytial virus (hRSV) detection in nasopharyngeal aspirates (NPAs) from infants with acute respiratory tract infection (ARTI) does not prove the hRSV etiology of the current ARTI episode. HRSV RNA quantification may help in affording this issue. Objectives hRSV was detected by quantitative reverse transcription-PCR in NPAs taken upon admission to hospital and, whenever possible, at discharge and subsequent medical visits. Study design Prospective study, including 63 infants affected by either hRSV upper or lower ARTI. Results Based on the kinetics of viral load, hRSV etiology was identified in 25 infants in whom hRSV load dropped from 2.5×106 upon admission (presence of respiratory symptoms) to 7.5×102 RNAcopies/ml NPA upon discharge (absence of symptoms) after a median time of 5 days, and in 19 infants, in whom hRSV load was determined at admission only, in association with clinical symptoms (2.4×106 copies/ml). Furthermore, low levels of hRSV RNA (<1×105 copies/ml NPA) identified 14 patients with non-hRSV ARTI. Finally, in 14 infants with hRSV coinfections or sequential infections, hRSV quantification defined the hRSV role in the current ARTI episode. Conclusions hRSV RNA quantification is critical in defining the hRSV role in respiratory infections.

Isochromosome (7)(q10) in Shwachman syndrome without MDS/AML and role of chromosome 7 anomalies in myeloproliferative disorders.

Shwachman syndrome (SS) is an autosomal recessive disorder in which bone marrow dysfunction is observed, with development of myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML) in up to one third of the cases. Inconclusive data are available as to increased chromosome breakage in SS, while chromosome 7 anomalies, and often an isochromosome (7)(q10), are frequent in cases with MDS/AML. We report on the consistent presence of an i(7)(q10) in the bone marrow and blood lymphocytes in one of two sisters affected with SS without any clinical or cytological signs of MDS/AML. Thus, this patient was either a case of constitutional mosaicism for the i(7)(q10), or this had to be acquired in a nondysplastic and non-neoplastic marrow clone. DNA polymorphism analysis demonstrated the paternal origin of the i(7q). We postulate that the SS mutation acts as a mutator gene, and causes karyotype instability; abnormal clones would thus arise in the marrow, and chromosome 7 anomalies, i(7q) in particular, will in turn lead to MDS/AML. If this interpretation is correct, it would be also an indication to consider chromosome 7 anomalies in general, out of SS, as primary changes in MDS/AML pathogenesis.