Distribution of VP7 serotypes and VP4 genotypes among rotavirus strains recovered from Italian children with diarrhea

Summary.  108 rotavirus strains obtained from children with diarrhea hospitalized in Palermo, Italy, in the years 1990–1994, were examined by seminested PCR to study the relative frequency and distribution of the four most common alleles of the gene 4. Such strains were selected from 344 human rotavirus strains recovered in Palermo during those years after characterization by electropherotyping, subgrouping and G serotyping. One hundred and seven of the 108 strains could be classified into P types, the P[8], G1 (38.3%) and the P[8], G4 (52.3%) types being predominant. The unique strain whose P genotype could not be identified showed an unusual combination of long migration electrophoretic pattern and subgroup I specificity. Received January 13, 1997 Accepted June 11, 1997     

Relative frequencies of G (VP7) and P (VP4) serotypes determined by polymerase chain reaction assays among Japanese bovine rotaviruses isolated in cell culture.

The relative frequencies of both the G (VP7) and P (VP4) serotypes of 40 bovine rotaviruses isolated in cell culture from diarrheic calves in Japan between January 1983 and February 1991 were determined by recently developed polymerase chain reaction assays. Isolates with G serotype 6 and P serotype 5 (UK-like strains) were most frequently found (42.5%) followed by isolates with G6P11 (17.5%), G6P1 (10%), or G10P5 (10%). Isolates with G10P11 (B223-like strains) were least frequently found (7.5%). The presence of various combinations of G and P serotypes suggests frequent reassortment in nature among bovine rotaviruses.     

Molecular epidemiology of Pseudomonas aeruginosa in intensive care units over a 10-year period (1998-2007)

Pseudomonas aeruginosa is one of the leading nosocomial pathogens in intensive care units (ICUs). The source of this microorganism can be either endogenous or exogenous. The proportion of cases as a result of transmission is still debated, and its elucidation is important for implementing appropriate control measures. To understand the relative importance of exogenous vs. endogenous sources of P. aeruginosa, molecular typing was performed on all available P. aeruginosa isolated from ICU clinical and environmental specimens in 1998, 2000, 2003, 2004 and 2007. Patient samples were classified according to their P. aeruginosa genotypes into three categories: (A) identical to isolate from faucet; (B) identical to at least one other patient sample and not found in faucet; and (C) unique genotype. Cases in categories A and B were considered as possibly exogenous, and cases in category C as possibly endogenous. A mean of 34 cases per 1000 admissions per year were found to be colonized or infected by P. aeruginosa. Higher levels of faucet contamination were correlated with a higher number of cases in category A. The number of cases in category B varied from 1.9 to 20 cases per 1000 admissions. This number exceeded 10/1000 admissions on three occasions and was correlated with an outbreak on one occasion. The number of cases considered as endogenous (category C) was stable and independent of the number of cases in categories A and B. The present study shows that repeated molecular typing can help identify variations in the epidemiology of P. aeruginosa in ICU patients and guide infection control measures.     

Tuberculous meningitis. 23 cases from a 12-year period (1976-1987)

Twenty-three patients with tuberculous meningitis were reviewed to see whether clinical features or initial laboratory findings could discriminate between these patients and other patients with bacterial meningitis. Nineteen patients were Danes and four immigrants. Preexisting diseases were found in eight cases. Duration of symptoms could be related to neurological sequelae, but not to death. The initial clinical picture was indistinguishable from meningitis of other causes. Microscopy of the cerebrospinal fluid (CSF) was negative in all but two cases, where acid fast bacilli were found. CSF cytology and biochemistry could not discriminate from other causes bacteria of meningitis although CSF/blood glucose ratio in 56% was below 0.3. One of the most important pieces of information in establishing an early diagnosis in tuberculous meningitis is the anamnestic information, and therapy often has to be started without knowing the microbiological data.     

Streptococcus pneumoniae serotype 1 causing invasive disease among children in Barcelona over a 20-year period (1989–2008)

Fifty-six isolates of serotype 1 were identified during a 20-year prospective study (1989–2008), including all children with culture-proven invasive pneumococcal disease (IPD) admitted to a children's hospital in Barcelona. Forty-eight of them (85.7%) were in children aged >2 years. Complicated pneumonia (n = 28) and non-complicated pneumonia (n = 20) were the main clinical presentations. The frequency of serotype 1 IPD increased from 1999–2003 to 2004–2008: 1.2 to 4.4 episodes/100 000 children (p <0.001). The ST306 clone were identified in 70.4% of isolates. As IPD caused by serotype 1 is mainly detected in older children, a vaccination programme for children >2 years should be considered.     

Rare AU-1-like G3P[9] human rotaviruses with a Kun-like NSP4 gene detected in children with diarrhea in Italy

Three G3P[9] rotaviruses, detected in children hospitalized with gastroenteritis in Palermo, Italy, were found to be genetically related to strains of either human or feline origin in the VP7, VP4, and VP6 genes. In contrast, in the NSP4 gene the viruses resembled G2P[4] human strains, suggesting a reassortment between AU-1-like and Kun-like strains.     

Reactivity of VP4-specific monoclonal antibodies to a serotype 4 porcine rotavirus with distinct serotypes of human (symptomatic and asymptomatic) and animal rotaviruses.

Thirteen hybridomas secreting VP4-specific monoclonal antibodies against the Gottfried strain of porcine rotavirus (serotype 4) were produced and characterized. Nine of the hybridomas secreted neutralizing monoclonal antibodies (N-MAbs) against Gottfried rotavirus. These N-MAbs were divided into five distinct groups (groups I to V) according to their patterns of reactivity with different serotypes of human and animal rotaviruses. Group I N-MAbs (n = 3) were cross-reactive with five different serotypes of human rotavirus examined by a plaque reduction virus neutralization test. Group II N-MAbs (n = 3) neutralized all symptomatic human rotavirus serotypes tested and asymptomatic human rotavirus serotype 4 to a low titer. The single group III N-MAb neutralized mainly symptomatic human rotavirus serotypes 2 and 9 and none of the asymptomatic human rotavirus serotypes. The one N-MAb in group IV reacted at low titers with only asymptomatic human rotavirus serotypes 1 through 4. A group V N-MAb recognized serotype 4 porcine rotaviruses (Gottfried and SB-2) but no other human or animal rotaviruses examined. None of the N-MAbs recognized any animal rotaviruses tested (SA-11, RRV, OSU, NCDV, and B223), except for the Gottfried and SB-2 rotaviruses. The failure of N-MAbs (groups I to IV) to react with any animal rotaviruses tested but their ability to react variably with all human rotaviruses tested suggest that neutralizing epitopes on the VP4 protein are highly conserved between the Gottfried porcine and human rotaviruses. The Gottfried rotavirus may possibly represent a naturally occurring reassortant between pig and human rotaviruses or a rotavirus which is human in origin but pathogenic for swine.     

Analysis of human rotavirus G1P[8] strains by RFLP reveals higher genetic drift in the VP7 than the VP4 gene during a 4-year period in Mexico

Several studies have demonstrated that rotaviruses of the G1P[8] genotype are among the most important worldwide. Sequence analysis of G1P[8] strains has revealed high genetic variability of VP4 and VP7 genes. The aim of this study was to investigate by restriction fragment length polymorphism (RFLP) analysis the genetic variability of the VP7 and VP4 genes within rotaviruses of the G1P[8] genotype. A total of 60 rotavirus-positive fecal samples genotyped as G1P[8], were collected from children with acute diarrhea under 5 years of age, between October 1995 and October 1998. The VP7 and VP4 genes were amplified by RT/PCR, using the Beg9/End9 primer pair and the Con3 and Con2 primers, respectively. VP7 amplicons were digested with three restriction enzymes Hae III, Taq I and Rsa I in separate reactions and VP4 amplicons were digested similarly with endonucleases Hinf I, Sau96 I and Rsa I. Analysis of the digested VP7 and VP4 amplicons showed a higher genetic drift for the VP7 gene (18 RFLPs) compared to the VP4 gene (9 RFLPs). The combination of profiles for both VP7 and VP4 amplicons, showed 27 different patterns, none of them similar to the Wa-1 strain. Furthermore, RFLP analysis of these G1P[8] strains, clearly differentiated the viruses into two main clusters, both of them sharing the same restriction pattern for the VP4 gene, and a different one for the VP7 gene.     

Papillary cystadenoma lymphomatosum (Warthin's tumor) in patients in a general hospital over a 24-year period.

One hundred thirty-seven Warthin's tumors from 120 patients seen during a 23 1/2 year period were studied as two groups. The first group consisted of 32 tumors removed by the otolaryngology department. Material from these cases was re-examined microscopically for evidence of tumor occurrence within lymph nodes located inside and outside the parotid gland. This group of 32 tumors was also studied as part of the second group, 105 additional lesions removed by the surgery department. Data on patient age, sex, and race, as well as tumor multiplicity, location, and size were incorporated into a single statistical analysis. Seventy-two percent of the tumors in the first group were intranodal in location. Second primary Warthin's tumors occurred in 13 (11%) of the patients. Eight occult tumors were identified, and their importance is discussed. A single Negro, a man, was found among 118 patients. The observed frequency of the tumor was twentyfold less than expected among the Negro population.     

Nontyphoidal Salmonella causing focal infections in patients admitted at a Spanish general hospital during an 11-year period (1991-2001)

In focal infections (FI) caused by nontyphoidal Salmonella serotypes and recorded at a Spanish hospital 1991-2001, clinical and microbiological features were analyzed. Thirty-five revised episodes were related to infections of the digestive (10), urinary (10), pulmonar (4), vascular (4), osteoarticular (3) and central nervous (3) systems, and with a submaxillary lymph node. At least 16 episodes were associated with previous or concomitant gastroenteritis, 19 with primary or secondary bacteremia, and 18 with underlying diseases of different severity. Eighteen patients were male and 14 female (data were not available for three patients), while 1, 4, 12 and 15 patients were, respectively, categorized as children, young adults, senior adults and elderly. Sources of Salmonella strains were urine (13), blood (11), purulent abscess (8), cerebrospinal fluid (3), peritoneal fluid, pleural fluid, wound exudates, aneurism (2 of each), ascitic fluid, sputum, tracheal aspirate, needle aspirate, bone and lymph node (1 of each) samples. Only 28 Salmonella strains involved in FIs were available for further analysis. They were discriminated into 6 serotypes, and into 13 XbaI macrorestriction, 6 virulence, 11 antimicrobial resistance, 5 integron and 10 plasmid profiles. Broadly, the pattern of serotype distribution of salmonellas involved in FIs matched that of those causing gastroenteritis, with the pandemic Enteritidis and Typhimurium (18 and 6 strains, respectively) being clearly predominant. Within serotype, the same lineages (as revealed by XbaI-macrorestriction analysis as well as R- and V-profiles) were represented in both disease groups, with host-related factors apparently playing a more critical role than the individual strain in the outcome of the disease.     

VP7, VP4, and NSP4 genes of species a rotaviruses isolated from sewage in Nigeria, 2014/2015: partial sequence characterization and biophysical analysis of NSP4 (enterotoxin)

Virus Genes - Species A rotavirus are an important cause of childhood gastroenteritis, and the main contributor to its pathogenicity is the enterotoxin (NSP4) protein. Some biophysical properties...     

Single gene substitution rotavirus reassortants containing the major neutralization protein (VP7) of human rotavirus serotype 4.

A series of reassortants was isolated from coinfection of cell cultures with wild-type bovine rotavirus (UK strain [serotype 6]) or rhesus rotavirus (strain MMU18006 [serotype 3]) and a tissue culture-adapted human rotavirus strain, ST3 (serotype 4). Monospecific antiserum or a set of monoclonal antibodies to the major outer capsid neutralization glycoprotein, VP7, of the animal rotavirus parent was used to select for reassortants with human rotavirus serotype 4 neutralization specificity. The majority of reassortants contained only gene 9 of the human rotavirus parent, ST3, whereas the remaining genes were derived from the animal rotavirus parent. These single human rotavirus gene substitution reassortants were neutralized to high titer by hyperimmune serum directed at ST3, thus demonstrating that gene 9 of ST3 codes for the major neutralization protein of this strain. Moreover, these single gene substitution, reassortants were also neutralized to low titer by antiserum directed at their animal rotavirus parent, probably because they derived gene 4, which codes for another outer capsid protein, VP3, from their animal rotavirus parent. None of the reassortants derived gene 4, which had previously been shown to be responsible for host range restriction of human rotaviruses in tissue culture, from ST3, despite the fact that the ST3 strain used for gene reassortment had been tissue culture adapted.     

Gender differences in memory and learning in children with insulin-dependent diabetes mellitus (IDDM) over a 4-year follow-up interval

OBJECTIVE: To examine demographic and disease predictors of memory and learning performance for children with diabetes and controls. METHOD: Children with diabetes (N = 95) and demographically similar control children (N = 100) were administered the Rey Auditory Verbal Learning Test (RAVLT) initially and 4 years later. RESULTS: Unlike other groups, boys with diabetes did not make expected developmental gains on the learning trials of the RAVLT. Boys with diabetes showed a plateau in words learned from the primacy position, and girls with diabetes appeared to lose their relative gender advantage for verbal information. Longer disease duration predicted poorer learning over time. CONCLUSIONS: Subtle difficulties were found in learning related to longer disease duration for a predominantly middle-class group of children with diabetes over a 4-year follow-up interval. It will be important to monitor children's educational development to help avoid a cumulative toll on classroom performance.     

Molecular epidemiology of human respiratory syncytial virus subgroup A over a six-year period (1999-2004) in Argentina

Human respiratory syncytial virus (HRSV) is the main viral cause of acute lower respiratory tract infections in children. Little information about the molecular epidemiology of HRSV in developing countries, such as Argentina, is available. By RT-PCR, we subgrouped 353 HRSV isolates over six consecutive epidemic seasons (1999-2004) and few isolates from 1997. Between them, 232 (65.7%) belonged to subgroup A and 121 (34.3%) to subgroup B. Therefore, the nucleotide, amino-acid variability and phylogenetic relations of 78 HRSV subgroup A isolates, were analyzed using RFLP and sequence analysis of the G-protein gene. The results showed that there were two main restriction patterns (PA1 and PA2) and two previously described genotypes (GA2 and GA5) cocirculating in Buenos Aires, without evidence of alternation between them during the studied period. The Argentine sequences were compared with previously reported molecular data from other countries. It showed that viruses genetically related circulated the same years within neighboring countries and the sequences from long-distant places were closely related to Argentine sequences, but they belonged to different sampling years. The data reported here support the growing database on the molecular diversity of HRSVA circulating in Latin America in children under 2 years of age and contributes to describe the pattern of global spread of this virus.     

Predictive and pre-natal testing for Huntington Disease in Australia: results and challenges encountered during a 10-year period (1994-2003)

This study summarizes 10-years' experience of predictive and pre-natal testing and pre-implantation genetic diagnosis (PGD) for Huntington disease (HD) in Australia. Results are presented from 2036 direct mutation predictive tests conducted between January 1994 and December 2003. Thirty-eight per cent of results (776/2036) were positive, 56% (1140/2036) were negative, and 6% (120/2036)) were in the mutable normal (27-35 CAG repeats) or in the reduced penetrance (36-39 CAG repeats) ranges. Ninety-four per cent (1908/2036) and 6% (128/2036) of those tested had prior genetic risks of 50% and 25%, respectively. Twenty-seven per cent (34/128) of those at 25% risk had their genetic status changed to positive, thus revealing the positive status of their at-risk parent. During this period, 63 pre-natal tests were also conducted, and 13 children were born following PGD for HD. Social workers specializing in predictive testing counselling over this 10-year period across Australia identified and summarized particularly challenging counselling issues. These included the interpretation of mutable normal and reduced penetrance range test results, potential conflicts of interest between family members regarding testing decisions, unanticipated consequences of both predictive and pre-natal testing decisions, the importance of following protocols for predictive testing to facilitate long-term adjustment to results, and the potential for genetic discrimination. The identified issues highlight the importance of the protocols for predictive testing and indicate that extension of the international guidelines published in 1994 may be timely.     

Characterization of vancomycin-heteroresistant Staphylococcus aureus from the metropolitan area of Detroit, Michigan, over a 22-year period (1986 to 2007)

We screened for heteroresistant, vancomycin-intermediate Staphylococcus aureus (hVISA) among clinical isolates of methicillin-resistant S. aureus collected from three hospitals (two urban teaching hospitals and one community hospital) in the Detroit metropolitan area over a 22-year period. The Macro Etest method was used to screen all available isolates. Confirmation of hVISA-positive screens were confirmed by population-area under the concentration-time curve (AUC) analysis. A total of 1,499 isolates revealed hVISA/VISA rates of 2.2/0.4% (n = 225; 1986 to 1993), 7.6/2.3% (n = 356; 1994 to 2002), and 8.3/0.3% (n = 917; 2003 to 2007). Population-AUC analysis confirmed 92.6% of the hVISA-positive strains determined by the Macro Etest method. For the isolates with known sources (1,208), the predominant source of hVISA was blood (60%), followed by lung (21%), skin and wound infections (14%), abscess (1%), and other (4%). The percentage of hVISA-positive strains appeared to increase as a function of the vancomycin MIC. Staphylococcal cassette chromosome mec (SCCmec) typing revealed that the majority (56.9%) of the hVISA strains were SCCmec type II and 39.4% were type IV; the majority of these strains were collected from 2000 to 2007. Our data indicate that the prevalence of hVISA may be increasing. Based on the association of vancomycin treatment failure in patients with hVISA, surveillance of hVISA strains is warranted.     

Comprehensive serotyping and epidemiology of human adenovirus isolated from the respiratory tract of Korean children over 17 consecutive years (1991–2007)

Among the 53 serotypes of human adenoviruses (HAdVs), identified to date, only a limited number have been associated with human respiratory infections. The epidemiology of each of the serotypes differs depending on the location and/or time of surveillance. This study was performed to elucidate the epidemiology of HAdV respiratory infections by comprehensive serotyping of the HAdVs isolated from the respiratory tract of Korean children. HAdVs isolated from respiratory specimens of Korean children over 17 consecutive years (1991–2007) were typed by the neutralization test or molecular methods, including two‐sets of multiplex PCR assays, and/or sequence analysis of the hexon gene. From January 1991 through December 2007, a total of 741 isolates were obtained from nasal aspirates of children hospitalized or requiring medical treatment in the emergency room. All isolates were type‐determined successfully and 13 different serotypes were identified, which included HAdV serotypes 1–8, 11, 19, 34, 37, and 41. HAdV‐3 (n = 285, 37.7%) and HAdV‐7 (n = 181, 23.9%) were the predominant serotypes; HAdV‐8, ‐11, ‐19, ‐34, ‐37, and ‐41 were not usually associated with respiratory diseases. HAdV‐3 was present both during outbreaks and in sporadic cases. HAdV‐7 emerged in a very large outbreak, followed by smaller outbreaks. HAdV‐1, ‐2, ‐4, ‐5, and ‐6 were isolated sporadically throughout the study period. In conclusion, a total of 13 different serotypes of HAdV were detected among Korean children with respiratory tract infections. HAdV‐3 and HAdV‐7 were the most common serotypes, and they were associated with HAdV outbreaks. J. Med. Virol. 82:624–631, 2010. © 2010 Wiley‐Liss, Inc.     

The principal protein antigens of isolates of Mycoplasma pneumoniae as measured by levels of immunoglobulin G in human serum are stable in strains collected over a 10-year period.

To determine whether antigenic variation in protein antigens of Mycoplasma pneumoniae occurred over time, 12 isolates obtained from pneumonia patients over a 10-year period (1964 to 1974) were compared by immunoblotting (Western blotting) against acute and convalescent human serum samples obtained from the same patients. The strains selected were isolated from patients who had low anti-lipid complement-fixing antibody titers in their acute-phase serum samples and high titers in their convalescent-phase serum samples. The polypeptide composition of the strains was closely similar by protein staining even when compared with prototype FH-Liu. On immunoblotting, all strains showed five bands (170, 130, 90, 45, and 35 kilodaltons [kDa]) which were stained more intensely by convalescent-phase than by acute-phase specimens. A sixth band (62 kDa) was detected by the conjugate alone. In FH-Liu, one band (110 kDa) was prominently stained by convalescent-phase specimens; this band was much less apparent in all of the clinical isolates. Two isolates possessed an additional band (92 kDa) which was stained more prominently by some but not all convalescent-phase specimens. Because of its known antigenic relationships and culture similarities, Mycoplasma genitalium was used for comparison. More polypeptides of M. genitalium than of M. pneumoniae were recognized by acute-phase serum samples, and 4 of 12 convalescent-phase serum samples showed increases in antibodies to certain M. genitalium polypeptides. However, these reactive polypeptides did not correspond in molecular mass to polypeptides recognized in M. pneumoniae; thus the signature profile of human convalescent-phase specimens with M. pneumoniae was distinct. These five polypeptides, individually or in combination, are especially promising for use in detection of human serum antibodies by enzyme-linked immunosorbent assay because they were found in all M. pneumoniae isolates tested.     

The influence of clinical features mimicking primary immunodeficiency diseases (mPID) on children with Langerhans cell histiocytosis (LCH) — Four with mPID among 39 LCH children from one referral center during 18-year period

BackgroundRecurrent or refractory infections can be a warning sign of primary immunodeficiency diseases (PID). Such mimicking PID (mPID) can occur in patients with Langerhans cell histiocytosis (LCH). Because some cases with refractory molluscum contagiosum-like lesions and persistent otorrhea are finally diagnosed with LCH, we wondered whether such mPID can occur in LCH children and affect on their prognosis.MethodsWe retrospectively reviewed all children with LCH at our institute from 2001 to 2018. A complete medical review of sex, age, symptoms, treatment course, and outcome comparison was performed.ResultsOf 39 enrolled LCH patients, three had persistent otorrhea and one had refractory molluscum contagiosum-like lesions despite aggressive antibiotic therapy. These four cases with mPID had significantly higher rates of multi-system involvement, recurrence and 5-month more lag time, but no risk organ (liver, spleen and bone marrow) involvement compared to those without mPID, although bone and skin were the most involved in both groups. Overall, the lag-time in multi-system was longer than that in single-system involvement (median 2.5 vs. 1.0 months; p = 0.003). The diagnosis-age of risk organ involvement was younger than those without (median 8 vs. 43 months; p = 0.004). There were no significant differences in diagnosis-age, single/multi-system and risk organ involvement between remission and recurrence groups. All were alive excluding four who were lost to follow-up.ConclusionsThe LCH children with mPID had greater lag time, multi-system involvement, recurrence and more refractory treatment including transplantation despite the ratio of bone and skin lesions equal to those without mPID.