Alterations in biochemical markers in adenovirus infection.

BACKGROUND: Analyze possible relationships between HAdV and markers for inflammation, specifically the C-reactive protein (CRP) and procalcitonin (PCT) tests, along with other haematological markers. METHODS: Retrospective study of 487 children presenting with fever and/or acute respiratory symptoms in the Paediatric Emergency Department. Analyses included viral presence/absence (both HAdV and other respiratory viruses) in respiratory exudates, CRP and PCT alterations in plasma, and haematological markers in whole blood. RESULTS: Viral load was >500 copies/103 cells of HAdV in 127 cases (26.1%), of which 66 (52%, P<0.0001) had alterations in PCT, and 112 (88.1%, P<0.0001) in CRP. Haematological markers were similar either HAdV was present or not, although many HAdV positive patients demonstrated leukocytosis (66%). Bacterial cultures from 141 samples showed altered PCT in 27 (60%) with HAdV infection, in 3 (18.7%) with bacterial infection, and 13 (26.5%) without either viral or bacterial infection (P<0.05). CRP was altered in 88.9% of HAdV infected children and in 87% infected with bacteria, although the percentage was greater than in cases where other respiratory viruses were present (61.3% P<0.05). CONCLUSIONS: Results demonstrate a clear relationship between HAdV infection and alterations in PCT and CRP which should be taken into account in paediatric patient management.

Simultaneous detection of Dengue virus, Chikungunya virus, Zika virus, Yellow fever virus and West Nile virus.

Although certain mosquito-borne virus, such as Dengue virus (DENV), Chikungunya virus (CHIKV), Zika virus (ZIKV), Yellow fever virus (YFV) and West Nile virus (WNV), are an important public health concern in those countries where transmitter mosquitoes are endemic, several cases of travelers from those endemic countries have been recently reported in Europe. Thus, early diagnosis of these viruses is essential for patient management and adoption of preventive measures. An assay for the simultaneous detection of DENV, CHIKV, ZIKV, YFV and WNV based on a multiplex real-time (RT)-PCR and its usefulness for diagnosis in infection screenings and surveillance of arbovirus in non-endemic countries are described.

Human respiratory syncytial virus load normalized by cell quantification as predictor of acute respiratory tract infection.

Human respiratory syncytial virus (HRSV) is a common cause of respiratory infections. The main objective is to analyze the prediction ability of viral load of HRSV normalized by cell number in respiratory symptoms. A prospective, descriptive, and analytical study was performed. From 7307 respiratory samples processed between December 2014 to April 2016, 1019 HRSV-positive samples, were included in this study. Low respiratory tract infection was present in 729 patients (71.54%). Normalized HRSV load was calculated by quantification of HRSV genome and human ß-globin gene and expressed as log10 copies/1000 cells. HRSV mean loads were 4.09 ± 2.08 and 4.82 ± 2.09 log10 copies/1000 cells in the 549 pharyngeal and 470 nasopharyngeal samples, respectively (P < 0.001). The viral mean load was 4.81 ± 1.98 log10 copies/1000 cells for patients under the age of 4-year-old (P < 0.001). The viral mean loads were 4.51 ± 2.04 cells in patients with low respiratory tract infection and 4.22 ± 2.28 log10 copies/1000 cells with upper respiratory tract infection or febrile syndrome (P < 0.05). A possible cut off value to predict LRTI evolution was tentatively established. Normalization of viral load by cell number in the samples is essential to ensure an optimal virological molecular diagnosis avoiding that the quality of samples affects the results. A high viral load can be a useful marker to predict disease progression.

Detection and quantification of Merkel cell polyomavirus. Analysis of Merkel cell carcinoma cases from 1977 to 2015.

This study investigates the presence of Merkel cell polyomavirus (MCPyV) in skin lesions of patients with Merkel cell carcinoma (MCC). MCPyV was quantified using quantitative Real-Time-PCR (qRT-PCR) in 34 paraffinized MCC samples (resected/biopsied) originally taken between 1977 and 2015, and six non-MCC samples. In 31 (91.2%) MCC-individuals, MCPyV was detected. No virus was observed in any non-MCC tumor. Average age at diagnosis was 78.2 ± 9.35 (55-97) years for women (n = 19) and 69.5 ± 14.7 (45-91) for men (n = 15) (P = 0.04). MCC tumor location, known in 25 cases, was: 11 (44%) in the head region, 6 (24%) in upper limbs, 4 (16%) in lower limbs, and 4 (16%) in the trunk. All but one patient had received some sort of treatment: 15 (45.45%) underwent both radio and chemotherapy, 13 (39.39%) only surgery, 2 (6.06%) surgery, plus radio and chemotherapy, 2 (6.06%) surgery and chemotherapy, and 1 (3.03%) only radiotherapy. Follow up data were available for 21/34 patients: recurrence was recorded for 4 (19.04%), and metastasis for 13 (61.9%). Recorded data showed that 10 men and 5 women (total 44.1%) died during follow up, 7 (46.7%) of them within 2 years of diagnosis. Viral load was 5.8 ± 1.4 log copies/105 cells (3.1-8.6), independent of any variable. MCPyV was very frequent in MCC. It was principally associated with head and limb tumors, it more commonly affected men, who in this study were, on average, younger than women, and had high rates of recurrence and mortality. The amplification techniques described here are easily applied and suitable for detecting the presence of MCPyV virus in MCC.

Utility of normalized genome quantification of Helicobacter pylori in gastric mucosa using an in-house real-time polymerase chain reaction.

Traditional diagnostic assays for Helicobacter pylori detection have their limitations. Molecular methods can improve both diagnosis and understanding of gastric diseases. Here we describe an in-house quantitative real-time polymerase chain reaction (q-rt-PCR) for the detection of H. pylori in gastric biopsies which has been developed and has a detection limit of 10 copies, the specificity of which was tested against other gastric colonizer bacteria. In this study, 199 gastric biopsies from adults with different clinical gastric symptoms were examined. Biopsies were obtained during endoscopy and the following tests performed: rapid urease testing (RUT), culture and q-rt-PCR. H. pylori bacterial load expressed as bacterial load per 105 cells was calculated using a standard curve. H. pylori was isolated in 41% of patients, RUT was positive in 32% and bacterial genome was detected in 45% (p = 0.010). Concordance between traditional invasive microbiological methods used together and q-rt-PCR was almost 100%. Bacterial load in patients with positive RUT was significantly higher than those where it was negative (p<0.0001). There were also significant differences between bacterial load in patients with more than one positive assay versus those where only one method was positive (p = 0.006). The in-house q-PCR developed here is quick and inexpensive, and allows accurate diagnosis of H. pylori infection. It also permits normalized bacterial load quantification, which is important to differentiate between asymptomatic colonisation and infection.

Human papillomavirus infection in a male population attending a sexually transmitted infection service.

OBJECTIVE: Human Papillomavirus (HPV) infection in men may produce cancer and other major disorders. Men play an important role in the transmission of the virus and act as a reservoir. The aim of this study was to determine the HPV-genotypes and their prevalence in a group of men attending a Sexually Transmitted Infection service. PATIENTS AND SAMPLES: Between July 2002 and June 2011, 1392 balanopreputial, 435 urethral, 123 anal, and 67 condyloma lesions from 1551 men with a mean age of 35.8±11.3 years old (range: 17-87) were collected for HPV-DNA testing. METHODS: A fragment of the L1-gene and a fragment of the E6/E7-genes were amplified by PCR. Positive samples were typed by hybridization. RESULTS: The HPV genome was detected in 36.9% (486/1318) balanopreputial and in 24.9% (101/405) urethral (p<0.0001) swabs from 38.1% (538) of 1469 men. Co-infections were present in 5.4% (80/1469) of cases. HPV was found in 43.9% (373/850) of men younger than 35 vs. 31.7% (187/589) of men aged >35. HPV was found in 59.4% (104) of 165 men with lesions (macroscopic or positive peniscopy), and in 22.8% (61/267) without clinical alterations. HPV was also detected in 71.4% (40/56) men with condylomata and in 58.7% (64/109) of men with positive peniscopy. CONCLUSIONS: HPV prevalence in men was high and decreased with age. HPV was found more frequently in balanopreputial than in urethral swabs. There was a low rate of co-infections. Low-risk HPV vaccine genotypes were the most recurrent especially in younger. Although HPV has been associated with clinical alterations, it was also found in men without any clinical presentation. Inclusion of men in the national HPV vaccination program may reduce their burden of HPV-related disease and reduce transmission of the virus to non-vaccinated women.

MAVS: a new weapon in the fight against viral infections.

In addition to their participation in metabolic processes and control of programmed cell death, the role of mitochondria as a fundamental hub for innate anti-viral immunity is now emerging. The participation of the mitochondrial antiviral signaling protein (MAVS) as a central regulator of a complex signaling cascade, which results in the induction of antiviral and inflammatory responses has been described. A patent based on this role of MAVS is highlighted in this review.

Overview of molecular biological methods for the detection of pathogens causing sexually transmitted infections.

We review here different state-of-the-art molecular methods currently used in the diagnosis of sexually transmitted infections.

The role of the clinical microbiology laboratory in solid organ transplantation programs.

Infections remain a major complication of solid organ transplantation. For this reason, the clinical microbiology laboratory plays a key role in the success of transplant programs, which must have the support of a qualified laboratory, both technically and professionally. Transplant programs strongly condition the structure and functionality of microbiology laboratories, but at the same time, benefit greatly from the knowledge generated from these programs. The laboratory must make a special effort to implement rapid methods that can respond to the broad spectrum of potential pathogens in solid organ transplant patients. The integration of microbiologists in multidisciplinary teams is highly recommended, as only then can they obtain the highest quality and efficiency in the diagnostic process. This article provides an updated review of the techniques to be used once transplantation has occurred. The role of the microbiologist is also crucial in the pretransplant period, as good microbiological candidate evaluation at this time strongly conditions the success of the transplantation program.

[Microbiology of transplants]. / Microbiología del trasplante.

Infections are one of the main complications that decisively affect the final outcome of transplants. Clinical microbiology laboratory has a key role in diagnosis, treatment and prevention of these complications. Centres with transplant programs must be technically supported with a well developed laboratory with special emphasis in rapid diagnostic techniques. In this article, we review the clinical background for the laboratory, its role in the evaluation of both donors and recipients, and the diagnostic methods for the main pathogens infecting transplant patients.

Noroviruses as cause of gastroenteritis in elderly patients.

BACKGROUND AND AIMS: Gastroenteritis is one of the infections which are particularly important in elderly people. Knowledge of the main pathogens causing gastroenteritis in this group of patients, whose number will dramatically increase in coming decades, is essential. The contribution of group A rotavirus, adenovirus types 41 and 42, norovirus and astrovirus as causes of gastroenteritis among patients of all ages, especially those with over 65 was evaluated over an extended time period. METHODS: A total of 4024 fecal samples, collected during seven seasons (October 2000 to September 2007), were tested with a commercial immunoassay (rotavirus) and an "in house" nested RT-PCR (adenovirus, norovirus and astrovirus). RESULTS: Although norovirus was the second most common cause of gastroenteritis (7.9%) in the total population, it was predominant in the age group over the age of 6, causing 7.2% of gastroenteritis in the 6-16-year-old group, 8.6% in the 16-64-year-old group, and 11.1% in the >65-year group (p=0.001). In the last age group, norovirus was the most frequently detected (11.1%), followed by adenovirus (7.4%), astrovirus (3.6%) and rotavirus (3.3%) (p<0.0001). In addition, norovirus was rarely found in association with other viruses. CONCLUSIONS: Our data suggest that the elderly are highly vulnerable to certain infections, and indicate the need to introduce simple tests for an early identification of norovirus in cases of gastroenteritis affecting elderly patients, improving patient care by reducing unnecessary treatments and hospital stays.

Prevalence, evolution, and features of infection with human papillomavirus: a 15-year longitudinal study of routine screening of a women population in the north of Spain.

Determination of the prevalence of type-specific human papillomavirus (HPV) is important for the development of new vaccines and to prevent malignancy. The objective of this study was to determine HPV infection in two areas in the north of Spain, and their evolution in the last 15 years. Between 1991 and 2007, 7,930 fresh cervical swabs were obtained from 5,554 women (37.8 +/- 11.8 years old). From them, 425 have been followed-up for an average of 3.7 +/- 2.08 years after sampling (range 2-14.6), and 71 for 7.7 +/- 2.2 years (range 5-14). Methods based on polymerase chain reaction (PCR) were carried out. Samples from 1,598 (28.8%) women were positive for HPV: 40.9% were under 25 years of age, 34.2% in the 25-35 year age group, 27.2% in the 36-45 year age group, and 19.6% older than 45 years (P < 0.001). HPV was found in 34.4% of the women with cytological alterations versus 23% of women without cervical changes (P < 0.0001). HPV-16 was present in 25.8% of the women, although the study identified 26 different HPV genotypes. After 3 years of follow-up, HPV remained or became undetectable in 87% of the cases, and in 5 years 70.3%. The prevalence of HPV is associated with younger women and women with cytological changes in the cervix. Although HPV-16 is more prevalent, HPV types not included in available vaccines were found the most commonly. The low 3-year (even 5-year) cumulative incidence rate of HPV infection suggests that cervical screening every 3 (or even 5) years is safe and effective.

[Viral infection of herpes simplex, Epstein-Barr, varicela zoster, human papilloma, cytomegalovirus, or adenovirus are not related to sinonasal adenocarcinomas]. / Las infecciones por virus herpes simplex, Epstein-Barr, varicela zoster, papiloma humano, citomegalovirus o adenovirus no tienen relación con los adenocarcinomas nasosinusales.

OBJECTIVE: Several types of virus have been implicated in the development of head and neck tumors. However, until now sinonasal adenocarcinomas (ACN) have not been studied. The aim of this study is to screen a series of ACN for the presence of a number of viruses known to play a role in cancer. MATERIAL AND METHOD: Viral DNA sequences of herpes simplex virus, Epstein-Barr, varicela zoster, human papilloma, cytomegalovirus, and adenovirus were analysed by PCR in 37 primary ACN. RESULTS: Three tumors (8.1%) were positive for Epstein-Barr virus and 1 case (2.7%) for cytomegalovirus. CONCLUSIONS: Viral infections do not seem to play a role in the etiology of ACN.

Role of metapneumovirus in viral respiratory infections in young children.

The contribution of human metapneumovirus (hMPV) relative to that of other respiratory viruses as a cause of respiratory infections in children less than 1 year old has been evaluated. From October 2003 to April 2004, nasopharyngeal samples from 211 children less than 1 year old were analyzed to detect respiratory viruses. Respiratory syncytial virus (RSV) was the predominant virus isolated (96 children [45.5%]), followed by influenza A virus, parainfluenza virus, adenovirus, cytomegalovirus, and herpes simplex virus type 1, which were only occasionally detected. From January 2004 to April 2004, a nested retrotranscription-PCR, using in-house primers directed to the matrix protein gene of hMPV, was carried out on samples in which no other viruses were detected. hMPV was detected in 18 (16.2%) children, indicating that this virus was the second-most-frequent cause of viral respiratory infections in children less than 1 year old. The rate of hospitalization for RSV- and hMPV-infected children was higher than 75%. While RSV had a peak from December to February, hMPV was increasingly detected from January to April. The mean age of hMPV-infected children (6.44 +/- 3.64 [mean +/- standard deviation] months) was significantly higher than that of RSV-infected children (3.99 +/- 2.96 [mean +/- standard deviation] months). On the other hand, 64.3% of the RSV-infected children and 12.5% of the hMPV-infected children showed high levels of C-reactive protein. Although several authors have reported that clinical symptoms of hMPV-positive patients mirrored those of RSV-positive patients, differences between the two viruses can be found.

Comparison of cytomegalovirus pp-65 antigenemia assay and plasma DNA correlation with the clinical outcome in transplant recipients.

The relationship between quantitative antigenemia and plasma DNAemia was studied for monitoring cytomegalovirus (CMV) viremia in CMV infection (CMVI) or disease (CMVD), in 20 transplant recipients (13 CMD, seven CMVI). A total of 142 samples of blood were assayed for CMV-DNA and pp-65 antigenemia (CMV-Ag). A quantitative correlation between both markers was found (P < 0.0001). First CMV antigenemia as well as first plasma DNA viral load was similar in CMVI and CMVD (29 vs. 24 CMV-Ag+ cells/10(5) PMN; and 7445 vs. 12407 CMV-DNA copies/ml). The maximum antigenemia was higher in CMVD than in CMVI (146 +/- 87 vs. 61 +/- 54 +cells/10(5) PMN, P < 0.05), but the highest CMV plasma viral load was similar in both groups (62592 +/- 33000 vs. 42055 +/- 38600 copies/ml). In nine patients, maximum antigenemia coincided with highest plasma DNA viral load, but in 10 highest DNAemia occurred 6 days later. On the contrary, antigenemia declined faster than CMV-DNAnemia, after treatment.

Altered replicative capacity of recombinant HIV type 1 containing mutations at codons 26 and 29 of the viral protease.

HIV-1 protease activity is essential for viral replication. In spite of the high rates of HIV mutation, the active site of protease (residues 24-29) is a conserved site, where mutations have not been previously described. To determine the effect of mutations at positions T26 and A29 of the viral protease and its viability in recombinant HIV-1 strains, Sup-t1 cells were transfected by electroporation with PCR products from a protease containing the 26X or 29X mutation. These mutations were constructed by mutagenesis site directed with degenerative primers. Both changes modified the replicative capacity of the virus: viruses containing the 26X mutation have delayed replication as compared to the control virus HTLVIII B; the presence of the 29X mutation in the viral protease results in the absence of HIV-1 replication. These findings confirm that this region of the viral protease appears to be necessary for the viability of HIV-1, and it could be a good target for antiviral therapy.

Etiology of sporadic cases of pediatric acute gastroenteritis in asturias, Spain, and genotyping and characterization of norovirus strains involved.

From November 2000 to October 2001, a reverse transcription-PCR using primers directed to the norovirus RNA polymerase coding region was included in a viral and bacterial routine screening to diagnose sporadic cases of acute gastroenteritis among children in Asturias, Spain. The role of noroviruses (8.6% of the positively diagnosed cases) as the cause of sporadic pediatric gastroenteritis was evaluated with respect to the detection rates of other gastroenteritis-associated viruses and bacteria. The results indicated that noroviruses were less common than rotaviruses (36.9%), Campylobacter spp. (28.8%), and Salmonella spp. (18.4%) but more frequent than astroviruses (4.3%), adenoviruses (3.8%), and Yersinia spp. (2.2%). Mixed infections involving noroviruses were rarely observed (0.5%). The presence of a norovirus-associated pediatric gastroenteritis peak in summer, as well as the complete absence of norovirus-associated cases in colder months, challenges the view that norovirus infections exclusively have wintertime seasonality. On the other hand, phylogenetic analysis of the amplified fragments showed that the norovirus strains responsible were closely related. A further study using the full-length capsid region showed that these strains could be included into genogroup II, Bristol/Lorsdale cluster, and were closely related to the 1995 and 1996 U.S. subset of strains associated with outbreaks recorded worldwide between 1995 and 1996.