Comparison of hemagglutination inhibition tests, immunoperoxidase monolayer assays, and serum neutralizing tests in detecting antibodies against blue eye disease in pigs.

Blue eye disease (BED) of pigs was identified in the early 1980s in La Piedad, Michoacan, Mexico. The causal agent is Porcine orthorubulavirus (PRV), which affects pigs of all ages, producing nervous, respiratory, and reproductive disorders. BED is geographically endemic to the center of Mexico, where 75% of the country's swine industry is concentrated. Due to its adverse effects on the swine industry and the risk of dissemination to other countries, it is essential to have reliable diagnostic methods for BED. The objective of this study was to establish the optimal conditions for three serological tests, hemagglutination inhibition (HI), immunoperoxidase monolayer assay (IPMA), and serum neutralization (SN), and to compare their sensitivity, specificity, kappa coefficient, and predictive values. Twelve different HI protocols (9408 tests), one SN protocol and one IPMA protocol (784 tests, each) were evaluated. Forty-nine sera were analyzed, and thirty-seven sera showed true positive results, while twelve showed true negative results. The kappa coefficient was used to assess the variation in each test. The best HI protocol registered a sensitivity and specificity of 89 and 100%, respectively, the IPMA test showed values of 85 and 100%, and the SN test registered a sensitivity of 91% and a specificity of 96%. One of the disadvantages of the HI test is that when chicken red blood cells (RBCs) are used, elution occurs in a short incubation time, which would decrease the specificity. The use of bovine RBCs increases the specificity of the testy and makes it more stable, but it decreases the sensitivity. The results of HI and SN revealed the importance of eliminating the complement system of the serum and removing other inhibitors to avoid test nonspecificity. The IPMA test does not use an active virus; hence, it is considered safe and does not present any risk of disseminating PRV.

Human parainfluenza virus circulation, United States, 2011-2019.

BACKGROUND: Human parainfluenza viruses (HPIVs) cause upper and lower respiratory tract illnesses, most frequently among infants and young children, but also in the elderly. While seasonal patterns of HPIV types 1-3 have been described, less is known about national patterns of HPIV-4 circulation. OBJECTIVES: To describe patterns of HPIVs circulation in the United States (US). STUDY DESIGN: We used data from the National Respiratory and Enteric Virus Surveillance System (NREVSS), a voluntary passive laboratory-based surveillance system, to characterize the epidemiology and circulation patterns of HPIVs in the US during 2011-2019. We summarized the number of weekly aggregated HPIV detections nationally and by US census region, and used a subset of data submitted to NREVSS from public health laboratories and several clinical laboratories during 2015-2019 to analyze differences in patient demographics. RESULTS: During July 2011 - June 2019, 2,700,135 HPIV tests were reported; 122,852 (5 %) were positive for any HPIV including 22,446 for HPIV-1 (18 %), 17,474 for HPIV-2 (14 %), 67,649 for HPIV-3 (55 %), and 15,283 for HPIV-4 (13 %). HPIV testing increased substantially each year. The majority of detections occurred in children aged ≤ 2 years (36 %) with fluctuations in the distribution of age by type. CONCLUSIONS: HPIVs were detected year-round during 2011-2019, with type-specific year-to-year variations in circulation patterns. Among HPIV detections where age was known, the majority were aged ≤ 2 years. HPIV-4 exhibited an annual fall-winter seasonality, both nationally and regionally. Continued surveillance is needed to better understand national patterns of HPIV circulation.

Establishment of reverse transcription polymerase chain reaction assay for simultaneous detection of human parainfluenza virus types 1 to 4 in children with influenza like illnesses.

OBJECTIVES: The aim of this study was to develop an in-house multiplex reverse transcription polymerase chain reaction (mRT-PCR), which can recognize HPIV1-4 in clinical samples. BACKGROUND: Human parainfluenza virus (HPIV) is one of the major causes of viral respiratory infections and can affect people at any age, especially infants and young children. METHODS: Four sets of specific primers targeting conserved areas of hemagglutinin-neuraminidase (HN) genes of HPIV1-4, were designed and tested with type-related plasmid controls. Specificity and sensitivity of mPCR were tested. One-step mRT-PCR was set up using a viral panel containing 10 respiratory viruses, including HPIVs. One hundred nasopharyngeal samples of respiratory infection patients were tested using the set One-step mRT-PCR. RESULTS: The specificity of set mPCR for HPIV1-4 using plasmid positive controls was proved and reaction sensitivity was measured. The specificity of set mRT-PCR was confirmed and 4 and 5 out of 100 clinical samples were HPIV1 and HPIV2 positive, respectively. CONCLUSION: The developed one-step mRT-PCR in this study is an effective and specific assay for clinical diagnosis of HPIV1 to 4 (Tab. 1, Fig. 6, Ref. 28).

First report of severe parainfluenza virus 4B and rhinovirus C coinfection in a liver transplant recipient treated with immunoglobulin.

We describe the first reported case of severe pneumonia due to coinfection by parainfluenza virus type 4B and rhinovirus C in a liver transplant recipient. The patient responded promptly to intravenous immunoglobulin and timely infection control measures prevented spreading of the infections. This report highlights respiratory viral coinfections as a possible cause of severe morbidity in transplant recipients and the importance of efficient molecular diagnostic technologies with major impact on clinical practice in a transplant center. It also describes a potential therapeutic strategy for such patients.

Development of a real-time RT-PCR method for detection of porcine rubulavirus (PoRV-LPMV).

In order to provide a rapid and sensitive method for detection of the Porcine rubulavirus La Piedad-Michoacan-Mexico Virus (PoRV-LPMV), we have developed a specific real-time reverse transcriptase polymerase chain reaction assay. The detection of PoRV-LPMV, represents a diagnostic challenge due to the viral RNA being present in very small amounts in tissue samples. In this study, a TaqMan(®) real-time PCR assay was designed based on the phosphoprotein gene of PoRV-LPMV, to allow specific amplification and detection of viral RNA in clinical samples. Assay conditions for the primers and probe were optimized using infected PK15 cells and ten-fold serial dilutions of a plasmid containing the whole P-gene. The sensitivity of the developed TaqMan(®) assay was approximately 10 plasmid copies per reaction, and was shown to be 1000 fold better than a conventional nested RT-PCR. The performance of this real-time RT-PCR method enables studies of various aspects of PoRV-LPMV infection. Finally, the assay detects all current known variants of the virus.

[Clinical and epidemiological manifestations of parainfluenza infection in hospitalized children]. / Características clínicas y epidemiológicas de la infección por virus parainfluenza en niños hospitalizados.

BACKGROUND: Human parainfluenza viruses (hPIV) are a common cause of respiratory illness of children but published data on clinical characteristics of hPIV infection in South America is scarce. OBJECTIVE: To review the clinical presentation and epidemiological features of hPIV in a series of hospitalized children in Chile. PATIENTS AND METHODS: Retrospective review of clinical charts from all pediatric admissions with a diagnosis of respiratory disease (between January 2001 to December 2004) at the Catholic University Hospital, Santiago, Chile. Nasopharyngeal secretions were tested for hPIV in children admitted with suspected respiratory viral infections. RESULTS: A total of 3,043 respiratory admissions were recorded during the study period; 64 children (2.1%) were hPIV positive. Average age was 13 months (range: lm to 12y) and 77%> were younger than 2 years. HPIV-2 was the most common type identified (47%). A seasonal trend was noted for serotypes hPIV-2 and 3. Acute wheezing (40%o) and pneumonia (30%) were the most common clinical diagnosis in hPIV positive children and 17%> hPIV positive children (44%> for hPIV-1) were associated with laryngitis. All hPIV positive bronchiolitis were due to serotypes hPIV-2 and 3. CONCLUSION: hPIV can cause respiratory disease requiring hospitalization; serotypes hPIV-2 and 3 displayed a seasonal trend. Although hPIV is an uncommon cause of severe respiratory infecion requiring hospitalization in children, it should be considered in the differential diagnosis of laryngitis, bronchiolitis and pneumonia, especially in younger children.

Características clínicas y epidemiológicas de la infección por virus parainfluenza en niños hospitalizados / Clinical and epidemiological manifestations of parainfluenza infection in hospitalized children

Los virus parainfluenza del ser humano (VPIh) son patógenos importantes de enfermedad respiratoria en niños; pese a ello, existe escasa información publicada en Sudamérica dirigida a caracterizar esta infección. Objetivo: Describir las manifestaciones clínicas y epidemiológicas específicas de los VPIh en niños hospitalizados. Pacientes y Métodos: Se revisaron todas las hospitalizaciones respiratorias (HR) efectuadas en el Hospital de la Pontificia Universidad Católica, Santiago, Chile, durante el período 2001-2004 y sus respectivos estudios virales obtenidos de secreciones nasofaríngeas en aquellos con sospecha de infección viral. Resultados: Se identificaron 3.043 HR siendo 64 (2,1 por ciento) VPUrh La edad promedio fue 13 meses (rango: 1 m-12 a) siendo 77 por ciento) de edad inferior a dos años. VPIh-2 fue el serotipo prevalente (47 por ciento), observándose una tendencia estacional para los serotipos 2 y 3. Las presentaciones más frecuentes fueron sibilancias asociadas a virus (40 por cientoo) y neumonía (30 por ciento). Todas las bronquiolitis se presentaron asociadas a VPIh serotipos 2 y 3. Sólo 17 por ciento de los hospitalizados por VPIh+ (44 por ciento VPIh-1) desarrollaron laringitis. Conclusión: Virus parainfluenza humano puede ser responsable de HR en niños, mostrando una tendencia estacional VPIh-2 y el serotipo 3. Aunque son poco frecuentes como causa de HR, confirmamos su participación como etiología específica de laringitis, bronquiolitis y neumonía, especialmente en niños pequeños.

Background: Human parainfluenza viruses (hPIV) are a common cause of respiratory illness of children but published data on clinical characteristics of hPIV infection in South America is scarce. Objective: To review the clinical presentation and epidemiological features of hPIV in a series of hospitalized children in Chile. Patients and Methods: Retrospective review of clinical charts from all pediatric admissions with a diagnosis of respiratory disease (between January 2001 to December 2004) at the Catholic University Hospital, Santiago, Chile. Nasopharyngeal secretions were tested for hPIV in children admitted with suspected respiratory viral infections. Results: A total of 3,043 respiratory admissions were recorded during the study period; 64 children (2.1 percent) were hPIV positive. Average age was 13 months (range: lm to 12y) and 77 percent> were younger than 2 years. HPIV-2 was the most common type identified (47 percent). A seasonal trend was noted for serotypes hPIV-2 and 3. Acute wheezing (40 percento) and pneumonia (30 percent) were the most common clinical diagnosis in hPIV positive children and 17 percent> hPIV positive children (44 percent> for hPIV-1) were associated with laryngitis. All hPIV positive bronchiolitis were due to serotypes hPIV-2 and 3. Conclusion: hPIV can cause respiratory disease requiring hospitalization; serotypes hPIV-2 and 3 displayed a seasonal trend. Although hPIV is an uncommon cause of severe respiratory infecion requiring hospitalization in children, it should be considered in the differential diagnosis of laryngitis, bronchiolitis and pneumonia, especially in younger children.

Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.

A pan-viral DNA microarray, the Virochip (University of California, San Francisco), was used to detect human parainfluenzavirus 4 (HPIV-4) infection in an immunocompetent adult presenting with a life-threatening acute respiratory illness. The virus was identified in an endotracheal aspirate specimen, and the microarray results were confirmed by specific polymerase chain reaction and serological analysis for HPIV-4. Conventional clinical laboratory testing using an extensive panel of microbiological tests failed to yield a diagnosis. This case suggests that the potential severity of disease caused by HPIV-4 in adults may be greater than previously appreciated and illustrates the clinical utility of a microarray for broad-based viral pathogen screening.

Human parainfluenza virus 4 outbreak and the role of diagnostic tests.

Owing to the difficulties in isolating the virus and the lack of routine surveillance, the clinical significance of human parainfluenza virus 4 (HPIV-4) is less well defined than that of the other human parainfluenza viruses. We describe the first outbreak of HPIV-4 infection in a developmental disabilities unit, involving 38 institutionalized children and three staff members, during a 3-week period in autumn 2004. Most subjects had upper respiratory tract infections (URTI), while lower respiratory tract infections (LRTI) occurred in three children (7%), one complicated by respiratory failure requiring ventilation support. All patients recovered. Nasopharyngeal aspirates tested for HPIV-4 were positive by reverse transcriptase PCR (RT-PCR) in all 41 cases (100%), by direct immunofluorescence in 29 of 39 tested cases (74%), and by cell cultures in 6 of 37 cases (16%), and serum was positive for antibodies against HPIV-4 in all 35 cases (100%) with serum samples available. In addition, RT-PCR detected HPIV-4 in four children (three LRTI and one URTI) out of 115 patients with community-acquired respiratory tract infection. Molecular analysis of the 1,198-bp phosphoprotein sequences showed that HPIV-4 isolates among the cases were genetically similar, whereas the community controls were more genetically distant, supporting nosocomial transmission of a single HPIV-4 genotype during the outbreak. Moreover, the HPIV-4 causing the outbreak is more closely related to HPIV-4A than HPIV-4B. HPIV-4 may be an important cause of more severe respiratory illness in children. The present RT-PCR assay is a sensitive, specific, and rapid method for the diagnosing HPIV-4 infection. To better define the epidemiology and clinical spectrum of disease of HPIV-4 infections, HPIV-4 should be included in the routine panels of respiratory virus detection on respiratory specimens.

Fatal hemorrhagic pneumonia concomitant with Chlamydia pneumoniae and parainfluenza virus 4 infection.

CONTEXT: Cases of fatal hemorrhagic pneumonia need to be investigated for highly contagious viral causes. While not all hemorrhagic pneumonias are caused by very contagious agents, the etiology must be correctly determined in order to administer appropriate patient care. OBJECTIVE: To determine whether chlamydia, paramyxovirus, or mycoplasma was the causative agent in a case of fatal hemorrhagic pneumonia, and to evaluate the possibility that this was the first case of hantavirus pulmonary syndrome in Illinois. DESIGN: Nonroutine virological and molecular analyses were performed on lung tissue taken during an unrestricted autopsy of a patient who died in 2002. SETTING AND PATIENT: An elderly, male, Chicago-area resident with a 3-week history of nonspecific, mild upper respiratory tract infection was admitted for hospital treatment of the respiratory infection and viral myositis without cardiac involvement. The patient became febrile, hypoxic, developed hemorrhagic pneumonia, and died. Because he had proven exposure to mice and had developed hemorrhagic pneumonia, hantavirus pulmonary syndrome was suspected as the cause of death. Mice known to carry hantaviruses live in Illinois, including the Chicago area. INTERVENTIONS: Gatifloxacin and heparin anticoagulation were initiated because community-acquired pneumonia and pulmonary embolism were considered likely etiologies for an acute exacerbation of hypoxemia. RESULTS: Two respiratory pathogens were isolated and identified: Chlamydia pneumoniae and human parainfluenza virus 4a. CONCLUSIONS: A mixed (polymicrobial) infection contributed to the patient's death. Hemorrhage was likely a result of anticoagulation therapy superimposed on lung tissues damaged by pneumonia. The uncommon nature of this case and the pathogens involved underscore the challenges in infection control and clinical evaluation that hospitals will face when confronted with possibly new and potentially deadly communicable diseases.

Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza a and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4.

Laboratory diagnosis of viral respiratory infections is generally performed by virus isolation in cell culture and immunofluorescent assays. Reverse transcriptase PCR is now recognized as a sensitive and specific alternative for detection of respiratory RNA viruses. A rapid real-time multiplex PCR assay was developed for the detection of influenza A and influenza B viruses, human respiratory syncytial virus (RSV), parainfluenza virus 1 (PIV1), PIV2, PIV3, and PIV4 in a two-tube multiplex reaction which used molecular beacons to discriminate the pathogens. A total of 358 respiratory samples taken over a 1-year period were analyzed by the multiplex assay. The incidence of respiratory viruses detected in these samples was 67 of 358 (19%) and 87 of 358 (24%) by culture and real-time PCR, respectively. Culture detected 3 influenza A virus, 2 influenza B virus, 57 RSV, 2 PIV1, and 2 PIV3 infections. All of these culture-positive samples and an additional 5 influenza A virus, 6 RSV, 2 PIV1, 1 PIV2, 1 PIV3, and 3 PIV4 infections were detected by the multiplex real-time PCR. The application of real-time PCR to clinical samples increases the sensitivity for respiratory viral diagnosis. In addition, results can be obtained within 6 h, which increases clinical relevance. Therefore, use of this real-time PCR assay would improve patient management and infection control.

[Prevalence of respiratory viruses in elderly people]. / Wystepowanie wirusów oddechowych u ludzi w podeszlym wieku.

We analysed the presence of respiratory tract viruses in 206 elderly people (more than 65 years old). To identify viruses we used immunofluorescence method. The study revealed respiratory virus infection in about one third of examined patients. We found the most frequent infection with parainfluenza viruses. We found also more frequent prevalence of respiratory viruses in the oldest subgroup (more than 75 years old). No correlation between presence of respiratory viruses and smoking, diabetes or congestive heart failure was found.

[Parainfluenza virus type 4 infections]. / Infecciones por el virus parainfluenza tipo 4.

OBJECTIVE: To describe the clinical and epidemiological characteristics of parainfluenza virus type 4 infections in pediatric patients. METHODS: Inpatients and outpatients with lower respiratory tract infections were studied. Recorded data were age, sex, clinical diagnosis, temperature, oxygen saturation, chest radiograph and length of hospital stay. Nasopharyngeal aspirates were studied through indirect immunofluorescence, tissue culture and a new multiplex reverse-transcriptase polymerase chain reaction (RT-PCR) able to identify the four types of human parainfluenza virus in the same reaction. RESULTS: Two hundred thirty specimens from 191 patients were analyzed. Parainfluenza viruses were diagnosed in 35.1 %: 37.3 % type 1, 10.4 % type 2, 38.9 % type 3 and 13.4 % type 4. RNA from parainfluenza virus type 4 was amplified in 10 aspirates from nine patients. Of these, all except one were aged less than two years (range: 26 days to 23 months). The clinical diagnosis was bronchiolitis or wheezing associated with lower respiratory tract infection. Six patients required admission, with a mean oxygen saturation of 89.5 % and a mean length of hospital stay of 7.6 days. DISCUSSION: This is the first description of the characteristics of parainfluenza virus type 4 in Spain. Infections associated with this virus are not as mild as previously thought. Application of a multiplex RT-PCR allows identification of respiratory infections due to parainfluenza virus type 4 that would otherwise be underdiagnosed.