Background Recent outbreaks of avian influenza A (H5N1) in poultry throughout Asia have had major economic and health repercussions. Human infections with this virus were identified in Vietnam in January 2004. Methods We report the clinical features and preliminary epidemiologic findings among 10 patients with confirmed cases of avian influenza A (H5N1) who presented to hospitals in Ho Chi Minh City and Hanoi, Vietnam, in December 2003 and January 2004. Results In all 10 cases, the diagnosis of influenza A (H5N1) was confirmed by means of viral culture or reverse transcriptase–polymerase chain reaction with primers specific for H5 and N1. None of the 10 patients (mean age, 13.7 years) had preexisting medical conditions. Nine of them had a clear history of direct contact with poultry (median time before onset of illness, three days). All patients presented with fever (temperature, 38.5 to 40.0°C), respiratory symptoms, and clinically significant lymphopenia (median lymphocyte count, 700 per cubic millimete...

https://www.nejm.org/doi/pdf/10.1056/NEJMoa040419

Avian Influenza A (H5N1) in 10 Patients in Vietnam

BACKGROUND
Recent outbreaks of avian influenza A (H5N1) in poultry throughout Asia have had major economic and health repercussions. Human infections with this virus were identified in Vietnam in January 2004.


METHODS
We report the clinical features and preliminary epidemiologic findings among 10 patients with confirmed cases of avian influenza A (H5N1) who presented to hospitals in Ho Chi Minh City and Hanoi, Vietnam, in December 2003 and January 2004.


RESULTS
In all 10 cases, the diagnosis of influenza A (H5N1) was confirmed by means of viral culture or reverse transcriptase-polymerase chain reaction with primers specific for H5 and N1. None of the 10 patients (mean age, 13.7 years) had preexisting medical conditions. Nine of them had a clear history of direct contact with poultry (median time before onset of illness, three days). All patients presented with fever (temperature, 38.5 to 40.0 degrees C), respiratory symptoms, and clinically significant lymphopenia (median lymphocyte count, 700 per cubic millimeter). The median platelet count was 75,500 per cubic millimeter. Seven patients had diarrhea. In all patients, there were marked abnormalities on chest radiography. There was no definitive evidence of human-to-human transmission. Eight patients died, one patient has recovered, and one is recovering.


CONCLUSIONS
Influenza A (H5N1) infection, characterized by fever, respiratory symptoms, and lymphopenia, carries a high risk of death. Although in all 10 cases the infection appears to have been acquired directly from infected poultry, the potential exists for genetic reassortment with human influenzaviruses and the evolution of human-to-human transmission. Containment of influenza A (H5N1) in poultry throughout Asia is therefore urgently required.

World health organization international avian influenza investigative team. avian influenza a (h5n1) in 10 patients in vietnam

Since influenza viruses can cause severe illness, timely diagnosis is important for an adequate intervention. The available rapid detection methods either lack sensitivity or require complex laboratory manipulation. This study describes a rapid, sensitive detection method that can be easily applied to routine diagnosis. This method simultaneously detects influenza viruses A and B in specimens of patients with respiratory infections using a TaqMan-based real-time PCR assay. Primers and probes were selected from highly conserved regions of the matrix protein gene of influenza virus A and the hemagglutinin gene segment of influenza virus B. The applicability of this multiplex PCR was evaluated with 27 influenza virus A and 9 influenza virus B reference strains and isolates. In addition, the specificity of the assay was assessed using eight reference strains of other respiratory viruses (parainfluenza viruses 1 to 3, respiratory syncytial virus Long strain, rhinoviruses 1A and 14, and coronaviruses OC43 and 229E) and 30 combined nose and throat swabs from asymptomatic subjects. Electron microscopy-counted stocks of influenza viruses A and B were used to develop a quantitative PCR format. Thirteen copies of viral RNA were detected for influenza virus A, and 11 copies were detected for influenza virus B, equaling 0.02 and 0.006 50% tissue culture infective doses, respectively. The diagnostic efficacy of the multiplex TaqMan-based PCR was determined by testing 98 clinical samples. This real-time PCR technique was found to be more sensitive than the combination of conventional viral culturing and shell vial culturing.

Simultaneous Detection of Influenza Viruses A and B Using Real-Time Quantitative PCR

There is increasing evidence that chronic obstructive pulmonary disease (COPD) is associated with chronic inflammation in the airways and lung parenchyma; however, little is known about the inflammatory response during acute COPD exacerbation. The objectives of this study were (1) to determine if inflammatory markers associated with neutrophilic inflammation and activation increase at times of acute COPD exacerbation relative to the clinically stable state, and (2) to determine whether the presence of acute bacterial or viral infection at the time of COPD exacerbation could be correlated with increases in sputum markers of inflammation. Induced sputum was collected from patients with COPD when they were clinically stable, during the time of an acute exacerbation, and 1 mo later. Sputum was analyzed at each time point for soluble markers associated with neutrophilic inflammation; myeloperoxidase (MPO), tumor necrosis factor-alpha (TNF-alpha), and interleukin-8 (IL-8). Serologic assays on acute and convalescent sera were performed for respiratory viruses, and induced sputum was also subject to quantitative bacterial cultures, viral cultures, and polymerase chain reaction (PCR) for detection of respiratory viruses. Fourteen of the 50 patients enrolled in the study met predetermined criteria for an acute COPD exacerbation over the 15-mo study period. TNF-alpha and IL-8 were significantly elevated in the sputum of patients during acute COPD exacerbation compared with when they were clinically stable (p = 0.01 and p = 0.05, respectively). Concentrations of these cytokines declined significantly 1 mo after the exacerbation. Three of 14 patients (21%) had confirmed bacterial or viral respiratory tract infections. Patients with documented infection did not demonstrate greater increases in sputum levels of inflammatory cytokines during exacerbations compared with patients without demonstrable infection. We conclude that markers of airway neutrophilic inflammation increase at the time of acute COPD exacerbation and then decline 1 mo later, and that this acute inflammatory response appears to occur independently of a demonstrable viral or bacterial airway infection.

https://www.atsjournals.org/doi/pdf/10.1164/ajrccm.163.2.2003122

Granulocyte inflammatory markers and airway infection during acute exacerbation of chronic obstructive pulmonary disease.

Identification and subtyping of avian influenza viruses by reverse transcription-PCR.

Type A and B influenza viruses can cause a wide spectrum of illness, and these viruses are responsible for considerable mortality and morbidity. Rapid typing of isolates is desirable when amantadine treatment or prophylaxis of contacts of type A influenza virus carriers is considered, but the available rapid techniques lack sensitivity and standard diagnostic methods require expansion of virus in tissue culture or embryonated hens' eggs. We developed a series of oligonucleotide primers able to detect, type, and subtype type A influenza viruses in a single reverse transcription-PCR. RNA was isolated from clinical specimens, and cDNA was generated with random primers. PCR was carried out with a mixture of primers specific for influenza viruses of types B, A/H1 and A/H3, and subtyping of the neuraminidase was carried out on the same cDNA template under identical conditions. Amplified products were detected by ethidium bromide staining of amplified products after agarose gel electrophoresis. When it was used to test 98 clinical specimens, this method was comparable to standard culture techniques in the detection, typing, and subtyping of influenza viruses.

D. Tan

Papers

Typing and subtyping of influenza viruses in clinical samples by PCR.