Improved detection of microbiological pathogens: role of partner and non-governmental organizations.

BACKGROUND: Proper detection of disease-causing organisms is very critical in controlling the course of outbreaks and avoiding large-scale epidemics. Nonetheless, availability of resources to address these gaps have been difficult due to limited funding. This report sought to highlight the importance of in-country partners and non-governmental organizations in improving detection of microbiological organisms in Ghanaian Public Health Laboratories (PHLs). METHODS/CONTEXT: This study was conducted between June, 2018 to August, 2019. U. S CDC engaged the Centre for Health Systems Strengthening (CfHSS) through the Association of Public Health Laboratories to design and implement strategies for strengthening three PHLs in Ghana. An assessment of the three PHLs was done using the WHO/CDS/CSR/ISR/2001.2 assessment tool. Based on findings from the assessments, partner organizations (CfHSS/APHL/CDC) serviced and procured microbiological equipment, laboratory reagents and logistics. CfHSS provided in-house mentoring and consultants to assist with capacity building in detection of epidemic-prone infectious pathogens by performing microbiological cultures and antimicrobial susceptibility tests. RESULTS: A total of 3902 samples were tested: blood (1107), urine (1742), stool (249) and cerebrospinal fluid (CSF) (804). All-inclusive, 593 pathogenic bacteria were isolated from blood cultures (70; 11.8%); urine cultures (356; 60%); stool cultures (19; 3.2%) and from CSF samples (148; 25%). The most predominant pathogens isolated from blood, urine and stool were Staphylococcus aureus (22/70; 31%), Escherichia coli (153/356; 43%) and Vibrio parahaemolyticus (5/19; 26.3%), respectively. In CSF samples, Streptococcus pneumoniae was the most frequent pathogen detected (80/148; 54.1%). New bacterial species such as Pastuerella pneumotropica, Klebsiella oxytoca, Vibrio parahaemolyticus, and Halfnia alvei were also identified with the aid of Analytical Profile Index (API) kits that were introduced as part of this implementation. Streptococcus pneumoniae and Neisseria meningitidis detections in CSF were highest during the hot dry season. Antimicrobial susceptibility test revealed high rate of S. aureus, K. pneumoniae and E. coli resistance to gentamicin (35-55%). In urine, E. coli was highly resistant to ciprofloxacin (39.2%) and ampicillin (34%). CONCLUSION: Detection of epidemic-prone pathogens can be greatly improved if laboratory capacity is strengthened. In-country partner organizations are encouraged to support this move to ensure accurate diagnosis of diseases and correct antimicrobial testing.

Effectiveness of Seasonal Influenza Vaccination in Children in Senegal During a Year of Vaccine Mismatch: A Cluster-randomized Trial.

BACKGROUND: The population effects of influenza vaccination in children have not been extensively studied, especially in tropical, developing countries. In rural Senegal, we assessed the total (primary objective) and indirect effectiveness of a trivalent inactivated influenza vaccine (IIV3). METHODS: In this double-blind, cluster-randomized trial, villages were randomly allocated (1:1) for the high-coverage vaccination of children aged 6 months through 10 years with either the 2008-09 northern hemisphere IIV3 or an inactivated polio vaccine (IPV). Vaccinees were monitored for serious adverse events. All village residents, vaccinated and unvaccinated, were monitored for signs and symptoms of influenza illness using weekly home visits and surveillance in designated clinics. The primary outcome was all laboratory-confirmed symptomatic influenza. RESULTS: Between 23 May and 11 July 2009, 20 villages were randomized, and 66.5% of age-eligible children were enrolled (3918 in IIV3 villages and 3848 in IPV villages). Follow-up continued until 28 May 2010. There were 4 unrelated serious adverse events identified. Among vaccinees, the total effectiveness against illness caused by the seasonal influenza virus (presumed to all be drifted A/H3N2, based on antigenic characterization data) circulating at high rates among children was 43.6% (95% confidence interval [CI] 18.6-60.9%). The indirect effectiveness against seasonal A/H3N2 was 15.4% (95% CI -22.0 to 41.3%). The total effectiveness against illness caused by the pandemic influenza virus (A/H1N1pdm09) was -52.1% (95% CI -177.2 to 16.6%). CONCLUSIONS: IIV3 provided statistically significant, moderate protection to children in Senegal against circulating, pre-2010 seasonal influenza strains, but not against A/H1N1pdm09, which was not included in the vaccine. No indirect effects were measured. Further study in low-resource populations is warranted. CLINICAL TRIALS REGISTRATION: NCT00893906.

Ebola Laboratory Response at the Eternal Love Winning Africa Campus, Monrovia, Liberia, 2014-2015.

West Africa experienced the first epidemic of Ebola virus infection, with by far the greatest number of cases in Guinea, Sierra Leone, and Liberia. The unprecedented epidemic triggered an unparalleled response, including the deployment of multiple Ebola treatment units and mobile/field diagnostic laboratories. The National Institute of Allergy and Infectious Diseases and the Centers for Disease Control and Prevention deployed a joint laboratory to Monrovia, Liberia, in August 2014 to support the newly founded Ebola treatment unit at the Eternal Love Winning Africa (ELWA) campus. The laboratory operated initially out of a tent structure but quickly moved into a fixed-wall building owing to severe weather conditions, the need for increased security, and the high sample volume. Until May 2015, when the laboratory closed, the site handled close to 6000 clinical specimens for Ebola virus diagnosis and supported the medical staff in case patient management. Laboratory operation and safety, as well as Ebola virus diagnostic assays, are described and discussed; in addition, lessons learned for future deployments are reviewed.

Ebola Virus Disease Diagnostics, Sierra Leone: Analysis of Real-time Reverse Transcription-Polymerase Chain Reaction Values for Clinical Blood and Oral Swab Specimens.

During the Ebola virus outbreak of 2013-2016, the Viral Special Pathogens Branch field laboratory in Sierra Leone tested approximately 26 000 specimens between August 2014 and October 2015. Analysis of the B2M endogenous control Ct values showed its utility in monitoring specimen quality, comparing results with different specimen types, and interpretation of results. For live patients, blood is the most sensitive specimen type and oral swabs have little diagnostic utility. However, swabs are highly sensitive for diagnostic testing of corpses.

Plasmodium Parasitemia Associated With Increased Survival in Ebola Virus-Infected Patients.

BACKGROUND: The ongoing Ebola outbreak in West Africa has resulted in 28 646 suspected, probable, and confirmed Ebola virus infections. Nevertheless, malaria remains a large public health burden in the region affected by the outbreak. A joint Centers for Disease Control and Prevention/National Institutes of Health diagnostic laboratory was established in Monrovia, Liberia, in August 2014, to provide laboratory diagnostics for Ebola virus. METHODS: All blood samples from suspected Ebola virus-infected patients admitted to the Médecins Sans Frontières ELWA3 Ebola treatment unit in Monrovia were tested by quantitative real-time polymerase chain reaction for the presence of Ebola virus and Plasmodium species RNA. Clinical outcome in laboratory-confirmed Ebola virus-infected patients was analyzed as a function of age, sex, Ebola viremia, and Plasmodium species parasitemia. RESULTS: The case fatality rate of 1182 patients with laboratory-confirmed Ebola virus infections was 52%. The probability of surviving decreased with increasing age and decreased with increasing Ebola viral load. Ebola virus-infected patients were 20% more likely to survive when Plasmodium species parasitemia was detected, even after controlling for Ebola viral load and age; those with the highest levels of parasitemia had a survival rate of 83%. This effect was independent of treatment with antimalarials, as this was provided to all patients. Moreover, treatment with antimalarials did not affect survival in the Ebola virus mouse model. CONCLUSIONS: Plasmodium species parasitemia is associated with an increase in the probability of surviving Ebola virus infection. More research is needed to understand the molecular mechanism underlying this remarkable phenomenon and translate it into treatment options for Ebola virus infection.

The Merits of Malaria Diagnostics during an Ebola Virus Disease Outbreak.

Malaria is a major public health concern in the countries affected by the Ebola virus disease epidemic in West Africa. We determined the feasibility of using molecular malaria diagnostics during an Ebola virus disease outbreak and report the incidence of Plasmodium spp. parasitemia in persons with suspected Ebola virus infection.

Outbreak of variant influenza A(H3N2) virus in the United States.

BACKGROUND: Variant influenza virus infections are rare but may have pandemic potential if person-to-person transmission is efficient. We describe the epidemiology of a multistate outbreak of an influenza A(H3N2) variant virus (H3N2v) first identified in 2011. METHODS: We identified laboratory-confirmed cases of H3N2v and used a standard case report form to characterize illness and exposures. We considered illness to result from person-to-person H3N2v transmission if swine contact was not identified within 4 days prior to illness onset. RESULTS: From 9 July to 7 September 2012, we identified 306 cases of H3N2v in 10 states. The median age of all patients was 7 years. Commonly reported signs and symptoms included fever (98%), cough (85%), and fatigue (83%). Sixteen patients (5.2%) were hospitalized, and 1 fatal case was identified. The majority of those infected reported agricultural fair attendance (93%) and/or contact with swine (95%) prior to illness. We identified 15 cases of possible person-to-person transmission of H3N2v. Viruses recovered from patients were 93%-100% identical and similar to viruses recovered from previous cases of H3N2v. All H3N2v viruses examined were susceptible to oseltamivir and zanamivir and resistant to adamantane antiviral medications. CONCLUSIONS: In a large outbreak of variant influenza, the majority of infected persons reported exposures, suggesting that swine contact at an agricultural fair was a risk for H3N2v infection. We identified limited person-to-person H3N2v virus transmission, but found no evidence of efficient or sustained person-to-person transmission. Fair managers and attendees should be aware of the risk of swine-to-human transmission of influenza viruses in these settings.

Highly pathogenic avian influenza A(H7N3) virus in poultry workers, Mexico, 2012.

We identified 2 poultry workers with conjunctivitis caused by highly pathogenic avian influenza A(H7N3) viruses in Jalisco, Mexico. Genomic and antigenic analyses of 1 isolate indicated relatedness to poultry and wild bird subtype H7N3 viruses from North America. This isolate had a multibasic cleavage site that might have been derived from recombination with host rRNA.

Myocardial injury and bacterial pneumonia contribute to the pathogenesis of fatal influenza B virus infection.

BACKGROUND: Influenza B virus infection causes rates of hospitalization and influenza-associated pneumonia similar to seasonal influenza A virus infection and accounts for a substantial percentage of all influenza-related hospitalizations and deaths among those aged <18 years; however, the pathogenesis of fatal influenza B virus infection is poorly described. METHODS: Tissue samples obtained at autopsy from 45 case patients with fatal influenza B virus infection were evaluated by light microscopy and immunohistochemical assays for influenza B virus, various bacterial pathogens, and complement components C4d and C9, to identify the cellular tropism of influenza B virus, characterize concomitant bacterial pneumonia, and describe the spectrum of cardiopulmonary injury. RESULTS: Viral antigens were localized to ciliated respiratory epithelium and cells of submucosal glands and ducts. Concomitant bacterial pneumonia, caused predominantly by Staphylococcus aureus, was identified in 38% of case patients and occurred with significantly greater frequency in those aged >18 years. Pathologic evidence of myocardial injury was identified in 69% of case patients for whom cardiac tissue samples were available for examination, predominantly in case patients aged <18 years. CONCLUSIONS: Our findings suggest that bacterial pneumonia and cardiac injury contribute to fatal outcomes after infection with influenza B virus and that the frequency of these manifestations may be age related.

Human infections with novel reassortant influenza A(H3N2)v viruses, United States, 2011.

During July-December 2011, a variant virus, influenza A(H3N2)v, caused 12 human cases of influenza. The virus contained genes originating from swine, avian, and human viruses, including the M gene from influenza A(H1N1)pdm09 virus. Influenza A(H3N2)v viruses were antigenically distinct from seasonal influenza viruses and similar to proposed vaccine virus A/Minnesota/11/2010.

Surveillance and laboratory collaboration in response to an outbreak of Vibrio parahaemolyticus, Plesiomonas shigelloides, and Aeromonas hydrophila in Sekondi-Takoradi, Ghana: a case series.

BACKGROUND: The detection of epidemic-prone pathogens is important in strengthening global health security. Effective public health laboratories are critical for reliable, accurate, and timely testing results in outbreak situations. Ghana received funding as one of the high-risk non-Ebola affected countries to build and strengthen public health infrastructure to meet International Health Regulation core capacities. A key objective was to build laboratory capacities to detect epidemic-prone diseases. CASE PRESENTATION: In June 2018, a local hospital received eight patients who presented with acute diarrhea. A sample referral system for Ghana has not been established, but the Sekondi Zonal Public Health Laboratory staff and mentors collaborated with Disease Surveillance Officers (DSOs) to collect, package, and transport stool specimens from the outbreak hospital to the Public Health Laboratory for laboratory testing. The patients included seven females and one male, of Fante ethnicity from the Fijai township of Sekondi-Takoradi Municipality. The median age of the patients was 20 years (interquartile range: 20-29 years). Vibrio parahaemolyticus was identified within 48 hours from four patients, Plesiomonas shigelloides from one patient, and Aeromonas hydrophila from another patient. There was no bacteria growth from the samples from the two other patients. All patients were successfully treated and discharged. CONCLUSION: This is the first time these isolates have been identified at the Sekondi Zonal Public Health Laboratory, demonstrating how rapid response, specimen transportation, laboratory resourcing, and public health coordination are important in building capacity towards achieving health security. This capacity building was part of the United States Centers for Disease Control and Prevention engagement of international and local partners to support public health laboratories with supplies, diagnostic equipment, reagents, and logistics.

Design and performance of the CDC real-time reverse transcriptase PCR swine flu panel for detection of 2009 A (H1N1) pandemic influenza virus.

Swine influenza viruses (SIV) have been shown to sporadically infect humans and are infrequently identified by the Influenza Division of the Centers for Disease Control and Prevention (CDC) after being received as unsubtypeable influenza A virus samples. Real-time reverse transcriptase PCR (rRT-PCR) procedures for detection and characterization of North American lineage (N. Am) SIV were developed and implemented at CDC for rapid identification of specimens from cases of suspected infections with SIV. These procedures were utilized in April 2009 for detection of human cases of 2009 A (H1N1) pandemic (pdm) influenza virus infection. Based on genetic sequence data derived from the first two viruses investigated, the previously developed rRT-PCR procedures were optimized to create the CDC rRT-PCR Swine Flu Panel for detection of the 2009 A (H1N1) pdm influenza virus. The analytical sensitivity of the CDC rRT-PCR Swine Flu Panel was shown to be 5 copies of RNA per reaction and 10(-1.3 - -0.7) 50% infectious doses (ID(50)) per reaction for cultured viruses. Cross-reactivity was not observed when testing human clinical specimens or cultured viruses that were positive for human seasonal A (H1N1, H3N2) and B influenza viruses. The CDC rRT-PCR Swine Flu Panel was distributed to public health laboratories in the United States and internationally from April 2009 until June 2010. The CDC rRT-PCR Swine Flu Panel served as an effective tool for timely and specific detection of 2009 A (H1N1) pdm influenza viruses and facilitated subsequent public health response implementation.

Household transmission of pandemic (H1N1) 2009, San Antonio, Texas, USA, April-May 2009.

To assess household transmission of pandemic (H1N1) 2009 in San Antonio, Texas, USA, during April 15-May 8, 2009, we investigated 77 households. The index case-patient was defined as the household member with the earliest onset date of symptoms of acute respiratory infection (ARI), influenza-like illness (ILI), or laboratory-confirmed pandemic (H1N1) 2009. Median interval between illness onset in index and secondary case-patients was 4 days (range 1-9 days); the index case-patient was likely to be < or =18 years of age (p = 0.034). The secondary attack rate was 4% for pandemic (H1N1) 2009, 9% for ILI, and 13% for ARI. The secondary attack rate was highest for children <5 years of age (8%-19%) and lowest for adults > or =50 years of age (4%-12%). Early in the outbreak, household transmission primarily occurred from children to other household members and was lower than the transmission rate for seasonal influenza.

Identification of plasmid-mediated AmpC beta-lactamases in Escherichia coli, Klebsiella spp., and proteus species can potentially improve reporting of cephalosporin susceptibility testing results.

The goal of this study was to determine if the interpretations of extended-spectrum and advanced-spectrum cephalosporins (ESCs and ASCs, respectively) for isolates of Enterobacteriaceae would be impacted by the results of aminophenylboronic acid (APBA) testing. Fifty-three isolates of Escherichia coli, 21 Klebsiella species, and 6 Proteus species that were resistant to at least one ESC were tested by disk diffusion with ceftazidime and cefotetan disks with and without APBA. Ceftazidime disks with and without clavulanic acid (CLAV) were also tested to confirm extended-spectrum beta-lactamase (ESBL) carriage. Twenty-nine (36.3%) isolates were only APBA test positive, 27 were only CLAV test positive, 2 were positive with both substrates, and 22 were negative with both substrates. Thirteen (41.9%) of the 31 APBA-test-positive isolates (all E. coli) tested susceptible to cefotaxime, ceftriaxone, or ceftazidime. Since clinical data suggest that AmpC-producing isolates should be reported as resistant to all ESCs, APBA testing can be helpful in identifying such organisms. Screening for AmpC-producing organisms using nonsusceptibility to cefoxitin and amoxicillin-clavulanate was less specific than APBA testing; it identified ESBL as well as AmpC-producing organisms. Only 18 of 31 APBA-positive isolates were positive by PCR for an AmpC beta-lactamase gene. Thus, testing with APBA could improve the accuracy of reporting ESCs, especially for E. coli. However, results of APBA and CLAV testing did not correlate well for isolates containing both AmpC beta-lactamases and ESBLs. Thus, additional data are needed before formal recommendations can be made on changing the reporting of ASC test results.

A novel coronavirus associated with severe acute respiratory syndrome.

BACKGROUND: A worldwide outbreak of severe acute respiratory syndrome (SARS) has been associated with exposures originating from a single ill health care worker from Guangdong Province, China. We conducted studies to identify the etiologic agent of this outbreak. METHODS: We received clinical specimens from patients in seven countries and tested them, using virus-isolation techniques, electron-microscopical and histologic studies, and molecular and serologic assays, in an attempt to identify a wide range of potential pathogens. RESULTS: None of the previously described respiratory pathogens were consistently identified. However, a novel coronavirus was isolated from patients who met the case definition of SARS. Cytopathological features were noted in Vero E6 cells inoculated with a throat-swab specimen. Electron-microscopical examination revealed ultrastructural features characteristic of coronaviruses. Immunohistochemical and immunofluorescence staining revealed reactivity with group I coronavirus polyclonal antibodies. Consensus coronavirus primers designed to amplify a fragment of the polymerase gene by reverse transcription-polymerase chain reaction (RT-PCR) were used to obtain a sequence that clearly identified the isolate as a unique coronavirus only distantly related to previously sequenced coronaviruses. With specific diagnostic RT-PCR primers we identified several identical nucleotide sequences in 12 patients from several locations, a finding consistent with a point-source outbreak. Indirect fluorescence antibody tests and enzyme-linked immunosorbent assays made with the new isolate have been used to demonstrate a virus-specific serologic response. This virus may never before have circulated in the U.S. population. CONCLUSIONS: A novel coronavirus is associated with this outbreak, and the evidence indicates that this virus has an etiologic role in SARS. Because of the death of Dr. Carlo Urbani, we propose that our first isolate be named the Urbani strain of SARS-associated coronavirus.

Efficacy of a Russian-backbone live attenuated influenza vaccine among children in Senegal: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Live attenuated influenza vaccines have been shown to significantly reduce influenza in diverse populations of children, but no efficacy studies have been done in resource-poor tropical settings. In Senegal, we assessed the efficacy and safety of a live attenuated influenza vaccine based on Russian-derived master donor viruses and licensed as a single dose. METHODS: In this double-blind, placebo-controlled, parallel group, single-centre trial done near Niakhar, Senegal, generally healthy children aged 2-5 years were randomly allocated (2:1) to receive a single intranasal dose of masked trivalent live attenuated influenza vaccine or placebo. The allocation sequence was computer-generated by PATH with block sizes of three. The manufacturer provided vaccine and placebo in coded vials to preserve blinding. Participants were monitored through the predictable influenza season in Senegal for adverse events and signs and symptoms of influenza using weekly home visits and surveillance in clinics. The primary outcome was symptomatic laboratory-confirmed influenza caused by any strain and occurring from 15 days post-vaccination to the end of the study. The primary analysis was per protocol. This study is registered with ClinicalTrials.gov, number NCT01854632. FINDINGS: Between May 23, and July 1, 2013, 1761 children were randomly assigned, 1174 to receive live attenuated influenza vaccine and 587 to receive placebo. The per-protocol set included 1173 vaccinees and 584 placebo recipients followed up to Dec 20, 2013. Symptomatic influenza was laboratory-confirmed in 210 (18%) of 1173 recipients of live attenuated influenza vaccine and 105 (18%) of placebo recipients, giving a vaccine efficacy of 0·0% (95% CI -26·4 to 20·9). Adverse events were balanced between the study groups. Two girls who had received live attenuated influenza vaccine died, one due to anasarca 12 days postvaccination and one due to malnutrition 70 days postvaccination. INTERPRETATION: Live attenuated influenza vaccine was well tolerated in young children in Senegal, but did not provide protection against influenza. Further study in such populations, which might experience extended periods of influenza circulation, is warranted. FUNDING: US Centers for Disease Control and Prevention and Bill & Melinda Gates Foundation.

Multiple reassortment events among highly pathogenic avian influenza A(H5N1) viruses detected in Bangladesh.

In Bangladesh, little is known about the genomic composition and antigenicity of highly pathogenic avian influenza A(H5N1) viruses, their geographic distribution, temporal patterns, or gene flow within the avian host population. Forty highly pathogenic avian influenza A(H5N1) viruses isolated from humans and poultry in Bangladesh between 2008 and 2012 were analyzed by full genome sequencing and antigenic characterization. The analysis included viruses collected from avian hosts and environmental sampling in live bird markets, backyard poultry flocks, outbreak investigations in wild birds or poultry and from three human cases. Phylogenetic analysis indicated that the ancestors of these viruses reassorted (1) with other gene lineages of the same clade, (2) between different clades and (3) with low pathogenicity avian influenza A virus subtypes. Bayesian estimates of the time of most recent common ancestry, combined with geographic information, provided evidence of probable routes and timelines of virus spread into and out of Bangladesh.

Emergence of multiple clade 2.3.2.1 influenza A (H5N1) virus subgroups in Vietnam and detection of novel reassortants.

Phylogenetic analyses of 169 influenza A(H5N1) virus genomes were conducted for samples collected through active surveillance and outbreak responses in Vietnam between September 2010 and September 2012. While clade 1.1 viruses persisted in southern regions, three genetically distinct subgroups of clade 2.3.2.1 were found in northern and central Vietnam. The identification of each subgroup corresponded with detection of novel reassortants, likely due to their overlapping circulation throughout the country. While the previously identified clade 1.1 and A/Hubei/1/2010-like 2.3.2.1 genotypes remained the predominant viruses detected, four viruses were found to be reassortants between A/Hubei/1/2010-like (HA, NA, PB2, PB1, PA, NP) and A/duck/Vietnam/NCVD-885/2010-like (M, NS) viruses and one virus was identified as having A/duck/Vietnam/NCVD-885/2010-like HA, NA, PB1, and NP with A/Hubei/1/2010-like PB2 and PA genes. Additionally, clade 2.3.2.1 A/Hong Kong/6841/2010-like viruses, first detected in mid-2012, were identified as reassortants comprised of A/Hubei/1/2010-like PB2 and PA and A/duck/Vietnam/NCVD-885/2010-like PB1, NP, NA, M, NS genes.

Genetic analysis and antigenic characterization of swine origin influenza viruses isolated from humans in the United States, 1990-2010.

Swine influenza viruses (SIV) have been recognized as important pathogens for pigs and occasional human infections with swine origin influenza viruses (SOIV) have been reported. Between 1990 and 2010, a total of twenty seven human cases of SOIV infections have been identified in the United States. Six viruses isolated from 1990 to 1995 were recognized as classical SOIV (cSOIV) A(H1N1). After 1998, twenty-one SOIV recovered from human cases were characterized as triple reassortant (tr_SOIV) inheriting genes from classical swine, avian and human influenza viruses. Of those twenty-one tr_SOIV, thirteen were of A(H1N1), one of A(H1N2), and seven of A(H3N2) subtype. SOIV characterized were antigenically and genetically closely related to the subtypes of influenza viruses circulating in pigs but distinct from contemporary influenza viruses circulating in humans. The diversity of subtypes and genetic lineages in SOIV cases highlights the importance of continued surveillance at the animal-human interface.

Diagnosis of influenza from respiratory autopsy tissues: detection of virus by real-time reverse transcription-PCR in 222 cases.

The recent influenza pandemic, caused by a novel H1N1 influenza A virus, as well as the seasonal influenza outbreaks caused by varieties of influenza A and B viruses, are responsible for hundreds of thousands of deaths worldwide. Few studies have evaluated the utility of real-time reverse transcription-PCR to detect influenza virus RNA from formalin-fixed, paraffin-embedded tissues obtained at autopsy. In this work, respiratory autopsy tissues from 442 suspect influenza cases were tested by real-time reverse transcription-PCR for seasonal influenza A and B and 2009 pandemic influenza A (H1N1) viruses and the results were compared to those obtained by immunohistochemistry. In total, 222 cases were positive by real-time reverse transcription-PCR, and of 218 real-time, reverse transcription-PCR-positive cases also tested by immunohistochemistry, only 107 were positive. Although formalin-fixed, paraffin-embedded tissues can be used for diagnosis, frozen tissues offer the best chance to make a postmortem diagnosis of influenza because these tissues possess nucleic acids that are less degraded and, as a consequence, provide longer sequence information than that obtained from fixed tissues. We also determined that testing of all available respiratory tissues is critical for optimal detection of influenza virus in postmortem tissues.