Genomic Diversity of Haemophilus influenzae Serotype a in an Outbreak Community-Alaska, 2018.

BACKGROUND: Haemophilus influenzae serotype a (Hia) can cause severe invasive disease, especially in young children. In 2018, 4 invasive Hia cases occurred in an Alaska community. We used whole-genome sequencing (WGS) to evaluate the relationship of the bacteria from this community and other Alaska patients with invasive Hia. METHODS: All carriage (n = 15) and invasive (n = 4) Hia isolates from the outbreak community, together with 15 nonoutbreak Alaska invasive Hia surveillance isolates from 2018, were tested for antimicrobial susceptibility and characterized using WGS. RESULTS: Phylogenetic analysis of both invasive and carriage Hia isolates revealed 2 major clades that differed by an average of 300 core single-nucleotide polymorphisms (SNPs). All isolates from the outbreak community were clustered in 1 subclade, within a larger clade containing 3 nonoutbreak invasive Hia isolates. Comparative genomics did not reveal any genetic mutations that distinguished carriage from invasive isolates. Three (20%) community isolates were rifampin resistant and had a previously unreported mutation in the rpoB gene. CONCLUSIONS: In the outbreak community, Hia isolates from carriers were indistinguishable from the invasive Hia isolates. Overall, invasive Hia isolates from Alaska in 2018 were genetically similar. The rifampin resistance mutation is concerning as rifampin is the first-line medication for Hia prophylaxis.

Baseline Asymptomatic Malaria Infection and Immunogenicity of Recombinant Vesicular Stomatitis Virus-Zaire Ebola Virus Envelope Glycoprotein.

BACKGROUND: The effect of malaria infection on the immunogenicity of the recombinant vesicular stomatitis virus-Zaire Ebola virus envelope glycoprotein (GP) vaccine (rVSVΔG-ZEBOV-GP) (ERVEBO) is unknown. METHODS: The Sierra Leone Trial to Introduce a Vaccine Against Ebola (STRIVE) vaccinated 7998 asymptomatic adults with rVSVΔG-ZEBOV-GP during the 2014-2016 Ebola epidemic. In STRIVE's immunogenicity substudy, participants provided blood samples at baseline and at 1, 6, and 9-12 months. Anti-GP binding and neutralizing antibodies were measured using validated assays. Baseline samples were tested for malaria parasites by polymerase chain reaction. RESULTS: Overall, 506 participants enrolled in the immunogenicity substudy and had ≥1 postvaccination antibody titer. Of 499 participants with a result, baseline malaria parasitemia was detected in 73 (14.6%). All GP enzyme-linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT) geometric mean titers (GMTs) at 1, 6, and 9-12 months were above baseline, and 94.1% of participants showed seroresponse by GP-ELISA (≥2-fold rise and ≥200 ELISA units/mL), while 81.5% showed seroresponse by PRNT (≥4-fold rise) at ≥1 postvaccination assessment. In participants with baseline malaria parasitemia, the PRNT seroresponse proportion was lower, while PRNT GMTs and GP-ELISA seroresponse and GMTs showed a trend toward lower responses at 6 and 9-12 months. CONCLUSION: Asymptomatic adults with or without malaria parasitemia had robust immune responses to rVSVΔG-ZEBOV-GP, persisting for 9-12 months. Responses in those with malaria parasitemia were somewhat lower.

Haemophilus influenzae Serotype a (Hia) Carriage in a Small Alaska Community After a Cluster of Invasive Hia Disease, 2018.

BACKGROUND: Between May and July 2018, 4 Haemophilus influenzae serotype a (Hia) infections occurred in a remote Alaska community. We performed a public health response to prevent further illness and understand Hia carriage. METHODS: We collected oropharyngeal samples community-wide to evaluate baseline carriage. Risk factors were evaluated by interview. We offered prophylactic rifampin to individuals in contact with invasive Hia patients (contacts) and to all children aged <10 years. Oropharyngeal samples were collected again 8 weeks after rifampin distribution. Samples were tested using real-time polymerase chain reaction and culture. RESULTS: At baseline, 4 of 27 (14.8%) contacts and 7 of 364 (1.9%) noncontacts (P < .01) carried Hia. Contacts aged <10 years were more likely to carry Hia at any timepoint (11/18 [61%]) compared to contacts aged ≥10 years (3/34 [8.8%]), noncontacts aged <10 years (2/139 [1.4%]), and noncontacts ≥10 years (6/276 [2.2%]) (P < .001 for all). Hia carriers were clustered in 9 households (7% of total households). At the household level, carriage was associated with households with ≥1 contact (prevalence ratio [PR], 5.6 [95% confidence interval {CI}, 1.3-21.6]), crowding (PR, 7.7 [95% CI, 1.1-199.5]), and ≥3 tobacco users (PR, 5.0 [95% CI, 1.2-19.6]). Elevated carriage prevalence persisted in contacts compared to noncontacts 8 weeks after rifampin distribution (6/25 [24%] contacts, 2/114 [1.8%] noncontacts; P < .001). CONCLUSIONS: Hia carriage prevalence was significantly higher among contacts than noncontacts. Rifampin prophylaxis did not result in a reduction of Hia carriage prevalence in this community.

Haemophilus influenzae serotype a invasive disease, Alaska, USA, 1983-2011.

Before introduction of Haemophilus influenzae type b (Hib) vaccines, rates of Hib disease in Alaska's indigenous people were among the highest in the world. Vaccination reduced rates dramatically; however, invasive H. influenzae type a (Hia) disease has emerged. Cases of invasive disease were identified through Alaska statewide surveillance during 1983-2011. Of 866 isolates analyzed for serotype, 32 (4%) were Hia. No Hia disease was identified before 2002; 32 cases occurred during 2002-2011 (p<0.001). Median age of case-patients was 0.7 years; 3 infants died. Incidence of Hia infection (2002-2011) among children <5 years was 5.4/100,000; 27 cases occurred in Alaska Native children (18/100,000) versus 2 cases in non-Native children (0.5/100,000) (risk ratio = 36, p<0.001). From 12/2009 to 12/2011, 15 cases of Hia disease occurred in southwestern Alaska (in children <5 years, rate = 204/100,000). Since introduction of the Hib conjugate vaccine, Hia infection has become a major invasive bacterial disease in Alaska Native children.

Epidemiology of invasive Haemophilus influenzae serotype a disease in the North American Arctic, 2006-2017.

Invasive Haemophilus influenzae type a (iHia) disease was detected in Alaska and Northern Canada in 2002 and 2000, respectively. From 2006 to 2017, 164 iHia cases (Alaska=53, Northern Canada=111) were reported. Rates of iHia disease per 100,000 persons were higher in Northern Canada compared to Alaska and were significantly higher in Indigenous (Alaska 2.8, Northern Canada 9.5) compared to non-Indigenous populations (Alaska 0.1, Northern Canada=0.4). Disease rates were highest in Indigenous children <2 years of age (Alaska 56.2, Northern Canada=144.1) and significantly higher than in non-Indigenous children <2 (Alaska 0.1, Northern Canada 0.4). The most common clinical presentation in children <5 years was meningitis of age and pneumonia in persons ≥5 years old. Most patients were hospitalised (Alaska=87%, Northern Canada=89%) and fatality was similar (Alaska=11%, Northern Canada=10%). MLST testing showed sequence types ST23 and ST576 in Northern Canada and ST576, ST23 and ST56 in Alaska. Alaska and Northern Canada have high rates of iHia disease. A vaccine is needed in these regions to protect young children.

Serotyping of Streptococcus pneumoniae isolates from nasopharyngeal samples: use of an algorithm combining microbiologic, serologic, and sequential multiplex PCR techniques.

We evaluated nasopharyngeal carriage of Streptococcus pneumoniae (pneumococci) in nine Alaskan communities and used an algorithm combining microbiologic, serologic, and sequential multiplex PCR (MP-PCR) techniques to serotype the isolates. After microbiological identification as pneumococci, isolates (n = 1,135) were serotyped using latex agglutination and Quellung tests (LA/Q) as well as a series of six sequential MP-PCR assays. Results from the two methods agreed for 94% (1,064/1,135) of samples. Eighty-six percent (61/71) of the discordant results were resolved. Discordant results occurred because (i) the MP-PCR gel was misread (31/61 [51%]), (ii) the LA/Q agglutination was misinterpreted (13/61 [21%]), (iii) two serotypes or sets of serotypes were identified by MP-PCR and only one of the two was identified by LA/Q (9/61 [15%]), (iv) different serotypes or sets of serotypes were identified by LA/Q and MP-PCR and both were correct (7/61 [11%]), and (v) the capsular polysaccharide locus (cps) did not amplify during the initial MP-PCR but was present upon retesting (1/61 [2%]). Overall, isolation of pneumococci followed by MP-PCR quickly and accurately identified pneumococcal serotypes in >97% of samples and made available isolates for additional tests such as antimicrobial susceptibility. Misinterpretation of the MP-PCR gel was identified as the main source of discordance. Increasing the number of MP-PCRs from six to seven and reducing the number of serotypes in each reaction may reduce this error. This method may be of use to laboratories characterizing large numbers of S. pneumoniae samples, especially when antimicrobial susceptibility data are needed.

Transplacental Respiratory Syncytial Virus and Influenza Virus Antibody Transfer in Alaska Native and Seattle Mother-Infant Pairs.

BACKGROUND: Alaska Native (AN) infants are at risk for severe disease due to respiratory syncytial virus (RSV) and influenza. Maternal immunization protects young infants through transplacental antibody transfer. RSV- and influenza-specific transplacental antibody transfer in mother-infant pairs has not previously been evaluated in the AN population. METHODS: Serum samples collected during pregnancy and at birth from AN mother-infant pairs in the Yukon-Kuskokwim Delta region (YKD) of Alaska (2000-2011; n = 75) and predominantly white pairs in Seattle, Washington (2014-2016; n = 57), were tested for RSV and influenza antibody using a microneutralization and hemagglutination inhibition assay, respectively, and compared between sites. RESULTS: Mean RSV antibody concentrations in pregnant women in YKD and Seattle were similar (log2 RSV antibody 10.6 vs 10.7, P = .86), but cord blood RSV antibody concentrations were significantly lower in infants born to mothers in YKD compared with Seattle (log2 RSV antibody 11.0 vs 12.2, P < .001). Maternal and cord blood influenza antibody concentrations were lower for women and infants in YKD compared with Seattle for all 4 influenza antigens tested (all P < .05). The mean cord to maternal RSV antibody transfer ratio was 1.15 (standard deviation [SD], 0.13) in mother-infant pairs in Seattle compared with 1.04 (SD, 0.08) in YKD. Mean cord blood to maternal antibody transfer ratios for influenza antigens ranged from 1.22 to 1.42 in Seattle and from 1.05 to 1.59 in YKD. CONCLUSIONS: Though the transplacental antibody transfer ratio was high (>1.0) for both groups, transfer ratios for RSV antibody were significantly lower in AN mother-infant pairs. Further studies are needed to elucidate the impact of lower transplacental antibody transfer on infant disease risk in rural Alaska.Alaska Native and continental US mother-infant pairs have high transplacental antibody transfer ratios (>1.0) for influenza and respiratory syncytial virus, but anti-respiratory syncytial virus antibody levels are significantly lower in Alaska Native pairs than in those from the continental US.

Viral respiratory infections in hospitalized and community control children in Alaska.

Respiratory syncytial virus (RSV) in Alaska Native children from the Yukon Kuskokwim (YK) Delta is associated with a hospitalization rate five times higher than that reported for the general US child population. The role of other viral respiratory pathogens has not been studied in this population. YK Delta children <3 years of age hospitalized with respiratory infections and same aged community control children were prospectively enrolled between October 2005 and September 2007. Polymerase chain reaction detection of viruses was performed on nasopharyngeal samples. Characteristics of hospitalized and asymptomatic control children were analyzed. From October 2005 to September 2007, 440 hospitalized and 425 control children were analyzed. Respiratory viruses were detected in 90% (395) of hospitalized children: 194 (44%) rhinovirus, 131 (30%) adenovirus, 102 (23%) RSV, 77 (18%) para influenza viruses (PIV), 66 (15%) human metapneumovirus (hMPV), 23 (5%) influenza, and 25 (6%) coronavirus. Fifty-two percent (221) of control children had a virus detected, most commonly rhinovirus (33%), and adenovirus (16%). RSV, PIV, hMPV, and influenza were significantly more common in hospitalized cases than control children, but rhinovirus, adenovirus, and coronavirus were not. RSV and hMPV were associated with higher severity of illness. In this study, RSV remains the most important virus associated with respiratory hospitalization, although hMPV and PIV were also common. RSV and hMPV were associated with more severe illness. Rhinovirus and adenovirus were detected in two-thirds of hospitalized children, but their frequent detection in control children made their role in respiratory hospitalization uncertain.

Respiratory syncytial virus and influenza hospitalizations in Alaska native adults.

BACKGROUND: Alaska Native (AN) infants from Yukon Kuskokwim Delta (YKD) have the highest U.S. infant hospitalization rate for respiratory syncytial virus (RSV). RSV can cause significant morbidity and mortality in adult populations, although the RSV burden in AN adults is unknown. Here we investigate RSV, influenza, and human metapneumovirus (hMPV) in hospitalized rural AN adults. METHODS: YKD AN adults, hospitalized with acute respiratory illness between November 2016 and October 2018 were enrolled prospectively. Nasopharyngeal (NP) swabs were tested for RSV, influenza and hMPV using polymerase chain reaction. Hospitalization rates were calculated. RESULTS: Of 251 patients who had an NP swab, RSV was detected in 8 (3.2 %), influenza in 31 (12.4 %), and hMPV in no patients. Weighted annual rates of lower respiratory tract infection (LRTI), RSV and influenza hospitalization were 192.0 (95 % CI: 176.5-208.4), 9.1 (6.0-13.3), and 42.2 (35.1-50.2) per 10,000. The most common discharge diagnosis was pneumonia (57.0 %), followed by chronic obstructive pulmonary disease (51.4 %). Ninety-eight percent (246/251) had a medical co-morbidity and 49.8 % (125/251) lived in a house with a smoker. Overall, 6.4 % (16/251) required mechanical ventilation, and 3.6 % (9/251) died during hospitalization. Only 35.7 % (66/185) of patients admitted during influenza season had received the annual influenza vaccine. DISCUSSION: We examined adult LRTI, influenza, and RSV hospitalization rates in an AN population with high infant RSV hospitalization rates. While we confirmed a high rate of hospitalization from LRTIs and influenza, we did not find a high rate due to RSV or hMPV. Improving influenza vaccination rates, and addressing co-morbidities could reduce respiratory hospitalizations.

Evaluation of Two Matrices for Long-Term, Ambient Storage of Bacterial DNA.

BACKGROUND: Culture-independent molecular analyses allow researchers to identify diverse microorganisms. This approach requires microbiological DNA repositories. The standard for DNA storage is liquid nitrogen or ultralow freezers. These use large amounts of space, are costly to operate, and could fail. Room temperature DNA storage is a viable alternative. In this study, we investigated storage of bacterial DNA using two ambient storage matrices, Biomatrica DNAstable® Plus and GenTegra® DNA. METHODS: We created crude and clean DNA extracts from five Streptococcus pneumoniae isolates. Extracts were stored at -30°C (our usual DNA storage temperature), 25°C (within the range of temperatures recommended for the products), and 50°C (to simulate longer storage time). Samples were stored at -30°C with no product and dried at 25°C and 50°C with no product, in Biomatrica DNAstable Plus or GenTegra DNA. We analyzed the samples after 0, 1, 2, 4, 8, 16, 32, and 64 weeks using the Nanodrop 1000 to determine the amount of DNA in each aliquot and by real-time PCR for the S. pneumoniae genes lytA and psaA. Using a 50°C storage temperature, we simulated 362 weeks of 25°C storage. RESULTS: The average amount of DNA in aliquots stored with a stabilizing matrix was 103%-116% of the original amount added to the tubes. This is similar to samples stored at -30°C (average 102%-121%). With one exception, samples stored with a stabilizing matrix had no change in lytA or psaA cycle threshold (Ct) value over time (Ct range ≤2.9), similar to samples stored at -30°C (Ct range ≤3.0). Samples stored at 25°C with no stabilizing matrix had Ct ranges of 2.2-5.1. CONCLUSION: DNAstable Plus and GenTegra DNA can protect dried bacterial DNA samples stored at room temperature with similar effectiveness as at -30°C. It is not effective to store bacterial DNA at room temperature without a stabilizing matrix.

Risk factors for hospitalization with lower respiratory tract infections in children in rural Alaska.

OBJECTIVE: Lower respiratory tract infections (LRTIs) are a major cause of morbidity for children worldwide and particularly for children from developing and indigenous populations. In this study, we evaluated risk factors for hospitalization with LRTI in a region in southwest Alaska. METHODS: The study was conducted from October 1, 2006, to September 30, 2007, in the Yukon Kuskokwim Delta region of Alaska. Cases were recruited from children <3 years of age hospitalized with LRTI. Controls were recruited during visits to the surrounding communities in the region and matched posthoc to cases on the basis of subregion, season, and age. Parents were interviewed for potential risk factors, and medical records were reviewed. Participants had a nasopharyngeal swab sample taken for polymerase chain reaction (PCR) testing for a panel of respiratory viruses. Samples positive for respiratory syncytial virus, human metapneumovirus, or parainfluenza type 3 were quantitated by reverse transcriptase real-time quantitative PCR. RESULTS: One hundred twenty-eight cases were matched to 186 controls. In a multivariable conditional logistic regression model, significantly (P < .05) increased risk of hospitalization was associated with medically high-risk status, having a woodstove in the house, being bottle fed, and vomiting after feeding; living in a house that had 2 or more rooms with sinks was a protective factor. Viral loads in hospitalized cases were significantly higher than those in controls, but a strict cutoff level was not observed. CONCLUSIONS: Several risk factors for LRTI hospitalization were identified in this high risk population. Some factors are amenable to environmental and behavioral interventions.

Comparison of nasopharyngeal flocked swabs and nasopharyngeal wash collection methods for respiratory virus detection in hospitalized children using real-time polymerase chain reaction.

This paper describes the molecular detection of respiratory viruses from nasopharyngeal flocked swabs (flocked swabs) and nasopharyngeal washes (washes) in a clinical setting. Washes and flocked swabs collected from children<3 years old hospitalized with a lower respiratory tract infection were tested for parainfluenza virus 1-3, respiratory syncytial virus, influenza A and B and metapneumovirus (Group 1) and adenovirus, rhinovirus and coronavirus (Group 2) using real-time reverse transcriptase PCR (rRT-PCR). A consensuses standard was used to determine sensitivity and compare cycle thresholds (C(T)) of washes and flocked swabs for each virus and group of viruses. Sensitivities ranged from 79 to 89% and 69 to 94% for flocked swabs and washes, respectively, excluding AdV which had a sensitivity of 35% for flocked swabs. When the flocked swabs and washes of Group 1 viruses were collected on the day of admission, the sensitivity of both sample types was 100%. Wash specimens had a lower C(T) value and higher sensitivity than flocked swabs; however there was no statistical difference in the sensitivity of a flocked swab (89%) versus wash (93%) for the detection of Group 1 viruses, particularly when samples were collected on the same day. Flocked swabs may be a useful alternative to washes for detection of respiratory viruses in clinical settings.

Performance of a rapid antigen test (Binax NOW® RSV) for diagnosis of respiratory syncytial virus compared with real-time polymerase chain reaction in a pediatric population.

BACKGROUND: Infants from Alaska's Yukon-Kuskokwim Delta (YKD) have a high respiratory syncytial virus (RSV) hospitalization rate (104/1000/yr). Appropriate patient management requires rapid and accurate RSV diagnosis. Antigen-based methods are often used in clinical settings, but these tests can lack sensitivity. OBJECTIVE: We compared Binax NOW(®) RSV (BN) used for RSV diagnosis in the YKD hospital with a real-time polymerase chain reaction assay (RT-qPCR) used for viral surveillance. STUDY DESIGN: Between October 2005 and September 2007 we obtained nasopharyngeal washes (NPW) from children <3 years hospitalized with a lower respiratory tract infection. The NPW were tested using BN and RT-qPCR. RESULTS: 79/311 (25%) children had RSV infection as determined by RT-qPCR. As compared with RT-qPCR, sensitivity and specificity of BN were 72% and 97%, respectively. The sensitivity of BN was higher in children <1 year compared with children ≥ 1 year (79% vs. 52%; p=0.025), children with bronchiolitis compared with children without bronchiolitis (89% vs. 38%; p<0.001), and children with a shorter duration of symptoms before testing (0-1 (92%) vs. 2-4 (78%) vs. 5+ (65%) days; p=0.04). The median RSV viral load in NPW positive by BN and RT-qPCR was 1.01 × 10(9)copies/mL vs. a median of 5.25 × 10(7)copies/mL for NPW positive by RT-qPCR only (p<0.001). CONCLUSION: RT-qPCR is more sensitive than BN in detecting RSV infection. BN sensitivity is high in children with bronchiolitis, but the sensitivity is low when children present with a non-bronchiolitis illness, especially after a longer duration of symptoms before testing.

Epidemiology of Haemophilus influenzae serotype a, North American Arctic, 2000-2005.

Before the introduction of Haemophilus influenzae type b (Hib) conjugate vaccines, rates of invasive H. influenzae disease among indigenous people of the North American Arctic were among the highest in the world. Routine vaccination reduced rates to low levels; however, serotype replacement with non-type b strains may result in a reemergence of invasive disease in children. We reviewed population-based data on invasive H. influenzae in Alaska and northern Canada from 2000-2005; 138 cases were reported. Among 88 typeable isolates, 42 (48%) were H. influenzae type a (Hia); 35 (83%) occurred in indigenous peoples. Among Hia patients, median age was 1.1 years; 62% were male; 1 adult died. Common clinical manifestations included meningitis, pneumonia, and septic arthritis. Overall annual incidence was 0.9 cases per 100,000 population. Incidence among indigenous children <2 years of age in Alaska and northern Canada was 21 and 102, respectively. Serotype a is now the most common H. influenzae serotype in the North American Arctic; the highest rates are among indigenous children.

The Alaska Haemophilus influenzae type b experience: lessons in controlling a vaccine-preventable disease.

OBJECTIVE: Before 1991, Alaska Native children experienced one of the highest rates of invasive Haemophilus influenzae type b disease. H influenzae type b vaccine has led to a near-elimination of invasive H influenzae type b disease in the United States. We describe challenges encountered in controlling H influenzae type b disease in Alaska and update the current status of H influenzae disease and carriage in Alaska as lessons to other populations. PATIENTS AND METHODS: We reviewed data from statewide H influenzae disease surveillance conducted during 1980-2004. Vaccine coverage data were based on audits from tribal facilities and the National Immunization Survey. H influenzae type b colonization data were based on 6 carriage studies. RESULTS: After universal infant vaccination in 1991, H influenzae type b disease among Alaska Native and non-Native children < 5 years of age decreased by 94% and 96%, respectively. After a 1996 change in H influenzae type b vaccine from polyribosylribitol phosphate-outer membrane protein conjugate vaccine to H influenzae type b oligosaccharide-CRM197 vaccine, the incidence of H influenzae type b disease increased in rural Alaska Natives from 19.8 to 91.1 cases per 100000 per year < 5 years of age. During 2001-2004, with use of polyribosylribitol phosphate-outer membrane protein conjugate vaccine, the rate of H influenzae type b disease in Alaska Native and non-Native children aged < 5 years decreased to 5.4 and 0 per 100000 per year, respectively. In postvaccine studies, H influenzae type b carriage has decreased in Alaska Native children < 5 years of age. CONCLUSIONS: H influenzae type b vaccination has resulted in a dramatic decrease in invasive H influenzae type b disease in Alaska; however, despite high rates of H influenzae type b vaccine coverage, H influenzae type b disease rates among rural Alaska Native children < 5 years of age remain higher than the rates among non-Native Alaska and other US children. Equity in disease rates may not be achieved in indigenous populations with the current vaccines unless other environmental and household factors contributing to disease transmission are addressed.