We used polymerase chain reaction (PCR) to identify bacterial infections in culture-negative pleural fluid specimens from Alaska Native children hospitalized with empyema. PCR identified ≥1 organism in 11 (79%) of 14 specimens. Streptococcus pneumoniae serotype 3 was detected in six specimens; all six participants had received 13-valent pneumococcal conjugate vaccine.

BACKGROUND: Haemophilus influenzae (Hi) can cause severe disease in children. This study aimed to identify risk factors related to invasive Hi disease in Alaska children and evaluate carriage in people around them. METHODS: From 2005 to 2011, we investigated episodes of invasive, typeable Hi disease in Alaska children <10 years old. Three age-matched control children were enrolled for each case-patient. We evaluated oropharyngeal Hi carriage in people in close contact with Hi case-patients (contacts) as well as control children and their household members. Individual and household risk factors for illness and carriage were evaluated using questionnaires and chart reviews. RESULTS: Thirty-eight of 44 (86%) children with invasive, typeable Hi disease were recruited: 20 Hi serotype a (53%), 13 serotype b (Hib) (34%) and 5 serotype f (13%). Children with the invasive Hi disease were more likely than controls to have underlying health problems (67% vs. 24%, P = 0.001), other carriers of any Hi in their household (61% vs. 15%, P < 0.001), and inadequate Hib vaccination (26% vs. 9%, P = 0.005). People who carried Hi were younger than noncarriers (mean 12.7 vs. 18.0 years, P = 0.008). The carriage was clustered within case-patient households, with carriage in 19% of household contacts, while only 6.3% of nonhousehold contacts and 5.5% of noncontacts carried the Hi serotype of interest ( P < 0.001). CONCLUSIONS: Factors associated with invasive Hi disease in children included underlying health problems, household carriage and inadequate Hib vaccination. The high level of carriage in case-patient households is important to consider when evaluating treatment and prophylaxis strategies.

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.

BACKGROUND: Haemophilus influenzae serotype a (Hia) can cause severe invasive disease, especially in young children. In 2018, four 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 (15) and invasive (4) Hia isolates from the outbreak community, together with 15 non-outbreak 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 two major clades that differed by an average of 300 core single nucleotide polymorphisms (SNPs). All isolates from the outbreak community were clustered in one subclade, within a larger clade containing 3 non-outbreak 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.

BACKGROUND: The effect of malaria infection on rVSVΔG-ZEBOV-GP (ERVEBO®) immunogenicity is unknown. METHODS: The Sierra Leone Trial to Introduce a Vaccine against Ebola (STRIVE) vaccinated 7998 asymptomatic adults with rVSVΔG-ZEBOV-GP during the 2014-6 Ebola epidemic. In STRIVE's immunogenicity sub-study, participants provided blood samples at baseline, 1, 6, and 9-12 months. Anti-glycoprotein (GP) binding and neutralizing antibodies were measured using validated assays. Baseline samples were tested for malaria parasites by PCR. RESULTS: Overall, 506 participants enrolled in the immunogenicity sub-study and had ≥1 post-vaccination antibody titer. Of 499 participants with a result, baseline malaria parasitemia was detected in 73(14.6%). All GP-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 seroresponded by GP-ELISA (≥2-fold rise AND ≥200 EU/ml), while 81.5% seroresponded by PRNT (≥4-fold rise) at ≥1 post-vaccination 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 and 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.

BACKGROUND: Between May and July 2018, four invasive Haemophilus influenzae serotype a (iHia) infections occurred in a remote Alaska community. We performed a public health response to prevent further illness and understand Hia carriage in the community. METHODS: We collected oropharyngeal (OP) samples community-wide from untreated individuals to evaluate baseline carriage. Risk factor data was collected by interview. To prevent additional illness, we offered prophylactic rifampin to individuals in contact with iHia patients (contacts) and to all children aged <10 years. OP samples were collected again eight weeks post-rifampin distribution. Samples were tested using real-time PCR and culture. RESULTS: At baseline, Hia was carried by 4/27 (14.8%) contacts and 7/364 (1.9%) non-contacts (p<0.01). Contacts aged <10 years were more likely to carry Hia at any timepoint (11/18, 61%) than contacts aged >/=10 years (3/34, 8.8%) or non-contacts aged <10 years (2/139, 1.4%) and >/=10 years (6/276, 2.2%)(p<0.001 for all). Hia carriers were clustered in nine households (7% of total households). At the household level, carriage was associated with households with >/=1 contact (PR=5.6, CI:1.3-21.6), crowding (PR=7.7, CI:1.1-199.5) and >/=3 tobacco users (PR=5.0, CI:1.2-19.6). Sixty-six percent (40/61) of contacts and 90% (111/124) of non-contacts aged <10 years received rifampin. Elevated carriage prevalence persisted in contacts when retested eight weeks after rifampin distribution (contacts 6/25 (24%), non-contacts 2/114 (1.8%), p<0.001). CONCLUSIONS: Hia carriage prevalence was significantly higher among people who had contact with iHia patients than the general community. Rifampin prophylaxis did not result in a reduction of Hia carriage prevalence in this community.

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.

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.

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(R) Plus and GenTegra(R) DNA. METHODS: We created crude and clean DNA extracts from five Streptococcus pneumoniae isolates. Extracts were stored at -30 degrees C (our usual DNA storage temperature), 25 degrees C (within the range of temperatures recommended for the products), and 50 degrees C (to simulate longer storage time). Samples were stored at -30 degrees C with no product and dried at 25 degrees C and 50 degrees 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 degrees C storage temperature, we simulated 362 weeks of 25 degrees 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 degrees 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 degrees C (Ct range ≤3.0). Samples stored at 25 degrees 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 degrees C. It is not effective to store bacterial DNA at room temperature without a stabilizing matrix.

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.

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 79-89% and 69-94% 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.

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.

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.

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((R)) 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 1.01x10(9)copies/mL vs. a median of 5.25x10(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.

We read with interest of three Neisseria meningitidis strains recovered in Germany that reacted with commercial (Oxoid, Wesel, Germany) serogroupY and serogroup W135 antisera (3). The molecular basis of this dual antigenic specificity has been determined to be due to a single amino acid change at position 310 in the EX7E motif of the capsule polymerase enzyme synG (also referred to as siaDY) (from glycine to serine) or synF (siaDW-135) (from proline to serine) (2). Both studies confirmed our earlier report of such unusual strains causing invasive meningococcal disease (IMD) (10).

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.