Genetic and Antigenic Analysis of the First A/New Caledonia/20/99-like H1N1 Influenza Isolates Reported in the Americas

From February through May of 1999, 13 cases of Influenza A virus (FLUAV), type H1N1 were reported at a Department of Defense influenza surveillance sentinel site in Lima, Peru. Genetic and antigenic analysis by hemagglutination inhibition and direct nucleotide sequencing of the HA1 region of the hemagglutinin gene were performed on two isolates, A/Peru/1641/99 and A/Peru/1798/99. Both isolates were distinct from the Bayern/7/95-like viruses circulating in the Americas and closely related to a Beijing/262/95-like variant, A/New Caledonia/20/99. With the exception of travel-related cases, the detection of these isolates represents the first appearance of New Caledonia/20/99-like viruses in the Americas. Since the characterization of these Peru isolates, a number of New Caledonia/20/99-like viruses have been reported worldwide. For the 2000/01 and 2001/02 influenza seasons, the World Health Organization (WHO) has recommended the inclusion of A/New Caledonia/20/99 as the H1N1 vaccine component for both the southern and northern hemispheres

Identifying Influenza Viruses with Resequencing Microarrays

Resequencing microarrays rapidly identify influenza viruses

Influenza A (H3N2) Outbreak, Nepal

Worldwide emergence of variant viruses has prompted a change in the 2005–2006 H3N2 influenza A vaccine strain

Improved detection by next-generation sequencing of pyrazinamide resistance in mycobacterium tuberculosis isolates

Technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis (MTB) isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multi-drug resistant tuberculosis (MDR-TB) . In this study, a 606 base pair fragment (comprising the pncA coding region plus promoter) was sequenced using Ion Torrent next generation sequencing (NGS) for detecting associated PZA resistance mutations in 90 re-cultured, MDR-TB isolates from an archived series collected in 2001. These 90 isolates were previously Sanger sequenced, with 55 (62%) designated as carrying wild type pncA gene and 33 (38%) showing mutations. Also earlier, PZA susceptibility of the isolates was determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were re-cultured and susceptibility testing performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 87% (n = 90), and with the Bactec 460, Wayne test, and pncA gene Sanger sequencing, 82% (n = 88), 83% (n = 88), and 89% (n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing, but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151-360 region, and warrants further exploration. In these isolates, with known resistance to rifampicin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard, and helped to resolve discordant results from conventional methodologies.University of Pretoria, the South African Medical Research Council, and the National Research Foundation of South Africa.http://jcm.asm.org2016-06-30hb201

Next-generation sequencing for identifying pyrazinamide resistance in Mycobacterium tuberculosis

No abstract available.http://cid.oxfordjournals.orgam201

Opsonic monoclonal antibodies enhance phagocytic killing activity and clearance of Mycobacterium tuberculosis from blood in a quantitative qPCR mouse model

BACKGROUND : Patients with impaired immunity often have rapid progression of tuberculosis (TB) which can lead to highly lethal Mycobacterium tuberculosis (MTB) sepsis. Opsonic monoclonal antibodies (MABs) directed against MTB that enhance phagocytic killing activity and clearance of MTB from blood may be useful to enhance TB immunity. METHODS : BALB/c mice were immunized with ethanol-killed MTB (EK-MTB) and MABs were produced and screened by ELISA for binding to killed and live Mycobacterium smegmatis (SMEG) and MTB. MAB opsonophagocytic killing activity (OPKA) was examined using SMEG with HL60 and U-937 cells and MTB with U-937 cells. Clearance of MTB from blood was evaluated in Institute of Cancer Research (ICR) mice given opsonic anti-MTB MABs or saline (control) 24 h prior to intravenous infusion with 108 CFUs gamma-irradiated MTB (HN878). MTB levels in murine blood collected 0.25, 4 and 24 h post-challenge were assessed by qPCR. MAB binding to peptidoglycan (PGN) was examined by ELISA using PGN cell wall mixture and ultra-pure PGN. RESULTS : Two MABs (GG9 and JG7) bound to killed and live SMEG and MTB (susceptible and resistant), and promoted OPKA with live MTB. MAB JG7 significantly enhanced OPKA of MTB. Both MABs significantly enhanced clearance of killed MTB from murine blood at 4 and 24 h as measured by qPCR. These opsonic MABs bound to PGN, a major cell wall constituent. CONCLUSIONS : Anti-MTB MABs that promote bactericidal phagocytic activity of MTB and enhance clearance of killed MTB from the blood, may offer an immunotherapeutic approach for treatment of MTB bacteremia or sepsis, and augment treatment of multi-drug resistant (MDR) or extensively drug resistant (XDR) TB.This work was supported by Longhorn Vaccines and Diagnostics, LLC. Live MTB work was additionally supported from post-graduate student bursaries to Bong-Akee Shey from the University of Pretoria and from grant 105830 to PBF by the National Research Foundation of South Africa.Post-graduate student bursaries to Bong-Akee Shey from the University of Pretoria and from grant 105830 to PBF by the National Research Foundation of South Africa.https://www.heliyon.comam2020Medical Microbiolog

Multi- and extensively drug resistant mycobacterium tuberculosis in South Africa : a molecular analysis of historical isolates

Modern advances in genomics provide an opportunity to reinterpret historical bacterial culture collections. In this study, genotypic antibiotic resistance profiles of Mycobacterium tuberculosis isolates from a historical 20-year-old multidrug-resistant tuberculosis (MDR-TB) culture collection in South Africa are described. DNA samples extracted from the phenotypically MDR-TB isolates (n = 240) were assayed by Hain line probe assay (LPA) for the confirmation of MDR-TB and by Illumina Miseq whole-genome sequencing (WGS) for the characterization of mutations in eight genes (rpoB, katG, inhA, rpsL, pncA, embB, gyrA, and rrs) that are known to code for resistance to commonly used anti-TB agents. LPA identified 71.3% of the TB isolates as MDR-TB, 18.3% as rifampin (RIF) monoresistant, 2% as isoniazid (INH) monoresistant, and 8.3% as susceptible to both RIF and INH (RIF+INH). In a subset of 42 randomly selected isolates designated as RIF+INH resistant by Löwenstein-Jensen (LJ) culture in 1993, LPA and WGS results confirmed MDR-TB. In all five INH-monoresistant isolates by LPA and in all but one (the wild type) of the 34 successfully sequenced RIF-monoresistant isolates, WGS revealed matching mutations. Only 26% of isolates designated as susceptible by LPA, however, were found to be wild type by WGS. Novel mutations were found in the rpoB (Thr480Ala, Gln253Arg, Val249Met, Val251Tyr, Val251Phe), katG (Trp477STOP, Gln88STOP, Trp198STOP, Trp412STOP), embB (Thr11Xaa, Gln59Pro), and pncA (Thr100Ile, Thr159Ala, Ala134Arg, Val163Ala, Thr153Ile, DelGpos7, Phe106Ser) genes. Three MDR-TB isolates showed mutations in both the gyrA and rrs genes, suggesting that extensively drug-resistant tuberculosis existed in South Africa well before its formal recognition in 2006.The Global Infectious Diseases Research Training Fogarty Fellowship, the University of Pretoria; the South African Medical Research Council and the National Research Foundation of South Africa.http://jcm.asm.org2018-10-31hj2018Medical Microbiolog

Molecular detection of Mycobacterium tuberculosis from sputum transported in PrimeStore(®) from rural settings

SETTING : Mopani District, South Africa. OBJECTIVE : To explore remote, molecular detection of Mycobacterium tuberculosis from sputum transported using PrimeStorew Molecular Transport Medium (PSMTM) compared to settings where microscopy or Xpertw MTB/RIF is used as the baseline test. DESIGN : Two sputum specimens were collected from patients with cough of72 weeks at clinics in rural South Africa. Shortly after expectoration and before processing using Xpert, microscopy and liquid culture, a flocked swab was swirled in each of these specimens and placed in PS-MTM. Swabs were stored and transported to the United States at ambient temperature for real-time PrimeMixw polymerase chain reaction (PM-PCR). RESULTS : Of 132 patients, 23 (17%) were positive on microscopy, 39 (30%) on Xpert and 44 (33%) by PSMTM/PSMTM/ PM-PCR. Concordance of PS-MTM/PM-PCR with positive microscopy and Xpert was respectively 96% and 85%. Of 107 microscopy-negative samples, 22 (21%) were positive using PS-MTM/PM-PCR, while 11/91 (12%) Xpert-negative samples were PS-MTM/ PM-PCR-positive. PS-MTM/PM-PCR positivity was significantly higher than smear microscopy positivity (P , 0.001), but similar to Xpert (P ¼ 0.33). CONCLUSION: PCR testing of specimens transported in PS-MTM would enhance TB diagnosis in settings where smear microscopy is the baseline diagnostic test, and could provide an alternative in settings where Xpert testing is not available.http://www.ingentaconnect.comcontent/iuatld/ijtld2015-11-30hb201

Next-generation ion torrent sequencing of drug resistance mutations in Mycobacterium tuberculosis strains

A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes—rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)—were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin- resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug- resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.http://jcm.asm.org/am201

Deep-Inelastic Inclusive ep Scattering at Low x and a Determination of alpha_s

A precise measurement of the inclusive deep-inelastic e^+p scattering cross section is reported in the kinematic range 1.5<= Q^2 <=150 GeV^2 and 3*10^(-5)<= x <=0.2. The data were recorded with the H1 detector at HERA in 1996 and 1997, and correspond to an integrated luminosity of 20 pb^(-1). The double differential cross section, from which the proton structure function F_2(x,Q^2) and the longitudinal structure function F_L(x,Q^2) are extracted, is measured with typically 1% statistical and 3% systematic uncertainties. The measured partial derivative (dF_2(x,Q^2)/dln Q^2)_x is observed to rise continuously towards small x for fixed Q^2. The cross section data are combined with published H1 measurements at high Q^2 for a next-to-leading order DGLAP QCD analysis.The H1 data determine the gluon momentum distribution in the range 3*10^(-4)<= x <=0.1 to within an experimental accuracy of about 3% for Q^2 =20 GeV^2. A fit of the H1 measurements and the mu p data of the BCDMS collaboration allows the strong coupling constant alpha_s and the gluon distribution to be simultaneously determined. A value of alpha _s(M_Z^2)=0.1150+-0.0017 (exp) +0.0009-0.0005 (model) is obtained in NLO, with an additional theoretical uncertainty of about +-0.005, mainly due to the uncertainty of the renormalisation scale.Comment: 68 pages, 24 figures and 18 table