Mortality in H1N1: A comparison of patient attributes in outbreaks due to A/California/7/2009 and A/Michigan/45/2015 strains.

INTRODUCTION: The resurgence of epidemic of Influenza A (H1N1) pdm 09 was phenomenal in 2015 and has become an annual phenomenon. Antigenic drift and reassortment is the rule rather than exception, conferring survival benefit to the virus. As this disease has high mortality, we compared the clinico-epidemiological profile of patients expired in the year 2015 due to "A/California/7/2009" strain with those of expired in the year 2018 due to "A/Michigan/45/2015" strain. MATERIAL AND METHOD: We collected data of all expired patients in our institute in the year 2015 from 1st January to 30th may as well as 2018 in the same time period. The data of 116 patients who expired in 2015 due to "A/California/7/2009" H1N1 strain were compared with similar data of 30 patients expired in 2018 due to "A/Michigan/45/2015" strain of H1N1. Patients of pneumonia, having age >18 years, positive for H1N1by real-time reverse-transcriptase-polymerase- chain-reaction (RT-PCR) and died in our hospital were included in this study. Clinical features and laboratory data were obtained from the hospital records of the patients. Data analysis was done using SPSS software. RESULT: In 2015 total number of hospitalized patients due to "A/California/7/2009" strain were 571 and 116(20.31%) out of them died, in 2018 those due to "A/Michigan/45/2015" total admission were 177 and 30(16.94%) out of them died (p-0.032). Though it was not statistically significant but it is lesser than in 2015 despite the fact that more patients with co morbidities were affected in 2018. Duration in ICU was significantly longer in 2018(MS) group [5(1-7)] compared to 2015 (CS) group [3(1-17)] with p value of 0.017 (i.e. < 0.05). But both groups were not different in terms of duration on mechanical ventilator. (p-0.257).The 2015 (CS) group had 74.1% with other co-morbidities versus 96.7% of those in 2018 (MS) group (p- 0.015). This implies that the mortality with "A/Michigan/45/2015" infection was mainly seen in the patients who already had one or more co-morbidities unlike "A/California/7/2009" infection.The 2018 (MS) group had significantly higher proportion (60%) of patients with acute kidney injury compared to 34.5% in 2015(CS) (p-0.019). 50% of dead patients in 2018(MS) had anemia compared to 11.2% in 2015(CS) (p<0.001). Deranged liver function test was seen in 46.7% patients in 2018(MS) compared to only 15.5% patients in 2014(CS) (p<0.001).The only reverse trend was shown in case of diabetes, A/California/7/2009 strain affected 27% diabetics compared with 6.7% affected by A/Michigan/45/2015 strain (p=0.030) (Table 5). CONCLUSION: The study showed that though "A/Michigan/45/2015" affected higher number of patients with co morbidities compared to "A/California/7/2009" but had slightly lesser mortality.

Progressive Disseminated Histoplasmosis with Coomb's Positive Hemolytic Anemia in an Immunocompetent Host.

Progressive Disseminated Histoplasmosis (PDH) is mainly described in immuno-compromised individuals and rare in immuno-competent subjects. Here we report a case of progressive disseminated histoplasmosis with Comb's positive hemolytic anemia, which is infrequently reported from a country like India where histoplasmosis is not an endemic mycosis.

A study of an influenza A (H1N1)pdm09 outbreak in pregnant women in Rajasthan, India.

OBJECTIVES: To describe the clinical and epidemiological factors of influenza A (H1N1)pdm09-infected patients who were pregnant or in the puerperal period during a recent influenza outbreak in Rajasthan, India. METHODS: A retrospective, observational study was performed using hospital records of individuals with confirmed influenza A (H1N1)pdm09 infection admitted to a large tertiary care center in Rajasthan, India, between January 1 and March 15, 2015. RESULTS: The study cohort included 44 patients who were pregnant or in the puerperal period. The mean age was significantly lower in the study cohort than in other female patients treated for influenza A (H1N1)pdm09 infection at the hospital during the study period (25.45±3.45years vs 40.46±13.99 years; P<0.001). Mortality was significantly higher in the study cohort than in the comparison female patients (36% vs 17%; P=0.003). Factors found to be associated with mortality were delayed initiation of oseltamivir, poor oxygen saturation at admission, and more than 50% lung involvement under radiographic examination. None of the patients in the study cohort had received a vaccination against seasonal influenza. CONCLUSION: Influenza A (H1N1)pdm09 infection during pregnancy causes more severe illness. Early recognition and timely administration of antiviral therapy can improve outcomes. The role of influenza vaccination in this high-risk population cannot be overemphasized.

Profiling the Mortality due to Influenza A (H1N1) pdm09 at a Tertiary Care Hospital in Jaipur during the Current Season--January & February 2015.

BACKGROUND: The onset of winter of 2014-2015 saw an alarming spurt in influenza A (H1N1) pdm 09 leading to a significant mortality. Rajasthan was one of the foremost affected state bearing the frontal attack in which majority of deaths occurred early and in the young. OBJECTIVE: To sketch out the mortality profile with respect to demographic and clinical progression with an aim to identify the groups, this virus conspicuously picked up with a perspective to control some of the avoidable factors. METHODS: We analyzed the epidemiological data in 76 RT-PCR confirmed deaths of H1N1 patients that occurred between 1st January 2015 and 28 February 2015 over a period of 59 days at SMS Medical College Hospital, Jaipur. RESULTS: A total of 412 patients got hospitalized during two months period from 1st January 2015 to 28th February 2015, out of which 76 fatal cases presenting with category C symptoms along with radiological evidence of bilateral pneumonia were analyzed. 48.6% deaths occurred in the 18-40 years of age group. The mean age being 44.01 ± 15.07 years. Females had a marginally increased mortality rate (F: M-1.23:1). The mean time of onset of symptoms to hospitalization was 6.79 ± 4.63 days. Fifty-one (67.1% 0) patients were from urban areas, whereas 25 (32.89%) belonged to rural areas. Only 7.83% patients presented within 24 - 48 hours whereas 46% presented within 5 days of onset of symptoms. 66.9% succumbed within 5 days of hospitalization, despite of starting Oseltamivir in a dose of 150 mg/bd on the day of admission. 64.5% had predisposing risk factors. Bilateral pneumonia was observed in all the 76 patients, septicemia in 21.12%, MODS in 30.26% and AKI in 9.21%. CONCLUSIONS: The in-hospital mortality of 17.79% despite of starting Oseltamivir has raised concern about identifying the so called "Rapid Progressors" [66.9% succumbing within 5 days of hospitalization]. As a corollary of this analysis the authors are of the opinion that a rejig of the existing guidelines to identify and treat influenza like illness be made available at the national level. What factors promote rapid progression especially in a group without any predisposing risk condition should form the focus of future studies. As risk group individuals formed a major chunk of deaths, the need to vaccinate this group should form a scaffold on which future directions and interventions have to be built up to combat the morbidity and mortality.

Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya (Carica papaya L.).

BACKGROUND: Genetically engineered (GE) ringspot virus-resistant papaya cultivars 'Rainbow' and 'SunUp' have been grown in Hawai'i for over 10 years. In Hawai'i, the introduction of GE papayas into regions where non-GE cultivars are grown and where feral non-GE papayas exist have been accompanied with concerns associated with transgene flow. Of particular concern is the possibility of transgenic seeds being found in non-GE papaya fruits via cross-pollination. Development of high-throughput methods to reliably detect the adventitious presence of such transgenic material would benefit both the scientific and regulatory communities. RESULTS: We assessed the accuracy of using conventional qualitative polymerase chain reaction (PCR) as well as real-time PCR-based assays to quantify the presence of transgenic DNA from bulk samples of non-GE papaya seeds. In this study, an optimized method of extracting high quality DNA from dry seeds of papaya was standardized. A reliable, sensitive real-time PCR method for detecting and quantifying viral coat protein (cp) transgenes in bulk seed samples utilizing the endogenous papain gene is presented. Quantification range was from 0.01 to 100 ng/µl of GE-papaya DNA template with a detection limit as low as 0.01% (10 pg). To test this system, we simulated transgene flow using known quantities of GE and non-GE DNA and determined that 0.038% (38 pg) GE papaya DNA could be detected using real-time PCR. We also validated this system by extracting DNA from known ratios of GE seeds to non-GE seeds of papaya followed by real-time PCR detection and observed a reliable detection limit of 0.4%. CONCLUSIONS: This method for the quick and sensitive detection of transgenes in bulked papaya seed lots using conventional as well as real-time PCR-based methods will benefit numerous stakeholders. In particular, this method could be utilized to screen selected fruits from maternal non-GE papaya trees in Hawai'i for the presence of transgenic seed at typical regulatory threshold levels. Incorporation of subtle differences in primers and probes for variations in cp worldwide should allow this method to be utilized elsewhere when and if deregulation of transgenic papaya occurs.

High-throughput functional marker assay for detection of Xa/xa and fgr genes in rice (Oryza sativa L.).

We apply CE for high-throughput analysis of functional markers for marker-assisted selection in rice. The accuracy, throughput and reproducibility of CE analysis for sequence-tagged site (STS) and simple sequence repeat (SSR) markers for bacterial blight resistance and aroma genes are demonstrated by using a CE system. Multiplex PCR products displayed well-differentiated allelic variants using different STS and SSR markers for identification of xa13, Xa21 and fgr genes using the CE system compared to 1.2% agarose gel images. Moreover, consumption of PCR product is much less in the CE system compared to traditional agarose gel systems. Sample consumption is less than 0.1 µL per analysis, thereby conserving samples for further downstream analysis. Out of 29 genotypes in BC(1)F(3) generation, 16 plants were found homozygous for all the three genes, viz., xa13, Xa21 and fgr. These homozygous lines can be used as potential donors in rice breeding programmes.

mRNA expression of canine ATP10C, a P4-type ATPase, is positively associated with body condition score.

Mouse and human Atp10c genes are strong candidates for changes in bodyweight and glucose homeostasis. Using comparative genomic analysis, a novel canine P4-type ATPase, ATP10C, was identified. Expression of ATP10C was compared between sex-matched lean (body condition score, BCS<8; n=7) and obese (BCS⩾8, n=8) client-owned dogs of comparable ages. Canine ATP10C is highly expressed in visceral and subcutaneous fat at approximately 3-fold levels compared to the omental adipose depot. There was a 5-fold significant increase (P<0.0001) in mRNA expression of ATP10C in dogs with a BCS⩾8.

A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings.

BACKGROUND: Grasses are relatively recalcitrant to genetic transformation in comparison to certain dicotyledons, yet they constitute some of the most important biofuel crops. Genetic transformation of switchgrass (Panicum virgatum L.) has previously been reported after cocultivation of explants with Agrobacterium and biolistics of embryogenic calli. Experiments to increase transient gene expression in planta may lead to stable transformation methods with increased efficiency. RESULTS: A high-throughput Agrobacterium-mediated transient gene expression system has been developed for in planta inoculation of germinating switchgrass seedlings. Four different Agrobacterium strains were compared for their ability to infect switchgrass seedlings, and strain AGL1 was found to be the most infective. Wounding pretreatments such as sonication, mixing by vortex with carborundum, separation by centrifugation, vacuum infiltration, and high temperature shock significantly increased transient expression of a reporter gene (GUSPlus, a variation of the beta-glucuronidase (GUS) gene). The addition of L-cysteine and dithiothreitol in the presence of acetosyringone significantly increased GUS expression compared with control treatments, whereas the addition of 0.1% surfactants such as Silwet L77 or Li700 decreased GUS expression. 4-Methylumbelliferyl beta-D-galactopyranoside (MUG) assays showed a peak of beta-glucuronidase (GUS) enzyme activity 3 days after cocultivation with Agrobacterium harboring pCambia1305.2, whereas MUG assays showed a peak of enzyme activity 5 days after cocultivation with Agrobacterium harboring pCambia1305.1. CONCLUSION: Agrobacterium strains C58, GV3101 and EHA105 are less able to deliver transfer DNA to switchgrass seedlings (cultivar Alamo) compared with strain AGL1. Transient expression was increased by double or triple wounding treatments such as mixing by vortex with carborundum, sonication, separation by centrifugation, and heat shock. The addition of thiol compounds such as L-cysteine and dithiothreitol in combination with acetosyringone during cocultivation also increased transient expression. The combination of multiple wounding treatments along with the addition of thiol compounds during cocultivation increased transient expression levels from 6% to 54%. There were differences in temporal GUS expression induced by pCambia1305.1 and pCambia1305.2.

Floral transformation of wheat.

A method is described for the floral transformation of wheat using a protocol similar to the floral dip of Arabidopsis. This method does not employ tissue culture of dissected embryos, but instead pre-anthesis spikes with clipped florets at the early, mid to late uninucleate microspore stage are dipped in Agrobacterium infiltration media harboring a vector carrying anthocyanin reporters and the NPTII selectable marker. T1 seeds are examined for color changes induced in the embryo by the anthocyanin reporters. Putatively transformed seeds are germinated and the seedlings are screened for the presence of the NPTII gene based on resistance to paromomycin spray and assayed with NPTII ELISAs. Genomic DNA of putative transformants is digested and analyzed on Southern blots for copy number to determine whether the T-DNA has integrated into the nucleus and to show the number of insertions. The nonoptimized transformation efficiencies range from 0.3 to 0.6% (number of transformants/number of florets dipped) but the efficiencies are higher in terms of the number of transformants produced/number of seeds set ranging from 0.9 to 10%. Research is underway to maximize seed set and optimize the protocol by testing different Agrobacterium strains, visual reporters, vectors, and surfactants.