Suppression of Bean Yellow Mosaic Virus by Plant Extracts in Faba Bean Plants- Juniper Publishers

 

Organic & Medicinal Chemistry- Juniper Publishers

Abstract

The virus was obtained from faba bean plants that were naturally infected. The studied virus was identified as Bean yellow mosaic virus (BYMV) according to symptomology, host range, virus stability, modes of transmission, ELISA, and RT-PCR. ELISA test confirmed the presence of BYMV in all the collected leaf samples at different times after inoculation. A fragment of coat protein gene (352 bp) was amplified using PCR from infected faba bean plants. Growth and seed characters were significantly lower in infected plants compared with healthy faba bean plants. Plant extracts promoted systemic resistance to BYMV. Disease severity and BYMV titre were substantially decreased in faba bean plants treated with extracts of Cinnamomum zeylanicum bark (CZ), Syzygium aromaticum, Matericaria chamomilla L., Foeniculum vulgare, Zingiber officinale before or after BYMV inoculation. The lowest disease severity and virus concentration were achieved using cinnamon extracts treated at 1 day before or after virus inoculation. In faba bean plants treated with plant extracts, the expression of pathogenesis-related genes (PR1 and PR2) was substantially higher than in control plants. These results explain the role of PR1 and PR2 genes in enhanced resistance against BYMV by plant extracts.

Keywords: Faba bean; Bean yellow mosaic virus; Induced resistance; ELISA; Real time PCR

Introduction

In Egypt, faba bean (Vicia faba L., Fabaceae) is a main food legume crop. It represents an important source of protein, carbohydrate, vitamins, essential amino acids, and mineral salts [1]. It is used as a human food in developing countries and as animal feed in industrialized ones, mostly for pigs, horses, poultry, and pigeons. It may be eaten raw or cooked, fresh, or preserved. After infecting the susceptible host plant, viruses cause a sequence of physiological changes that may lead to disease symptoms such as systemic and local symptoms [2,3]. BYMV (Bean Yellow Mosaic Potyvirus) is a widespread disease of beans and other hosts that may be found all over the globe [4]. It is a severe disease wherever susceptible crops grown because it is transmitted through seeds and Aphids [5]. Recently, Skelton et al. [6] isolated and identified BYMV from Dactylorhiza foliosa (a hardy orchid species, native to the Island of Madeira) that showed symptoms of chlorotic mottle and streaking. Finally, the virus was mechanically inoculated into two indicator species. Chenopodium quinoa (chlorotic local lesions) and Nicotiana benthamiana (distortion and mosaic) developed leaf symptoms. The presence of BYMV was verified by ELISA testing of plants exhibiting symptoms.

Several investigators found that faba bean was the most susceptible host to BYMV causing the considered losses in the grain yield [7]. This virus was isolated, either alone or mixed with other viruses, from faba bean. Systemic symptoms produced by BYMV infection may not kill faba bean plants, but they can spread quicker and deeper throughout the crop, causing a larger total yield loss, despite inducing milder symptoms [8,9]. A long-known virus inhibitor from carnation leaves has now been shown to be an inducer of systemic resistance to virus as well [10]. Thus, it is possible that many of the other well-known virus inhibitors of plant origin may in fact be proven to be inducers of systemic resistance. The earliest evidence that plants possess inhibitory compounds came from virus-infected plants, and then viral inhibitory substances were discovered in a variety of healthy plants from several Angiosperm families, including Amaranthaceae, Caryophyllaceae, Chenopodiaceae, Solanaceae, and Verbenaceae [11].

While working on resistance induced by leaf extracts from carnation plants, they observed that a very short period was required to the induced antiviral state in host tissue [10]. Pyrethrin (Pyrethrum) is a source of synthetic pyrethroid insecticides that is generated in the flowers of Chrysanthemum cinerariaefolium. Pyrethrin is approved for the use against a wide range of pests. One formulation of pyrethrin was shown to be moderately to very efficient (61-100 % control) against the fruit pests, such as grape leafhoppers, potato leafhoppers, leaf curl plum aphids, blueberry flea beetles, blueberry thrips, and blueberry sawfly. It also works against the cranberry fruit worm. It decomposes rapidly in the environment and may be used up to harvest day [12].

Ribosome-inactivating proteins (RIPs), which have antiviral effects, are found in many plant species [13]. A RIP from pokeweed (Phytolacca americana) was shown to provide broad-spectrum viral resistance in tobacco and potato. All the tested RIPs exhibited strong antiviral action [14]. Purified antiviral proteins from roots, shoots, leaves, fruits, and seeds of Mirabilis jalapa were successful in protecting economically significant crops (tobacco, maize, and potatoes) against a wide range of plant viruses such as Tobacco mosaic virus and Tomato spotted wilt virus [15,16]. This study aims to isolate of the most common faba bean virus found in collected samples and to evaluate the induced systemic resistance activities of some medicinal plant extracts against Bean yellow mosaic virus.

Materials and Methods

Isolation and identification of the isolated virus

One type of naturally infected faba bean samples showing virus-like symptoms was collected from faba bean fields, Legume Department, Sakha Agricultural Research Station, Kafr EL-Sheikh Governorate. According to Elsharkawy et al. [17], mechanical inoculation was employed to inoculate the test plants (faba bean). The viral isolate was biologically purified using Chenopodium amaranticolor L. and the local lesion method was used [18]. The Sakha Agricultural Research Station in the Kafr El-Sheikh Governorate, Egypt, provided sixty plant species and cultivars from ten distinct families that were mechanically inoculated by the isolated virus to study the host range.

Isolation and identification of the isolated virus

One type of naturally infected faba bean samples showing virus-like symptoms was collected from faba bean fields, Legume Department, Sakha Agricultural Research Station, Kafr EL-Sheikh Governorate. According to Elsharkawy et al. [17], mechanical inoculation was employed to inoculate the test plants (faba bean). The viral isolate was biologically purified using Chenopodium amaranticolor L. and the local lesion method was used [18]. The Sakha Agricultural Research Station in the Kafr El-Sheikh Governorate, Egypt, provided sixty plant species and cultivars from ten distinct families that were mechanically inoculated by the isolated virus to study the host range.

Serological diagnosis of the isolated virus using indirect ELISA

Faba bean samples showing symptoms suggested to be due to Bean yellow mosaic virus (BYMV) was tested with their specific antiserum by indirect ELISA technique. This method was described by Elsharkawy et al. [19]. Leaves of faba bean were ground in a mortar with 0.02 M phosphate buffer (1:10) and mix the sap 1:1 with 2x concentrated coating buffer, pH 9.6, fill the wells with 200μ1 aliquots of test samples. The plates were covered and incubated at 30- 37 °C for 2 hrs. Washing with PBS-Tween was repeated 3 times. Cross-absorbed antiserum (200 μ1) was added to each well then Incubated for 1-1.5h at 30-37°C followed by washing with PBS-Tween. Goat anti-rabbit antibody conjugated to alkaline phosphatase (200 μ1) was added and incubated for one hour at 37°C followed by washing with PBS-Tween. P-nitrophenyle phosphate (200 μ1) was added and the absorbance values was measured at 405nm using Vniskan ELISA reader.

Extraction of total nucleic acid from plant tissues infected with BYMV

The Spin/ vacuum SV total RNA isolation system (Promega Corporation, Maison, WI) has been used successfully to isolate RNA from leaves of healthy or BYMV infected faba bean plants. Thirty mg of infected and uninfected faba bean leaves were powdered in liquid nitrogen. One hundred seventy-five μl of RNA lysis buffer (4M Guanidine thiocyanate, 0.01 Tris-HCl, pH 7.5, 0.97% β- mercaptoethanol) was added and the tube was mixed by inversion. The dilution buffer was added, and the tube was mixed by inversion followed by centrifugation at 15000rpm in a microcentrifuge for 15minutes. RNA precipitation and DNase treatment were carried out as described by Elsharkawy et al. [17]. RT-PCR primers used in this study were designed to detect the coat protein gene of BYMV as described by Shooman [20].

The selection of the primers was performed according to the primer analysis software Oligo 4.1 (National Bioscience Inc., Plymounth MN, USA). The primer bought from integrated DNA Technologies, Inc. The primer pair used for the detection of BYMV (NIF-5’-GAGCGCATCGTTTCAATTCT-3’ and NIR-5’- AGCATGGGGCTATCCAACT-3’) was designed based on GenBank accession AM884180. The PCR primers utilized for cDNA synthesis and amplification of the BYMV coat protein gene were based on the conserved sequence of other BYMV isolates from all over the world. The purified total RNAs extracted from infected and healthy faba bean plants by using SV total RNA isolation system were used as starting material in the Reverse transcriptase polymerase chain reaction (RT-PCR) process as described by Shooman [20] and Hataya et al. [21]. the PCR products were examined on a 1% agarose gel electrophoresis and the size of the full length BYMV DNA fragment was determined in accordance with the DNA molecular weight markers.

Induction of systemic resistance against BYMV by plant extracts

Extracted oils from five medicinal plants (Cinnamomum zeylanicum withbark (Cinnamon), Syzygium aromaticum (Clove), Matericaria chamomilla L. (Chamomile), Foeniculum vulgare (Fennel), Zingiber officinale (Ginger) were kindly provided by Horticulture Department, Faculty of Agriculture, Kafrelsheikh University. Plant extracts (oils) were stored in the refrigerator at 4°C until use. The isolated virus inoculum was prepared by triturating 1 g of young viral symptomatic faba leaves cv. “Giza 843” in a sterilized mortar by adding 20ml (1:20 w/v) of sterile distilled water. The extract was filtered through muslin cloth then centrifuged for 20min at 3000rpm. The supernatant was used as inoculum.

Plastic pots (25cm) were filled with suitable amount of sandclay- loamy sterilized soil mixture. Ten seeds per pot were planted and subsequently trimmed to three seedlings after 15 days from planting. When needed, pots were irrigated with tap water in equal quantities. NPK fertilizers were used (0.6g of urea/pot, 0.75g of Ca-super-phosphate/pot, and 0.25g of K-sulphate/pot). Phosphorus was supplied before sowing during soil preparation. N and K were supplied in two equal dosages at thinning and two weeks afterwards.

Faba bean plants were divided into eleven groups (15 plants for each in 5 pots), five of them sprayed 24 hours pretreatment (virus-inoculation) with medicinal plants. Five groups were sprayed 24 hours post BYMV-inoculation with medicinal plants. The last group (15 plants) served as control, pretreated with water, and subsequently inoculated with the sap from virus-infected plants. Crude extracts of selected medicinal plants adjusted as 1:5 w/v and sprayed using pressure sprayer (2L). After two weeks from virus inoculation, virus concentration was determine using indirect ELISA as described previously and also disease severity of BYMV was measured based on the following scale: 0=no symptoms; 2=mild mosaic of the youngest two leaves; 4 = pronounced leaf deformation and mosaic of the youngest two leaves; 6=pronounced leaf deformation and mosaic with progression of symptoms into sequentially older leaves; 8=pronounced leaf deformation and mosaic, with all leaves expressing some forms of BYMV induced symptoms and 10=similar symptoms as described for a rating of 8, with plants also being stunted in growth.

Molecular investigation of pathogenesis-related genes expression

The kit (Thermo Scientific, Fermentas, #K0731) was used for RNA extraction. Pure RNA was reverse transcribed using the reverse transcription kits (Thermo Scientific, Fermentas, #EP0451). RNA was quantified using Nanodrop. The Q5000 (Uv- Vis’s spectrophotometer Q5000/USA) automatically performs all necessary measurements and calculations. For pure RNA, the OD260/OD280 ratio is less than 2. Protein contamination (which has a maximum absorbance of 280 nm) or phenol contamination will result in a ratio that is considerably lower than these values. The expression of target genes (PR1 and PR2) was assessed using real-time PCR with SYBR Green, with elongation factor 1 alpha (EF1α) as an internal reference following the manufacturer protocol (Thermo scientific, USA, # K0221) and gene specific primers. Table 1 lists the primers used in the amplification. The housekeeping gene (EF1α) is utilized to determine the relative gene expression or fold change in the target gene. Therefore, the 2-ΔΔCt technique was used to normalize the amounts of the target gene’s critical threshold (Ct) with the quantities of the housekeeping gene (EF1α) [22].

Statistical Analysis

The data were all presented as means (±S.E.). Comparisons were obtained using Duncan’s multiple range test (DMRT) and statistical significance was determined using one-way analysis of variance (ANOVA) by SPSS 18.0 software, 2011. When p<0.05, values were determined statistically significant.

Results

Isolation and identification of virus isolate

BYMV has a narrow host rang. Symptoms induced by the virus isolate in experimentally inoculated faba bean plants were like those expressed by field plants. BYMV induced severe and mild mosaic on all Vicia faba L. cultivars. The host range of the isolated virus was restricted to family leguminosae (Cicer arietinum, Lupinus termis, Phaseolus vulgaris, Trifolium alexandrinum and Vicia faba) and some members of Chenopodiaceae (Chenopodium album, C. amaranticolor and C. quinoa) (Figures 1&2). Stability of the isolated virus in sap indicated that, the thermal inactivation point was 60 to 65°C for BYMV, dilution end point was at 10-4 -10-5 and the longevity in vitro was 2 to 3 days. The isolated virus suggestive Bean yellow mosaic virus was mechanical transmissible readily (90–95%) with the infectious sap. Both green peach aphid (Myzus persicae Sulz.) and faba aphid (Aphis fabae) were found to be vectors of BYMV in a non-persistent manner with the average rate 80% and 40%, respectively. Seeds were harvested from mechanically inoculated faba bean (Vicia faba L.) plants and showed obvious viral infections with the isolated virus. The percentage of seed transmission was 10-13 % for BYMV in faba bean cultivar 843. BYMV was identified by ELISA. The experimental results confirmed the presence of BYMV in the infected samples. Positive reaction was obtained using BYMV-specific antiserum.

Detection of BYMV-E in infected faba bean plants was accomplished through the amplification of the viral RNA by RTPCR (Fig. 3). Isolation of RNA from plant tissues was done by SV total-RNA extraction method. The total RNA yield obtained was 137.7μg/100μl elution volume at 260nm, indicating high yield of RNA and the purity of total RNA also exhibited an A260/A280 absorbance ratio of 2.37 indicating high purity of total RNA isolated by the method described previously. Following PCR, the amplification products were analyzed by gel electrophoresis (Figure 3). The fragment was amplified from infected faba bean leaf tissues by using primer set. It appears from these data that the isolated virus is Bean yellow mosaic virus.

Effect of Bean yellow mosaic virus on faba bean growth

Chlorophyll a, b and total chlorophyll were significantly lower in infected plants (7.96, 8.37 and 15.36, respectively) compared with healthy plants (6.31, 7.13 and 13.58, respectively) at 60 days from sowing. Shoot and root length were significantly lower in infected plants compared to healthy plants. Similarly, seedling dry weight was significantly reduced due to BYMV infection in faba bean plants at 8 days after planting (Data not shown). Weight and volume of 100 seeds from infected faba bean were significantly lower compared with seeds from healthy plants. Moreover, relative density of seeds and germination percentage were also reduced in infected plants in comparison with healthy plants (Data not shown). Crude protein percentage in infected seeds was higher in infected faba bean plants (38.5) compared with the healthy seeds (33.5).

Effect of plant extracts application on BYMV disease severity

The lowest disease severity was achieved using cinnamon extracts under pot experiments. Ginger and fennel extracts (2.64, 2.81, respectively) were more effective than the extracts of clove and chamomile (3.69, 3.83, respectively). The protective activity of oil extracts was significantly effective compared with the control (8.00) (Figure 4A). On the other hand, the curative treatments of plant extracts after BYMV inoculation were also significant compared with the control (Figure 4B). Treatment with ginger extract achieved the best results (3.08) compared to the control (7.40). The lowest virus concentration was recorded in cinnamon (Cinnamomum zeylanicum) treatment (0.23) compared to control treatment (1.2). Similarly, BYMV concentration in plants treated with extracts before virus inoculation was also lower than the control (Figure 5A). Faba bean leaves sprayed with ginger extract showed the best results in this respect (0.39), compared to control treatment being 1.4 (Figure 5B).

Effect of plant extracts treatments before BYMV inoculation on the relative expression of defense genes

Our results revealed a significant (P≤0.05) increase of PR1 gene expression level in plant extract treated faba bean plants before BYMV inoculation as compared to the control (Figure 6A). Plants treated with cinnamon extracts showed the highest expression values (5.21), as compared to non-treated plants (1.01). As compared to control treatment, PR2 gene expression level was significantly increased in plant extract treated faba bean plants before BYMV inoculation (Figure 6B). In addition, plants treated with cinnamon extracts resulted in the highest upregulation of PR2 gene expression followed by fennel, chamomile, ginger, and clove (5.78, 4.56, 3.56, 2.81 and 2.28, respectively).

Effect of plant extracts treatments after BYMV inoculation on the relative expression of defense genes

Our results revealed a significant (P≤0.05) increase in PR1 gene expression level in plants treated with plant extracts as compared to the control (Figure 7A). However, no significant difference in gene expression was found between clove, chamomile, and fennel treatments (1.71, 1.69 and 1.47, respectively). When comparing all treatments, treatment with cinnamon (3.43) showed the highest expression of PR1 gene than other treatments. PR2 gene expression level was significantly increased in plants treated with plant extracts as compared to control (Figure 7B). In all groups, cinnamon and fennel treated plants (2.89 and 2.28, respectively) showed a significant higher expression than other treatments. Moreover, treatment with cinnamon resulted in higher upregulation of PR2 gene expression than fennel sprayed plants (Figure 7B).

Discussion

Recently, faba bean crop in Egypt suffered great losses due to infection with mosaic diseases. Bean yellow mosaic potyvirus (BYMV) was reported to be one of the most prevalent viruses affecting leguminous crops in Egypt. Approximately 75% of aphidvectored potyviruses are transmitted in a non-persistent (noncirculative) manner [23]. The results confirmed the presence of BYMV in the infected samples of faba bean using indirect ELISA. ELISA proved to be reliable and sensitive method for detecting and identifying BYMV in infected faba bean extracts [6,24]. Bean yellow mosaic virus (BYMV) is composed of a protein molecular weight of 30-35kd. In this study, the detection and quantification of BYMV in infected plant tissues was based on semi-quantification reverse transcription polymerase chain reaction (RT-PCR). RT-PCR was used to detect low concentration of BYMV in total RNA extracts of infected gladiolus leaves [25].

In the present work, the decrease in growth, pigment contents and seed characters and viability probably explained based on an extensive production of excitation energy that occurred under virus stress, leading to photo-inhibitory damage in the reaction centers within chloroplasts [26]. Moreover, the lower number of chloroplasts is thought to be directly responsible for the decrease of chlorophyll content detected in BYMV-infected leaves. In compatibility with the previous results, a chloroplast is the most sensitive plant cell organelle to viral infection [27]. The application of antiviral principles derived from higher plants as biological virus control agents seems to be very promising. Antiviral proteins (AVPs) are most of these compounds, which are proteinaceous in origin.

However, deletion of the C-terminal of Pokeweed antiviral protein (PAP) mutants reduces viral infection but do not depurinate host ribosomes, indicating that PAP’s antiviral action may be separated from its ribosome inactivating proteins (RIPs) activity [28]. In the current study, faba bean plants were sprayed with plant extracts of Cinnamomum zeylanicum bark (Cinnamon), Syzygium aromaticum (Clove), Matericaria chamomilla L. (Chamomile), Foeniculum vulgare (Fennel), Zingiber officinale (Ginger) at 1 day before or after BYMV inoculation, to control BYMV in faba bean plants. Cinnamon extracts achieved the best results in reduction of disease severity, BYMV titre and activation of PR1 and PR2 gene expressions in treated plants compared with the control. Clerodendrum inerme contains basic protein which is resistant to proteases. Induced systemic resistance by antiviral agents has been introduced against many plant viruses [17,19,29,30]. A minimum concentration of 400Rg/ml of MAP (Mirabilis jalapa antiviral protein) was sufficient to inhibit TSWV [16].

In addition, Mirabilis antiviral protein (MAP) was isolated from roots and leaves of Mirabilis jalapa L. which possess repellent properties against aphids and white flies. MAP showed antiviral activity against mechanically transmitted viruses but not against aphid transmitted viruses [15]. Barakat et al. [14] reported that, Endogenous proteins have been discovered as ribosomeinactivating proteins (RIPs) such as enzymes that operate on ribosomes in a very particular manner in a variety of plant species. Six RIPs were isolated from leaves and/or seeds of several plant species, including types I and 2. Tobacco necrosis virus (TNV) on Phaseolus vulgaris plants, Tobacco mosaic virus (TMV) on Chenopodium amaranticolor plants, and Bean yellow mosaic virus (BYMV) on its systemic host (Vicia faba), all tested RIPs exhibited strong antiviral action.

Conclusion

In conclusion, the isolated virus was identified as Bean yellow mosaic virus based on host range, virus stability in crude sap, transmission, ELISA, and RT-PCR. Plant extracts was successfully used to control BYMV. The expression of pathogenesis-related genes (PR1 and PR2) was substantially higher in faba bean plants treated with plant extracts before or after BYMV inoculation which confirmed that the induced resistance was involved in disease control mechanisms.

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Acute Hematocrit Change as an Indicator of Intracranial Hemorrhage on Neonatal Head Ultrasound- Juniper Publishers

 

Intellectual & Developmental Disabilities- Juniper Publishers

Abstract

Purpose: To determine whether an indication related to a decrease in hematocrit is predictive of an intracranial hemorrhage (ICH) on neonatal head ultrasound (HUS).

Methods: This was a single center retrospective study of 215 neonatal head ultrasounds (HUS). The association between an ICH on HUS and indication related to hematocrit, a change in hematocrit in the 48 hours prior to HUS, gestational age, number of days since birth at time of HUS, designation of prematurity, or abnormal neurologic exam were examined.

Results: Thirty-seven (37/215, 17.2%) neonates had an ICH on their HUS. There was no significant association between studies that were ordered with an indication related to hematocrit (or similar indication) and the likelihood of having an ICH on HUS (OR 0.92, p=0.82). Lower gestational age, prematurity, and lower birthweight were associated with an ICH on HUS (p<0.05), but only gestational age remained statistically significant in a multivariable model (p<0.005).

Conclusion: Neither a decrease in hematocrit in the preceding 48 hours, nor an indication related to a decrease in hematocrit, was predictive of an ICH on HUS. Rather, prematurity, lower birth weight, early gestational age, and younger age were significantly associated with the presence of an ICH on neonatal HUS.

Keywords: Hemorrhage; Neonate; Ultrasound; Hematocrit; Prematurity

Introduction

Intracranial hemorrhage (ICH) is a devastating cause of neonatal morbidity and mortality that can result in substantial adverse outcomes during an important window for neurodevelopment. Prior investigations have shown that ICH is typically associated with prematurity (defined as a neonate born prior to 37 weeks), low birth weight, and anticoagulation [1,2]. Subdural and intraparenchymal hemorrhages are more commonly found in term infants, primarily related to birth trauma, while the most common type of intracranial hemorrhage in premature infants are germinal matrix hemorrhages (GMH) [3-5]. GMHs occur in preterm infants due to the thin-walled vessels found at the germinal matrix which are more susceptible to hemorrhage in the setting of hemodynamic changes [6-10]. The clinical data has shown that the majority of GMHs are identified during the first 4 days of life, with 40% detected within the first 5 hours of birth [2, 11-12]. GMHs are graded based on their severity as described in a landmark paper in 1978 (see Figure 1) [13]. Although most grade 1 and 2 GMHs are managed with observation, higher grade hemorrhages are associated with substantial neurodevelopmental sequelae, significant disability, and poor survival rates [14,15].

Head ultrasound (HUS) is the most common first line technique for imaging neonates suspected of having an ICH [16]. HUS affords real-time assessment of the brain using the fontanelles as a sonographic window. However, it is not always clear which neonates warrant an evaluation with HUS for ICH. We have observed that HUS are often ordered to look for an ICH when a change in hematocrit or hemoglobin has occurred on routine labs. But to our knowledge, no study to date has evaluated whether HUS ordered for indications related solely to a decrease in hematocrit are more likely to reveal an ICH than those ordered for alternative indications. The purpose of this study was to determine whether a decrease in hematocrit or a similar HUS indication (e.g., decrease in hematocrit or hemoglobin, bleeding, anemia, etc.) was predictive of an ICH on neonatal HUS.

Materials and Methods

This was a retrospective study of neonates that underwent HUS from 2009-2018. This study was conducted in accordance with the Declaration of Helsinki with approval by our Institutional Review Board. A waiver of informed consent was granted due to the retrospective nature of this work.

The PACS (Picture Archive and Communication System) at our institution (Centricity, General Electric) was searched using two search filters: head ultrasound, and the year (2009-2018). The indications for the head ultrasound examinations (HUS) were consecutively reviewed for any terms that were related to a decrease in hematocrit (or similar indication), including the following: “low”, “decreased”, “drop in”, “fall in”, or “dropping” hematocrit or hemoglobin, “hemoglobin drop”, “hematocrit drop”, “HH drop”, “bleeding”, or “anemia.” This resulted in approximately 116 patients with an indication for HUS that was related to a decrease in hematocrit or hemoglobin. A random sample of 101 patients from the same time period with indications unrelated to hematocrit, hemoglobin, bleeding, or anemia were selected for inclusion as a comparison group. The images of all HUS were initially reviewed, and a radiology report was generated by a board-certified radiologist to determine whether or not an ICH had occurred. The reports and images were then later reviewed by another board-certified radiologist for agreement of imaging findings. If there was concurrence of HUS findings, the examination was included in the study; if there was disagreement on HUS findings, the examination was excluded from the study. Only two HUS examinations were excluded from this study (2/217 = 0.9%). Those patients with an ICH on HUS (37/215 =17.2%) were then further classified into one of four groups based on known GMH grading (see Figure 1) [13].

The electronic medical records of the 215 patients included in this study were then reviewed and the following clinical data were collected: the change in hematocrit in the 48 hours prior to and at the time of HUS, whether or not there was a decrease in the hematocrit lab value in the 48 hours prior to HUS, the gestational age (GA) in weeks, whether the patient was classified as premature (< 37 weeks) or not (≥37 weeks), the birth weight in kilograms, the number of days since birth, and whether the result of a neurologic examination was abnormal or normal when it was performed. Premature neonates were further subclassified as term (>37 weeks), moderately preterm (32-37 weeks), very preterm (28-32 weeks), and extremely preterm (<28 weeks).

Statistical Analysis

The association between the presence or absence of an ICH on the HUS and each of the clinical and demographic independent variables was then analyzed using separate univariable logistic regression models to estimate the odds ratios (OR) and 95% confidence intervals. A multivariable model was constructed to evaluate the association between the likelihood of an ICH on HUS and an indication related to a decrease in hematocrit (or similar indication) as well as any clinical or demographic factors that had been statistically significant in univariable analysis.

Separate linear regression models were used to assess whether the magnitude of the change in hematocrit was associated with the category of prematurity (i.e., moderately or very or extremely preterm vs. term) or the grade of ICH on HUS (i.e., Grade 4 or 3 or 2 vs. 1). Similarly, a logistic regression model was constructed to assess whether the presence or absence of a measured drop in the hematocrit lab value was associated with the category of prematurity. A p-value of <0.05 was considered statistically significant. All statistical analyses were completed in Stata (version 16.0, College Station, TX).

Results

A total of 215 patients were included in our study, 112 male (52%) and 103 (48%) female. A total of 37/215 (17.2%) of patients had an ICH on HUS. The mean gestational age (GA) at birth was 32.6±5.4 weeks with a median of 34 weeks (Interquartile range 27, 37). The mean age at the time of HUS was 21.3±41.6 days with the median at 7 days (Interquartile range 2, 18). A majority of patients included in the study were premature, or < 37 weeks old at the time of birth) (145/192, 75.5%), and the average birthweight was 2.05±1.39 kg with a median of 1.9 kg (Interquartile range 0.9, 2.9) (Table 1).

There were 114/215 (53%) patients with an indication related to a decrease in hematocrit (or similar indication) and 101 patients with indication unrelated to hematocrit drop (Figure 2). Of those with a decrease in hematocrit, 19/114 (17%) had an intracranial hemorrhage and 95/114 (83%) did not. Most of the patients with an ICH were premature (32/36, 89%), and most patients with an ICH were ≤ 7 days old at the time of HUS (28/37, 75.7%).

Table 2 shows the association of each of the clinical or demographic factors with the presence or absence of an ICH on HUS. Studies that were ordered with an indication related to a decrease in hematocrit (or similar indication) were no more likely to have an ICH on HUS than those ordered for an alternative indication (OR 0.92, p=0.82). Also, there was no significant association between the likelihood of having an ICH on HUS and a decrease in the measured hematocrit lab value (OR 0.74, p=0.44), or the magnitude of the change in the hematocrit lab value (OR 1.0, p=0.90) during the 48 hours preceding HUS. The actual hematocrit lab value at the time of HUS also did not significantly differ between those with an ICH and without an ICH, suggesting no significant association between anemia and ICH (OR 0.97, p=0.14).

We also evaluated whether an abnormal neurologic examination was associated with an ICH on HUS and found that 12 patients had missing data, 25 patients were sedated at the time of HUS, 22 had abnormal neurologic exams, and 156 had normal neurologic exams. Thus, 6/37 (16.2%) of patients with ICH had an abnormal neurologic examination, and 16/178 (9.0%) of patients without an ICH had an abnormal neurologic examination. The odds of having an abnormal neurologic examination was higher in the group with an ICH compared to those without an ICH (OR 1.71, p=0.30), but the association was not statistically significant. Reasons for an abnormal neurologic examination included “hypotonia,” “decreased tone and flexation,” “hyperactive reflexes,” “decreased tone and reflexes,” “minimal response to stimulation,” “no spontaneous movement,” “no withdrawal to painful stimuli,” and “facial droop.”

In univariable logistic regression analyses, prematurity (OR 3.04, p=0.04), lower gestational age in weeks (OR 0.83, p<0.001), younger age in days at the time of HUS (OR 0.96, p=0.02), and lower birth weight (OR 0.41, p<0.001) were each significantly associated with an ICH on HUS. However, only gestational age remained significantly predictive of ICH (p<0.005) in a multivariable logistic regression model that included indication related to hematocrit (or similar indication), and each of the factors that had been statistically significant (p<0.05) in univariable analysis (i.e., prematurity, younger age, and lower birth weight).

We next examined whether the level of prematurity was associated with a greater likelihood of having an ICH on HUS (Table 3). Patients that were very preterm (OR 4.96, p=0.02) or extremely preterm (OR 8.50, p<0.001) were at a significantly greater risk than those that were term. Moderately preterm infants did not have an increased risk of ICH (OR 1.1, p=0.89). However, there was no association between the level of prematurity and a measured drop in the hematocrit lab value (all p>0.60), nor the magnitude of that change in hematocrit in the 48 hours prior to HUS (all p>0.70).

Finally, we evaluated whether the grade of hemorrhage on HUS was related to a drop in hematocrit (all p>0.20) or the magnitude of the change in hematocrit during the 48 hours preceding the HUS (all p>0.40), and there was no significant association or trend to suggest any relationship between the change in the hematocrit lab value and the grade of ICH.

Discussion

Our study demonstrates that neither a decrease in the hematocrit lab value during the 48 hours prior to HUS nor an indication for HUS related to a decrease in hematocrit (or similar indication) is predictive of having an ICH on HUS. There was also no significant association between the change in hematocrit and the grade of ICH on HUS. Rather, younger age at the time of ultrasound, lower gestational age at birth, and prematurity were all associated with a greater likelihood of finding an ICH on HUS, regardless of whether the patient also had a decrease of hematocrit. Of these factors, lower gestational age was the strongest predictor of an ICH and was the only variable that remained significantly associated with ICH in a multivariable model. Infants with a higher degree of prematurity, such as very preterm or extremely preterm, were also at a substantially increased risk of having an ICH, but there was no relationship between the degree of prematurity and the change in hematocrit in the 48 hours prior to HUS. Thus, a decline in hematocrit alone on laboratory testing is unlikely to be explained by an ICH on HUS and may not be a sufficient indication for ordering this test. However, higher degrees of prematurity or lower gestational age are strong risk factors for an ICH and warrant thorough evaluation with HUS.

Head ultrasound is an inexpensive adjunct to MRI because of its wide availability, lack of side effects, and wealth of anatomical and functional information [17,18]. The threshold to screen neonates for abnormalities with HUS have been historically low since HUS is relatively inexpensive compared to brain MRI. In fact, in 2002 the American Academy of Neurology (AAN) and Child Neurology Society (CNS) recommended routine screening with HUS in all preterm neonates <30 weeks, and at 36- and 40-weeks gestational age, as well as routine Noncontrast CT in encephalopathic term infants with a history of birth trauma, low hematocrit, or coagulopathy [19]. However, this recommendation was retired in 2018 because the AAN and CNS did not update or reaffirm their recommendations. Thus, it may be time to reexamine whether such low thresholds for HUS examinations should be routinely recommended in most neonates [20].

Moreover, with rising healthcare costs, there is increasing concern regarding over-utilization of imaging studies in medicine. Since a hospital’s resources do not simply encompass the costs of imaging tests, but also the time and energy spent by experienced sonographers, support staff, and radiologists to acquire and interpret these tests, it is important that clinicians have clear guidelines regarding the appropriate indications for ordering radiologic tests for their patients. The results of our study suggest that ordering a HUS solely because the hematocrit has decreased or because there is a concern for new anemia may not be a costeffective use of hospital resources. We found that head ultrasounds ordered for an indication related to a decrease in hematocrit (or a similar indication) were no more likely to have an ICH on HUS than those ordered for other indications. Moreover, neonates that demonstrated a decrease in hematocrit in the 48 hours preceding the HUS were no more likely to have an ICH, nor were they at greater risk of having a higher-grade ICH, compared to those with a stable hematocrit.

On the other hand, our study confirmed several well-known risk factors for neonatal ICH, including lower gestational age, especially prematurity, younger age at the time of HUS, and low birthweight [21-23]. We found that gestational age was the most significant predictor of ICH, independent of the other factors in a multivariable model. There was a notable dose response with higher levels of prematurity, such as very and extremely preterm infants, showing increasing odds of having an ICH compared to those who were only moderately preterm or term. This result confirms the findings of recent studies investigating the incidence of GMH in premature neonates [2,3,6]. One such study evaluated a total of 101 neonates and found that those born at <30 weeks gestational age had a 47% chance of ICH, and those born weighing <1000 grams at birth had a 54% chance of ICH [24]. Even though we also found that neonates with lower gestational age (especially higher levels of prematurity) were more likely to have an ICH, we did not find that a change in hematocrit was associated with either the level of prematurity or the likelihood of an ICH. Thus, change in hematocrit neither confounds nor modifies the relationship between prematurity and ICH on HUS.

Our study also suggests that an abnormal neurologic examination may be associated with an increased likelihood of ICH, but the association was not very strong and did not reach statistical significance. Dubowitz et al. have previously shown that multiple neurologic signs can correlate with ICH [25]. Our study did not evaluate whether the many different types of neurologic examination findings correlate with the results of HUS because the neurologic examination findings were not consistently documented in the medical record, and a substantial proportion of patients were either sedated or on a ventilator at the time of HUS and thus lacked documentation of specific neurologic signs. Furthermore, since our sample size was small and our primary question was whether an indication related to hematocrit alone was associated with an ICH on HUS, our study was not powered to adequately study the predictive power of abnormal neurologic examinations for ICH.

Some organizations, such as the Choosing Wisely initiative and American College of Physicians, have attempted to identify imaging tests and procedures that are overused by medical professionals [20]. Moreover, the American College of Radiology (ACR) has developed appropriateness criteria for the use of several imaging examinations in an attempt to limit and properly direct the use of imaging resources [26]. Given the findings in our study, we suggest that a decline in hematocrit, hemoglobin or other laboratory indication of new anemia is unlikely to be predictive of an ICH and is not a sufficient indication for ordering a HUS in the absence of other well-known risk factors as described in this and several other studies [22,24].

This study has several limitations. The types and quality of the data available for review in the electronic medical record were limited by the retrospective nature of this study. Data regarding anticoagulation at the time of HUS was not consistently available and thus was not evaluated in this study. Although abnormal neurologic examination was associated with an ICH on HUS, missing data may have limited our power to detect a statistically significant p-value. Also, since we could not know the neurologic examination of patients that were sedated at the time of HUS, our analysis of this variable was limited. It can be difficult to compare the results of neurologic examinations among patients with ICH and those without ICH given the great variability in expertise and experience amongst neonatal care providers, and the level of expertise could not be ascertained from the medical record. Finally, though the included cases represent nearly a decade’s worth of data, the relatively small sample size of patients with an intracranial hemorrhage within a single institution are limiting factors. A larger sample of patients with ICH on HUS collected across multiple medical centers would allow greater generalizability as well as additional subgroup analyses.

Conclusion

In conclusion, this study demonstrates that a decrease in hematocrit (or similar indication) is not predictive of an ICH and should not be the sole indication for ordering a HUS to rule out ICH. When additional factors are present in the neonate, such as low gestational age, prematurity, lower birth weight, younger age at the time of HUS, or abnormal neurologic examination, then a HUS is more likely to detect an ICH. Infants who are very or extremely preterm appear to be at a substantially greater risk for disability resulting from intracranial hemorrhage than those who are moderately preterm or term, and thus a HUS should always be considered in these patients whether or not there is a clinically reported drop in hematocrit.

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Effective converting ratio when switching between Ona-botulinumtoxin-A and Abo-botulinumtoxin-A in cervical dystonia. A patient assessment cross over study- Juniper Publishers

 

Neurology & Neurosurgery- Juniper Publishers

Introduction

In the 1950s it was hypothesized that Botulinum Neurotoxins (BoNT-A’s) could be used to reduce activity in muscle disorders [1]. Botulinum neurotoxin is made of a gram-positive anaerobic bacterial exotoxin-

clostridium botulinum [2]. When injected in the skeletal muscle, the botulinum toxin inhibits vesicular neurotransmitter (acetylcholine) release at the neuromuscular junction at the presynaptic membrane and a local chemo-denervation appears [1]. Thereby the toxin can reduce muscular contraction, which is the desire to reduce overactive muscle activity [3-5]. The medical therapies for dystonia are effective, and studies have shown that botulinum toxin is the most effective treatment for cervical dystonia [6]. The evidence from several Class I and Class II studies has led to a level A recommendation for use of BoNT-A for treating focal dystonia such as cervical dystonia, hemifacial spasm and blepharospasm [7]. Three main BoNT-A products are available on the market today: Ona-botulinumtoxin-A (Botox®), Abo-botulinumtoxin-A (Dysport®) and Inco-botulinumtoxin (Xeomin®). Due to different production methods, the biological nature of the three BoNT-A’s are different (e.g. the protein load, formulation, pH, dose and immunogenicity). Moreover, the BoNT-A’s are not interchangeable on an equivalent-dose basis [1, 7].

Study background

The goal of this study was to find the optimal conversion ratio for patients with cervical dystonia when switching treatment between Abo-botulinumtoxin-A and Ona-botulinumtoxin-Awhile obtaining a stable satisfying symptom improvement based on a subjective patient assessment. There is heterogeneity in treatment strategies and on how doses and the effect of BoNT-As are reported until now. This study represents a retrospective (follow-up), cross-over study (each patient acts as his/her own control). Effect of treatment was based on individual patient assessment. Individual based conversion rates were calculated using the optimal treatment dose for Abo-botulinumtoxin-A and Ona-botulinumtoxin-A. Due to the length of the present study with a follow-up time from 1991 to end 2014, it was possible to find stable toxin doses where each patient felt the best effect (min. 90%, VAS-score).

Methods

Study design

The clinical data was sampled from the patients’ medical records. Dystonic muscles and injection sites were identified by using electromyography (EMG). The neurologist determined the toxin doses based on each patient´s disease condition and the dystonic muscles activity (EMG). Each treatment (dosing) pr. clinical visit was individualized. At each consultation the neurologist reported the data (doses of toxin and the patient assessed effect and side effects) in the internal IT-system (Danish electronical patient journal) and also used standard sketches of the face/neck-regions to note which muscles have been injected

Botulinum toxin treatment

Ona-botulinumtoxin-A was diluted with saline to a concentration of 40U/ml. Abo-botulinumtoxin-A was dilutedwith saline to a concentration of 200 U/ml. For cervical dystonia the injection sites were between 2-8 sites. The injection doses in the specific muscles pr. patient and the total botulinum toxin doses pr. consultation were registered.

Effect of the procedure

The patients received treatment with BoNT-A´s every 3rd month at the neurological movement disorder clinic by an experienced neurologist. The subjective assessment of maximal treatment effect on dystonia symptoms between each treatment visits, were evaluated at each visit by the patient on a horizontal 100-mm Visual Analogue Scale (VAS) ; where the left end point indicated 0% treatment effect (no change in dystonia symptoms and no effect of the botulinum toxin) and the right end point indicated a total 100% treatment effect (indicate no dystonia symptoms and a maximal effect of botulinum toxin) between treatments)[8-11].

Data collection

In this study, data was only obtained from patients with a stable idiopathic cervical dystonia, and no change in relevant medical treatment over the data recording period. (Patient study entry, Table 1). The mean individual dose of Onabotulinumtoxin- A was obtained from 4 continuous consultations from each patient with a symptom relief of >90%. The treatment was then changed to Abo-botulinumtoxin-A after filtration to maximal effect and the mean individual dose of this toxin was obtained in the same manner as Ona-botulinumtoxin-A. The conversion ratio of Ona- & Abo-botulinumtoxin-A could then be calculated for each individual by dividing the mean individual Ona-botulinumtoxin-A dose with the mean Abobotulinumtoxin- A dose. The final average conversion ratio (Ona- BoNT-A: Abo-BoNT-A) in the study was found by using all the individual conversion ratio data from the 48 patients (Figure 1).

Statistical analysis

For our statistical analysis of our data we used the software package SPSS Statistics. All averages are in mean± standard deviation.

Results

625 out-patients in total were registered at the movement disorder clinic and received treatments with botulinum toxins. Of these, 48 patients with cervical dystonia patients met the inclusion criteria (Figure 2).

Cervical treatment was determined by the level of dystonia and ranged between 25- 200 units (Ona-botulinumtoxin-A) and 125- 775 units (Abo-botulinumtoxin-A) pr. clinical visit. More females had dystonia and most of the patients’ ages were between middle 50s to middle 70s (Table 2). All data wascollected in a period of 25 years from the movement disorder clinic. The VAS-outcome for each patient was minimum 90%. This retrospective data formed a cross-over design as each patient acted as his/her own control. Therefore, patients with confounders such as smoking, obesity etc. and comorbidities such as heart and lung diseases were not excluded, due to the cross over design. The average treatment time for patients with cervical dystonia receiving Ona-botulinumtoxin-A and Abobotulinumtoxin- A was 15,8±4,6 years. This average treatment time is the total period before and after switching botulinum toxin-A. 2546 injection series (total) were analyzed. Doses were 126 ± 38,8 IE-U for Ona-botulinumtoxin-A and 381,6 ± 151,1 IE-U for Abo-botulinumtoxin-A (Table 3). The mean conversion ratio for cervical dystonia was 1: 2.56. (Table 4).

Discussion

We are reporting 55 patients who converted treatment between Abo- and Ona-botulinumtoxin-A. All data wascollected in a period of 25 years from the movement disorder clinic. Patients have been stabilized with the Ona- or Abobotulinumtoxin- A treatment for at least a year before crossing over to treatment with the other toxin. Due to the long followup and moderate amount of data, we were able to find the average conversion ratio based on each patient´s subjective assessment. Former reports show no statistically significant difference on duration of the effect or adverse effects between Ona-botulinumtoxin-A and Abo-botulinumtoxin-A [12]. Data in these topics were excluded and not evaluated here. Treatment effect of Ona-botulinumtoxin-A and Abo-botulinumtoxin-A was in this study assessed by the treated patients. Due to the routine feedback from each of the treated patients over several years it was possible to achieve regular measurement of treatment effect from a subjective patient perspective without extra time for objective observations and evaluation in between toxin treatment sessions. The conversion ratio between the toxins (Ona-BoNT´s and Abo-BonT´s) using a patient-based assessment has in the present study shown to be equal to the study where they were using multicenter double-blind randomized trials [13]. Due to risk of different treatment techniques, toxin diffusion and especially individual variation in differentiating on relevant pain, a 90% margin was chosen as cut off for registration of desired effect.

Botulinum toxin effect & adverse effect

One unit of Ona-botulinumtoxin-A is not bioequivalent to one unit of Abo-botulinumtoxin-A. The ratio- Ona-botulinumtoxin-A: Abo-botulinumtoxin-A varies between 1:3 and 1:6 [14]. An earlier double-blinded study showed that the conversion ratio of Ona-botulinumtoxin-A and Abo-botulinumtoxin-A was 1:3 [14]. Abo-botulinumtoxin-A has been shown to have a doserelated significant longer duration of the effect rather than Onabotulinumtoxin- A, with a ratio 1:4 (Ona-botulinumtoxin-A: Abobotulinumtoxin- A, p-value= 0,02) [15]. Similar to former reports 12, this report have shown that the effect on dystonia, stabilizes after a period of treatments when changing between different toxin doses. In the present study the toxin doses were stabilized when the patients felt an effect of at least 90% measured on a VAS-score (0-100%) minimum at 4 continuous consultations.

Experimental results have shown that Abo-botulinumtoxin-A diffuses faster than Ona-botulinumtoxin-A which was explained by Abo-botulinumtoxin-A´s lighter molecular weight; Abobotulinumtoxin- A 3-400 kDa vs. Ona-botulinumtoxin-A 900kDa [16], and therefore the side effects in Abo-botulinumtoxin-A compared to Ona-botulinumtoxin-A was assumed due to a higher diffusion rate [17-19]. Other studies stated that botulinum toxin products dissociate under physiological conditions, and therefore the time of diffusion is not related to the toxin size [20]. In a study using Abo- and Ona-botulinumtoxin-A in the same patient with palmar hyperhidrosis, the patient was treated with Ona-botulinumtoxin-A in one palm, and Abo-botulinumtoxin-A in the other palm at the same session for 8 months. The patientswere their own control- like in the present study. The conclusion of that study was that the efficacy and safety of treating with Ona-botulinumtoxin-A and Abo-botulinumtoxin-A was by using the toxins in the conversion ratio of 1: 2.5 [21] which is similar to our conclusion. It has also been shown that conversion ratios of 1:3 or lower could be appropriate for the treatment of spasticity, cervical dystonia, hemifacial spasm, and blepharospasm 22. In the present study we found the conversion ratio of cervical dystonia to be 1:2, 56 which is similar to the former findings [21, 22].

The repeated administration and documentation of the usage of toxin doses (every 3rd month) at the movement disorder clinic reduced the information and selection bias. Due to the long follow-up period with a positive outcome and rare expected side effects, it was found to be effective to use a conversion ratio of 1: 2,56 (Ona-BoNT-A : Abo-BoNT-A) for botulinum toxin-A. The present study did not exclude any patients if they had confounders or comorbidities in behalf of that each patient was his/her own control in this cross-over design. The study design intended to prevent informational bias when evaluating the effect shortly after the toxin injections. To prevent selection and treatment variation bias the use of data was from patients who have been stabilized first with either Ona- or Abo-botulinumtoxin-A treatment for at least a year before crossing over to treatment with the other BoNT-A.

Conclusion

Considering the positive treatment effect, and the long followup period, we concluded that it is safe and clinically useful to use a conversion ratio at 1: 2,56 based on a patient assessment when changing treatment in between Ona- and Abo-botulinumtoxin-A for cervical dystonia. We consider the conversion ratios in our study applicable in other settings as well thus the effect of the botulinum toxin treatments was evaluated on a VAS-score (each patient is his/her own control) in this long follow-up study on dystonic out-patients.

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