A family with Robertsonian translocation: a potential mechanism of speciation in humans

Jieping Song
†
Xi Li
†
Lei Sun
†
Shuqin Xu
Nian Liu
Yanyi Yao
Zhi Liu
Weipeng Wang
Han Rong
Bo Wang
A family with Robertsonian translocation: a potential mechanism of speciation in humans
10.1186/s13039-016-0255-7C A S E R E P O R T Open Access
Background: Robertsonian translocations occur in approximately one in every 1000 newborns. Although most Robertsonian translocation carriers are healthy and have a normal lifespan, they are at increased risk of spontaneous abortions and risk of producing unbalanced gametes and, therefore unbalanced offspring. Here we reported a previously undescribed Robertsonian translocation. Case Presentation: We identified three Robertsonian translocation carriers in this family. Two were heterozygous translocation carriers of 45,XX or XY,der(14;15)(q10;q10) and their son was a homozygous translocation carrier of a 44,XY,der(14;15)(q10;q10), der(14;15)(q10;q10) karyotype. Chromosomal analysis of sperm showed 99.7 % of sperm from the homozygous translocation carrier were normal/balanced while only 79.9 % of sperm from the heterozygous translocation carrier were normal/balanced. There was a significantly higher frequency of aneuploidy for sex chromosome in the heterozygous translocation carrier. Conclusions: The reproductive fitness of Robertsonian translocation carriers is reduced. Robertsonian translocation homozygosity can be a potential speciation in humans with 44 chromosomes.

Background

Robertsonian translocations, fusions between two acrocentric chromosomes, are the most common structural chromosomal rearrangements in humans and occur in approximately 1 in every 1000 newborns [1]. It occurs in the five acrocentric chromosomes, 13, 14, 15, 21, and 22, which have very small short arms that contain no unique genes. During a Robertsonian translocation, the participating chromosomes break at their centromeres and the long arms fuse to form a single chromosome with a single centromere. The short arms also join to form a smaller reciprocal product, which typically contains nonessential genes and is usually lost within a few cell divisions. This type of translocation is cytologically visible and can reduce chromosome number if the smaller chromosome that results from a translocation is lost over cellular divisions. However, the smaller chromosome lost may carry so few genes that it can be lost without an ill effect to the individual [2,3].

When the translocation is balanced, the person with it is called a Robertsonian translocation carrier. As carriers are healthy and have a normal lifespanand, many never discover their unusual chromosome rearrangement. Therefore, the translocation can be passed down in families for many generations without anyone discovering. Robertsonian translocation carriers produce six types of gametes. At the end of meiosis I, segregation of the translocated and nontranslocated chromosomes from the two different chromosome pairs involved leads to the formation of either balanced gametes via alternate segregation or unbalanced gametes via adjacent segregation during anaphase [1,2]. Meiotic tetravalent configuration tends to segregate in alternate way, resulting in preferential production of normal/balanced spermatozoa. However, certain percentages of unbalanced gametes derived from adjacent segregation are also produced, leading to the increased risk of miscarriage and chromosomally unbalanced fetus. In this study, we investigated a Robertsonian translocation family and analyzed the meiotic segregation patterns and interchromosomal effects in sperm from two Robertsonian translocation carriers.

Case presentation

Clinical information

A 25-year-old Chinese man (IV-1) married to a nonconsanguineous woman with normal chromosomes (IV-2). This couple had a son (V-1) who died at 6-month old without an autopsy. Previous chromosome analysis indicated V-1 had a 45,XY,der(14;15)(q10;q10) karyotype. The parents of the propositus (IV-1) are phenotypically normal first cousins (III-1, III -2). Their parents, a mutual uncle and both grandparents were not available for cytogenetic analysis ( Fig. 1). Mental state examination was performed on IV-1 by a psychiatrist.

Karyotype analysis

G-banding karyotype analysis was performed on all the four members in the family. The previously undescribed karyotype, 44,XY,der(14;15)(q10;q10), der(14;15)(q10;q10), was further authenticated by the Chinese Academic Committee of the State Key Laboratory of Medical Genetics (Fig. 2).

Fluorescence in situ hybridization (FISH)

Sperm chromosomes from patient III-1 and IV-1 were analyzed by FISH. Semen samples were assessed for volume, concentration and motility according to the World Health Organization criteria. After semen analysis, progressively mobile sperm were isolated and washed twice in phosphate buffered saline (pH 7.4) and centrifuged at 1500 rpm for 11 min. Pellets were fixed with 5 ml of acetic acid/methanol mixture (1:3) for at least 30 min at 4°C. Aliquots (40-50 μl) of the resulting suspension of nuclei were smeared on cold pre-cleaned slides. Nuclei decondensation was performed in 1 N NaOH for 2 min. The slides were then degraded by a graded ethanol series (70 %, 90 %, 100 %) and denatured with 0.25 % formamide in 2xSSC followed by overnight hybridization with a combination of commercially available probes. Two sets of probe mixtures were used in this study. Dual-color FISH was carried out using locusspecific probes (LSP) and Tel probes from Vysis (Vysis, Downers Grove, IL, USA). For patient IV-1 and III-1,TelVysion Probe 14q (D14S1420, Spectrum Red) was used for 14q32.33 and TelVysion Probe 15q(D15S120, Spectrum Green) was used for 15q26.3.

To investigate the presence of interchromosomal effect, triple-color FISH was performed using the second probe mixture, which consists of commercial satellite (DNA) probes from Vysis, including chromosomes 18, X and Y (CEP 18, Spectrum Blue/CEP X, Spectrum Green/CEP Y, Spectrum Red). Post-hybridization washes included 2 min in 0.4xSSC/0.3%NP-40 (pH = 7) at 72°C, followed by 1 min in 2xSSC/0.1%NP-40 (pH = 7) at room temperature. Slides were covered with DAPI II (Vysis). Only intact spermatozoa bearing a similar degree of decondensation and clear hybridization signals were scored. All disrupted or overlapping spermatozoa were excluded from analysis. Slides with hybridization efficiency of 99 % and more were analyzed. 1,000 sperm nuclei were analyzed per patient.

Array comparative genomic hybridization (aCGH)

Array comparative genomic hybridization (aCGH) has been introduced in clinical diagnosis to rapidly detect genomewide gains and losses with higher resolution [4]. It is a high throughput method which detects copy number changes to a resolution of even as low as 1 Kb. For aCGH analysis, DNA was extracted from peripheral blood [5]. aCGH analysis was performed on using the

Data analysis

The statistical analysis was performed using the SAS system (2002-2003, SAS Institute Inc., Cary, NC). Chi-squared test was used for meiotic segregation analysis. A probability value of less than 0.05 (P < 0.05) was considered to be statistically significant.

Results

The karyotype analysis was performed on four members in the family and detected two different karyotypes of Robertsonian translocation. The results are shown in below. III-1 and III-2 were heterozygous while IV-1 was homozygous.

III-1: 45,XY,der(14;15)(q10;q10) III-2: 45,XX,der(14;15)(q10;q10) IV-1: 44,XY,der(14;15)(q10;q10), der(14;15)(q10;q10) IV-2: 46,XX Because the patient (IV-1) has a symptom of apathy, we referred him to see a psychiatrist for a thorough mental status examination. The patient had no psychiatric history and remained cooperative during the psychiatric interview. His thought processes were linear, logical, and consistently goal directed and his judgment was intact. Therefore, no psychiatric symptoms were observed. In addition, neurology consultation revealed no focal neurological deficits. The results of Craniocerebral Computed Tomography (CT) and lumbar puncture (LP) were normal. Electroencephalography (EEG) showed no signs of seizure activity.

The aCGH analysis on IV-1 detected a 2.03 Mb duplication in the region of q11.1-q11.2 on chromosome 15 (Fig. 3). According to the Copy number variation database (CNVdb), this duplication is a chromosomal polymorphism in normal population. Because IV-1 is a is Homologous Robertsonian translocation carrier and his two derivative chromosomes came from the same Robertsonian translocation carrier, we speculated that each of the two derivative chromosomes should have a 2.03 Mb duplication in the region of q11.1-q11.2 on chromosome 15.

To analysis the sperm of III-1 and IV-1, semen samples from the two different Robertsonian translocation carriers (III-1 and IV-1) were assessed for volume, concentration and motility according to the World Health Organization criteria. The volume, concentration and mobility of sperm in III-1 are significantly reduced due to his old age. In contrast, they are all normal in IV-1 (Table 1). We further analyzed the meiotic segregation patterns in these two different Robertsonian translocation carriers. In the heterozygote Robertsonian translocation carrier (III-1), the frequency of normal/balanced spermatozoa resulting from alternate segregation was 79.9 % while the frequency of unbalanced spermatozoa resulting from adjacent segregation was 20.1 %, which is significantly higher than normal population [5][6][7][8] ( Table 2). In contrast, in the homozygote Robertsonian translocation (IV-1), the frequency of balanced spermatozoa was 99.7 % while the frequency of unbalanced spermatozoa was only 0.3 %, which is similar to normal populations [5][6][7][8] (Table 2). These data demonstrated there nearly all sperm of Robertsonian translocation homozygotes were balanced while those of heterozygotes had a percentage of unbalanced. In addition, chromosome analysis of IV-1's sperm showed either a 22,X,der (14;15) karyotype or 22,Y,der(14;15) karyotype supporting our speculation. As shown in Fig. 4, the arrow pointed a sperm with four fluorescence signals (two red and two green) suggesting this sperm has two derivative chromosomes of der(14;15). These data suggest a potential mechanism of speciation in humans via Robertsonian translocation.

The influence of translocated chromosomes on the synapses and disjunction of other chromosomes is called an interchromosomal effect (ICE) [9]. To determine whether ICE was present, the nullisomy, disomy and diploid rates for chromosomes 18, X and Y were analyzed in both Robertsonian translocation carriers (Table 3). In the heterozygote Robertsonian translocation carrier (III-1), there was a significant increase in the rate of sex chromosome aneuploidy and diploid. In contrast, in the homozygote Robertsonian translocation (IV-1), the incidence of spermatozoa with nullisomy, disomy and diploid for the sex chromosomes remained similar to normal populations (Table 3) [5][6][7][8]. This data suggest that ICE only presents in certain Robertsonian translocation carriers.

Discussion

Rob translocation carries are phenotypically normal since they involve loss only of short arm material. Many  such rearrangments in natural populations of different species are known. Robertsonian rearrangments can increase polymorphisms in a species, provide material for natural selection and even lead to speciation [10,11]. It has been argued that the types of chromosome rearrangments that occur as polymorphisms or as fixed permanent heterozygotes invariably involve meta-or submetacentric chromosomes. Those that distinguish species and serve to isolate these species involve telocentric or acrocentric chromosomes, which are self-sterilizing [12]. In our study, IV-1, who is a homologous Robertsonian translocation carrier, was healthy and had a balanced chromosomal complement. Assessment of a semen sample from showed normal sperm morphology and motility. More importantly, 99.7 % of his sperm are normal or balanced (Table 2) and there was no interchromosomal effect (Table 3). Multicolor FISH on decondensed sperm nuclei allows for a rapid analysis of meiotic segregation in sperm of translocation carriers, providing information on the exact amount of normal/balanced sperm [13]. Such information is undoubtedly important not only for basic cytogenetic research but also for reproductive counseling of Robertsonian translocation carriers. t(14;15) are rare Robertsonian translocations. Our data confirmed previous studies showing that alternate segregation was more frequently than adjacent segregations in Robertsonian translocations carriers [5,8]. However, the meiotic segregation patterns and interchromosomal effects in two Robertsonian translocation carriers are different ( Table 2). These findings are consistent with previously published data [14][15][16]. The high prevalence of the alternate segregation is presumably due to cis-configuration of the trivalent during meiosis, which favors an alternate segregation in all Robertsonian translocations [2]. Although a Robertsonian translocation carrier has a full genetic complement, their productive fitness is reduced due to high probability of genetically imbalanced gametes.

The interchromosomal effect could be explained by the formation of heterosynapses between chromosomes involved in the translocation and the sex vesicle, which could also involve other chromosomes [17]. ICE remains controversial in the literature. Some publications have indicated its presence with Robertsonian translocation [18,19] while others demonstrated no evidence of this phenomenon [4,20]. In our study, we observed higher incidence of aneuploidy for sex chromosomes in spermatozoa in the heterozygote Robertsonian translocation but not the homozygote Robertsonian translocation suggesting that ICE only presents in some Robertsonian translocation carriers.

Robertsonian rearrangements are common chromosomal changes that can lead to rapid and efficient reproductive isolation between karyotypically similar populations, especially when many Robertsonian metacentric chromosomes display monobrachial homologies [10]. Homozygous Robertsonian translocations have reported by several groups [21][22][23] and systemically reviewed by the O'Neill group [24]. Dallapiccola et al. [21] reported a fetus with two t(14;21) chromosomes. The related parents were heterozygous for the same translocation. Martinez et al. [22] reported three adult siblings homozygous for t (13;14). Their parents were first-cousins and both were heterozygous carriers. Rajangam et al. [23] found a unique DS karyotype 45,  XY, der(14;21)pat, der(14;21)mat, +21mat. Although translocation heterozygosity is associated with meiotic disturbances, which will lead to infertility and subfecundity, it should not have any effect on meiosis. Here we reported a man with a 44,XY,der(14;15)(q10;q10),der(14;15)(q10;q10) karyotype. Robertsonian translocation may provide material for evolution. Long term isolation of a group of individuals who are homozygous for a particular Robertsonian translocation chromosome could theoretically lead to the establishment of a new human subspecies having a full genetic complement in 44 chromosomes. Although karyotypic differences between species have long been recognized, the question of whether these mutations play a causal role in speciation remains unanswered [25][26][27]. In our study, all sperm of Robertsonian translocation homozygotes were balanced while those of heterozygotes had a percentage of unbalanced ones implicating of a potential of the homozygosity of Robertsonian translocations for speciation.

Conclusions

To conclude, in this study we reported a previously described Robertsonian translocation karyotype. The homozygosity of Robertsonian translocation for speciation may be a potential mechanism of speciation in Humans.

Availability of data and materials

The dataset(s) supporting the conclusions of this article are available in the folder named raw data for the manuscript at https://www.dropbox.com/sh/ izfb6bdmyz43gz7/AAB4nVTovhquFbSqA3kqtTtHa?dl=0

Fig. 1
1Pedigree of the family transmitting Rob translocation chromosome t(14; 15)(q10;q10) Open hexagon designates a presumed carrier of t(14; 15)(q10;q10). Filled hexagon designates a known carrier of t(14;15)(q10;q10). The proband, IV-1 (arrow), has disomy t(14;15)(q10;q10). The proband's wife, IV-2, had a normal karyotype. Their deceased son, V-1, was a carrier of karyotype 45,XY,der(14;15)(q10;q10) 8 × 60 K commercial arrays (Agilent Technologies, Santa Clara, CA, USA).

Fig. 2
2Karyotype of the proband with 44,XY,der(14;15)(q10;q10),der(14;15)(q10;q10) chromosomal constitution The arrows indicated disomy of chromosome der(14;15)

Fig. 3
3aCGH analysis of IV-1 revealed a 2.03 Mb duplication at chromosome 15 q11.1-q11.2 (arr 15q11.1q11.2(20,481,702-22,509,254)x4)

aCGH, Array comparative genomic hybridization; CT, Craniocerebral Computed Tomography; EEG, Electroencephalography; FISH, Fluorescence in situ hybridization;; ICE, interchromosomal effect; LP, lumbar puncture Funding This work is supported by a scientific research and technology development grant of Guangxi province (#1598012-35)

Table 1
1Cytogenetic and spermiologic results of III-1 and IV-1Patient
Age (years)
Sperm concentration
(×10 6 /ml)

Mobility (a + b) (%)

III-1
48
13
22

IV-1
25
56
53

Table 2
2The number of spermatozoa scored, the alternate mode of segregation, incidence of sperm nullisomy, disomy and 3:0/diploid for the chromosomes involved in the Rob translocation in two Rob translocation carriersFig. 4Sperm cells after hybridization with TelVysion 14q32.33 (Spectrum Red) and TelVysion 15q26.3 (Spectrum Green). The sperm with red arrow may be a diploid spermSegregation modes
III-1
IV-1

normal or balanced
799
997

nullisomy 14
41
2

disomy 14
49
0

nullisomy 15
55
0

disomy 15
45
0

3:0 or diploid
11
1

Total
1000
1000

Table 3
3Incidence of sperm nullisomy, disomy and diploid for chromosomes 18, X and Y in two Rob translocation carriersSegregation modes
III-1
IV-1

normal or balanced
949
994

nullisomy 18
3
0

disomy 18
2
1

nullisomy Sex chromosome
15
2

disomy Sex chromosome
10
3

3:0 or diploid
21
0

Total
1000
1000

Authors' contributions B.W., J.S. and L.S designed the study. B.W. and J.S. analyzed the results and wrote the manuscript. L.S. revised the manuscript. X. L. perfomred the sperm FISH. S. X. participated in genetic counseling. N. L. and Z. L. performed array CGH anaysis. Y. Y. and W.W. performed chromosome analysis. H. R. perforemed psychiatric interview. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Ethics approval and consent to participateThe affected patient were informed about the study, and written informed consent, approved by the Ethical committee of Hubei Maternal and Child Health Hospital, was obtained. Submit your next manuscript to BioMed Central and we will help you at every step:
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The ΔF/F m ′-guided supply of nitrogen in culture medium facilitates sustainable production of TAG in Nannochloropsis oceanica IMET1

Jiao Liu
Changhong Yao
Yingying Meng
Xupeng Cao
Peichun Wu
Song Xue
The ΔF/F m ′-guided supply of nitrogen in culture medium facilitates sustainable production of TAG in Nannochloropsis oceanica IMET1
10.1186/s13068-018-1168-yLiu et al. Biotechnol Biofuels (2018) 11:168 RESEARCHSemi-continuous cultivationΔF/F m ′ monitoringNitrogen stress indexMicroalgae
Background: Triacylglycerol (TAG) from photosynthetic microalgae is a sustainable feedstock for biodiesel production. Physiological stress triggers microalgal TAG accumulation. However excessive physiological stress will impair the photosynthesis system seriously thus decreasing TAG productivity because of the low biomass production. Hence, it is critical to quantitatively and timely monitor the degree of the stress while the microalgal cells growing so that the optimal TAG productivity can be obtained.Results:The lack of an on-line monitored indicator has limited our ability to gain knowledge of cellular "health status" information regarding high TAG productivity. Therefore, to monitor the degree of nitrogen stress of the cells, we investigated the correlation between the photosynthetic system II (PS II) quantum yield and the degree of stress based on the high relevancy between photosynthetic reduction and nitrogen stress-induced TAG accumulation in microalgal cells. ΔF/F m ′, which is the chlorophyll fluorescence parameter that reflects the effective capability of PS II, was identified to be a critical factor to indicate the degree of stress of the cells. In addition, the concept of a nitrogen stress index has been defined to quantify the degree of stress. Based on this index and by monitoring ΔF/F m ′ and guiding the supply of nitrogen in culture medium to maintain a stable degree of stress, a stable and efficient semi-continuous process for TAG production has been established.Conclusion:The results indicate that the semi-continuous cultivation process with a controlled degree of stress by monitoring the ΔF/F m ′ indicator will have a significant impact on microalgal TAG production, especially for the outdoor controllable cultivation of microalgae on a large scale.

Background

Photosynthetic microalgae have received increasing attention as the most promising biofuel feedstock as humans are facing increasing problems related to climate and fossil energy [1][2][3]. Microalgae can use light energy and CO 2 to produce energy-storage compounds such as triacylglycerol (TAG), which is the precursor of biodiesel [4]. Physiological stress is usually applied to microalgal cultures to trigger TAG accumulation. When confronted with physiological stress, such as nitrogen stress (N-stress), microalgal cells make adjustments such as enhancing the energy-storage compounds (e.g., TAG) accumulation, to acclimate to unfavourable conditions. However, the physiological stress affects both photosynthesis and C-storage mechanism. When under excessive physiological stress, the photosynthetic efficiency progressively decreases and the cell growth is significantly diminished. Therefore, it is quite challenging to achieve maximum TAG productivity by balancing the TAG content and high productivity of microalgal biomass because

Open Access

Biotechnology for Biofuels *Correspondence: firstname.lastname@example.com 1 Marine Bioengineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 16023, China Full list of author information is available at the end of the article TAG production largely relies on stress conditions such as nitrogen stress (N-stress) and high irradiance etc. [5].

N-stress is the most effective means to trigger TAG accumulation [4,6,7]. When subjected to N-stress, in addition to enhanced TAG accumulation, protein synthesis in microalgal cells is affected as well. Protein synthesis in microalgae is immediately suppressed upon nitrogen shortage, which mostly hinders the protein turnover of photosynthetic apparatus, especially the photosystem (PS) II D1 reaction centre protein [8]. This will lead to a decline in the photosynthetic electron transport rate (ETR) and, consequently, a reduction in photochemical energy conversion [8,9]. Moreover, limited nitrogen supply causes impairment of photosynthetic CO 2 fixation by degradation of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) for the recycling of nitrogen. Limitation of CO 2 fixation then decreases the consumption of ATP and NADPH and leads to an excess of NADPH and electrons [8]. The sufficient supply of NADPH is essential for TAG accumulation [10], whereas excessive electrons will lead to the formation of reactive oxygen species (ROS), which exposes microalgae to oxidative stress and is also believed to be a signal trigger for TAG formation [11][12][13]. In addition, the remodelling of the photosynthetic membranes caused by N-stress also contributes to a considerable fraction of TAG production by providing a fatty acid acyl moiety [5,14,15]. Therefore, it is closely linked to photosynthetic reduction and N-stress-induced TAG accumulation in microalgal cells. Therefore, it is very important to make systematic research on it.

Photosynthesis is a coordinated physiological process that exclusively provides both energy and the material foundation for photoautotrophic microalgae [16]. Therefore, it could be considered the most important cellular metabolism in algae. With no exception, photosynthesis provides the energy as well as fixes the carbon used for TAG synthesis in photoautotrophic microalgae. However, as stated above, TAG accumulation is inevitably accompanied by photosynthetic reduction under excessive stress in photoautotrophic oleaginous microalgae, which means that excessive stress could cause inhibition of growth and reduce biomass and overall TAG yield. Optimal TAG productivity can be achieved only if the photosynthetic performance is properly maintained. Therefore, quantitative and timely monitoring of the stress status of the subjected microalgal cells is vital so that the optimal TAG productivity can be attained in time. Because of the tight relationship between photosynthesis and TAG synthesis, monitoring of the photosynthetic performance should be a plausible method.

Chlorophyll fluorescence analysis is a powerful tool for the study of photosynthesis in both plants and algae [17][18][19]. It allows a non-invasive and nearly instantaneous measurement of performance in photosynthetic light capture and electron transport [17]. The PS II quantum yield, which is a chlorophyll fluorescence parameter that measures the proportion of the light absorbed by chlorophyll related to PS II and is used in photochemistry, provides an estimation of the linear ETR and hence the PS II performance [18,19]. It is widely used as the indicator to assess nutrient limitations in situ in microalgae [9,20,21]. However, to the best of our knowledge, little research has been conducted hitherto to apply this fluorescence parameter for the control of TAG production. In our previous work, a decreased PS II quantum yield was found to be highly related to the degree of N-stress. In both Tetraselmis subcordiformis and Isochrysis zhangjiangensis, critical values of chlorophyll fluorescence existed where the maximum productivities of energy storage substances, such as carbohydrates, were obtained [22,23]. Therefore, in this work, we proposed the use of this fluorescence parameter as an N-stress indicator to monitor the stress degree and TAG production during cultivation by controlling the nitrogen supply strategy. To obtain an efficient and stable production of lipid from Nannochloropsis oceanica IMET1, we established a reproducible TAG production process by monitoring photosynthetic activity to maintain a constant degree of N-stress under semi-continuous cultivation.

Methods

Strain and Cultivation conditions

Nannochloropsis oceanica IMET1 from the University of Maryland Biotechnology Institute was cultivated in seawater with modified F/2 medium at the ambient temperature of 25 ± 1 °C. In the present study, a 500-mL bubble column bioreactor (5 cm in diameter) was used with the air flow at 100 mL/min (2% CO 2 , v/v) filtered by a 0.22-μm membrane as described by Pan et al. [24]. Additionally, 140 μmol/m 2 s cool white fluorescent lights were provided from one side of the bioreactor under a 14-h/10-h light/dark cycle. After pre-culture in the bioreactor with sufficient nutrient elements until the cells reached exponential growth phase, the cells were inoculated into new bioreactors at an initial biomass concentration of ~ 0.18 mg/mL (dry weight, DW).

For the batch culture, the initial N amount was 15 mg/L in the medium, and no additional N was added into the medium until harvest. For the semi-continuous cultivation, the inoculum was the N-stressed cells from the batch. During cultivation, the fluorescence parameter ΔF/F m ′ was monitored. During the semi-continuous, ΔF/ F m ′ was used as an indicator of the degree of N-stress. When ΔF/F m ′ reached the predetermined level, which was determined in batch cultivation as the monitoring point, harvest and dilution were conducted to start a new cycle of cultivation. During the dilution, the relative amount of fresh medium replenished to the system was defined as the "renewal rate". For each cycle during the same semi-continuous cultivation process, the initial biomass concentration and supplementary N amount were the same.

Measurements

Microalgal pellets from centrifugation were washed twice with 0.5 M NH 4 HCO 3 and were then dried at 60 °C to achieve a constant weight. The DW was the difference between the final weight and the weight of the empty tube as described by Zhu et al. [25]. According to Chi et al. [26], the nitrate concentrations in the medium were measured using a UV/VIS spectrophotometer with a predrawn standard curve of the nitrate absorption of light. The light intensity was measured by an Optometer P9710 using a photosynthetically active radiation detector (Gigahertz Optik Corporation, Germany). The nitrogen quota of the biomass was determined using an elemental analysis instrument (Vario EL cube, Elementar Analysensysteme GmbH Germany). The fluorescence parameter ΔF/F m ′ was measured using a chlorophyll fluorometer (Water-PAM WALZ, Germany) by the method described by Yao et al. [22]. After applying a saturating light pulse to the light-adapted cells, F m ′ and F′ were obtained, and F m ′ refers to the maximum fluorescence at the light-adapted state. The effective photosynthesis capability of PS II, ΔF/ F m ′, was calculated according to Eq. (1).

The TAG content was calculated using the method developed by Wang and Shen and based on the concept of characteristic fatty acid, which provided a way to quantify TAGs in microalgae precisely by immediate transesterification from the wet biomass and required smaller
(1) �F /F ′ m = (F ′ m − F ′ )/F ′ m
samples than conventional methods [27,28]. Shen et al. identified EPA as the characteristic fatty acid of N. oceanica and confirmed the highly linear correlation between the TAG content and the relative percentage content of EPA obtained by normalizing the total fatty acids (regression equation: y = 36.737 − 1.212x, where R 2 = 0.97, y is the TAG content, and x is the relative percent content of EPA). The fatty acid composition was determined by gas chromatography after transesterification and according to Liu et al. [29]. TAG productivity was calculated by Eq. (2), where DW i and C TAG−i represent the DW and TAG content at the end of cycle i, and the symbols with the subscript of cycle 0 represent the corresponding data of the initial state of cycle i.

Results and discussion

Verification of ΔF/F m ′ as an N-stress indicator for TAG production

The N-stress indicator should be automatically and rapidly measured. It should be sensitive to N-stress, including the content of intracellular N and other parameters of physiology or biochemistry, such as the metabolite content, elemental content, photosynthesis activity, and respiration rate. Therefore, during cell growth and TAG accumulation under N-stress, the physiological characteristics and cellular biochemical properties were investigated including the DW, TAG content, intracellular N content and content of N in the medium and the chlorophyll fluorescence parameter ΔF/F m ′. As shown in Fig. 1a, the N in the medium was completely consumed in one day, and the intracellular N decreased over time from 8.09 to 2.72%, which indicated that the cells became N-stressed from day 1. Another parameter, ΔF/F m ′, which reflects the effective photosynthesis capability of PS II, decreased consistently with changes in intracellular N from 0.56 to  Fig. 1b, the TAG content increased rapidly in the first 4 days, from 3 to 27.3%, and increased slowly after day 5, from 28.0 to 28.4%; the growth rate of DW decreased over time from 0.17 mg/ mL day at day 4 to 0.13 mg/mL day at day 5 and further to 0.08 mg/mL day at day 6. The daily TAG productivity shown in Fig. 1b reached the highest level at day 4 and then obviously decreased. Because the TAG productivity was related to the DW and TAG content, the increase in the DW and TAG content became slower and thus led to the decrease in TAG productivity after day 4. This indicates that the improvement in TAG content by strengthening N-stress will conversely lead to the slowdown or even the stoppage of cell growth, which results in lower TAG productivity. When comparing all of the parameters, intracellular N is a critical parameter to trigger the initiation of TAG accumulation. There were studies that made efforts on the determining the proper N-stress and emphasized the importance of accurate control of intracellular N when to optimize the lipid productivity [30,31]. However, the lack of a fast detection method for intracellular N limits its application as an online indicator to control microalgal cells at a proper degree of N-stress and to balance cell growth and TAG accumulation. Among the measured parameters, ΔF/ F m ′ is a promising indicator of N-stress in microalgae due to the high correlation between intracellular N and ΔF/F m ′. By investigating the growth of N. oceanica IMET1 under N-stress, it was found that during the period of day 3 to day 6 when the highest TAG productivity under N-stress was obtained, there was a high correlation between intracellular N and ΔF/F m ′. The correlation coefficient between intracellular N and ΔF/F m ′ reached 0.97 (y = 0.1083x + 0.1258, where R 2 = 0.9713, y refers to ΔF/F m ′, and x refers to intracellular N). In the other species of microalgae, a correlation also exists between intracellular N and ΔF/ F m ′. For T. subcordiformis and I. zhangjiangensis, the correlations were y = 0.0741x + 0.151 (R 2 = 0.9779) and y = 0.168x + 0.1597 (R 2 = 0.9302), respectively (unpublished data). In short, it is not a special case where the ΔF/F m ′ and intracellular N synchronously change while cells are under the N-stress condition. The great advantages of ΔF/F m ′ are its characteristic of in situ and rapid measurement. In addition, the photosystem (whose activity ΔF/F m ′ reflects) plays the role of the hub in microalgal metabolism. Therefore, we propose monitoring the degree of N-stress using ΔF/F m ′ as an on-line indicator and then making the unsteady status of high TAG productivity reproducible to realize continuous TAG production.
(2) P i = (DW i × C TAG−i − DW i0 × C TAG−i0 )/t a b

Determination of the proper degree of N-stress of microalgal cells for TAG production

High TAG productivity is highly related to the degree of N-stress, and determination of the proper degree of N-stress of microalgal cells is required. The proper degree of N-stress could not only induce TAG accumulation but also maintain the cells at a relatively high photosynthesis capability for growth and thus ensure the balance of the contradiction between TAG accumulation and fast growth. From day 3 to day 5, as shown in Fig. 1, both the TAG productivity and ΔF/F m ′ stayed at a relatively high level, above 63 mg/mL day and 0.45, which was 78% of the maximum value of 0.58, respectively, as shown in Fig. 1b. Therefore, it could be concluded that from day 3 to day 5 the cells would reach the status with the proper degree of N-stress. To determine the proper degree of N-stress as the monitoring point and to realize semi-continuous cultivation under the constant N-stress condition and investigate the recovery of N-stressed cells after N replenishment, three batches of cultivation were conducted using N-stressed cells obtained at day 3, day 4 and day 5 as inoculum (day3-cell, day4-cell and day5cell). Before recultivating the N-stressed cells, a new concept of the N-stress Index (NSI) was defined to quantify the degree of N-stress, which is never named. So far, the degree of stress has never been maintained by controlling any parameter. The NSI is calculated based on the intracellular N according to Eq. (3), where N max is the maximum intracellular N content of the cells under N-replete conditions, N min refers to the minimum intracellular N for the cells to only keep alive subjected to severe N-stress for a long time, and N i is the intracellular N of the cells at day i. Therefore, NSI ranges from 0 to 1 and a greater value of NSI indicates a higher degree of N-stress of the cells. Then, the NSIs of the day3-cell, day4-cell and day5-cell were 0.62, 0.72 and 0.81, respectively, and represent aggravated N-stress of the cells.

During the recultivation, ΔF/F m ′ was used as an N-stress indicator in situ. The cultivation of the day4-cell is used as an example to describe in detail the actual operation. The ΔF/F m ′ of the day4-cell was 0.503 at the inoculation, and this value was set as the critical point of monitoring. After supplying N into the medium and culturing for a few days, when the ΔF/F m ′ reached the same level as the preset critical point at inoculation, the cultivation was ended. Specifically, as shown in Fig. 2a, after 4 days of cultivation, when the ΔF/F m ′ reached 0.507, which was the same level as the initial ΔF/F m ′, that is, 0.503 for the day4-cell, the cultivation was ended. The operations of the day3-cell and day5-cell were the same as those of the day4-cell, and actual changes in ΔF/F m ′ can be seen Fig. 2a. The daily intracellular N was also measured and depicted in Fig. 2a. Moreover, the TAG content also reached the expected level, as shown in Fig. 2b. The above results confirmed that it is feasible to reproduce cells at a specified status to produce TAG by monitoring ΔF/F m ′. In addition, from the results of the TAG productivity obtained by cultivation of the 3 types of N-stressed cells shown in Fig. 3c, it was obvious that the TAG productivity of the day4-cell was higher than those of the other two types of cells and reached 49 mg/mL day. For the day3-cell, even though the ΔF/F m ′ was higher and indicated a faster growth rate than the other two kinds of cells, the TAG accumulation was lower because the cells encountered a lower degree of N-stress, which ultimately resulted in less TAG productivity than the day4-cell. The result of the day3-cell indicated that the NSI of 0.62 was not large enough. Different from the day3-cell, a lower growth rate of day5-cells led to a lower TAG productivity. In contrast to the day3-cell, day5-cells encountered excessive N-stress with a large NSI of 0.81, which resulted in too much impairment of the cell growth. Therefore, according to the above results, the day4-cell experienced the proper degree of N-stress with an NSI of 0.72, and the corresponding ΔF/F m ′ of 0.503 could be considered the critical monitoring point.
(3) NSI i = (N max − N i )/(N max − N min ) in

Stable reproduction of TAG under semi-continuous cultivation by monitoring ΔF/F m ′

The proper degree of N-stress has been disclosed for TAG stable production. Using ΔF/F m ′ as an N-stress indicator, a semi-continuous cultivation of the proper N-stressed day4-cell was established by controlling the N supplying strategy. The initial N concentration in the medium and the renewal rate were optimized as the key operating parameters to improve the preliminary established semi-continuous process since the two operating parameters were reported to greatly affect the specific growth rate of the cells and productivity of the target products in a semi-continuous cultivation [32][33][34]. The optimization experiments are listed in Table 1, and the results are shown in Figs. 3 and 4. During the cultivation, ΔF/F m ′ was monitored daily to guide semi-continuous recycling, and the critical point of monitoring for ΔF/ F m ′ was 0.503. When the ΔF/F m ′ reached the preset critical point of monitoring, a new cycle was restarted. The harvest and additional proportion of medium were the same and were operated according to the renewal rate in Table 1. Additionally, the initial N concentration was adjusted as required in Table 1.

To keep the culture conditions consistent with that of the batch cultivation in "Determination of the proper degree of N-stress of microalgal cells for TAG production" section, in experiment a, the initial N concentration Experiments b and c were performed to investigate the effects of the initial N concentration in medium on TAG productivity. As shown in Fig. 4b, c, the TAG productivity of each cycle during experiments b and c were lower than that during experiment a. It is common sense that the DW increment of each cycle is dependent on the total N in the medium. For example, regarding cycle 2 in experiment a, the intracellular N at the end point was set to be equal to the initial intracellular N of 3.82%, and the total N amount added into the medium was fixed to be 22.9 mg/L; thus, theoretically, the DW increment of cycle 2 should be 0.60 mg/mL (the amount of N added into the medium each time divided by the initial intracellular N). Practically, the actual DW increment was 0.65 mg/mL in accordance with the theoretical value. In other words, when the degree of N-stress of the cells at the end point was fixed, which indicated that the intracellular N was set to be at a fixed value, the total N amount added into the medium would decide the absolute increment of DW.   4 Optimization of the N supply amount and the renewable rate Therefore, it was the cell growth rate that actually determined the TAG productivity of each cycle. Additionally, the DW increase rates in experiments b and c were 0.13 and 0.09 mg/mL day, respectively, and were lower than that of experiment a, which was 0.18 mg/mL day. The lower cell growth rate was caused by the lower photosynthetic activity of the cells. In semi-continuous cultivation, the cells used as inoculum were N-stressed with the "sub-health" status. After resupplying the N nutrient, the stressed cells started to recover towards the "health" status. The photosynthetic activity of the cells was recovered as one of the important physiological features. As shown in Fig. 4b-1, c-1, the ΔF/F m ' recovered to the peak of 0.55 and 0.52 during each cycle in experiment b and c after N replenishment and recovered to 0.58 in experiment a. Obviously, resupplying the N amount has a large effect on the level of photosynthetic activity recovery. In experiments b and c, the cells did not recover to the health status as in experiment a, leading to the lower cell growth and lower TAG productivity.

Another critical operating factor was the renewal rate of the semi-continuous cultivation mode [34]. In this work, three experiments with different renewal rates of 0.8, 0.6 and 0.3 were conducted. Though different from changing the initial N concentration, adjusting the renewal rate obviously improved the stability of TAG productivity. Both the TAG productivity in experiments d and e of each cycle remained at approximately 50 mg/L day, as shown in Fig. 4. Additionally, in Fig. 4, other parameters such as the DW and TAG content showed high reproducibility between cycles. Furthermore, the fatty acid composition of the cells at the beginning and end point of each cycle was analysed. As shown in Fig. 5, after N replenishment at day 0 and day 4 of cultivation, all of the fatty acid compositions were the same at each beginning and end point of the two cycles. The constant fatty acid composition at the end point of each cycle indicated a high consistency of TAG quality. In contrast, the reported semi-continuous cultivation of microalgae under N-stress, which was controlled by other parameters such as biomass concentration or pH et al., showed that the lipid productivity and composition were usually unstable and fluctuated [32,35,36].

Compared to the healthy cells with sufficient nutrition, there was an obvious reduction in the photosynthesis activity of the proper N-stressed cells as shown in Fig. 1a, which hindered the cell growth. However, the rate of TAG accumulation of the stressed cells received a tremendous boost instead and remedied the sacrifice of cell growth. Hence, via the semi-continuous cultivation with maintenance of the degree of N-stress by controlling the N supply strategy, stable, sustainable TAG production was achieved.

Taken together, the data indicate that ΔF/F m ′ is a rapid and in situ N-stress indicator and could precisely guide the control of the degree of N-stress during the cultivation of microalgae for efficient and stable TAG production.

Conclusion

It is vital to quantify the degree of N-stress when it is applied to obtain maximum TAG productivity during microalgal cultivation. The concept of the nitrogen stress index, NSI, was defined. The photosynthetic activity parameter of microalgae, ΔF/F m ', was identified as a perfect online indicator for the degree of N-stress of the cells. Based on NSI, a novel semi-continuous cultivation strategy of precise N-stress control by the accurate monitoring of ΔF/F m ' as an N-stress indicator was established for stable and efficient TAG production. The renewal rate and initial N concentration were optimized as 0.6 and 24 mg/mL, respectively. This new cultivation strategy provides significant guidance for outdoor microaglal cultivation in industrial applications and controlled indoor cultivation.

Fig. 1
1N-limited cultivation of N. oceanica IMET1 0.42. As shown in

Fig. 2 N
2-limited cultivation of different N-stressed cells was set to 24 mg/L. The initial cell density and total amount of N, including the intracellular N and free N in the medium, were regulated to the same level as that in the batch cultivation. It should be noted that cycle 1 was started at day 0 and ended at day 4; at the same time, day 4 was also the beginning point of cycle 2, which ended at day 8. The intracellular N at day 0, day 4 and day 8 coincided with each other as shown inFig. 3a-1. The NSIs were maintained as almost equal, with values of 0.72, 0.71 and 0.70, respectively. The DW and TAG content also showed the same trend between cycles. The results indicated the successful repeat of the proper N-stressed cells defined as day4-cell with an NSI of 0.72 in "Determination of the proper degree of N-stress of microalgal cells for TAG production" section.

Fig. 3
3Semi-continuous cultivation of day4-cell

Table 1
1Operating conditions of five semi-continuous experiments a to e represents the five experimentsFig.Experiment
a
b
c
d
e

Renewal rate
0.8
0.8
0.8
0.6
0.3

Initial N concentra-
tion (mg/L)

24
12
5
24
24

AcknowledgementsAll authors acknowledge the support by the National Nature Science Foundation of China and the Ministry of Science and Technology of China. The authors thank Prof. Qiang Hu from the Institute of Hydrobiology, Chinese Academy of Sciences and for valuable comments on this study.Authors′ contributionsSX, JL, CY and XC designed the research. SX, JL and CY wrote the paper. SX, JL and XC analyzed the data. JL, YM and PW performed the research and provided technical support. All authors read and approved the final manuscript.Author detailsCompeting interestsThe authors declare that they have no competing interests.Fig. 5 Fatty acid composition of the day4-cell cultured in semi-continuous modeAvailability of data and materialsThe data sets supporting the results of this article are included within the article.Consent for publicationNot applicable.Ethics approval and consent to participateNot applicable.FundingThis work was supported by the National High Technology Research and Development Program "863" (2012AA052101).Publisher′s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Impact of rapid antigen detection testing on antibiotic prescription in acute pharyngitis in adults. FARINGOCAT STUDY: a multicentric randomized controlled trial

Jordi Madurell
Montse Balagué
Mónica Gómez
Josep M Cots
Carl Llor
Impact of rapid antigen detection testing on antibiotic prescription in acute pharyngitis in adults. FARINGOCAT STUDY: a multicentric randomized controlled trial
S T U D Y P R O T O C O L Open Access
Background: Acute pharyngitis is one of the most frequent consultations to the general practitioner and in most of the cases an antibiotic is prescribed in primary care in Spain. Bacterial etiology, mainly by group A betahemolytic streptococcus (GABHS), accounts for 10-20% of all these infections in adults. The purpose of this study is to assess the impact of rapid antigen detection testing (RADT) to identify GABHS in acute pharyngitis on the utilization of antibiotics in primary care. Methods/design: Multicentric randomized controlled trial in which antibiotic prescription between two groups of patients with acute pharyngitis will be compared. The trial will include two arms, a control and an intervention group in which RADT will be performed. The primary outcome measure will be the proportion of inappropriate antibiotic prescription in each group. Two hundred seventy-six patients are required to detect a reduction in antibiotic prescription from 85% in the control group to 75% in the intervention group with a power of 90% and a level of significance of 5%. Secondary outcome measures will be specific antibiotic treatment, antibiotic resistance rates, secondary effects, days without working, medical visits during the first month and patient satisfaction.Discussion: The implementation of RADT would allow a more rational use of antibiotics and would prevent adverse effects of antibiotics, emergence of antibiotic resistance and the growth of inefficient health expenses.

Background

Acute pharyngitis is frequently seen in primary care for which uncertain etiology may result in inappropriate management. Acute pharyngitis includes rhinitis, the most common cause of visit in primary care in Spain [1]. Most pharyngotonsillitis are viral in origin while group A β-hemolytic Streptococcus (GABHS) is the most common bacterial cause of acute pharyngitis, accounting for approximately 15% to 30% of the cases in children and 5% to 15% of cases in adults [2]. GABHS is the only commonly occurring form of acute pharyngitis for which antibiotic therapy is indicated [3]. Nonetheless, a total of 78-98% of adults with acute pharingytis is treated with antibiotics, especially if sore throat is present [4,5]. Pharyngeal infection is also a frequent cause of absenteeism, with an estimated average of 6.5 days of sick leave per episode [2,6]. In streptococcal pharyngitis, antibiotic treatment shortens the transmission and dissemination of GABHS in the community, reduces the symptomatology compared to the non treated group by a mean of 16 hours and prevents suppurative complications. According to the Cochrane review, for each 100 patients treated with antibiotics compared to the placebo assigned group, one case less of rheumatic fever, two cases less of acute otitis media and 3 cases less of quinsy are produced [7,8].

Overuse of antibiotics can lead to side effects and emergence of antibiotic resistance [9]. The main reason for antibiotic overprescription in acute pharyngitis is the difficulty to obtain a rapid and correct etiological diagnosis. In addition, physicians often believe that patients expect an antibiotic and, in case of doubt, general practitioners are more prone to prescribe antibiotics. In Spain, rapid antigen detection tests (RADT) are seldom used in general practice and physicians still rely on the Centor criteria -fever, tonsillar exudates, tender vertical nodes, and/or absence of cough-for prescribing antibiotic therapy [10]. Sore throat culture is the gold standard for the diagnosis of streptococcal pharyngitis. However, the delay in obtaining results makes its performance useless in clinical practice.

Over the last years, low-cost and rapid immunological techniques targeting the detection of GABHS antigens have been developed for use in the clinical setting. Their high specificity (>95) make the probability of obtaining false positive results very low [11][12][13]. A recent paper published in Greece shows that the use of these tests can reduce the prescription of antibiotics in pharyngotonsillitis in children [14]. However, very few studies have been carried out to know the impact of RADT on the pattern of how general practitioners prescribe antibiotics for patients with pharyngotonsillitis in adults.

Methods/Design

Objectives The main objective of this investigation is to assess the impact of the RADT in adults with acute pharyngitis on the percentage of antibiotics prescribed by general practitioners

The secondary objectives are:

a. To describe the antibiotics prescribed. b. To describe GABHS resistance to the antibiotics prescribed. c. To assess clinical recurrence within the first month.

Design

Multicenter randomized clinical trial designed to compare antibiotic prescription in patients with acute pharyngitis in two groups of physicians, one with the use of RADT and the other without these tests.

Setting

The trial will be conducted in Primary Health Care Centers in Catalonia (Spain) in which the clinical history records are accessed electronically.

Participants

Study population

Eligible patients will be consecutively selected by general practitioners. All patients who agree to participate will be asked to provide written informed consent and an informative sheet of the study.

Inclusion criteria

1. Men and women aged 14 to 60 years. 2. More than one symptom of acute pharyngitis, such as fever, sore throat, tonsillar exudates, tender cervical nodes and/or absence of cough.

Exclusion criteria

1. Patients who do not consent to participate. 2. Patients younger than 14 or older than 60 years of age.

3. More than five episodes of pharyngitis over the last year. 4. Immunosuppressed condition, such as active neoplasm, acquired immunodeficiency syndrome or reception of chemotherapy, radiotherapy, corticosteroids and/ or immunosuppressive therapy. 5. Heart valve disease. 6. Rheumatic fever. 7. Episode of pharyngitis treated with antibiotics in the previous fifteen days. 8. Pharyngitis of gonococcal etiology. 9. Tonsillectomy.

Intervention

Participating primary health care centers will be randomized to the intervention or to the control arm of the study. Physicians allocated to the intervention group will use the RADT and those assigned to the control group will make the diagnosis of streptococcal pharyngitis with only clinical criteria. Treatment will be decided upon by the general practitioners in both groups, including antithermic drugs, non-steroidal anti-inflammatory drugs, corticosteroids, antibiotic therapy or none. Samples will be taken by the family physicians who will be previously trained to perform the technique correctly with vigorous rotation of the tonsils and the posterior pharynx without touching the tongue, teeth or gums. RADT will be undertaken with all the samples with the OSOM StrepA (Genzyme) following the manufacturer's instructions. The other sample will be sent to the Department of Microbiology of the two hospitals of the area with AMIES (Copan Innovation, Italy) as medium. Samples will be seeded on a plate of blood agar and will be incubated at 37°C in an atmosphere of CO 2 at 5% during 48 hours. A culture will be considered positive for GABHS with a growth of any number of β-hemolytic colonies, Gram staining with streptococcal morphology and a catalase negative test with posterior identification with an automated panel for WIDER Gram positive cocci (Soria Melguizo). Results will be confirmed with posterior serogrouping with the Streptococcal Grouping Kit (Oxford, UK). The culture will be considered negative after 48 hours of incubation with the absence of β-hemolytic colonies.

Outcome measures

Primary outcome: proportion of inappropriate antibiotic prescription, use of antibiotic treatment, use of RADT, RADT result, and culture result.

Secondary outcomes

-Both intervention and control groups:

a. Age, sex, clinical symptoms (fever, sore throat, tender cervical nodes, absence of cough), specific antibiotic treatment, culture result, antibiotic resistance, side effects, days without working, change of antibiotic, and medical visits within the first month. b. Patient satisfaction. Patients will be required to score the degree of satisfaction, from 0 (no satisfaction at all) to 10 (very satisfied), for the following items: ease of obtaining a visit the day the patient wants, duration of the visit, doctor clinical ability, doctor kindness, doctor predisposition to listen, the way the problem is solved, the feeling of being in "good hands", and overall satisfaction with the doctor. Patients will be asked if they have been attended by their usual doctor or not and if they would recommend the attending doctor to a friend or relative. The open questions include: What is the most satisfying aspect of the visit? What is the worst aspect of the visit? Would you like to make any comment on anything else?

Ascertainment of visits Baseline data collection

The baseline questionnaire at the clinical practice will include age, sex, clinical symptoms, specific antibiotic treatment, use of RADT and RADT result.

Follow-up data collection

• 48-72 h data collection by means of a phone call that will include side effects, days without working, and change of antibiotic or not.

• Three-week data collection: The questionnaire will include the number of visits, if there has been any complication, culture result and antibiotic resistance to GABHS if the culture is positive.

• The satisfaction interview will be given one month after the baseline visit.

Sample size

Two hundred seventy-six patients are required in each arm to detect a reduction of 10% in antibiotic prescription from 85% in the control group to 75% in the intervention group, assuming a rate of 85% of prescription in the control group, allowing 10% for losses in the followup visits, with a power of 90% and a level of significance of 5% (2-sided) and performance values of the RADT given by the manufacturer.

Randomization

The unit of randomization will be the primary health care center, which will be allocated to the control or intervention group by a random sequence generated by a computer program. This unit of randomization will be taken into account in order to minimize contamination between these two groups. It may not be appropriate to randomize individuals since patients who attend the same primary health care center may be treated in the same way. Patients who meet the inclusion criteria will be consecutively selected by the participating physicians and allocated to the treatment group corresponding to the center.

Blinding

It is not possible to blind participants, patients or doctors. Data will be analyzed blinded to treatment group allocation.

Withdrawals

Patients will be free to withdraw from the study at any time. For obtaining the patient satisfaction data the patients will be phoned at least five times in three different timetables before being considered as withdrawal.

Analysis

Data will be analyzed in accordance with CONSORT guidelines. The primary end point for the trial will be at baseline and at three weeks (prescription or not of antibiotic, RADT use and result, and culture result). Descriptive statistics of the outcome measures will be performed as well as baseline characteristics and clinic measures, calculating means and standard deviations or percentages, with 95% confidence intervals at each assessment for all the patients and for each group. Chisquare tests will be carried out to assess the impact of RADT on the percentage of antibiotic prescription in the intervention and control arms. For assessing the cost-effectiveness study of RADT direct costs such as antibiotics prescribed, RADT costs, and number of follow-up visit and indirect costs such as sick leave days will be taken into account. The different antibiotics prescribed, the GABHS resistance rates, the clinical recurrences and the clinical criteria will also be determined to assess the secondary objectives. The level of significance of all models will be set at 5%. Statistical analysis will be carried out with SPSS v. 15 (SPSS Inc., Chicago, IL) and the SAS v. 9.1.3 for Windows (SAS Institute Inc., Cary, NC, USA).

Risks

We do not believe that the participants will be exposed to excessive risk by participation in this study since the visit schedule dictated by the protocol is similar to that undertaken in daily clinical practice. Neither do we foresee any risk to the patients in obtaining the pharyngeal sample.

Study limitations

Acute pharyngitis is a very common process and we will be able to obtain the sample size required in this protocol without difficulty. In addition, the protocol is adapted to the way in which primary care physicians work and therefore implies neither ethical nor legal problems for its undertaking in our setting. The only difference with the usual practice will be the performance of RADT and the collection of the sample for culture. However, both procedures are relatively rapid and simple and are widely used in other countries. We hope there will not be any logistic problems with the submission of samples from the health care centers to the laboratory for culture since we will be personally responsible for this task. There are no major differences in the GABHS detection technique in the different laboratories of our setting and thus, no bias in this sense is expected. Centers and practitioners willing to participate may be more motivated professionals and will more likely to follow current guidelines and follow better clinical practice. This aspect could reduce the impact of the results obtained in relation to the optimization of treatment of acute pharyngitis with RADT. We do not expect many dropouts since the protocol is very similar to usual practice.

Ethical and organizational review

The study will be conducted according to guidelines of the Helsinki Declaration and the Good Clinical Research Practice. The Project has been approved by the Ethical and Clinical Research Committee of the Jordi Gol Institute of Research in Primary Health Care. The certificate number is P06/03. Informed consent: The information will be provided orally as well as written. Study subjects will have sufficient opportunity to ask questions regarding study details. Informed consent follows the guidelines of Helsinki Declaration and the rules of Title I, Article 12 of the Spanish Royal Decree 561 of the 16th April 1993

Data confidentiality: Individual data will be coded to ensure anonymity. Only researchers and monitors will have access to the data.

Discussion

This article presents a detailed description of a randomized clinical trial designed to explore the impact of RADT on antibiotic prescribing in acute pharyngitis in adults. The trial proposed is sufficiently powered and, to the best of our knowledge, the first to allow conclusions on the effect of RADTs in adults with pharyngitis. Pharyngotonsillitis is a very prevalent disease and is one of the most frequent causes of medical visits in primary care. Many patients in our country visit the physician in search of antibiotic prescription since they believe that this is the treatment of choice, particularly among patients with multiple disease recurrence. In other cases, patients seek home remedies or go directly to the pharmacy to obtain medications to alleviate the sore throat that accompanies pharyngitis. Primary care physicians are aware that when the patients visit they wish to receive effective treatment with medications which rapidly alleviate the accompanying symptomatology and, in many cases, what they want is treatment with an antibiotic. This is probably the main reason why physicians in our country mainly prescribe these medications together with the fact that it is clinically difficult to distinguish streptococcal from viral etiology. We believe that this clinical trial will be of great use to know whether the RADT are truly effective in reducing the percentage of antibiotic prescription in our setting.

Abbreviations (RADT): Rapid antigen detection test; (GABHS): Group A betahemolyticstreptococcus.
Madurell et al. BMC Family Practice 2010, 11:25 http://www.biomedcentral.com/1471-2296/11/25 Page 2 of 5
AcknowledgementsThe study is funded by the Fondo de Investigaciones Sanitarias, the University and Innovation Department of Spain (Reference number: PI061782), and from the Catalan Society of Family Medicine.Authors' contributions JC and CL had the original idea of the study, obtained funding from the University and Innovation Department and from the Catalan Society of Family Medicine and helped with the drafting of the protocol. MB, JM and MG obtained funding from the University and Innovation Department and from the Catalan Society of Family Medicine and helped with the drafting the protocol. JM is main responsible as well as coordinator study group, CL helped with the drafting of the protocol. All authors have read and approved the final manuscript.Competing interestsThe Rapid Test Device OSOM StrepA of Genzyme will be kindly provided by Leti.
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Pre-publication history The pre-publication history for this paper can be accessed here. 10.1186/1471-2296-11-25Pre-publication history The pre-publication history for this paper can be accessed here: http://www. biomedcentral.com/1471-2296/11/25/prepub doi:10.1186/1471-2296-11-25

Impact of rapid antigen detection testing on antibiotic prescription in acute pharyngitis in adults. FARINGOCAT STUDY: a multicentric randomized controlled trial. Madurell, BMC Family Practice. 1125Cite this article asCite this article as: Madurell et al.: Impact of rapid antigen detection testing on antibiotic prescription in acute pharyngitis in adults. FARINGOCAT STUDY: a multicentric randomized controlled trial. BMC Family Practice 2010 11:25.