Keywords: Pneumocystis jirovecii, Pneumocystis pneumonia, immunocompromised patients, molecular diagnosis, PCR

Purpose of review: Pneumocystis pneumonia (PCP) is a frequent opportunistic infection associated with a high mortality rate. PCP is of increasing importance in non-HIV immunocompromised (IC) patients, who present with severe respiratory distress with low fungal loads. Molecular detection of Pneumocystis in broncho-alveolar lavage (BAL) has become an important diagnostic tool, but quantitative PCR (qPCR) needs standardization.

Recent findings: Despite a high negative predictive value, the positive predictive value of qPCR is moderate, as it also detects colonized patients. Attempts are made to set a cut-off value of qPCR to discriminate between PCP and colonization, or to use non-invasive samples or combined strategies to increase specificity.

Summary: It is easy to set a qPCR cut-off for HIV-infected patients. In non-HIV IC patients, a gain in specificity could be obtained by combining strategies, i.e. qPCR on BAL and a noninvasive sample, or qPCR and serum beta-1,3-D-glucane dosage.

Molecular diagnosis of Pneumocystis pneumonia in immunocompromised patients

 [START_REF] Thomas | Pneumocystis Pneumonia[END_REF][START_REF] Morris | Colonization by Pneumocystis jirovecii and its role in disease[END_REF]and in HIV-infected patients who do not comply with antiretroviral therapy or anti-Pneumocystis prophylaxis [START_REF] Magne | Pneumocystosis: a network survey in the Paris area 2003-2008[END_REF]. Over the last decade, PCP has been also recognized as a major opportunistic infection in non-HIV immunocompromised patients, such as transplant patients, patients with hematological malignancies or solid cancers, and patients receiving corticosteroid therapy or other immunosuppressive drugs [START_REF] Fillatre | Incidence of Pneumocystis jiroveci Pneumonia among Groups at Risk in HIV-negative Patients[END_REF]. The growing number of immunosuppressive procedures led to an increasing frequency of PCP diagnoses in non-HIV compared to HIV-infected patients [START_REF] Magne | Pneumocystosis: a network survey in the Paris area 2003-2008[END_REF][START_REF] Roux | Pneumocystis jirovecii pneumonia in patients with or without AIDS, France[END_REF][START_REF] Robert-Gangneux | Diagnosis of Pneumocystis jirovecii Pneumonia in Immunocompromised Patients by Real-Time PCR: a 4-Year Prospective Study[END_REF]. The heterogeneity of immune background in this non-HIV population can influence the clinical and radiological signs, which can be misleading, since they are often less typical than in HIV patients [START_REF] Morris | Colonization by Pneumocystis jirovecii and its role in disease[END_REF][START_REF] Azoulay | Polymerase chain reaction for diagnosing pneumocystis pneumonia in non-HIV immunocompromised patients with pulmonary infiltrates[END_REF].

Mortality in the non-HIV-infected immunosuppressed population is generally higher than that in HIV infection, reaching 50% in some studies [START_REF] Enomoto | Differences in the clinical characteristics of Pneumocystis jirovecii pneumonia in immunocompromized patients with and without HIV infection[END_REF][START_REF] Monnet | Critical care management and outcome of severe Pneumocystis pneumonia in patients with and without HIV infection[END_REF]. Whether this poor outcome is due to the patient immune background itself, or to gaps in diagnosis or management of patients at risk, is unclear. However, it is now widely recognized that evolution of the disease is more acute than in HIV-infected patients, with rapid clinical deterioration even with low fungal loads [START_REF] Morris | Colonization by Pneumocystis jirovecii and its role in disease[END_REF].

Therefore, rapid and reliable diagnosis is of critical importance.

As Pj cannot be cultured, diagnosis relies on its detection in broncho-alveolar lavage (BAL) or other lung specimens, by microscopic examination and PCR. While microscopy is helpful to diagnose PCP in HIV-infected patients who usually harbor high fungal loads, molecular diagnosis is critical to detect low Pj counts in non-HIV patients [START_REF] Robert-Gangneux | Diagnosis of Pneumocystis jirovecii Pneumonia in Immunocompromised Patients by Real-Time PCR: a 4-Year Prospective Study[END_REF], and PCR has been included in the guidelines for the diagnosis of PCP in patients with hematological malignancies [START_REF] Alanio | ECIL guidelines for the diagnosis of Pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients[END_REF]. Pj PCR is assumed to have a 100% negative predictive value (NPV). Meanwhile, these sensitive methods have highlighted the possible colonization of hospitalized patients with Pj, thus limiting the positive predictive value (PPV) of PCR result. Therefore, continuous efforts are ongoing to improve the interpretation of molecular results or to develop new strategies of amplification that would be more specific of acute infection.

A c c e p t e d m a n u s c r i p t

In this article, we provide a review of the literature on the molecular diagnosis of PCP, with special emphasis on the interpretation of results and possible complementary diagnostic tools.

MOLECULAR DIAGNOSIS OF PCP: TECHNICAL ASPECTS AND DIAGNOSTIC IMPACT

Gene targets and PCR methods: what's new?

Many gene targets have been described for the molecular diagnosis of PCP and have been assessed in clinical series (Table 1) . The mitochondrial large sub-unit of ribosomal RNA (mtLSU) has been the first target for PCP diagnosis [START_REF] Wakefield | Detection of Pneumocystis carinii with DNA amplification[END_REF] and it is still the most widely used and reported in the literature. Among the multiple gene targets described, mtLSU, mtSSU and the major surface glycoprotein (MSG) are the only gene targets to be repeated in Pneumocystis genome, and thus are associated to a very high sensitivity of detection, compared to singlecopy targets. Actually, single copy loci such as 5.8SrRNA, DHFR, DHPS, TS and beta-tubulin have been also investigated for PCP diagnosis, but they are more often used for Pj genotyping in epidemiological studies [START_REF] Nevez | Pneumocystis jirovecii and Cystic Fibrosis in Brittany, France[END_REF]. The use of conventional amplification techniques has been supplanted by real-time PCR, which offers improved specificity and allows to quantify fungal loads in fluid samples. However, no clear correlation has been made to date between parasite concentration and severity of infection, probably, at least partially, because of a variable number of copies of the targeted genes (32)**.

A reverse-transcriptase PCR assay targeting the mRNA from a gene encoding a Pneumocystis HSP70 was also investigated with the aim to detect only viable organisms [START_REF] Maher | Development and Characterization of a Molecular Viability Assay for Pneumocystis carinii f sp hominis[END_REF][START_REF] De Oliveira | Performance of a molecular viability assay for the diagnosis of Pneumocystis pneumonia in HIV-infected patients[END_REF], to monitor treatment. It was evaluated on a HIV-infected cohort, but despite good performances on BAL compared to microscopy (100% concordance), the detection rate strongly decreased in induced sputum (IS) and oral wash (OW) samples (65% and 35%, respectively), thus hampering the use of less invasive specimens to monitor treatment efficacy after initial diagnosis.

Over the last decade, several commercial quantitative PCR (qPCR) assays have been developed, all targeting mtLSU or MSG gene (Table 2) [START_REF] Hauser | Prospective Clinical Evaluation of Respiratory Samples from Subjects at Risk for Pneumocystis jirovecii Infection by Use of a Commercial Real-Time PCR Assay[END_REF][START_REF] Orsi | Performance of 2 commercial real-time polymerase chain reaction assays for the detection of Aspergillus and Pneumocystis DNA in bronchoalveolar lavage fluid samples from critical care patients[END_REF][START_REF] Orsi | Detection of Pneumocystis jirovecii and Aspergillus spp. DNA in bronchoalveolar lavage fluids by commercial real-time PCR assays: comparison with conventional diagnostic tests[END_REF][START_REF] Sasso | Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia[END_REF][START_REF] Mctaggart | Validation of the MycAssay Pneumocystis Kit for Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Specimens by Comparison to a Laboratory Standard of Direct Immunofluorescence Microscopy, Real-Time PCR, or Conventional PCR[END_REF]. MycAssay Pneumocystis [START_REF] Hauser | Prospective Clinical Evaluation of Respiratory Samples from Subjects at Risk for Pneumocystis jirovecii Infection by Use of a Commercial Real-Time PCR Assay[END_REF][START_REF] Orsi | Performance of 2 commercial real-time polymerase chain reaction assays for the detection of Aspergillus and Pneumocystis DNA in bronchoalveolar lavage fluid samples from critical care patients[END_REF][START_REF] Orsi | Detection of Pneumocystis jirovecii and Aspergillus spp. DNA in bronchoalveolar lavage fluids by commercial real-time PCR assays: comparison with conventional diagnostic tests[END_REF][START_REF] Sasso | Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia[END_REF][START_REF] Mctaggart | Validation of the MycAssay Pneumocystis Kit for Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Specimens by Comparison to a Laboratory Standard of Direct Immunofluorescence Microscopy, Real-Time PCR, or Conventional PCR[END_REF], BioEvolution PCR [START_REF] Sasso | Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia[END_REF][START_REF] Montesinos | Comparison of 2 real-time PCR assays for diagnosis of Pneumocystis jirovecii pneumonia in human immunodeficiency virus (HIV) and non-HIV immunocompromised patients[END_REF] and Amplisens [START_REF] Sasso | Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia[END_REF][START_REF] Huh | Comparative Evaluation Between the RealStar Pneumocystis jirovecii PCR Kit and the AmpliSens Pneumocystis jirovecii (carinii)-FRT PCR Kit for Detecting P. jirovecii in Non-HIV Immunocompromised Patients[END_REF] have been the most evaluated. Sasso et A c c e p t e d m a n u s c r i p t al. compared these 3 assays with an in-house RT PCR targeting MSG and found excellent performances of all assays excepted BioEvolution kit, to detect very low fungal burdens [START_REF] Sasso | Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia[END_REF].

Over the past 2 years, a growing number of assays have been commercialized and evaluated in clinical series, yielding heterogenous performances with sensitivity and specificity ranging from 60 to 100% and from 82 to 100%, respectively [START_REF] Huh | Comparative Evaluation Between the RealStar Pneumocystis jirovecii PCR Kit and the AmpliSens Pneumocystis jirovecii (carinii)-FRT PCR Kit for Detecting P. jirovecii in Non-HIV Immunocompromised Patients[END_REF][START_REF] Guillaud-Saumur | Comparison of a commercial real-time PCR assay, RealCycler® PJIR kit, progenie molecular, to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections[END_REF][START_REF] Issa | Pneumocystosis and quantitative PCR[END_REF][START_REF] Hoarau | Evaluation of quantitative FTD-Pneumocystis jirovecii kit for Pneumocystis infection diagnosis[END_REF][START_REF] Montesinos | Evaluation of a new commercial real-time PCR assay for diagnosis of Pneumocystis jirovecii pneumonia and identification of dihydropteroate synthase (DHPS) mutations[END_REF].

To date, only 2 real-time assays have been adapted on fully automated platforms combining DNA extraction and multiplex real-time PCR, allowing on-demand molecular testing (47-49*). The BD MAX system was prospectively evaluated and yielded similar sensitivity as a reference qPCR assay [START_REF] Dalpke | Development and Evaluation of a Real-Time PCR Assay for Detection of Pneumocystis jirovecii on the Fully Automated BD MAX Platform[END_REF]. Moreover, samples from patients with colonization had significantly higher median amplification cycle threshold (Ct) values than samples from patients with PCP (32.0 vs 25.7; p = 0.002). The Genecube platform was evaluated on BAL and IS samples and yielded similar sensitivity compared with "in-house" qPCR assay (92.3% versus 94.9%) (49)*.

As an alternative method to PCR, the loop-mediated isothermal amplification (LAMP) technique has been described to amplify Pneumocystis DNA [START_REF] Uemura | Development of a loopmediated isothermal amplification method for diagnosing Pneumocystis pneumonia[END_REF]. Nakashima et al.

retrospectively assessed LAMP performances in BAL and IS from 78 non-HIV immunocompromised patients and demonstrated LAMP to have sensitivity and positive predictive value of 95.4% and 91.3%, respectively, higher than that of nested PCR (72.7%, 84.2%, respectively) [START_REF] Nakashima | Loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia in HIV-uninfected immunocompromised patients with pulmonary infiltrates[END_REF]. However, despite these interesting performances, clinical evaluations on larger cohorts are still needed and the absence of fungal load quantification does not help interpretation of Pj presence as colonizing or pathogenic agent.

Sample types: which are best suited?

BAL is considered as the "gold standard" procedure to diagnose PCP, since i) it provides the most appropriate sample to perform microscopic staining to detect trophic forms and cysts of Pj, and ii) it allows to retrieve Pj from the lower respiratory tract (LRT), thus most likely responsible for infection. Therefore, a fair amount of literature evaluated molecular diagnosis in BAL samples.

However, if the patient cannot stand BAL which is an invasive procedure associated to potential complications, tracheal aspirates, induced sputum (IS), or oral washes (OW) can be A c c e p t e d m a n u s c r i p t used, as well as nasopharyngal aspirates (NPA) for neonates [START_REF] Juliano | Use of Oropharyngeal Washes to Diagnose and Genotype Pneumocystis jirovecii[END_REF][START_REF] Fischer | The use of oral washes to diagnose Pneumocystis carinii pneumonia: a blinded prospective study using a polymerase chain reactionbased detection system[END_REF][START_REF] Van Halsema | Short Communication: Diagnosis of Pneumocystis jirovecii Pneumonia by Detection of DNA in Blood and Oropharyngeal Wash, Compared with Sputum[END_REF][START_REF] Nyamande | Low Sensitivity of a Nested Polymerase Chain Reaction in Oropharyngeal Washings for the Diagnosis of Pneumocystis Pneumonia in HIV-Infected Patients[END_REF][START_REF] Samuel | Improved detection of Pneumocystis jirovecii in upper and lower respiratory tract specimens from children with suspected pneumocystis pneumonia using real-time PCR: a prospective study[END_REF][START_REF] To | Use of Nasopharyngeal Aspirate for Diagnosis of Pneumocystis Pneumonia[END_REF]. However, these samples from the upper respiratory tract are not suitable for microscopic examination, thus molecular diagnosis is essential, but must be carefully evaluated in clinical practice. Few studies have addressed prospectively a comparative evaluation of molecular diagnosis on paired samples of BAL and other sample types (Table 3). Alanio et al. found no significant difference in fungal DNA loads between IS and BAL fluid samples from immunocompromised patients (mixed HIV status)(57). Teh et al. in cancer patients obtained higher levels of Pj DNA detection in IS than in BAL, probably because it is less diluted, but found no link with the severity of infection [START_REF] Teh | Molecular diagnosis of Pneumocystis jirovecii in patients with malignancy: Clinical significance of quantitative polymerase chain reaction[END_REF].

When comparing PCR on OW and BAL paired samples, the sensitivity was shown to be significantly reduced in the former, but the PCR specificity increased (Table 3). However, it is awkward that Juliano et al. found 11 false-negative results of PCR on OW out 61 paired BAL with positive microscopy, suggesting that this sample might be inadequate for the diagnosis of PCP in HIV-negative patients, who present frequently with PCP with negative microscopy [START_REF] Juliano | Use of Oropharyngeal Washes to Diagnose and Genotype Pneumocystis jirovecii[END_REF].

On the other hand, Samuel et al. [START_REF] Samuel | Improved detection of Pneumocystis jirovecii in upper and lower respiratory tract specimens from children with suspected pneumocystis pneumonia using real-time PCR: a prospective study[END_REF] reported NPA to be of interest for PCP confirmation in young children. On a series of 147 NPA, they reported roughly similar DNA detection rates in NPA, compared to paired IS or BAL (49.6% versus 55.1%).

Apart from respiratory specimens, the detection of circulating Pj DNA in peripheral blood has been considered in a few reports, based on the Pj transient passage from lung alveoli towards the blood compartment, that would occur during PCP. Whether Pj PCR in blood sample is a valuable tool to diagnose PCP remains controversial. Wang et al. [START_REF] Wang | Diagnosis of Pneumocystis jirovecii pneumonia with serum cell-free DNA in non-HIV-infected immunocompromised patients[END_REF]* recently demonstrated that despite a moderate sensitivity compared to BAL or sputum (68.6% versus 91.4-97.1%), PCR on serum had a higher PPV (96% versus 86.5-87.2%). Therefore, when BAL is not available, they emphasized the relevance of combining PCR on sputum and serum to reach similar performances.

PERFORMANCE OF MOLECULAR DIAGNOSIS

How to evaluate clinical performance?

A c c e p t e d m a n u s c r i p t

The evaluation of the clinical performance of molecular diagnosis of PCP is a difficult task and should be made in each individual institution. Indeed, data from the literature are barely comparable, because i) lack of assays standardization (gene target, PCR method, type of sample and processing before DNA extraction), and ii) lack of standardized score to classify patients for the diagnosis of PCP (definite PCP, probable PCP or colonization), both influence the evaluation of PCR performances. Some authors use radiological and clinical findings associated to a positive microscopic and/or molecular detection of Pj to classify the diagnosis as PCP, while others also consider the clinical improvement after cotrimoxazole therapy. The interpretation of a positive PCR when direct examination is negative is particularly challenging when there is another possible cause for respiratory symptoms, and authors use variable criteria to define PCP in this setting. Additionally, the proportion of patients with probable PCP or colonization may vary according to the population study, i.e. the patients' immune background. Whether Pj detection represents an aggravating factor leading to shortened survival, or only the reflection of colonization, may be difficult to appreciate, and can lead to underestimate the PPV of a molecular assay. Therefore it is important to keep in mind these limitations, as they might greatly impact the estimation of PCR specificity in clinical studies. In any case, the use of microscopic detection as gold standard, should now be definitely avoided.

Sensitivity of PCR on BAL samples is usually considered to be as high as 100%, although unpredictable mutation at the site of probe hybridization can be an anecdotic source of falsenegative result [START_REF] Gal | A misleading falsenegative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: A French multicenter study[END_REF]. Consequently, the 100% NPV allows to use qPCR as an exclusion test, but positive qPCR results need careful interpretation when microscopy is negative. Therefore, attempts have been made to set up a threshold to distinguish between probable PCP and colonization, which is feasible on BAL samples only, provided that lavage volumes instilled are somewhat standardized. Given the absence of Pj international standard material, these cutoffs are assay-specific and are expressed in various units (Ct of amplification or number of copies). As stated above, due to the variability in the number of copies per genome and the unknown proportion of cysts and trophic forms in a given sample, the significance of quantification is still debatable.

Over the past decade, several studies have proposed to define cut-off values in relation to clinical diagnosis. Alanio et al [START_REF] Alanio | Real-time PCR assaybased strategy for differentiation between active Pneumocystis jirovecii pneumonia and A c c e p t e d m a n u s c r i p t colonization in immunocompromised patients[END_REF] estimated the sensitivity and specificity of qPCR, using different qPCR cut-off values to discriminate between PCP and colonization: a >1900 trophic A c c e p t e d m a n u s c r i p t form equivalent (TFEq) /ml value giving 100% PPV, and a <120 TFEq/mL value yielding a 100% NPV. A limited number of samples quantified within the grey zone were considered to be undetermined. Other authors proposed cut-off values expressed as cycle threshold (Ct) of amplification or number of gene copies per mL. As shown in Table 4, these thresholds are highly variable, depending on the gene target, the sample input for DNA extraction, the HIV status and the bronchial lavage volume according to local procedures of pneumologists [START_REF] Robert-Gangneux | Diagnosis of Pneumocystis jirovecii Pneumonia in Immunocompromised Patients by Real-Time PCR: a 4-Year Prospective Study[END_REF][START_REF] Montesinos | Comparison of 2 real-time PCR assays for diagnosis of Pneumocystis jirovecii pneumonia in human immunodeficiency virus (HIV) and non-HIV immunocompromised patients[END_REF][START_REF] Fauchier | Detection of Pneumocystis jirovecii by Quantitative PCR To Differentiate Colonization and Pneumonia in Immunocompromised HIV-Positive and HIV-Negative Patients[END_REF][START_REF] Louis | Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii[END_REF][START_REF] Maillet | Pneumocystis jirovecii (Pj) quantitative PCR to differentiate Pj pneumonia from Pj colonization in immunocompromised patients[END_REF][START_REF] Matsumura | Quantitative realtime PCR and the (1 → 3)-β-d-glucan assay for differentiation between Pneumocystis jirovecii pneumonia and colonization[END_REF][START_REF] Chumpitazi | Characteristics and clinical relevance of the quantitative touch-down major surface glycoprotein polymerase chain reaction in the diagnosis of Pneumocystis pneumonia[END_REF]. These findings underline the need to standardize analytical procedures to make comparison possible between hospital centers. Overall, the use of a threshold value is easier to handle for HIV-infected patients, but is awkward for non-HIV patients [START_REF] Robert-Gangneux | Diagnosis of Pneumocystis jirovecii Pneumonia in Immunocompromised Patients by Real-Time PCR: a 4-Year Prospective Study[END_REF][START_REF] Montesinos | Comparison of 2 real-time PCR assays for diagnosis of Pneumocystis jirovecii pneumonia in human immunodeficiency virus (HIV) and non-HIV immunocompromised patients[END_REF][START_REF] Louis | Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii[END_REF].

Recently, Valero et al. [START_REF] Valero | Copy Number Variation of Mitochondrial DNA Genes in Pneumocystis jirovecii According to the Fungal Load in BAL Specimens[END_REF]** quantified 4 different mitochondrial (mt) genes, and compared their copy numbers to two nuclear single-copy genes in 84 BAL samples from patients with various background. Interestingly, they observed a high variation in the copy numbers of mt genes and found no correlation with the immunofluorescence (IF) detection of Pj, nor the PCP diagnosis. For the six genes tested, there was an overlap in the Ct between IF-positive and IFnegative samples, ranging from 34 to 54%, and a variation of the number of copies according to the fungal load in 5 out of 6. Taken together, mt small sub-unit (mtSSU) rRNA displayed the highest copy number and seemed to be the most stable, thus could be a more sensitive target to detect Pj and probably more robust gene to quantify fungal loads.

In conclusion, as no qPCR assay currently allows to safely distinguish a colonized patient from an infected patient, the absence of Pj DNA detection can be used to rule out PCP, but the presence of Pj DNA in pulmonary samples must be interpreted according to the severity of clinical signs and the immune background of the patient.

Use of qPCR result as a prognosis marker

Although attributable mortality is difficult to evaluate, the co-infection or colonization with Pj could be an aggravating factor, possibly worsening prognosis or reducing survival of patients with cancer, and should be taken into account to improve survival [START_REF] Robert-Gangneux | Diagnosis of Pneumocystis jirovecii Pneumonia in Immunocompromised Patients by Real-Time PCR: a 4-Year Prospective Study[END_REF]. Additionally, several studies indicated that colonization with Pj could be the first step before the development of acute PCP in the following weeks or months, particularly in HIV-negative immunocompromised patients.

A c c e p t e d m a n u s c r i p t

On the other hand, Choi et al. reported that negative conversion of PCR result after specific therapy was associated with lower mortality rates (HR 0.433 [0.203-0.928], p<0.05) in HIVnegative patients with severe PCP [START_REF] Choi | Pneumocystis jirovecii pneumonia (PCP) PCR-negative conversion predicts prognosis of HIV-negative patients with PCP and acute respiratory failure[END_REF]. They suggested that qPCR could be used to monitor treatment efficacy and to guide a switch of treatment, if first-line treatment does not result in negative PCR. Overall, they observed that patient outcome was poorer when time-to PCR negativation was longer. However, we presently lack other studies to validate these findings and to recommend patient monitoring by qPCR at a larger scale.

FUTURE PERSPECTIVE: COMBINED DIAGNOSTIC STRATEGIES?

Beta 1,3-D glucan (BDG) is a polysaccharide of the walls of most fungi, including Pneumocystis cysts. It can be detected in serum specimens from patients with PCP, thus has triggered recent interest as a noninvasive diagnostic test for PCP. Several studies have reported the performance characteristics of commercial BDG assays, with a sensitivity ranging from 90 to 100% and a specificity of 78 to 96% (67), depending on the BDG kit and the patient population.

As BDG is not specific for Pj, it should be associated to Pj qPCR to improve its specificity. Morjaria et al. (68)** recently reported a retrospective study on 438 cancer patients with suspicion of PCP, and found that in patients with a positive Pj PCR, BDG had a sensitivity, specificity, and PPV of 88%, 85%, and 96%, respectively. Therefore, combining qPCR and BDG would allow to benefit from the 100% NPV of qPCR, and to increase the PPV when qPCR and BDG are both positive. If such strategy is confirmed in other patient groups, it would greatly improve care management.

Finally, new commercial real-time multiplex assays combine the detection of Pneumocystis together with dihydropteroate synthase (DHPS) point mutations responsible for resistance to sulfonamides drugs used for prophylactic and curative PCP therapy. PneumoGenius ® assay (PathoNostics) has been recently shown to outpace "in-house" DHPS sequencing in terms of DHPS successful amplification (89 versus 72 BAL), and gave 100% concordant results [START_REF] Montesinos | Evaluation of a new commercial real-time PCR assay for diagnosis of Pneumocystis jirovecii pneumonia and identification of dihydropteroate synthase (DHPS) mutations[END_REF]. However, its sensitivity for diagnosing PCP was only moderate (70%).

In conclusion, despite a significant body of literature on molecular diagnosis of PCP, there is still a need for innovative strategies to improve the PPV. Further studies would be welcome

Florence

  Guegan, Florence Robert-Gangneux Univ Rennes, CHU Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé Environnement et Travail) -UMR_S 1085, F-35000 Rennes, France Corresponding author : .robert-gangneux@univ-rennes1.fr This is a non-final version of an article published in final form in Current Opinion in Infectious Diseases: August 2019 -Volume 32 -Issue 4 -p 314-321 A c c e p t e d m a n u s c r i p t INTRODUCTION Pneumocystis jirovecii (Pj) is responsible for Pneumocystis pneumonia (PCP), a life-threatening fungal infection in immunocompromised patients, ranking first among opportunistic pathogens revealing the HIV-positive status

A

  c c e p t e d m a n u s c r i p t to precise the interest of combining qPCR on pulmonary samples with serum BDG, and their respective cut-off to allow a reliable discrimination between colonization and active disease. Meanwhile, the search for new specific markers on non-invasive samples would be highly welcome. Negative qPCR in BAL can rule out PCP  qPCR cut-off values are unreliable for patients with hematological malignancies  Negative qPCR result from non-invasive samples cannot rule out PCP whatever the patient background  Positive qPCR in BAL and BDG detection in serum is a promising strategy to target specific therapy Conflicts of interest: none Sources of funding: none A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t 68. **Morjaria S, Frame J, Franco-Garcia A, Geyer A, Kamboj M, Babady NE. Clinical Performance of (1,3) Beta-D-Glucan for the diagnosis of Pneumocystis Pneumonia in cancer patients tested with PCP PCR. Clin Infect Dis. 2018; **Well-conducted study with thoroughly analyzed data, using different cut-off values for serum beta 1,3-D glucan A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t

Table 1 .

 1 Gene targets used in molecular diagnosis of Pneumocystis pneumonia (excluding typing studies)

	a n u s c r i p t
	m
	A c c e p t e d

Table 2 .

 2 Performances of commercial quantitative PCR assays for Pneumocystis jirovecii diagnosis

Table 3 .

 3 Clinical evaluations of PCR performances on oral washes, nasopharyngeal aspirates and blood, compared with bronchoalveolar fluid or induced sputum samples

Table 4 .

 4 Clinical studies defining cut-off values for quantitative PCR interpretation and corresponding performances of the assays


Danilo Rosa Nunes

Manuel Reche

Emilie Ressouche

Matthieu Raynal

Benjamin Isare

Pascale Foury-Leylekian

Pierre-Antoine Albouy
email: pierre-antoine.albouy@u-psud.fr

Patrick Brocorens

Roberto Lazzaroni
email: roberto.lazzaroni@umons.ac.be

Laurent Bouteiller
email: laurent.bouteiller@sorbonne-universite.fr

Organogel formation rationalized by Hansen solubility parameters: shift of the gelation sphere with the gelator structure

Keywords: organogel, powder XRD, crystal habit, Hansen solubility parameters

In order to rationalize how the gelation ability of a low molecular weight gelator is influenced by its molecular structure, we performed extensive solubility tests of a group of thiazole based gelators and made use of Hansen solubility parameters formalism. We observe that the increase of a linear alkyl chain in these gelators promotes an increase of the radius of the gelation sphere as well as a gradual shift of its center to lower values of the polar (δP) and hydrogen bonding (δH) components. The molecular packing within the fibers and the crystal habit were determined by a combination of X-ray diffraction and molecular modeling. We attribute the gradual and linear shift of the gelation sphere to the fact that all the studied gelators share the same molecular packing, so that an increasing length of the alkyl chain reduces the proportion of polar groups at the surface, resulting in a gradual increase in the contact between apolar parts of the fiber and the solvent.

Introduction

Organogels are solid-like materials resulting from the self-assembly of low molecular weight gelators (LMWG) into anisotropic structures within a liquid. [START_REF] Terech | Low Molecular Mass Gelators of Organic Liquids and the Properties of Their Gels[END_REF][START_REF] George | Soft Matter Comprised of Low-Molecular-Mass Organic Gelators and Organic Liquids[END_REF][START_REF] Dastidar | Supramolecular Gelling Agents: Can They Be Designed?[END_REF] These anisotropic structures (usually fibrillar) entrap the liquid altering the mechanical properties of the system. Several examples of LMWG have been reported in the literature showing a high structural variety between them with different levels of complexity. Such molecules are usually discovered by serendipity, and their gelation properties studied by time-consuming gelation tests. In an attempt to speed up this process we [START_REF] Raynal | Organogel Formation Rationalized by Hansen Solubility Parameters[END_REF][START_REF] Bonnet | Organogel Formation Rationalized by Hansen Solubility Parameters: Dos and Don'ts[END_REF][START_REF] Nunes | Organogel Formation Rationalized by Hansen Solubility Parameters: Improved Methodology[END_REF] and others [START_REF] Gao | Harnessing Hansen Solubility Parameters to Predict Organogel Formation[END_REF][START_REF] Xu | Estimation of Organogel Formation and Influence of Solvent Viscosity and Molecular Size on Gel Properties and Aggregate Structures[END_REF][START_REF] Huang | Preparation of Efficient Organogelators Based on Pyrazine-2,5-Dicarboxylic Acid Showing Room Temperature Mesophase[END_REF][START_REF] Lai | Internal Probing of the Supramolecular Organization of Pyrene-Based Organogelators[END_REF][START_REF] Wang | Robust, Self-Healing, and Multistimuli-Responsive Supergelator for the Visual Recognition and Separation of Short-Chain Cycloalkanes and Alkanes[END_REF][START_REF] Xiao | Aliphatic-Alcohol-Induced Opaque-to-Transparent Transformation and Application of Solubility Theory in a Bis-Dipeptide-Based Supramolecular Gel[END_REF][START_REF] Lan | Comparing and Correlating Solubility Parameters Governing the Self-Assembly of Molecular Gels Using 1,3:2,4-Dibenzylidene Sorbitol as the Gelator[END_REF][START_REF] Yin | Gelation Phenomenon during Antisolvent Crystallization of Cefotaxime Sodium[END_REF][START_REF] Zhang | Structural and Solubility Parameter Correlations of Gelation Abilities for Dihydroxylated Derivatives of Long-Chain, Naturally Occurring Fatty Acids[END_REF][START_REF] Liu | Self-Assembly of 12-Hydroxystearic Acid Molecular Gels in Mixed Solvent Systems Rationalized Using Hansen Solubility Parameters[END_REF][START_REF] Diehn | Insights into Organogelation and Its Kinetics from Hansen Solubility Parameters. Toward a Priori Predictions of Molecular Gelation[END_REF][START_REF] Lan | Do Molecular Gelators Cluster in Hansen Space?[END_REF][START_REF] Aparicio | Supramolecular Polymerization of C3-Symmetric Organogelators: Cooperativity, Solvent, and Gelation Relationship[END_REF][START_REF] Edwards | Dynamic Evolving Two-Component Supramolecular Gels -Hierarchical Control over Component Selection in Complex Mixtures[END_REF][START_REF] Wu | Influence of Solvent on the Supramolecular Architectures in Molecular Gels[END_REF][START_REF] Edelsztein | Self-Assembly of 2,3-Dihydroxycholestane Steroids into Supramolecular Organogels as a Soft Template for the in-Situ Generation of Silicate Nanomaterials[END_REF][START_REF] Ando | Gelation Properties of Poly(Aryl Ether) Dendrons Bearing Long Alkyl Chains[END_REF][START_REF] He | The Readout of Base-Pair Information in Adenine-Thymine α-D -Arabinonucleosides[END_REF][START_REF] Tong | Application of Solubility Parameters in a D-Sorbitol-Based Organogel in Binary Organic Mixtures[END_REF][START_REF] Shen | Application of Solubility Parameters in 1,3:2,4-Bis(3,4-Dimethylbenzylidene)Sorbitol Organogel in Binary Organic Mixtures[END_REF] have applied the Hansen solubility parameter (HSP) framework to try and rationalize the gelation proprieties of several LMWG families. To apply this methodology, we need to explore the Hansen space defined by the three HSP cohesive energies of the tested liquids: dispersive interactions (δD), polar interactions (δP) and hydrogen bonds (δH). Following the exploration of the Hansen space, it is possible to determine a gelation domain for individual LMWGs. This can then be used to predict whether an untested liquid could be gelated by a particular LMWG. [START_REF] Raynal | Organogel Formation Rationalized by Hansen Solubility Parameters[END_REF] The next challenge in the field is to evaluate how small changes in the structure of a LMWG will affect the gelation domain. [START_REF] Yan | Correlations of Gel Properties with Gelator Structures and Characterization of Solvent Effects[END_REF][START_REF] Edelsztein | Self-Assembly of 2,3-Dihydroxycholestane Steroids into Supramolecular Organogels as a Soft Template for the in-Situ Generation of Silicate Nanomaterials[END_REF][START_REF] Yan | How Do Liquid Mixtures Solubilize Insoluble Gelators? Self-Assembly Properties of Pyrenyl-Linker-Glucono Gelators in Tetrahydrofuran-Water Mixtures[END_REF][START_REF] Zhang | Structural and Solubility Parameter Correlations of Gelation Abilities for Dihydroxylated Derivatives of Long-Chain, Naturally Occurring Fatty Acids[END_REF][START_REF] Li | Synthesis and Properties of Novel Organogelators Functionalized with 5-Iodo-1,2,3-Triazole and Azobenzene Groups[END_REF][START_REF] Singh | Molecular Nuances Governing the Self-Assembly of 1,3:2,4-Dibenzylidene-D-Sorbitol[END_REF][START_REF] Bonnet | Organogel Formation Rationalized by Hansen Solubility Parameters: Influence of Gelator Structure[END_REF] In particular, we previously probed the influence of the length of an alkyl chain within a family of related gelators, but showed that it is difficult to rationalize the results without knowledge of the molecular packing. [START_REF] Bonnet | Organogel Formation Rationalized by Hansen Solubility Parameters: Influence of Gelator Structure[END_REF] To better correlate the molecular structure with the gelation domain, we selected a family of amide-based thiazole derivatives bearing a long aliphatic chain, for which the elucidation of the molecular packing seemed to be within reach (Figure 1). Within these gelators the amide group is expected to provide supramolecular H-bond interactions while the long alkyl chain contributes to the dispersion interactions. This group of LMWGs was first reported by Ballabh et al. together with the single-crystal structure of two members of the family [START_REF] Yadav | Probing the Role of Weaker Interactions in Immobilization of Solvents in a New Class of Supramolecular Gelator[END_REF][START_REF] Yadav | Odd-Even Effect in a Thiazole Based Organogelator: Understanding the Interplay of Non-Covalent Interactions on Property and Applications[END_REF] . In the present work, the gelation domains of several members of this family, with the alkyl chain length ranging from 6 to 16 CH2 groups, are determined, together with structural characterization (X-ray diffraction measurements and scanning electronic microscopy). To extract detailed structural information from the X-ray powder patterns, a molecular modeling protocol is applied, in which the most probable packing structure is identified by comparing the simulated patterns of dozens of candidates to the experimental data. The crystal habit is then evaluated using the Bravais-Friedel-Donnay-Harker method [START_REF] Bravais | Etudes Crystallographiques[END_REF][START_REF] Friedel | Etudes sur la loi de Bravais[END_REF] , and the Growth Morphology Method [START_REF] Berkovitch-Yellin | Toward an ab initio derivation of crystal morphology[END_REF][START_REF] Docherty | Application of Bravais-Friedel-Donnay-Harker attachment energy and Ising models to predicting and understanding the morphology of molecular crystals[END_REF] (GMM) based on attachment energy of the crystal faces, in order to determine along which direction crystalline fibers are expected to grow preferentially. With this study, we thus aim at correlating the gelation domain with the molecular packing and the fiber morphology of the gels.

Experimental section

Gel preparation

The samples were prepared by adding 20 mg of LMWG and 1 mL of liquid in a screw-cap vial.

The suspensions were heated up to 100° C until dissolution and left to cool to room temperature on the bench. After 24 hours the vials were turned upside-down, and the aspect of the samples was noted as gel (G), solution (S) or precipitate (P). A material is considered a gel if no deformation is observed after turning the vial upside-down.

Hansen sphere determination

All solubility and gelation spheres were calculated according to our recently proposed general procedure, [START_REF] Nunes | Organogel Formation Rationalized by Hansen Solubility Parameters: Improved Methodology[END_REF] using the generic algorithm provided in the HSPiP software. [START_REF] Hansen | Solubility Parameters A User's Handbook[END_REF][START_REF] Abbott | Solubility Parameters in Practice[END_REF] In short, the solubility sphere was calculated in a way that as many S points as possible lie inside the sphere, but as many G and P points as possible lie outside. The gelation sphere was determined so that as many G points as possible lie inside the sphere, but as many P points as possible lie outside. S points are not taken in consideration for the gelation sphere.

X-ray diffraction measurements

Xerogels were obtained by extracting gel fibers by filtration of a gel and drying overnight. The measurements were performed with a Rigaku SmartLab X-Ray Diffractometer using a rotating Cu anode X-ray source (40 kV, 200 mA).

Modeling of the molecular packing

As the first step, the X-ray patterns were indexed using Dicvol 42 and the symmetry groups were obtained using the Check group program incorporated in the FullProf suite. [START_REF] Rodríguez-Carvajal | Recent Advances in Magnetic Structure Determination by Neutron Powder Diffraction[END_REF] In parallel, the atomic positions were obtained by molecular modeling simulations, through Simulated Annealing using the Polymorph module, implemented in the Materials Studio software. 44 The generated molecular packings were then optimized by Molecular Mechanics (MM). All the MM calculations were performed with the Dreiding force field (in which the hydrogen bond parameters have been adapted to properly reproduce van der Waals interactions in the solid state) and assigning PCFF atomic charges. The best models were then refined using the Reflex module from the Materials Studio software by fitting the experimental XRD pattern using Rietveld refinement coupled with the force field to avoid unrealistic changes in the molecular geometry. This joint modelingexperimental approach (see SI for more technical details) has been fully implemented for the Th12 compound, and the agreement between the simulations and the single crystal X-ray data has been found to be excellent (see below). That structure was then used as a starting point for determining the crystal structure of the other members of the series, by increasing the chain length, adapting the cell parameter along the chain direction, and refining the structure using Reflex.

Crystal habit calculation

The calculations were performed in the Materials Studio software on crystals that were fully relaxed (including the cell parameters) by MM using the modified Dreiding force field. Due to the force field approximations, this full relaxation slightly changes the cell parameters of the proposed cell but ensures that energy discrepancies are avoided (the repulsive wall of van der Waals interactions is a few tenths of Å far from the minimum). The long-range interactions were treated by Ewald for electrostatics (accuracy of 10 -5 kcal/mol) and an atom-based cutoff for van der Waals (cutoff distance of 14 Å, spline width of 3 Å).

Scanning electron microscopy (SEM)

The samples for SEM observation (Zeiss Sigma VP microscope) were prepared by freeze-drying the gel samples in liquid nitrogen and vacuum-pumping the solvent.

Results and discussion

Determination of the gelation domain

First, the gelation domain in Hansen space of LMWG Th12 was determined using our previously established methodology. [START_REF] Nunes | Organogel Formation Rationalized by Hansen Solubility Parameters: Improved Methodology[END_REF] The gelator concentration was varied between 3 and 0.75 wt% (Figure S1). We observed that: (i) below 1 wt% Th12 formed very few gels in the tested liquids and (ii) at 3 wt% almost none of the tested liquids fully solubilized the LMWG. Therefore, we selected an intermediate concentration (2 wt%) to be able to detect any effect of the length of the alkyl chain on the gelation domain. The gelation domain of LMWGs Th8 to Th18 was then determined using the same methodology at the concentration of 2 wt% (Figure 2). The evolution of the size of the solubility and gelation spheres is summarized in Figure 3a. The solubility sphere dramatically shrinks with the alkyl chain length, in such a way that for Th16 and Th18, solubility is no longer observed. In clear contrast, the increase in length of the alkyl chain leads to an expansion of the gelation sphere from Th12 to Th18. 45 As expected, the center of the solubility sphere shifts gradually to a less polar region (smaller dH and dP) and to a less polarisable region of the Hansen space (smaller dD) as the alkyl chain length increases (Figure 3b). More interestingly, a very similar trend also occurs for the gelation sphere, which is in contrast to our previous study. [START_REF] Bonnet | Organogel Formation Rationalized by Hansen Solubility Parameters: Influence of Gelator Structure[END_REF] It can be seen from the trend lines (see SI) that adding one CH2 decreases the parameters for the center of the solubility sphere by 2dD = 0.43; dP = 0.25; dH = 0.40 MPa 1/2 , while it decreases the parameters for the center of the gelation sphere by 2dD = 0.51; dP = 0.23; dH = 0.41 MPa 1/2 . Therefore, a longer alkyl chain increases the contribution of the the gelation sphere. Finally, one can notice that the shift of the center of the gelation sphere to a less polar region is not accompanied by a loss of gelation in the polar region because the shift in the center occurs together with the increase of the radius of the gelation sphere. HSPiP software. [START_REF] Hansen | Solubility Parameters A User's Handbook[END_REF][START_REF] Abbott | Solubility Parameters in Practice[END_REF] ) and center of the gelation spheres (green) represented in Hansen space. Data from Figure 2; for Th14, a light blue color has been selected to highlight the limited precision of that particular data (only 4 S points); the lines are drawn as explained in SI.

In order to try and rationalize these observations, we investigated the morphology and the structure of the fibers.

SEM observation of fibers

The morphology of Th12 to Th18 gel fibers was observed by scanning electronic microscopy (SEM). Th12 xerogels obtained from various liquids (toluene, cyclohexane and 1-butanol) (Figure 4.a to c) all show rectilinear shaped fibers with very few differences in size, indicating that the different liquids do not affect the morphology of the fibers. Moreover, the fibers of the various gelators Th12 to Th18 (Figure 4.a and d to f) obtained from toluene also share a very similar morphology, even if the Th18 sample appears more blurred than the others. Therefore, no major effect is detected at this length scale.

X-ray measurements in transmission

X-ray measurements were performed on xerogels obtained from Th12 to Th18 from various liquids to determine whether the nature of the liquid influences the packing in the fibers. Xerogels were grinded to obtain a fine powder and then measured in transmission mode with the X-ray beam set-up in the parallel beam configuration. The data from the Th12 and Th14 xerogels were compared to the powder pattern simulated from the previously published single-crystal structure. Similar X-ray powder patterns were obtained from the Th12 xerogels regardless of the liquid in which they were formed (Figure 5a 1,2,3). Small differences in diffraction peak intensity can be attributed to small effects of preferential orientation within the samples. This observation also applies to Th14, Th16, and Th18 (Figure 5b,c,d). This indicates that in this family of LMWGs, the molecular packing within the fibers is independent of the liquid in which the gel is formed. In fact, different patterns for a given gelator were only observed for Th12 when precipitates were obtained instead of gels (Figure S2). Thus, within each gelation sphere the molecular packing can be considered to be similar. The xerogels obtained from Th12 also present a high resemblance with the powder pattern simulated from the published single-crystal structure of Th12 (Figure 5a C) [START_REF] Yadav | Probing the Role of Weaker Interactions in Immobilization of Solvents in a New Class of Supramolecular Gelator[END_REF] . This indicates that the gel fibers of Th12 share the same packing as the corresponding singlecrystal.

Th14 shows the same X-ray pattern for all the measured xerogels (Figure 5b 1,2,3) but small differences appear with respect to the powder pattern simulated from the single-crystal data [START_REF] Yadav | Probing the Role of Weaker Interactions in Immobilization of Solvents in a New Class of Supramolecular Gelator[END_REF] (Figure 5b C): the measured xerogels show a good match to the single-crystal pattern in the small angle region (0 to 15 degrees) and a constant small shift of +0.5 degrees in the wide-angle region (above 15 degrees). This small shift is due to a slightly denser packing (3.9%) in the case of the single crystal (see Table S9), but the conformations are identical and the molecular packings are very similar (see below and Figure S4).

Modeling of the molecular packing

To better understand how the length of the alkyl chain affects the crystal structure within the gel fibers, the molecular packing was determined based on the powder patterns measured on the xerogels and molecular modeling simulations. The validation of the methodology was performed by comparing the structure obtained for Th12 with the published single-crystal structure of the same molecule. Overlaying the two structures, it was found that they are similar, with cell parameters differing by less than 0.05 Å and 0.1 °, and atoms overlapping almost perfectly (Figure S3). The very good agreement between the modelled and published single-crystal structure validates the applicability of the established procedure to determine the molecular packing within the gel fibers. The structure proposed for the Th14 xerogels also presents a high resemblance with the previously published single-crystal structure (Figure S4). The indexation of the powder patterns for all these thiazole-based gelators points to monoclinic unit cells, with the cell parameter "a" increasing with the length of the alkyl chain and the other cell parameters remaining constant (see Table 1). Figure 6 illustrates the high structural similarity between gelators Th12 to Th18, suggesting that differences in the length of the alkyl chain do not change the packing of these gelators within the gel fibers. The structures are characterized by the presence of H-bonded dimers forming a cyclic H-bond topology (see Figures S7 andS8), where the amide group is the donor of hydrogen bond and the nitrogen of thiazole is the acceptor (NH…N bonds), as reported previously [START_REF] Yadav | Probing the Role of Weaker Interactions in Immobilization of Solvents in a New Class of Supramolecular Gelator[END_REF] . The thiazole moieties of the dimers form planar objects that organize in a g packing, i.e., a combination of a parallel-displaced arrangement along the [001] direction and a herringbone arrangement along the [010] direction (see Figure S7). The sulfur atoms of neighboring stacks are in van der Waals contact, with a distance of 3.57 Å. All alkyl chains organize in an extended all-trans conformation, interdigitating along the (100) plane (Figure 6). The detailed structures and fitted XRD patterns are in Supporting Information.

X-ray measurements on oriented fibers

Usually, a strong directional interaction is responsible for the anisotropic growth of the organogel fibers. Here, this feature is not obvious since the amide groups are involved in dimer formation instead of chains of hydrogen bonds. To unambiguously identify which axis of the unit cell lies along the fiber growth direction, we performed X-ray diffraction on oriented fibers. By keeping the gel fibers intact (i.e., no grinding was performed) and compacting them as a film on a flat surface, we expect that most fibers lie parallel to the substrate. In these conditions, only reflections associated to lattice planes parallel to the fiber axis can be visualized in symmetric diffraction geometry (see Figure 7, which shows the difference in the experimental setup between the measurements performed on randomized crystals and oriented fibers). Figure 8 illustrates the difference between the patterns obtained from randomized and oriented fibers for Th12 in toluene. The diffraction peaks that are present in both patterns can be easily identified as due to diffraction by (hk0) lattice planes. Reflections of the type 00L corresponding to the diffraction of the (00L) planes are not present in the oriented fibers pattern. Thus the (00L) planes are never in reflection condition which means that the [00L]* direction, which deviates from the [00L] axis by only few degrees, is never orthogonal to the sample plane. This is an indication of a preferred orientation of the fibers in a 2D powder, where the [00L] axis is in the sample plane, and should thus correspond to the fiber axis. This point is illustrated in Figure 9 where some (hk0) planes are represented. A further proof of the growth direction was given by calculations with the Morphology module of Materials Studio. Two simulation methods were used: the Bravais-Friedel-Donnay-Harker [START_REF] Bravais | Etudes Crystallographiques[END_REF][START_REF] Friedel | Etudes sur la loi de Bravais[END_REF] (BFDH) and the Growth Morphology Method [START_REF] Berkovitch-Yellin | Toward an ab initio derivation of crystal morphology[END_REF][START_REF] Docherty | Application of Bravais-Friedel-Donnay-Harker attachment energy and Ising models to predicting and understanding the morphology of molecular crystals[END_REF] (GMM). The BFDH method is based on purely geometrical rules using crystal lattice and symmetry elements, with the growth rate of a given face inversely proportional to the thickness of the molecular layer added to it. Even though energy effects are neglected, BFDH is often reliable for weakly interacting molecules, and is helpful in showing prevailing faces in crystal growth. In the GMM method, the growth rate of a face is proportional to the attachment energy (Eatt), i.e. the potential energy per unit cell gained when a new molecular layer is attached to the face. Faces with the strongest attachment energies grow fastest and have the lowest morphological dominance.

With GMM, we find that the crystal habit is elongated along the [001] direction, with {100}, {110} (and {020} for Th12) lateral faces, and is capped by {11-1} and {011} faces (Table 2).

These results confirm the X-ray measurements on oriented fibers, where the signal of planes parallel to the long axis, here the {hk0} planes, is intensified, in contrast to the signal of planes tilted with respect to the long axis, i.e. planes with L different from 0, which are attenuated. A similar crystal habit was found with BFDH (see SI), suggesting that simple geometry constrains are sufficient to induce a high anisotropy of the crystal, in a system where it is not straightforward to pinpoint strong intermolecular interactions along a particular direction (the hydrogen bonds lead to the formation of dimers, not rows of H-bonded molecules).

As the length of the alkyl chain only affects the a cell parameter, only faces with a dominant h character grow slower as the molecules get longer. As a result, from Th12 to Th18, the facet area of {100} increases with respect to that of {110}, from 1.3:1 to 1.9:1 with BFDH, and from 0.6:1 to 0.7:1 with GMM, and the {020} face disappears from Th14 on. In addition, the anisotropy grows slightly from Th12 to Th18, with an aspect ratio evolving from 4.3 to 5.7 with BFDH, and from 2.5 to 2.9 with GMM. This stronger anisotropy could be the reason why the radius of the gelation sphere increases with the length of the alkyl chain (Figure 3a). Indeed, more anisotropic fibers have a higher tendency to form a percolating network.

Due to symmetry operations, there are two or four ways to build a face, depending on the part of the molecule that is exposed to the environment; this influences the surface stability and thus the facet growth. With the GMM method, the face covering, i.e., the molecular groups present at the outer surface, that grows slowest is always selected as the limiting factor. In Table 2, the characteristics of the relevant faces relate to the most stable covering, except for the {100} faces, where information for the two possible coverings (alkyl chains or thiazole groups) are indicated.

The {100} faces can be covered either by alkyl chains or thiazole groups protruding perpendicular to the surface. The alkyl chain covering and its Eatt are strongly affected by the molecular size, as the length of alkyl chain dangling in the environment increases (from Th12 to Th18, Eatt evolves from -71.3 to -104.2 kcal/mol), while the thiazole covering and its Eatt are not (from Th12 to Th18, Eatt drifts only from -82.3 to -83.3 kcal/mol, respectively). As a result, the face covering could change depending on the size of the alkyl chain. Here, the face covering appears more stable with alkyl chains below Th16, while from Th16 on it is the thiazole termination that is most stable.

However, we must keep in mind that in this approach the effects of the relaxation of molecules and interactions with the solvent were neglected. The relaxation of the long dangling alkyl chains could significantly reduce the attachment energies, slow down the growth of the {100} faces, and further increase the anisotropy of the fibers. The {100} faces could also adapt their covering in response to the environment, either exposing polar groups or apolar groups. Furthermore, apolar groups present at the surface could either extend into the solvent or adsorb on the crystal face.

These protruding molecular groups are typical of {100} faces and could help stabilizing the fibers; for other crystal faces, they are less conspicuous or do not exist at all, e.g. for {020}, where both thiazole groups and alkyl chains lie flat at the surface. Due to this orientation, the relative occupancy of the {020} surfaces by the apolar alkyl chains increases with the size of the molecule.

This increase of the apolar character is also observed for the four possible coverings of the dominant {110} faces, as the molecules are tilted with respect to the surface (see SI).

In summary, our calculations show that the crystal habit of Th12 to Th18 are very similar and that when the alkyl chain length is increased, the proportion of polar and apolar groups at the surface changes, likely resulting in a gradual increase in the contact between apolar parts of the fiber and the solvent. We propose that this is the reason why the gelation sphere shifts regularly toward the less polar region of the Hansen space from Th12 to Th18. The {100}-th and {100}-alk notations correspond to thiazole-terminated and alkyl chainterminated {100} faces, respectively.

Conclusion

This study presents an in-depth crystallographic analysis of the molecular packing and crystal habit of a set of thiazole-based gelators. In terms of solid-state structure, we show that the four molecules differing only in the length of an alkyl chain crystallize in the same symmetry group, and with the same crystal habit. In terms of gelation behavior, the increase in alkyl chain length in these gelators: (i) increases the size of the gelation sphere in the Hansen space and (ii) gradually shifts its center. We attribute the first effect to an increased anisotropy for fiber growth when the alkyl chain length is increased. We attribute the second effect to the fact that, since all the studied gelators share the same molecular packing, an increasing length of the alkyl chain gradually increases the contact between apolar parts of the fiber and the solvent and therefore reduces the relative influence of polar and hydrogen bond interactions.

Figure 1 .

 1 Figure 1. Chemical structure of the thiazole-based LMWGs.

Figure 2 .

 2 Figure 2. Gelation data plotted in Hansen space for the thiazole-based LMWGs at 2wt%. The

Figure 3 .

 3 Figure 3. a) Radius of the solubility (blue) or the gelation (green) spheres for the thiazole-based

Figure 4 .

 4 Figure 4. SEM images of the thiazole-based xerogels: a -Th12 from toluene, b -Th12 from

34

 34

Figure 5 .

 5 Figure 5. X-ray patterns of xerogels of the thiazole-based gelators obtained from (1) toluene, (2)

Figure 6 .

 6 Figure 6. Molecular packing representation, with the cell parameters a in red, b in green, c in blue.

Figure 7 .

 7 Figure 7. Comparison of the X-ray methodology to measure randomized or oriented gel fibers.

Figure 8 .

 8 Figure 8. Comparison between the X-ray patterns of oriented (top) and randomized (bottom) gel

Figure 9 .

 9 Figure 9. Representation of the diffraction planes observed in the X-ray patterns of the oriented

Figure 10 .

 10 Figure 10. Crystal habit of Th12 built from the Growth Morphology Method. Views along the 010

Table 1 .

 1 Proposed unit cell parameters of the thiazole-based gelators.

	compound	Th12	Th14	Th16	Th18
	crystal system	monoclinic	monoclinic	monoclinic	monoclinic
	space group	P21/c	P21/c	P21/c	P21/c
	a (Å)	20.00	22.30	24.60	26.99
	b (Å)	15.17	15.18	15.19	15.19
	c (Å)	5.54	5.55	5.55	5.56
	α (deg)	90	90	90	90
	β (deg)	92.37	90.86	93.54	95.74
	γ (deg)	90	90	90	90
	volume (Å 3 )	1697.40	1889.64	2081.88	2274.12
	Z	4	4	4	4
	density (gcm -3 )	1.173	1.147	1.132	1.115

Table 2 .

 2 List of relevant faces of thiazole based LMWG obtained by the GMM method, sorted out by attachment energy (Eatt in kcal/mol).

			Multiplicity	Eatt total	% total facet area
		{100}-alk	2	-71.3	27.8
		{100}-th	2	-82.3	-
		{110}	4	-76.6	44.0
	Th12	{020}	2	-87.2	4.8
		{11-1}	4	-144.8	16.1
		{011}	4	-154.1	7.3
		{100}-alk	2	-82.3	26.2
		{100}-th	2	-82.6	-
		{110}	4	-82.3	50.9
	Th14
		{020}	2	-98.1	-
		{11-1}	4	-161.1	14.6
		{011}	4	-170.4	8.3
		{100}-th	2	-82.9	29.9
	Th16	{100}-alk	2	-93.3	-
		{110}	4	-88.0	48.3

Acknowledgements

This work was supported by the French Agence Nationale de la Recherche (project ANR-16-CE08-0012 HSPgel). Research in Mons was supported by the UMONS research council and FNRS, through the EOS 2Dto3D project and the 'Consortium des Equipements de Calcul Intensif' (CECI -grant No. 2.5020.11). We acknowledge the provision of facilities and technical support by Aalto University at OtaNano -Nanomicroscopy Center (Aalo-NMC).


Abdulrashid Iliya
email: abdulrashid.iliya@aun.edu.ng

Chidi G Ononiwu

Muhammadou M O Kah

Ago K M Quaye

Mobile Phone Use for Empowerment and Well-Being of the Physically Challenged in Nigeria

Keywords: Mobile Phones, Physically challenged, Critical realist ethnography, Capability Approach

National and economic benefits of mobile phone use in developing countries has been a well-articulated research domain over an extended period. This can be attributed to the ubiquitous nature of mobile phones and their increased penetration in developing nations. However, the potential benefits of mobile phones for empowerment and well-being of people with disability (PWD) has been ignored. This paper focuses on the well-being of the physically challenged in Nigeria and how mobile phones can be employed to empower them. The link between ICT and human development has been well researched, but minimal research has attempted to link ICT, mobile phone and disability using the Capability Approach as a theoretical lens. The critical realist ethnographic study approach is employed in this study to show how mobile phones can be used to empower and impact on the well-being of the physically challenged. Data were collected from the Adamawa skill acquisition center for persons with disability, Nigeria. It is argued that mobile phones have the capabilities to empower and impact on the well-being of the physically challenged. Thus, the findings illustrate that mobile phones play significant roles in the well-being and empowerment of the physically challenged.

Introduction

Mobile phones are regarded as an important tool for national development and poverty reduction (Bhavnani, Chiu, Janakiram, Silarszky, & Bhatia, 2008; Hoan, Chib, & Mahalingham, 2016). The contributions of mobile phones have generated numerous studies in developing countries. As such, these studies are generally attributed to economic development [START_REF] Shirazi | Information and communication technology and women empowerment in Iran[END_REF], gender empowerment (Hoan, Chib, & Mahalingham, 2016), improvement of maternal health (Nyemba-Mudenda, & Chigona, 2015) and poverty reduction [START_REF] Islam | Women in rural Bangladesh: empowered by access to mobile phones[END_REF]. Similarly, there are many studies on mobile phone use in developing countries [START_REF] Donner | Research approaches to mobile use in the developing world: A review of the literature[END_REF]) that focus on marginalized populations. Most of these studies focus on marginalized women [START_REF] Shirazi | Information and communication technology and women empowerment in Iran[END_REF], marginalized youth (Sam, 2017) and well-being of blind micro-entrepreneurs [START_REF] Anwar | Mobile phones and the well-being of blind micro-entrepreneurs in Indonesia[END_REF]. However, in-depth and/or extensive studies on physically challenged persons have not been carried out. [START_REF] Walsham | ICT4D research: reflections on history and future agenda[END_REF], indicate the need for more focused studies on disability in the ICT4D domain. The term 'empowerment' can mean different things to different people, but in this research, empowerment refers to the ability for people with disability to overcome their deficiencies in society. It is the ability to exhibit social, economic, political and cultural freedoms (Sen, 2014). Lately, technological advancements have been touted to enhance and empower People with disabilities (PWDs) especially in the provision of access to information and services, as well as, the physical environment (D' Aubin, 2007). ICT has proven to play a vital role in the spheres of PwDs. It has removed barriers which prevent PwDs from participating in daily activities [START_REF] D'aubin | Working for Barrier Removal in the ICT Area: Creating a More Accessible and Inclusive Canada: A Position Statement by the Council of Canadians with Disabilities[END_REF]. Although, a number of studies have linked ICT with disability (e.g., [START_REF] Goggin | Disability, Mobiles, and Social Policy: New Modes of Communication and Governance[END_REF] The question suggests that the study is focusing on how mobile phone use support economic and social development, including its impact in contributing to some aspects of human well-being. Secondly, the study sets out to investigate how CA and ICT are linked to empowerment, especially, of the physical challenged.

The paper is structured as follows: the next section will discuss the research context of empowerment and mobile phones in developing countries, followed by the methodology. Preliminary results of the findings are presented. Finally, the study concludes with a discussion of how CA evaluates the contributions of mobile phones to empowerment and well-being of the physically challenged. In Nigeria, the number of mobile phone subscribers have increased from 10.2 to 144 Million from 2005 to 2017 (NCC, 2017). Mobile phones have become an integral part of the day-to-day activities in Nigeria. People engage their mobile phones in sending text messages, making calls and sometimes listening to music or FM radio. Due to its ubiquity, mobile phones have penetrated all of life including the poor, marginalized and the disabled. Mobile phone has become the IT artefact that seems to provide the social inclusion of people below the pyramid.

Research Methodology

This study focus on understanding participants' experiences and views, using critical realist ethnographic approach. Critical realist ethnographic approach provides understandings of participants' perceptions, beliefs and experiences. Critical realism regards reality as objective and stratifies ontology into empirical, actual and real domains. The empirical domain includes what we see and observe, the actual domain comprises events that are generated by mechanism while the real domain contains mechanisms and structures with enduring properties [START_REF] Mingers | Introduction [special issue: Critical realism in information systems research[END_REF]. Critical realist ethnography is associated with reflexivity and has the prospects to be a dominant methodological framework about the nature of reality [START_REF] Barron | The potential and challenges of critical realist ethnography[END_REF]. The adequacy and representation of the world of the empirical requires audio, visual and written representations. Writing of field notes and photographs are essential in capturing the world of the empirical without being intrusive [START_REF] Barron | The potential and challenges of critical realist ethnography[END_REF]. For adequate representation, sharing of the field notes and photographs with participants will enhanced, challenge and provide additional empirical detail where necessary. At the 'actual' world, it is imperative to consider the events that may not have been experience and what had been experience related to had not. This is where reflexivity plays a vital role in enabling the researcher challenge straightforward outcomes and consider "as many events as possible in the world of the actual". This include the use of photography as a 'text'.

Research method

The research approach (ethnography) constructs theory from the data obtained in the field through participants' observation. Participant's observation and in-depth interviews were used as the core data collection method. The field notes constituted the primary data, together with semi-structured interview were analyzed. Various questions were developed to facilitate the semi-structured interviews. The investigations carried out in the field addressed questions on how mobile phones contribute to empowerment and well-being of the physically challenged.

We conducted our field work from February 2018 to September 2018 in the city of Yola, Nigeria. The location represents the northern part of Nigeria. A total of 10 participants were observed and interviewed. Been a critical realist ethnographic study, attention is given at the empirical domain for this study. Further work will focus on the actual and real domains. We envisage that the data collection will continue for about 2 years.

Semi-Structured Interviews

We conducted in-depth interviews with the members of the Adamawa skill acquisition center for persons with disabilities. Our focus was on the physically challenged. The interviews varied in duration but with an average length of 30 minutes. For the purpose of this study, 10 interviews were conducted. Each interview was intensively written (note taking).

Focus Group Interviews

The focus group interview occurred in May 2018 where the 10 participants were brief about the 1 st order categories of our findings. A unanimous agreement was reach about the about the first-order concepts (those important to the participants)

Participant Observations

We were allowed access to the Adamawa skill acquisition center over the course of our field study. This has provided valuable understandings into the day-day activities of the physically challenged. Detailed notes were taken during the events to complement the semi-structured interviews and the focus group interviews. Discussion and note taking were conducted within the research team to arrive at the participants' expressions. The names used in this study were given pseudonyms so as to protect the security and privacy of the research subjects.

Data Analysis

The data was deductively analyzed following the naturalistic inquiry method [START_REF] Lincoln | Naturalistic Inquiry[END_REF] under the CA theoretical underpinnings. These methods of data analysis "provide rigor in analyzing qualitative data to simultaneously assist in determining the sampling and content foci of future data collection efforts" [START_REF] Harrison | Clean Climbing, Carabiners, and Cultural Cultivation: Developing an Open-Systems Perspective of Culture[END_REF])

Figure 1 at the appendix represent the data structure we analyzed. We began by analyzing the initial transcribed data to grouped them as the 1 st order categories. Next, we examined the relationships among these categories where we arrived at the 2 nd order themes. The similar themes were further reduced into the aggregate dimensions that led to the emergent of our model. Been a critical realist ethnographic study, the 1 st categories, 2 nd order themes and the aggregate dimensions has only focused on the empirical domain. This is where our study intends to contribute. A further study will focus on the underlying mechanism that must exist for empowerment and well-being to occur.

The figure shows the data structure, including first-order concepts (those important to the participants) and second-order themes (induced by the researchers), that led to the generation of the aggregate dimensions.

4

The participants: Physically challenged

Preliminary Findings

In this section, preliminary findings of the study are discussed, including the concept of well-being and empowerment as understood by the participants. How the physically challenged use mobile phones for personal and business activities is also discussed.

The well-being of the Physically challenged

For the well-being of the physically challenged, it is necessary to understand their perceptions and what is vital to their well-being. Numerous concepts were advanced to explain what constitutes the term 'well-being' to the physically challenged. This is necessary to successfully define an accurate representation of well-being to the physically challenged. The empirical location where this study was conducted consists of participants' mostly of the Fulani tribe, who are mostly Muslims. They speak Fulfulde and they view well-being as 'Nanugobeldum'. Nanugobeldum meaning enjoyment, peace of mind and satisfaction. Clearly, this has no similar meaning in the English word well-being. Although, Nanugobeldum was interpreted differently between the participants, a common perception was achieved. Nanugobeldum was comprehended as a form of spiritual and physical fulfillments. Spiritual context here depicts freedom to practice their religion and physical fulfillment is linked to healthy lifestyle and wealth. Being valuable to family and society was also perceived as a form of Nanugobeldum. In summary, it is evident from the interviews that what matter to the well-being of the physically challenged can be categorized as follows: being healthy, family, peaceful co-existence and being independent.

Being Healthy

Participants in this study believe that being healthy is an integral part of their well-being. In their context, being healthy means psychological ability to be able to provide for their family. Getting ill is an added disadvantage to their disability, as such they use mobile phone to contact their doctor or pharmacist whenever they feel sick.

All I want in life is to be healthy so that I can be able to provide food for my children. I call my doctor whenever I feel sick (Amadu)

Family

The participants believe that the happiness of their family is paramount to their wellbeing. Family is important to them. In fact, some participants believe their well-being is not their own, but for their family. They use mobile phone to communicate to their family whenever it is necessary.

My family is everything to me; I work hard every day to provide for them. I just called my wife to check on her and our new baby. (Moses)

Peaceful co-existence

All the participants believe that societal peace is paramount to their well-being. They lament on the increase in-security cause by Boko-haram as a drawback to their wellbeing. Their mobile phone serves as a form of news channel.

Boko-haram have crippled my business; I hardly make money for my upkeep. I am however, grateful to Allah. Thank God for my phone, it has help me follow the news on boko haram crisis. (Adamu)

Being independent

The physically challenged perceived being independent as an important aspect to their well-being. They see dependency as a form of inferiority and as such they prefer to do everything by themselves if they can. Mobile phone provide support for the physically challenged though physical mobility.

My husband died in 2012 during the Boko-haram crisis. He left me with 4 children and I have to come out here every day to sell my products so that I can be able to feed my children. If I am not feeling too good, I call on my sister to take me to the hospital. But I don't like troubling her all the time (Asabe).

The business and personal use of Mobile phones

The other categories that emerged from the interview data are the use of mobile phones for business and personal use. These can be categorized into the following themes: mobile phone use for business networking, maintaining kingship amongst family members and mobile phones for sales/customers' relationships. These are discussed below.

Mobile Phone use for Business Networking

Participants agreed to use mobile phones to communicate amongst themselves. They used mobile phones to schedule for business meetings between themselves.

Before, it is difficult to have everybody participate in a meeting. But now that we all have a mobile phone, I called everyone on the phone whenever we are having a meeting. For example, I called everyone to let them know you (researcher) are coming to interview them. (Sera)

It also helps the participant to agree on a collective price for their products

Due to the recent recession, we all receive a message from our coordinator to review our prices so as to be in line with each other. (Shuaibu)

Maintaining Kingship amongst Family Members

Mobile phone is an integral artifact in the life of the physically challenged. As a ubiquitous device, it has facilitated communication amongst relatives, family and friends. The participants believe that the mobile phone is important in maintaining relationships amongst them.

Mobile phone has helped me communicate with my son who is a soldier. He is at the battle grounds fighting with boko-haram. I am always happy when he calls me. (umma)

However, other participants believe maintaining a mobile phone is very expensive. For example, I always call my wife in the village to find out how she is doing with our children. She doesn't have a phone; I always call my neighbor to help me take the phone to her so that I can talk to her. I want to buy her a phone but it is expensive. I will buy her a phone soon. In sha Allah.

(Idris)

Mobile Phones for Sales and customer relationships

All the participants have access to mobile phones. They agreed that mobile phone help them maintain relationship with their customers.

In fact, since I started using a mobile phone, my customers have increased dramatically. They can now call to order for their type of bags and shoes. I am very happy to use my mobile phone all the time. (Ibrahim)

The mobile phone also help them to showcase their products:

I have been using my phone for almost three years now. However, I was recently introduced to WhatsApp messenger which has made my business so easy. I can now snap pictures of my products and send it to my customers. But the data price is very high. (Saleh)

Discussion

How Mobile Phones impact on Well-being and Empowerment

As described in the findings, mobile phones assist the physically challenged in communicating with their friends, family and improving their business strategies and network. But most importantly, it helps them to be independent and brings them to be an inclusive part of the society. The ubiquity of mobile phones which provide information sharing and connectedness offer opportunities that may have been challenging to realized. The effective use of mobile phones for business activities has resulted in increased income, more customers thereby improving the livelihood of the physically challenged. It also facilitated freedom and independence in conducting business and personal activities. In the case of the physically challenged, mobile phones are seen as the more preferred tool for communication amongst the group. The lack of assistance and flexibility from government agencies promotes networking within the group. These virtual interaction benefits the physically challenged by offering efficient and more effective avenues for business interaction thereby leading to empowerment. However, this comes with its own disadvantage as mobile phones and their services are very expensive to maintain. The ability to contact family members at any given time enhanced their social empowerment. Being able to convey their feelings freely through the mobile phones have surely given them a sense of self-expression among one another. All these can be attributed to the participants' goals in achieving well-being and empowerment.

The findings indicate that the physically challenged often experience inferiority around able people. Certainly, this has led people with disability to be culturally, economically and socially marginalized. In the case of the physically challenged, dependency is being attributed to a form of inferiority. Removing dependency and improving the physically challenged independence proves vital in achieving a better life and empowerment. Despite the barriers of costs involved in the maintenance of mobile phones, participants still utilized the capability of mobile phones for their empowerment.

Drawing from the concept of the CA, a theoretical framework is proposed as shown in figure 2. The framework is illustrated in the form of a diagram that depicts the relationships between well-being, mobile phone use and empowerment. It shows that mobile phone use has an impact on the well-being and can empower the physically chal-lenged. The framework also shows that well-being lead to empowerment of the physically challenged.

Conclusion and Future Work

This paper has demonstrated that the primary effects of mobile phones to the wellbeing and empowerment of the physically challenged include their family, being healthy, peaceful co-existence and being independent. The paper has also shown how mobile phone is used for personal and business purposes of the physicallyc hallenged. It can be concluded that mobile phones use has expanded the capabilities of the physically challenged to live the type of life they value. Although, the results from the study has depict the positive effects of mobile phones to the physically challenged, there might be negative elements that may affect the well-being of the physically challenged.

The findings have shown that there is a strong relationship between well-being and empowerment. For the physically challenged, the categories of well-being such as being healthy, family, being independent and peaceful co-existence as discovered from this research has the capability to empower the physically challenged. And as such, well-being leads to empowerment to the physically challenged.

Additionally, the paper has contributed to the ignored topic of disability and mobile phone in the ICT4D domain. As a timely contribution, it has given insights into how mobile phone can be linked to human development frameworks, particularly, the capability approach. It has reported with empirical evidence how mobile phones have the capabilities of expanding the well-being and empower the physically challenged in a developing country. The findings from the study is significant to policy and policy makers in addressing the challenges faced by disabled in Nigeria.

Future work needed in this research domain is to understand the underlying mechanisms that affect disabled people from using mobile phones to achieve empowerment. Mechanisms such as these can then be addressed to enhance the well-being of the physically challenged. The research could be important to a wider social domain, as findings from the research can be used to address social marginalization and inequalities among people living with disability. To this end, the prevailing notion of technology in the CA has not been harmonized empirically. A further work which this study intends to continue with is how to ex-plicitly justify the inclusion of technology (mobile phone) and disabilism into the CA, thus, contribute to the theoretical development of the CA. We believe that our contribution will fill an important theoretical gap in the CA and add to the current contribution by Haenssgen & Ariana (2017), who examines the concept of technology in the CA with the aim of understanding the technical objects, social structures and individuals. By putting disabilism within the CA, we are not adding another framework. Instead, our goal is to contribute to the harmonizing discussion on technology and disabilism in the CA.

  ;[START_REF] Kane | Freedom to Roam: A Study of Mobile Device Adoption and Accessibility for People with Visual and Motor Disabilities[END_REF][START_REF] Toboso | Rethinking Disability in Amartya Sen's Approach: ICT and Equality of Opportunity[END_REF][START_REF] Chaudhry | Rethinking the Digital Divide in Relation to Visual Disability in India and the United States: Towards a Paradigm of "Information Inequity[END_REF][START_REF] Pal | Technology for Employability in Latin America: Research with At-risk Youth and People with Disabilities[END_REF], there are no empirical evidences of studies on the use of mobile phones in empowering the physically challenged. Studies have categories the PwDs into the virtually impaired (Hashemi, Khabazkhoob, Saatchi & Yekta, 2018), hearing impaired[START_REF] Qi | Large scale academic achievement testing of deaf and hard of hearing students: Past, present and future[END_REF], mental health conditions[START_REF] Kleinman | Global mental health: a failure of humanity[END_REF], intellectual disability[START_REF] Lindsay | Research and literature on sex offenders with intellectual and developmental disabilities[END_REF], autism spectrum disorder (Virnes, Karna & Vellonen, 2015) and the physical disability or physically challenged[START_REF] Miyahara | Self-Esteem of Children and Adolescents with Physical Disabilities: Quantitative Evidence from Meta-Analysis[END_REF]. To contextualize disability in this study, our focus is on the physically

	physically challenged persons
	in Nigeria. Thus, the study asked the following question:
	What are the effects of mobile phone use in empowering and well-being of people
	with physical disabilities in Nigeria?

challenged (Wheelchair users). Therefore, we define the physically challenged people as those who have significant limitations in functioning and often experience exclusion from participation in their societies due to their impairments (Krahn, Walker, & Correa-De-Araujo, 2015). This research is part of a larger PhD work that is looking at how the use of mobile phones can empower the physically challenged in Nigeria. Our emphasis is on understanding how the use of mobile phones can empower the physically challenged. This is accomplished by understanding and framing technology use from the human development point of view. Human development stresses on a community where individual dreams can be actualized. This perspective is similar in view to that expressed in Amartya Sen's work

(1985, 1999)

on freedom of choice. Sen developed a framework for evaluating individual well-being called the Capability Approach (CA). The CA has been widely used by various researchers (Grunfeld, Hak, & Pin, 2011; Zheng & Walsham, 2008; Robeyns, 2005; Nussbaum, 2000, Dasuki et al. 2017) and organizations as published in the United Nations Development Programme (UNDP) report on human development. In this study, a number of concepts including empowerment, well-being, human development, the CA and ICT4D are employed with emphasis on disability. Consequently, the study's focus is to explore the capabilities of mobile usage in empowering and improving the well-being of the

2 Research context: Physically challenged and Mobile phones in Nigeria

	There are approximately 25 million people living with disabilities in Nigeria
	(Eleweke, 2013; Obiakor & Eleweke, 2014), of these, those using mobile phones for
	their daily activities is not known. The Nigerian government has provided support to
	the people with disability through services offered by the skill acquisition centers es -
	tablished in every state of the country. The skill acquisition centers are saddled with
	the responsibilities of empowering people with disabilities including the physically
	challenged. Skill training offered include making of shoes, bags and soaps. This ini-
	tiative has no doubt empowered people with disability. Nonetheless, evidence have
	shown that PwDs continue to face widespread discrimination, barriers in accessing
	service and societal exclusion (Eleweke, 2013; Obiakor & Eleweke, 2014).

Appendix