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Background Visceral leishmaniasis (VL), a life-threatening neglected tropical disease, is targeted for elimination from Nepal by the year 2026. The national VL elimination programme is still confronted with many challenges including the increasingly widespread distribution of the disease over the country, local resurgence and the questionable efficacy of the key vector control activities. We assessed the status and risk of Leishmania donovani transmission based on entomological indicators including seasonality, natural Leishmania infection rate, and feeding behavior of vector sand flies, Phlebotomus argentipes in three VL endemic districts in two epidemiologically contrasting settings; each district with one village with and one without reported VL cases in recent years.Methods Adult sand flies were collected using CDC light traps and mouth aspirators in each village for 12 consecutive months from July 2017 to June 2018. Sand fly species level identification was based on morphological characters and verified by the DNA barcoding method. We assessed P. argentipes abundance and seasonality based on the light trap collections, considering it as the standard method of collection. Leishmania infection was assessed in gravid sand flies targeting the small-subunit ribosomal RNA gene of the parasite (SSU-rRNA) and further sequenced for species identification. A segment (~ 350 bp) of the vertebrate cytochrome b (cytb) gene was amplified from blood-fed P. argentipes from dwellings shared by both humans and cattle and sequenced to identify the preferred host.Results Vector abundance varied among districts and village types and peaks in sand fly density was observed in June-July and September-November. The estimated Leishmania infection rate in vector sand flies was 2.2% (1.1% — 3.7% at 95% credible interval) and 0.6% (0.2% — 1.3% at 95% credible interval) in VL and non-VL villages respectively. The common source of blood meal was humans, 52.7% in VL villages and 74.2% in non-VL villages, followed by cattle.Conclusion Our findings highlight the risk of ongoing L. donovani transmission in VL endemic districts even in villages not reporting the presence of the disease over the past many years and emphasize the remaining threats to the VL elimination programme in Nepal.

Background: Malaria is still a major health problem in Nepal as 43.3% of population are at risk of infection. However, the national priorities have been given to achieve malaria elimination in Nepal, the question of diag­nostic accuracy could jeopardize the elimination target. Among others, multiplex PCR could be the reliable alternative tool to detect the low num­ber of parasitemia and circulating asymptomatic infection in low endemic countries like Nepal. Hence, this paper aims to analyse the diagnostic pre­cision among microscopy, rapid diagnostic test (RDT) and multiplex real time PCR assay. Methods: A total of 63 archived blood samples obtained from patient with clinical suspicion of malaria were subjected to multiplex PCR and their re­sults were compared with microscopy and rapid diagnostic test. The La­tent Class Analysis (LCA) approach was used to compare the diagnostic precision among three different assays. Results: Our study documented that PCR had 97.4% sensitivity and 83.9% specificity. Among the negative microscopy results, 10 (15.9%) were false negatives whereas 4 (6.3%) RDT results were false negatives based on PCR results. Conclusions: PCR could be the useful alternative tool to detect the low number of parasite in low endemic settings which could be crucial to rule out the false diagnosis determined by microscopy or RDT.

Antibiotic Resistance and mecA Gene Characterization of Coagulase-negative Staphylococci Isolated from Clinical Samples in Nepal

Background. Carbapenem resistance among Gram-negative isolates caused by the production of the metallo-β-lactamase (MBL) enzyme is being increasingly reported worldwide. One of the newly emerged metallo-β-lactamases is New Delhi metallo-β-lactamase. Data regarding its occurrence in hospital setting and percentage prevalence among different Gram-negative bacterial isolates are lacking in our part. This study has been undertaken for determining the presence of the bla NDM-1 gene among the clinical isolates of imipenem-resistant Gram-negative bacteria in a tertiary care center in Dharan, Nepal. Methods. A total of 75 imipenem-resistant Gram-negative isolates were studied. These were screened for metallo-β-lactamase (MBL) production by phenotypic assays such as double-disc synergy test (DDST) and combined disc diffusion test (CDDT). PCR was performed for the molecular detection of gene NDM-1. Ten-disc method was performed to detect the presence of ESBL, AmpC, carbapenamase, and K1 β-lactamase production. Results. Using the molecular method, bla NDM-1 was detected in 36% of the isolates. Phenotypically, double-disc synergy test (DDST) and combined disc diffusion test (CDST) detected MBL production in 38.7% and 37.3% of the isolates, respectively. Ten-disc method detected ESBL in 26.6% of the isolates, but none of the isolates was found to be AmpC, carbapenamase, and K1 β-lactamase producers. Conclusion. A high percentage of the NDM-1 producer was noted among imipenem-resistant GNB. Apart from performing only antimicrobial sensitivity test, phenotypic and molecular screening should be employed to find out the actual number of metallo-β-lactamase producers and the existence of the resistance gene.

The Acinetobacter species is an important hospital-acquired pathogen. The rapid development of resistance to multiple drugs and the ability to form biofilm make these bacteria more adaptable to survive in healthcare facilities, thus posing a challenge to their effective management.This study aimed to characterize clinical isolates of Acinetobacter spp and to study their antimicrobial susceptibility patterns and ability to form biofilm. Resistant Acinetobacter was further analyzed for the detection of extended-spectrumA total of 324 Acinetobacter species were isolated from various clinical specimens which were submitted to the Department of Microbiology, B.P. Koirala Institute of Health Sciences, Dharan, Nepal, and were studied for antibiotic susceptibility testing, detection of ESBL and MBL production, and formerly biofilm formation was performed by standard microbiological methods. PCR was carried out to determine the presence of the blaNDM-1 gene.The predominant Acinetobacter species isolated was A calcoaceticus-baumannii Complex (Acb complex) 167 (51.5%). Among those, all A. species 128 (40%) were multidrug resistant (MDR). In which 13 (4.0%) were ESBL producers, 70 (61.9%) were MBL, and 12 (10.6%) were carbapenemases producers. The blaNDM1 gene was present in 33 isolates. Thirty-seven percent (121/324) of isolates formed biofilm. The majority of A. species were resistant to cefotaxime 73.8% (239) and cefepime 74.4% (241). A significant proportion of biofilm producers were MDR (Drug-resistant Acinetobacter formed a substantial proportion of this hospital's samples with a large presence of the

Post-kala-azar dermal leishmaniasis (PKDL) is a skin manifestation of visceral leishmaniasis (VL) which develops after apparent cure in some patients. PKDL is considered as the potential reservoir for the VL infection. Molecular epidemiological characterization ofL. donovaniisolates obtained from VL and PKDL isolates is essentially required in order to understand the transmission dynamics of the VL infection. To date, genetic variation among the VL and PKDLL. donovaniisolates was not fully elucidated. Therefore, 14 clinical isolates from VL and 4 clinical isolates from PKDL were speciated byhsp70and rDNA genes. Further characterization ofL. donovanibyhaspBPCR demonstrates two different genotypes. All PKDL isolates have the same genetic structure. kDNA PCR-RFLP assay revealed 18 different genotypes; however, structural analysis showed the two distinct kDNA genotype population (k = 2). The kDNA fingerprint patterns of parasites from hilly districts were clustered separately from low-land districts. Therefore, further study with a large number of samples is urgently required for systematic characterization of the clinical isolates to track the molecular epidemiology of theLeishmania donovanicausing VL and the role of PKDL as a reservoir.

Background For ongoing malaria elimination programme, available methods like microscopy and rapid diagnostic test (RDT) are not able to detect all the cases of malaria in acute febrile illness. These methods are entirely dependent on the course of infection, parasite load and skilled technical resources thus the study was carried out to compare performance of microscopy and RDT which are commonly used in a low resource country along with reference to real-time PCR. Methods Altogether 52 blood samples collected from patients with acute febrile illness were tested by microscopy, RDT and real-time PCR. The results were compared in terms of sensitivity and specificity. Results The test results were as follows: 5.8% positivity by Microscopy, 13.5% positivity by RDT and 27% by real time PCR. Taking into consideration of PCR as a gold standard method microscopy showed 21.4% sensitivity and 100% specificity. Likewise, RDT results revealed 28.6% sensitivity and 92.1% specificity. Conclusion Despite of various diagnostic tools available, microscopy stills remains the gold standard for the diagnosis and RDT is the user friendly to execute the test under the tree, but our preliminary results emphasized the need to implement the test with the higher sensitivity and specificity in context of malaria elimination programme which could be another important opportunity to understand the parasite circulation in case of low endemic region. However, these results should be further verified with the large study cohort in order to document the submicroscopic infection.

Background. Helicobacter pylori infection is most prevalent in developing countries. It is an etiological agent of peptic ulcer, gastric adenocarcinoma, and mucosal-associated lymphoid tissue (MALT) lymphoma. Despite the development of different assays to confirm H. pylori infection, the diagnosis of infection is challenged by precision of the applied assay. Hence, the aim of this study was to understand the diagnostic accuracy of PCR and microscopy to detect the H. pylori in the gastric antrum biopsy specimen from gastric disorder patients. Methods. A total of 52 patients with gastric disorders underwent upper gastrointestinal endoscopy with biopsy. The H. pylori infection in gastric biopsies was identified after examination by microscopy and 23S rRNA specific PCR. The agreement between two test results were analysed by McNemar’s test and Kappa coefficient. Result. H. pylori infection was confirmed in 9 (17.30%) patients by both assays, 6.25% in antral gastritis, 22.22% in gastric ulcer, 100% in gastric ulcer with duodenitis, 50% in gastric ulcer with duodenal ulcer, and 33.33% in severe erosive duodenitis with antral gastritis. Out of nine H. pylori infection confirmed patients, 3 patients were confirmed by microscopy and 8 patients by PCR. In case of two patients, both microscopy and PCR assay confirmed the H. pylori infection. The agreement between two test results was 86.54% and disagreed by 13.46% (p value > 0.05). Conclusion. We found that PCR assay to detect H. pylori is more sensitive than microscopy. However, we advocate for the combination of both assays to increase the strength of diagnostic accuracy due to the absence of the gold standard assay for H. pylori infection.

Background: Acinetobacter species is an important hospital acquired pathogen. Rapid development of resistance to multiple drug and ability to form biofilm make these bacteria more adaptable to survive in health care facilities, thus posing a challenge for its effective management. This study was aimed to characterize clinical isolates of Acinetobacter spp, study their antimicrobial susceptibility pattern and ability to form biofilm. Resistant Acinetobacter was further analyzed for the detection of extended spectrum β-lactamases (ESBLs), metallo β-lactamases (MBLs) production and presence of blaNDM-1 (New Delhi metallo-beta-lactamase-1) gene.Results: A total of 324 Acinetobacter species isolated from various clinical specimens submitted to Department of Microbiology, B. P. Koirala Institute of Health Sciences, Dharan, were studied Antibiotic susceptibility testing, detection of ESBL and MBL production, and biofilm formation was performed by standard microbiological methods. PCR was done to determine the presence of blaNDM-1 gene.Predominant Acinetobacter species isolated was A calcoaceticus-baumannii Complex (Acb complex) 167 (51.5%). 13 (4.0%) were ESBL producers, 61.9% (70/113) were MBL, 10.6% (12/113) were carbapenemase producers. About 40% were Multi-drug resistant (MDR). Among MDR, producers of ESBL, MBL and carbapenemase were 1.6%, 57% and 9.6%, respectively. Thirty seven percent isolates formed biofilm. A significant proportion of biofilm producers were MDR (p

The diagnosis of extra-pulmonary tuberculosis by Mycobacterium tuberculosis infection in retro­pharyngeal and thyroid abscess is challenging due to a lack of rapid, sensitive and specific diag­nostic assay. Hence, we evaluated the performance of highly sensitive polymerase chain reaction (PCR) to detect the M. tuberculosis in the retropharyngeal and thyroid abscess. The microscopy and culture were negative test result however; PCR result had demonstrated the presence of M. tuberculosis in the same specimens with retropharyngeal and thyroid abscess. Therefore, it is rec­ommended to perform PCR to detect undiagnosed cases, which were not detected by convention­al approaches for the diagnosis, of extra-pulmonary tuberculosis.

Post-kala-azar dermal leishmaniasis (PKDL) is a skin manifestation of visceral leishmaniasis (VL) which develops after apparent cure in some patients. PKDL is considered as the potential reservoir for the VL infection. Molecular epidemiological characterization of L. donovani isolates obtained from VL and PKDL isolates is essentially required in order to understand the transmission dynamics of the VL infection. To date, genetic variation among the VL and PKDL L. donovani isolates was not fully elucidated. Therefore, 14 clinical isolates from VL and 4 clinical isolates from PKDL were speciated by hsp70 and rDNA genes. Further characterization of L. donovani by haspB PCR demonstrates two different genotypes. All PKDL isolates have the same genetic structure. kDNA PCR-RFLP assay revealed 18 different genotypes; however, structural analysis showed the two distinct kDNA genotype population (k = 2). The kDNA fingerprint patterns of parasites from hilly districts were clustered separately from low-land districts. Therefore, further study with a large number of samples is urgently required for systematic characterization of the clinical isolates to track the molecular epidemiology of the Leishmania donovani causing VL and the role of PKDL as a reservoir.

Author Correction: A multicentric evaluation of dipstick test for serodiagnosis of visceral leishmaniasis in India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain. Scientifc Reports. 2021; 11:3967. DOI: 10.1038/s41598-021-83332-8. PMID: 33574485 Visceral leishmaniasis (VL) is one of the leading infectious diseases affecting developing countries. Colloidal gold-based diagnostic tests are rapid tools to detect blood/serum antibodies for VL diagnosis. Lack of uniformity in the performance of these tests in different endemic regions is a hurdle in early disease diagnosis. This study is designed to validate a serum-based dipstick test in eight centres of six countries, India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain with archived and fresh sera from 1003 subjects. The dipstick detects antibodies against Leishmania donovani membrane antigens (LAg). The overall sensitivity and specificity of the test with 95% confidence intervals were found to be 97.10% and 93.44%, respectively. The test showed good sensitivity and specificity in the Indian subcontinent (>95%). In Brazil, Ethiopia, and Spain the sensitivity and specificity of the dipstick test (83.78-100% and 79.06-100%) were better as compared to the earlier reports of the performance of rK39 rapid test in these regions. Interestingly, less cross-reactivity was found with the cutaneous form of the disease in Spain, Brazil, and Sri Lanka demonstrating 91.58% specificity. This dipstick test can therefore be a useful tool for diagnosing VL from other symptomatically similar diseases and against cutaneous form of leishmaniasis. Sí

Background Leishmania donovani is the etiological agent of visceral leishmaniasis (VL) in the Indian subcontinent. However, it is also known to cause cutaneous leishmaniasis (CL) in Sri Lanka. Sri Lankan L. donovani differs from other L. donovani strains, both at the molecular and biochemical level. To investigate the different species or strain-specific differences of L. donovani in Sri Lanka we evaluated sequence variation of the kinetoplastid DNA (kDNA). Methods Parasites isolated from skin lesions of 34 CL patients and bone marrow aspirates from 4 VL patients were genotyped using the kDNA minicircle PCR analysis. A total of 301 minicircle sequences that included sequences from Sri Lanka, India, Nepal and six reference species of Leishmania were analyzed. Results Haplotype diversity of Sri Lankan isolates were high (H d = 0.757) with strong inter-geographical genetic differentiation (F ST > 0.25). In this study, L. donovani isolates clustered according to their geographic origin, while Sri Lankan isolates formed a separate cluster and were clearly distinct from other Leishmania species. Within the Sri Lankan group, there were three distinct sub-clusters formed, from CL patients who responded to standard antimony therapy, CL patients who responded poorly to antimony therapy and from VL patients. There was no specific clustering of sequences based on geographical origin within Sri Lanka. Conclusion This study reveals high levels of haplotype diversity of L. donovani in Sri Lanka with a distinct genetic association with clinically relevant phenotypic characteristics. The use of genetic tools to identify clinically relevant features of Leishmania parasites has important therapeutic implications for leishmaniasis. Electronic supplementary material The online version of this article (10.1186/s12879-017-2883-x) contains supplementary material, which is available to authorized users.

Leishmania parasite isolation from the human aspirates is always challenging due to most probability of the fungal contamination and the use of antifungal drug which could support the selective growth of the Leishmania parasite. In this study, we examine the effect of antifungal drug caspofungin on the promastigote stage of Leishmania donovani. Promastigote parasite was cultivated in M199 + 20% heat-inactivated fetal calf serum and plated in 96-well plates. Seven different concentrations of caspofungin (512 µg/ml to 8 µg/ml) were exposed to parasites, and 50% inhibitory concentration (IC50) was calculated. Candida spp. was used in the experiments to know the efficacy of caspofungin to inhibit fungal growth. The IC50 values of Leishmania strains ranged from 23.02 to 155.80 µg/ml (mean 90.25 ± 39.01 µg/ml), and it was significantly higher (P value = 0.02) than IC50 values of Candida spp. (ranged from 0.001 to 0.12 µg/ml, mean = 0.05 ± 0.05 µg/ml). The reduced growth rate of the parasite was found with exposure to 50 µg/ml of caspofungin. Growth inhibition of Leishmania donovani is significantly lower with caspofungin and could be used to protect the parasite cultivation from fungal contamination.

The isolation by distance analysis showed the linear correlation between genetic diversity and geographic distance. Three populations were defined such as Sri Lanka, India and Nepal. However, this correlation was not statistically significant. (DOC 175Â kb)

Background. Miltefosine (MIL), the only oral drug for visceral leishmaniasis (VL), is currently the first-line therapy in the VL elimination program of the Indian subcontinent. Given the paucity of anti-VL drugs and the looming threat of resistance, there is an obvious need for close monitoring of clinical efficacy of MIL. Methods. In a cohort study of 120 VL patients treated with MIL in Nepal, we monitored the clinical outcomes up to 12 months after completion of therapy and explored the potential role of drug compliance, parasite drug resistance, and reinfection. Results. The initial cure rate was 95.8% (95% confidence interval [CI], 92.2-99.4) and the relapse rate at 6 and 12 months was 10.8% (95% CI, 5.2-16.4) and 20.0% (95% CI, 12.8-27.2), respectively. No significant clinical risk factors of relapse apart from age

Leishmania donovani is an intracellular protozoan parasite that causes leishmaniasis, which can range from a self-healing cutaneous disease to a fatal visceral disease depending on the infecting species. Miltefosine is currently the latest and only oral antileishmanial that came out of drug discovery pipelines in the past few decades, but recent reports indicate a significant decline in its efficacy against visceral leishmaniasis (also known as kala-azar) in the Indian subcontinent. This relapse rate of up to 20% within 12 months after treatment was shown not to be related to reinfection, drug quality, drug exposure, or drug-resistant parasites. We therefore aimed to assess other phenotypes of the parasite that may affect treatment outcome and found a significant association between the number of metacyclic parasites, parasite infectivity, and patient treatment outcome in the Indian subcontinent. Together with previous studies on resistance of L. donovani against pentavalent antimonials, these data suggest that the infectivity of the parasite, or related phenotypes, might be a more determinant factor for treatment failure in visceral leishmaniasis than drug susceptibility, warranting a reassessment of our current view on treatment failure and drug resistance in leishmaniasis and beyond., IMPORTANCE The high miltefosine relapse rate poses a major challenge for the current Kala-Azar Elimination Program in the Indian subcontinent and other leishmaniasis control programs worldwide. This relapse rate could not be related to reinfection, drug-resistant parasites, or reduced treatment quality. Here we report that an increased infectivity of the parasite is associated with miltefosine relapse of visceral leishmaniasis (VL) patients. These results supplement those obtained with antimonial-resistant L. donovani where an increased infectivity was also observed. This challenges the current view of Leishmania drug susceptibility being the biggest parasitic factor that contributes to treatment failure in leishmaniasis. These selected more infectious parasites may pose an additional burden to leishmaniasis control programs, highlighting the importance of multifaceted control measures to achieve leishmaniasis elimination in the Indian subcontinent and other regions where leishmaniasis is endemic.

Background Detection of Mycobacterium leprae in slit skin smear (SSS) is a gold standard technique for the leprosy diagnosis. Over recent years, molecular diagnosis by using PCR has been increasingly used as an alternative for its diagnosis due to its higher sensitivity. This study was carried out for comparative evaluation of PCR and SSS microscopy in a cohort of new leprosy cases diagnosed in B. P. Koirala Institute of health Sciences, Dharan, Nepal. Methodology/Principal Findings In this prospective crossectional study, 50 new clinically diagnosed cases of leprosy were included. DNA was extracted from SSS and PCR was carried out to amplify 129 bp sequence of M. leprae repetitive element. Sensitivity of SSS and PCR was 18% and 72% respectively. Improvement of 54% case detection by PCR clearly showed its advantage over SSS. Furthermore, PCR could confirm the leprosy diagnosis in 66% of AFB negative cases indicating its superiority over SSS. In the paucibacillary (PB) patients, whose BI was zero; sensitivity of PCR was 44%, whereas it was 78% in the multibacillary patients. Conclusions/Significance Our study showed PCR to be more sensitive than SSS microscopy in diagnosing leprosy. Moreover, it explored the characteristic feature of PCR which detected higher level of early stage(PB) cases tested negative by SSS. Being an expensive technique, PCR may not be feasible in all the cases, however, it would be useful in diagnosis of early cases of leprosy as opposed to SSS., Author Summary Although leprosy has been eliminated at the national level, but region wise eastern Nepal has still the higher number of leprosy cases. Early diagnosis and then timely treatment is very crucial to prevent disabilities due to leprosy. Slit-skin smear (SSS), the routine diagnostic method was compared with the molecular method polymerase chain reaction (PCR) for the detection of new clinically diagnosed cases at BPKIHS. PCR was found to be much more superior than SSS in its diagnostic efficacy detecting more than half number of cases missed by SSS. Moreover our finding of PCR in detection of early stages was nearly 50% where SSS are mostly negative on such spectrum. Therefore, this costly but sensitive diagnostic tool PCR was found to be a better option in detection of early cases of leprosy in comparison to SSS.

Irradiation of 2-formylbenzylamine 7 in the presence of fumarates affords adducts corresponding to trapping of quinodimethane intermediate 9, while photolysis of 2-formylbenzamides leads to amino lactones 16.

Irradiation of 2-formylbenzylamine 7 in the presence of fumarates affords adducts corresponding to trapping of quinodimethane intermediate 9, while photolysis of 2-formylbenzamides leads to amino lactones 16.

Multiple sequence alignment. Multiple sequence alignment of the Sri Lankan, Indian and Nepalese isolates along with the reference sequences using CLUSTAL 2.1 multiple sequence alignment. Dashes(â ) represent gaps introduced to optimize alignment. Asteriks (*) represent consensus nucleotides in the sequence. Lb(P):L. braziliensis [Peru] (Skin);Ld(DD8)/Ld(IN):L. donovani [India] (Bone marrow); Li(SP):L. infantum [Spain] (Unknown); Lm(A1):L. major [Libya]; Lt(TK):L. tropica [Turkey] (Skin); Lam: L. amazonenis (Skin); Li(TK):L. infantum [Turkey] (Spleen); SL-VL (52â 55): VL Sri Lankan isolate (Bone marrow); SL_CL-S:Leishmania CL Sri Lankan isolate (Skin); SL_CL-DR:Leishmania CL Sri Lankan that failed to respond to antimonial drugs (Skin); IN-VL: L. donovani [India] (Bone marrow); IN-PKDL: Leishmania from post-kala azar dermal leishmaniasis (PKDL) [India] (Skin); NP-VL: Leishmania VL Nepalian isolate (Bone marrow); NP-PKDL:Leishmania cause PKDL [Nepal] (Skin). (DOCX 25Â kb)

Multiple sequence alignment. Multiple sequence alignment of the Sri Lankan, Indian and Nepalese isolates along with the reference sequences using CLUSTAL 2.1 multiple sequence alignment. Dashes(â ) represent gaps introduced to optimize alignment. Asteriks (*) represent consensus nucleotides in the sequence. Lb(P):L. braziliensis [Peru] (Skin);Ld(DD8)/Ld(IN):L. donovani [India] (Bone marrow); Li(SP):L. infantum [Spain] (Unknown); Lm(A1):L. major [Libya]; Lt(TK):L. tropica [Turkey] (Skin); Lam: L. amazonenis (Skin); Li(TK):L. infantum [Turkey] (Spleen); SL-VL (52â 55): VL Sri Lankan isolate (Bone marrow); SL_CL-S:Leishmania CL Sri Lankan isolate (Skin); SL_CL-DR:Leishmania CL Sri Lankan that failed to respond to antimonial drugs (Skin); IN-VL: L. donovani [India] (Bone marrow); IN-PKDL: Leishmania from post-kala azar dermal leishmaniasis (PKDL) [India] (Skin); NP-VL: Leishmania VL Nepalian isolate (Bone marrow); NP-PKDL:Leishmania cause PKDL [Nepal] (Skin). (DOCX 25Â kb)