Whole genome analysis of Human Mastadenovirus D causing Keratoconjunctivitis in India - A multicentre study.

INTRODUCTION: Human mastadenovirus (HAdV) types 8, 37, 64 have been considered the major contributors in Epidemic keratoconjunctivitis (EKC) epidemics, but recent surveillance data have shown the involvement of emerging recombinants, including HAdV-53, HAdV-54, and HAdV-56. In our initial work, positive samples for adenovirus revealed that our strains were closer to HAdV-54 than HAdV-8. Hence, the current study aimed to use whole genome technology to identify the HAdV strain correctly. METHODOLOGY: Oxford Nanopore technique was used, wherein a Targeted sequencing approach using long-range PCR amplification was performed. Primers were designed using HAdV-54 (AB448770.2) and HAdV-8 (AB897885.1) as reference sequences. Amplicons were sequenced on the GridION sequencer. Sequences were annotated using Gatu software, and similarities with standard reference sequence was calculated using Bioedit software. The phylogenetic tree was built after alignment in MEGA v7.0 using Neighbour joining method for each of the genes: Penton, Hexon, and Fiber. The effect of novel amino acid changes was evaluated using the PROVEAN tool. The Recombination Detection Program (RDP) package Beta 4.1 was used to identify recombinant sequences. RESULTS: Of the five samples sequenced, OL450401, OL540403, and OL540406 showed nucleotide similarity to HAdV-54 in the penton region. Additionally, OL450401 showed a statistically significant recombination event with HAdV-54 as minor and HAdV-8 as major parents. This was further supported by phylogenetic analysis as well. CONCLUSIONS: In the present study, we have found evidence of a shift from HAdV-8 towards HAdV-54, thus stressing the need for surveillance of HAdVs and to stay updated on the rise of new recombinants.

Humoral Immune Response Profile of COVID-19 Reveals Severity and Variant-Specific Epitopes: Lessons from SARS-CoV-2 Peptide Microarray.

The amaranthine scale of the COVID-19 pandemic and unpredictable disease severity is of grave concern. Serological diagnostic aids are an excellent choice for clinicians for rapid and easy prognosis of the disease. To this end, we studied the humoral immune response to SARS-CoV-2 infection to map immunogenic regions in the SARS-CoV-2 proteome at amino acid resolution using a high-density SARS-CoV-2 proteome peptide microarray. The microarray has 4932 overlapping peptides printed in duplicates spanning the entire SARS-CoV-2 proteome. We found 204 and 676 immunogenic peptides against IgA and IgG, corresponding to 137 and 412 IgA and IgG epitopes, respectively. Of these, 6 and 307 epitopes could discriminate between disease severity. The emergence of variants has added to the complexity of the disease. Using the mutation panel available, we could detect 5 and 10 immunogenic peptides against IgA and IgG with mutations belonging to SAR-CoV-2 variants. The study revealed severity-based epitopes that could be presented as potential prognostic serological markers. Further, the mutant epitope immunogenicity could indicate the putative use of these markers for diagnosing variants responsible for the infection.

Immune profile and responses of a novel dengue DNA vaccine encoding an EDIII-NS1 consensus design based on Indo-African sequences.

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1-4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy.

Opposing roles for sMAdCAM and IL-15 in COVID-19 associated cellular immune pathology.

Immune cell dysregulation and lymphopenia characterize COVID-19 pathology in moderate to severe disease. While underlying inflammatory factors have been extensively studied, homeostatic and mucosal migratory signatures remain largely unexplored as causative factors. In this study, we evaluated the association of circulating IL-6, soluble mucosal addressin cell adhesion molecule (sMAdCAM), and IL-15 with cellular dysfunction characterizing mild and hypoxemic stages of COVID-19. A cohort of SARS-CoV-2 infected individuals (n = 130) at various stages of disease progression together with healthy controls (n = 16) were recruited from COVID Care Centres (CCCs) across Mumbai, India. Multiparametric flow cytometry was used to perform in-depth immune subset characterization and to measure plasma IL-6 levels. sMAdCAM, IL-15 levels were quantified using ELISA. Distinct depletion profiles, with relative sparing of CD8 effector memory and CD4+ regulatory T cells, were observed in hypoxemic disease within the lymphocyte compartment. An apparent increase in the frequency of intermediate monocytes characterized both mild as well as hypoxemic disease. IL-6 levels inversely correlated with those of sMAdCAM and both markers showed converse associations with observed lympho-depletion suggesting opposing roles in pathogenesis. Interestingly, IL-15, a key cytokine involved in lymphocyte activation and homeostasis, was detected in symptomatic individuals but not in healthy controls or asymptomatic cases. Further, plasma IL-15 levels negatively correlated with T, B, and NK count suggesting a compensatory production of this cytokine in response to the profound lymphopenia. Finally, higher levels of plasma IL-15 and IL-6, but not sMAdCAM, were associated with a longer duration of hospitalization.

sMAdCAM: IL-6 Ratio Influences Disease Progression and Anti-Viral Responses in SARS-CoV-2 Infection.

The role of sMAdCAM, an important gut immune migratory marker, remains unexplored in COVID-19 pathogenesis considering recent studies positing the gut as a sanctuary site for SARS-CoV-2 persistence. Thus, assimilating profiles of systemic inflammatory mediators with sMAdCAM levels may provide insights into the progression of COVID-19 disease. Also, the role of these markers in governing virus specific immunity following infection remains largely unexplored. A cohort (n = 84) of SARS-C0V-2 infected individuals included a group of in-patients (n = 60) at various stages of disease progression together with convalescent individuals (n = 24) recruited between April and June 2020 from Mumbai, India. Follow-up of 35 in-patients at day 7 post diagnosis was carried out. Th1/Th2/Th17 cytokines along with soluble MAdCAM (sMAdCAM) levels in plasma were measured. Also, anti-viral humoral response as measured by rapid antibody test (IgG, IgM), Chemiluminescent Immunoassay (IgG), and antibodies binding to SARS-CoV-2 proteins were measured by Surface Plasmon Resonance (SPR) from plasma. IL-6 and sMAdCAM levels among in-patients inversely correlated with one another. When expressed as a novel integrated marker-sMIL index (sMAdCAM/IL-6 ratio)-these levels were incrementally and significantly higher in various disease states with convalescents exhibiting the highest values. Importantly, sMAdCAM levels as well as sMIL index (fold change) correlated with peak association response units of receptor binding domain and fold change in binding to spike respectively as measured by SPR. Our results highlight key systemic and gut homing parameters that need to be monitored and investigated further to optimally guide therapeutic and prophylactic interventions for COVID-19.

A novel ORF1a-based SARS-CoV-2 RT-PCR assay to resolve inconclusive samples.

BACKGROUND: India bears the second largest burden of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. A multitude of reverse transcription polymerase chain reaction (RT-PCR) detection assays with disparate gene targets, including automated high-throughput platforms, are available. Varying concordance and interpretation of diagnostic results in this setting can result in significant reporting delays, leading to suboptimal disease management. This article reports the development of a novel ORF1a-based SARS-CoV-2 RT-PCR assay - Viroselect - that shows high concordance with conventional assays and the ability to resolve inconclusive results generated during the peak of the epidemic in Mumbai, India. METHODS: A unique target region within SARS-CoV-2 ORF1a - the non-structural protein 3 (nsp3) region - was used to design and develop the assay. This hypervariable region (1923-3956) between SARS-CoV-2, SARS-CoV-1 and Middle East respiratory syndrome coronavirus was utilized to design the primers and probes for the RT-PCR assay. The concordance of this assay with commonly used emergency use authorization (US Food and Drug Administration) manual kits and an automated high-throughput testing platform was evaluated. Further, a retrospective analysis was carried out using Viroselect on samples reported as 'inconclusive' between April and October 2020. RESULTS: In total, 701 samples were tested. Concordance analysis of 477 samples demonstrated high overall agreement of Viroselect with both manual (87.6%) and automated (84.7%) assays. Also, in the retrospective analysis of 224 additional samples reported as 'inconclusive', Viroselect was able to resolve 100% (19/19) and 93.7% (192/205) of samples which had inconclusive results on manual and automated high-throughput platforms, respectively. CONCLUSION: Viroselect had high concordance with conventional assays, both manual and automated, and has potential to resolve inconclusive samples.

Ileal Perforation and Enteric Fever: Implications for Burden of Disease Estimation.

BACKGROUND: Ileal perforation occurs in about 1% of enteric fevers as a complication, with a case fatality risk (CFR) of 20%-30% in the early 1990s that decreased to 15.4% in 2011 in South East Asia. We report nontraumatic ileal perforations and its associated CFR from a 2-year prospective enteric fever surveillance across India. METHODS: The Surveillance for Enteric Fever in India (SEFI) project established a multitiered surveillance system for enteric fever between December 2017 and March 2020. Nontraumatic ileal perforations were surveilled at 8 tertiary care and 6 secondary care hospitals and classified according to etiology. RESULTS: Of the 158 nontraumatic ileal perforation cases identified,126 were consented and enrolled. Enteric fever (34.7%), tuberculosis (19.0%), malignancy (5.8%), and perforation of Meckel diverticulum (4.9%) were the common etiology. In those with enteric fever ileal perforation, the CFR was 7.1%. CONCLUSIONS: Enteric fever remains the most common cause of nontraumatic ileal perforation in India, followed by tuberculosis. Better modalities of establishing etiology are required to classify the illness, and frame management guidelines and preventive measures. CFR data are critical for comprehensive disease burden estimation and policymaking.

Laboratory preparedness for SARS-CoV-2 testing in India: Harnessing a network of Virus Research & Diagnostic Laboratories.

Background & objectives: An outbreak of respiratory illness of unknown aetiology was reported from Hubei province of Wuhan, People's Republic of China, in December 2019. The outbreak was attributed to a novel coronavirus (CoV), named as severe acute respiratory syndrome (SARS)-CoV-2 and the disease as COVID-19. Within one month, cases were reported from 25 countries. In view of the novel viral strain with reported high morbidity, establishing early countrywide diagnosis to detect imported cases became critical. Here we describe the role of a countrywide network of VRDLs in early diagnosis of COVID-19. Methods: The Indian Council of Medical Research (ICMR)-National Institute of Virology (NIV), Pune, established screening as well as confirmatory assays for SARS-CoV-2. A total of 13 VRDLs were provided with the E gene screening real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay. VRDLs were selected on the basis of their presence near an international airport/seaport and their past performance. The case definition for testing included all individuals with travel history to Wuhan and symptomatic individuals with travel history to other parts of China. This was later expanded to include symptomatic individuals returning from Singapore, Japan, Hong Kong, Thailand and South Korea. Results: Within a week of standardization of the test at NIV, all VRDLs could initiate testing for SARS-CoV-2. Till February 29, 2020, a total of 2,913 samples were tested. This included both 654 individuals quarantined in the two camps and others fitting within the case definition. The quarantined individuals were tested twice - at days 0 and 14. All tested negative on both occasions. Only three individuals belonging to different districts in Kerala were found to be positive. Interpretation & conclusions: Sudden emergence of SARS-CoV-2 and its potential to cause a pandemic posed an unsurmountable challenge to the public health system of India. However, concerted efforts of various arms of the Government of India resulted in a well-coordinated action at each level. India has successfully demonstrated its ability to establish quick diagnosis of SARS-CoV-2 at NIV, Pune, and the testing VRDLs.

Pulmonary cystic echinococcosis: A case report from tertiary care hospital.

Human echinococcosis is a zoonotic infection transmitted by dogs in livestock-raising areas. We present a case of a 30-year-old female with respiratory symptoms.

Respiratory Pathogens in Infants Diagnosed with Acute Lower Respiratory Tract Infection in a Tertiary Care Hospital of Western India Using Multiplex Real Time PCR.

OBJECTIVE: To determine the frequency of respiratory pathogens in infants diagnosed with acute lower respiratory tract infections. METHODS: A prospective cross-sectional observational study was conducted in infants hospitalized with a diagnosis of acute lower respiratory tract infection (ALRTI), in a tertiary care hospital in a metropolitan city of Western India. Nasopharyngeal swabs were analyzed by multiplex real time polymerase chain reaction, for 18 viruses and 3 bacteria (H. influenzae type b, C. pneumoniae and M. pneumoniae). The entire data was entered in Microsoft excel sheet and frequencies were determined. RESULTS: One hundred eligible infants were enrolled. Pathogens were detected in 82 samples, which included Respiratory syncytial viruses (RSV) A / B (35.4%), Human rhinovirus (25.6%), Adenovirus (22%), Human Parainfluenza viruses (11%), Human bocavirus (9.8), Human metapneumovirus A / B (8.5%), Influenza A (H1N1) pdm 09 (6.1%), Parechovirus (3.7%), Human coronaviruses (3.66%), Haemophilus influenzae type b (6.1%), Chlamydia pneumoniae (2.4%) and Mycoplasma pneumoniae (2.4%). Influenza A (other than H1N1), Influenza B, Human Coronavirus 229E and Enterovirus were not detected. The rate of coinfection was 34% and rhinovirus was the most common of the multiple pathogens. CONCLUSIONS: Spectrum of viral etiologies of ALRTI is highlighted. Etiological diagnosis of ALRTI would enable specific antiviral therapy, restrict antibiotic use and help in knowing burden of disease.

Prevalence of non A to E hepatitis in Mumbai, India / Prevalencia de la hepatitis no A /E en Mumbai, India

Introduction: Acute viral hepatitis is a common problem in India. World wide data shows that 5 to 20 percent of this is caused by non A-E hepatitis. There is no data in India regarding non A-E hepatitis. We carried out this study to evaluate the epidemiology, clinical features, risk factors and outcome of non A-E hepatitis. Material and methods: In this single centre study, we evaluated all patients admitted with features of acute viral hepatitis at our hospital between the period of February to July 2015. A detailed history about the epidemiology, risk factors and clinical features was done. Patients were evaluated with bilirubin, transaminases and prothrombin time. Each patient was investigated for IgM HAV, IgM HEV, HBsAg and Antibody against hepatitis C. Patients turning out negative were investigated for presence of autoimmune hepatitis or Wilson's disease. All viral markers were repeated a week later to confirm non A-E status. Results: A total 265 patients were included of which 41 (15.4%) patients were non A-E hepatitis. They had higher age (28.55 vs 34.99, p<0.05) but similar gender and sub urban location. Median SEC classification was A2 in hepatitis A/E group as compared to A3 in non A-E group. The duration of symptoms and clinical features between the two groups were similar with Anorexia, Malasie, Nausea/vomiting being most common. The risk factors between the two groups were similar. The bilirubin and transaminases were non significantly lower than hepatitis A/E patients while albumin levels were significantly lower. The outcomes of both groups were similar with no mortality or fulminant hepatitis. Conclusion: Non A-E hepatitis patients tends to be older, lower SEC class and had lower albumin levels as compared to hepatitis A/E

Introdución: La hepatitis viral aguda es un problema común en la India. Los datos mundiales indican que el 5 al 20% es causada por hepatitis no A-E. No hay datos en la India sobre hepatitis no A-E. Objetivo: Se realiza este estudio para evaluar la epidemiología, clínica, factores de riesgo y pronóstico de la hepatitis no A-E. Material y métodos: En este estudio de un solo centro evaluamos a todos los pacientes que se admitieron con clínica de hepatitis viral aguda en nuestro hospital en el periodo de febrero a julio del 2015. Se realizó una historia detallada para evaluar la epidemiología, características clínicas. Se les tomó bilirrubinas, transaminasas y tiempo de protrombina. A cada paciente se le realizó HAV IgM, HEV IgM, HbsAg y anticuerpo anti hepatitis C. Los que fueron negativos se les estudió para hepatitis autoinmune y enfermedad de Wilson. Todos los marcadores virales se repitieron a la semana para confirmar hepatitis no A-E. Resultados: Se incluyeron 256 pacientes, 41 de ellos (15,4%) fueron hepatitis no A-E. Tuvieron más edad (28,55 vs 34,99, p<0,05), pero el mismo género y ubicación urbana. La clasificación media SEC fue A2 en el grupo hepatitis A/E, comparada con 3 en el grupo de no A-E. La duración de los síntomas y el desarrollo clínico fue similar en ambos grupos, siendo la anorexia el malestar general, las náuseas y los vómitos los más frecuentes. El factor de riesgo fue similar, al igual que las transaminasas, mientras que la albúmina fue significativamente menor. El resultado fue similar sin caso alguno de hepatitis fulminante. Conclusión: Los pacientes con hepatitis no A-E tienden a ser mayores, de clase SEC más baja y con valores de albumina más bajos que los pacientes con hepatitis A-E

An outbreak of Burkholderia cepacia complex in the paediatric unit of a tertiary care hospital.

INTRODUCTION: Burkholderia cepacia complex (Bcc) has emerged as a serious nosocomial pathogen worldwide especially in patients with indwelling catheters and cystic fibrosis. Bcc is a common contaminant of pharmaceutical products. We describe an outbreak of Bcc bacteraemia amongst children admitted in Paediatric Intensive Care Unit (PICU) and paediatric ward at a tertiary care hospital, Mumbai, in Western India. MATERIALS AND METHODS: Blood culture samples from paediatric patients yielded growth of non-fermenting, oxidase positive, motile, Gram negative bacilli (NFGNB) (76/909) over a period of 8 months. Based on conventional biochemical tests and antimicrobial susceptibility testing, these isolates were provisionally identified as Bcc. The increased, repeated and continued isolation of Bcc alerted the possibility of an outbreak confined to PICU and paediatric ward. Active surveillance was undertaken to trace the source and contain the outbreak. Isolates were subjected to recA polymerase chain reaction (PCR) and Expanded multilocus sequence typing (EMLST). RESULTS: Surveillance revealed the presence of Bcc on the upper surface of rubber stopper of sealed multidose amikacin vials. Isolates from blood culture and rubber stoppers were confirmed as Bcc by recA PCR. EMLST revealed that these isolates shared an identical novel sequence type 824 proving clonality. Timely interventions instituted led to control of the outbreak. CONCLUSION: This study highlights the importance of identification and molecular characterization of Bcc to establish its role in infection and outbreak.

Multilocus sequence analysis reveals high genetic diversity in clinical isolates of Burkholderia cepacia complex from India.

Burkholderia cepacia complex (Bcc) is a complex group of bacteria causing opportunistic infections in immunocompromised and cystic fibrosis (CF) patients. Herein, we report multilocus sequence typing and analysis of the 57 clinical isolates of Bcc collected over the period of seven years (2005-2012) from several hospitals across India. A total of 21 sequence types (ST) including two STs from cystic fibrosis patient's isolates and twelve novel STs were identified in the population reflecting the extent of genetic diversity. Multilocus sequence analysis revealed two lineages in population, a major lineage belonging to B. cenocepacia and a minor lineage belonging to B. cepacia. Split-decomposition analysis suggests absence of interspecies recombination and intraspecies recombination contributed in generating genotypic diversity amongst isolates. Further linkage disequilibrium analysis indicates that recombination takes place at a low frequency, which is not sufficient to break down the clonal relationship. This knowledge of the genetic structure of Bcc population from a rapidly developing country will be invaluable in the epidemiology, surveillance and understanding global diversity of this group of a pathogen.

Subungual hyperkeratosis nail biopsy: a better diagnostic tool for onychomycosis.

BACKGROUND: Onychomycosis is one of the most common nail disorders. Mycological examination by potassium hydroxide (KOH) mount and fungal culture is the most commonly used diagnostic method. However, it is associated with a low sensitivity. AIMS: To evaluate the technique of subungual hyperkeratosis nail biopsy in diagnosing onychomycosis in HIV-infected and immunocompetent adults and compare it with mycological examination. METHODS: 34 HIV-positive patients who presented clinically with onychomycosis were recruited in the study from the beginning. There was no screening done for patients with onychomycosis. This has been clarified in manuscript under the heading of methods. RESULTS: All the fungal cultures yielded dermatophytes correlating with the biopsy findings. Only hyphal form of fungus was detected in KOH examination, indicating it was not a contaminant. Clinical types of onychomycosis are stated in discussion. CONCLUSIONS: PAS stain of subungual hyperkeratosis nail biopsy was the most sensitive in the diagnosis of onychomycosis in both HIV-infected and non-infected groups.

Malaria evolution in South Asia: knowledge for control and elimination.

The study of malaria parasites on the Indian subcontinent should help us understand unexpected disease outbreaks and unpredictable disease presentations from Plasmodium falciparum and Plasmodium vivax infections. The Malaria Evolution in South Asia (MESA) research program is one of ten International Centers of Excellence for Malaria Research (ICEMR) sponsored by the US National Institutes of Health. In this second of two reviews, we describe why population structures of Plasmodia in India will be characterized and how we will determine their consequences on disease presentation, outcome and patterns. Specific projects will determine if genetic diversity, possibly driven by parasites with higher genetic plasticity, plays a role in changing epidemiology, pathogenesis, vector competence of parasite populations and whether innate human genetic traits protect Indians from malaria today. Deep local clinical knowledge of malaria in India will be supplemented by basic scientists who bring new research tools. Such tools will include whole genome sequencing and analysis methods; in vitro assays to measure genome plasticity, RBC cytoadhesion, invasion, and deformability; mosquito infectivity assays to evaluate changing parasite-vector compatibilities; and host genetics to understand protective traits in Indian populations. The MESA-ICEMR study sites span diagonally across India and include a mixture of very urban and rural hospitals, each with very different disease patterns and patient populations. Research partnerships include government-associated research institutes, private medical schools, city and state government hospitals, and hospitals with industry ties. Between 2012 and 2017, in addition to developing clinical research and basic science infrastructure at new clinical sites, our training workshops will engage new scientists and clinicians throughout South Asia in the malaria research field.

Malaria in South Asia: prevalence and control.

The "Malaria Evolution in South Asia" (MESA) program project is an International Center of Excellence for Malaria Research (ICEMR) sponsored by the US National Institutes of Health. This US-India collaborative program will study the origin of genetic diversity of malaria parasites and their selection on the Indian subcontinent. This knowledge should contribute to a better understanding of unexpected disease outbreaks and unpredictable disease presentations from Plasmodium falciparum and Plasmodium vivax infections. In this first of two reviews, we highlight malaria prevalence in India. In particular, we draw attention to variations in distribution of different human-parasites and different vectors, variation in drug resistance traits, and multiple forms of clinical presentations. Uneven malaria severity in India is often attributed to large discrepancies in health care accessibility as well as human migrations within the country and across neighboring borders. Poor access to health care goes hand in hand with poor reporting from some of the same areas, combining to possibly distort disease prevalence and death from malaria in some parts of India. Corrections are underway in the form of increased resources for disease control, greater engagement of village-level health workers for early diagnosis and treatment, and possibly new public-private partnerships activities accompanying traditional national malaria control programs in the most severely affected areas. A second accompanying review raises the possibility that, beyond uneven health care, evolutionary pressures may alter malaria parasites in ways that contribute to severe disease in India, particularly in the NE corridor of India bordering Myanmar Narayanasamy et al., 2012.

Acanthamoeba on Sabouraud's agar from a patient with keratitis.

A 25-year-old transgender patient came with complaints of watery discharge, red eye and photophobia in the left eye since 2 days. The patient had a history of wearing colored contact lenses since 4 years and cleaning the lens with tap water. Culture of lenses on Mac Conkey and blood agar yielded Klebsiella pneumoniae and Pseudomonas aeruginosa. Sabouroud's agar showed yeast cells and double-walled cysts of Acanthamoeba species. On further incubation of Sabouroud's agar, the cysts transformed to trophozoites. Parallel results were obtained on tap water agar. The previous therapy of moxifloxacin was changed to local Neosporin application.