Current perspective of adrenal histoplasmosis in India: A prospective study in a tertiary care hospital, Eastern India.

PURPOSE: Adrenal histoplasmosis (AH) is an uncommon form of disseminated histoplasmosis caused by the dimorphic fungus Histoplasma capsulatum. Though, India is considered to be a non-endemic area for histoplasmosis, a high rise of AH cases is reported currently from various parts of India. Our study aimed to evaluate the current perspective of adrenal histoplasmosis in India by reviewing its clinical course, differential diagnosis, treatment, and mortality of our eleven confirmed cases of AH along with the review of authentic reported AH cases from India. MATERIAL &METHOD: Clinical materials were taken from radiologically suspected all 15 cases either with unilateral or bilateral adrenal enlargement, referred between 2018 and 2020 for microbiological investigations. Fungal stain and fungal culture along with other tests for possible differential diagnosis with AH were conducted. RESULT: Out of fifteen incidentaloma detected by radio-imaging, eleven cases of AH had been diagnosed in our hospital with yield of Rhodotorula spp. in one mimicking case. Nine of them were male (82%) and all were HIV nonreactive, which corroborates with the literature review. All of them had nonspecific clinical presentation of chronic abdominal pain, fever, weight loss, and anorexia. Four developed primary adrenal insufficiency, which are similar to the literature review (41%). On treatment with itraconazole and/or amphotericin B, all patients survived except one lost in follow-up. CONCLUSION: Male preponderance and non-compromised immune status are two special characteristics of most AH though reasons are ill understood. So, mycological investigations are to be done for every such case.

Preparation of serum capped silver nanoparticles for selective killing of microbial cells sparing host cells.

Following access into the cell, colloidal silver nanoparticles exhibit generalized cytotoxic properties, thus appear as omnipotent microbicidal, but not suitable for systemic use unless are free of toxic effects on host cells. The AgNP-Serum-18 when prepared from silver nitrate, using dextrose as reducing and group-matched homologous serum as a stabilizing agent, selective endocytosis, and oxidative stress-dependent bio-functional damages to the host are mostly eliminated. For their bio-mimicking outer coat, there is the least possibility of internalization into host cells or liberation of excess oxidants in circulation following interaction with erythrocytes or vascular endothelial cells. The presence of infection-specific antibodies in the serum can make such nano-conjugates more selective. A potent antimicrobial action and a wide margin of safety for mammalian cells in comparison with very similar PVA-capped silver nanoparticles have been demonstrated by the in-vitro challenge of such nanoparticles on different microbes, human liver cell-line, and in-vivo study on mice model. This may open up wide-range therapeutic prospects of colloidal nanoparticles.

Conidiobolomycosis: An Unusual Fungal Disease-Our Experience.

Conidiobolomycosis is a rare mycotic disease caused by Conidiobolus coronatus. Very few cases have been reported in English literature. Often it is clinically misdiagnosed as soft tissue tumour. A prospective case study was done from 2006 to 2015 in a tertiary care hospital of West Bengal, India. The objectives of our study were to describe the epidemiological and clinical features and treatment of Conidiobolomycosis to prevent disfigurement. Patients clinically suspected to be suffering from Conidiobolomycosis were subjected to biopsy followed by histopathological and mycological examinations. Then they were treated with oral saturated solution of potassium iodide along with other drugs. Total six cases were histopathologically proved to be suffering from Conidiobolomycosis. Fungus was isolated and identified in one case. Complete resolution was seen in five patients. Conidiobolomycosis should be brought into mind as differential diagnosis of subcutaneous swelling in the rhinofacial region.

Periodicities in the roughness and biofilm growth on glass substrate with etching time: Hydrofluoric acid etchant.

Adherence of the microorganism to submerged solid surfaces leads to biofilm formation. Biofilm formation modifies the surfaces in favor of bacteria facilitating the survival of the bacteria under different stressed conditions. On the other hand, the formation of biofilm has a direct adverse economic impact in various industries and more importantly in medical practices. This adherence is the reason for the failure of many indwelling medical devices. Surface biofilm adhesion is the key to biofilm growth and stability. Hence this adhesion needs to be substantially lowered to inhibit biofilm stability. Both chemical and physical properties of the surface influence biofilm formation and modulating these properties can control this formation. In this study, we have investigated the effect of Hydrofluoric acid (HF), at a specific concentration as an etchant, on the surface morphology of substrates and the growth of biofilms of Pseudomonas aeruginosa. and Staphylococcus aureus. We find that the bacterial counts on the etched surfaces undergo a periodic increase and decrease. This, on one hand, shows the close correlation between the biofilm growth and the particular roughness scale, and on the other hand, explains the existing contradictory results regarding the effects of etching on substrate roughness and biofilm growth. We propose a simple model of a sequence of hole formation, hole expansion and etching away of the hole walls to form a new, comparatively smooth surface, coupled with the preferential accumulation of bacteria at the hole edges, to explain these periodicities.

VIPER regulates naive T cell activation and effector responses: Implication in TLR4 associated acute stage T cell responses.

Naive T cells are known to express the modest level of TLR4 while it is known to go down during TCR activation. However, information towards the requirement of TLR4 signaling during TCR or mitogenic activation of naive wild-type T cells remains scanty. Here we have investigated the endogenous functional expression of TLR4 in naive mice T cells during TCR and mitogenic stimulation in presence of VIPER peptide (VP), an established inhibitor of TLR4 signaling. As expected we found that TLR4 expression goes down during TCR and mitogenic activation. Interestingly, we observed that VP treatment restores TLR4 expression on those activated T cells. Moreover, VP was found to regulate such activation of naive T cell as evident by reduction of CD25, CD69 expression, effector cytokines (IL-2, IFN-γ, TNF) production, T cell proliferation and down-regulation of T cell activation-dependent Fas (CD95), FasL (CD95L) expression. Together, our current observation highlights a possible requirement of TLR4 responses in T cells, which might have possible implication towards the pathogenic acute phase activation of naive T cells.

Biofilm colonization of Mycobacterium abscessus: New threat in hospital-acquired surgical site infection.

INTRODUCTION: Rapidly growing non-tuberculous mycobacteria (NTM) are hazardous cause of post-operative soft tissue infection leading to nosocomial outbreaks following various surgical procedures, especially laparoscopic surgeries using heat sensitive, non-autoclavable surgical instruments. METHODOLOGY: Surgery department of our hospital noticed increase in rate of post-laparoscopic abdominal port site infection (PSI) and informed the Microbiology Department. A prospective investigational study of defined cases with the aim of source tracing and formulation of infection control measures was initiated. Pus or wound scrapings were collected and processed for aerobic, anaerobic bacteria and Mycobacterium, both by staining and culture. Environmental samples were collected from laparoscopic instruments, and different parts of operation theatre (OT). Mycobacterial isolates were speciated by line probe assay. All the cases were treated with clarithromycin and ofloxacin±amikacin. RESULTS: Among 15 cases of PSI, 11 patients had undergone laparoscopic cholecystectomy, 3 had laparoscopic mesh hernioplasty and one had laparoscopic orchidopexy. Of the 13 pus/discharge specimens examined, 11 revealed growth of NTM. All the isolates were identified as Mycobacterium abscessus by line probe assay. Scraping of biofilm from the disinfectant tray also produced growth of the same organism. Plastic trays used for disinfectants were replaced with metal trays and instructed to do mechanical scrubbing before autoclaving at regular interval. No similar PSI cases were notified after those measures were taken, till date. CONCLUSIONS: This study has shown the need of culture and identification of pathogens causing persistent post surgical wound infections and illuminated importance of rapid source tracing in resource constraint situation which could control outbreak.

Isolation of bacteria from diabetic foot ulcers with special reference to anaerobe isolation by simple two-step combustion technique in candle jar.

BACKGROUND & OBJECTIVES: Although polymicrobial infections involving both aerobic and anaerobic bacteria are very common in diabetic foot ulcers, in many centres of developing countries, anaerobes are rarely isolated due to technical difficulties. This can be overcome by using a new simple, innovative technique of a combination of candle combustion and use of acidified copper-coated steel wool, as reported here. METHODS: In-house developed method was used in a prospective clinico-microbiological study for anaerobes from randomly selected 43 patients with diabetic foot ulcers along with conventional method of anaerobic culture in GasPak system and aerobic culture by standard laboratory procedures. For primary isolation of anaerobes, Brucella blood agar supplemented with hemin (5 µg/ml) and menadione (1 µg/ml) was used. Antibiotic sensitivity tests were performed by the standard disc diffusion method for aerobes and E-test method for anaerobes. RESULTS: All the 43 samples were culture positive, of which aerobic Gram-negative bacteria (GNB) predominated, followed by Staphylococcus aureus, Enterococcus and diphtheroids. Anaerobes isolated from 21 samples were Peptostreptococcus, Bacteroides, Porphyromonas, Veillonella spp. and Clostridium perfringens by both GasPak and in-house developed and modified candle jar techniques. Imipenem and metronidazole were most sensitive while clindamycin, penicillin and cefoxitin were least sensitive drugs for anaerobes. Aerobic GNB were found to be multidrug resistant, especially to penicillin and cephalosporins. The most sensitive drug was piperacillin-tazobactam. INTERPRETATION & CONCLUSIONS: For isolation of anaerobes from clinical specimens such as diabetic foot ulcers, modified candle jar technique was found to be as reliable as GasPak system. This modified technique needs to be tested for many other clinical materials which are not yet evaluated.

Biofilm colonization in chronic treatment refractory infections presenting with discharging sinuses: A study in a tertiary care hospital of Eastern India.

INTRODUCTION: Treatment refractory chronic recurrent infections mean those chronic infections which recur by same causal agents with similar drug responsiveness after apparent relief following full course of recommended antimicrobial management. MATERIALS AND METHODS: Fifty different samples were collected from patients with chronic surgical site infections, laparoscopic port site infections, anal fistula, mesh hernioplasty, chronic dacryocystitis, chronic osteomyelitis, and chronic burn wounds. Samples were processed for culture, identification, antibiotic sensitivity testing using standard microbiological techniques. Biofilm (BF) forming capacity for aerobic organisms were tested by tissue culture plate method. Those for anaerobes and atypical mycobacteria were studied by a novel method using atomic force microscopy (AFM). In vivo BF colonization in lacrimal mucosae of chronic dacryocystitis, patients were studied from histopathological sections by Gram staining, H and E, and fluorescent in situ hybridization (FISH). RESULTS: Out of fifty different samples, sixty-three isolates were obtained in pure culture as follows: Staphylococcus aureus (25.39%), Escherichia coli (14.28%), Klebsiella pneumonia (14.28%), Mycobacterium abscessus (12.69%), Citrobacter spp. (9.52%), Bacteroides fragilis (6.3%), Pseudomonas aeruginosa (4.7%), Proteus spp. (4.7%), Staphylococcus epidermidis (3.1%), Enterobacter spp. (1.5%), Morganella morganii (1.5%), and Peptostreptococcus spp. (1.5%). Among the isolates, 74% were found to be BF producers in the following frequency: P. aeruginosa 100%, S. epidermidis 100%, B. fragilis 100%, Klebsiella spp. 88.88%, S. aureus 81.25%, M. abscessus 75%, Citrobacter spp. 83.33%, Proteus spp. 66.66%, E. coli spp. 33.33%, and Enterobacter spp. 0%. CONCLUSION: AFM has been proven to be a useful method for detection of in vitro grown BF including those for anaerobes and atypical Mycobacteria. In vivo BF detection becomes possible by FISH. S. aureus was the most common isolate. Among the aerobic isolates, P. aeruginosa and S. epidermidis were found to be the most common BF producers. Atypical mycobacteria were also found to be BF producers. Diagnosis of BF s in chronic infections significantly changes the management strategy as these infections can no longer be dealt simply with antibiotics alone but require mechanical removal of the foci along with antibiotic coverage for complete cure.

Port-site infections by nontuberculous mycobacterium: A retrospective clinico-microbiological study.

BACKGROUND: Port-site infection (PSI) is a prevailing, chronic, nagging, treatment refractory complication of laparoscopic surgery (LS). It neutralizes the advantages of minimally invasive surgery and increases morbidity, treatment cost of patient, leading to loss of confidence on operating surgeon. PSIs are preventable with appropriate preoperative, intraoperative, and postoperative measures. Atypical mycobacterium is most commonly associated with nonhealing postlaparoscopic wound infections, causing outbreaks or sporadic cases worldwide. PURPOSE: We retrospectively studied the occurrence of nontuberculous mycobacterium (NTM) from PSIs following LS that did not respond to antibiotics used for pyogenic infections and having sterile routine aerobic cultures and their antimicrobial susceptibility pattern to guide proper management. METHODS: The study was done in a tertiary care hospital of Eastern India over a 1-year period which included PSI cases with delayed onset not responding to antibiotics, following different types of LS. Pus/discharge from 32 patients was collected and examined for isolation and identification of the causative agents. Gram stain and Ziehl-Neelsen staining methods were used for direct examination. Culture media included blood agar, Robertson's cooked meat broth, MacConkey agar, and Lowenstein-Jensen medium. Isolates from the cases were identified using biochemical tests or molecular methods and studied the antimicrobial susceptibility pattern by the standard microbiologic procedures. RESULTS: Mycobacterium abscessus (13) and Mycobacterium fortuitum (2) were isolated from 15 serosanguinous drainage obtained from 32 cases by routine microbiological techniques. All isolates analyzed for antimicrobial susceptibility pattern were highly sensitive to clarithromycin (93.3%), amikacin (93.3%), and imipenem (80%) but were variable to ciprofloxacin, ofloxacin, and linezolid. CONCLUSIONS: Our present study shows frequent association of NTM with laparoscopic port-site nonhealing chronic infection or wound dehiscence. Although direct microscopy can give us a clue to diagnosis, culture isolation is required for speciation and antimicrobial susceptibility testing, which helps formulate therapeutic regimen.

Experience of Varied Presentation of Chronic Progressive Disseminated Histoplasmosis in Immunocompetent Patients: A Diagnostic Conundrum.

We report two cases of chronic progressive disseminated histoplasmosis with unusual and rare clinical picture in a patient with no underlying risk factor. One 50-year-old male, presented with hoarseness of voice, chronic cough, with a history of nonresponding anti-tubercular therapy, revealed mucocutaneous lesions on examination. Fungating vocal cord lesions were visualized on bronchoscopy, raised suspicion of carcinoma. The second case, a 22-year-old female, referred to hospital with suspected vasculitis, with complaints of "off and on" fever with decreased oral intake, arthralgia, who later developed generalized nodular skin eruptions. On investigation, human immunodeficiency virus test was found to be negative in both the cases. Histopathological findings of skin biopsy, adrenal and bone marrow aspirates raised suspicion, whereas fungal cultures confirmed Histoplasma infection. Although diagnosis was delayed, but both of them were successfully treated with amphotericin B.

Biofilm in Osteomyelitis caused by a Rare Pathogen, Morganella morganii : A Case Report.

Morganella morganii is a member of Enterobacteriaceae family, whose natural habitat is the human gastrointestinal tract. It rarely causes infection alone and is generally encountered in immunosuppressed patients. Osteoarticular pathologies are not commonly observed with Morganella morganii and infections by it have high mortality rate. Biofilm colonization is a causative factor behind the chronicity and/or refractoriness of certain infections. Biofilms colonize on inert medical devices, prosthesis, fibrosed tissues, sinus tracts as well as dead bones as in case of chronic osteomyelitis. Morganella morganii is not a common pathogen to produce biofilm. In this case report, we present a 56-year-old male patient with chronic osteomyelitis of right proximal tibia caused by biofilm producing strain of Morganella morganii, following trauma.

Optimization, purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA.

Actinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2 °C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn(2+) and strongly inhibited by Ba(2+). All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.

Development of novel antibodies against non-structural proteins nsP1, nsP3 and nsP4 of chikungunya virus: potential use in basic research.

Chikungunya virus (CHIKV) has reemerged recently as an important pathogen, causing several large epidemics worldwide. This necessitates the development of better reagents to understand its biology and to establish effective and safe control measures. The present study describes the development and characterization of polyclonal antibodies (pAbs) against synthetic peptides of CHIKV non-structural proteins (nsPs; nsP1, nsP3 and nsP4). The reactivity of these pAbs was demonstrated by ELISA and Western blot. Additionally, in vitro infection studies in a mammalian system confirmed that these pAbs are highly sensitive and specific for CHIKV nsPs, as these proteins were detected very early during viral replication. Homology analysis of the selected epitope sequences revealed that they are conserved among all of the CHIKV strains of different genotypes, while comparison with other alphavirus sequences showed that none of them are 100% identical to the epitope sequences (except Onyong-nyong and Igbo Ora viruses, which show 100% identity to the nsP4 epitope). Interestingly, two different forms of CHIKV nsP1 and three different forms of nsP3 were detected in Western blot analysis during infection; however, further experimental investigations are required to confirm their identity. Also, the use of these antibodies demonstrated faster and enhanced expression profiles of all CHIKV nsPs in 2006 Indian outbreak strains when compared to the CHIKV prototype strain, suggesting the epidemic potential of the 2006 isolate. Accordingly, it can be suggested that the pAbs reported in this study can be used as sensitive and specific tools for experimental investigations of CHIKV replication and infection.

A Clinico-Mycological Study on Suspected Cases of Chromoblastomycosis: Challenges in Diagnosis and Management.

INTRODUCTION: Verrucous plaques mimicking chromoblastomycosis are frequently seen in dermatology outpatient departments (OPD). However, no scientific evaluation has been carried out till date from eastern India. So this present endeavour is aimed at a thorough study of those cases to readdress the challenges in diagnosis and management in chromoblastomycosis from this part of the country. AIM: The study is to observe the incidence of proved chromoblastomycosis cases from clinically mimicking conditions and to note therapeutic prospects by use of different antifungal agents. MATERIALS AND METHODS: Twenty clinically suspected cases attending dermatology OPD were included in this study. Relevant histories were taken. Apart from routine hematological and biochemical investigations, scrapings from lesions were examined by direct microscopy with KOH wet mount, calcoflour white mount and fungal culture. Histopatholgical examination was also done. Any fungal growth was identified by growth characteristics and morphological features. RESULTS: Sclerotic bodies were detected in five samples. Of them three were found to be culture positive. Two growths were identified as Fonsecaea pedrosoi and one as Cladosporium carrionii. Rest 12 cases were diagnosed as either lupus vulgaris (3 cases; 15%), atypical mycobacterial infections (3 cases, 15%), Hypertrophic lichen planus (2, 10%), Hypertrophic DLE plaque (2; 10%), Wart (1, 5%) or fixed cutaneous sporothricosis (1, 5%), zygomycosis (1, 5%). Three cases (15%) were lost in follow up. Cases of chromoblastomycosis were managed with prolonged use of antifungal alone or in combination with saturated solution of potassium iodide and/or debridement. They were followed up for at least six months post treatment. CONCLUSION: A database comprising diagnostic clues and effective therapeutic intervention have been proposed for these rare subcutaneous mycoses.

A novel tool for capture and detection of typhoid fever using Ag-labeled nanocomposites.

This work investigated a simple and versatile modification to a solid substrate to develop antibody recognition using nanoparticles. The new immobilized metal ion affinity adsorbent containing nanoparticles and hydrophilic resins is proposed here to improve the binding of antigen on its surface. The light-scattering properties of submicroscopic metal particles ranging from 100 to 120 nm in diameter were confirmed by scanning electron microscopy. We found that synthesized nanoparticles have an inherent enzyme mimetic activity similar to that found in natural peroxidases. The synthesized nanoparticles were coated with Salmonella typhi and Salmonella paratyphi antigens and were allowed to react with Salmonella-infected serum. Positive reactions were detected visually with the naked eye. The color changes of substrate (TMB) were observed even in 1:800 dilutions. This report helps in developing a specific immunoassay using nano-conjugated antigen for the rapid detection of S. typhi and S. paratyphi antibodies in infected serum.

Development and characterization of monoclonal antibody against non-structural protein-2 of Chikungunya virus and its application.

The recent epidemics of Chikungunya viruses (CHIKV) with unprecedented magnitude and unusual clinical severity have raised a great public health concern worldwide, especially due to unavailability of vaccine or specific therapy. This emphasizes the need to understand the biological processes of this virus in details. Although CHIKV associated research has been initiated, the availability of CHIKV specific reagents for in-depth investigation of viral infection and replication are scanty. For Alphavirus replication, non-structural protein 2 (nsP2) is known to play a key regulatory role among all other non-structural proteins. The current study describes the development and characterization of nsP2 specific monoclonal antibody (mAb) against a synthetic peptide of CHIKV. Reactivity and efficacy of this mAb have been demonstrated by ELISA, Western blot, Flow cytometry and Immunofluorescence assay. Time kinetic study confirms that this mAb is highly sensitive to CHIKV-nsP2 as this protein has been detected very early during viral replication in infected cells. Homology analysis of the selected epitope sequence reveals that it is conserved among all the CHIKV strains of different genotypes, while analysis with other Alphavirus sequences shows that none of them are 100% identical to the epitope sequence. Moreover, using the mAb, three isoforms of CHIKV-nsP2 have been detected in 2D blot analysis during infection in mammalian cells. Accordingly, it can be suggested that the mAb reported in this study can be a sensitive and specific tool for experimental investigations of CHIKV replication and infection.

Entomophthoromycosis in South Bengal (Eastern India): a 9 years study.

Subcutaneous mycoses caused by the family Entomophthoraceae is very rare type of disease and is being reported sporadically from various Tropical countries including India. Here we report 8 cases of rhinoentomophthoromycosis caused by Conidiobolous coronatus and 7 cases of chronic subcutaneous phycomycosis caused by Basidiobolus ranarum. Cases were detected during a span of 9 years between 1991 to 1999, from 9 districts in and around Kolkata (Eastern India). Former type of lesions were detected among 20 to 65 age group of healthy individuals, predominantly males (7:1). In the latter type, male-female ratio was 2:5, and except for one all cases belonged to below 20 years age group of healthy individuals. Several cases were detected only after examination of repeat biopsy samples. With high degree of clinical suspicion, right approach is needed for laboratory confirmation of diagnosis.