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Introduction: Antinuclear Antibodies (ANA) detection by Human Epithelial Substrate (Hep2) is the recommended screening test for the diagnosis of ANA-Associated Rheumatic Diseases (AARD). ANA is ordered by various specialists in a tertiary care hospital, and a positive ANA result is followed by testing for specific autoantibodies. High pretest probability, pattern type with intensity, and referral departments are key factors determining the autoimmune diagnosis. Aim: To analyse the ANA referral patterns among the various departments and also to estimate the prevalence and clinical significance of rare ANA patterns. Materials and Methods: A retrospective observational study was conducted at Nizam’s Institute of Medical Sciences, Hyderabad, a tertiary care hospital, in Hyderabad, Telangana, India where ANA test reports (n=16,994) from various departments over three years (November 2017 to October 2020) were evaluated between November 2022 to December 2022. ANA tests were performed on Hep2 substrate at a 1:100 dilution, and ANA patterns were reported according to the International Consensus on ANA Patterns (ICAP) nomenclature. Statistical analysis of department-wise ANA positivity and fluorescence intensity was conducted, and the final diagnoses of patients with rare ANA patterns (

Introduction: The most important pathogen isolated from Skin and Soft Tissue Infections (SSTIs) is a gram positive organism, Staphylococcus aureus (S. aureus). Wide range of emerging Methicillin Resistant Staphylococcus aureus (MRSA) infections is leading to global threat causing Community Acquired-MRSA (CA-MRSA) or Hospital Acquired MRSA (HA-MRSA). Minimum Inhibitory Concentration (MIC) is done to ensure that antibiotics are chosen efficiently in the clinical settings by Vitek 2, Epsilometertest (E-test) and Broth Microdilution (BMD) method. Aim: To determine the clinical spectrum of MRSA and comparison of vancomycin MICs obtained by E-test, Vitek 2C and BMD method. Materials and Methods: This was a cross-sectional study conducted at Nizam’s Institute of Medical Sciences, Hyderabad, Telangana, India, during the period of November 1st 2019 to December 31st, 2020 from Inpatient Department (IPD) and Outpatient Department (OPD). Among 464 SSTIs, 132 were S. aureus, out of which 38 isolates were MRSA. Identification and sensitivity of the isolates such as pus, wound swabs and tissues were identified through Vitek 2 compact system. Statistical analysis of the demographic and clinical characteristics was represented as frequency and percentages. Results: Among the total 464 SSTIs, S. aureus were 132 of which MRSA were 38 (29%) and Methicillin sensitive Staphylococcus aureus (MSSA) were 94 (71%) with male predominance of 29 (76.3%) with MRSA. Most of the patients were in the age group of 21-30 years (26%). Amongst the total 38 patients analysed, 25 were from IPD and 13 were from OPD. Around 23/38 were wound swabs (60%) 12/38 of pus (31.5%) and 3/38 (8%) were tissues. The predominant risk factor observed was Surgical Site Infections (SSI) in 19 cases (50%) followed by prior antibiotic therapy in 17 (44.7%) cases. The median duration of hospitalisation was 31.5 days. Vancomycin susceptibility by all three methods with an MIC range of 0.5-2 μg/m by all three methods, except for one isolate where the MIC was >32 ug/mL by Vitek 2C and 8 ug/mL by E-test, which was sensitive by BMD with an MIC of 0.25 µg/mL. Conclusion: Implementing infection control practices and controlling the risk factors will help in management of MRSA infections. Drug resistance to glycopeptides can be avoided by regular screening of vancomycin creeps by different susceptibility methods in order to avoid treatment failures.

Introduction: Intestinal coccidian parasites known to cause opportunistic infections have increased since the past years and are often indistinguishable from other forms of communityacquired diarrhoea, which suggests a need for proper and rapid diagnostic techniques to recover and identify the organism. Aim: To investigate if Auramine Rhodamine (AR) staining is a sensitive and time conserving staining technique in contrast to modified acid fast stain. Materials and Methods: A descriptive observational study was conducted at Nizam’s Institute of Medical Sciences, Hyderabad, India, over a period of three months (i.e., from July to September 2019). A total of 100 stool samples received by the Department of Microbiology were subjected to macroscopic and microscopic examination by saline mount, iodine mount, kinyoun acid fast stain, modified AR staining. Results: In the present study the prevalence of coccidian infections were observed to be 6%. Coccidian parasites reported were Cryptosporidium species and Cystoisospora species immunocompromised patients were found to be more prone to infections with coccidian parasites. The AR stain showed 100% agreement with modified acid fast stain. Conclusion: This study concluded that the AR stain is a rapid and better stain than kinyoun acid-fast stain for the detection of coccidian parasites.

Background: Nontuberculosis mycobacterium (NTM) is the emerging group of organisms being recognized as etiological agents for diverse clinical conditions such as lymphadenitis, cutaneous, and pulmonary or disseminated lesions. Diverse background patients can acquire these infections such as immunocompetent, immunocompromised patients, or postoperative settings. Rapid addition of newer strains to this group necessitates heightened suspicion in the clinical settings. Specific requirements for cultures, biochemical testing, and molecular methods are needed to diagnose these organisms. Methods: The prospective study conducted at Nizam's Institute of Medical Sciences from January 2019 to December 2021 using various clinical samples using molecular techniques such as line probe assay and hsp-65 gene sequencing to discover new NTM species. The management is challenging since it requires prolonged treatment, multiple drugs, drug resistance, and individualization of treatment in the combination of surgery if needed. In this article, we describe three different NTM species which were not reported in India and highlight to consider these organisms in adequate clinical situation. Results: Mycobacterium iranicum is a rare strain with quick growth and scotochromogenic colonies that are orange-colored. Eight distinct strains were discovered in clinical samples from six different countries: Two each from Iran, Italy, Greece, the Netherlands, Sweden, and the United States. Two of the strains were recovered from cerebrospinal fluid, which is unusual. Mycobacterium species AW6 is an unidentified and unclassified Mycobacterium according to NCBI taxonomy. Mycobacteria malmoense has been linked to lymphadenitis, notably cervical adenitis in children, and pulmonary infection in the majority of cases. Using Line Probe Assay and hsp-65 gene sequencing, novel and uncommon species of NTM were detected from a clinical samples, including sputum and tissue. Conclusion: We report three unusual species of NTMs: M. iranicum, M. species-AW6, and M. malmoense for the first time in India. Novel and rare emerging species of NTMs need to be considered in diverse clinical situations for appropriate therapy and good clinical outcomes.

Introduction: Blood cultures play an important role in the early diagnosis of sepsis and its management. Early detection of pathogens in Blood Stream Infections (BSI) and their Antimicrobial Susceptibility Testing (AST) pattern, plays a vital role in the diagnosis of sepsis and is important for guidance of appropriate therapy. Aim: To evaluate the accuracy of shortly incubated blood cultures in comparison with standard method for an early Identification (ID) and AST. Materials and Methods: This was a prospective observational study undertaken from July 2015 to June 2016 at Nizam’s Institute of Medical Sciences, Hyderabad, Telangana, India. The blood cultures were loaded in the BacT/Alert system. A total of 92 patients with two sets of blood cultures that flagged positive within 24 hours of collection were included in the study. Grams stain and subcultures of the broths were done. The culture plates were examined after four hours and then at hourly intervals for the presence of growth. Once the growth was sufficient it was processed immediately for ID and AST by Vitek 2C. Incubation of the plates was continued for the rest of the 24 hours at 37oC and was processed again. The mean time for detection were compared between short and standard cultures. Results: Gram negative pathogens were the predominant organisms isolated in 82/92 (89.1%) followed by gram positive in 10/92 (10.9%). The short and standard cultures had comparable results with respect to ID of the isolates. But, the AST results were comparable only in 88/92 (95.6%) patients. Of the remaining four patients, the AST showed Very Major Error (VME) in 3 (3.3%) patients and Major Error (ME) in 1 (1.08%) patient. Conclusion: Short incubation of cultures enabled earliest ID and AST of the isolates from blood cultures than standard incubation.

Introduction: Non-tuberculous Mycobacteria (NTM) pulmonary disease is often unrecognised or misdiagnosed as Mycobacterium tuberculosis (MTB), Multi-Drug Resistant Tuberculosis (MDRTB), because of similar clinical presentation in counties with high burden of Tuberculosis (TB) including India. In India due to lack of awareness among clinicians and lack of laboratory facilities to diagnose these infections, its prevalence is largely unknown. Aim: To evaluate the efficacy of identification of NTM species by Matrix Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF) and Heat Shock Protein (hSP65) gene sequencing and to determine their Antimicrobial Susceptibility Testing (AST). Materials and Methods: All the clinical specimens from pulmonary and extra-pulmonary TB suspects at Nizam’s Institute of Medical Sciences, Hyderabad, Telangana, India, over a period of one year i.e., from June 2017 to May 2018 were included in the study. The specimens were subjected to microscopy, culture and GeneXpert. The NTMs isolated in the culture were further characterised genotypically by MALDI-TOF and hsp65 gene sequencing. The identified NTM isolates were subjected to AST. All the methods were followed as per the standard protocols. Data was analysed using SPSS 25. Results: A total of 1085 samples were processed out of which Mycobacteria was detected in 201 cases (18.5%). Among the culture positives, MTB complex was detected in 146 cases (13.5%) and NTM in 55 (5.06%). Mycobacterium abscessus was the predominant isolate. The most common co-morbidities were bronchiectasis and Chronic Obstructive Pulmonary Disease (COPD). Linezolid, clarithromycin, moxifloxacin and amikacin showed high sensitivity. Conclusion: Molecular assay helps in rapid identification which can lead to targeted therapy and can thus combat antimicrobial resistance. The MALDI-TOF and hsp65 gene sequencing also offers quick results at a low cost and is easy to perform hence it can be considered as an alternate diagnostic tool for identification.

Background and Aims: Tuberculosis kills more than 1 million people every year, most of them in low-income and middle-income countries. An understanding of the trends in tuberculosis incidence, prevalence, and mortality s crucial to track the success of tuberculosis control programs. Microbiological diagnosis of diseases caused by Mycobacteria should be fast and effective to prevent contagions and optimize the management of infections. Materials and Methods: A total of 1412 clinical pulmonary and extra pulmonary specimens were studied from January 2017 to December 2017 at Nizam's Institute of Medical Sciences, Hyderabad. All specimens were processed according to standard operating procedures. All the specimens were subjected to microscopy, culture, GeneXpert. Results: Among 1412 samples received 813 were males (57.6%) and 599 females (42.4%). Among these 818 (57.9%) were pulmonary samples and 594 extra pulmonary samples. Mycobacterium prevalence was (21.6%) out of which Mycobacterium tuberculosis was found in 18.3% and Non tuberculous Mycobacteria (3.25%). The contamination rate was 2.6% (37 out of 1412). Among the positives, the most common affected age group was 21-30 yrs (22.2%). About 64 (4.53%) were smear positive. A total of 200 isolates (14.16%) were recovered by at least one culture LJ medium or BacT Alert 3D system. MTB was recovered in 216 (15.29%) by GeneXpert. MDRTB was detected in 8 (3.7%) by GeneXpert. Conclusion: M. tuberculosis complex is responsible for immense worldwide morbidity and mortality. Delays in diagnosis may postpone administration of appropriate treatment and be detrimental to patient outcomes. Since traditional culture methods are slow, newer molecular techniques allow more rapid and sensitive laboratory diagnosis of tuberculosis.

Introduction: Postoperative infections in orthopaedic implants is a major concerning factor. It can lead to the failure of implants and in severe cases can even lead to amputation and mortality. Total joint replacements and fracture fixations do help by reducing the agony of many patients but postoperative infections are a detrimental complication. It is an economic disaster for hospitals that treat large number of these patients therefore identification of risk factors, common pathogens involved, early initiation and prompt use of empirical therapy is necessary to prevent implant failures and also collection of deep tissue samples improves the diagnostic reliability and microbiological yield of implant related infections. Hence, appropriate collection of specimens is a key factor for early diagnosis and differentiating colonisers from true pathogens. Aim: To evaluate the rate of implant related infections, sensitivity of Gram’s stain and its accuracy in tissue samples over wound swabs and also to determine the trends in the antibiogram. Materials and Methods: A retrospective analysis was performed from the samples (tissues and wound swabs) received to the Department of Microbiology from October 1st 2017-October 30th 2018 (a period of one year) in patients with orthopaedic implants. Identification and susceptibility patterns of the bacterial pathogens were identified by the automated Vitek 2 compact system (biomeriux). The data was analysed in terms of frequency and percentage. Results: A total of 141 cases were analysed out of which 45.3% (64/141) were tissue samples and 54.6% (77/141) were wound swabs. Culture positivity observed in tissues was 43.7% (28/64), in wound swabs 50.6% (39/77) and mixed infections were observed in 7.14% (2/28) of tissues and 5.12% (2/39) of wound swabs. Colonisers were observed in 14.2% (11/77) of wound swabs. Specificity of Gram’s stain in comparison with tissue cultures was found to be 83.3% and sensitivity was 50%. Methicillin Sensitive Staphylococcus aureus (MSSA) 17.8% (05/28) and Escherichia coli 21.4% (6/28) were the major pathogens isolated from tissue cultures. Methicillin Resistant Staphylococcus aureus (MRSA) was isolated from 33.3% (13/39) of wound swabs followed by Escherichia coli (28.2%) (11/39). In the present study 25% (7/28) of tissue isolates showed Multidrug Resistance (MDR) and 3.5% (1/28) were Extended Spectrum Beta Lactamase (ESBL) producers. Among wound swabs 23% (9/39) isolates were MDR and 10.2% (4/39) were ESBL producers. Conclusion: In the present study it was observed that tissue samples were found to be better specimens in comparison with wound swabs for the diagnosis of implant infections. Hence, appropriate collection of sample helps in accurate isolation of pathogens in implant infections.

INTRODUCTION: Urinary tract infections (UTIs) are the most common infectious diseases occurring in either the community or healthcare setting. Turnaround time for urine culture is about 24 h, and antimicrobial susceptibility testing (AST) requires another 24 h. Consequently, initial antibiotic therapy is mostly empirical. MATERIALS AND METHODS: This study was conducted at Nizam’s Institute of Medical Sciences, Hyderabad. Turbid urine samples which showed pus cells and Gram‑negative (GN) bacilli of single morphotype were included. The turbidity of the urine was adjusted to 0.5 McFarland and uploaded directly in the VITEK 2 identification (ID) GN and N‑280 panel for AST. The specimen was also inoculated on CHROMagar, and the ID and AST of the isolates from the agar plate were repeated on VITEK 2, and the results were compared. RESULTS: Out of 844 turbid urines screened, 62 met the inclusion criteria. Escherichia coli was the most common isolate (71.9%). Complete agreement for ID was 80.7%, misidentified were 12.2%, and unidentified were 7%. Complete agreement with AST was 94.3%, very major errors 0.5%, major errors 2.2%, and minor errors 3%. Conclusions: With a 94.3% agreement for AST and a reduced turnaround time by 24 h, the direct inoculation had a potential clinical benefit for initiating timely and appropriate antibiotic therapy for UTI.