Diagnostic Stewardship in Clinical Microbiology: An Indispensable Component of Patient Care.

Emerging infectious diseases and increasing resistance to available antimicrobials are mapping the evolution of clinical microbiology and escalating the nature of undertakings required. Rapid diagnosis has become the need of the hour, which can affect diagnostic algorithms and therapeutic decisions simultaneously. Subsequently, the concept of 'diagnostic stewardship' was introduced into clinical practice for coherent implementation of available diagnostic modalities to ensure that these new rapid diagnostic technologies are conserved, rather than consumed as part of health care resources, with a view to improve the patient care and reduce Turnaround Time (TAT) and treatment expense. The present study highlights the requisite of diagnostic stewardship and outlines the infectious disease diagnostic modalities that can assist in its successful implementation. Diagnostic stewardship promotes precise, timely diagnostics, from the initial specimen collection and identification to reporting with appropriate TAT, so as to enable timely management of the patient. The main aim of diagnostic stewardship is to optimize the right choice of diagnostic test for the right patient to attain clinically significant reports with the least possible TAT for timely management and the least expected adverse effects for the patient, community, and the healthcare system. This underlines the requisite of a multifaceted approach to make technological advancements effective and successful for implementation as a part of diagnostic stewardship for the best patient care.

Insights into Changing Patterns of Extrapulmonary Tuberculosis in North India.

PURPOSE: Tuberculosis is one of the dreadful infections and India contributes to substantial burden of TB cases globally. Though majority of cases are pulmonary, extra-pulmonary tuberculosis (EPTB) share significant burden, more in HIV-positive persons. Despite the striking burden, very few studies have been conducted in India and present study was undertaken to determine trends of EPTB at our tertiary care centre. METHODS: This was a retrospective study conducted over a period of 4 years 3 months. Diagnosis of EPTB was based on suspected clinical features, with positive micobiological evidence with cartridge based nucleic acid amplification test (CBNAAT) with/without microscopy. RESULTS: A total of 10,560 samples (pulmonary and extra-pulmonary) were received during the study period, of which 3,972 were extrapulmonary. Of these, a total of 18% were noted to be positive for EPTB. Trend of positivity revealed highest burden in in 2018 and a decline was noted over the years, however, rise in cases was noted in 2022. Pleural, meningitis, musculoskeletal, peritoneal and pericardial TB was more common in males, while lymphadenitis was more common in females (p value: <0.0001). Pleural TB (31%) was the most common presentation, followed by lymphadenitis. A gradual decline in lymphadenitis was noted with significantly increasing trend only for musculoskeletal TB. Rifampicin resistance was detected in 7.45% of positive samples, of which the maximum rate of resistance was noted in lymph node aspirates (11.11%), followed by musculoskeletal and pleural samples. CONCLUSION: The present study showed a gradual decline in positivity of EPTB cases over the years. Younger productive age group with more propensity to transmit infection was the most commonly affected, with pleural TB as the most common presentation. Rare presentations of EPTB also contributed major share. Higher rates of resistance underline requisite to strengthen ongoing programs, to achieve the End TB strategy by 2025.

Clinical spectrum, phenotypic and molecular characterization, and antifungal susceptibility of an emerging human pathogen, Acrophialophora, from India.

Acrophialophora is implicated in superficial and invasive infections, especially in immunosuppressed individuals. The present study was undertaken to provide clinical, microbiological, phylogenetic, and antifungal susceptibility testing (AFST) profile of Acrophialophora isolated from India. All the isolates identified as Acrophialophora species at National Culture Collection for Pathogenic Fungi, Chandigarh, India were revived. Phenotypic and molecular characterization was performed, followed by temperature studies, scanning electron microscopy (SEM) and AFST. We also performed systematic review of all the cases of Acrophialophora species reported till date. A total of nine isolates identified as Acrophialophora species were identified by molecular method as A. fusispora (n = 8) and A. levis (n = 1), from brain abscess (n = 4), respiratory tract (n = 3) and corneal scraping (n = 2). All patients but two had predisposing factors/co-morbidities. Acrophialophora was identified as mere colonizer in one. Temperature studies and SEM divulged variation between both species. Sequencing of the ITS ribosomal DNA and beta-tubulin loci could distinguish species, while the LSU ribosomal DNA locus could not. AFST showed lowest MICs for triazoles and highest for echinocandins. Systematic literature review revealed 16 cases (11 studies), with ocular infections, pulmonary and central nervous system infections, and A. fusispora was common species. All the patients except three responded well. High MICs were noted for fluconazole, micafungin and caspofungin. This is the first study delineating clinical, phenotypic, and genotypic characteristics of Acrophialophora species from India. The study highlights microscopic differences between both species and emphasizes the role of molecular methods in precise identification. Triazoles appear to be the most effective antifungals for managing patients.

We describe clinical, phenotypic, and genotypic characteristics of Acrophialophora species. This species causes mild infection to fatal infection in immunosuppressed individuals. Triazoles are effective in treating such infections.

Performance of Cephid CT/NG Xpert rapid PCR test for detection of Chlamydia trachomatis and Neisseria gonorrhoeae at a tertiary care centre in North India.

INTRODUCTION: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are prevalent causes of sexually transmitted infections (STIs) globally, leading to substantial morbidity and transmission risks. METHODS: This study evaluates the diagnostic efficacy of Xpert CT/NG compared to conventional PCR and culture methods in 121 patients at a tertiary care centre in North India. RESULTS: Xpert CT/NG demonstrated high sensitivity (85.8%) and specificity (96.3%) outperforming conventional PCR. Xpert CT/NG's rapidity and accuracy underscore its utility in timely diagnosis and control of STIs. CONCLUSION: As sexually transmitted infections pose a serious health concern implementation of such rapid diagnostic methods/point of care testing methods are to be implemented for early diagnosis.

Risk factor and resistance profile of colistin resistant Acinetobacter baumannii and Klebsiellapneumoniae.

PURPOSE: Antimicrobial resistance is one of the major global health concerns, which is relentless despite multipronged measures. Carbapenems and colistin, drug of choice for multi drug resistant Klebsiella pneumoniae and Acinetobacter species, have also been rendered of less use. This underlines the need to decipher prevalence of colistin resistance comprehensively for formulation of hospital and country-wise antibiogram. We conducted this study to decipher the prevalence of colistin resistance in our tertiary care centre of North India. MATERIALS AND METHODS: This was a prospective, case control study conducted over a period of one and half years. All carbapenem resistant Klebsiella pneumoniae and Acinetobacter isolates were included. Kirby-Bauer method of disc diffusion was used for all antibiotics, except colistin for which broth microdilution was performed and interpreted using CLSI guidelines. Demographic details, risk factors and outcome details were recorded. Genotypic characterization was performed using representative strains, for blaNDM, blaKPC and blaOXA-48. RESULTS: Of 103 carbapenem resistant isolates, 7 were found to be colistin resistant. Median age was 43 years, with male:female ratio of 1.1:1. 35% isolates were from pus samples, followed by endotracheal aspirate. Colistin resistance was more in ICUs than wards. Presence of indwelling devices was noted as the most common risk factor, followed by previous antibiotic exposure and use of steroids/immunosuppressants. Indwelling devices, steroids/immunosuppressants usage, length of hospital stay, COPD, prior usage of carbapenems, piperacillin-tazobactam and colistin, usage of ampicillin-sulbactam during hospital stay, were statistically significant. Mortality was noted in 4 cases, with statistical difference between control and case arm. The blaNDM and blaOXA-48 were noted in 3 and 2 isolates respectively, with absence of blaKPC. CONCLUSION: The present study unravels incidence, risk factors and resistance encoding genes at our centre. This is of immense help in formulation of antibiotic policies and guidance for infection control measures.

Triple Burden: The Incorrigible Threat of Tuberculosis, HIV, and COVID-19.

The Coronavirus-19 (COVID-19) hasn't seen the dawn since its emergence, however waxing and waning has resulted in the emergence of deadly variants. The effects of the pandemic have not been limited to its virulence, but have rather conferred multiple collateral effects, especially in developing countries; thereby, designating it as a SYNDEMIC. The same culminated in neglect of non-COVID-19 conditions like tuberculosis (TB) and human immunodeficiency virus-acquired immunodeficiency syndrome (HIV/AIDS). Besides being the prognostic factor for severe COVID-19, these infections in hidden pockets served as a reservoir for the emergence of the deadly Omicron. Another significant impact of this juxtaposition was on the delivery of healthcare services for TB and HIV. The unanticipated COVID-19 pandemic turned the path of ongoing progress of elimination programs. Direct consequences of the COVID-19 pandemic were pronounced on diagnosis, treatment, and services for patients with TB and HIV. Essential TB services were reallocated to the COVID-19 rapid response task force. However, despite escalating the tribulations, this triple burden has simultaneously taught lessons to escalate the progress of halted programs. The pandemic has catalyzed an unusual level of collaboration among scientists, which can be exploited for TB and HIV. Fast-track diagnostics, digitalization, contact tracing, and vaccine development have enabled the world to envision the same for TB/HIV.

Cladosporium halotolerans: Exploring an Unheeded Human Pathogen.

BACKGROUND: Cladosporium halotolerans is a saprobic fungus, rarely implicated in human infections. The identification is challenging due to non-specific phenotypic features. OBJECTIVE: To decipher clinical spectrum, microbiological and susceptibility profile of clinical and environmental isolates of Cladosporium halotolerans. METHOD: All the isolates identified as Cladosporium halotolerans deposited in National Culture Collection for Pathogenic Fungi (NCCPF), Postgraduate Institute of Medical Education and Research, Chandigarh, India were revived. Phenotypic and molecular characterization targeting internal transcribed spacer (ITS) region of ribosomal DNA, large subunit of ribosomal DNA (LSU; NL1 and NL4), actin (ACT) and beta-tubulin (TUB) was done. Scanning electron microscopy (SEM) was performed to determine any phenotypic variations. Antifungal susceptibility testing (AFST) was carried out for eight antifungal agents as per CLSI M38 Ed3 guidelines. We also performed systematic literature review of all the cases of Cladosporium halotolerans reported till date. RESULTS: A total of four isolates (clinical, n = 3; soil, n = 1) identified as Cladosporium halotolerans were included in the study. The clinical sites were skin, maxillary tissue and nail. All patients were apparently immunocompetent, and history of trauma was recorded in one patient. All patients improved on antifungal therapy. The cultures revealed growth of black mycelial fungus and microscopic examination demonstrated dematiaceous septate hyphae with erect conidiophores and conidia in branched acropetal chains. Based on molecular methods, all the four isolates were identified as C. halotolerans. SEM revealed no variation in length and width of the conidia, conidiophores, ramoconidium and hyphae among the isolates. All molecular targets, such as ITS region, LSU (partially sequenced), ACT and TUB were able to differentiate the isolates. Minimum inhibitory concentrations for antifungals were: triazoles (0.12-2 µg/ml), amphotericin B (4 µg/ml) and echinocandins (2-8 µg/ml). CONCLUSION: We report role of the rarely isolated dematiaceous fungus, C. halotolerans, in causing human infections. The study emphasizes the role of molecular methods in precisely identifying these species. Triazoles are more active against these black fungi compared to polyenes or echinocandins.

Utility of CDC DENV1-4 real time PCR assay and trioplex assay for the diagnosis of dengue in patients with acute febrile illness.

Nucleic acid amplification tests (NAATs) have revolutionized reliable detection of dengue virus (DENV) during acute phase of infection. The study evaluated performance of CDC DENV-1-4 real-time assay, trioplex RT-PCR and heminested conventional RT-PCR assay in the diagnosis of DENV. The three NAATs were performed on 107 consecutive samples collected from patients suspected of DENV infection during acute phase of illness. Their performance was compared against composite reference standard, consisting of DENV NS1 antigen ELISA and DENV IgM ELISA. 88/107 study samples were positive by DENV ELISA, either NS1Ag (80), IgM (3) or both (5). The overall sensitivity of CDC DENV-1-4 RT-PCR assay, trioplex RT-PCR assay and conventional multiplex RT-PCR was 68.18%, 54.55% and 38.64%, respectively in diagnosing dengue during acute phase, with an area under the curve of 0.841, 0.773 and 0.693 respectively when compared against composite reference standard. The sensitivity was 82.93%, 73.17% and 51.22%, respectively within three days of illness and 60%, 42.86% and 28.57%, respectively between 4 and 5th day of illness. All the three molecular assays had 100% specificity. Maximum concordance values of 86.9% were recorded among CDC DENV-1-4 rRT-PCR assay and trioplex assay with kappa value of 0.74, suggestive of substantial agreement. CDC DENV-1-4 rRT-PCR assay can be used as a reliable and accurate test for diagnosis of DENV during acute phase of illness.

Emergence of COVID-19 Variants: An Update.

Severe acute respiratory disease virus-2 (SARS CoV-2) is one of the deadliest global threats faced by mankind to date. Despite the colossal efforts, the viral pandemic swept across all boundaries. Besides the virulence and susceptible population, the low proofreading capacity and error-prone mechanism of RNA-dependent RNA polymerase (RdRp) have contributed to new variants and reinfections. The World Health Organization has officially categorized these variants as variants of concern or variants of interest. This nomenclature is not merely to suffice the surveillance but also to have effective treatment and vaccine options in place. Coronavirus disease 2019 (COVID-19) variants have the propensity to render the available treatment strategies futile owing to the mutations they acquire. The futility of treatment strategies can be attributed either to the ineffectiveness or the shortage of supply given the skyrocketing increase in the number of cases. Presently, the Omicron variant is the most widespread one and is known to escape the protection, be it immune-derived, vaccination-derived, or hybrid. WHO has recommended modification in vaccine development policies and few companies have introduced Omicron-adapted vaccine jabs. Keeping in view the unending tale of COVID-19 variants and the huge data available on the same, this review focuses on providing insight into the emergence and ongoing dynamics of these new COVID-19 variants.

Stable isotope labeling as a promising tool for rapid drug susceptibility testing in Neisseria gonorrhoeae.

The world is heading towards an era of intractable and impending untreatable N. gonorrhoeae, thereby underlining the significance of rapid and accurate prediction of drug resistance as an indispensable need of the hour. In the present study, we optimized and evaluated a stable isotope labeling-based approach using the MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry) for rapid and reliable detection of ciprofloxacin and azithromycin resistance in N. gonorrhoeae. All the isolates were cultured under three varied condition setups viz. medium supplemented with normal lysine, heavy lysine (isotope), and heavy lysine along with the antibiotics (ciprofloxacin/azithromycin), respectively. After incubation, spectra were acquired using the MALDI-TOF MS which were further screened for unique patterns (media-specific spectra) to differentiate drug-susceptible and resistant isolates. The results of the stable isotope labeling assay were comparable to the results of phenotypic methods used for susceptibility testing.

Mucoid Staphylococcus haemolyticus: an unheeded multidrug-resistant pathogen.

Coagulase-negative Staphylococci (CoNS) are among the most abundant members of human skin microbiome. CoNS have lately been recognized as substantial agents in plethora of infections, especially nosocomial infections in preterm infants and immunocompromised patients. Staphylococcus haemolyticus is the second most common species isolated from blood, and identification is further hindered when there is a deviation in morphology from the classical one. Here, we report an uncommon case of multidrug resistant mucoid S. hemolyticus isolated from blood in a patient of polytrauma. The patient was managed with ceftriaxone-sulbactam, gentamicin, and meropenem as empirical therapy, which was subsequently changed to intravenous vancomycin. The patient showed favorable response to treatment. Mucoid isolates are known to be more virulent and multi-drug resistant than the classical morphotypes. We also conducted systematic review to decipher the prevalence of mucoid S. hemolyticus and linezolid (LZD) resistance in the same. This case highlights the significance of awareness of mucoid phenotypes of Gram-positive cocci for clinical microbiologists to reach accurate identification. Resistance to LZD further underscores the need of restriction policies in hospitals and to roll out antimicrobial stewardship program stringently, so that the growing resistance could be contained.

Bacterial diversity of hospital water tanks and the efficacy of hydrogen peroxide based cleaning: Experience in a tertiary care center in India.

BACKGROUND: Hospital water is often an overlooked yet preventable source of hospital-acquired infections. CDC recommends annual cleaning of water reservoirs in health care settings. In our tertiary care hospital, periodic disinfection and microbiological surveillance of all the water tanks in hospital premises is carried out. The aim of this paper is to report the diversity of bacterial flora noted and the role of cleaning method adopted in our hospital. METHODS: This retrospective study was carried out from July 2015 to September 2020. Tanks were cleaned using hydrogen peroxide based method and swabs were collected, pre- and post-cleaning. Any growth noted was identified using MALDI-TOF MS. RESULTS: A total of 398 swabs were collected during this period. In pre-cleaning samples, 144 (72%) showed growth of 219 microorganisms. Gram-negative organisms (53.7%, 116/216) were more frequently isolated than Gram-positive organisms (46.3%, 100/216). Although the overwhelming majority is generally regarded as non-pathogenic, a few pathogenic bacteria were also recovered. No bacteria were isolated in any of the post-cleaning samples. CONCLUSIONS: Diverse bacteria colonize water tanks over time, some of which are known to cause infections. Hydrogen peroxide is a simple and highly efficacious method of water tank disinfection. More such studies are required with other disinfectants to generate evidence with the ultimate aim of increasing safety of water supplied in hospitals.

Disseminated Cryptococcosis and Pulmonary Mucormycosis: Dealing with Dual Mycoses in a Renal Transplant Patient.

We describe a 36-year-old post-renal transplant patient diagnosed with a rare dual infection by Cryptococcus neoformans and Lichtheimia ramosa. The case highlights the significance of invasive samples and accurate interpretation of fungal biomarkers in the diagnosis of fungal infections in immunosuppressed patients. The mortality in such cases is high owing to delay in diagnosis, inability to perform surgical management or antifungal resistance.

Trend of pediatric cryptococcosis in a tertiary care centre and review of literature.

PURPOSE: Cryptococcosis is one of the most significant systemic fungal infections worldwide. Epidemiological data for pediatric cryptococcosis is very limited. Therefore, we planned this study to determine the burden of cryptococcosis in the pediatric population at our tertiary care center and performed review of literature. MATERIAL AND METHODS: In this retrospective study, all the patients less than 18 years of age were diagnosed with cryptococcosis, from January 2015-June 2021 were included. Demographic, clinical, and laboratory details of all the patients were noted. Furthermore, PubMed and MedLine databases were comprehensively searched for cases of pediatric cryptococcosis till June 2021. RESULTS: Of the total 5420 samples from suspected cryptococcosis cases processed at mycology laboratory, a total of 21 episodes of cryptococcosis (0.39%) were identified in 15 pediatric patients. The majority of the patients were apparently immunocompetent (10/15). Central nervous system (CNS) cryptococcosis was the most common presentation, followed by disseminated disease. All the isolates were identified as Cryptococcus neoformans (formerly referred to as C. neoformans var grubii), except one that was identified as Papiliotrema laurentii (formerly referred to as Cryptococcus laurentii). A standard treatment regimen inclusive of induction and maintenance therapy was provided in only five patients. The literature review revealed a total of 125 studies describing 1134 cases, of which 76.4% are reported from outside Asia, the majority (65.7%) restricted to CNS with C. neoformans as the commonest species. The management profile divulged a significantly higher use of standard drug regimen in Asia as compared to the rest of the world. Mortality of 13.32% was noted worldwide. CONCLUSION: To the best of our knowledge, this is the first clinico-epidemiological study of pediatric cryptococcosis from India and the largest retrospective study worldwide. The rising incidence among immunocompetent individuals, especially in Asia, is a matter of concern. Clinical suspicion and early diagnosis are the cornerstones for the management of cases.

Quantification of diurnal variation in "glove hygiene" compliance in COVID ICUs: An exploratory study.

BACKGROUND: Hand hygiene compliance (HHC) monitoring is almost always done in daytime. Documentation of HHC in health care workers (HCWs) is limited during odd hours and nighttime. The objective of the study was to determine diurnal variation in HHC in different categories of health care workers in tertiary care hospital in North India. METHODS: A prospective, observational study was conducted in 3 COVID-19 intensive care units (ICUs) with closed-circuit television (CCTV) cameras. Dedicated infection control nurses monitored HHC among various HCWs (doctors, nursing staff, technicians, hospital and sanitary attendants) during day and nighttime, in 20-minute durations. The difference in HHC by-professional category and for each WHO moment was assessed using χ² test and P value. RESULTS: A total of 705 opportunities were observed over a period of 7 days, with overall compliance of 53%. Day and nighttime compliance was recorded to be 60.7% and 42.1%, respectively (P < .001). HCC was highest amongst resident doctors with little diurnal variation. However, nurses and housekeeping staff exhibited significant diurnal variation. The compliance at "after" moments was much higher than "before" moments in all professional categories. CONCLUSION: There was a significant decrease in compliance during nighttime, amongst all HCWs, with maximum variation exhibited by nursing staff. The present study underlines the importance of monitoring HHC at odd hours, to elicit a more accurate picture round the clock. Health care facilities monitoring compliance only during the daytime may substantially overestimate HHC.

Initial Case Report of Cladorrhinum samala Mycotic Keratitis.

PURPOSE: The purpose of this study was to report the first case of keratitis caused by Cladorrhinum samala and review of the literature. METHODS: This was a case report and literature review. RESULTS: A 35-year-old immunocompetent man presented with pain, redness, and watering in the right eye 7 days after trauma with some foreign body. He was diagnosed with infectious keratitis, and a thorough microbiological workup was performed. Corneal scrapings were subjected to a potassium hydroxide (KOH) examination, Gram staining, bacterial (blood agar and Robertson cooked meat broth), and fungal culture (Sabouraud dextrose agar and brain-heart infusion agar). The KOH mount revealed septate fungal hyphae with irregular margins. Yellow-white nonsporulating mycelial growth was noted on the Sabouraud dextrose agar, which was identified as C. samala by sequencing. The patient responded to 5% natamycin and 1% voriconazole eye drops, and there was a formation of a corneal opacity in a period of 3 weeks. CONCLUSIONS: We report the first case of keratitis by C. samala, highlighting the emergence of a rare dematiaceous fungi causing keratitis and the role of molecular modalities in the diagnosis of nonsporulating fungi in suspected cases.

Haemophilus influenzae bacteremia: A 5-year (2016-2020) retrospective study analysing the clinical and laboratory features.

Haemophilus influenzae (H. influenzae) causes invasive disease like bacteremia which is rarely diagnosed. We conducted this retrospective study of H. influenzae bacteremia diagnosed between January 2016 and December 2020. Nineteen patients were identified. Majority were children ≤5 years of age (84.2%), inpatients (89.5%), males (78.9%) and admitted in ICUs (26.6%). The most common underlying primary diagnosis was malignancy. These isolates were most susceptible to carbapenems (100%) followed by cefotaxime (83.3%) and ampicillin (82.4%). The overall mortality rate was 33.3%. Increased mortality resulted in those admitted in ICUs with H. influenzae bacteremia episode along with polymicrobial/co-isolates infection.

First case of endophthalmitis by Corynespora cassiicola.

Corynespora cassiicola is an environmental phytopathogen implicated in various human infections and has been isolated from one case of keratitis. We report here the first case of endophthalmitis caused by Corynespora cassiicola in a 54-year-old diabetic patient, who developed diminished vision, redness, and watering in the right eye, six weeks after intravitreal triamcinolone acetonide for non-proliferative diabetic retinopathy and macular oedema. Direct microscopy of the vitreous tap revealed septate hyphae, and culture grew white mycelium that turned olive-grey to black within two days and was confirmed as C. cassiicola after sequencing of the ITS region of 28S rDNA. The patient was started on oral voriconazole 200 mg twice a day. The patient is on regular follow-up without recurrence. To our knowledge, this case represents the first case of endophthalmitis due to this fungus across the globe and the first case of C. cassiicola infection from India.