Utility of a multiplex pathogen detection system directly from respiratory specimens for treatment and diagnostic stewardship.

The availability of syndrome-based panels for various ailments has widened the scope of diagnostics in many clinical settings. These panels can detect a multitude of pathogens responsible for a particular condition, which can lead to a timely diagnosis and better treatment outcomes. In contrast to traditional identification methods based on pathogen growth on culture, syndrome-based panels offer a quicker diagnosis, which can be especially beneficial in situations requiring urgent care, such as intensive care units. One such panel is the Biofire Filmarray Pneumonia plus Panel (BFP), which we have compared against microbiological culture and identification. The lower respiratory samples from patients were tested with BFP, culture, and identification with culture considered the gold standard. The phenotypic antibiotic susceptibility results (Vitek 2) were compared with the antimicrobial resistance (AMR) genes detected in BFP. Statistical analysis was carried out using GraphPad 7.0 and MS Excel (Microsoft Inc.). The results showed a positive percent agreement of 100% and a negative percent agreement of 47.8% with an overall agreement of 76.72% compared to culture. BFP was better at identifying fastidious bacteria, and the agreement with culture was higher for high bacterial identification numbers (107 and 106). There was also a correlation between the number of pathogens detected and growth in culture. Carbapenemase genes were detected in around 80% of phenotypically resistant samples and correlated with in-house PCR 60% of the time. Hence, BFP results need to be interpreted with caution especially when multiple pathogens are detected. Similarly, the presence or absence of AMR genes should be used to guide the therapy while being watchful of unusual resistance or susceptibility. The cost constraints and low throughput call for patient selection criteria and prioritization in emergency or resource-limited conditions.IMPORTANCEApplication of syndrome-based panels in clinical microbiology is of huge support in infectious conditions requiring urgent interventions, such as pneumonia. Interpreting the results requires caution; hence, we have compared the results obtained from Biofire Filmarray Pneumonia plus Panel with standard microbiological methods.

Kodamaea ohmeri: A rare yeast causing invasive infections in immunocompromised patients.

INTRODUCTION: Kodamaea ohmeri is a rare, recognized pathogen that has previously been isolated from environmental sources. The patients commonly affected by this yeast include immunocompromised as well as immunocompetent patients having several associated risk factors. METHODOLOGY: We report three cases in which K. ohmeri was isolated from blood using Bact T/ALERT. Identification was carried out by MALDI-TOF MS (Vitek-MS, BioMérieux, Marcy-l'Etoile, France) in addition to color characteristics on chromogenic media. The patients had diminished immune response on account of a multitude of comorbidities. RESULTS: K. ohmeri can be misidentified as Candida tropicalis, Candida albicans, or Candida hemolounii by conventional methods; correct and timely identification can be achieved by MALDI-TOF MS. Antifungal susceptibility breakpoints for K. ohmeri are currently not defined. An Echinocandin was added to the treatment regimen of all three of the cases. CONCLUSIONS: Identification of K. ohmeri using conventional methods is difficult and unusual yeasts should be carefully observed, especially upon prolonged incubation.

Utility of an in-house real-time PCR in whole blood samples as a minimally invasive method for early and accurate diagnosis of invasive mould infections.

INTRODUCTION: Invasive mould infections (IMIs) are a leading cause of death in patients with compromised immune systems. Proven invasive mould infection requires detection of a fungus by histopathological analysis of a biopsied specimen, sterile culture, or fungal DNA amplification by PCR in tissue. However, the clinical performance of a PCR assay on blood samples taken from patients suspected of invasive mould disease has not been fully evaluated, particularly for the differential diagnosis of invasive aspergillosis (IA) and invasive Mucormycosis (IM). OBJECTIVES: To assess the diagnostic utility of our previously validated in-house real-time PCR in blood samples for diagnosis of invasive aspergillosis and mucormycosis in patients with suspected invasive mould infection. METHODS: All patients with suspected invasive mould infection were prospectively enrolled from May 2021 to July 2021. Conventional fungal diagnosis was performed using tissue and respiratory samples. In-house PCR was performed on blood samples and its diagnostic performance evaluated. RESULTS: A total of 158 cases of suspected invasive mould infection were enrolled in the study. The sensitivity and specificity of in-house PCR performed on blood samples was found to be 92.5% and 81.4% respectively for diagnosis of probable IA, and 65% and 84.62% respectively for diagnosis of proven and probable IM. It was also able to detect 3 out of 5 cases of possible IM where no other microbiological evidence of IM was obtained. CONCLUSIONS: This assay could be helpful in minimally invasive diagnosis of IMIs for patients in whom invasive sampling is not feasible, especially as a preliminary or screening test. It can help in early diagnosis, anticipating conventional laboratory confirmation by days or weeks. Possible correlation between fungal load and mortality can help in initiating aggressive treatment for patients with high initial fungal load.

Utility of in-house and commercial PCR assay in diagnosis of Covid-19 associated mucormycoss in an emergency setting in a tertiary care center.

Introduction. Invasive mucormycosis (IM) is a potentially fatal infection caused by fungi of the order Mucorales. Histopathology, culture, and radiology are the mainstays of diagnosis, but they are not sufficiently sensitive, resulting in delayed diagnosis and intervention. Recent studies have shown that PCR-based techniques can be a promising way to diagnose IM.Hypothesis/Gap Statement. Early diagnosis of fungal infections using molecular diagnostic techniques can improve patient outcomes, especially in invasive mucormycosis.Aim. The aim of this study was to evaluate the utility of our in-house mould-specific real time PCR assay (qPCR) in comparison with the commercially available real time PCR (MucorGenius PCR), for the early diagnosis of mucormycosis in tissue samples from patients with suspicion of invasive mucormycosis (IM). This in-house assay can detect and distinguish three clinically relevant mould species, e.g. Aspergillus spp., Mucorales and Fusarium spp. in a single reaction with only one pair of primers, without the need for sequencing.Methodology. We enrolled 313 tissue samples from 193 patients with suspected IM in this prospective study. All cases were classified using EORTC/MSGERC guidelines. All samples were tested using traditional methods, in-house qPCR, and MucorGenius PCR.Results. Using direct microscopy as a gold standard, the overall sensitivity and specificity of in-house qPCR for detection of IM was 92.46% and 80% respectively, while that of the MucorGenius PCR was 66.67% and 90% respectively. However, co-infection of IM and IA adversely affected the performance of MucorGenius PCR in detection of IM.The in-house PCR detected Aspergillus spp. in 14 cases and Fusarium spp. in 4 cases which showed clinical and radiological features of fungal sinusitis. The in-house qPCR also performed better in detecting possible cases of IM. This aids early diagnosis and appropriate treatment to improve patient outcomes.Conclusion. Because the in-house PCR is not only sensitive and specific, but also entirely based on SYBR Green for detection of targets, it is less expensive than probe-based assays and can be used on a regular basis for the diagnosis of IM in resource-constrained settings. It can be used to distinguish between mucormycosis and fungal sinusitis caused by Aspergillus and Fusarium in high-risk patients, as well as to accurately detect Mucorales in fungal co-infection cases.

E. hirae Causing Biliary Tract Infection in a Patient with Cholangiocarcinoma: A Case Report.

INTRODUCTION: E. faecium and E. faecalis are the common species of Enterococcus responsible for the majority of infections. Earlier, species other than the common ones were usually unidentified and reported as Enterococcus species. However, modern equipment, like MALDI-TOF and VITEK2, have been utilitarian, helping us to identify the previously unidentified species. E. hirae is an organism seldom reported to cause human infections. Here, we report a case of a biliary tract infection in a female patient with cholangiocarcinoma caused by E. hirae. CASE: A 56-year-old female presented with fever and abdominal pain. Bile aspirated during the ERCP was received in our laboratory. The gram stain of the bile sample revealed abundant polymorphonuclear leucocytes along with gram-positive diplococci. The organism failed to grow on MacConkey agar. On blood agar, non-hemolytic colonies grew. The organism was identified as E. hirae by MALDI-TOF MS. The antibiotic susceptibility performed using VITEK2 revealed it to be resistant to high-level gentamicin and susceptible to all remaining drugs. She was successfully treated with oral ciprofloxacin for the infection. DISCUSSION: Bile is colonized with bacteria due to obstruction in the biliary tree, leading to cholangitis. This causes bacterial proliferation and translocation of bacteria into the systemic circulation. Our case was resistant to high-level gentamicin, while all previously reported cases were susceptible. The resistant isolates of E. hirae being isolated from cattle and their surroundings amidst the rampant use of antibiotics in livestock can pose a difficult situation for humans. Thus, there should be regulations on antibiotic usage in livestock. Cases like these should be reported and recognized for their potential to cause outbreaks if they remain unreported. CONCLUSION: Thus, E. hirae, when encountered, should not be ignored but considered a pathogen and reported. The presence of drug-resistant organisms in cattle and their surroundings, their zoonotic potential to cause infections in humans, and the uncontrolled usage of antibiotics in livestock are causes for concern. Thus, we need to be more vigilant regarding it in the future.

Infection Trends, Susceptibility Pattern, and Treatment Options for Stenotrophomonas maltophilia Infections in Trauma Patients: A Retrospective Study.

Introduction Stenotrophomonas maltophilia is an emerging environmental, gram-negative, multidrug-resistant organism, associated with risk factors such as prolonged hospitalization, invasive procedures, admission to the intensive care unit, mechanical ventilation, use of indwelling catheters, administration of immunosuppressants or corticosteroids, human immunodeficiency virus infection, underlying malignancy, and organ transplantation. The organism, despite being of low invasiveness in immune-competent individuals, is difficult to treat because of intrinsic resistance to several antimicrobial agents. Materials and Methods This study focuses on commonly encountered resistance from among the isolates over a duration of 7 years from 2012 to 2018, analyzed retrospectively. Identification and susceptibility testing were performed using Vitek 2 (BioMérieux, Marcy-l'Etoile, France). Results Bloodstream infections were found to be most common (52.02%), followed by respiratory infections (35.83%). The median age of the patients was 36 years, and male to female ratio was 143:27. The median duration of hospital stay was 18 days, and mortality was seen in 18.82% of patients. Susceptibility to cotrimoxazole and levofloxacin was seen in 97.1% of isolates (168 out of 173) and 90.1% of isolates (156 out of 173), respectively. Conclusion Despite being effective in a majority of S . maltophilia isolates, both cotrimoxazole and levofloxacin have their shortcomings. Cotrimoxazole is bacteriostatic and can cause bone marrow suppression and resistance to levofloxacin sometimes develops during therapy. Thus, the therapy should be decided considering the characteristics of both of these drugs.

Microbiological Profile and the Resistance Pattern of Pathogens in Neurosurgical Patients from a New Delhi Trauma Center.

BACKGROUND: Neurosurgical patients are considered to be at higher risk for infections including nosocomial infections compared with other critically ill individuals. Empirical antimicrobial therapy is of utmost importance for the survival of infected neurosurgical patients. METHODS: The microbial distribution and antimicrobial resistance patients from January 2012 to December 2021 (10 years) were analyzed retrospectively. Identification was done using VITEK-2 and MALDI-TOF systems. Antimicrobial susceptibility testing was determined by the Kirby Bauer Disk Diffusion Agar method (Clinical and Laboratory Standards Institute) and VITEK-2. RESULTS: A total of 48,474 samples were received, out of which 10,134 (21%) had growth. Respiratory specimens showed the maximum isolation of pathogens (42% n = 4292). The predominant bacterial pathogens were gram negative (n = 8972; 88.5%), whereas gram positives were only 11.5% (n = 1162) of the total organisms. Among the gram positives, the most common was Staphylococcus aureus (64.6%), and among gram negatives, the most common pathogen was Acinetobacter baumanni (38.6%). The weighted average of the drug-resistance profile across all gram positives was >50% for fluoroquinolones (levofloxacin, ciprofloxacin), gentamicin, erythromycin, and ampicillin, and in the case of gram negatives it was >90% for ampicillin-sulbactam, ticarcillin, cefazolin, cefotaxime, and ceftriaxone. Thirty-two patients were found to have candidemia, out of which 6 were C. albicans and the rest were nonalbican. Six neurosurgery patients had infection with C. auris, 4 from blood samples and 2 from urine. CONCLUSIONS: This study will add to the current knowledge and provide a better understanding of pathogen profile and resistance patterns in traumatic brain injury patients.

Therapeutic Drug Monitoring of Voriconazole in Children with Hematologic Malignancy and Invasive Fungal Infections: An RCT from a Tertiary Care Centre in India.

INTRODUCTION: Voriconazole is a triazole anti-fungal with non-linear kinetics and a narrow therapeutic range. The objective of our study was to monitor the voriconazole serum levels in children with hematological malignancy and clinically suspected invasive fungal infections. METHODS: The study was a prospective, randomized controlled trial conducted from June 2016 to December 2017. All children who had haematologic malignancies with clinically suspected invasive fungal infections and received voriconazole as the only anti-fungal were included in the study. The children were randomly allotted into two groups; one was the group that underwent TDM, and the other, TDM, was not done. Bioassay was the method employed for TDM. The trough levels were evaluated on a sample obtained on the fifth day of starting the drug. The institute's ethics committee approved the study. RESULT: A total of 30 children were included in the study: 15 in the TDM group and 15 in the non-TDM group. The most common underlying malignancy was AML. Neutropenia due to chemotherapy sessions was these patients' most common risk factor. A favorable outcome was seen in 13/15 (86.7%) in the TDM group and 11/15 in the non-TDM group (73.3%). CONCLUSION: Only five out of 15 (33.3%) children had voriconazole serum levels within the therapeutic range. Alterations in dose had to be done in the remaining to achieve the recommended serum levels. Thus, we recommend TDM for all children of hematologic malignancy receiving voriconazole for better management. Our findings also revealed that children with AML had lower than recommended levels of voriconazole on TDM evaluation, whereas those with ALL had normal to elevated levels of voriconazole.

Secondary Bacterial Infections in Mucormycosis-COVID-19 Cases: Experience during the Second COVID-19 Wave in India.

In the second wave of COVID-19 in India, there was a new challenge in the form of mucormycosis. Coinfection with mucormycosis was perilous as both conditions required a prolonged hospital stay, thus serving as an ideal platform for secondary infections. Using a retrospective observational study, we studied secondary infections and their impact on the outcome in COVID-19 patients with mucormycosis. The outcome in these patients was evaluated and compared with COVID-19 patients with mucormycosis but without any secondary infection. SPSS V-20 was used for data analysis. Fifty-five patients tested positive for mucormycosis (55/140; 39.28). Twelve out of these 55 (21.8%) developed secondary infections during their hospital stay. Bloodstream infection was the most common (42.86%) secondary infection. The Gram-negative (GN) organisms were more common (11/16; 68.75%) compared with the Gram-positives (GP) (5/16; 31.25%). But the most common isolate was Enterococcus faecium (5/16; 31.25%). A high percentage of microorganisms isolated were multidrug-resistant (15/16; 93.75%). Two out of five (40%) isolates of Enterococcus faecium were vancomycin-resistant (VRE). High resistance to carbapenems was noted in the GN isolates (9/11; 81.81%). The comparison of length of stay in both subgroups was statistically significant (P value <0.001). When compared, the length of stay in people with adverse outcomes was also statistically significant (P value <0.001). Procalcitonin (PCT) had a positive predictive value for the development of secondary bacterial infections (P value <0.001). Antimicrobial stewardship and strict infection control practices are the need of the hour. IMPORTANCE Although our knowledge about COVID-19 and secondary infections in patients is increasing daily, little is known about the secondary infections in COVID-19-mucormycosis patients. Thus, we have intended to share our experience regarding this subgroup. The importance of this study is that it brings to light the type of secondary infections seen in COVID-19-mucormycosis patients. These secondary infections were partially responsible for the mortality and morbidity of the unfortunate ones. We, as health care workers, can learn the lesson and disseminate the knowledge so that in similar situations, health care workers, even in other parts of the world, know what to expect.

High Prevalence of Fungal and NDM-OXA Producing Gram-Negative Bacterial Superinfections in the Second Wave of Coronavirus Disease 2019 in India: Experience from a Dedicated Coronavirus Disease 2019 Hospital in North India.

Introduction: During the second wave of coronavirus disease 2019 (COVID-19), superinfection caused by fungus and multidrug-resistant bacteria worsened the severity of illness in COVID-19 patients. Limited studies from India reported the antimicrobial resistance pattern of secondary infections. In this study, we aim to study the epidemiology of pathogens causing superinfections and genotyping of Gram-negative isolates in COVID-19 patients. Methods: This retrospective study was conducted at a dedicated COVID-19 center, India. The identification of bacteria/fungi was done by Vitek2® and matrix-assisted laser desorption/ionization-time of flight mass spectrometry system. Identification of beta-lactamase genes was done using thermal cycler. The diagnosis of mucormycosis was based on 10% potassium hydroxide direct microscopy. Statistical analyses were performed using STATA version 15.1 (StataCorp., College Station, TX, USA). For continuous variables, mean and standard deviation were computed. For comparing proportions of secondary infections across admission location and outcomes, the Chi-squared test of independence was used. To compare the mean and median between intensive care units and outcomes, an independent t-test and a Mann-Whitney test were used. Results: Of all the clinical samples, 45.4% of samples were cultured positive for secondary infections. Acinetobacter baumannii (35%) was the most common Gram-negative pathogen, while among Gram positive, it was Enterococcus faecium (40%). Among fungus, Candida spp. (61%) predominates followed by molds. Colistin and tigecycline proved effective against these pathogens. blaNDM was the most prevalent gene followed by the blaOX among the carbapenemase genes. Conclusions: The mortality rate among COVID-19 patients with secondary infection was significantly higher compared to the overall mortality rate in COVID-19 patients.

Evaluation of COVID-19 Antigen Fluorescence Immunoassay Test for Rapid Detection of SARS-CoV-2.

INTRODUCTION: Tests detecting SARS-CoV-2-specific antigen have recently been developed, and many of them are now commercially available. However, the real-world performance of these assays is uncertain; therefore, their validation is important. In this study, we have evaluated the performance of STANDARD F COVID-19 antigen fluorescence immunoassay (FIA) kit. METHODS: Nasopharyngeal samples collected from patients were subjected to the test as per manufacturer's instructions. The performance of the kit was compared with the gold standard real-time polymerase chain reaction. RESULTS: A total of 354 patients were tested with STANDARD F COVID-19 antigen FIA test kit. The overall sensitivity, specificity, positive predictive value, and negative predictive value of this test were found to be 38%, 99%, 96.2%, and 72%, respectively, with a diagnostic accuracy of 75.7%. CONCLUSION: STANDARD F COVID-19 antigen FIA showed high specificity and positive predictive value but low sensitivity and negative predictive value.

Profile of co-infections & secondary infections in COVID-19 patients at a dedicated COVID-19 facility of a tertiary care Indian hospital: Implication on antimicrobial resistance.

BACKGROUND: The COVID-19 pandemic has raised concerns over secondary infections because it has limited treatment options and empiric antimicrobial treatment poses serious risks of aggravating antimicrobial resistance (AMR). Studies have shown that COVID-19 patients are predisposed to develop secondary infections. This study was conducted to ascertain the prevalence and profiles of co- & secondary infections in patients at the COVID-19 facility in North India. METHODS: We studied the profile of pathogens isolated from 290 clinical samples. Bacterial and fungal pathogens were identified, and antimicrobial susceptibility was determined by the Vitek2® system. Additionally, respiratory samples were tested for any viral/atypical bacterial co-infections and the presence of AMR genes by FilmArray test. The clinical and outcome data of these patients were also recorded for demographic and outcome measures analyses. RESULTS: A total of 151 (13%) patients had secondary infections, and most got infected within the first 14 days of hospital admission. Patients aged >50 years developed severe symptoms (p = 0.0004) and/or had a fatal outcome (p = 0.0005). In-hospital mortality was 33%.K.pneumoniae (33.3%) was the predominant pathogen, followed by A. baumannii (27.1%). The overall resistance was up to 84%.Majority of the organisms were multidrug-resistant (MDR) harbouring MDR genes. CONCLUSION: A high rate of secondary infections with resistant pathogens in COVID-19 patients highlights the importance of antimicrobial stewardship programs focussing on supporting the optimal selection of empiric treatment and rapid-de-escalation, based on culture reports.

Rapid chromatographic immunoassay-based evaluation of COVID-19: A cross-sectional, diagnostic test accuracy study & its implications for COVID-19 management in India.

Background & objectives: Coronavirus disease 2019 (COVID-19) has so far affected over 41 million people globally. The limited supply of real-time reverse transcription-polymerase chain reaction (rRT-PCR) kits and reagents has made meeting the rising demand for increased testing incompetent, worldwide. A highly sensitive and specific antigen-based rapid diagnostic test (RDT) is the need of the hour. The objective of this study was to evaluate the performance of a rapid chromatographic immunoassay-based test (index test) compared with a clinical reference standard (rRT-PCR). Methods: A cross-sectional, single-blinded study was conducted at a tertiary care teaching hospital in north India. Paired samples were taken for RDT and rRT-PCR (reference standard) from consecutive participants screened for COVID-19 to calculate the sensitivity and specificity of the RDT. Further subgroup analysis was done based on the duration of illness and cycle threshold values. Cohen's kappa coefficient was used to measure the level of agreement between the two tests. Results: Of the 330 participants, 77 were rRT-PCR positive for SARS-CoV-2. Sixty four of these patients also tested positive for SARS-CoV-2 by RDT. The overall sensitivity and specificity were 81.8 and 99.6 per cent, respectively. The sensitivity of RDT was higher (85.9%) in participants with a duration of illness ≤5 days. Interpretation & conclusions: With an excellent specificity and moderate sensitivity, this RDT may be used to rule in COVID-19 in patients with a duration of illness ≤5 days. Large-scale testing based on this RDT across the country would result in quick detection, isolation and treatment of COVID-19 patients.