Culture-negative Samples Research Articles

Evaluation of Sysmex-UF 5000-BF Module for Sterile Body Fluids. An Alternative for Conventional Methods?

Infections of cerebrospinal fluid (CSF), peritoneal and pleural fluids often result in severe morbidity and mortality. Cytometric analysis, cell count, and cell differentiation is supportive in rapid clinical decision. The aim of this study is to evaluate the performance of Sysmex UF-5000 (Sysmex Co., Kobe, Japan) in body fluid (BF) mode for white blood cells and bacterial counting and compare the results with manual microscopy, gram staining, and culture as reference method. A total of 634 body fluid samples (433 CSF, 100 pleural, and 101 peritoneal fluid) submitted to our clinical microbiology laboratory between December 2018 and January 2020 were enrolled in the study. Specimens were analyzed for white blood cell (WBC) counts with Sysmex UF-5000-BF mode. The results were grouped into five categories and compared with the conventional methods as reference method. The relationship between culture results and Sysmex UF5000-BF bacterial load and WBC count were evaluated for the detection of infection. The categorical agreement of UF 5000-BF mode was very strong for CSF (correlation coefficient: 0.70, p < 0.05) and good agreement was found for pleural and peritoneal samples (correlation coefficient: 0.63, p < 0.05). Sensitivity was 100% and specificity was 64% in pleural samples. For ascites sensitivity and specificity were found as 94.1% and 77.4%, and for CAPD samples as 100% and 81,2% respectively. Bacterial and WBC count with flow cytometry was higher among culture positive samples (p < 0.05). The same significant difference was detected between the UF 5000-BF bacterial counts and the samples in which bacteria were detected in the gram preperation (p < 0.05). WBC count by UF-5000-BF was also higher among culture positive compared to culture negative samples (p < 0.05). Our results demonstrated that the flow cytometric method, Sysmex UF-5000 body fluid (BF) mode, can be an alternative particularly in CSF samples. Detection of 100% sensitivity in peritoneal and pleural samples can prevent unnecessary antibiotic treatment and possible resistance development. Besides, automated bacterial counting can be used for rapid prediction of a positive body fluid culture.

Advancing Microbe Detection for Lower Respiratory Tract Infection Diagnosis and Management with Metagenomic Next-Generation Sequencing.

Due to limitations of traditional microbiological methods and the presence of the oropharyngeal normal flora, there are still many pathogens that cause lower respiratory tract infections (LRTIs) cannot be detected. Metagenomic next-generation sequencing (mNGS) has the potential capacity to solve this problem. This retrospective study successively reviewed 77 patients with LRTI and 29 patients without LRTI admitted to Tianjin Medical University General Hospital, China from August 2020 to June 2021. Pathogens in bronchoalveolar lavage fluid (BALF) specimens were detected adopting mNGS and traditional microbiological assays. The diagnostic performance of pathogens was compared between mNGS and BALF culture. The value of mNGS for aetiological and clinical impact investigation in LRTI was also evaluated. Among 77 patients with LRTI, 22.1%, 40.3%, and 65.0% of cases were detected as definite or probable pathogens by culture, all conventional microbiological tests, and mNGS, respectively. Using the final diagnosis as a gold standard, mNGS exhibited a sensitivity of 76.6% (95% confidence interval [CI], 65.6-85.5%), which was considerably superior to that of BALF culture (76.6% vs 18.2%; P < 0.01); specificity of 79.3% (95% CI, 60.3-92.0%), which was similar (79.3% vs 89.7%; P = 0.38); positive-predictive value of 90.8% (95% CI, 81.0-96.5%), and negative-predictive value of 56.1% (95% CI, 39.7-71.5%). According to our data, mNGS identified potential microorganisms in 66.7% (42/63) of culture-negative samples. Among 59 patients with pathogens identified by mNGS, conventional microbiological methods confirmed pathogenic infections in less than half (28/59) cases. Within the 77 patients, 34 (44.2%) patients received pathogen-directed therapy, 7 (9.1%) patients underwent antibiotic adjustment, and 3 (3.9%) patients stopped using antibiotics due to mNGS results. mNGS exhibits high accuracy in diagnosing LRTI, and combine with traditional microbiological tests, causative pathogens can be detected in approximately 70.0% of cases, thus yields a positive effect on antibiotic application.

IDENTIFICATION OF PATHOGENS IN THE INTRAOCULAR FLUID SAMPLES OF PATIENTS WITH ENDOGENOUS ENDOPHTHALMITIS USING RAPID NANOPORE TARGETED SEQUENCING.

Nanopore targeted sequencing showed a higher positivity rate and a shorter turnaround time than did traditional culture in identifying pathogens in the intraocular fluid samples of patients with endogenous endophthalmitis. To evaluate the feasibility of clinical application of nanopore targeted sequencing (NTS) for the identification of pathogens in patients with endogenous endophthalmitis, especially those with fungus-associated endophthalmitis. In this retrospective study, medical records and etiological results of 27 patients (34 eyes) with endogenous endophthalmitis were reviewed. The intraocular fluid samples were examined using both NTS and microbial culture. The results included the differences in detection time, positivity rate of pathogen detection, and positivity rate of fungus identification between two methods. NTS and microbial culture enabled the detection of etiologic agents in 89.28% and 35.71% of the samples, respectively. The difference of positivity rate between these methods was statistically significant ( P < 0.001). NTS also showed high sensitivity in both culture-positive and culture-negative samples (100% and 83.33%, respectively). Regarding culture-positive samples, the NTS results displayed a strong match with culture results. NTS showed a significantly higher positivity rate for fungal infection than did microbial culture (46.43% vs. 7.14%, P = 0.002). The average detection time of NTS was 1.11 ± 0.31 days, which was shorter than that of microbial culture (2.50 ± 0.58 days, Z = -4.686, P < 0.001). NTS technology facilitated an informed switch of intravitreal antimicrobial agents in 13 eyes. NTS, as a sensitive, specific, and timely complementary method, can be used along with traditional methods for the identification of pathogenic microorganisms in the intraocular fluid of patients with endogenous endophthalmitis.

Diagnostic yield of broad-range 16s rRNA gene PCR varies by sample type and is improved by the addition of qPCR panels targeting the most common causative organisms.

Introduction. Molecular techniques are used in the clinical microbiology laboratory to support culture-based diagnosis of infection and are particularly useful for detecting difficult to culture bacteria or following empirical antimicrobial treatment.Hypothesis/Gap Statement. Broad-range 16S rRNA PCR is a valuable tool that detects a wide range of bacterial species. Diagnostic yield is low for some sample types but can be improved with the addition of qPCR panels targeting common bacterial pathogens.Aim. To evaluate the performance of a broad-range 16S rRNA gene PCR and the additional diagnostic yield of targeted qPCR applied to specimens according to a local testing algorithm.Methodology. In total, 6130 primary clinical samples were collected as part of standard clinical practice from patients with suspected infection during a 17 month period. Overall, 5497 samples were tested by broad-range 16S rRNA gene PCR and a panel of targeted real-time qPCR assays were performed on selected samples according to a local testing algorithm. An additional 633 samples were tested by real-time qPCR only. The 16S rRNA gene PCR was performed using two assays targeting different regions of the 16S rRNA gene. Laboratory developed qPCR assays for seven common bacterial pathogens were also performed. Data was extracted retrospectively from Epic Beaker Laboratory Information Management System (LIMS).Results. Broad-range 16S rRNA gene PCR improves diagnostic yield in culture-negative samples and detects a large range of bacterial species. Streptococcus spp., Staphylococcus spp. and the Enterobacteriaceae family are detected the most frequently in samples with a single causative organism, but mixed samples frequently contained anaerobic species. The highest diagnostic yield was obtained from abscess, pus and empyema samples; 44.9 % were positive by 16S and 61 % were positive by the combined 16S and targeted qPCR testing algorithm. Samples with a particularly low diagnostic yield were blood, with 3.3 % of samples positive by 16S and CSF with 4.8 % of samples positive by 16S. The increased diagnostic yield of adding targeted qPCR is largest (~threefold) in these two sample types.Conclusion. Broad-range PCR is a powerful technique that can detect a very large range of bacterial pathogens but has limited diagnostic sensitivity. The data in this report supports a testing strategy that combines broad-range and targeted bacterial PCR assays for maximizing diagnosis of infection in culture-negative specimens. This is particularly justified for blood and CSF samples. Alternative approaches, such as metagenomic sequencing, are needed to provide the breadth of broad-range PCR and the sensitivity of targeted qPCR panels.

328. Universal PCR Testing- Is It Worth It?

Abstract Background Universal PCR and Next Generation Sequencing (uPCR/NGS) is a major advancement in microbiology. It is a highly sensitive and specific test that amplifies ribosomal RNA in samples to detect bacterial and fungal pathogens. We investigated the uPCR/NGS tests sent from Henry Ford Health (HFH) and the effect obtaining this test had on patient care. Methods We completed a retrospective, observational study assessing all consecutive uPCR/NGS tests obtained from at HFH from 2016-2021. This included uPCR/NGS for detection of bacterial, fungal, mycobacterium tuberculosis (MTB), and non-tuberculosis mycobacterium (NTM). All samples were of non-blood fluids and tissue samples. Patients concurrent tissue cultures and blood cultures from day of uPCR/NGS, and within 6 months of obtaining the uPCR/NGS sample were evaluated. Primary outcomes included if uPCR/NGS testing resulted in a change of choice or duration of antibiotic therapy. Results At HFH, 226 uPCR/NGS tests from 111 samples were analyzed. This included 83 bacterial, 51 MTB, 51 NTM, and 41 fungal uPCR/NGS tests. Of the 226 uPCR/NGS tests, 31 tests (13.7%) resulted with positive result on uPCR/NGS. A total of 31 tests (13.7%) were ordered on a known culture positive sample, and 195 (86.3%) were ordered on a culture negative sample. Of samples sent from a culture positive sample, 7 (22.6%) resulted with positive uPCR/NGS results. 3 (42.9%) matched the tissue cultures, and 2 (28.6%) matched the patients’ blood cultures. None of the patients had any change in choice or duration of antibiotics based on uPCR/NGS results. Of the samples sent from a culture negative sample, 24 (12.3%) samples resulted with positive uPCR/NGS results. 2 (8.3%) matched a patient’s tissue cultures and none matched blood cultures. Change in antibiotic choice or duration occurred in 16 (17.6%) patients. The samples with the highest rates of change in management included cerebrospinal fluid (42.9%), brain tissue (36.4%), cardiac valvular tissue (27.3%), lymph nodes (25%), synovial fluid (20%). Figure 1:Flowchart of Results and Impact on Management Conclusion Our results show that uPCR/NGS has an overall low percent of impact in clinical outcomes, but it is a useful test in when used on a culture negative sample in certain sample types, along with a high index of suspicion for infection. Disclosures All Authors: No reported disclosures.

Characterizing SARS-CoV-2 Transcription of Subgenomic and Genomic RNAs During Early Human Infection Using Multiplexed Droplet Digital Polymerase Chain Reaction.

Control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission requires understanding SARS-CoV-2 replication dynamics. We developed a multiplexed droplet digital polymerase chain reaction (ddPCR) assay to quantify SARS-CoV-2 subgenomic RNAs (sgRNAs), which are only produced during active viral replication, and discriminate them from genomic RNAs (gRNAs). We applied the assay to specimens from 144 people with single nasopharyngeal samples and 27 people with >1 sample. Results were compared to quantitative PCR (qPCR) and viral culture. sgRNAs were quantifiable across a range of qPCR cycle threshold (Ct) values and correlated with Ct values. The ratio sgRNA:gRNA was stable across a wide range of Ct values, whereas adjusted amounts of N sgRNA to a human housekeeping gene declined with higher Ct values. Adjusted sgRNA and gRNA amounts were quantifiable in culture-negative samples, although levels were significantly lower than in culture-positive samples. Daily testing of 6 persons revealed that sgRNA is concordant with culture results during the first week of infection but may be discordant with culture later in infection. sgRNA:gRNA is constant during infection despite changes in viral culture. Ct values from qPCR correlate with active viral replication. More work is needed to understand why some cultures are negative despite presence of sgRNA.

Identification of microbial agents in culture-negative brain abscess samples by 16S/18S rRNA gene PCR and sequencing.

Despite clinical suspicion of an infection, brain abscess samples are often culture-negative in routine microbiological testing. Direct PCR of such samples enables the identification of microbes that may be fastidious, non-viable, or unculturable. Brain abscess samples (n = 217) from neurosurgical patients were subjected to broad range 16S rRNA gene PCR and sequencing for bacteria. All these samples and seven formalin-fixed paraffin-embedded tissue (FFPE) samples were subjected to species-specific 18S rRNA PCR for neurotropic free-living amoeba that harbour pathogenic bacteria. The concordance between smear and/or culture and PCR was 69%. One-third of the samples were smear- and culture-negative for bacterial agents. However, 88% of these culture-negative samples showed the presence of bacterial 16S rRNA by PCR. Sanger sequencing of 27 selected samples showed anaerobic/fastidious gram negative bacteria (GNB, 38%), facultative Streptococci (35%), and aerobic GNB (27%). Targeted metagenomics sequencing of three samples showed multiple bacterial species, including anaerobic and non-culturable bacteria. One FFPE tissue revealed the presence of Acanthamoeba 18S rRNA. None of the frozen brain abscess samples tested was positive for 18S rRNA of Acanthamoeba or Balamuthia mandrillaris. The microbial 16/18S rRNA PCR and sequencing outperformed culture in detecting anaerobes, facultative Streptococci and FLA in brain abscess samples. Genetic analyses of 16S/18S sequences, either through Sanger or metagenomic sequencing, will be an essential diagnostic technology to be included for diagnosing culture-negative brain abscess samples. Characterizing the microbiome of culture-negative brain abscess samples by molecular methods could enable detection and/or treatment of the source of infection.

Evaluation of Nested broad-range PCR for Pathogen Detection in Negative Blood Cultures

Background: Bloodstream infections are a major cause of morbidity and mortality among all age groups. In the laboratory, it is routinely diagnosed by performing a blood culture which is considered an imperfect gold standard. The diagnostic limitations and uncertainties of blood cultures are related to low sensitivity, particularly in antibiotic-exposed cases, and extended time for pathogen detection that increases turn-around-time of the results. An alternative and recently introduced nucleic acid amplification method for 16S rDNA, a conserved DNA sequence common to all bacteria, has gained significant. It has high sensitivity and an ability to detect organisms that are non-cultivable or nonviable owing to prior antibiotic treatment and where the organism fails to grow on a culture medium. Objectives: To determine the utility of broad-range PCR in the detection of pathogens from negative blood culture was the aim of our study. Materials and Methods: A total of 50 negative blood cultures after 5 days of incubation on BacT/Alert continuously monitored blood culture system were included in this study. From each blood broth sample, DNA was isolated which was subjected to a broad range 16S rDNA nested PCR. The PCR products obtained were visualized on agarose gel electrophoresis and the results were interpreted. Results: Broad range bacterial 16S rDNA nested PCR detected the desired amplicon in 4/50 (8%) of the blood culture-negative samples. Conclusion: The broad range bacterial nested PCR method if used in combination with routine culture techniques in clinical microbiology laboratories will increase the detection of Bloodstream pathogens.

Culture-Independent Genotyping Improves Surveillance of Neisseria gonorrhoeae, Especially in Oropharyngeal Samples, the Netherlands, 2017 to 2018.

It is important i to monitor the transmission and antimicrobial resistance of Neisseria gonorrhoeae (NG). Current surveillance relies on culturing, which frequently fails. Previously, a culture-independent genotyping method was developed based on NG multi-antigen sequence typing (NG-MAST). To determine whether crucial sequence types (STs) are missed during culture-dependent surveillance, NG-positive NAAT samples were genotyped, and the results of the culture-positive and culture-negative samples were compared. In total, 196 NG-positive NAAT samples, from January 2017 until August 2018, which were also routinely cultured, were retrospectively included. Genotyping was successful in 152 NAAT samples (77.0%), 33 NAAT samples failed, and 11 NAAT samples showed possible mixed strain infections. Oropharyngeal samples (n = 16) showed the largest increase in typing rate from 6.3% (1/16) success in culture-dependent genotyping to 81.3% (13/16) in culture-independent genotyping. Nine genogroups (n ≥ 5 samples) were found; all included both culture-positive and culture-negative NG. However, culture-independent surveillance revealed 14 additional STs in the culture-negative samples. Overall, culture-dependent surveillance could detect all genogroups, indicating that major trends could be identified with culture-dependent surveillance. However, culture-independent surveillance provides more STs, mixed infections and more oropharyngeal samples, giving a more detailed view and could result in an earlier detection of outbreaks and transmission.

Diagnostic utility of adenosine deaminase in bronchoalveolar lavage (BAL) in pulmonary tuberculosis from a tertiary care hospital in North India

Adenosine Deaminase (ADA) has been acclaimed as a biochemical marker in numerous studies in the diagnosis of tuberculosis of pleura, peritoneum, meninges etc because of its high sensitivity and specificity. As a diagnostic test, ADA offers several advantages; it is rapid, simple, low cost, non-invasive and can be performed easily in most clinical laboratories. The current retrospective study was carried out on a total of 91 specimens of bronchoalveolar lavage collected on bronchoscopy. The samples were collected and sediments were confirmed for presence of tubercular bacilli through Lowenstein Jensen(LJ) media (Gold standard), GeneXpert(CBNAAT).Simultaneously the ADA estimation was done from supernatant fluid obtained after centrifugation of sample.The Mean ADA level for Culture Positive samples in BAL was 5.899± 1.723 and from Culture Negative Samples the Mean ADA was1.217 ± 1.439. The ADA cut off levels of &amp;#62;4.0 IU/L in BAL when compared with LJ culture media (gold standard) showed a sensitivity of 90.0% and a specificity of 97.2%. Upon ROC analysis a high rate of accuracy was recorded in diagnosis of TB through ADA estimation with sensitivity and specificity reaching 100% and 97.2% at a ADA cut off 3.76 IU/L when compared with LJ culture media (gold standard). However, the sensitivity and specificity achieved was slightly lower when ADA results were compared with GeneXpert results.The determination of ADA levels may help in early diagnosis, improve the prognosis and reduce the spread of disease and thereby the test should be included in routine investigations in patients suspected of tuberculosis.

P395 Clinical utility of semi-nested conventional PCR for diagnosis of mucormycosis in fresh clinical samples

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesWe aim to assess the clinical utility of the semi-nested conventional PCR in smear positive, culture-negative clinical samples for diagnosis of mucormycosis.MethodsThis prospective study was conducted for a period of 3 months (April-June 2021). A total of 218 clinical samples were included from patients in whom smear was positive for aseptate hyphae, but the culture failed to grow within 2 days of incubation or smear was negative but had high suspicion of mucormycosis. Molecular diagnosis was attempted using semi-nested PCR with Mucorales-specific primers targeting 18S region, described previously by Bialek et al.1. Phenol-chloroform based manual DNA extraction protocol was optimized in the mycology laboratory of the department of medical microbiology, PGIMER, Chandigarh. This method was applicable to different types of clinical samples to yield good quality DNA with minimal chances of extraneous contamination (Fig. 1). Amplified PCR products were further sequenced to identify the causative species (Fig. 2).ResultsAmong 218 patients with suspected mucormycosis included in this study, the majority were rhino-orbito-cerebral mucormycosis (ROCM), (77.7%, n = 169), followed by pulmonary mucormycosis (19.2%, n = 42), cutaneous (0.02%, n = 4), and gastro-intestinal (GI) mucormycosis (0.01%, n = 3). In 24 samples, the presence of both septate and aseptate hyphae was seen under microscopic examination raising the possibility of mixed infection. On microscopic examination, 90.3% samples (197/218) had aseptate hyphae while the remaining samples were smear-negative but had strong clinical suspicion of mucormycosis. The molecular technique was able to identify causative agent in 154 culture-negative samples (81.9%, 154/188) and 52.4% (11/21) in smear-negative cases. Among 218 patients, only 20 samples show delayed growth of Mucorales, and on comparison with molecular results 100% concordance was observed. However, 10 patients with strong clinical suspicion for mucormycosis were negative by both conventional and molecular methods. The low culture positivity necessities the molecular diagnosis based on in-house semi-nested PCR using above-mentioned primers followed by Sanger sequencing. In the case of 24 mixed infections with aseptate-septate hyphae, Mucorales-specific PCR correctly identified 23/24 (95.8%) Mucorales as a causative agent. The overall turn-around-time from the sample receiving to diagnosis was ˂48 h. Overall, R. arrhizus (85/143, 59.4%) was most commonly associated with ROCM, while R. microsporus (13/38, 34.2%), and R. homothallicus (5/38, 13.1%) were seen mainly with pulmonary mucormycosis. Apophysomyces and Saksenaea genus were associated with GI and cutaneous mucormycosis.ConclusionsThe molecular technique utilizing semi-nested PCR, followed by Sanger sequencing was able to identify Mucorales species 81.9% of culture-negative cases. Optimized manual DNA extraction protocol is suitable for different sample types, with minimum chances of extraneous contamination and offers low cost with a shorter turnaround time (TAT).

P418 qPCR is a useful tool for management and surveillance of hospital contacts in the context of a Candida auris infection

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objectives Candida auris is an emerging fungal pathogen responsible for hospital outbreaks. It represents a serious threat due to its drug resistance profile and its potential spread within healthcare facilities. Since the global alert by the CDC in 2016, specific control measures are now available to prevent the further spread of the pathogen. These measures should be implemented immediately as soon as a case is identified to prevent patient-to-patient transmission. Until recently culture was the main technique used for the detection of C. auris from patient and environmental samples. Nevertheless, PCR protocols have been reported and commercial kits are now available. Our objectives were to compare culture and PCR in routine for the management of a case of C. auris infection in a hospital setting.MethodsWe report here the case of a patient infected by C. auris following injuries in a public road traffic accident in Dubai. Following the medical evacuation and transfer of the patient to our hospital in Paris, C. auris was isolated from several surgical specimens from the elbow. Identification of the species level was initially performed by MALDI-TOF Mass spectrometry and confirmed by ITS sequencing. Antifungal susceptibility testing was performed by Etest and EUCAST. Surveillance of the index case included bi-weekly surveillance for 2 weeks and then once a week. Contacts were also screened for C. auris colonization once a week by swabbing axilla and groin. Samples were analyzed by standard mycological cultures and a specific C. auris qPCR kit (kit Fungiplex Candida auris®, Bruker).ResultsIn total 133 samples were analyzed for the patient and 52 contacts. For the index case, 14/22 samples were positive in culture for C. auris including elbow biopsies, urine, and axilla, groin, and rectal swabs. Other Candida species (C. albicans, C. krusei) were also recovered from the same samples for the patient. For the contacts, all 111 samples were negative for C. auris by culture, but retrieved several other yeast species (C. albicans, C. glabrata, C. kefyr, C. paraspsilosis, C. tropicalis, Saccharomyces cerevisiae, Trichosporon inkin, and Wickerhamomyces anomalus). By using qPCR, all culture-positive samples were positive (Ct ranged from 29.7 to 38.0, with a median at 31.4). Two culture-negative samples (one biopsy and one axillary swab) were also qPCR-positive. All samples from contacts were negative by qPCR. The strain was resistant to fluconazole (>256 μg/ml) and susceptible to all other tested antifungals (amphotericin B, flucytosine, voriconazole, and caspofungin). Whole genome sequencing of the C. auris strain is in progress to determine the clade.DiscussionThe Fungiplex C. auris qPCR kit showed good sensitivity and specificity, even for the frequent situation of samples growing with two or three Candida species. These results highlight the usefulness of the PCR for surveillance of infected patients as well as for contacts.

S2.3c High-resolution digital DNA melting: a breakthrough in diagnosing IMI?

S2.3 Novel diagnostic tools for invasive mold infection, September 21, 2022, 3:00 PM - 4:30 PMInvasive mold infections (IMIs) such as Aspergillosis, Mucormycosis, Fusariosis, and Lomentosporiosis have emerged as important pathogens in immunocompromised patients, with mortality rates as high as 50% to nearly 80% for these infections. Outcomes can be substantially improved with early initiation of appropriate antifungal therapy, yet early diagnosis remains difficult to establish and often requires multidisciplinary teams evaluating clinical and radiological findings plus supportive microbiologic findings. Conventional fungal culture and PCR techniques are limited by low sensitivity, long turn-around time, and are insufficient for differentiating between infection and colonization. Other non-culture-based tests, such as the Galactomannan test, are available only for invasive aspergillosis and are limited by imperfect test performance. Better and more rapid diagnostics are needed to enable early diagnosis and targeted treatment of IMIs and improve survival. We have developed a new technology called digital high resolution melting analysis (dHRM) to enable a rapid and robust diagnosis of IMI. This technology accomplishes fast genotyping of fungal genomic sequences in clinical samples by: (1) conducting broad-based amplification of fungal genes in a digital PCR format, and (2) conducting high-resolution melting of the DNA amplicons in each digital reaction. High resolution melting measures the fluorescence of a saturating intercalating dye as dPCR-amplified fungal DNA fragments are rapidly heated and disassociated, producing sequence-defined melt curves in a closed reaction format. These melt curves serve as unique fungal ‘fingerprints’, which allows us to correctly identify disease-causing pathogen(s) with an accuracy of 99%-100% through the use of machine learning methods trained to tolerate variations in reaction conditions. This approach provides a simple, low-cost, fast, and robust method for pathogen identification. Here, we will present the performance of this new technology on clinical bronchoalveolar lavage (BAL) samples from patients with and without IMI. A total of 75 patient BAL samples were tested, coming from 30 patients with proven (n = 1), probable (n = 25), or putative (n = 4) invasive pulmonary aspergillosis (IPA) infections, and from 45 patients classified as not having IPA (n = 4 possible IPA and n = 41 no IPA). Our results show that dHRM was able to reliably differentiate between different Aspergillus spp. and was also able to identify Aspergillus spp. in BAL samples from patients with probable IPA where qPCR had resulted in negative. In a few instances, it also detected rare molds in culture-negative samples and multiple Aspergillus spp. within one BAL sample, a phenomenon that has been previously described in cultures. Interestingly, there was no clear correlation between dHRM results and BAL galactomannan levels, suggesting that dHRM may serve as an independent method allowing for a combined diagnostic approach. While further validation and optimization are needed, these results suggest that dHRM is a promising new diagnostic technology capable of providing clinically relevant information within 2-3 h.

Nanopore-based metagenomic sequencing for the rapid and precise detection of pathogens among immunocompromised cancer patients with suspected infections.

Cancer patients are at high risk of infections and infection-related mortality; thereby, prompt diagnosis and precise anti-infectives treatment are critical. This study aimed to evaluate the performance of nanopore amplicon sequencing in identifying microbial agents among immunocompromised cancer patients with suspected infections. This prospective study enlisted 56 immunocompromised cancer patients with suspected infections. Their body fluid samples such as sputum and blood were collected, and potential microbial agents were detected in parallel by nanopore amplicon sequencing and the conventional culture method. Among the 56 body fluid samples, 47 (83.9%) samples were identified to have at least one pathogen by nanopore amplicon sequencing, but only 25 (44.6%) samples exhibited a positive finding by culture. Among 31 culture-negative samples, nanopore amplicon sequencing successfully detected pathogens in 22 samples (71.0%). Nanopore amplicon sequencing showed a higher sensitivity in pathogen detection than that of the conventional culture method (83.9% vs. 44.6%, P<0.001), and this advantage both existed in blood samples (38.5% vs. 0%, P=0.039) and non-blood samples (97.7% vs. 58.1%, P<0.001). Compared with the culture method, nanopore amplicon sequencing illustrated more samples with bacterial infections (P<0.001), infections from fastidious pathogens (P=0.006), and co-infections (P<0.001). The mean turnaround time for nanopore amplicon sequencing was about 17.5 hours, which was shorter than that of the conventional culture assay. This study suggested nanopore amplicon sequencing as a rapid and precise method for detecting pathogens among immunocompromised cancer patients with suspected infections. The novel and high-sensitive method will improve the outcomes of immunocompromised cancer patients by facilitating the prompt diagnosis of infections and precise anti-infectives treatment.

P181 Mucor in the land of the liver: a case report

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Introduction and ObjectivesMucormycosis is a rare, highly lethal opportunistic fungal infection affecting immune-compromised patients. It accounts for about 2% of invasive fungal infections occurring within 1 year after solid organ transplantation. Among renal recipients, Rhinocerebral is the commonest manifestation and Rhizopus species is the most frequent pathogen.The objectives are:To report a case of Hepatic mucormycosis in a renal transplant recipient.To resolve the dilemma in identification of Mucorale species detected on microscopy but culture negative.MethodA 29-year-old male patient was admitted with severe abdominal pain on day 28 post-live related renal transplantation. Patient was hypertensive, on oral steroids for idiopathic thrombocytopenic purpura, chronic kidney disease with venous thromboembolism-presumed chronic interstitial nephritis, and maintained on hemodialysis for the last 1 year. Post-transplant he was on triple immunosuppressants.Initial ultrasound of abdomen and transplant doppler were normal. CT of abdomen in view of worsening hypotension and non-resolving abdominal pain, revealed hepatomegaly with multiple discrete, coalescing hypodense areas in both lobes of liver with minimal subcapsular extension. Initial cultures included interventional radiology-guided liver aspirate and blood culture. Patient was started on antibiotics and antifungals. Patient continued to deteriorate and a repeat abdominal CT revealed an increase in liver size and rupture of liver abscess. Sample collected on pigtail catheter insertion was also sent for culture.ResultsThe pus aspirate was received for bacterial and fungal culture. Aerobic bacterial culture yielded Klebsiella species. Examination with KOH Calcofluor revealed broad aseptate fungal hyphae. However, fungal culture did not yield any growth. Pain resistant (PDR) Klebsiella species was isolated from blood culture. Pigtail catheter sample also yielded the same. Aseptate fungal hyphae were observed in the pigtail catheter sample also. The team decided on conservative management and patient was started on intravenous amphotericin B deoxycholate(50 mg/day), tigecycline, and meropenem. Immunosuppressants were withheld. The clinical course worsened and the patient succumbed to illness after 18 days of antifungal therapy. The pus sample was then sent to the referral center PGIMER for PCR and sequencing where it was identified as Lichtheimia species.ConclusionMucormycosis is an increasingly emerging invasive fungal infection especially in immune-compromised patients, including solid organ transplant recipients. Though uncommon it is frequently a fatal mycosis in transplant patients. Hepatic mucormycosis in renal transplant recipients has been reported in three patients so far. None are due to Lichtheimia species. The diagnosis of hepatic mucormycosis was confirmed as microscopy was positive in two successive samples. Our patient also had bacteremia with PDR Klebsiella.Diagnosis is challenging and often delayed, as the clinical presentation is non-specific. Although mucormycosis in a renal transplant recipient is rare, a high index of suspicion and critical microscopic examination is warranted for early initiation of specific therapy which includes liposomal amphotericin B and surgery. Often cultures yield no growth and gene sequencing proves to be efficient and time-saving. This probably is the way ahead, especially for culture-negative samples to establish the etiological diagnosis.

Clinical laboratory diagnostics in dentistry: Application of microbiological methods.

Diagnosis and treatment in dentistry are based on clinical examination of the patients. Given that the major oral diseases are of microbial biofilm etiology, it can be expected that performing microbiological analysis on samples collected from the patient could deliver supportive evidence to facilitate the decision-making process by the clinician. Applicable microbiological methods range from microscopy, to culture, to molecular techniques, which can be performed easily within dedicated laboratories proximal to the clinics, such as ones in academic dental institutions. Periodontal and endodontic infections, along with odontogenic abscesses, have been identified as conditions in which applied clinical microbiology may be beneficial for the patient. Administration of antimicrobial agents, backed by microbiological analysis, can yield more predictable treatment outcomes in refractory or early-occurring forms of periodontitis. Confirming a sterile root canal using a culture-negative sample during endodontic treatment may ensure the longevity of its outcome and prevent secondary infections. Susceptibility testing of samples obtained from odontogenic abscesses may facilitate the selection of the appropriate antimicrobial treatment to prevent further spread of the infection.

Improving the Diagnosis of Bacterial Infections: Evaluation of 16S rRNA Nanopore Metagenomics in Culture-Negative Samples.

While 16S rRNA PCR-Sanger sequencing has paved the way for the diagnosis of culture-negative bacterial infections, it does not provide the composition of polymicrobial infections. We aimed to evaluate the performance of the Nanopore-based 16S rRNA metagenomic approach, using both partial and full-length amplification of the gene, and to explore its feasibility and suitability as a routine diagnostic tool for bacterial infections in a clinical laboratory. Thirty-one culture-negative clinical samples from mono- and polymicrobial infections based on Sanger-sequencing results were sequenced on MinION using both the in-house partial amplification and the Nanopore dedicated kit for the full-length amplification of the 16S rRNA gene. Contamination, background noise definition, bacterial identification, and time-effectiveness issues were addressed. Cost optimization was also investigated with the miniaturized version of the flow cell (Flongle). The partial 16S approach had a greater sensitivity compared to the full-length kit that detected bacterial DNA in only 24/31 (77.4%) samples. Setting a threshold of 1% of total reads overcame the background noise issue and eased the interpretation of clinical samples. Results were obtained within 1 day, discriminated polymicrobial samples, and gave accurate bacterial identifications compared to Sanger-based results. We also found that multiplexing and using Flongle flow cells was a cost-effective option. The results confirm that Nanopore technology is user-friendly as well as cost- and time-effective. They also indicate that 16S rRNA targeted metagenomics is a suitable approach to be implemented for the routine diagnosis of culture-negative samples in clinical laboratories.

Comparison of Light Emitting Diode (LED) Fluorescent Microscopy with Ziehl-Neelsen Microscopy on Sputum Specimens for Diagnosis of Pulmonary Tuberculosis Keeping Culture as ‘Gold Standard’

Objective: To compare LED fluorescence microscopy and Ziehl-Neelsen staining in terms of their diagnostic performance in diagnosing pulmonary tuberculosis, taking sputum specimens from patients suspected of pulmonary tuberculosis.&#x0D; Study Design: Prospective longitudinal study.&#x0D; Place and Duration of Study: Microbiology Department, Armed Forces Institute of Pathology Rawalpindi, from Jan 2019 to Dec 2019.&#x0D; Methodology: Sputum samples from patients with clinical suspicion of pulmonary tuberculosis were stained using Ziehl-Neelsen (ZN) stain, fluorescent stain with Auramine O staining (AO) stain and Mycobacterial culture on Mycobacterial Growth Indicator Tube (MGIT 960), to detect Mycobacterium tuberculosis. WHO guidelines were followed to grade positive smears.&#x0D; Results: Among 206 patients with suspicion of tuberculosis, 143 (69%) were male, and 63 (30%) were female patients. The mean age of the patients was 53.67 ± 14.73 years. Out of 206 sputum samples, 64 were negative by all three techniques used. 142 (68%) specimens detected Mycobacterium tuber-culosison MGIT960. Within 142 culture-positive samples, only 40 samples were positive on Ziehl-Neelsen microscopy, whereas 97 samples were detected positive by LED fluorescent microscopy. In culture-negative samples, three were missed on Ziehl-Neelsen staining, which was positive with Fluorescent microscopy. Sensitivity and specificity for Ziehl-Neelsen smear microscopy were 26.7% and 96.8%, respectively. Sensitivity and specificity for Fluorescent smear microscopy were 64.8% and 92.2%, respectively.&#x0D; Conclusion: We concluded that the efficacy of LED fluorescence microscopy has proven to have many potential advantages over conventional Ziehl-Neelsen microscopy.

Streptococcus agalactiae and Prototheca spp. induce different mammary gland leukocyte responses in Holstein cows

In this study, we investigated the association between natural subclinical intramammary infection (IMI) caused by Streptococcus agalactiae and Prototheca spp. and milk differential cell counts assessed by cytofluorimetric analysis in Holstein cows. After an initial bacteriological screening on 188 animals and a second assessment carried out 2 wk later aimed at confirming the bacteriological status, we collected milk samples from 47 animals and performed (1) milk composition analyses; (2) somatic cell counts and differential somatic cell counts (DSCC); and (3) cytofluorimetric analyses. Before statistical analyses, animals with co-infections were filtered out. Bacteriological status (negative, positive for Strep. agalactiae, or positive for Prototheca spp.) significantly affected the investigated traits. Compared with culture-negative samples, those that were positive for Strep. agalactiae and Prototheca spp. had higher SCS (+61% and +49%, respectively), DSCC (+4% and +19%, respectively), log polymorphonuclear neutrophil (PMN)-lymphocyte (LYM) counts (+59% and +71%, respectively), and log macrophage (MAC) counts (+63% and +72%, respectively). The individual leukocyte populations determined by cytofluorimetric analysis confirmed that mastitis infection increased the proportion of PMN in the milk samples compared with culture-negative samples, particularly when caused by Strep. agalactiae (+51%). In the case of MAC, the 2 pathogens behaved in opposite ways: Strep. agalactiae increased MAC by 41%, whereas Prototheca decreased MAC by 25%. Prototheca infection strongly increased the proportion of total T lymphocytes (TL; +87%) and T-helper lymphocytes (+83%). Accordingly, the (PMN+MAC):TL ratio increased with Strep. agalactiae infection (+95%) and decreased with Prototheca infection (−43%) compared with culture-negative samples. These results suggest the prevalence of an adaptive immune response and chronic inflammation in Prototheca infection, and an innate immune response to Strep. agalactiae. This knowledge might provide an important contribution to the development of novel and effective diagnostics and therapeutics.

Viral culture and immunofluorescence for the detection of SARS-CoV-2 infectivity in RT-PCR positive respiratory samples.

BackgroundKnowing how long SARS-CoV-2-positive individuals can remain infective is crucial for the design of infection prevention and control strategies. Viral culture is the gold standard for detecting an active-replicative virus and evaluating its infectious potential.ObjectiveTo assess the correlation of SARS-CoV-2 infectivity with the number of days from symptom onset and the Ct value, using culture as a reference method. Also, to describe a detailed protocol for SARS-CoV-2 culture and immunofluorescence confirmation based on our experience with other respiratory viruses.Study design100 consecutive respiratory samples positive for SARS-CoV-2 by RT-PCR from different subjects were inoculated into VERO E6 cells.ResultsViral isolation was successful in 58% of samples. The median number of days from symptom onset for culture-positive samples was 2, and 15 for culture-negative samples. Six positive cultures were obtained in patients ≥14 days after symptom onset, all of whom were immunocompromised or with severe COVID-19. The mean Ct value was 12.64 units higher in culture-negative than in culture-positive samples. The probability of successfully isolating SARS-CoV-2 in samples with a Ct value <22 was 100%, decreasing to 3.1% when >27.ConclusionsOur findings show a significant positive correlation between the probability of isolating SARS-CoV-2 in culture, fewer days of symptoms and a lower RT-PCR Ct value. SARS-CoV-2 infectivity lasts no more than 14 days from symptom onset in immunocompetent individuals. In contrast, in immunocompromised patients or those with severe COVID-19 infectivity may remain after 14 days. Ct value <22 always indicates infectivity.