Salvaging lives in carbapenem-resistant Gram-negative osteoarticular and soft tissue infections: early compromises save lives.

PURPOSE: Incidence of carbapenem-resistant Gram-negative infections has risen alarmingly all across the globe, both in developed and developing countries alike. The purpose of this study was to assess whether challenges of life-threatening infections with very high resistance pattern can be successfully addressed by a modified approach. METHODS: This is a retrospective study of 26 patients with osteoarticular and soft tissue infections with carbapenem-resistant Gram-negative bacilli treated between 2001 and 2017 with at least two year follow-up after stopping antibiotics. All were treated by a multispecialty team approach with primary aim of "source control at the earliest and avoiding recurrence at all cost". The protocol involved opting for early compromises especially in at "risk individuals", such as resorting to early amputations, especially if salvage meant multiple bony and soft tissue reconstructive procedures, explanation of prosthesis than staged revision, avoiding internal fixations, opting for shortest possible time in external fixators with reshaping and telescoping bone ends to get bony stability and increase surface area even if it meant compromising length. RESULTS: There were five amputations, two excision arthroplasty of hip, many minor but acceptable malunions and shortening. However, lives of 24/26 patients could be salvaged, much better than most of the published data. The two patients who died had peri-prosthetic joint infection after total hip arthroplasty and presented very late in sepsis and died within days of explantation. Infection remission could be achieved in remaining patients. CONCLUSION: These "risk to life" cases can be successfully treated by lowering the aims and expectations from "excellent function to salvage of life and infection remission". Therein lies the "success" in these complex high-risk cases.

Immune Modulation and Cytomegalovirus Reactivation in Sepsis-induced Immunosuppression: A Pilot Study.

INTRODUCTION: Sepsis is a life-threatening condition caused due to dysregulated immune response to an infection and progressive immunosuppression. Reactivation of cytomegalovirus (CMV) occurs frequently in sepsis and is found associated with adverse outcomes. The study objective was to evaluate the association between incidence of CMV reactivation and immune alteration in sepsis-induced immunosuppression in patients with prolonged sepsis. PATIENTS AND METHODS: Patients admitted to intensive care unit (ICU), with severe sepsis and CMV immunoglobulin G (IgG) seropositivity, were prospectively enrolled. Other manifest immune suppression causes were excluded. Samples were collected on enrolment and further once a week until day 21 or death/discharge. CMV viral load was quantified using qPCR. Lymphocyte subset analysis (CD3+, CD4+, CD8+, CD19+, CD16+/CD56+, and CD25+CD127- regulatory T cells), human leukocyte antigen-DR isotype (HLA-DR) expression on monocytes, programmed death-1 (PD-1) expression on T lymphocytes, and proinflammatory (interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ)), anti-inflammatory cytokines levels (IL-2, IL-4, and IL-10) were analyzed by flow cytometry as markers for immunosuppression. RESULTS: A total of 25 CMV IgG-positive patients and 11 healthy controls were included. CMV reactivation occurred in 20 patients. Patients with CMV reactivation had T-cell lymphopenia. PD-1 expression on CD4+ and CD8+ T cells was markedly elevated (p <0.02) in CMV-reactivated patients compared to nonreactivated patients. HLA-DR expression was significantly low on monocytes in all septic patients (p <0.01) compared to healthy controls. IL-6 levels showed elevation at day 7, whereas IL-10 was found to be significantly higher from day 0 in CMV-reactivated group. CONCLUSION: Our study concluded that immune suppression markers and cytokine levels in patients with severe sepsis were found to be significantly associated with the incidence of CMV reactivation. HOW TO CITE THIS ARTICLE: Lambe G, Mansukhani D, Khodaiji S, Shetty A, Rodrigues C, Kapadia F. Immune Modulation and Cytomegalovirus Reactivation in Sepsis-induced Immunosuppression: A Pilot Study. Indian J Crit Care Med 2022;26(1):53-61.

Linezolid resistance in Mycobacterium tuberculosis isolates at a tertiary care centre in Mumbai, India.

Background & objectives: Linezolid (LZD) is increasingly being used in tuberculosis (TB) treatment. However, LZD resistance has already been reported, which is highly alarming, given its critical therapeutic role. This study was aimed to phenotypically and genotypically assess LZD resistance in Mycobacterium tuberculosis (MTB) isolates at a laboratory in a tertiary care centre in Mumbai, India. Methods: A sample of 32 consecutive LZD-resistant MTB isolates identified by liquid culture susceptibility testing was subjected to whole-genome sequencing (WGS) on the Illumina NextSeq platform. Sequences were analyzed using BioNumerics software to predict resistance for 12 antibiotics within 15 min. Results: Sixty eight of the 2179 isolates tested for LZD resistance by MGIT-based susceptibility testing (June 2015 to June 2016) were LZD-resistant. Thirty two consecutive LZD-resistant isolates were analyzed by WGS to screen for known mutations conferring LZD resistance. WGS of 32 phenotypically LZD-resistant isolates showed that C154R in the rplC gene and G2814T in the rrl gene were the major resistance determinants. Interpretation & conclusions: LZD resistance poses an important risk to the success of treatment regimens, especially those designed for resistant isolates; such regimens are extensively used in India. As LZD-containing regimens increase in prominence, it is important to support clinical decision-making with an improved understanding of the common mutations conferring LZD resistance and their frequency in different settings.

Laboratory Diagnosis of Tropical Infections.

HIGHLIGHTS: (1) Blood culture is the gold standard for the diagnosis of bacterial infections. (2) Bone marrow culture is more sensitive than blood culture even in patients with enteric fever receiving antibiotics. (3) Microscopic agglutination test is considered the gold standard for diagnosing leptospirosis; however, now IgM ELISA and polymerase chain reaction (PCR) are more frequently used for diagnosis. (4) Tuberculosis is diagnosed with the help of nucleic acid amplification tests like Xpert MTB/RIF Ultra which also detects rifampicin resistance. Other tests include microscopy, Lowenstein-Jensen and mycobacteria growth indicator tube culture, line probe assay. (5) Tropical rickettsial infections are diagnosed by serological reactions (Weil-Felix, ELISA for antibodies) and PCR. (6) For Brucellosis culture from blood, bone marrow or tissue specimens remain the mainstay in diagnosis. (7) Dengue, Zika, Crimean-Congo hemorrhagic fever, Ebola, hantavirus, rabies are diagnosed with reverse transcriptase-polymerase chain reaction. Serological tests like IgM ELISA or paired sera samples for IgG are also used for diagnosis. HOW TO CITE THIS ARTICLE: Basu S, Shetty A. Laboratory Diagnosis of Tropical Infections. Indian J Crit Care Med 2021;25(Suppl 2):S122-S126.

Characteristics, outcome and risk factors for mortality of paediatric patients with ICU-acquired candidemia in India: A multicentre prospective study.

BACKGROUND: The epidemiology, clinical profile and outcome of paediatric candidemia vary considerably by age, healthcare settings and prevalent Candida species. Despite these differences, few comprehensive studies are undertaken. This nationwide study addresses this knowledge gap. METHODS: 487 children who contracted ICU-acquired candidemia at 23 Indian tertiary care centres were assessed for 398 variables spanning demography, clinical characteristics, microbiology, treatment and outcome. RESULTS: Both neonates (5.0 days; range = 3.0-9.5) and non-neonatal children (7.0 days; range = 3.0-13.0) developed candidemia early after ICU admission. Majority of neonates were premature (63.7%) with low birthweight (57.1%). Perinatal asphyxia (7.3%), pneumonia (8.2%), congenital heart disease (8.4%) and invasive procedures were common comorbidities, and antibiotic use (94.1%) was widespread. C tropicalis (24.7%) and C albicans (20.7%) dominated both age groups. Antifungal treatment (66.5%) and removal of central catheters (44.8%) lagged behind. Overall resistance was low; however, emergence of resistant C krusei and C auris needs attention. The 30-day crude mortality was 27.8% (neonates) and 29.4% (non-neonates). Logistic regression identified admission to public sector ICUs (OR = 5.64), mechanical ventilation (OR = 2.82), corticosteroid therapy (OR = 8.89) and antifungal therapy (OR = 0.22) as independent predictors of 30-day crude mortality in neonates. Similarly, admission to public sector ICUs (OR = 3.62), mechanical ventilation (OR = 3.13), exposure to carbapenems (OR = 2.18) and azole antifungal therapy (OR = 0.48) were independent predictors for non-neonates. CONCLUSIONS: Our findings reveal a distinct epidemiology, including early infection with a different spectrum of Candida species, calling for appropriate intervention strategies to reduce candidemia morbidity and mortality. Independent factors identified in our regression models can help tackle these challenges.

Multiplex PCR to detect pAmpC ß-lactamases among enterobacteriaceae at a tertiary care laboratory in Mumbai, India.

Drug-resistance due to AmpC ß-lactamases represents a growing problem worldwide. In this study, a previously collected sample of 108 cefoxitin-resistant clinical isolates was assessed for AmpC ß-lactamase production through routine phenotypic testing and double-disc cefoxitin/cloxcallin (DD-CC), cefoxitin/phenylboronic acid (CDT-PBA) and AmpC disc tests. The same isolates were characterized by a novel multiplex polymerase chain reaction molecular assay to detect the presence of blaACT, blaDHA, blaCIT, blaFOX, blaMIR and blaMOX. By phenotypic analysis, 56%, 55% and 48 % were detected as being AmpC ß-lactamase producers by the CDT-PBA, DD-CC and AmpC disc tests, respectively. By molecular analysis, 57  % were determined to be AmpC ß-lactamase producers, including 34 % blaFOX, 8 % blaCIT and 1.6 % blaDHAas mono-AmpC producers. The production of multiple AmpC molecular types was common, including 30 % with both blaCIT+FOX and 1.6 % each of blaCIT+DHA, blaACT+MIR, blaACT+FOX, blaACT+DHA and blaMIR+FOX. Molecular characterization of AmpC would help detect the prevalence of AmpC ß-lactamase producers, facilitate proper patient management and implement infection control practices.

Trichosporon-Blood Stream Infection.

AIM: Trichosporon species are the major emerging opportunistic pathogen in immunocompromised patients. Its diverse refractoriness to conventional antifungal drugs and association with high mortality rate is worrisome. The present study aims to determine the risk factors, treatment outcome and antifungal susceptibility pattern of Trichosporon species in blood stream infections. MATERIAL AND METHODS: All patients with blood culture positive for Trichosporon species from January 2012 to August 2016 at PD Hinduja National Hospital and research centre were evaluated retrospectively. Species identification and antifungal susceptibility by broth microdilution method for various drugs was determined using Vitek2 compact automated system. RESULTS: 12 patients were found to have Trichosporon blood stream infection. 9 isolates that were speciated all were T. asahii. All patients had central venous catheter and received prior antibiotics. Overall mortality rate was 50%. CONCLUSION: Higher mortality was associated with central venous catheter and voriconazole should be used as drug of choice for treatment. Identification of Trichosporon species along with its sensitivity and proper treatment of patients is of utmost importance.

Indian Antimicrobial Prescription Guidelines in Critically Ill Immunocompromised Patients.

How to cite this article: Kulkarni AP, Sengar M, Chinnaswamy G, Hegde A, Rodrigues C, Soman R, Khilnani GC, Ramasubban S, Desai M, Pandit R, Khasne R, Shetty A, Gilada T, Bhosale S, Kothekar A, Dixit S, Zirpe K, Mehta Y, Pulinilkunnathil JG, Bhagat V, Khan MS, Narkhede AM, Baliga N, Ammapalli S, Bamne S, Turkar S, Bhat KV, Choudhary J, Kumar R, Divatia JV. Indian Journal of Critical Care Medicine 2019;23(Suppl 1): S64-S96.

Gastric bacterial Flora in patients Harbouring Helicobacter pylori with or without chronic dyspepsia: analysis with matrix-assisted laser desorption ionization time-of-flight mass spectroscopy.

BACKGROUND: The gastric microbiota has recently been implicated in the causation of organic/structural gastroduodenal diseases (gastric and duodenal ulcers, gastric cancer) in patients with Helicobacter pylori (H. pylori) infection. We aimed to ascertain, in patients harbouring H. pylori, the role of the gastric microbiota in the causation of symptoms (chronic dyspepsia) in the absence of organic disease. METHODS: Seventy-four gastric biopsy samples obtained at endoscopy from patients with (n = 21) or without (n = 53) chronic dyspepsia, and that tested positive by the bedside rapid urease test for H. pylori infection, were cultured for detection of H. pylori and non-H. pylori organisms. The cultured organisms were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF MS). RESULTS: A total of 106 non-H. pylori isolates were obtained from 74 patients' samples. This included 33 isolates (median 2, range 1-2 per patient) from dyspeptic and 73 (median 2, range 1-2 per patient) from non-dyspeptic patients. These were identified from the Bruker Biotyper 2 database as Staphylococcus spp., Streptococcus spp., Lactobacillus spp., Micrococcus spp., Enterococcus spp., Pseudomonas spp., Escherichia spp., Klebsiella spp. and Bacillus spp., Staphylococcus and Lactobacillus were identified significantly more commonly in dyspeptics and Streptococcus, Pseudomonas, Escherichia coli and Klebsiella pneumoniae in non-dyspeptics. All identified organisms belonged to the phyla Firmicutes and Proteobacteria. CONCLUSIONS: There is a qualitative difference in the gastric microbial spectrum between patients harbouring H. pylori with and without chronic dyspepsia. Whether these organisms have an independent role in the development or prevention of dyspepsia or act in concurrence with H. pylori needs study.

Paradoxical Response in Cerebral Nocardiosis in a Renal Transplant Recipient.

Paradoxical response (PR) in patients on anti-tuberculosis drugs and immune reconstitution inflammatory syndrome (IRIS) in patients started on antiretroviral therapy are well known phenomenon. We encountered a case of a paradoxical response in cerebral nocardiosis in a renal transplant recipient. To our knowledge this phenomenon in cerebral nocardiosis has not been reported earlier in literature.

Implications for Diagnosis and Treatment of Infective Endocarditis: Eight year Experience of an Infectious Disease Team in a Private Tertiary Care Centre.

Background: The profile of Infective endocarditis (IE) has been evolving continuously. Like other infectious Diseases (ID) syndromes, IE has not escaped from antibiotic resistance issues. The aim of this study was to determine the implications for diagnosis and treatment by studying the clinical profile and outcome of patients admitted with IE in a tertiary care centre in Mumbai during the period from 2007-2015. Methods: 53 patients having definite or possible IE as per Modified Duke's Criteria (MDC), that were referred to the ID division, were included in this study. Results: 44 (83%) patients had definite IE and 9 (17%) patients had possible IE. 77.4% of the patients were above 40 years of age. 3 patients presented as euthermic IE. Vegetations were not seen on transthoracic echocardiography (TTE) in 3 patients and were seen only on transesophageal echocardiography (TEE). 15 patients had prosthetic valve IE. 7 patients had rheumatic heart disease. 3 patients had bicuspid aortic valve and 4 had ventricular septal defect (VSD). The rest had no apparent underlying heart disease (45.3%). 41 patients (77.3%) had culture-positive IE and 12 patients (22.6%) had culture-negative IE. Streptococcus spp. was found in 14 (26.4%) patients, Enterococcus spp. in 9 patients (17%). Other organisms isolated were methicillin-sensitive S. aureus (3), Methicillin Resistant S. aureus (1), Eikenella corrodens (1), B. cepacia (2), Salmonella Typhi (1), P. aeruginosa (1), M. abscessus (2) and other rapidly growing mycobacteria (RGM) (5), Candida parapsilosis (1), Candida pelliculosa (1) and Aspergillus fumigatus (1). Notably there was only one case of MRSA. Among the Streptococcus spp., Penicillin MIC testing was done in 11 cases of the 14 cases of Strep spp. 3 of them showed intermediate resistance and 2 were resistant. Among enterococcal IE, 3 had high level aminoglycoside resistance (HLAR) and 2 had ß-lactamase producing enterococci with HLAR and 1 had Vancomycin resistance. These were successfully treated with combinations of Ampicillin with Ceftriaxone, Ampicillin-Sulbactam with Imipenem and Daptomycin respectively. The only case of MRSA prosthetic valve endocarditis was successfully treated with Vancomycin and Rifampicin in addition to surgery. Surgery for IE was performed in 26 out of 53 (49%) patients. Early valve surgery (within 15 days of hospital admission) was performed in 6 of these 26 patients. . Conclusion: There is a change in the spectrum and antimicrobial susceptibility of organisms causing IE. We encountered several difficulties with the use of the MDC as 43.5% patients had no predisposing factors for IE and blood cultures were negative in 22.6% cases. In our study, PVE was the most common predisposing condition for IE. VGS followed by enterococci were found to be the commonest cause for IE in our setting. Both organisms show variable drug resist patterns. MRSA was isolated in 1 patient only. Thus vancomycin may not be required as empiric treatment in our setting. This is important from the perspective of antimicrobial stewardship Good infection control practices are essential to prevent nosocomial IE due to pathogens such as non-tuberculous mycobacteria (NTM). Important changes in the disease characteristic, treatment, and outcome are noted. Surgery, whenever indicated, helps in improving outcome in these patients thus reiterating the need for a team approach for optimal management of this complex, challenging condition..

Fosfomycin Susceptibility in Urinary Tract Enterobacteriaceae.

INTRODUCTION: Antibiotic treatment of Urinary Tract Infections (UTI) is becoming increasingly difficult due to emergence of multi-drug resistant (ESBLs, AmpC, CRE) uropathogens. Fosfomycin is an old antibiotic that has evoked renewed interest with unique properties of not sharing any structural similarity and lack of cross-resistance with other antimicrobial agents. Our aim is to evaluate in-vitro activity of Fosfomycin against urinary tract Enterobacteriaceae. MATERIAL & METHODS: The study period was March 2014 to September 2015. All 72 isolates were identified using conventional biochemical tests. Antimicrobial susceptibility testing was performed using the automated broth microdilution system Vitek 2 (bio- Mérieux, Inc., Durham, NC). Fosfomycin susceptibility was determined by the E-test (bioMérieux, Inc., Durham, NC) method. Interpretive criteria from the Clinical and Laboratory Standards Institute (CLSI) for fosfomycin susceptibility are not available for the Enterobacteriaceae other than Escherichia coli. Therefore, results were interpreted according to criteria for E. coli (i.e., susceptible at a MIC of ≤ 64 µg/ml), as has been reported previously. RESULTS: Overall, 79.16% (57/72) isolates were susceptible to fosfomycin w i t h 92.00% (23/25) susceptibility in ESBL producing enterobacteriaceae and 72.34% (34/47) in CRE. One CRE isolate has developed resistant while on treatment. There was not much difference in number of susceptible isolates CLSI:EUCAST = 57:53,but number of resistant isolates was more with EUCAST (CLSI:EUCAST = 10:19). CONCLUSION: Study demonstrate that, a considerable proportion (79.16%) of the multidrug-resistant Enterobacteriaceae with diverse resistance mechanisms, including ESBL and CRE, found susceptible to fosfomycin. Consequently, fosfomycin may currently be considered a useful antibiotic agent in the treatment armamentarium of UTIs.

Successful Management of Elizabethkingia meningoseptica Meningitis with Intraventricular Vancomycin.

Elizabethkingia meningoseptica, formerly Chryseobacterium meningosepticum usually causes neonatal meningitis and is a rare cause of nosocomial meningitis in adults. E. meningoseptica is resistant to most antibiotics, and the use of inactive drugs as empirical therapy may contribute to poor outcome in many patients. Vancomycin, alone or in combination with rifampicin, has been successful in the treatment of meningitis in infants1. We present a case of E. meningoseptica meningitis in an adult who was treated initially with intravenous vancomycin and oral rifampicin, but did not respond to the treatment. Thereafter, intraventricular vancomycin was added which resulted in good treatment response.

Antibiotic Lock Therapy in the Era of Gram-Negative Resistance.

BACKGROUND: Central-line-associated blood-stream infection (CLABSI) is a highly consequential nosocomial infection. The most effective management includes the removal of the infected catheter. Retention of the catheter and antibiotic lock therapy (ALT) along with systemic antibiotics may be attempted only if there are unusual extenuating circumstances. CLABSIs due to Gram-negative bacteria (GNB) is more common in our setting and the organisms are often highly resistant. Hence, there is a need to explore the use of novel antimicrobials for catheter lock solutions along with antibiofilm agents. PATIENTS AND METHODS: We report the use of antibiotic lock therapy in the first 29 patients who had 37 episodes of bacteremia (CLABSI/symptomatic colonization) due to long-term catheters in our unit from February 2008 to September 2014. Patients received ALT if they had CLABSI or were symptomatic with a colonized catheter. Patients who needed removal of the catheter were ineligible for ALT. Patients received systemic antibiotic therapy and lock solutions were kept in the catheter for dwell times of 24 hours, and therapy was continued for 14 days. Successful treatment was defined as any of the following: 1) Clinical cure with disappearance of signs of sepsis 2) Microbiological cure with resolution of bacteremia (confirmed by a negative blood culture which was obtained through the catheter 2-5 days after stopping therapy. RESULTS: Among the 37 episodes treated with ALT, 30 episodes were caused by GNB and four episodes were caused by Gram-positive cocci (GPC); Enterococcus, methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), and methicillin-sensitive coagulase-negative staphylococcus (CoNS). There were three episodes of CRBSI due to Candida and one episode each due to L. monocytogens and Bacillus spp. Of the other 30 episodes due to GNB, Acinetobacter baumannii were isolated in eight episodes, Stenotrophomonas (n=6), E. coli (n=5), Flavobacterium (n=2), and P. aeruginosa (n=4), and B. cepacia in three episodes. The other organisms isolated were K. pneumoniae, and non-typhoidal Salmonella (1 episode each). Successful treatment with ALT was observed in 30 (81.08%) of the 37 episodes. CONCLUSIONS: In patients with CLABSI due to Gram-negative pathogens, the use of ALT along with systemic antibiotics has an excellent catheter salvage rate. Newer antibiotics (tigecycline and colistin) may be useful options as antibiotic lock solutions along with antibiofilm agents especially in the setting of resistant Gram-negative bacilli producing CLABSI.

Is it Time to Rethink the Use of Steroids for Pulmonary Leptospirosis?

Pulmonary involvement is a fairly common complication of leptospirosis. A high dose of steroids is often used in the treatment of pulmonary leptospirosis. Here we report two cases who developed severe invasive fungal infections following the use of steroids for pulmonary leptospirosis. Routine use of steroids for pulmonary leptospirosis may do more harm than good as the evidence for this practice is sparse.

The Concepts that Revolutionized the Field of Infectious Diseases.

The fields of Microbiology and Infectious diseases have developed tremendously in the 19th and the 20th centuries. Four revolutionary concepts that evolved during this period form the cornerstones on which these fields have developed further.

Intravascular Stent-related Endocarditis due to Rapidly Growing Mycobacteria : A New Problem in the Developing World.

We report five patients with infective endocarditis (IE) due to rapidly growing mycobacteria (RGM) which we postulate are related to the reuse of percutaneous transluminal angioplasty balloon catheters prior to the intravascular stent placement. The index procedure was performed at various institutes in different parts of the country. We call attention to diagnostic and therapeutic difficulties and the uniformly dismal outcome of RGM IE as well as the potential hazards of re-use of single-use devices.

Successful management of highly drug resistant tuberculosis with individualised drug susceptibility testing.

INTRODUCTION: Many tuberculosis (TB) patients have resistance patterns intermediate between multidrug-resistant (MDR) and extensively drug-resistant (XDR). We defined MDR+ as resistance to rifampin (RMP), isoniazid (INH) and at least one more drug other than fluoroquinolone (FQ) and second-line injectable agent (IA); and Pre-XDR as MDR with additional resistance to either FQ or IA. Such patients too, have compromised treatment options that require various combinations of second line drugs (SLD). The aim of our study was to assess the clinical outcome of patients having such resistance patterns, managed on the basis of prior drug exposure and drug susceptibility testing (DST). METHODOLOGY: 52 consecutive patients were studied. Treatment regimen was devised as per DST and predominantly consisted of a second-line injectable agent (IA), para-aminosalicylic acid (PAS) and clofazimine. Additionally, cycloserine, linezolid, co-amoxiclav and clarithromycin were used to complete a regimen of four to five drugs. Clinical and radiological outcome was evaluated at follow-up and at the end of treatment. RESULTS: 49/52 (94%) patients had good outcome. However, 34 different regimens had to be used, as options for individual patients were limited. CONCLUSION: Management on the basis of prior drug exposure and individualised DST led to good clinical outcomes. No single regimen emerged as having a wide applicability. This study supports the clinical relevance of DST of oral second line drugs.

In vitro evaluation of antifungal combinations against neurotropic dematiaceous fungi associated with primary cerebral phaeohyphomycosis.

Primary cerebral phaeohyphomycosis is a life-threatening disease caused by neurotropic dematiaceous fungi. At present, there are no consensus guidelines regarding optimal antifungal therapy in such cases. Generally, a combination of antifungal agents is recommended for treatment. However, the activities of antifungal combinations against these fungi have not been investigated. In this study, we evaluated the in vitro activities of 13 double and five triple antifungal combinations against clinical isolates of Cladophialophora bantiana (n = 7), Fonsecaea monophora (n = 2), and Cladosporium cladosporioides (n = 1), using a simplified checkerboard procedure. The minimum inhibitory concentrations (MICs) of nine antifungal drugs were determined by the broth microdilution method, and the interaction between antifungal agents in each combination was assessed by the fractional inhibitory concentration index. Excellent activity was observed for posaconazole and itraconazole. Flucytosine had potent activity against C. bantiana but was ineffective against F. monophora, and C. cladosporioides. The echinocandins demonstrated high MICs for all the isolates. Synergistic interactions were observed for all the double combinations, except when itraconazole was combined with either amphotericin B or flucytosine. The combination of amphotericin B with caspofungin showed synergistic interactions against 40% of the isolates. Antagonism was observed with isavuconazole-flucytosine combination against two C. bantiana isolates. The triple combinations of caspofungin and flucytosine with amphotericin B or posaconazole were synergistic against one isolate of F. monophora. For C. cladosporioides, synergy was observed for the triple combination of amphotericin B with caspofungin and flucytosine. Our results indicate that combination of caspofungin with amphotericin B or a triazole, with or without 5-flucytosine has great potential against neurotropic dematiaceous fungi.IMPORTANCEThis research uses a modified version of the checkerboard assay to standardize the in vitro testing of double and triple combinations of antifungal agents against neurotropic dematiaceous fungi. Antifungal combination therapy is associated with improved outcomes in cerebral phaeohyphomycosis. In this study, we demonstrate that posaconazole is the single most active antifungal drug against this group of fungi. The double combination of amphotericin B with caspofungin or a trizole, and the triple combinations of caspofungin and flucytosine with amphotericin B or posaconazole might hold promise in the treatment of cerebral phaeohyphomycosis. Our findings will guide in developing optimal therapeutic strategies for these refractory infections.

Targeted Next Generation Sequencing (tNGS) for detection of drug-resistant tuberculous meningitis: Is this sequencing technology ready for prime time?

PURPOSE: Tuberculous meningitis (TBM) is the most severe form of tuberculosis (TB). Difficulty in diagnosing the condition along with other factors, increases its potential for high morbidity and mortality. Targeted Next Generation Sequencing (tNGS) generates high quality sequence read depths, enabling the identification of low-frequency alleles linked to Drug resistance (DR). The paucibacillary nature of tuberculous meningitis is a challenge for making a definitive diagnosis. METHODS: tNGS was performed on 20 cerebrospinal fluid (CSF) samples where, MGIT has shown Positive MTB Cultures. We simultaneously performed pyrosequencing (PSQ) and phenotypic Drug susceptibility testing (pDST) for these 20 samples. RESULTS: Sequencing results (from tNGS and PSQ) were compared with reference standards i.e. pDST. tNGS detected MTB in 7/20 (35%) CSF samples whereas, PSQ detected MTB in 17/20 (85%). CONCLUSION: Although tNGS has ability to detect minority variants along with detection of additional targets than PSQ, PSQ remains the diagnostic choice in our tertiary lab.