Pott's puffy tumor: An unusual complication of rhino-orbito-cerebral mucormycosis.

Objective: To describe clinicoradiological features and surgical outcomes in a series of nine patients with rhino-orbito-cerebral mucormycosis (ROCM) who presented with Pott's puffy tumor (ROCM-PPT). Methods: The records of nine patients with ROCM-PPT seen between March 2020 and December 2021 were analysed. Clinical features, radiology, histopathology, operative findings, management and outcome were noted. Frontal sinus pneumatisation and outflow tract configuration was compared between patients and controls with ROCM and no PPT. Results: ROCM-PPT was diagnosed in 9 of 284 (3.2 %) patients with ROCM seen during the study period. There were six (66.7 %) males and the median age was 54 (IQR 46-60) years. Eight (88.9 %) patients had diabetes mellitus and seven (77.8 %) had been COVID-19 positive. Radiological features of osteomyelitis, subperiosteal abscess formation and dural enhancement were seen in all patients. No significant differences in pneumatisation or frontal sinus outflow tract configuration were noted between patients and controls. All patients underwent a craniectomy with frontal bone debridement and frontal sinus exteriorisation. All patients were treated with anti-fungal agents for several months. All patients had symptomatic improvement at a median follow-up of 21 (IQR 18-23) months. Repeat CT/MRI scans showed disease regression/resolution in six out of eight (75 %) patients with follow-up imaging, and stable disease in two others. Conclusions: ROCM-PPT is a rare, delayed complication of mucormycosis that was seen in larger numbers during the recent COVID-19 pandemic. Aggressive debridement of osteomyelitic bone and antifungal therapy results in a good outcome.

Challenging the gold standard: the limitations of molecular assays for detection of Mycobacterium tuberculosis heteroresistance.

OBJECTIVES: Heteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has been linked with poor treatment outcomes. We compared the analytical sensitivity of molecular methods, such as GeneXpert and whole genome sequencing (WGS) in detecting heteroresistance when compared with the 'gold standard' phenotypic assay: the agar proportion method (APM). METHODS: Using two rounds of proficiency surveys with defined monoresistant BCG strains and mixtures of susceptible/resistant M. tb, we determined the limit of detection (LOD) of known resistance associated mutations. RESULTS: The LOD for rifampin-R (RIF-R) detection was 1% using APM, 60% using GeneXpert MTB/RIF, 10% using GeneXpert MTB/RIF Ultra and 10% using WGS. While WGS could detect mutations beyond those associated with RIF resistance, the LOD for these other mutations was also 10%. Additionally, we observed instances where laboratories did not report resistance in the majority population, yet the mutations were present in the raw sequence data. CONCLUSION: The gold standard APM detects minority resistant populations at a lower proportion than molecular tests. Mycobacterium bovis BCG strains with defined resistance and extracted DNA from M. tb provided concordant results and can serve in quality control of laboratories offering molecular testing for resistance. Further research is required to determine whether the higher LOD of molecular tests is associated with negative treatment outcomes.

Effectiveness of Adjunctive High-Dose Infliximab Therapy to Improve Disability-Free Survival Among Patients With Severe Central Nervous System Tuberculosis: A Matched Retrospective Cohort Study.

BACKGROUND: Few treatment options exist for patients with severe central nervous system (CNS) tuberculosis (TB) worsening due to inflammatory lesions, despite optimal antitubercular therapy (ATT) and steroids. Data regarding the efficacy and safety of infliximab in these patients are sparse. METHODS: We performed a matched retrospective cohort study based on Medical Research Council (MRC) grading system and modified Rankin Scale (mRS) scores comparing 2 groups of adults with CNS TB. Cohort A received at least 1 dose of infliximab after optimal ATT and steroids between March 2019 and July 2022. Cohort B received only ATT and steroids. Disability-free survival (mRS score ≤2) at 6 months was the primary outcome. RESULTS: Baseline MRC grades and mRS scores were similar between the cohorts. Median duration before initiation of infliximab therapy from start of ATT and steroids was 6 (IQR: 3.7-13) months and for neurological deficits was 4 (IQR: 2-6.2) months. Indications for infliximab were symptomatic tuberculomas (20/30; 66.7%), spinal cord involvement with paraparesis (8/30; 26.7%), and optochiasmatic arachnoiditis (3/30; 10%), worsening despite adequate ATT and steroids. Severe disability (5/30 [16.7%] and 21/60 [35%]) and all-cause mortality (2/30 [6.7%] and 13/60 [21.7%]) at 6 months were lower in cohort A versus cohort B, respectively. In the combined study population, only exposure to infliximab was positively associated (aRR: 6.2; 95% CI: 2.18-17.83; P = .001) with disability-free survival at 6 months. There were no clear infliximab-related side effects noted. CONCLUSIONS: Infliximab may be an effective and safe adjunctive strategy among severely disabled patients with CNS TB not improving despite optimal ATT and steroids. Adequately powered phase 3 clinical trials are required to confirm these early findings.

Short intravenous amphotericin B followed by oral posaconazole using a simple, stratified treatment approach for diabetes or COVID-19-associated rhino-orbito-cerebral mucormycosis: a prospective cohort study.

OBJECTIVES: To evaluate the efficacy and safety of short-course intravenous amphotericin B followed by sustained release posaconazole tablets for diabetes or COVID-19-associated rhino-orbito-cerebral mucormycosis. METHODS: This prospective, pragmatic study included adults with diabetes or COVID-19-associated rhino-orbito-cerebral mucormycosis. Patients received short (7-14 days) or long (15-28 days) intravenous antifungal therapy (short intravenous antifungal treatment [SHIFT] or long intravenous antifungal treatment [LIFT], respectively) depending on the presence or absence of brain involvement. All patients received step-down posaconazole tablets, debridement, and glycemic control. The primary outcome was the treatment success at week 14, which was determined by assessing survival and the absence of disease progression through clinical evaluation and nasal endoscopy. Log-binomial regression analysis (risk ratio and 95% CI) was performed to assess factors associated with the primary outcome. RESULTS: Intravenous therapy was administered to 251 participants: SHIFT, 205 (median duration, 13 days); LIFT, 46 (median duration, 22 days). Treatment success at 3 months was 88% (217/248; 95% CI, 83-91%): SHIFT group, 93% (189/203; 89-96%); LIFT group, 62% (28/45; 47-76%). All-cause mortality was 12% (30/251): SHIFT group, 6% (13/205); LIFT group, 37% (17/46). Age (aRR [95% CI]: 1.02 [1.00-1.05]; p 0.027), diabetic ketoacidosis at presentation (2.32 [1.20-4.46]; p 0·012), glycated haemoglobin A1c (1.19 [1.03-1.39]; p 0.019), stroke (3.93 [1.94-7.95]; p 0·0001), and brain involvement (5.67 [3.05-10.54]; p < 0.0001) were independently associated with unsuccessful outcomes. DISCUSSION: Short intravenous amphotericin B with step-down posaconazole tablets should be further studied as primary treatment option for diabetes or COVID-19-associated mucormycosis in randomized controlled trials.

Pulmonary nocardiosis: Risk factors and species distribution from a high burden centre.

Nocardiosis is a clinical and diagnostic challenge. This was a retrospective study carried out on cases of pulmonary nocardiosis presenting over 15 years. Clinical data was retrieved using the electronic patient records. Vitek MS 3.2 (MALDI TOF MS) was carried out on 22 isolates and sequencing on another 9 isolates. Of 71 patients presenting with pulmonary nocardiosis, 58 (81.6%) were on immunosuppressant therapy, 26 (46%) had a previous lung pathology, 11 (8%) were HIV associated. Disseminated disease was seen in 6 (8.4%). There were 8 (11.26%) deaths in this cohort of patients. Of 31/71 identified to species, the most common were Nocardia cyriacigeorgica (n â€‹= â€‹11) followed by Nocardia farcinica (n â€‹= â€‹9).

Cryptic aspergillosis: a rare entity and a diagnostic challenge.

Introduction: Cryptic aspergillosis, caused by cryptic species of Aspergillus, is increasingly reported in humans and causes significant morbidity and mortality in immunocompromised individuals. The main aim of this study was to describe the occurrence of this entity at a large tertiary care centre and analyse the challenges in identifying them in a routine diagnostic laboratory. Methods: This was a retrospective case review of all patients diagnosed with cryptic Aspergillus species from April 2019 to February 2020. The isolates were identified using conventional microbiological techniques, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF MS), 28S rRNA and internal transcribed spacer (ITS) sequencing. Results: The species identified were Aspergillus tamarii, Aspergillus lentulus and Aspergillus sydowii. Identification by MALDI- TOF MS and sequencing was concordant for all except A. sydowii, with MALDI- TOF MS misidentifying it as Aspergillus thermomutans. All isolates showed low minimum inhibitory concentrations (MICs) for the panel of antifungal drugs. Conclusion: Aspergillosis caused by cryptic Aspergillus species presents a diagnostic challenge. This study confirms the importance of molecular methods for accurate identification.

Accuracy of Xpert MTB/RIF Ultra for the diagnosis of tuberculosis in adult patients: a retrospective cohort study.

OBJECTIVES: The value of the "trace" result in Xpert Ultra for diagnosing active tuberculosis (TB) remains unclear. Our study evaluated the diagnostic accuracy of Xpert MTB/RIF Ultra (Xpert Ultra) (Cepheid, Sunnyvale, USA) over Xpert MTB/RIF (Xpert) (Cepheid, Sunnyvale, USA) and mycobacterial culture when compared with a composite reference standard (CRS). METHODS: A retrospective single-center observational study was conducted in a tertiary care hospital in South India. Over three months, patients (aged ≥15 years) data on Xpert Ultra tests and mycobacterial culture of pulmonary and extrapulmonary samples were extracted from their electronic medical records. Patients were defined as TB cases based on the CRS criteria. Sensitivity, specificity, positive and negative predictive values of diagnostic tests were calculated by comparing them to the CRS. RESULTS: Xpert Ultra was more sensitive (87.8%) than Xpert (72.1%) and culture (44.1%). The specificity of Xpert Ultra was lower (98.1%) than those of Xpert (100%) and culture (100%). The sensitivity (92%) and specificity (100%) of Xpert Ultra were highest when performed on pus samples. CONCLUSIONS: Xpert Ultra with the trace category is superior to the conventional Xpert, and mycobacterial culture in identifying TB.

Chronic Mucocutaneous Candidiasis due to Signal Transducer and Activator of Transcription 1 (STAT 1) Mutation in an Indian Patient - A Case Report.

Chronic mucocutaneous candidiasis (CMC) is a primary immunodeficiency due to defect in various genes leading to an increase in susceptibility to skin and mucosal infection. Mutation in signal transducer and activator of transcription 1 (STAT 1) gene being the most common cause of CMC can lead to increased risk of infections, multisystem abnormalities, and malignancy. We describe a 27 year old Indian woman with clinical features of CMC including esophageal stenosis, gangrene of the finger, endocrinological and immunological abnormalities and STAT1 mutation (p.Leu407Val). She was treated with antifungals which led to symptomatic improvement.

Effectiveness of a real-time PCR for diagnosis of Pneumocystis pneumonia in immunocompromised patients - Experience from a tertiary care center, India.

Pneumocystis jirovecii pneumonia (PCP) is a life-threatening fungal infection in immunocompromised patients. Traditionally, the laboratory diagnosis of PCP relied on the visualization of organisms by microscopy as Pneumocystis cannot be readily cultured in the laboratory. The polymerase chain reaction (PCR) method is preferred over the conventional microscopic methods as PCR is rapid and found to have higher sensitivity. This retrospective study aimed to analyze the diagnostic value of a real-time PCR (qPCR) for routine diagnosis of PCP in immunocompromised patients with various underlying conditions. The qPCR targets a 121 bp fragment of P.jirovecii mitochondrial large subunit rRNA gene. The study was conducted in a 2600-bed tertiary care hospital between January and December 2019. All patients whose respiratory samples were tested for PCP by qPCR were included. The clinical diagnosis was made for each patient and categorized into PCP and non-PCP based on multi-component clinical criteria by a multi-disciplinary team. The performance characteristics of qPCR were analyzed using clinical diagnosis as the reference. A total of 339 respiratory samples from 289 patients were tested for PCP by qPCR during the study period. The overall sensitivity and specificity of qPCR were 84.75% (95% CI, 73.01% to 92.78%) and 96.1% (95% CI, 92.7 to 98.2), respectively. The sensitivity was slightly higher among HIV-infected patients (91%) than the non- HIV group (81%). The PCR exhibited higher sensitivity in bronchoalveolar lavage (BAL) (94%) than in sputum samples (81%). The colonization can be ruled out with the cycle threshold (CT) value of below 34 with a sensitivity and specificity of 100% and 78%, respectively. The real-time PCR showed good sensitivity and specificity for routine diagnosis of PCP in patients with various underlying conditions. In addition, a cut-off CT value (≤ 34) was determined to exclude colonization from active pneumonia.

Candida utilis: a rare cause of septicemia in children.

Candida utilis is an emerging fungal pathogen in blood. The main aim of this study was to describe the prevalence, methods of speciation and antifungal susceptibility of Candida utilis at a tertiary care centre. METHODS: This was a retrospective study carried out at a tertiary care centre in South India. Over a period of 1 year, three Candida utilis were isolated from blood culture identified by MALDI-TOF MS Version 3.2 and were confirmed by ITS sequencing. Susceptibility testing was carried out by micro broth dilution. RESULTS: All three patients had a common risk factor of prolonged ICU stay but the source of infection could not be identified. Candida utilis isolates were identified by MALDI-TOF and confirmed by ITS sequencing. They were pansusceptible to all tested antifungal drugs. Among these, two patients who were treated in hospital had good clinical outcome and response to antifungal drugs. A third patient was lost to follow up. CONCLUSION: Candida utilis was predominantly seen between 0-3 month olds. Conventional methods of speciation were unable to identify C. utilis to species level. Rapid identification was done by MALDI-TOF MS and confirmed by sequencing. Rapid identification leads to prompt treatment and favours a good clinical outcome.

Cryptococcosis with Tuberculosis: Overlooked Coinfections.

Tuberculosis and cryptococcosis are important opportunistic pathogens causing significant morbidity and mortality in immunocompromised individuals. Concurrent infections of these two agents are rarely reported. We report five cases of culture-proven coinfection of Mycobacterium tuberculosis and Cryptococcus neoformans during inpatient admission at a tertiary care hospital in southern India between 2007 and 2019. Four patients were persons living with HIV infection and one was on immune suppression for chronic renal disease. Maintaining a high degree of clinical suspicion will ensure early diagnosis and appropriate management of coinfections in the immunocompromised individual.

Challenges in the management of slowly growing non-tuberculous mycobacteria causing pulmonary disease: Perspectives from a high burden country.

PURPOSE: There is a dearth of data on epidemiology, diagnosis and management of slow growing non tuberculous mycobacteria(NTM) in India, despite being a TB endemic country. This study aims to describe the geographic distribution, risk factors, and the challenges in management of slow growing NTM causing pulmonary infections. METHODS: Over a period of 3 years, all slow growing NTM received from pulmonary specimens at a tertiary care centre were further studied from electronic hospital records to correlate non tuberculous mycobacteria species with demographics, geographic location, describe comorbidities including immunosuppression, radiologic findings and treatment regimes. RESULTS: M.intracellullare was found in the majority of isolates with significant geographic variation and M.simiae the second commonest found exclusively in southern states. Common comorbidities included a previous history of treatment for tuberculosis, structural lung disease as well as systemic risk factors. Disseminated disease only occurred in immunocompromised hosts as was expected, but at a high rate of 44%. Treatment completion and outcomes were difficult to attain in our population. CONCLUSION: The burden of NTM infection and its management in India remain a challenge. Ensuring it is made a notifiable disease may improve the current situation.

Risk factors associated with the mucormycosis epidemic during the COVID-19 pandemic.

This study was performed to assess the risk factors driving the epidemic of coronavirus disease 2019 (COVID-19)-associated mucormycosis (COVID-Mucor) in India that has accompanied the COVID-19 pandemic, particularly during the second wave. Risk factors were analysed among 164 participants: 132 COVID-Mucor (cases) and 32 non-COVID-Mucor (controls). Data from a prospective cohort study of mucormycosis over a period of 1 year were used. Diabetes mellitus remained a significant risk factor in both groups (97%), while uncontrolled diabetes mellitus (odds ratio (OR) 4.6; P = 0.026) and newly detected diabetes (OR 3.3; P = 0.018) were more common among the cases. Most patients with COVID-Mucor had mild COVID-19. Steroid use, often unwarranted, was highly associated with COVID-Mucor after adjusting for other risk factors (OR 28.4; P = 0.001). Serum ferritin was significantly higher (P = 0.041), while C-reactive protein was not, suggesting that alterations in iron metabolism may predispose to COVID-Mucor. Oxygen was used only in a small minority of patients with COVID-Mucor. The in-hospital mortality in both groups was low. In conclusion, the Indian COVID-Mucor epidemic has likely been driven by a convergence of interlinked risk factors: uncontrolled diabetes mellitus, unwarranted steroid use, and perhaps COVID-19 itself. Appropriate steroid use in patients with severe COVID-19 and screening and optimal control of hyperglycaemia can prevent COVID-Mucor.

Clinical implications of high-risk mutations in drug resistant tuberculosis (DR-TB): An observational cohort study.

Genotype MTBDRsl [SL-LPA] was endorsed as a tool for early diagnosis of fluoroquinolones (FQ) and injectable second-line TB drugs (SLID) resistance in DR-TB. Correlation between specific genetic mutations using this tool and clinical outcome has not hitherto been studied in India. We conducted a observational cohort study to evaluate the predictive value of specific mutations for bad outcome. Our study identified 15 different types of gyrA mutations, commonest being A90V and D94G. Poor outcome was associated with mutations D94G and D94N/D94Y.Most XDR-TB patients harbored the high risk mutation of A1401G. Hence information of specific mutations using SL-LPA can help prognosticate and design appropriate treatment regimens.

Reconsidering Mycobacterium bovis as a proxy for zoonotic tuberculosis: a molecular epidemiological surveillance study.

BACKGROUND: Zoonotic tuberculosis is defined as human infection with Mycobacterium bovis. Although globally, India has the largest number of human tuberculosis cases and the largest cattle population, in which bovine tuberculosis is endemic, the burden of zoonotic tuberculosis is unknown. The aim of this study was to obtain estimates of the human prevalence of animal-associated members of the Mycobacterium tuberculosis complex (MTBC) at a large referral hospital in India. METHODS: We did a molecular epidemiological surveillance study of 940 positive mycobacteria growth indicator tube (MGIT) cultures, collected from patients visiting the outpatient department at Christian Medical College (Vellore, India) with suspected tuberculosis between Oct 1, 2018, and March 31, 2019. A PCR-based approach was applied to subspeciate cultures. Isolates identified as MTBC other than M tuberculosis or as inconclusive on PCR were subject to whole-genome sequencing (WGS), and phylogenetically compared with publicly available MTBC sequences from south Asia. Sequences from WGS were deposited in the National Center for Biotechnology Information Sequence Read Archive, accession number SRP226525 (BioProject database number PRJNA575883). FINDINGS: The 940 MGIT cultures were from 548 pulmonary and 392 extrapulmonary samples. A conclusive identification was obtained for all 940 isolates; wild-type M bovis was not identified. The isolates consisted of M tuberculosis (913 [97·1%] isolates), Mycobacterium orygis (seven [0·7%]), M bovis BCG (five [0·5%]), and non-tuberculous mycobacteria (15 [1·6%]). Subspecies were assigned for 25 isolates by WGS, which were analysed against 715 MTBC sequences from south Asia. Among the 715 genomes, no M bovis was identified. Four isolates of cattle origin were dispersed among human sequences within M tuberculosis lineage 1, and the seven M orygis isolates from human MGIT cultures were dispersed among sequences from cattle. INTERPRETATION: M bovis prevalence in humans is an inadequate proxy of zoonotic tuberculosis. The recovery of M orygis from humans highlights the need to use a broadened definition, including MTBC subspecies such as M orygis, to investigate zoonotic tuberculosis. The identification of M tuberculosis in cattle also reinforces the need for One Health investigations in countries with endemic bovine tuberculosis. FUNDING: Bill & Melinda Gates Foundation, Canadian Institutes for Health Research.

Treatment outcomes with daily self-administered treatment and thrice-weekly directly-observed treatment in two cohorts of newly-diagnosed, sputum-positive adults with pulmonary tuberculosis.

BACKGROUND: The Revised National Tuberculosis Control Program (RNTCP) envisages shifting from thrice-weekly to a daily anti-tuberculosis treatment (ATT) regimen. The potential merits and demerits of both regimens continue to be debated. METHODS: This retrospective study compared treatment outcomes in 191 HIV-negative, newly diagnosed, sputum-positive adults with pulmonary tuberculosis from Vellore district of Tamil Nadu who were treated at a private medical college during 2009 to 2012 with intermittent Directly Observed Treatment Short Course (intermittent DOTS cohort, n=132) or who opted for daily Self-Administered Treatment (daily SAT cohort, n=59). Treatment outcomes obtained from medical records were supplemented by interviews with consenting, traceable patients. RESULTS: The rates for the RNTCP-recommended sputum smear examinations were suboptimal (42% for daily SAT and 72% for intermittent DOTS). However, treatment success with daily SAT and intermittent DOTS (76.2% vs. 70.4%); default (11.9% vs. 18.2%); death (6.8% vs. 5.3%); treatment failure (5.1% vs. 4.6%); and relapse (0% vs. 1.5%) did not significantly differ. CONCLUSIONS: While evaluable treatment outcomes were not significantly different with daily SAT and intermittent DOTS, rates for timely smear examinations and for treatment success were lower, and for default higher, in both cohorts than comparable RNTCP data from Vellore district. Further strengthening of RNTCP facilities within private medical colleges and regular, real-time audits of performance and outcomes are needed if daily ATT regimen under the RNTCP is to succeed.

Candida auris: Clinical profile, diagnostic challenge and susceptibility pattern: Experience from a tertiary-care centre in South India.

OBJECTIVES: Candida auris is an emerging, multidrug-resistant yeast transmitted in healthcare settings. Conventional methods of speciation are unable to identify C. auris to species level. Three methods, namely VITEK® MS v.3.0, VITEK®2 and target sequencing of the internal transcribed spacer (ITS) region and 28S rRNA gene, were evaluated. Antifungal susceptibility testing (AFST) was also performed, and risk factors for acquisition of C. auris candidaemia were studied. METHODS: Between November 2016 and November 2017, 203 Candida spp. were isolated from blood cultures, of which 11 isolates that were unidentifiable by conventional methods were further tested by VITEK® MS v.3.0 and VITEK®2 and were confirmed by sequencing. AFST was carried out on all 11 isolates by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Clinical and epidemiological data of all patients retrieved from electronic patient records were reviewed. RESULTS: Of the 11 isolates identified as C. auris both by ITS and 28S rRNA sequencing, VITEK®2 identified only 5 as C. auris and VITEK® MS v.3.0 was not able to identify any of them as C. auris. Ten isolates (91%) were resistant to fluconazole, whereas all isolates were susceptible to amphotericin B and caspofungin. CONCLUSION: Candida auris can be misidentified in routine microbiology laboratories. Sequencing remains the gold standard if commercial identification systems are not updated.

A modular paper-and-plastic device for tuberculosis nucleic acid amplification testing in limited-resource settings.

We present a prototype for conducting rapid, inexpensive and point-of-care-compatible nucleic acid amplification tests (NAATs) for tuberculosis (TB). The fluorescent isothermal paper-and-plastic NAAT (FLIPP-NAAT) uses paper-based loop mediated isothermal amplification (LAMP) for DNA detection. The cost of materials required to build a 12-test-zone device is $0.88 and the cost of reagents per reaction is $0.43. An inexpensive imaging platform enables filter-free fluorescence detection of amplified DNA using a cell-phone camera. FLIPP-NAAT can be operated by an untrained user and only requires a regular laboratory incubator as ancillary equipment. All reagents can be dry-stored in the device, facilitating storage and transportation without cold chains. The device design is modular and the assay demonstrated high specificity to Mycobacterium tuberculosis (Mtb), analytical sensitivity of the order of 10 copies of Mtb gDNA, and tolerance to complex samples. The clinical sensitivity and specificity of sputum-based FLIPP NAAT tests were 100% (zero false negatives) and 68.75% (five false positives), respectively (N = 30), using Xpert MTB/RIF assay as the reference standard. FLIPP-NAAT has the potential to provide affordable and accessible molecular diagnostics for TB in low- and middle-income countries, when used in conjunction with an appropriate sample preparation technique. Although demonstrated for the detection of TB, FLIPP-NAAT is a platform technology for amplification of any nucleic acid sequence.

Robustness of the Line Probe Assay for the Rapid Diagnosis and Characterization of Mutations in Extensively Drug-Resistant Tuberculosis.

Introduction: Extensively drug-resistant tuberculosis (XDRTB) is a public health concern. We evaluated the diagnostic accuracy of Genotype® MTBDRsl for detection of resistance to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs) and characterized mutations seen. Materials and Methods: MTBDRsl was carried out either directly on sputum samples or indirectly on culture isolates (n = 100) from known multidrug-resistant tuberculosis (MDRTB) patients from July 2015 to September 2017. Diagnostic accuracy for the detection of resistance to FQs and SLIDs was calculated in comparison with conventional culture-based drug susceptibility testing. Mutations at the gyrA and rrs loci, as well as discrepant phenotypic and genotypic results, were studied. A subset of isolates underwent pyrosequencing. Results: Out of 100 MDRTB samples/isolates tested, 59% were pre-XDRTB and 7% were XDRTB. The sensitivity and specificity for the detection of resistance to FQs were 96.6% [95% confidence interval (CI): 88.3-99.6] and 80% [95% CI: 64.4-90.9] and those for SLIDs were 70% [95% CI: 34.8-93.3] and 100% [95% CI: 95.9-100]. The most frequent mutations were the absence of wild type 3 with corresponding mutation 3c (20/66) at the gyrA locus, and absence of wild type 1 and corresponding mutation 1 (6/7) at the rrs locus. The absence of a wt2 band with a corresponding mutation at the gyrA locus was seen in four of eight patients with discrepant genotypic and phenotypic results for FQ resistance. All isolates tested by pyrosequencing (n = 5) were concordant with the line probe assay for FQ resistance with identical mutations (D94G) and four of five isolates were concordant with SLIDs with identical mutations (A1401G). Conclusion: The MTBDRsl is a useful test for accurate diagnosis of XDRTB and may help to tailor therapy.