Intestinal mucormycosis initially identified by next-generation sequencing of cell-free DNA.

Mucormycosis is a rare fungal infection that typically affects severely immunocompromised individuals, often resulting in significant morbidity and mortality. Although early and aggressive intervention is necessary to prevent poor outcomes, diagnosis of this infection remains difficult. We report the first case, to our knowledge, of invasive gastrointestinal mucormycosis initially identified by next-generation sequencing of cfDNA from the blood, and discuss the various benefits and challenges which come with new molecular diagnostic techniques.

Carbapenem Resistant Aeromonas hydrophila Carrying blacphA7 Isolated From Two Solid Organ Transplant Patients.

Aeromonas hydrophila resides in a variety of aquatic environments. Infections with A. hydrophila mainly occur after contact with fresh or brackish water. Nosocomial infections with A. hydrophila can also occur. A. hydrophila infections can be difficult to treat due to both intrinsic and acquired antimicrobial resistance (AMR) mechanisms. In 2018-19, we isolated multi-drug resistant (MDR) A. hyrodphila from two solid organ transplant patients with intra-abdominal infections. We aimed to characterize their AMR mechanisms and to determine their genetic relatedness to aid epidemiological investigation. We performed whole genome sequencing (WGS) using Illumina MiSeq and Nanopore MinIon on 3 A. hydrophila isolates, with one isolate from Patient A (blood) and two isolates from Patient B (abdominal and T-tube fluid, isolated 2 weeks apart). Phenotypic assays included: Broth Microdilution (BMD), Modified Hodge Test (MHT), Modified Carbapenem Inactivation Method (mCIM), and EDTA Carbapenem Inactivation Method (eCIM). Data analyses were performed using CLCbio and Geneious. AMR genomic analysis revealed that all three isolates possess chromosomally encoded genes including blaOXA-12(oxacillinase), blacepS (AmpC), and blacphA7(metallo-beta-lactamase). All isolates tested strongly positive by MHT and mCIM, but only Patient B's second isolate (after 2 weeks of meropenem treatment) tested positive by eCIM. More intriguingly, Patient B's first isolate (before meropenem treatment) tested falsely susceptible to carbapenems by BMD, suggesting blacphA7 gene was not expressed constitutively. Phylogenetic analysis showed the two isolates from Patient B were highly similar with only 1 SNP difference. The isolate from Patient A only differed from Patient B's isolates by 35 and 36 SNPs, respectively, suggesting close genetic relatedness. Further epidemiological investigation is undergoing. We report the first cases of CphA-mediated carbapenem resistant A. hydrophila in the U.S. It is concerning that 1 out of 3 isolates tested falsely susceptible to carbapenems by BMD despite clear carbapenemase production shown by strongly positive MHT and mCIM. In both cases, meropenem was initially used to treat the patients. Clinicians and microbiologists in the US should be aware of the emerging MDR Aeromonas nosocomial infections and the potential false carbapenem susceptible results due to CphA-type carbapenemase, which may be induced during treatment.

Disseminated Coccidioidomycosis Treated with Interferon-γ and Dupilumab.

We describe a case of life-threatening disseminated coccidioidomycosis in a previously healthy child. Like most patients with disseminated coccidioidomycosis, this child had no genomic evidence of any known, rare immune disease. However, comprehensive immunologic testing showed exaggerated production of interleukin-4 and reduced production of interferon-γ. Supplementation of antifungal agents with interferon-γ treatment slowed disease progression, and the addition of interleukin-4 and interleukin-13 blockade with dupilumab resulted in rapid resolution of the patient's clinical symptoms. This report shows that blocking of type 2 immune responses can treat infection. This immunomodulatory approach could be used to enhance immune clearance of refractory fungal, mycobacterial, and viral infections. (Supported by the Jeffrey Modell Foundation and the National Institutes of Health.).

Coinfections of Two Strains of NDM-1- and OXA-232-Coproducing Klebsiella pneumoniae in a Kidney Transplant Patient.

We report here a fatal case of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in a renal transplant patient without a travel history in the prior year, from whom 2 genetically different CRKP (sequence type 14 [ST14] and ST2497) strains carrying the same plasmids and antimicrobial resistance genes, including blaNDM-1, blaOXA-232, blaCTX-M-15, armA, and tet(D), were isolated from blood and the abdominal cavity. The isolates were susceptible to colistin, tigecycline, eravacycline, and cefiderocol, which was used to treat the CRKP in combination with ceftazidime-avibactam and polymyxin B and resulted in bacterial clearance. Despite the aggressive treatment, the patient died of ischemic colitis and multiorgan failure.

Impact of the introduction of the Haemophilus influenzae type b conjugate vaccine in an urban setting in southern India.

INTRODUCTION: Haemophilus influenzae type b was the leading cause of bacterial meningitis in infants and children below the age of two years prior to the introduction of H. influenzae type b conjugate vaccines. In December 2011, the Indian government introduced H. influenzae b vaccine in the state of Tamilnadu. A prospective surveillance for bacterial meningitis was established at the Institute of Child Health in Chennai to evaluate the etiology of meningitis and impact of the vaccine. MATERIAL AND METHODS: Infants aged one to 23 months who were admitted to the hospital with symptoms of suspected bacterial meningitis were enrolled and lumbar puncture was performed. Cerebrospinal fluid samples were analyzed for white blood cells, protein, and glucose. Bacterial culture and a latex agglutination test for common bacterial pathogens were performed. RESULTS: Between January 2009 and March 2014, 4,770 children with suspected bacterial meningitis were enrolled. Prior to the introduction of the vaccine, an average of 11.7 cases of H. influenzae b meningitis and 31.1 cases of probable meningitis with no etiology were identified each year. After introduction, the number of cases were reduced by 79% and 44% respectively. The average H. influenzae b vaccine coverage after introduction was 69% among all children with clinically suspected meningitis. In contrast, the mean number of aseptic meningitis and pneumococcal meningitis cases remained stable throughout the pre and post vaccination period; 28.2 and 4.8 per year, respectively. CONCLUSIONS: H. influenzae b conjugate vaccine reduced the number of cases of H. influenzae b meningitis and probable meningitis within the first two years of its introduction. The impact against meningitis was higher than the vaccination rate, indicating indirect effects of the vaccine. India has recently scaled up the use of Hib conjugate vaccine throughout the country which should substantially reduce childhood meningitis rates further in the country.

Efficacy of Oral Zinc Supplementation in Radiologically Confirmed Pneumonia: Secondary Analysis of a Randomized Controlled Trial.

Objective: To evaluate the effect of zinc as an adjuvant therapy in radiologically confirmed pneumonia in children 2-24 months of age. Patients and Methods: We analyzed data of 212 children with pneumonia for whom chest X-ray films were available at enrollment and at least two radiologists agreed on the diagnosis of pneumonia. We compared the time to recovery in the two groups (n = 121, zinc group and n = 91, placebo group) using a Cox proportional hazards regression model. Results: Time to recovery was similar in both groups [median interquartile range: zinc, 84 h (64, 140 h); placebo, 85 h (65, 140 h)]. The absolute risk reduction for treatment failure was 5.2% (95% confidence interval: -4.8, 15.1) with zinc supplementation. Conclusion: There was no significant beneficial effect of zinc on the duration of recovery or risk of treatment failure in children with radiologically confirmed pneumonia.

Group B streptococcal meningitis in infants beyond the neonatal period.

OBJECTIVES: To describe the clinico-bacteriological profile, and early outcomes of infants diagnosed with Group B streptococcus (GBS) meningitis. METHODS: This was a retrospective review of infants (aged 1 mo to 2 y) diagnosed with GBS meningitis in a tertiary care hospital in New Delhi from October 2010 through January 2012. The clinico-bacteriological data and early outcomes of infants with suspected bacterial meningitis and a positive CSF latex agglutination test for GBS were studied. The CSF samples were subjected to PCR for broad spectrum 16s ribosomal DNA and the GBS species specific gene, the scpB. RESULTS: Twenty seven patients (13 boys, and 14 girls) were diagnosed with GBS meningitis during the study period. Broad spectrum 16s ribosomal DNA PCR was performed on 18 of the 27 CSF samples. Sixteen were positive. All these 16 were also positive for the species specific scpB gene. The median duration of hospital stay was 7 d (range 1-72 d). Nine patients died. One patient each developed ventriculitis, optic atrophy and hydrocephalus. Overall, 12 patients had a complete recovery at discharge. CONCLUSIONS: GBS must be considered in the etiology of bacterial meningitis in Indian infants.

Bacterial meningitis in children <2 years of age in a tertiary care hospital in South India: an assessment of clinical and laboratory features.

OBJECTIVES: To assess the clinical and laboratory features of suspected meningitis to assist in the accurate diagnosis of bacterial meningitis in young Indian children. STUDY DESIGN: Children <2 years of age with clinical suspicion of meningitis were enrolled. Clinical and laboratory information was collected, and cases were classified based on cerebrospinal fluid findings as clinical, aseptic, or probable and confirmed bacterial meningitis. RESULTS: A total of 2564 children with suspected meningitis were enrolled over 45 months; 156 cases of aseptic and 51 cases of bacterial meningitis were identified. Stiff neck and bulging fontanelle were more common in bacterial meningitis (P < .05), but were present in <15% of patients. The World Health Organization and American Academy of Pediatrics classifications for high suspicion of bacterial meningitis were met in 84% and 88% of cases of bacterial meningitis, respectively, but were also present in 54% and 74% cases of aseptic meningitis. Culture and gram stain were positive in 7 (14%) and 4 (8%) cases of bacterial meningitis. CONCLUSIONS: Signs of bacterial meningitis and proposed criteria for high suspicion of bacterial meningitis are non-specific in this population. Standard microbiological tests for bacteria are insensitive in this setting, necessitating highly sensitive methods to identify bacterial meningitis.

Efficacy of zinc given as an adjunct in the treatment of severe and very severe pneumonia in hospitalized children 2-24 mo of age: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Pneumonia is a leading cause of death; in India, an estimated 370,000 children die of pneumonia each year. Zinc has multiple influences on the immune response to infections. Zinc supplementation has been shown to prevent diarrhea and pneumonia in children. However, zinc's therapeutic effect on respiratory infections is less clear. OBJECTIVE: We evaluated the role of zinc as an adjunct to antibiotics in the treatment of children hospitalized for severe or very severe pneumonia. DESIGN: In this randomized, double-blind, placebo-controlled trial, we enrolled 550 children aged 2-24 mo with severe or very severe pneumonia. Within each hospital and pneumonia-severity stratum, children were randomly assigned to receive zinc (20 mg elemental zinc/d) or a placebo in addition to antibiotics and supportive care. RESULTS: The time to recovery from severe or very severe pneumonia was similar in both groups (HR: 0.98; 95% CI: 0.82, 1.17). In the stratified analysis, zinc was shown to be efficacious in reducing the time to recovery in children with very severe pneumonia (HR: 1.52; 95% CI: 1.03, 2.23); however, the effect was no longer statistically significant after adjustment for differences in severely underweight children in the 2 groups. CONCLUSIONS: This study showed no overall benefit of the addition of zinc to antibiotics in reducing the time to recovery from pneumonia but showed a possible benefit of zinc supplementation in a subgroup of children with very severe pneumonia. Additional research is needed in specific subgroups such as children with very severe pneumonia. This trial was registered at http://www.controlled-trials.com as ISRCTN48954234.

Prospective multi-centre sentinel surveillance for Haemophilus influenzae type b & other bacterial meningitis in Indian children.

BACKGROUND & OBJECTIVES: Haemophilus influenzae type b (Hib) is one of the leading bacterial causes of invasive disease in populations without access to Hib conjugate vaccines (Hib-CV). India has recently decided to introduce Hib-CV into the routine immunization programme in selected States. Longitudinal data quantifying the burden of bacterial meningitis and the proportion of disease caused by various bacteria are needed to track the impact of Hib-CV once introduced. A hospital-based sentinel surveillance network was established at four places in the country and this study reports the results of this ongoing surveillance. METHODS: Children aged 1 to 23 months with suspected bacterial meningitis were enrolled in Chennai, Lucknow, New Delhi, and Vellore between July 2008 and June 2010. All cerebrospinal fluid (CSF) samples were tested using cytological, biochemical, and culture methods. Samples with abnormal CSF (≥10 WBC per µl) were tested by latex agglutination test for common paediatric bacterial meningitis pathogens. RESULTS: A total of 708 patients with abnormal CSF were identified, 89 of whom had a bacterial pathogen confirmed. Hib accounted for the majority of bacteriologically confirmed cases, 62 (70%), while Streptococcus pneumoniae and group B Streptococcus were identified in 12 (13%) and seven (8%) cases, respectively. The other eight cases were a mix of other bacteria. The proportion of abnormal CSF and probable bacterial meningitis that was caused by Hib was 74 and 58 per cent lower at Christian Medical College (CMC), Vellore, which had a 41 per cent coverage of Hib-CV among all suspected meningitis cases, compared to the combined average proportion at the other three centres where a coverage between 1 and 8 per cent was seen (P<0.001 and P= 0.05, respectively). INTERPRETATION & CONCLUSIONS: Hib was found to be the predominant cause of bacterial meningitis in young children in diverse geographic locations in India. Possible indications of herd immunity was seen at CMC compared to sites with low immunization coverage with Hib-CV. As Hib is the most common pathogen in bacterial meningitis, Hib-CV would have a large impact on bacterial meningitis in Indian children.

Second-line anti-tuberculosis drug concentrations for susceptibility testing in the MODS assay.

Multidrug-resistant tuberculosis (TB) threatens TB control worldwide. The microscopic observation drug susceptibility (MODS) assay is a low-cost, high-performance TB diagnostic tool for rapid liquid culture and direct isoniazid and rifampicin drug susceptibility testing (DST). The objective of this study was to explore the potential for extending the MODS assay to rapid second-line DST and to identify critical concentrations of candidate drugs for prospective testing. Sputum samples from 94 TB culture-positive patients receiving second-line TB agents were cultured following standardised MODS protocols, with a range of titrations of antimicrobial drugs added. Critical concentrations were determined using a modified Kaplan-Meier survival curve analysis. Candidate critical concentrations were determined for capreomycin (10 µg·mL(-1)), ciprofloxacin (1.25 µg·mL(-1)), cycloserine (40 µg·mL(-1)), ethambutol (10 µg·mL(-1)), ethionamide (5 µg·mL(-1)), kanamycin (5 µg·mL(-1)), para-aminosalicylic acid (10 µg·mL(-1)) and streptomycin (10 µg·mL(-1)). No cut-off point was identified for the other second-line drugs or for pyrazinamide. At particular concentrations of some second-line TB drugs this novel Kaplan-Meier analysis clearly differentiated populations that were susceptible or resistant. These candidate critical concentrations should now be tested in a range of epidemiological settings to define the performance of direct, second-line TB DST with MODS, offering potential low-cost second-line TB DST capacity.

The worldwide impact of the seven-valent pneumococcal conjugate vaccine.

BACKGROUND: Pneumococcal conjugate vaccines (PCV) are emerging as one of the most promising means to prevent pediatric disease. The 7-valent PCV (PCV-7) has been extensively evaluated in clinical trials, and recent evidence from the introduction of PCV-7 through national immunization programs has demonstrated impact on pneumococcal disease. METHODS: Clinical trials have shown PCV-7 to be effective against the more severe forms of pneumococcal infections: pneumonia and invasive pneumococcal disease (IPD), as well as overall child mortality. A review shows the tremendous impact PCV-7 has had to date, and the potential further benefits of the emerging multi-valent vaccines. RESULTS: Since its introduction, the PCV-7 has substantially reduced the incidence of IPD, hospital admissions due to pneumonia and acute otitis media in numerous, mostly high income, low-disease burden countries. The reductions in IPD and pneumonia have also been observed among unvaccinated age groups in countries with routine use of PCV-7, demonstrating that PCV-7 provides herd immunity. Some settings observed an increase in rate of nonvaccine serotype IPD, yet rates of overall and vaccine-serotype IPD show marked reductions post-PCV-7 introduction. Limited data are available on the impact of PCV-7 in lower income countries. The available data from efficacy trials from The Gambia and South Africa suggest that PCV-7 will have substantial impact on reducing pneumococcal disease. CONCLUSION: PCV-7 has shown dramatic reduction in disease and mortality rates in the countries in which it has been introduced. The newly introduced 10-valent and 13-valent pneumococcal vaccines are expected to have substantial disease impact, but monitoring is essential to determine their true impact and sustain further introduction of pneumococcal conjugate vaccines.

High rates of colonization with drug resistant hemophilus influenzae type B and Streptococccus Pneumoniae in unvaccinated HIV infected children from West Bengal.

OBJECTIVE: To determine nasopharyngeal colonization rates of two vaccine preventable bacterial pathogens Hemophilus influenzae type b (Hib), and Streptococcus pneumoniae (Pneumococcus), antibiotic susceptibility of isolates, factors associated with their colonization, and immunization history in a cohort of HIV infected children. METHODS: The authors conducted a cross-sectional nasopharyngeal swab survey of 151 children affected with HIV presenting for routine outpatient care in West Bengal, India. RESULTS: 151 HIV affected children were enrolled. The median age was 6, 148/151 children were HIV positive, 65% had moderate to severe malnutrition, 53% were moderately to severely immunosuppressed, 17% were on antiretroviral therapy (ART), 90% were on cotrimoxazole prophylaxis (TMP/SMX). None had received the pneumococcal or Hib conjugate vaccines. Hib prevalence was 13% and pneumococcal prevalence was 28%. Children with normal or moderate immune suppression had high rates of colonization compared to those with severe immunosuppression (71% Hib, 61% pneumococcus). Hib and pneumococcal isolates had high rates of resistance to tested antibiotics including TMP/SMX and third generation cephalosporins. Neither ART nor TMP/SMX prevented colonization. Children colonized with multidrug resistant isolates had high rates of exposure to TMP/SMX. CONCLUSIONS: HIV infection, late access to ART, high rates of colonization to resistant organisms and lack of access to vaccines makes this population vulnerable to invasive disease from Hib and pneumococcus.

Haemophilus influenzae type b conjugate vaccines: considerations for vaccination schedules and implications for developing countries.

Prior to widespread vaccination, Haemophilus influenzae type b was a leading cause of severe childhood bacterial infection, including meningitis, worldwide. Over the last decade the world has taken great strides towards controlling Hib disease through routine use of conjugate vaccines in developed and developing countries. Currently there is no consensus on the appropriate schedule by which to use Hib vaccine. Vaccination schedules around the world vary greatly, particularly between high and low income countries. Questions remain as to the most effective and efficient schedule of primary doses, the need for a booster dose, and the implications of using combination vaccines. Here, we present a synthesis of data supporting various Hib vaccine schedules, with a focus on the implications for developing countries.

Prevention of rotavirus gastroenteritis in infants and children: rotavirus vaccine safety, efficacy, and potential impact of vaccines.

Rotavirus infection is the most common cause of severe gastroenteritis globally, with greater than 86% of deaths occurring in low-income and middle-income countries. There are two rotavirus vaccines currently licensed in the United States and prequalified by the World Health Organization. RV1 is a monovalent attenuated human rotavirus strain, given orally in two doses. RV5 is a pentavalent human-bovine reassortant rotavirus vaccine, given orally in three doses. A third rotavirus vaccine, LLV, is a lamb rotavirus strain given orally as a single dose, which is currently available only in China. RV1 and RV5 have been shown to be highly efficacious in developed countries, and initial results from trials in Africa and Asia are promising as well. At least three other vaccines are in development, which are being developed by manufacturers of developing countries. Further studies are needed to clarify issues including administration of oral rotavirus vaccines with breastfeeding and other oral vaccines, and alterations in dosing schedule. Using new data on global diarrheal burden, rotavirus is estimated to cause 390,000 deaths in children younger than 5 years. Should rotavirus vaccines be introduced in the routine immunization programs of all countries, a potential of 170,000 deaths could be prevented annually. The largest impact on mortality would be seen in low-income and middle-income countries, despite poor immunization coverage and lower efficacy. Therefore, international efforts are needed to ensure that rotavirus vaccines reach the populations with highest burden of rotavirus disease.

Prolonged infectiousness of tuberculosis patients in a directly observed therapy short-course program with standardized therapy.

BACKGROUND: Effective tuberculosis control is compromised by a lack of clarity about the timeframe of viable Mycobacterium tuberculosis shedding after treatment initiation under programmatic conditions. This study quantifies time to conversion from smear and culture positivity to negativity in unselected tuberculosis patients receiving standardized therapy in a directly observed therapy short-course (DOTS) program. METHODS: Longitudinal cohort study following up 93 adults initiating tuberculosis therapy in Lima, Peru. Baseline culture and drug susceptibility tests (DSTs) were performed using the MBBacT, proportion, and microscopic observation drug susceptibility (MODS) methods. Smear microscopy and MODS liquid culture were performed at baseline and weekly for 4 weeks then every other week for 26 weeks. RESULTS: Median conversion time from culture positivity to culture negativity of 38.5 days was unaffected by baseline smear status. Patients with fully susceptible tuberculosis had a median time to culture conversion of 37 days; 10% remained culture positive at day 60. Delayed culture conversion was associated with multidrug resistance, regardless of DST method used; non-multidrug resistance as defined by the proportion method and MODS (but not MBBacT) was also associated with delay. Persistent day 60 smear positivity yielded positive and negative predictive values of 67% and 92%, respectively, for detecting multidrug resistance. CONCLUSIONS: Smear and culture conversion in treated tuberculosis patients takes longer than is conventionally believed, even with fully susceptible disease, and must be accounted for in tuberculosis treatment and prevention programs. Persistent day 60 smear positivity is a poor predictor of multidrug resistance. The industrialized-world convention of universal baseline DST for tuberculosis patients should become the standard of care in multidrug resistance-affected resource-limited settings.

Progress and barriers for the control of diarrhoeal disease.

Discovery of intestinal sodium-glucose transport was the basis for development of oral rehydration solution, and was hailed as potentially the most important medical advance of the 20th century. Before widespread use of oral rehydration solution, treatment for diarrhoea was restricted to intravenous fluid replacement, for which patients had to go to a health-care facility to access appropriate equipment. These facilities were usually neither available nor reasonable to use in the resource-poor settings most affected by diarrhoea. Use of oral rehydration solution has stagnated, despite being effective, inexpensive, and widely available. Thus, diarrhoea continues to be a leading cause of child death with consistent mortality rates during the past 5 years. New methods for prevention, management, and treatment of diarrhoea-including an improved oral rehydration formulation, zinc supplementation, and rotavirus vaccines-make now the time to revitalise efforts to reduce diarrhoea mortality worldwide.

Polymerase chain reaction for chronic Trypanosoma cruzi infection yields higher sensitivity in blood clot than buffy coat or whole blood specimens.

Trypanosoma cruzi polymerase chain reaction (PCR) is widely used, but sensitivity varies widely. We compared PCR using 121/122 primers targeting kinetoplast minicircle DNA in whole blood, buffy coat, and clot from Bolivian women. Sensitivity was significantly higher in clot (60.1%) than buffy coat (46.5%) or whole blood (40%). The use of clot could simplify specimen collection while improving sensitivity.

Detection and measurement of alternative splicing using splicing-sensitive microarrays.

Splicing and alternative splicing are major processes in the interpretation and expression of genetic information for metazoan organisms. The study of splicing is moving from focused attention on the regulatory mechanisms of a selected set of paradigmatic alternative splicing events to questions of global integration of splicing regulation with genome and cell function. For this reason, parallel methods for detecting and measuring alternative splicing are necessary. We have adapted the splicing-sensitive oligonucleotide microarrays used to estimate splicing efficiency in yeast to the study of alternative splicing in vertebrate cells and tissues. We use gene models incorporating knowledge about splicing to design oligonucleotides specific for discriminating alternatively spliced mRNAs from each other. Here we present the main strategies for design, application, and analysis of spotted oligonucleotide arrays for detection and measurement of alternative splicing. We demonstrate these strategies using a two-intron yeast gene that has been altered to produce different amounts of alternatively spliced RNAs, as well as by profiling alternative splicing in NCI 60 cancer cell lines.