Towards national systems for continuous surveillance of antimicrobial resistance: Lessons from tuberculosis.

In a Perspective on the research article from Jacobson and colleagues, Amitabh Suthar and colleagues from the Centers for Disease Control and Prevention discuss the importance of and considerations for developing real-time and large-scale reporting systems for tracking and controlling antimicrobial resistance.

Performance of Clinical Screening Algorithms for Tuberculosis Intensified Case Finding among People Living with HIV in Western Kenya.

OBJECTIVE: To assess the performance of symptom-based screening for tuberculosis (TB), alone and with chest radiography among people living with HIV (PLHIV), including pregnant women, in Western Kenya. DESIGN: Prospective cohort study. METHODS: PLHIV from 15 randomly-selected HIV clinics were screened with three clinical algorithms [World Health Organization (WHO), Ministry of Health (MOH), and "Improving Diagnosis of TB in HIV-infected persons" (ID-TB/HIV) study], underwent chest radiography (unless pregnant), and provided two or more sputum specimens for smear microscopy, liquid culture, and Xpert MTB/RIF. Performance of clinical screening was compared to laboratory results, controlling for the complex design of the survey. RESULTS: Overall, 738 (85.6%) of 862 PLHIV enrolled were included in the analysis. Estimated TB prevalence was 11.2% (95% CI, 9.9-12.7). Sensitivity of the three screening algorithms was similar [WHO, 74.1% (95% CI, 64.1-82.2); MOH, 77.5% (95% CI, 68.6-84.5); and ID-TB/HIV, 72.5% (95% CI, 60.9-81.7)]. Sensitivity of the WHO algorithm was significantly lower among HIV-infected pregnant women [28.2% (95% CI, 14.9-46.7)] compared to non-pregnant women [78.3% (95% CI, 67.3-86.4)] and men [77.2% (95% CI, 68.3-84.2)]. Chest radiography increased WHO algorithm sensitivity and negative predictive value to 90.9% (95% CI, 86.4-93.9) and 96.1% (95% CI, 94.4-97.3), respectively, among asymptomatic men and non-pregnant women. CONCLUSIONS: Clinical screening missed approximately 25% of laboratory-confirmed TB cases among all PLHIV and more than 70% among HIV-infected pregnant women. National HIV programs should evaluate the feasibility of laboratory-based screening for TB, such as a single Xpert MTB/RIF test for all PLHIV, especially pregnant women, at enrollment in HIV services.

Evaluation of the Cepheid Xpert MTB/RIF assay.

The lack of capacity to provide laboratory confirmation of a diagnosis of tuberculosis disease (TB) is contributing to enormous gaps in the ability to find, treat and follow TB patients. WHO estimates that globally only about 57% of the notified new cases of pulmonary TB in 2012 and about 19% of rifampicin-resistant TB cases were laboratory confirmed. The Cepheid Xpert(®) MTB/RIF assay has been credited with revolutionizing laboratory testing to aid in the diagnosis of TB and rifampicin-resistant TB. This semi-automated test can detect both the causative agent of TB and mutations that confer rifampicin resistance from clinical specimens within 2 h after starting the test. In this article, we review the performance of the test, its pathway to regulatory approval and endorsement, guidelines for its use and lessons learned from the implementation of the test in low-burden, high-resource countries and in high-burden, low-resource countries.

An integrated approach to rapid diagnosis of tuberculosis and multidrug resistance using liquid culture and molecular methods in Russia.

OBJECTIVE: To analyse the feasibility, cost and performance of rapid tuberculosis (TB) molecular and culture systems, in a high multidrug-resistant TB (MDR TB) middle-income region (Samara, Russia) and provide evidence for WHO policy change. METHODS: Performance and cost evaluation was conducted to compare the BACTEC MGIT 960 system for culture and drug susceptibility testing (DST) and molecular systems for TB diagnosis, resistance to isoniazid and rifampin, and MDR TB identification compared to conventional Lowenstein-Jensen culture assays. FINDINGS: 698 consecutive patients (2487 sputum samples) with risk factors for drug-resistant tuberculosis were recruited. Overall M. tuberculosis complex culture positivity rates were 31.6% (787/2487) in MGIT and 27.1% (675/2487) in LJ (90.5% and 83.2% for smear-positive specimens). In total, 809 cultures of M. tuberculosis complex were isolated by any method. Median time to detection was 14 days for MGIT and 36 days for LJ (10 and 33 days for smear positive specimens) and indirect DST in MGIT took 9 days compared to 21 days on LJ. There was good concordance between DST on LJ and MGIT (96.8% for rifampin and 95.6% for isoniazid). Both molecular hybridization assay results correlated well with MGIT DST results, although molecular assays generally yielded higher rates of resistance (by approximately 3% for both isoniazid and rifampin). CONCLUSION: With effective planning and logistics, the MGIT 960 and molecular based methodologies can be successfully introduced into a reference laboratory setting in a middle incidence country. High rates of MDR TB in the Russian Federation make the introduction of such assays particularly useful.

Resistance of neisseria meningitidis to the toxic effects of heme iron and other hydrophobic agents requires expression of ght.

Several genetic systems that allow the use of iron-protoporphyrin IX (heme) have been described for the pathogenic bacterium Neisseria meningitidis. However, many questions about the process of heme acquisition and utilization remain to be answered. To isolate and analyze unidentified genes that play a role in heme iron uptake and utilization, a Himar1 transposon mutant library was screened in N. meningitidis serogroup A strain IR4162. One locus identified by transposon mutagenesis conferred protection against heme toxicity. A mutant with a deletion in a gene termed ght (gene of hydrophobic agent tolerance) within this locus was susceptible to heme and other hydrophobic agents compared to the parental strain. Transcriptional analysis indicated that ght is cotranscribed with an upstream open reading frame NMA2149. Uncharacterized orthologues of ght were identified in many other gram-negative bacteria. We present genetic evidence for the importance of ght in resistance to hydrophobic agents and its potential role in interaction with other hydrophobic agent resistance mechanisms within N. meningitidis.

Identification of Neisseria meningitidis genetic loci involved in the modulation of phase variation frequencies.

It has been proposed that increased phase variation frequencies in Neisseria meningitidis augment transmissibility and invasiveness. A Himar1 mariner transposon mutant library was constructed in serogroup A N. meningitidis and screened for clones with increased phase variation frequencies. Insertions increasing the frequency of slippage events within mononucleotide repeat tracts were identified in three known phase variation-modulating genes (mutS, mutL, and uvrD), as well as six additional loci (pilP, fbpA, fbpB, NMA1233, and two intergenic regions). The implications of these insertion mutations are discussed.

Natural transformation and phase variation modulation in Neisseria meningitidis.

Neisseria meningitidis has evolved the ability to control the expression-state of numerous genes by phase variation. It has been proposed that the process aids this human pathogen in coping with the diversity of microenvironments and host immune systems. Therefore, increased frequencies of phase variation may augment the organism's adaptability and virulence. In this study, we found that DNA derived from various neisserial co-colonizers of the human nasopharynx increased N. meningitidis switching frequencies, indicating that heterologous neisserial DNA modulates phase variation in a transformation-dependent manner. In order to determine whether the effect of heterologous DNA was specific to the Hb receptor, HmbR, we constructed a Universal Rates of Switching cassette (UROS). With this cassette, we demonstrated that heterologous DNA positively affects phase variation throughout the meningococcal genome, as UROS phase variation frequencies were also increased in the presence of neisserial DNA. Overexpressing components of the neisserial mismatch repair system partially alleviated DNA-induced changes in phase variation frequencies, thus implicating mismatch repair titration as a cause of these transformation-dependent increases in switching. The DNA-dependent effect on phase variation was transient and may serve as a mechanism for meningococcal genetic variability that avoids the fitness costs encountered by global mutators.

In vitro identification of Rns-regulated genes.

To identify Rns-regulated genes, a maltose binding protein (MBP)-Rns fusion protein was used to isolate DNA fragments from enterotoxigenic Escherichia coli genomic DNA that carry Rns binding sites. In vivo transcription fusion analysis shows that Rns positively regulates the expression of the open reading frame of yiiS, which lies immediately downstream of one MBP-Rns-bound fragment.