Much like the revolutionary impact on bacterial identifications, MS will likely impact resistance testing. Preliminary studies have demonstrated the rapid identification of MRSA, extended spectrum beta lactamase (ESBL) organisms, and carbapenemase-producing organisms by MALDI-TOF MS. Resistant organisms can be identified in two ways using MS. Similar to genetic approaches, resistant organisms can be identified by the presence or absence of characteristic mass peaks. This approach has been most widely used in the identification of MRSA by MS, though there are conflicting reports as to its effectiveness [45–47]. The other approach to identifying resistant organisms using MS is to apply a phenotypic approach such as measurement of substrate modification. For example, to determine the presence of a microbial carbapenemase, a carbapenem and test organism can be co-incubated followed by MS detection of native carbapenem drug peaks and/or peaks of its hydrolyzed products in the supernatant [48, 49]. Although this approach only detects resistance mechanisms that modify the substrate, it has the distinct advantage of looking for a phenotype instead of a particular resistance determinant. This can be especially useful in the cases of ESBLs and carbapenemases which have many genetic determinants that cause the same phenotype [50].

Screening patients for MRSA nasal colonization is a central strategy for preventing the spread of this organism in health care settings. The reference method used to accurately detect resistance due to altered PBP2 in S. aureus is NAA and detection of the mecA gene. Conventional and real-time PCR have been used to detect mecA both on bacterial isolates and directly on patient specimens. However, direct specimen testing has limitations, often including a lower positive predictive value than conventional methods based on the possible co-detection of MSSA and methicillin-resistant coagulase-negative staphylococci [51, 52]. Manufacturers have circumvented this problem through the detection of the SCCmec-orf junction in the S. aureus genome. However, strains that contain the SCCmec cassette but have a non-functional or deleted mecA (so-called “mecA-dropouts”) will be falsely positive. In addition, MRSA strains that carry mecC, a mecA homologue, will be falsely negative in these assays, though the prevalence of these strains is still low [53]. Several FDA-cleared molecular assays are available for the detection of MRSA with or without MSSA detection from nasal swabs and clinical specimens, such as positive blood cultures and swabs obtained from skin and soft tissue infections (Table 49.1). NAA detection of MRSA is at least equal in sensitivity to culture-based methods, but has the advantage of offering a faster turnaround time, which, when combined with appropriate infection control interventions, may significantly decrease hospital costs by decreasing the number of health-care-associated MRSA infections [54].

Although the incidence of Group B Streptococcus (GBS) neonatal disease has been declining since the 1990s due to enhanced prevention efforts, it is still the leading infectious cause of morbidity and mortality in neonates in the USA. In 2002, the Centers for Disease Control and Prevention (CDC), with the American College of Obstetricians and Gynecologists and the American Academy of Pediatrics, first published guidelines to perform vaginal–rectal screening of all pregnant women at 35–37 weeks gestation. Women who are colonized should be given intrapartum prophylactic treatment. Thus, accurate GBS results are critical to ensure appropriate antibiotic administration. Additionally, if a woman’s GBS colonization status is not known due to lack of prenatal care or premature delivery, she should receive prophylactic antibiotics based on risk assessment, specifically for gestation less than 37 weeks, membrane rupture more than 18 h prior to delivery, or a fever of greater than 38 °C [55]. Since antibiotic administration is not without risks to the mother and newborn, intrapartum rapid molecular tests for GBS colonization are beneficial.

The first molecular technique used for routine GBS screening was direct probe hybridization either to colonies or swab-inoculated Lim broth. Although this provided the advantage of decreased turnaround time and reduced technologist time [56], it is not cost-effective for routine antepartum screening. Further development of molecular technologies in GBS detection has resulted in seven FDA-cleared molecular tests (Table 49.2) and numerous laboratory-developed tests (LDTs). Notably, the BD GeneOhm StrepB test (BD GeneOhm Sciences, San Diego, CA) and the Cepheid Smart GBS and Xpert GBS offer detection of GBS directly from rectovaginal swabs for antepartum or intrapartum detection of GBS colonization. FDA-cleared in 2006, Xpert GBS performed on the GeneXpert (Cepheid) is a moderate-complexity test that is self-contained from extraction to result. This technology makes random access testing for intrapartum screening feasible. Given that approximately 10 % of women with negative cultures at 35–37 weeks’ gestation are GBS positive at the time of delivery [57], intrapartum testing is the most accurate test for colonization at the time of delivery. As an intrapartum screening test at one institution, the Xpert GBS had a sensitivity of 95.8 % and specificity of 64.5 %, whereas the antenatal culture was 83.3 % sensitive and 80.6 % specific, when intrapartum culture was used as the gold standard [58]. In a multicenter study of the IDI-StrepB assay (BD GeneOhm), when intrapartum culture was the gold standard, molecular detection at the time of labor was 94 % sensitive and 95.9 % specific [59]. Relative to either the sensitivity of antenatal cultures (54 %) or risk factor analysis (42 %), the sensitivity of the IDI-StrepB assay was superior [59]. The advantage in all these applications is the decreased turnaround time relative to culture in the intrapartum setting. Additional data regarding the sensitivity and specificity of molecular tests for GBS detection is shown in Table 49.2.

The use of molecular methods for the diagnosis of sepsis has been a challenging endeavor. Only one FDA-approved test is available for the identification of potential pathogens directly from blood obtained from septic patients, and this test is limited to candidemia. The gold standard remains automated blood cultures, and this reference method may be difficult to match owing to the large amount of blood that is cultured (typically 40 ml). Nonetheless, research use only products are available for direct testing of blood. Roche Molecular Systems SeptiFast (Branchburg, NJ) uses multiplex real-time PCR and melt curve analysis, while the Molzym SepsiTest (Bremen, Germany) uses multiplex PCR followed by sequencing, and the SIRS-Lab Vyoo (Jena, Germany) uses multiplex PCR followed by gel electrophoresis. These products vary in both the organisms and the resistance determinants detected, as well as analytical performance characteristics [60–64]. In general, these products suffer from both a lack of sensitivity and specificity, as well as requiring additional optimization before routine clinical use is possible.

Other shortcomings of NAA-based diagnosis of sepsis include the inconclusive clinical significance of the detection of pathogen DNA in the blood stream and the inability to obtain full antimicrobial susceptibility results [62]. However, blood culture is an imperfect reference method, suffering from a number of limitations including a prolonged time to pathogen identification, effects of variable blood volume, and lack of growth for fastidious pathogens or in the presence of prior antimicrobial therapy [62]. One limitation that can be addressed by molecular methods is the time to definitive identification.

A number of commercial molecular testing products are available for the identification of organisms and resistant determinants directly from positive blood culture bottles. This approach takes advantage of the culture amplification of bacteria from blood while adding molecular methods to lessen the time to identification. FDA-cleared tests for use directly with positive blood culture bottles include AdvanDx PNA-FISH (Woburn, MA), Cepheid (Sunnyvale, CA), Biofire FilmArray BC-ID (Salt Lake City, UT) and Nanosphere BC-GP and BC-GN panels (Northbrook, IL). The molecular targets for each of these products are listed in Table 49.3. The use of MALDI-TOF MS in direct pathogen detection directly from positive blood cultures also is being investigated [65, 66]. Recent data show identification rates of approximately 85 %, while reducing time to identification by more than a day [67]. Several studies have demonstrated the cost-effectiveness of utilizing rapid detection of organisms from positive blood culture bottles [8, 9, 11].