Antibiotic
Resistance gene
Nucleotide change
Amino acid change
Clarithromycin
23S rRNA
A2142G
NA
A2143G
NA
Ciprofloxacin
gyrA
C(T) to A
N87K
C(T) to G
N87K
A to G
D91G
G to T
D91Y
G to A
D91N
Tetracycline
16S rRNA
A926G
NA
A926C
NA
A926T
NA
A928C
NA
RFLP-based assays for the detection of clarithromycin resistance take advantage of the fact that antibiotic resistance mutations create restriction sites within the 23S rRNA gene not present in susceptible strains. Conventional PCR using 23S rRNA specific primers produces amplicons, which when cleaved with restriction endonucleases and visualized by agarose gel electrophoresis create a pattern composed of two bands instead of one. While conceptually simple, these methods are more time consuming than real-time PCR assays.
Real-time PCR methods can detect clarithromycin resistance mutations directly from biopsy specimens with excellent sensitivity and rapid turnaround times of 1–4 h [66, 83–85]. An assay described by Gibson et al. uses fluorescent-labeled probes complementary to the clarithromycin-sensitive 23S rRNA gene sequence [86]. Resistance mutations result in mismatched bases between the probe and target, and melting curve analysis reveals a lower peak melting temperature for the mismatched hybrid than a fully complementary probe and target hybrid [86]. This assay has good concordance with culture-based methods [66, 84], but also identified susceptibilities for an additional 28 patients whose cultures were negative. Of the 28 additional susceptibility results rendered, 21 had resistance genotypes. Another assay design using a biprobe system was tested on 200 patients who failed eradication therapy. The assay detected resistance genotypes with a sensitivity and specificity of 98.4 % and 94.1 %, respectively, when compared to culture-based testing [85]. Clarithromycin-resistant genotypes can also be detected in stool samples using real-time PCR methods; however, the sensitivity is lower [87]. Real-time PCR assays have also been developed to detect point mutations in the quinolone resistance-determining region of the gyrA gene resulting in resistance to ciprofloxacin and point mutations in the 16S rRNA gene conferring decreased susceptibility and resistance to tetracyclines [88, 89]. The assay for determining fluoroquinolone resistance identifies mutations using two hybridization biprobes designed to detect the most frequently occurring mutations at amino acid positions 87 or 91 [88]. Tetracycline resistance is detected using 16S rDNA primers and a fluorescently labeled probe complementary to the wild-type 16S rDNA allele. In both assays, melting curve analysis differentiates amplicons with resistance mutations from those with wild-type sequences [89]. While various mutations in the NADPH nitroreductase gene (rdxA) are associated with metronidazole resistance, detection of these mutations is not a reliable indicator of resistance [90].
Histopathologic diagnosis of H. pylori infection is a sensitive and specific method (>95 % and 100 %, respectively) under optimal conditions, yet ancillary molecular techniques such as FISH may help in difficult cases [56]. Visualization of the characteristic bacterial forms may be difficult when reduced numbers of bacteria are present, such as when biopsies are obtained after eradication therapy or if the patient has been on long-term acid suppression therapy with PPIs. These same conditions may change the typical morphology of H. pylori from a comma or S-shaped bacillus to a coccoidal form, obscuring a visual diagnosis. Several studies using fluorescently labeled, species-specific probes have demonstrated the ability of FISH to reliably detect H. pylori [91, 92]. Additionally, clarithromycin-resistant strains also can be detected using FISH performed on formalin-fixed tissue sections [92]. Fluorescent-labeled oligonucleotide probes designed to detect the most common mutations determining clarithromycin resistance are both sensitive and specific when compared to culture-based susceptibility testing [91]. FISH testing, however, may produce results more rapidly than culture.
Interpretation of Results
PCR tests can achieve equal or better performance when compared to non-molecular tests [58–62]. The sensitivity is highly dependent on the target gene and is discussed in greater detail in the “Laboratory Issues” section below.
The specificity of different PCR test methods varies and determining specificity is complicated by the lack of gold standard. Real-time PCR assays applied to tissue biopsies have detected H. pylori at low densities that were missed by histology, UBT, and RUT suggesting that the poorer sensitivity of these non-molecular assays is due to low numbers of organisms [61]. However, due to the high sensitivity of PCR-based methods and the amplification of DNA from nonviable organisms, isolated positive PCR results in the post-treatment period must be interpreted with caution. Positive results in this setting may represent continued presence of organisms at low levels or nonviable organisms, and, therefore, PCR may not have utility in determining eradication failures in the post-treatment period. Isolated positive PCR results in untreated patients may reflect true infection with a low H. pylori density, but may also be due to nonspecific amplification of non-H. pylori bacterial DNA [93]. Due to these factors, PCR-based methods should not be used as the sole diagnostic test.
Like PCR testing of biopsy specimens, the specificity of results obtained from PCR testing of stool samples may be decreased due to amplification of nonviable organisms. Studies examining the use of PCR-based testing of stool for determining eradication success rates in the early post-treatment period are conflicting [80, 94]. While negative PCR results within 12 days of treatment were obtained for a small group of infected patients, another study demonstrated false-positive results occurring in half the patients 1 month after treatment [94, 95]. False-positive PCR results decrease after longer follow-up periods, and approach zero after 12 weeks of therapy [94]. In addition, analytical specificity of PCR testing on stool samples may be reduced due to the presence of non-pylori Helicobacter species present in fecal material [56]. While clinical specificity for PCR-based assays is determined by comparison with UBT, culture, and RUTs, determinations of analytical specificity by testing of non-pylori Helicobacter species is rarely performed [96].
Antibiotic resistance genotype testing using real-time PCR can produce results indicating the presence of more than one genotype [85]. These results are interpreted as representing a mixed population and combinations of one or more distinct mutant strains among wild-type strains have been detected [85]. While some studies have detected mutant strains in the presence of wild-type strains down to a level of 10 % [85], other studies cite failed resistance detection due to high levels of susceptible strains [87].
Laboratory Issues
Sensitivity and specificity of PCR-based methods are greatly dependent on primer choice and target gene. Additionally, significant inter-study variation in sensitivity and specificity exists for several of the commonly used primers. Nucleotide differences among distinct H. pylori strains may partly explain this test performance variability [77]. A study comparing the diagnostic performance of several different primers demonstrated poor specificity for SSA gene primers and unsatisfactory sensitivity for the ureA gene and random H. pylori genome sequences [76]. While this study concluded that glmM gene PCR performed best, other studies have reported lower specificities [60, 77]. Assays using 16S rRNA sequences generally report excellent sensitivities, but the specificity of these primers is questionable. Several authors argue that 16S rRNA primers are inappropriate because of sequence conservation among different bacterial genera as well as the possibility for nonspecific amplification of human DNA [76, 77]. Assays targeting vacA have reported moderate sensitivity (89.5 %) but excellent specificity (99.0 %) [84]. The HSP60 gene is thought to be both well conserved and demonstrates species-specific variation [77]. A nested assay design using primers targeting HSP60 claims to have sensitivity and specificity approaching 100 % [77].
Determining whether tissue to be used for PCR assays is preserved by formalin fixation or cryopreservation represents an important variable in testing, but may ultimately be decided by proximity of laboratory and endoscopy suite. Both formalin-fixed and frozen tissue specimens may be used for PCR-based testing, although frozen samples are far superior [56]. Formalin fixation causes DNA to fragment; however, assays using formalin-fixed tissues may still perform acceptably if short DNA sequences are targeted.
PCR assays applied to stool specimens suffer from inconsistent results attributable to substances inhibitory to PCR amplification, low numbers of H. pylori organisms within fecal samples, as well as degradation of DNA during intestinal transit [53, 56]. To avoid false-negative results, complex purification and extraction steps to eliminate PCR inhibitors are required before DNA amplification. Performance of the different biochemical, immunologic, and physical purification methods varies due to degradation of target DNA and incomplete removal of inhibitors [96]. Frozen stocks of H. pylori strain NCTC 11637 and dried genomic DNA from that strain (American Type Culture Collection 43504D, Manassas, VA) as well as titered cultures of H. pylori (ZeptoMetrix Corp, Buffalo, NY) are commercially available reference materials. Proficiency testing exercises are not commercially available.
Viral Agents
The most common causes of viral gastroenteritis include adenovirus serotypes 40 and 41, rotavirus, astrovirus, and caliciviruses (noroviruses, sapoviruses). Conventional detection of these viruses is based on antigen detection and EM. Molecular methods have been primarily used for epidemiologic or research purposes but have also demonstrated significant improvement in the diagnosis of viral gastroenteritis and are becoming available in clinical laboratories.
Other viruses less frequently implicated as causes of acute gastroenteritis include coronaviruses and toroviruses. In addition, viruses such as cytomegalovirus and herpes simplex virus are opportunistic causes of enteric disorders in patient infected with the human immunodeficiency virus (HIV) or with compromised immunity but are diagnosed by examination or testing of gastric or intestinal biopsy tissues rather than examination of stool.
Adenovirus
Description of Pathogen
Adenoviruses are nonenveloped viruses with a linear, non-segmented, double-stranded DNA genome surrounded by an icosahedral protein capsid. The genome size varies among adenoviral groups and is between 26,000 and 45,000 nucleotides which theoretically provides the capacity for 22–40 genes. The genome consists of immediate early (E1A), early (E1-E4), intermediate, and late genes (L1-L5).
Adenoviruses are classified into five genera including Atadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus, and Siadenovirus. The adenoviruses that infect humans belong to the genus Mastadenovirus. Seven species of human adenovirus (A through G) within the genus Mastadenovirus are currently recognized. Species designations are determined by immunologic properties as well as DNA homology and oncogenicity [97]. Each species group contains several serotypes classified by neutralization reactions to specific antisera [97]. At present, over 50 serotypes have been described [98]. Serotype groups may be further subdivided into genomic types. Genotypes are assigned lowercase letters to differentiate them from the prototype strain as indicated by the letter “p” [97]. Interspecies DNA sequence variation may be as low as 4 % whereas genotypes within an adenovirus species may be 50 % to nearly 100 % homologous [99, 100]. Intraspecies recombination resulting in intermediate strains has been reported [101].
Species F serotypes 40 and 41 are the most frequent adenovirus serotypes isolated from patients with gastroenteritis and are referred to as the “enteric adenoviruses.” These serotypes are second only to rotavirus as the most common cause of acute diarrheal illness in children [102]. Adenoviruses of all serotypes are implicated in approximately 5–15 % of childhood diarrhea cases [97]. Gastroenteritis due to adenovirus occurs worldwide and the incidence does not demonstrate significant seasonal variation [103]. More than one serotype or species may be isolated in a given patient [104]. Serotypes infrequently associated with gastroenteritis include 1, 2, 3, 5, 7, 12–18, 21, 25, 26, 29, 31, and 52 [102].
Transmission is thought to occur by fecal-oral spread and the mean incubation period for gastroenteritis is 3–10 days [97, 103]. After clinical symptoms improve, enteric adenoviruses are shed in stool rarely for longer than a few days compared to patients with respiratory infections not involving enteric adenoviruses who may shed for 3–6 weeks and as long as 18 months [97]. The duration of viral shedding in the gastrointestinal tract may be prolonged in immunocompromised individuals [97]. Adenoviruses can also be shed in the stool of individuals with asymptomatic infections which are common, particularly in children [97].
Clinical symptoms include watery, non-bloody diarrhea accompanied by mild fever, vomiting, and abdominal pain. Gastroenteritis in immunocompetent patients usually resolves without complication; however, rare fatalities are documented [97].
Gastrointestinal infections in immunocompromised patients occur most often in hematopoietic stem cell (HSCT), bone marrow (BMT), and solid organ (SOT) transplant patients. Infections in these patients are frequently severe and can become disseminated. In pediatric allogeneic HSCT patients, detectable virus in stool almost always precedes systemic adenovirus infection [105]. Adenovirus species isolated most frequently in HSCT and BMT patients with gastrointestinal disease include species A (serotype 31), B (serotype 7), and C (serotype 2) [106]. The incidence of adenovirus infections in patients with HIV infection or acquired immunodeficiency syndrome (AIDS) has dropped due to effective treatment of the HIV infection with highly active antiretroviral therapy [10]. Serotypes within species D cause the majority of gastroenteritis in HIV-positive patients and include serotypes 9, 17, 20, 22, 23, 26, 27, and 42–51 [106].
Clinical Utility of Testing
Diagnosis of adenovirus gastroenteritis is primarily determined through testing of stool samples although tissue biopsy specimens also may be used. Conventional methods used to identify the presence of adenovirus in stool samples include shell vial cultures, direct fluorescent antibody assays, EIAs, and EM. Disadvantages of culture methods include delays of up to weeks and false-negative results with difficult to culture AV serotypes such as 40 and 41 [99]. Immunofluorescent and immunochromatographic methods, while rapid, are insensitive [107]. EM also is insensitive and is not routinely used in clinical laboratories. Indirect diagnosis using serology is limited by poor sensitivity especially in immunocompromised patients and by high seroprevalence among children preventing the ability to identify acute disease [107, 108]. Despite these diagnostic limitations, conventional methods may be sufficient to detect infection in immunocompetent patients with localized or benign gastrointestinal symptoms [109].
Adenovirus infection may become severe in immunocompromised patients and the ability to begin early treatment, such as reduction of immunosuppression or cidofovir therapy, requires rapid and sensitive diagnostic techniques [107]. Several PCR-based assays have been validated for stool specimens and are comparable or better than conventional methods [110–112]. Both qualitative and quantitative PCR assays are used. Qualitative assays vary in serotype detection of all or only some serotypes. While qualitative PCR methods are sensitive, quantitative assays assess stool viral load and proliferation kinetics. These parameters may become important in assessing the need for preemptive treatment of adenovirus infection in pediatric HSCT patients by detection of significant levels of virus in stools before the onset of viremia and disseminated infection in the majority of these patients [105, 113].
Multiplex assays differ in their clinical application. One particular assay provides qualitative, yet species-specific results [114], while others offer quantitative results and detect adenovirus in combination with other important gastrointestinal pathogens. Quantitative assays may be useful for determining which pathogen is responsible for disease in cases of mixed infections [115, 116].
In most cases, determination of adenovirus serotype is unnecessary for clinical management [117]. By contrast, serotyping is important for epidemiology studies, when investigating an especially severe infection, and in predicting clinical outcome [106]. Isolation of specific adenovirus serotypes from the gastrointestinal tract, such as 1, 2, 5, and 6, may raise concern for the possibility of disseminated disease since these serotypes have been documented to cause systemic infections in immunocompromised patients [107]. On the other hand, serotypes 40 and 41, while frequently isolated in cases of gastroenteritis, have not been recovered from immunocompromised patients with disseminated infections [107]. While conventional serotyping methods may take up to several weeks [97], molecular methods such as PCR have improved turnaround times and allow for the characterization of isolates at the species, serotype, and genotype level [107]. Molecular and serological typing results usually are concordant [97].
Available Assays
Tissue biopsies may be submitted for culture or for histological examination using hematoxylin-and-eosin or Wright-Giemsa stains and immunohistochemistry. Molecular techniques such as in situ hybridization may aid diagnosis by confirming characteristic microscopic findings [118]. Alternatively, biopsy specimens may be submitted directly for molecular testing using conventional or real-time PCR [102, 119].
Molecular methods offer increased sensitivity and shorter turnaround time compared with conventional methods [110–112]. Different PCR methods for the detection of adenovirus in stool or tissue biopsies include conventional PCR, real-time PCR, and multiplex PCR. Most clinical laboratories use LDTs because no US FDA-cleared tests are available for stool testing. Regardless of the PCR method, degenerate or non-degenerate primers and probes targeting the hexon or fiber genes or the VA RNA-encoding regions are typically used. These regions display homology across serotypes for consistent binding of primers and probes, yet also include hypervariable regions suitable for differentiating serotypes.
Conventional PCR assays range in their detection abilities. Some systems detect and report specific serotypes [110, 120, 121], while others report genus- or species-specific results and purport to detect all serotypes [107, 117]. These assays are qualitative and usually have a 1–2 day turnaround time. Methods used to detect PCR amplicons include ethidium bromide-stained gel electrophoresis, Southern blotting, or liquid phase hybridization quantitated by time resolved fluorometry [122]. These detection methods are time- and labor-intensive and necessitate handling of PCR products, thus potentiating the risk of contamination.
Real-time PCR methods offer quantitative results and are more rapid and involve less contamination risk than conventional PCR assays [102, 119, 123, 124]. Several LDTs and one commercial assay are validated for use on stool specimens. While some methods rely on a single probe and primer pair, most utilize more than one set of primers and multiple probes. Weighing of stool specimens before DNA extraction allows results to be quantitated in copies per gram of stool. This standardization permits assessment of serial stool specimens for viral load kinetics and facilitates comparison of results between assays.
Several multiplex PCR assays have been validated for stool testing and differ in their clinical applications and detection methods. One particular method allows for identification of all six adenoviral species in a single reaction mixture using species-specific hexon primers [110]. Species-specific results are visualized by agarose gel electrophoresis, which shows a different amplicon length for each species [110]. Other multiplex assays offer quantitative results and combine adenovirus detection with other common gastrointestinal viral pathogens [115, 116]. Detection techniques differ and involve either fluorescent-labeled sequence-specific probes or sequence-specific capture probes bound to microspheres, which are interrogated by flow cytometry.
Conventional typing may take weeks making such methods impractical for clinical use. Molecular typing methods greatly improve turnaround time and several assays have been tested for use with stool samples. Strategies for producing serotype or genotype specific results vary by assay, and may be performed from cultured isolates or directly from clinical specimens [125, 126]. Traditional molecular typing methods rely on REA and may be performed on adenoviral genome DNA or following PCR amplification of specific regions [117, 127]. Genotype or serotype is inferred from the band pattern on agarose gel electrophoresis. REA methods are still used to identify new strains or for type identification of an isolate causing severe disease [106]. Sequence-based typing may be used to determine both serotype and genotype and is usually performed after PCR amplification of hypervariable regions [128]. Generated sequences are compared to banked sequences of known serotypes.
Interpretation of Results
Similar to other clinical situations where highly sensitive molecular assays are applied, PCR methods used in the diagnosis of adenovirus gastroenteritis offer improved sensitivity over conventional methods, but may provide positive results in the absence of disease. The ability for adenoviruses to cause asymptomatic infection and the tendency for nonenteric adenoviruses to be shed in the stool for weeks to months after resolution of clinical symptoms make interpretation of positive results in patients without symptoms difficult [97, 103].
Diagnosis of adenovirus gastroenteritis in immunocompetent patients is straightforward when PCR results are positive for an enteric adenovirus species or serotype in the presence of characteristic symptoms. Viral shedding in the absence of symptoms is unusual for enteric adenoviruses, especially in immunocompetent individuals.
Shedding of nonenteric serotypes for long periods of time occurs more frequently in immunocompromised patients and makes it difficult to determine conclusively that a detected adenovirus serotype is the cause of the patient’s symptoms. Persistent viral shedding from a previous adenovirus infection may be difficult to distinguish from a newly acquired asymptomatic infection, which occurs often in the immunocompromised patient population. Further complicating interpretation in immunocompromised individuals is the frequent occurrence of coinfections.
The ability to determine serotype by sequencing is limited by incomplete reference databases containing sequence information for only certain serotypes [107]. The completeness of the reference database depends on the genome region sequenced. Serotype determination by methods relying on enzyme restriction patterns is limited to serotypes whose restriction patterns have been previously described. These methods are further hampered by the genetic variability created over time by recombination events between viruses of different serotypes. Such variation may alter cleavage sites and create unrecognizable restriction enzyme patterns. Infections caused by one or more serotypes may also create uninterpretable results.
As mentioned above, quantitative testing of stool allows clinicians to monitor stool viral load and proliferation kinetics. The mere presence of adenovirus in stool is common in pediatric HSCT patients, and does not necessitate treatment, as the majority will clear the virus spontaneously [129]. Quantitative measurements, however, have allowed investigators to identify rising stool viral loads in the majority of pediatric HSCT patients who go on to develop adenovirus viremia and disseminated disease [105, 113]. Quantitative serial stool measurements, therefore, may serve as a useful tool for predicting when early treatment is warranted and could prevent the overuse of the nephrotoxic antiviral drug cidofovir [113].
Laboratory Issues
Detection of all adenovirus serotypes is important because serotypes other than enteric adenoviruses cause a significant number of gastroenteritis cases in immunocompromised patients. The high degree of genetic heterogeneity among adenovirus serotypes makes detection of all known serotypes by a generic PCR assay difficult [106]. Genetic diversity also complicates identification of regions with sufficient homology to allow for uniform annealing of primers and probes in all serotypes. Currently, most assays use primers and probes that bind to the highly conserved hexon gene, which has only approximately 50 % nucleotide homology between serotypes (NCBI database, [105]). Uniform annealing of primers and probes is even more important for quantitative assays, to ensure equal amplification efficiency of all serotypes [108]. Concern that nucleotide mismatches between target and primer or probe would result in decreased sensitivity of detection for many serotypes has led to more optimal assay designs utilizing multiple primer and probe sets [123]. Lastly, multiplex assays that detect multiple viral pathogens can detect coinfections, although the test performance for adenovirus was occasionally negatively affected by coamplification of other viruses [116].
DNA from both adenovirus 40 and adenovirus 41 is available from ATCC (Manassas, VA). The Zeptometrix NATtrol™ gastrointestinal pathogens verification panel includes adenovirus among the other analytes. The Stool Pathogen Panel (SP) proficiency survey from the College of American Pathologists includes challenges for adenovirus 40/41.
Rotavirus
Description of Pathogen
Rotaviruses are non-enveloped viruses in the Reoviridae family, named because of their characteristic wheel-like appearance by EM. Rotaviruses are very stable in the environment and can remain infectious for several weeks. They have a triple-layered structure with concentric capsid layers that surround a core which contains the genome. The surface of the outermost capsid layer contains two major structural viral proteins, VP4, a protease-cleaved protein (P protein), and VP7, a glycoprotein (G protein). The middle layer of the capsid contains structural protein VP6. The inner capsid layer contains proteins VP1, VP2, and VP3. The rotavirus genome consists of 11 segments of double-stranded RNA with a complete genome length of 16,500–21,000 nucleotides. The genomes can reassort during dual infection of a single cell which results in co-circulation of a wide variety of strains.
Rotaviruses are classified into serogroups A through G based on the antigenic characteristics the VP6 protein. Only groups A, B, and C infect humans and animals. The remaining rotavirus groups have been found only in animals. The Group A human rotaviruses cause the majority of viral gastrointestinal infections in children. Group B rotaviruses were first identified as causing adult diarrhea in a large waterborne epidemic in China. Serologic evidence indicates that Group B rotavirus is also present in the UK and the US, and genome profiles consistent with Group B rotavirus have been detected as causes of diarrhea in India [130]. Group C rotaviruses are an emerging cause of gastroenteritis in both children and adults and have been identified in sporadic cases and outbreaks worldwide. The Group A rotaviruses are further classified into serotypes based on neutralizing serologic reactions against the P (VP4) and G (VP7) proteins. Also, because the two gene segments that encode the P and G proteins segregate independently, a genotyping system has been developed based on the sequences of both genes. The most prevalent Group A rotavirus genotypes in humans are G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]. A Rotavirus Classification Working Group has been formed to assist in classification of any newly described rotavirus genotypes based on sequence information for all 11 genomic RNA segments.
Rotavirus is endemic worldwide and is the single most common cause of diarrhea among infants and young children [131]. Most rotavirus infections are self-limiting but some children become very ill with severe vomiting, diarrhea, and life-threatening loss of fluids that requires hospitalization. Death due to rotavirus infection is relatively rare in the USA but is a significant concern in developing countries. Rotaviruses are estimated to cause more than half a million infant and young children deaths worldwide every year [131].
In the USA and other countries with temperate climates, annual outbreaks of rotavirus infection occur during winter and spring, with fewer cases in summer. However, with the introduction of rotavirus vaccines, the seasonality has shifted and the winter–spring trend in peak rotavirus activity is no longer consistently observed [132]. Seasonal variation is not seen in tropical climates.
Rotaviruses are shed in large quantities in the stools of infected children beginning 2 days before the onset of diarrhea and for up to about 10 days after the onset of symptoms. Immunocompromised individuals may shed detectable rotavirus for more than 30 days after infection. Rotaviruses are highly communicable, with a small infectious dose of less than 100 virus particles [133]. Rotaviruses are spread by fecal-oral transmission, both through close person-to-person contact and through fomites, and are common causes of diarrheal outbreaks in families, in childcare centers, and other institutions, and among hospitalized children [134]. The incubation period for rotavirus illness is about 2–3 days. Immunity after infection is incomplete, but repeat infections tend to have milder signs and symptoms than the initial infection.
A rotavirus vaccine is now included in the American Academy of Pediatrics recommended immunization schedule for infants. Two vaccines, RotaTeq® (Merck & Co., Inc., West Point, PA) and Rotarix® (GlaxoSmithKline, London, UK) were introduced in 2006 and 2008, respectively, and are currently licensed for use in the USA. A previous rotavirus vaccine was taken off the market in 1999 because of an increased risk for intussusception, which does not occur with either RotaTeq or Rotarix.
Adults and older children also can be infected with rotaviruses. Infection in adults is often subclinical or very mild. Clinically evident cases are most often seen in immunocompromised patients, the elderly, and travelers to developing countries [135].
Clinical Utility of Testing
Rotavirus infection cannot be diagnosed by clinical presentation because the clinical features of rotavirus gastroenteritis do not differ from those of gastroenteritis caused by other pathogens. Confirmation of rotavirus infection by laboratory testing is used for surveillance but also is useful in clinical settings to avoid inappropriate use of antimicrobial therapy.
Since rotavirus is present in high concentrations in the stool of infected children, stool is the preferred specimen for diagnosis. Rotaviruses can be cultured in Madin-Darby bovine kidney (MDBK), fetal African green monkey kidney cells (MA104 cell line), and some other cell lines in media containing trypsin or pancreatin, but culture is relatively inefficient and not performed in clinical laboratories. The most widely available method for detection of rotavirus antigen in stool is EIA directed at the VP6 antigen common to all group A rotaviruses. Several commercial EIA kits are available, which are inexpensive, easy to use, rapid, and sensitive (approximately 90–100 %). Latex agglutination is less sensitive and specific than EIA but is still used in some settings. Immunochromatographic point of care tests have reported sensitivities of 94–100 % and specificities of 96–100 % compared with clinical laboratory tests [136]. Other techniques, including EM, reverse transcription-polymerase chain reaction, nucleic acid hybridization, sequence analysis, and culture are used primarily in research settings.
Molecular methods have been used primarily for characterization of G and P genotypes in epidemiologic studies and to evaluate the impact of vaccination [137, 138]. RT-PCR has increased detection rates for rotavirus A by up to 48 % compared to EIA or EM [139]. Sensitivity of RT-PCR tests is estimated at 104 rotavirus particles per milliliter of stool, while EIA methods detect 106 rotavirus particles per milliliter of stool. While increased sensitivity is usually seen as a benefit, some have considered RT-PCR assays too sensitive for the detection of rotaviruses due to their ability to detect asymptomatic infections, which are common in infants and young children [140, 141].
Available Assays
Some commercial tests designed for testing food sources or environmental samples are available as research use only kits. Most reports of molecular tests used for diagnosis of human rotavirus infections are LDTs. The xTAG GPP gastrointestinal pathogen panel test kit (Luminex Corp., Austin, TX) is an US FDA-cleared qualitative RT-PCR multiplexed test that can be performed in about 5 h and simultaneously detects the most common parasitic, bacterial and viral gastrointestinal pathogens, including rotavirus Group A.
Interpretation of Results
Asymptomatic carriage of rotavirus can be detected by molecular tests and needs to be considered when interpreting positive results in clinical specimens [140, 141]. Asymptomatic carriage vs true mixed infection also needs to be considered in the rare event that rotavirus is detected in combination with another gastrointestinal pathogen in the same clinical sample or during an episode of diarrhea. Laboratories should be aware that rotaviruses can be detected by RT-PCR in clinical specimens for about 10 days after resolution of an acute diarrheal episode in healthy children [137].
Laboratory Issues
Ideally, diagnostic RT-PCR tests would be able to detect all three genogroups of human rotaviruses. Except for epidemiologic purposes, identification of the specific genogroup is probably not necessary. Laboratories should be aware that rotavirus RNA has been detected in serum, CSF, and throat swab specimens [142, 143].
The NATtrol™ (ZeptoMetrix Corp, Buffalo, NY) verification set includes reference material for rotavirus among other analytes. Human rotavirus in diarrheal stool samples is available from ATCC (Manassas, VA). The Gastrointestinal Panel for Molecular Multiplex Testing (GIP) and the Stool Pathogens (SP) survey, both from the College of American Pathologists, include proficiency testing challenges for rotavirus among other gastrointestinal pathogens.
Astrovirus
Description of Pathogen
Astroviruses are small, round, non-enveloped viruses with icosahedral cubic capsids that have a characteristic five or six-point star-like surface structure when viewed by EM. Astroviruses contain three structural proteins (VP26, VP29, and VP32). The genome is composed of non-segmented, positive-sense, single-stranded RNA. The total genome length is 6,800–7,900 nucleotides, excluding the poly (A) tract at the 3′ end and the genome has been sequenced.
The family Astroviridae contains two genera: Mamastroviruses which infect numerous types of mammals and Avastroviruses which infect birds (e.g., ducks, chickens, turkeys). Within each genus are species of astroviruses, which, according to International Committee on Taxonomy of Viruses guidelines, are named based on the host in which they replicate. The astroviruses are further subclassified within each species into serotypes. Three species of astroviruses are found in human stool: HAstV (serotypes 1–8), AstV-MLB, and HMOAstV (serotypes A to C). These species are more closely related to animal astroviruses than to each other, indicating phylogenetically separate origins of human astroviruses [144].
Simultaneous circulation of multiple types of astrovirus is not rare [144]. Human astrovirus serotype 1 (HAstV-1) is the most prevalent serotype detected worldwide. However, serotype 3 produces higher quantities of virus in stool and appears to cause a larger proportion of cases of persistent gastroenteritis [145].
Human astroviruses are endemic worldwide. Studies using sensitive detection techniques, such as RT-PCR, have demonstrated that astrovirus infection is a more common and important cause of viral gastroenteritis than previously known. Symptomatic illness is most common in children < 2 years of age, although infection in immunocompromised individuals and outbreaks among adults and the elderly have also been reported. Astroviruses are highly stable in the environment and are resistant to a wide range of detergents and lipid solvents. The fecal-oral route is thought to be the most common means of transmission and contaminated food, water, and fomites are common sources of virus. As with many other viral causes of gastroenteritis, astrovirus infection has a peak incidence in winter in temperate climates and is associated with the rainy season in tropical regions. Astrovirus is thought to replicate in the intestinal tissue of the jejunum and ileum and generally causes mild, self-limiting illness of short duration. Prevalence is likely under-estimated since surveillance and seroprevalence studies have demonstrated that astrovirus infection is common and is largely asymptomatic.
Immunity to astrovirus infection is not well understood. Prevalence of symptomatic infection among young children and institutionalized elderly populations suggests that antibody is acquired early in childhood, provides protection through adult life, and begins to decline later in life [146]. Heterologous protection does not occur across the human astrovirus serotypes [147].
Clinical Utility of Testing
No vaccine or anti-viral treatment is available for prevention or treatment of astrovirus infection, but diagnosis may be important to avoid unnecessary antibiotic use. Establishing an etiology also may be important in hospitalized patients for infection control purposes to prevent nosocomial spread [148, 149]. Further, diagnosing astrovirus gastroenteritis in patients with malnutrition, immunodeficiency, and underlying gastrointestinal disease, may be important because of the increased likelihood of complications that require hospitalization in these populations. The impact of astrovirus infection on the morbidity of infants and children may become increasingly important as the rotavirus vaccine becomes more widely used and the burden of rotavirus is reduced.
Available Assays
Astroviruses have been adapted to cell culture using CaCo-2 human colonic carcinoma cells in some research settings, but are not cultured for clinical diagnostic purposes. EM and immune EM (IEM) have been effectively used to detect astroviruses in clinical stool specimens but are not available in many clinical laboratories. Also, identification by EM can be difficult since only a small portion of astrovirus particles (about 10 %) display the characteristic star-like morphology [150].
EIA tests have been developed for the qualitative detection of astrovirus antigen in clinical specimens and are commercially available in Europe (e.g., RIDASCREEN® Astrovirus test, R-biopharm AG, Darmstadt, Germany, IDEIA™ Astrovirus, Dako Diagnostics Ltd, Ely, UK) but are not US FDA-cleared for diagnostic use in the USA.
Commercial real-time RT-PCR kits are available for testing environmental and food samples. Molecular assays are considered to be an improved diagnostic method over EM and EIA [151], but US FDA-cleared diagnostic tests for astrovirus are not available. Specifications for LDTs for detection of astrovirus in clinical samples using highly sensitive group-specific RT-PCR primers targeted to conserved genomic regions coding for the nonstructural proteins and untranslated regions are available [151]. Reported detection limits for these assays vary from 1 to 10 viral copies depending of the quality of the analyzed nucleic acid. Some tests utilize primers from the capsid coding region which can be less sensitive, but provide type information [151].
Interpretation of Results
Shedding of astrovirus is generally limited to about 1 week in immunocompetent individuals, but as with other viruses that cause gastroenteritis, prolonged shedding of astrovirus (e.g., 4 weeks) has been observed in immunocompromised patients [148, 152]. Although asymptomatic infection is common, determining the significance of astrovirus detection should not be problematic in most clinical settings since presumably only diarrheal stools from symptomatic patients would be tested.
Laboratory Issues
Astrovirus infections are generally limited to the gastrointestinal tract; however, astroviruses have been detected in plasma as a cause of febrile illness and in brain tissue of an immunocompromised patient [153]. Human gastrointestinal astrovirus infections have been limited to the eight closely related serotypes described above. Recently, several highly divergent astrovirus serotypes (MLB1, MLB2, VA1, VA2, and VA3) have been detected in stool samples from patients with and without diarrhea [153]. An association with gastrointestinal disease has not been definitively made for these newly described astroviruses [153]. Reference material and proficiency testing challenges are not currently commercially available.
Caliciviridae (Noroviruses, Sapoviruses)
Description of Pathogen
Human caliciviruses belong to the family caliciviridae and are small, round, non-enveloped viruses with a single-stranded, positive-sense RNA genome. The family currently includes the genera Norovirus (previously Norwalk and Norwalk-like viruses), Sapovirus (previously Sapporo and Sapporo-like viruses), Lagovirus, Vesivivirus, and the newly proposed Becovirus and Recovirus. The noroviruses and sapoviruses have recognized roles as causes of acute gastroenteritis in humans. Within each genus, strains are further grouped into genogroups and genotypes or clusters.
The noroviruses are partitioned into genogroups GI to GVII, each further subdivided into genotypes and subgenotypes. Porcine, bovine, and murine noroviruses belong to genogroups II, III, and V, respectively. The majority of human norovirus outbreaks are caused by genogroup II genotype 3 (GII-3) and genogroup II genotype 4 (GII-4) viruses. Human norovirus are thought to be specific to humans and transmission from an animal reservoir has not been described. However, at least three clusters of porcine noroviruses in genogroup II are genetically closely related to the human noroviruses in genogroup II, introducing the potential for zoonotic transmission [154].
The sapoviruses are similarly partitioned into five genogroups (GI to GV) with genotypes in each group. Human sapoviruses belong to genogroups GI, GII, GIV, and GV. GIII contains the porcine strains. Caliciviruses appear to naturally undergo recombination during normal replication of the virus, leading to the emergence of a continuous array of new variants [155].
Human caliciviruses are cannot be grown in standard in vitro cell culture assays and their role as agents of gastrointestinal diseases was under appreciated because clinical tests for the detection of caliciviruses were not commonly available. Molecular methods such as RT-PCR have revealed that caliciviruses are broadly distributed worldwide and are very common causes of epidemic and sporadic gastroenteritis in both children and adults [156]. The study of noroviruses is significantly more advanced than that of sapoviruses. Noroviruses are recognized as the leading cause of epidemic gastroenteritis, often causing large water- or food-borne outbreaks in all ages, while sapoviruses are implicated mainly in pediatric gastroenteritis [157].
Caliciviruses are presumed to replicate primarily in the upper intestinal tract and histopathologic lesions are seen in the jejunum of infected individuals. Symptoms of calicivirus infection are popularly known as “stomach flu” and include vomiting, abdominal cramps, diarrhea, headache, and fever. Symptoms generally last 1–4 days and most people recover completely without treatment. Infants, older adults and people with underlying disease can become severely dehydrated and require medical attention. Asymptomatic infections with shedding of virus are common, and could be the source of some outbreaks [158]. Protective immunity is thought to be short-lived and individuals who have been infected may or may not be immune to reinfection. Studies of immune response are complicated by the ability of the virus to produce naturally occurring variants which are difficult for the immune system to recognize.
Caliciviruses are stable in the environment and can survive freezing, heating to 60 °C, and in chlorinated water up to 10 ppm. They can also survive for several days on many types of surfaces (e.g., door knobs, counter tops, pens, and telephones). Caliciviruses are highly contagious with an estimated infectious dose as low as 10–100 virus particles [159]. These characteristics facilitate rapid spread of caliciviruses, especially in households and institutional settings such as schools, day care centers, hospitals, nursing homes, restaurants, and cruise ships [160]. Calicivirus infections occur year round, although a winter seasonal peak is frequently observed [161].
Transmission is thought to occur mainly through fecal-oral routes. Evidence also suggests that caliciviruses may be transmitted by close exposure to aerosols generated during vomiting episodes in infected individuals. The most frequent cause of norovirus infection appears to be consumption of food or beverages that are contaminated either at their source or by infected food handlers. Uncooked shellfish, particularly oysters, as well as raspberries and precooked foods, such as salad, ham, and sandwiches, are among the common foods that have been responsible for outbreaks [161]. Outbreaks resulting from contamination of municipal water are rare, but water-borne outbreaks associated with community or family water systems have been documented. Sapoviruses have been associated with food-borne outbreaks, but much less frequently than noroviruses. Sapovirus infections are not associated with eating seafood. Nosocomial infections due to caliciviruses are increasingly recognized and may be quite common.
Clinical Utility of Testing
Laboratory diagnosis of calicivirus infection is difficult and clinical diagnosis is often used, especially when other agents of gastroenteritis have been ruled out. Detection of caliciviruses may be important because of their biologic, physicochemical, and epidemiologic features, which present significant challenges for infection control in hospital environments. The regular turnover of patients leaving the hospital and being replaced by new patients provides an opportunity for introduction of the virus from the community and subsequent transmission within the hospital environment.
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