Social Networks, the COVID-19 Pandemic, and Emerging Adults' Mental Health: Resiliency Through Social Bonding and Cohesion.

Objectives. To assess how personal social network characteristics moderated mental health declines during the COVID-19 pandemic in emerging adults compared with other age groups. Methods. The Person to Person Health Interview Study, a representative, probability-based cohort study (n = 2485) in Indiana, collected data through face-to-face (baseline) and phone (follow-up) interviews before and during the pandemic. We used survey-weighted growth curve models to examine network effects on computer-adaptive testing measures of depression and anxiety severity. Results. Respondents reported significantly increased depression and anxiety in 2021, which returned almost to baseline levels for most age groups by 2022 (P < .001). Stronger ties to others and more interconnected ties were significantly associated with lower depression (B = -0.112 [P < .05]; B = -0.086 [P < .001]) and anxiety (B = -0.101 [P < .05]; B = -0.063 [P < .01]) severity across the pandemic. Interaction models revealed disproportionate protective effects of network characteristics on depression (B = -0.456 [P < .001]; B = -0.268 [P < .001]) and anxiety (B = -0.388 [P < .001]; B = -0.284 [P < .001]) for emerging adults. Conclusions. Cohesive and affectively strong personal networks promote resiliency to common mental health challenges during periods of crisis, particularly for emerging adults whose social roles and relationships were disrupted during a critical period of development. (Am J Public Health. 2024;114(S3):S258-S267. https://doi.org/10.2105/AJPH.2023.307426).

Substrate-mediated regulation of the arginine transporter of Toxoplasma gondii.

Intracellular parasites, such as the apicomplexan Toxoplasma gondii, are adept at scavenging nutrients from their host. However, there is little understanding of how parasites sense and respond to the changing nutrient environments they encounter during an infection. TgApiAT1, a member of the apicomplexan ApiAT family of amino acid transporters, is the major uptake route for the essential amino acid L-arginine (Arg) in T. gondii. Here, we show that the abundance of TgApiAT1, and hence the rate of uptake of Arg, is regulated by the availability of Arg in the parasite's external environment, increasing in response to decreased [Arg]. Using a luciferase-based 'biosensor' strain of T. gondii, we demonstrate that the expression of TgApiAT1 varies between different organs within the host, indicating that parasites are able to modulate TgApiAT1-dependent uptake of Arg as they encounter different nutrient environments in vivo. Finally, we show that Arg-dependent regulation of TgApiAT1 expression is post-transcriptional, mediated by an upstream open reading frame (uORF) in the TgApiAT1 transcript, and we provide evidence that the peptide encoded by this uORF is critical for mediating regulation. Together, our data reveal the mechanism by which an apicomplexan parasite responds to changes in the availability of a key nutrient.

Perceived discrimination and mental health among African American and Caribbean Black adolescents: ethnic differences in processes and effects.

Objectives: Although the relationship between perceived discrimination and mental health among Black youth is well-documented, the mechanisms underlying this association remain unclear. Drawing from the stress process model, this study examines two psychosocial mediators - self-esteem and self-efficacy - in the association between perceived discrimination and mental health among African American and Caribbean Black adolescents. This research addresses three primary research questions: First, how is perceived discrimination associated with mental health? Second, to what extent do self-esteem and self-efficacy mediate the relationship between perceived discrimination and mental health? Finally, do these relationships and processes differ between African American and Caribbean Black adolescents?Design: Data for these analyses come from the National Survey of American Life - Adolescent Supplement. We use generalized structural equation modeling to examine relationships among perceived discrimination, psychosocial resources, and mental health.Results: For both African American and Caribbean Black adolescents, more frequent perceptions of discriminatory events were associated with greater depressive symptoms and a higher likelihood of having an anxiety disorder diagnosis. However, the association between perceived discrimination and depressive symptoms was significantly stronger for Caribbean Black adolescents. Generally, self-esteem and self-efficacy were found to be significant mediators in the association between perceived discrimination and mental health, although mechanisms varied between African American and Caribbean Black youth.Conclusion: African American and Caribbean Black adolescents are highly susceptible to experiences of discrimination that negatively affect their mental health. Heterogeneity among Black youth populations must be considered when developing interventions to reduce exposure to and the effects of discrimination among these adolescents.

Examining the Relationship between Discrimination and Prescription Drug Misuse: Findings from a National Survey of Black Americans.

Background: Research shows that substance use may be a way individuals cope with psychosocial stressors. Less is known about whether discrimination contributes to prescription drug misuse. Methods: Using a national sample of Black Americans, we examined whether two psychosocial stressors (i.e., everyday and lifetime major discrimination) were associated with lifetime prescription drug misuse (i.e., opioids, tranquilizers, sedatives, or stimulants). Results: Our logistic regression models separately examining the influence of everyday and major discrimination controlling for relevant demographic, health, and other drug use variables showed that only everyday discrimination was associated with higher odds of prescription drug misuse. In the model simultaneously considering both types of discrimination, only unit increases in everyday discrimination were associated with higher odds of prescription drug misuse. Conclusions: Encounters with everyday discrimination may be an important psychosocial stressor linked to prescription drug misuse in Black adults and possibly other racial-ethnic minorities. Intervention strategies aiming to reduce prescription drug misuse should consider developing ways to curb the negative health-related consequences of discriminatory experiences. Strategies to combat discrimination-related prescription drug misuse and limitations of this study are discussed.

Association of length of hospital stay with delay to surgical fixation of hip fracture.

BACKGROUND: Previous research has shown increases in length of stay (LOS), morbidity and mortality when the standard for surgical fixation of hip fracture of 48 hours is not met. However, few investigators have analyzed LOS as a primary outcome, and most used time of diagnosis as opposed to time of fracture as the reference point. We examined the effect of time to surgical fixation of hip fracture, measured from time of fracture, on length of hospital stay; the secondary outcome was average 1-year mortality. METHODS: We conducted a retrospective cohort study of patients presenting to 1 of 2 tertiary care centres in St. John's, Newfoundland and Labrador, Canada, with a hip fracture from Jan. 1, 2014, to Dec. 31, 2018. We analyzed 3 groups based on timing of surgical fixation after fracture: less than 24 hours (group 1), 24-48 hours (group 2) and more than 48 hours (group 3). We assessed statistical significance using 1-way analysis of variance. RESULTS: Of the 692 patients included in the study, 212 (30.6%) were in group 1, 360 (52.0%) in group 2 and 120 (17.3%) in group 3. A delay to surgical fixation exceeding 48 hours was associated with a significantly longer LOS, by an average of 2.9 and 2.8 days compared to groups 1 and 2, respectively (p = 0.04); there was no significant difference in LOS between groups 1 and 2. A significant difference in average 1-year mortality was observed between groups 1 (11%) and 3 (26%) (p = 0.004), and groups 2 (13%) and 3 (p = 0.009). CONCLUSION: Surgical fixation beyond 48 hours after hip fracture resulted in significantly increased LOS and 1-year mortality. Further research should be conducted to evaluate reasons for delays to surgery and the effects of these delays on time to surgical fixation as measured from time of fracture.

Black-White disparities in women's physical health: The role of socioeconomic status and racism-related stressors.

Black women have elevated rates of multiple physical illnesses and conditions when compared to White women - disparities that are only partially explained by socioeconomic status (SES). Consequently, scholars have called for renewed attention to the significance of racism-related stress in explaining Black-White disparities in women's physical health. Drawing on the biopsychosocial model of racism as a stressor and the intersectionality perspective, this study examines the extent to which SES and racism-related stressors - i.e., discrimination, criminalization, and adverse neighborhood conditions - account for disparities in self-rated physical health and chronic health conditions between Black and White women. Results indicate that Black women have lower SES and report greater exposure to racism-related stressors across all domains. Moreover, I find that SES and racism-related stressors jointly account for more than 90% of the Black-White disparity in women's self-rated physical health and almost 50% of the Black-White disparity in chronic health conditions. Theoretical and policy implications of these findings are discussed.

Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes.

BACKGROUND: The apicomplexan parasite Toxoplasma gondii is cosmopolitan in nature, largely as a result of its highly flexible life cycle. Felids are its only definitive hosts and a wide range of mammals and birds serve as intermediate hosts. The latent bradyzoite stage is orally infectious in all warm-blooded vertebrates and establishes chronic, transmissible infections. When bradyzoites are ingested by felids, they transform into merozoites in enterocytes and expand asexually as part of their coccidian life cycle. In all other intermediate hosts, however, bradyzoites differentiate exclusively to tachyzoites, and disseminate extraintestinally to many cell types. Both merozoites and tachyzoites undergo rapid asexual population expansion, yet possess different effector fates with respect to the cells and tissues they develop in and the subsequent stages they differentiate into. RESULTS: To determine whether merozoites utilize distinct suites of genes to attach, invade, and replicate within feline enterocytes, we performed comparative transcriptional profiling on purified tachyzoites and merozoites. We used high-throughput RNA-Seq to compare the merozoite and tachyzoite transcriptomes. 8323 genes were annotated with sequence reads across the two asexually replicating stages of the parasite life cycle. Metabolism was similar between the two replicating stages. However, significant stage-specific expression differences were measured, with 312 transcripts exclusive to merozoites versus 453 exclusive to tachyzoites. Genes coding for 177 predicted secreted proteins and 64 membrane- associated proteins were annotated as merozoite-specific. The vast majority of known dense-granule (GRA), microneme (MIC), and rhoptry (ROP) genes were not expressed in merozoites. In contrast, a large set of surface proteins (SRS) was expressed exclusively in merozoites. CONCLUSIONS: The distinct expression profiles of merozoites and tachyzoites reveal significant additional complexity within the T. gondii life cycle, demonstrating that merozoites are distinct asexual dividing stages which are uniquely adapted to their niche and biological purpose.

RNA Seq analysis of the Eimeria tenella gametocyte transcriptome reveals clues about the molecular basis for sexual reproduction and oocyst biogenesis.

BACKGROUND: The protozoan Eimeria tenella is a common parasite of chickens, causing avian coccidiosis, a disease of on-going concern to agricultural industries. The high prevalence of E. tenella can be attributed to the resilient oocyst stage, which is transmitted between hosts in the environment. As in related Coccidia, development of the eimerian oocyst appears to be dependent on completion of the parasite's sexual cycle. RNA Seq transcriptome profiling offers insights into the mechanisms governing the biology of E. tenella sexual stages (gametocytes) and the potential to identify targets for blocking parasite transmission. RESULTS: Comparisons between the sequenced transcriptomes of E. tenella gametocytes and two asexual developmental stages, merozoites and sporozoites, revealed upregulated gametocyte transcription of 863 genes. Many of these genes code for proteins involved in coccidian sexual biology, such as oocyst wall biosynthesis and fertilisation, and some of these were characterised in more depth. Thus, macrogametocyte-specific expression and localisation was confirmed for two proteins destined for incorporation into the oocyst wall, as well as for a subtilisin protease and an oxidoreductase. Homologues of an oocyst wall protein and oxidoreductase were found in the related coccidian, Toxoplasma gondii, and shown to be macrogametocyte-specific. In addition, a microgametocyte gamete fusion protein, EtHAP2, was discovered. CONCLUSIONS: The need for novel vaccine candidates capable of controlling coccidiosis is rising and this panel of gametocyte targets represents an invaluable resource for development of future strategies to interrupt parasite transmission, not just in Eimeria but in other Coccidia, including Toxoplasma, where transmission blocking is a relatively unexplored strategy.

Strategies for anti-coccidial prophylaxis.

Coccidiosis, a serious disease resulting from infection with parasitic protozoa of the genus Eimeria, causes significant economic losses to the poultry industry, where intensive rearing facilitates transmission of infectious oocysts via the fecal/oral route. Current control relies primarily on prophylactic drugs in feed but, whilst cost effective, the rise of drug resistance and public demands for residue-free meat has encouraged development of alternative control strategies. Chickens that recover from infection with Eimeria develop solid immunity that is directed against the early asexual stages of the parasite life cycle. This has allowed development of a number of vaccines that utilize deliberate infection with controlled doses of virulent oocysts or reproductively attenuated lines of Eimeria. The latter are immunogenic but non-pathogenic. The realization that both prophylactic drugs and attenuated vaccines control but do not eradicate infection with Eimeria encouraged development of a vaccine based upon maternal immunity. Laying hens exposed to Eimeria are able to transfer protective antibodies to hatchlings via egg yolks and these antibodies have been used to identify parasite proteins that are conserved across the genus. When delivered maternally, these provide an economical means of preventing coccidiosis, offering immediate protection to newly hatched chicks.

Residential segregation and Black-White differences in physical and mental health: Evidence of a health paradox?

Ample research finds that residential segregation is detrimental to Black Americans' physical health and exacerbates Black-White physical health disparities. However, less is known about how residential segregation may influence Black Americans' mental health and Black-White differences in mental health. Drawing on U.S. census data and a state representative study of Indiana residents (N = 2,685), I examine associations between residential segregation and multiple dimensions of physical and mental health. Consistent with past research, I find that residential segregation has an adverse association with physical health among Black respondents. In contrast, I find residential segregation to have a salubrious association with Black respondents' mental health, producing a Black mental health advantage at higher levels of segregation. I conclude by discussing the implications of these findings for research on residential segregation and health and the Black-White mental health paradox.

Inter-rater and intrarater reliability of superior labrum anterior to posterior lesion classification using magnetic resonance arthrography.

Background: The glenoid labrum is a fibrocartilaginous ring that affixes the joint capsule and ligaments of the glenohumeral joint. Superior labrum anterior to posterior (SLAP) lesions are a subset of injuries that affect the superior glenoid labrum, most common in laborers and overhead-throwing athletes. In 1990, Snyder et al classified SLAP lesions into one of four types. Later, Maffet et al expanded this scale to include three additional subclassifications. At present, arthroscopy is considered the gold standard for SLAP tear diagnosis. Classification under arthroscopy has demonstrated low to moderate inter-rater reliability. Magnetic resonance arthrography (MRa) is an alternate, less invasive test for diagnosing SLAP lesions. The reliability of MRa for diagnosing slap tears is uncertain. Methods: Magnetic resonance arthrograms were identified using the Picture Archiving and Communication System (PACS). In total, 273 shoulder arthrograms were reviewed, and 20 were selected with the desired pathology. Three orthopedic surgeons and three musculoskeletal radiologists were asked to classify the SLAP lesions into one of seven categories (Snyder & Maffet classification systems). Data was collected on two separate occasions at an interval of at least two months. Inter-rater and intrarater reliability were calculated using Fleiss Kappa and Cohen's Kappa, respectively. Results: Between all raters, there was poor inter-rater reliability for each round of data collection (κ = .177, κ = .124 for rounds 1 and 2, respectively). Between orthopedic surgeons, there were poor levels of agreement (κ = -.056, κ = .114), whereas, between radiologists, there was fair to moderate agreement (κ = 0.479, κ = 0.340). Within orthopedic raters, κ values ranged from -0.059 to 0.125, indicating, at best, poor intrarater reliability. Within radiologists, κ values ranged from 0.545 to 0.553, indicating moderate agreement within raters. The analysis determined that none of the orthopedic values for inter or intrarater reliability could be deemed statistically different from zero. Conclusion: Overall, classification using MRa resulted in significant disagreement between and within raters. Trained radiologists demonstrated higher overall levels of agreement than orthopedic surgeons. In summary, when using MRa to assess SLAP lesions, Snyder and Maffet classification demonstrates poor reliability by orthopedic surgeons and moderate reliability when used by musculoskeletal radiologists.

The role of the P2X7 receptor in infectious diseases.

ATP is an extracellular signal for the immune system, particularly during an inflammatory response. It is sensed by the P2X7 receptor, the expression of which is upregulated by pro-inflammatory cytokines. Activation of the P2X7 receptor opens a cation-specific channel that alters the ionic environment of the cell, activating several pathways, including (i) the inflammasome, leading to production of IL-1ß and IL-18; (ii) the stress-activated protein kinase pathway, resulting in apoptosis; (iii) the mitogen-activated protein kinase pathway, leading to generation of reactive oxygen and nitrogen intermediates; and (iv) phospholipase D, stimulating phagosome-lysosome fusion. The P2X7 receptor can initiate host mechanisms to remove pathogens, most particularly those that parasitise macrophages. At the same time, the P2X7 receptor may be subverted by pathogens to modulate host responses. Moreover, recent genetic studies have demonstrated significant associations between susceptibility or resistance to parasites and bacteria, and loss-of-function or gain-of-function polymorphisms in the P2X7 receptor, underscoring its importance in infectious disease.

Sex and Eimeria: a molecular perspective.

Eimeria is a common genus of apicomplexan parasites that infect diverse vertebrates, most notably poultry, causing serious disease and economic loss. Like all apicomplexans, eimerians have a complex life cycle characterized by asexual divisions that amplify the parasite population in preparation for sexual reproduction. This can be divided into three events: gametocytogenesis, producing gametocytes from merozoites; gametogenesis, producing microgametes and macrogametes from gametocytes; and fertilization of macrogametes by microgametes, producing diploid zygotes with ensuing meiosis completing the sexual phase. Sexual development in Eimeria depends on the differential expression of stage-specific genes, rather than presence or absence of sex chromosomes. Thus, it involves the generation of specific structures and, implicitly, storage of proteins and regulation of protein expression in macrogametes, in preparation for fertilization. In Eimeria, the formation of a unique, resilient structure, the oocyst wall, is essential for completion of the sexual phase and parasite transmission. In this review, we piece together the molecular events that underpin sexual reproduction in Eimeria and use additional details from analogous events in Plasmodium to fill current knowledge gaps. The mechanisms governing sexual stage formation and subsequent fertilization may represent targets for counteracting parasite transmission.

Classification of congenital anomalies of the hand and upper limb: development and assessment of a new system.

The Oberg, Manske, and Tonkin (OMT) classification of congenital hand and upper limb anomalies was proposed in 2010 as a replacement for the Swanson International Federation of Societies for Surgery of the Hand classification system, which has been the accepted system of classification for the international surgical community since 1976. The OMT system separates malformations from deformations and dysplasias. Malformations are subdivided according to the axis of formation and differentiation that is primarily affected and whether the anomalies involve the whole limb or the hand plate. This review outlines the development of classification systems and explores the difficulty of incorporating our current knowledge of limb embryogenesis at a molecular level into current systems. An assessment of the efficacy of the OMT classification demonstrates acceptable inter- and intraobserver reliability. A prospective review of 101 patients confirms that all diagnoses could be classified within the OMT system. Consensus expert opinion allowed classification of those conditions for which there is not a clear understanding of the mechanism of dysmorphology. A refined and expanded OMT classification is presented.

Pandemic Precarity: COVID-19's Impact on Mexican and Central American Immigrant Families.

Objective: The study examines the association of gender, parenthood, and marriage with reports of perceived pandemic precarity among Mexican and Central American immigrants during the COVID-19 pandemic (Fall 2020) to understand predictors of vulnerability in periods of crisis. Background: Latinos/as, immigrants, parents, and women have faced significant challenges during the COVID-19 pandemic. Family structure, along with social expectations for gender (i.e., self-sacrificing femininity for women and hegemonic masculinity for men), parenthood, and marriage may explain perceptions of pandemic precarity - defined as the material deprivation and economic anxiety resulting from the COVID-19 pandemic. Method: This study used data from the Hispanic COVID-19 Rapid Response Study (n=400), a follow-up of the VidaSana Study of Mexican and Central American immigrants, to examine how family structure is associated with pandemic precarity (i.e., food, housing, and economic insecurity). Using linear regression models, average marginal effects (AMEs), and tests for group differences we investigate the independent and interactive effects of gender, parenthood, and marriage on pandemic precarity. Results: Men and parents reported the highest pandemic precarity. Fathers reported higher pandemic precarity than mothers. For men, marriage is associated with greater precarity, and for women, marriage is associated with less precarity, yet marriage increased precarity for those without children. Conclusion: We discuss the importance and implications of examining gender along with family structure to understand how immigrant families were faring in response to the pandemic.

Stage-specific expression of protease genes in the apicomplexan parasite, Eimeria tenella.

BACKGROUND: Proteases regulate pathogenesis in apicomplexan parasites but investigations of proteases have been largely confined to the asexual stages of Plasmodium falciparum and Toxoplasma gondii. Thus, little is known about proteases in other Apicomplexa, particularly in the sexual stages. We screened the Eimeria tenella genome database for proteases, classified these into families and determined their stage specific expression. RESULTS: Over forty protease genes were identified in the E. tenella genome. These were distributed across aspartic (three genes), cysteine (sixteen), metallo (fourteen) and serine (twelve) proteases. Expression of at least fifteen protease genes was upregulated in merozoites including homologs of genes known to be important in host cell invasion, remodelling and egress in P. falciparum and/or T. gondii. Thirteen protease genes were specifically expressed or upregulated in gametocytes; five of these were in two families of serine proteases (S1 and S8) that are over-represented in the coccidian parasites, E. tenella and T. gondii, distinctive within the Apicomplexa because of their hard-walled oocysts. Serine protease inhibitors prevented processing of EtGAM56, a protein from E. tenella gametocytes that gives rise to tyrosine-rich peptides that are incorporated into the oocyst wall. CONCLUSION: Eimeria tenella possesses a large number of protease genes. Expression of many of these genes is upregulated in asexual stages. However, expression of almost one-third of protease genes is upregulated in, or confined to gametocytes; some of these appear to be unique to the Coccidia and may play key roles in the formation of the oocyst wall, a defining feature of this group of parasites.

P2X7 receptor-mediated killing of an intracellular parasite, Toxoplasma gondii, by human and murine macrophages.

The P2X7R is highly expressed on the macrophage cell surface, and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this study, we show that macrophages from people with the 1513C (rs3751143, NM_002562.4:c.1487A>C) loss-of-function P2X7R single nucleotide polymorphism are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X7R knockout mice (P2X7R-/-) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production.

Evidence-based indications for distraction ankle arthroplasty.

BACKGROUND: The purpose of this study was to review the literature to provide a comprehensive description of the Level of Evidence (LOE) available to support the operative technique of distraction ankle arthroplasty for the current generally accepted indications and make a grade of recommendation for each. METHODS: A comprehensive review of the literature was performed (November 2010 to January 2011) using the PubMed database. The abstracts from these searches were reviewed to isolate literature that described therapeutic studies investigating the results of distraction ankle arthroplasty. All articles were reviewed and assigned a classification (I-V) of Level of Evidence. An analysis of the literature reviewed was used to assign a Grade of Recommendation for each current generally accepted indication for distraction ankle arthroplasty. RESULTS: There is insufficient evidence based literature (Grade I) to support or refute the procedure for either: post-traumatic ankle arthritis, arthritis associated with ligamentous instability, primary degenerative joint disease, chondrolysis, deformity associated with arthritis, osteochondral defects and congenital ankle abnormalities. CONCLUSION: Inadequate evidence based literature exists to support or refute all currently accepted indications for distraction ankle arthroplasty and further high quality, scientific studies are needed upgrade to these recommendations.

Correction of varus heel pad in patients with Syme's amputations.

Syme's amputations can provide a reliable alternative to more proximal amputations, but they are not without their occasional complication. Varus heel pad migration has been well documented as a complication following Syme's amputations. We describe a technique of resection of soft tissue and bone combined with anchoring of the lateral band of the plantar fascia in order to treat patients with the complication of varus heel pad migration.

Laboratory diagnostics for human Leishmania infections: a polymerase chain reaction-focussed review of detection and identification methods.

Leishmania infections span a range of clinical syndromes and impact humans from many geographic foci, but primarily the world's poorest regions. Transmitted by the bite of a female sand fly, Leishmania infections are increasing with human movement (due to international travel and war) as well as with shifts in vector habitat (due to climate change). Accurate diagnosis of the 20 or so species of Leishmania that infect humans can lead to the successful treatment of infections and, importantly, their prevention through modelling and intervention programs. A multitude of laboratory techniques for the detection of Leishmania have been developed over the past few decades, and although many have drawbacks, several of them show promise, particularly molecular methods like polymerase chain reaction. This review provides an overview of the methods available to diagnostic laboratories, from traditional techniques to the now-preferred molecular techniques, with an emphasis on polymerase chain reaction-based detection and typing methods.