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The main objective of this study was to determine the frequency and patterns of HIV drug resistance-associated mutations among children under 18 months of age born to HIV-1-positive mothers enrolled in the prevention of mother-to-child transmission services in Haiti.Between January 1, 2013 and December 31, 2014, HIV-positive remnant dried blood spots collected from children under 18 months of age for Early Infant Diagnosis at the National Public Health Laboratory were used for HIV-1 genotyping. HIV drug resistance mutations were analyzed using the Stanford Drug Resistance HIVdb program.Of the 3555 dried blood spots collected for Early Infant Diagnosis, 360 (10.1%) were HIV-positive and 355 were available for genotyping. Of these, 304 (85.6%) were successfully genotyped and 217 (71.4%) had ≥1 drug resistance mutation. Mutations conferring resistance to nucleoside reverse transcriptase inhibitor (NRTIs) and non-NRTIs were present in 40.5% (123) and 69.1% (210), respectively. The most frequent mutations were K103N/S (48.0%), M184V (37.5%), G190A/S (15.1%), and Y181C/G/V (14.1%). Predicted drug resistance analysis revealed that 68.8% of the children had high-level resistance to non-NRTIs and 11.5% had intermediate to high-level resistance to abacavir.This study showed high rates of resistance to NRTIs and non-NRTIs among newly HIV-diagnosed children in Haiti, suggesting that in the era of "Option B+" (initiation of lifelong combination antiretroviral therapy to pregnant women with HIV), the majority of children who acquire HIV infection through mother-to-child transmission of HIV have resistant HIV. These results have led the National HIV Program to revise the pediatric guidelines to include protease inhibitors in first-line regimens for all HIV-positive newborns.

Delayed HIV diagnosis in HIV-exposed infants (HEIs) results in missed opportunities for early antiretroviral therapy (ART), causing significant morbidity and mortality. Early infant diagnosis (EID) depends on the availability of accessible and reliable testing services. We explored the acceptability, appropriateness, and feasibility of deploying a targeted community-based point-of-care (POC) EID testing model (i.e., "community POC model") to reach high-risk mother-infant pairs (MIPs) in Lusaka, Zambia.We conducted in-depth interviews with a purposive sample of health care workers, study staff, and caregivers in high-risk MIPs at 6 health facilities included in a larger implementation research study evaluating the community POC model. We defined "high-risk MIPs" as mothers who did not receive antenatal testing or an attended delivery or infants who missed EID testing milestones. Interviews were audio-recorded, translated, and transcribed verbatim in English. Content and thematic analysis were done using NVivo 10 software.Health care workers (n=20) and study staff (n=12) who implemented the community POC model noted that the portability and on-screen prompts of the POC platform made it mobile and easy to use, but maintenance and supply chain management were key to field operations. Respondents also felt that the community POC model reached more infants who had never had EID testing, allowing them to find infants with HIV infection and immediately initiate them on ART. Caregivers (n=22) found the community POC model acceptable, provided that privacy could be ensured because the service was convenient and delivered close to home.We demonstrate the acceptability, appropriateness, and feasibility of implementing the community POC model in Zambia, while identifying potential challenges related to client privacy and platform field operations. The community POC model may represent a promising strategy to further facilitate active HIV case finding and linkage to ART for children with undiagnosed HIV infection in the community.

Early diagnosis of HIV infection in infants and children remains a challenge in resource-limited settings, with approximately half of all HIV-exposed infants receiving virological testing for HIV by the recommended age of 2 months in 2015. To reduce morbidity and mortality among HIV-infected children and close the treatment gap for HIV-infected children, there is an urgent need to evaluate existing programmatic and laboratory practices for early infant diagnosis and introduce strategies to improve identification of HIV-exposed infants and ensure access to systematic, early HIV testing, with early linkage to treatment for HIV-infected infants. This article describes progress made in follow-up of HIV-exposed infants since 2006, including remaining unmet laboratory and programmatic needs, and recommends strategies for improvement, especially those related to the implementation of point-of-care technology for early infant diagnosis.

The recent Ebola virus outbreak in West Africa clearly demonstrated the critical role of laboratory systems and networks in responding to epidemics. Because of the huge challenges in establishing functional laboratories at all tiers of health systems in developing countries, strengthening specimen referral networks is critical. In this review article, we propose a platform strategy for developing specimen referral networks based on 2 models: centralized and decentralized laboratory specimen referral networks. These models have been shown to be effective in patient management in programs in resource-limited settings. Both models lead to reduced turnaround time and retain flexibility for integrating different specimen types. In Haiti, decentralized specimen referral systems resulted in a 182% increase in patients enrolling in human immunodeficiency virus treatment programs within 6 months. In Uganda, cost savings of up to 62% were observed with a centralized model. A platform strategy will create a network effect that will benefit multiple disease programs.

Background. Acute gastroenteritis (AGE) remains a common cause of clinic visits and hospitalizations in the United States, but the etiology is rarely determined. Methods. We performed a prospective, multicenter emergency department–based study of adults with AGE. Subjects were interviewed on presentation and 3–4 weeks later. Serum samples, rectal swab specimens, and/or whole stool specimens were collected at presentation, and serum was collected 3–4 weeks later. Fecal specimens were tested for a comprehensive panel of viral, bacterial, and parasitic pathogens; serum was tested for calicivirus antibodies. Results. Pathogens were detected in 25% of 364 subjects, including 49% who provided a whole stool specimen. The most commonly detected pathogens were norovirus (26%), rotavirus (18%), and Salmonella species (5.3%). Pathogens were detected significantly more often from whole stool samples versus a rectal swab specimen alone. Nine percent of subjects who provided whole stool samples had .1 pathogen identified. Conclusions. Viruses, especially noroviruses, play a major role as agents of severe diarrhea in adults. Further studies to confirm the unexpectedly high prevalence of rotaviruses and to explore the causes of illness among patients from whom a pathogen cannot be determined are needed. Studies of enteric pathogens should require the collection of whole stool samples.

Nearly all human beings, by the time they reach adolescence, are infected with multiple herpesviruses. At any given time, this family of viruses accounts for 35–40 billion human infections worldwide, making herpesviruses among the most prevalent pathogens known to exist. Compared to most other viruses, herpesviruses are also unique in that infection lasts the life of the host. Remarkably, despite their prevalence and persistence, little is known about how these viruses interact with their hosts, especially during the clinically asymptomatic phase of infection referred to as latency. This review explores data in human and animal systems that reveal the ability of latent herpesviruses to modulate the immune response to self and environmental antigens. From the perspective of the host, there are both potentially detrimental and surprisingly beneficial effects of this lifelong interaction. The realization that latent herpesvirus infection modulates immune responses in asymptomatic hosts forces us to reconsider what constitutes a ‘normal’ immune system in a healthy individual.

Objectives. The role of noroviruses in both foodborne and person-to-person outbreaks of acute gastroenteritis (AGE) has been difficult to determine in the U.S. because of lack of routine norovirus testing and of national reporting of person-to-person outbreaks. We conducted a prospective study in one state in which enhanced testing for noroviruses was performed to better understand the relative contribution of all gastroenteric pathogens. Methods. During the two-year period, 2000–2001, we took all fecal specimens from AGE outbreaks reported in Georgia that were negative for bacteria and tested these for norovirus. Results. We investigated 78 AGE outbreaks, from which suitable fecal samples were collected from 57 of them. Norovirus was identified in 25 (44%) outbreaks, bacteria in 20 (35%) outbreaks, and parasites in one (2%) outbreak. Forty-three (75%) of the outbreaks tested were foodborne, of which 17 (40%) were attributable to norovirus and 18 (42%) were attributable to bacteria. Adjusting for incomplete testing, we estimated that 53% of all AGE outbreaks were attributable to norovirus. A total of 2,674 people were reported ill in the 57 outbreaks, and norovirus infections accounted for 1,735 (65%) of these cases. Norovirus outbreaks tended to be larger than bacterial outbreaks, with a median number of 30 vs. 16 cases per outbreak, respectively ( p=0.057). Conclusions. This study provides further evidence that noroviruses are, overall, the most common cause of AGE outbreaks in the U.S. Improved specimen collection, reporting person-to-person outbreaks, and access to molecular assays are needed to further understand the role of these viruses and methods for their prevention.

Introduction Surveillance of HIV drug resistance (HIVDR) is crucial to ensuring the continued success of antiretroviral therapy (ART) programs. With the concern of reduced genotyping sensitivity of HIV on dried blood spots (DBS), DBS for HIVDR surveillance have been limited to ART-naive populations. To investigate if DBS under certain conditions may also be a feasible sample type for HIVDR testing in ART patients, we piloted nationwide surveys for HIVDR among ART patients using DBS in two African countries with rapid scale-up of ART. Methods EDTA-venous blood was collected to prepare DBS from adult and pediatric ART patients receiving treatment during the previous 12–36 months. DBS were stored at ambient temperature for two weeks and then at -80°C until shipment at ambient temperature to the WHO-designated Specialized HIVDR Laboratory at CDC in Atlanta. Viral load (VL) was determined using NucliSENS EasyQ® HIV-1 v2.0 kits; HIVDR genotyping was performed using the ATCC HIV-1 Drug Resistance Genotyping kits. Results DBS were collected from 1,368 and 1,202 ART patients; 244 and 255 these specimens had VL ≥1,000 copies/mL in Kenya and Tanzania, respectively. The overall genotyping rate of those DBS with VL ≥1,000 copies/mL was 93.0% (95% CI: 89.1%-95.6%) in Kenya and 91.8% (87.7%-94.6%) in Tanzania. The turnaround times for the HIVDR surveys from the time of collecting DBS to completing laboratory testing were 6.5 months and 9.3 months for the Kenya and Tanzania surveys, respectively. Conclusions The study demonstrates a favorable outcome of using DBS for nationwide surveillance of HIVDR in ART patients. Our results confirm that DBS collected and stored at ambient temperature for two weeks, and shipped with routine courier services are a reliable sample type for large-scale surveillance of acquired HIVDR.

Without a virus culture system, genetic analysis becomes the principal method to classify norovirus (NoV) strains. Currently, classification of NoV strains beneath the species level has been based on sequences from different regions of the viral genome. As a result, the phylogenetic insights of some virus were not appropriately interpreted, and no consensus has been reached to establish a uniform classification scheme. To provide a consistent and reliable scientific basis for classifying NoVs, we analyzed the amino acid sequences for the major capsid protein of 164 NoV strains by first using an alignment based on the predicted 3D structures. A Bayesian tree was generated, and the maximum likelihood pairwise distances of the aligned sequences were used to evaluate the results from the uncorrected pairwise distance method. Analyses of the pairwise distances demonstrated three clearly resolved peaks, suggesting that NoV strains beneath the species level can be classified at three levels: strain (S), cluster (C), and genogroup (G). The uncorrected pairwise distance ranges for S, C, and G were 0–14.1%, 14.3–43.8%, and 44.9–61.4%, respectively. A scheme with 29 genetic clusters [8 in genogroup 1 (G1), 17 in G2, 2 in G3, and 1 each in G4 and G5] was defined on the basis of the tree topology with the standards provided and was supported by the distance analysis. Of these, five clusters in G2 and one in G1 are newly described. This analysis can serve as the basis for a standardized nomenclature to genetically describe NoV strains.

As more HIV-infected people gain access to antiretroviral therapy (ART), monitoring HIV drug resistance (HIVDR) becomes essential to combat both acquired and transmitted HIVDR. Studies have demonstrated dried blood spots (DBS) are a suitable alternative in HIVDR monitoring using DBS collected on Whatman 903 (W-903). In this study, we sought to evaluate two other commercially available filter papers, Ahlstrom 226 (A-226) and Munktell TFN (M-TFN), for HIVDR genotyping following ambient temperature storage. DBS were prepared from remnant blood specimens collected from 334 ART patients and stored at ambient temperature for a median time of 30 days. HIV-1 viral load was determined using NucliSENS EasyQ® HIV-1 v2.0 RUO test kits prior to genotyping of the protease and reverse transcriptase regions of the HIV-1 pol gene using an in-house assay. Among the DBS tested, 26 specimens had a viral load ≥ 1000 copies/mL in all three types of filter paper and were included in the genotyping analysis. Genotyping efficiencies were similar between DBS collected on W-903 (92.3%), A-226 (88.5%), and M-TFN (92.3%) filter papers (P = 1.00). We identified 50 DR-associated mutations in DBS collected on W-903, 33 in DBS collected on A-226, and 48 in DBS collected on M-TFN, resulting in mutation detection sensitivities of 66.0% for A-226 and 88.0% for M-TFN when compared to W-903. Our data indicate that differences among filter papers may exist at this storage condition and warrant further studies evaluating filter paper type for HIVDR monitoring.

We documented transmission by food and person-to-person contact; persistence of virus despite sanitization onboard, including introductions of new strains; and seeding of an outbreak on land., We describe an investigation of a norovirus gastroenteritis outbreak aboard a cruise ship affecting 6 consecutive cruises and the use of sequence analysis to determine modes of virus transmission. Noroviruses (NoV), are the most common cause of infectious acute gastroenteritis and are transmitted feco-orally through food and water, directly from person to person and by environmental contamination (1). These viruses are often responsible for protracted outbreaks in closed settings, such as cruise ships, nursing homes, and hospitals (2,3).

Analysis of foodborne outbreaks shows how advances in viral diagnostics are clarifying the causes of foodborne outbreaks and determining the high impact of norovirus infections., Efforts to prevent foodborne illness target bacterial pathogens, yet noroviruses (NoV) are suspected to be the most common cause of gastroenteritis. New molecular assays allow for better estimation of the role of NoV in foodborne illness. We analyzed 8,271 foodborne outbreaks reported to the Centers for Disease Control and Prevention from 1991 to 2000 and additional data from 6 states. The proportion of NoV-confirmed outbreaks increased from 1% in 1991 to 12% in 2000. However, from 1998 to 2000, 76% of NoV outbreaks were reported by only 11 states. In 2000, an estimated 50% of foodborne outbreaks in 6 states were attributable to NoV. NoV outbreaks were larger than bacterial outbreaks (median persons affected: 25 versus 15), and 10% of affected persons sought medical care; 1% were hospitalized. More widespread use of molecular assays will permit better estimates of the role of NoV illness and help direct efforts to control foodborne illness.

An infant with diarrhea attended a community playgroup. In the subsequent 48 hours, 6 of the 7 mothers and children reported gastroenteritis; fecal specimens from 5 persons tested positive for norovirus, with identical sequences. No breach of hygiene or contact with fecal matter was identified. Excluding the child with gastroenteritis from the playgroup could have prevented this outbreak.

Nearly all human beings, by the time they reach adolescence, are infected with multiple herpesviruses. At any given time, this family of viruses accounts for 35–40 billion human infections worldwide, making herpesviruses among the most prevalent pathogens known to exist. Compared to most other viruses, herpesviruses are also unique in that infection lasts the life of the host. Remarkably, despite their prevalence and persistence, little is known about how these viruses interact with their hosts, especially during the clinically asymptomatic phase of infection referred to as latency. This review explores data in human and animal systems that reveal the ability of latent herpesviruses to modulate the immune response to self and environmental antigens. From the perspective of the host, there are both potentially detrimental and surprisingly beneficial effects of this lifelong interaction. The realization that latent herpesvirus infection modulates immune responses in asymptomatic hosts forces us to reconsider what constitutes a ‘normal’ immune system in a healthy individual.

The gammaherpesviruses, including Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), establish latency in memory B lymphocytes and promote lymphoproliferative disease in immunocompromised individuals. The precise immune mechanisms that prevent gammaherpesvirus reactivation and tumorigenesis are poorly defined. Murine gammaherpesvirus 68 (MHV68) is closely related to EBV and KSHV, and type I (alpha/beta) interferons (IFNαβ) regulate MHV68 reactivation from both B cells and macrophages by unknown mechanisms. Here we demonstrate that IFNβ is highly upregulated during latent infection, in the absence of detectable MHV68 replication. We identify an interferon-stimulated response element (ISRE) in the MHV68 M2 gene promoter that is bound by the IFNαβ-induced transcriptional repressor IRF2 during latency in vivo. The M2 protein regulates B cell signaling to promote establishment of latency and reactivation. Virus lacking the M2 ISRE (ISREΔ) overexpresses M2 mRNA and displays uncontrolled acute replication in vivo, higher latent viral load, and aberrantly high reactivation from latency. These phenotypes of the ISREΔ mutant are B-cell-specific, require IRF2, and correlate with a significant increase in virulence in a model of acute viral pneumonia. We therefore identify a mechanism by which a gammaherpesvirus subverts host IFNαβ signaling in a surprisingly cooperative manner, to directly repress viral replication and reactivation and enforce latency, thereby minimizing acute host disease. Since we find ISREs 5′ to the major lymphocyte latency genes of multiple rodent, primate, and human gammaherpesviruses, we propose that cooperative subversion of IFNαβ-induced IRFs to promote latent infection is an ancient strategy that ensures a stable, minimally-pathogenic virus-host relationship., Author Summary Herpesviruses establish life-long infection in a non-replicating state termed latency. During immune compromise, herpesviruses can reactivate and cause severe disease, including cancer. We investigated mechanisms by which interferons alpha/beta (IFNαβ), a family of antiviral immune genes, inhibit reactivation of murine gammaherpesvirus 68 (MHV68). MHV68 is related to Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, human gammaherpesviruses associated with multiple cancers. We made the surprising discovery that during latency, MHV68 cooperates with IFNαβ to inhibit its own replication. Specifically, a viral gene required for reactivation has evolved to be directly repressed by an IFNαβ-induced transcription factor, IRF2. Once virus replication has triggered sufficient IFNαβ production, expression of this viral gene is reduced and reactivation efficiency decreases. This strategy safeguards the health of the host, since a mutant virus that cannot respond to IRF2 replicates uncontrollably and is more virulent. Viral sensing of IFNαβ is also potentially subversive, since it allows MHV68 to detect periods of localized immune quiescence during which it can reactivate and spread to a new host. Thus, we highlight a novel path of virus-host coevolution, toward cooperative subversion of the antiviral immune response. These observations may illuminate new targets for drugs to inhibit herpesvirus reactivation or eliminate herpesvirus-associated tumors.

Between July 2000 and June 2004, fecal specimens from 270 outbreaks of acute gastroenteritis were sent to the Centers for Disease Control and Prevention by local or state health departments for calicivirus testing. Of the 226 outbreaks that met the criteria for inclusion in the present study, caliciviruses were detected in 184 (81%) by reverse-transcription polymerase chain reaction and nucleotide sequencing. Nursing homes, retirement centers, and hospitals were the most frequently reported settings, and person-to-person contact was the most common mode of transmission, followed by foodborne spread. Overall, genogroup II norovirus (NoV) strains were the most abundant (79%), followed by genogroup I NoV strains (19%) and sapovirus (2%). Nucleotide-sequence analysis indicated a great diversity of NoV strains and implicated the emergence of one particular sequence variant in outbreaks occurring between July 2002 and June 2003. The public health impact of caliciviruses will not be fully appreciated, nor will interventions be completely evaluated, until methods to detect these viruses are more routinely used.

In 2002, a sharp increase in outbreaks of norovirus-associated illness, both on cruise ships and on land, encouraged us to examine the molecular epidemiology of detected noroviruses, to identify a common strain or source. Of 14 laboratory-confirmed outbreaks on cruise ships, 12 (86%) were attributed to caliciviruses; among these 12, outbreak characteristics included continuation on successive cruises in 6 (50%), multiple modes of transmission in 7 (58%), and high (>10%) attack rates in 7 (58%). Eleven of the 12 calicivirus outbreaks were attributed to noroviruses, 7 (64%) of which were attributed to a previously unreported lineage, provisionally named "the Farmington Hills strain." From May 2002 to December 2002, 10 (45%) of 22 land-based outbreaks also were attributed to this strain. Nucleotide-sequence analysis provided insights into norovirus transmission, by documenting links among outbreaks, the introduction of strains onto ships, and viral persistence on board (despite cleaning). Control measures for outbreaks should address all routes of transmission. Better outbreak surveillance and collection of data on sequences will help to monitor norovirus strains and to identify common sources.

In February 2001, episodes of acute gastroenteritis were reported to the Wyoming Department of Health from persons who had recently vacationed at a snowmobile lodge in Wyoming. A retrospective cohort study found a significant association between water consumption and illness, and testing identified Norwalk-like virus (NLV) in 8 of 13 stool samples and 1 well. Nucleotide sequences from the positive well-water specimen and 6 of the positive stool samples were identical. This multistrain NLV outbreak investigation illustrates the importance of NLV as a cause of waterborne illness and should encourage monitoring for NLVs in drinking water.

To better assess the risk for transmission of the severe acute respiratory syndrome–associated coronavirus (SARS-CoV), we obtained serial specimens and clinical and exposure data from seven confirmed U.S. SARS patients and their 10 household contacts. SARS-CoV was detected in a day-14 sputum specimen from one case-patient and in five stool specimens from two case-patients. In one case-patient, SARS-CoV persisted in stool for at least 26 days after symptom onset. The highest amounts of virus were in the day-14 sputum sample and a day-14 stool sample. Residual respiratory symptoms were still present in recovered SARS case-patients 2 months after illness onset. Possible transmission of SARS-CoV occurred in one household contact, but this person had also traveled to a SARS-affected area. The data suggest that SARS-CoV is not always transmitted efficiently. Laboratory diagnosis of SARS-CoV infection is difficult; thus, sputum and stool specimens should be included in the diagnostic work-up for SARS-CoV infection.