State Public Health Laboratories Research Articles

Improved Sensitivity of a Commercial Reverse Transcription-PCR Test for Subtyping of the 2009 H1N1 Influenza A Virus

Rapid detection of influenza A virus and determination of its subtype are important globally for public health surveillance and locally for the selection of antiviral treatment (1). We have used the ProFlu+ assay to test respiratory samples from children for influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) and the ProFlu ST test to subtype influenza A virus (Gen-Probe Prodesse, Inc., Waukesha, WI). ProFlu ST is a multiplex assay that detects the 2009 H1N1 virus by targeting the nucleoprotein gene and also detects the seasonal H1N1 and seasonal H3N2 viruses by targeting the hemagglutinin gene. Here we report that the new subtyping test from Prodesse, ProFAST, is more sensitive than ProFlu ST for the 2009 H1N1 virus. Between 2 October 2009 and 10 May 2010, we tested respiratory specimens using ProFlu+, and if influenza A virus was detected, subtyping was performed using the ProFlu ST test. RNA was extracted using the Qiagen QIAamp MinElute virus spin kit with the QiaCube system (Qiagen, Valencia, CA). The Prodesse ProFlu+ assay was performed according to the manufacturer's instructions using the Cepheid SmartCycler system (Cepheid, Sunnyvale, VA). Of the 2,249 specimens tested with ProFlu+, 269 (12.0%) were positive for influenza A virus. Of these, 210 (78.1%) were subtyped as the 2009 H1N1 virus and the remaining 60 samples (21.9%) were negative with the ProFlu ST test. Nearly all influenza A virus circulating in the United States during the 2009-2010 influenza season was 2009 H1N1 (FluView; www.cdc.gov/flu), so it was likely that the negative specimens from the ProFlu ST test were of this subtype. As such, the ProFlu+ results compared to the ProFlu ST data suggested an inadequate sensitivity in the subtyping assay. Recently, Prodesse introduced a new influenza A virus subtyping kit, ProFAST, to replace the ProFlu ST test. ProFAST targets the hemagglutinin gene for all three subtypes of influenza A virus. We used nucleic acid samples from selected specimens collected in previous influenza seasons and stored at −80°C to determine the performance of the ProFAST test. These samples had previously been tested by both the ProFlu+ and ProFlu ST tests. All of the specimens that were positive for 2009 H1N1, seasonal H1N1, or H3N2 viruses in the ProFlu ST assay were determined to be the same subtype in the ProFAST assay (Table). We also used the ProFAST assay to test 24 specimens from the 2009-2010 influenza season which were positive for influenza A virus in the ProFlu+ test but which did not give a detectable subtype in the ProFlu ST test. These same specimens were tested by the state public health laboratory using PCR reagents and protocols provided by the CDC (2). All 24 of these samples were positive for 2009 H1N1 using the CDC panels. More than half (14/24) were positive for 2009 H1N1 in the ProFAST assay (Table 1). There was no clear difference between the specimen types of the 27 samples that were subtyped with the ProFlu ST test (16 nasopharyngeal swabs, 6 nasopharyngeal aspirates, 4 nasopharyngeal washes, and 1 sputum sample) and the 24 that were not (16 nasopharyngeal swabs, 4 nasopharyngeal aspirates, 2 sputa, 1 nasopharyngeal wash, and 1 endotracheal tube aspirate sample). Our data support implementation of the new ProFAST subtyping test in our algorithm to significantly reduce the number of influenza virus specimens that are refractory to subtyping. Table 1. Results of subtyping of selected specimens that were positive for influenza A virus in the ProFlu+ test

The Michigan BioTrust for Health: Using Dried Bloodspots for Research to Benefit the Community While Respecting the Individual

The Michigan Department of Community Health (MDCH) stores almost 4 million dried blood spot specimens (DBS) in the Michigan Neonatal Biobank. DBS are collected from newborns under a mandatory public health program to screen for serious conditions. At 24 to 36 hours of age, a few drops of blood are taken from the baby’s heel and placed on a filter paper card. The card is sent to the state public health laboratory for testing. After testing, MDCH retains the spots indefinitely for the personal use of the patient and also, pursuant to a 2000 law, for possible research.

Laboratory Testing Practices for West Nile Virus in the United States

We surveyed state public health and commercial diagnostic reference laboratories regarding current testing practices for West Nile virus (WNV). The majority of WNV testing is now performed in commercial diagnostic reference laboratories using commercially available Food and Drug Administration-cleared kits labeled for the presumptive diagnosis of WNV. However, only 25% of surveyed state public health or commercial diagnostic reference laboratories currently have the capacity to perform the recommended confirmatory testing. These findings indicate the need for both manufacturers and laboratories to monitor the performance of these WNV test kits. Further, clinicians should be aware of the limitations of these kits and the need for additional testing to confirm a diagnosis of WNV disease.

Leadership Principles for Developing a Statewide Public Health and Clinical Laboratory System

In 1999, the Centers for Disease Control and Prevention (CDC), the Association of Public Health Laboratories (APHL), and the Federal Bureau of Investigation established the national Laboratory Response Network (LRN) for bioterrorism readiness. A more broad application of the LRN is the National Laboratory System (NLS), an effort to promote the 10 Essential Public Health Services and the Core Functions and Capabilities of State Public Health Laboratories (hereafter, Core Functions). State public health laboratories (PHLs) are responsible for leading the development of both the LRN and the NLS in their jurisdictions. Based on the experience of creating a laboratory network in Wisconsin, leadership principles are provided for developing and strengthening statewide laboratory networks of PHLs and clinical laboratories, which can also include point-of-care testing sites. Each state PHL, in the context of these Core Functions and leadership principles, sets its priorities, budgets, and strategic plans. For a limited investment of personnel and funds that will yield a large benefit to public health, a robust state laboratory system can be established.

The State Public Health Laboratory System

This article describes the development since 2000 of the State Public Health Laboratory System in the United States. These state systems collectively are related to several other recent public health laboratory (PHL) initiatives. The first is the Core Functions and Capabilities of State Public Health Laboratories, a white paper that defined the basic responsibilities of the state PHL. Another is the Centers for Disease Control and Prevention National Laboratory System (NLS) initiative, the goal of which is to promote public-private collaboration to assure quality laboratory services and public health surveillance. To enhance the realization of the NLS, the Association of Public Health Laboratories (APHL) launched in 2004 a State Public Health Laboratory System Improvement Program. In the same year, APHL developed a Comprehensive Laboratory Services Survey, a tool to measure improvement through the decade to assure that essential PHL services are provided.

Laboratory Services in Support of Public Health: A Status Report

To assess Healthy People 2010 Objective 23-13 and its related sub-objectives measuring comprehensive laboratory services in support of essential public health programs, the Association of Public Health Laboratories (APHL) collaborated with the Centers for Disease Control and Prevention (CDC) to create and administer a survey of state public health laboratories (PHLs). A committee of APHL, with representation from CDC, constructed the survey based on the 11 Core Functions of State Public Health Laboratories (hereafter, Core Functions)--the premise being that the extent to which they fulfilled these Core Functions would represent their level of providing or assuring comprehensive laboratory services in support of public health. The survey was distributed biennially to all state health agencies from 2004 to 2008, and respondents were given two months to complete it. The response rate for all surveys was > or = 90.2%. State PHLs were more likely to meet the sub-objectives relating to traditional functions (e.g., disease surveillance and reference testing) than other areas (e.g., food safety and environmental testing). Emergency preparedness fell in between. Overall, but most notably in the areas of food safety and training and education, there was improvement from 2006 to 2008, with the percentage of respondents who met more than half of the sub-objectives increasing from 58.7% in 2006 to 61.2% in 2008. The comprehensive laboratory services survey has been a valuable tool in measuring the laboratory infrastructure that underpins public health in the U.S. It will be necessary to continue monitoring laboratory infrastructure in this way to determine where the gaps in services exist and how they can best be addressed.

Origins and Development of the National Laboratory System for Public Health Testing

Although not recognized as such, a National Laboratory System (NLS) has existed since the inception of public health laboratory (PHL) testing more than a century ago. The NLS has always relied upon the participation of clinical laboratories, both to report test results that represent public health threats and to submit specimens and isolates to PHLs for additional or confirmatory testing. Historically, a number of factors have hindered the strengthening of the relationships between clinical laboratories and PHLs, but the reality of bioterrorism and subsequent focus on strengthening public-private relationships has stimulated the development of a more robust NLS. Since 2002, there has been substantial strengthening of the NLS through the sharing of lessons learned from several demonstration projects. There is a growing emphasis on defining critical elements of the NLS, including the State Public Health Laboratory System (SPH Laboratory System) and the functions of the Laboratory Program Advisor, a position that every state should have at the center of its laboratory system's capacity-building. Additional strengthening of the NLS is occurring through (1) national biennial measurement of state PHLs' abilities to meet the Core Functions and Capabilities of State PHLs, (2) the new Laboratory System Improvement Program (L-SIP) for the SPH Laboratory System, and (3) sharing ideas to integrate and improve the SPH Laboratory System (e.g., using the L-SIP Online Resource Center). Public health emergencies, such as the recent H1N1 epidemic, illustrate and reinforce the need for a strong NLS within which federal, public health, and clinical (i.e., hospital and private reference) laboratories function in close collaboration.

Public Health Laboratory System Improvement Program: Development and Implementation

Instruments designed to measure the performance of public health systems at state and local levels were supported by the Centers for Disease Control and Prevention (CDC) and implemented in 2002. This article describes the process and outcomes of a system and tool designed to measure performance of State Public Health Laboratory Systems, accomplished by the Association of Public Health Laboratories (APHL) in partnership with CDC. We describe the process used to develop the instrument and its subsequent pilot testing and field testing in 11 states. Throughout the field testing and early implementation phases, both CDC and APHL recognized that the core rationale for measuring system performance would be to provide the basis for subsequent system improvement. APHL implemented the Laboratory System Improvement Program (L-SIP) in 2007 and conducted an evaluation of the field testing of the instrument and related materials that same year. We conclude with a summary of future implications for L-SIP, the program's recognition as an international standard for laboratory systems, and the critical importance of its continuation.

The role of local public health laboratories.

Local public health laboratories (PHLs) serve many of the same roles as state PHLs and often perform many or portions of the 11 Core Functions and Capabilities of State Public Health Laboratories; however, they differ in several important ways. First, many local laboratories provide testing at the site of patient care (e.g., sexually transmitted infection clinics) or address local environmental issues (e.g., water quality). Second, local PHLs support the missions of local public health departments, which may differ from those at the state level. Third, local PHLs often serve as conduits, collecting specimens for various state-level screening and disease-control programs; and while they may not perform the testing, local PHLs are responsible for tracking specimens, ordering tests, and reporting results. Fourth, local PHLs often serve as surge capacity for state PHLs, particularly for testing to support emergency response. Last, local PHLs work with and are typically co-located in the local public health agency with other public health programs. Local PHL professionals work as a team with investigators, inspectors, and community and public health medical professionals and, thus, are poised to provide rapid and relevant responses to community needs.

Usefulness of the Paralens™ Fluorescent Microscope Adaptor for the Identification of Mycobacteria in Both Field and Laboratory Settings

The presence of acid-fast bacilli (AFB) in laboratories has traditionally been demonstrated using the fluorochrome method, which requires a fluorescent microscope or the Ziehl-Neelsen (ZN) method employing light microscopy. Low sensitivity of the ZN method and high costs of fluoroscopy make the need for a more effective means of diagnosis a top priority, especially in developing countries where the burden of tuberculosis is high. The QBC ParaLens™ attachment (QBC Diagnostic Inc., Port Matilda, PA) is a substitute for conventional fluoroscopy in the identification of AFB. To evaluate the efficacy of the ParaLens LED (light-emitting diode) system, the authors performed a two-part study, looking at usefulness, functionality and durability in urban/rural health clinics around the world, as well as in a controlled state public health laboratory setting. In the field, the ParaLens was durable and functioned well with various power sources and lighting conditions. Results from the state laboratory indicated agreement between standard fluorescent microscopy and fluorescent microscopy using the ParaLens. This adaptor is a welcome addition to laboratories in resource-limited settings as a useful alternative to conventional fluoroscopy for detection of mycobacterial species.

CDC Releases Updated Biomonitoring Report

Biomonitoring—the science of measuring environmental chemicals in human blood, urine, and other tissues—made another modest advance with the 10 December 2009 release of the Fourth National Report on Human Exposure to Environmental Chemicals by the Centers for Disease Control and Prevention (CDC). The report includes data on 75 substances not in the preceding report, for a total of 212 reported chemicals. Data for more than 45 additional pesticides and metabolites are expected to begin appearing on the CDC website (http://www.cdc.gov/exposurereport/) by spring 2010. This is almost a 10-fold increase over the 27 substances covered in the initial report in 2001, greatly expanding the data researchers can use in their continuing efforts to take the next step—determining what the reported concentrations mean from a health risk perspective. “Interpretation is key to making biomonitoring data more meaningful,” says Sarah Brozena, senior director for regulatory and technical affairs with the American Chemistry Council. Yet such understanding is extremely limited so far for most chemicals. But the science of biomonitoring may be nearing a short-term zenith for the number of substances assessed in the general population, and future CDC reports “will probably grow less than [they] have recently,” says John Osterloh, chief medical officer and toxicologist with the CDC Division of Laboratory Sciences. That’s due to technological limitations as well as capacity and budget constraints. As a result, in the foreseeable future it’s likely that data for only a tiny fraction of the 239,000 toxic substances listed by the Chemical Abstract Service as regulated or included in inventories worldwide would be included in biomonitoring efforts. The report draws on data from the National Health and Nutrition Examination Survey, an ongoing survey that every 2 years samples a small number of people intended to represent the U.S. general population. Chemicals included in the fourth report have been selected on the basis of likelihood of exposure in the U.S. population, seriousness of known or suspected health effects resulting from exposure, and availability of appropriate ways to measure the chemical, among other factors. Of the samples assessed, 90–100% had detectable levels of substances such as perchlorate, mercury, bisphenol A, acrylamide, multiple perfluorinated chemicals, and the flame retardant BDE-47. Osterloh knows of no breakthrough technologies that can substantially expand the CDC biomonitoring program. One option could be to have industries generating chemicals conduct such testing—a suggestion that could be presented in future legislative proposals or efforts to revise the Toxic Substances Control Act. In the meantime, biomonitoring programs in state public health laboratories, Canada, and Europe offer no near-term prospects for covering substantially more substances. Another important limitation of the CDC’s current effort is that it covers just one substance at a time. “In the real world, we’re exposed to mixtures,” says Anila Jacob, a senior scientist with the Environmental Working Group, an advocacy organization that has documented 414 substances in 186 people it has tested, many of whom carry scores of contaminants. While Osterloh acknowledges concerns about the combined effects of chemicals, he says the CDC’s current program isn’t designed to address mixtures. However, he adds the CDC plans to evaluate mixtures for some chemicals, such as the dioxin-like chemicals, in separate scientific endeavors. The current CDC biomonitoring methodology is not designed to assess geographic differences in exposure, nor can it be used to assess fetal contamination. The latter is a particular concern for Jacob, whose team found up to 232 contaminants in a recent study of cord blood from 10 newborns. “The developing fetus is one of the most vulnerable populations out there,” she says. However, a pilot study of cord blood from infants born to 525 women that is on tap as part of the blossoming National Children’s Study (of which CDC is a part) could begin to help remedy this shortcoming.

SURVEILLANCE AND MONITORING: CDC Releases Updated Biomonitoring Report

Biomonitoring—the science of measuring environmental chemicals in human blood, urine, and other tissues—made another modest advance with the 10 December 2009 release of the Fourth National Report on Human Exposure to Environmental Chemicals by the Centers for Disease Control and Prevention (CDC). The report includes data on 75 substances not in the preceding report, for a total of 212 reported chemicals. Data for more than 45 additional pesticides and metabolites are expected to begin appearing on the CDC website (http://www.cdc.gov/exposurereport/) by spring 2010. This is almost a 10-fold increase over the 27 substances covered in the initial report in 2001, greatly expanding the data researchers can use in their continuing efforts to take the next step—determining what the reported concentrations mean from a health risk perspective. “Interpretation is key to making biomonitoring data more meaningful,” says Sarah Brozena, senior director for regulatory and technical affairs with the American Chemistry Council. Yet such understanding is extremely limited so far for most chemicals. But the science of biomonitoring may be nearing a short-term zenith for the number of substances assessed in the general population, and future CDC reports “will probably grow less than [they] have recently,” says John Osterloh, chief medical officer and toxicologist with the CDC Division of Laboratory Sciences. That’s due to technological limitations as well as capacity and budget constraints. As a result, in the foreseeable future it’s likely that data for only a tiny fraction of the 239,000 toxic substances listed by the Chemical Abstract Service as regulated or included in inventories worldwide would be included in biomonitoring efforts. The report draws on data from the National Health and Nutrition Examination Survey, an ongoing survey that every 2 years samples a small number of people intended to represent the U.S. general population. Chemicals included in the fourth report have been selected on the basis of likelihood of exposure in the U.S. population, seriousness of known or suspected health effects resulting from exposure, and availability of appropriate ways to measure the chemical, among other factors. Of the samples assessed, 90–100% had detectable levels of substances such as perchlorate, mercury, bisphenol A, acrylamide, multiple perfluorinated chemicals, and the flame retardant BDE-47. Osterloh knows of no breakthrough technologies that can substantially expand the CDC biomonitoring program. One option could be to have industries generating chemicals conduct such testing—a suggestion that could be presented in future legislative proposals or efforts to revise the Toxic Substances Control Act. In the meantime, biomonitoring programs in state public health laboratories, Canada, and Europe offer no near-term prospects for covering substantially more substances. Another important limitation of the CDC’s current effort is that it covers just one substance at a time. “In the real world, we’re exposed to mixtures,” says Anila Jacob, a senior scientist with the Environmental Working Group, an advocacy organization that has documented 414 substances in 186 people it has tested, many of whom carry scores of contaminants. While Osterloh acknowledges concerns about the combined effects of chemicals, he says the CDC’s current program isn’t designed to address mixtures. However, he adds the CDC plans to evaluate mixtures for some chemicals, such as the dioxin-like chemicals, in separate scientific endeavors. The current CDC biomonitoring methodology is not designed to assess geographic differences in exposure, nor can it be used to assess fetal contamination. The latter is a particular concern for Jacob, whose team found up to 232 contaminants in a recent study of cord blood from 10 newborns. “The developing fetus is one of the most vulnerable populations out there,” she says. However, a pilot study of cord blood from infants born to 525 women that is on tap as part of the blossoming National Children’s Study (of which CDC is a part) could begin to help remedy this shortcoming.

H1N1: A Mexican Perspective

In April this year, a new influenza virus of swine origin emerged in Mexico and spread rapidly around the world. As the Northern hemisphere winter flu season kicks off, Laura Vargas-Parada reports on the measures that Mexico is taking to combat the H1N1 pandemic.

Pulmonary nontuberculous mycobacterial infections: Antibiotic treatment and associated costs

Recent studies suggest an increasing prevalence of pulmonary nontuberculous mycobacteria (NTM) disease. In the absence of prevalence and cost data, the public health burden of pulmonary NTM disease is difficult to assess. The goal of this study was to assess costs associated with NTM disease treatment and to identify risk factors associated with increased costs. Records from subjects with pulmonary NTM disease enrolled in a natural history protocol were abstracted for presenting symptoms, comorbidities, microbiology, and treatment histories. Antibiotic frequency, duration, adverse reaction, and costs were noted, the total antibiotic burden and cost were calculated, and risk factors associated with high costs were analyzed. From Jan 2004 to Dec 2005, 33 subjects were enrolled; 27 met disease criteria and had sufficient data to assess antibiotic use. Mycobacterium avium complex was present in 89% and Mycobacterium abscessus was present in 21% of subjects. Subjects received a median of 5 (1-10) antibiotics. Adverse effects were common seen in up to 50% with common antibiotics and up to 100% with uncommonly used antibiotics. Median burden of treatment was 2638 (84-7689) drug-days and the median total cost per patient was $19,876 ($398-70,917). Subjects with high treatment costs had an adjusted 9.5 fold (95% CI 1.5-97.2) likelihood of having M. abscessus and a 4.2 fold (95% CI 0.6-59.3) increased likelihood of having more extensive disease. Pulmonary NTM represent an underappreciated disease burden in the US population, with an associated treatment cost comparable to that for other chronic diseases of infectious origin such as HIV/AIDS.

Evaluation of a clinic‐based cholinesterase test kit for the Washington State Cholinesterase Monitoring Program

The Washington State Cholinesterase Monitoring Program for pesticide handlers requires blood draws at local clinics, with samples tested at a central laboratory. At present, workers with inhibited cholinesterase activity may be re-exposed before they can be removed from work. In this study we explored the option of on-site testing at local clinics using the EQM Test-mate Kittrade mark, a portable cholinesterase test kit. Test kit cholinesterase activity measurements were performed on 50 blood samples by our research staff, and compared to measurements on the same samples by the Washington State Public Health Laboratory. Another set of samples was also analyzed with the test kit by medical staff at an eastern Washington clinic. Triplicate measurements with the test kit had a 3.3% average coefficient of variation (CV) for plasma cholinesterase (PChE), and a 3.5% average CV for erythrocyte cholinesterase (AChE) measurements. The kit's PChE measurements were similar to PHL measurements (average ratio of 0.98) when performed in the laboratory, but had a tendency to underestimate activity when used in the clinic setting (average ratio of 0.87). The kit systematically overestimated AChE activity by 42-48% relative to the PHL measurements, regardless of where the samples were analyzed. This easy-to-use test kit appeared to be a viable method for clinic-based PChE measurements, but was less consistent for AChE measurements performed in the clinic. Absolute measurements with the kit need to be evaluated carefully relative to standardized methods.

The Laboratory Response Network: Its role in times of disaster

The Laboratory Response Network (LRN) was established by the Centers for Disease Control and Prevention. Today, the LRN is charged with the task of maintaining an integrated network of state and local public health, federal, military, and international laboratories that can respond to bioterrorism, chemical terrorism, and other public health emergencies. The more than 150 laboratories that make up the current LRN are affiliated with federal agencies, military installations, international partners, and state and local public health departments. Laboratories in the network may accept samples from hospitals, clinics, the Federal Bureau of Investigation, other law enforcement groups, emergency medical services, and the military and other agencies. All of the LRN laboratories use the same protocols and validated methods to ensure rapid and certain identification of dangerous biologic agents that cause anthrax, botulism, plague, tularemia, brucellosis, and other illnesses.

Exercising the Urgent Transport of Clinical Laboratory Biothreat and Pandemic Influenza Specimens to the Oregon State Public Health Laboratory

Exercising the Urgent Transport of Clinical Laboratory Biothreat and Pandemic Influenza Specimens to the Oregon State Public Health Laboratory

A Survey of State Public Health Laboratories for Pertussis Diagnostics

Pertussis (whooping cough) is a highly communicable disease caused by the bacterium Bordetella pertussis. Specific diagnosis is made using clinical symptoms and laboratory detection of the etiologic agent. Recent molecular techniques have been reported that allow more rapid results using polymerase chain reaction (PCR). A survey of state public health laboratories was done to determine test methods available for pertussis diagnosis with emphasis on PCR method characteristics. Results show that 70% of state public health laboratories offer PCR in conjunction with culture and direct fluorescent-antibody (DFA). A majority of public health laboratories offer PCR testing for pertussis, for a variety of possible reasons. Testing by PCR should be used in addition to culture, not as a replacement.

A Survey of State Public Health Laboratories for Pertussis Diagnostics

A Survey of State Public Health Laboratories for Pertussis Diagnostics

Increase in Nalidixic Acid Resistance among Non-Typhi Salmonella enterica Isolates in the United States from 1996 to 2003

Fluoroquinolones commonly are used to treat adult Salmonella infections. Fluoroquinolone treatment has failed for persons infected with nalidixic acid-resistant Salmonella. From 1996 to 2003, state public health laboratories forwarded 12,252 non-Typhi Salmonella enterica isolates to the Centers for Disease Control and Prevention for antimicrobial susceptibility testing; 203 (1.6%) of the isolates were nalidixic acid resistant, and 14 (7%) of those were ciprofloxacin resistant. Resistance to nalidixic acid significantly increased from 0.4% in 1996 to 2.3% in 2003. All ciprofloxacin-resistant isolates had at least one point mutation in the quinolone resistance determining region (QRDR) of gyrA and did not harbor qnr or have point mutations in the QRDR of gyrB, parC, or parE. Continued surveillance of antimicrobial resistance among non-Typhi S. enterica isolates is needed to mitigate the increasing prevalence of nalidixic acid resistance.