New Rapid, Low-Cost Test for Zika Virus Surveillance

Researchers developed a rapid and low-cost test for Zika virus that may permit on-site detection, according to a study published in Science Translational Medicine.

In adults, infection with Asian-lineage Zika virus — an arbovirus transmitted via mosquitoes — has been associated with nonspecific symptoms such as fever and arthralgia, as well as Guillain-Barré syndrome. Mother-to-child transmission can cause microcephaly in infants. The Zika virus outbreak currently encompasses the American continents, and Africa may soon be affected.

The available diagnostic tests for Zika virus include virus isolation, serology after exposure to the virus, and viral RNA detection by reverse transcription polymerase chain reaction (RT-PCR). However, these modalities are often costly, and rapid and inexpensive tests for Zika virus are needed for surveillance and patient care purposes.

Loop-mediated isothermal amplification (LAMP) is quick and simple to perform and has been implemented successfully for the detection of other arboviruses in affected regions. Researchers, led by Joel Rovnak, PhD, and Nunya Chotiwan, MS, from Colorado State University, developed a rapid, sensitive, low-cost test for Zika using LAMP.

The LAMP assay for Zika virus differentiates between Asian- and African-lineage Zika viruses, although it cannot identify other viruses such as dengue or yellow fever. The assay can also be used for direct detection of Zika virus in mosquitoes and human plasma, serum, and semen. Unlike RT-PCR, the LAMP assay does not require reverse transcription or RNA isolation.

The limits of Zika virus RNA detection for the LAMP assay are similar to the limits for RT-PCR, and the sensitivity of LAMP is comparable to that of quantitative RT-PCR (qRT-PCR). A major drawback of LAMP and RT-PCR, according to Dr Rovnak, is that they can only detect Zika virus during active infection. “The virus and its RNA are quickly cleared after production of neutralizing antibodies after about 10 to 14 days,” he told Infectious Disease Advisor. Current research efforts are attempting to identify sites of low-level virus persistence, which is detectable by both assays.

“A main advantage of the LAMP assay is cost of implementation,” Dr Rovnak said. An RT-PCR machine costs approximately $20,000, not including the training of personnel. In contrast, the LAMP assay, if commercially developed, may cost only $500 for the equipment and less than $2 per assay. Thus, the LAMP assay may “provide a simple, in-office or in-clinic assay.”

“The assay was initially designed as a field assay for detection of Zika virus in mosquitoes,” said Dr Rovnak. “Just as with clinical samples, detection of Zika in mosquito populations requires well equipped, regional laboratories. We intend to deploy the assay, in comparison with qRT-PCR, to determine its effectiveness in monitoring virus in mosquitoes in support of focused and timely efforts in mosquito control.”

“I believe that the increased availability and speed of such a diagnostic [tool] will have a significant, positive impact on public health. Although there is no specific therapy for these virus infections beyond palliative care, the range of possible disease agents can be clearly defined and the data on the extent and timing of outbreaks will inform public health official's decisions regarding disease control efforts in the community,” he added.

Dr Rovnak pointed out that the LAMP assay is not yet available for clinical use. He and his colleagues plan to further develop the test for the clinical setting. “We will be doing direct comparisons between the current, approved standard diagnostic, qRT-PCR, and the LAMP assay on patient samples with our collaborators in Managua, Nicaragua, over the next year. The goal is to obtain more data on its utility at points of care,” he said.

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Quidel Receives FDA Clearance for Solana Molecular Assay for Detecting C. difficile

Quidel Corporation, a provider of rapid diagnostic testing solutions, cellular-based virology assays and molecular diagnostic systems, announced that it has received 510(k) clearance from the United States Food and Drug Administration (FDA) to market its Solana® C. difficile Assay for the direct, qualitative detection of the Clostridium difficile DNA in unformed stool specimens of patients suspected of having Clostridium difficile-infection (CDI).

Clostridium difficile is the most frequently identified enteric pathogen in patients with antibiotic-associated diarrhea and colitis. Per the Centers for Disease Control and Prevention (CDC), C. difficile was responsible for approximately half a million infections in the United States in 2011, with 29,000 patient deaths occurring shortly after the initial diagnosis.

Clostridium difficile bacterial infections are life threatening, especially for the elderly, for the immunocompromised, and for patients on a prolonged antibiotic regimen. Typical CDI symptoms include nausea, fever, watery diarrhea and abdominal pain due to inflammation of the colon.
Traditional methods for diagnosing CDI, such as glutamate dehydrogenase (GDH) or toxin antigen tests, can lack sensitivity and increase lab costs due to additional confirmation testing. In addition to significant technical expertise, cytotoxicity assays and toxigenic culture require 24 to 48 hours and 3 to 5 days, respectively, before reliable results can be obtained. The Solana C. difficile assay will now enable laboratories to offer a fast and sensitive result generated by molecular methods, without an upfront nucleic acid extraction step.

The Solana C. difficile Assay is an easy-to-use, accurate, molecular diagnostic test that generates an accurate result in about 35 minutes.

The Solana molecular platform leverages the Helicase-Dependent Amplification (HDA) technology that is resident in Quidel's AmpliVue® molecular product line to generate a fast and accurate test result. Solana can process up to 12 different assays or patient samples in each batched run, and provides time-saving workflow advantages to healthcare professionals in moderately complex settings.

"We are excited to introduce a Solana assay that can make a profound difference in the lives of people that are affected by CDI through a quick and accurate diagnosis. The laboratorian will benefit from the Solana platform's ability to address the particular workflow needs of the moderately complex laboratory setting in a cost-effective manner by neatly balancing higher volume sample testing at scale with customizable, on-demand assay processing," said Douglas Bryant, president and chief executive officer of Quidel Corporation.

The Solana® C. difficile Assay is Quidel's first molecular diagnostic test to receive 510(k) clearance from the FDA in the scalable and versatile Solana format for diagnosis of a Healthcare Associated Infection (HAI).

With the Solana franchise, Quidel has broadened its molecular strategy to include instrumented systems, and grown the number of its molecular platforms that are both 510(k) cleared and available commercially. Quidel's other FDA cleared molecular solutions include the AmpliVue® non-instrumented system for lower-volume moderately complex labs, and Lyra® reagents for higher throughput, highly complex laboratories that are compatible with existing PCR infrastructure.

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FDA Reclassification of Rapid Flu Tests Forces Changes in Technology

When it comes to diagnosing viral infections, correctly identifying influenza in a patient can be harder than it sounds. Even as point-of-care diagnostics become more sophisticated, doctors still sometimes rely on methods that made their way to the clinic in the 1990s because they're faster than other tests. But these rapid influenza diagnostic tests (RIDTs) have been proven in recent years to have faults of their own.

 In a Cepheid-sponsored workshop at the Clinical Virology Symposium here this week, Columbia University professor Daniel Green outlined the virtues and weaknesses of RIDTs, noting that the US Food and Drug Administration recently changed its classification of these tests in order to ensure they are as sensitive and specific as possible.

There are three main methods for detecting flu in a patient sample, Green said — viral culture, antigen detection, and nucleic acid tests. Viral cultures, though accurate, take far too long to be useful in the clinic. When immediate treatment can significantly impact a patient, waiting 48 hours for test results makes no sense. And although nucleic acid tests are gaining in popularity because of their speed and accuracy, they're still prohibitively expensive to be very widely used, and their complexity can sometimes require skilled lab personnel that not all hospitals have, Green said.

That leaves RIDTs, which take only a few minutes to return a result and are relatively inexpensive. A sample is applied to a test strip, which detect viral particles using antibodies embedded in the strip. The newer tests even come with automated readers to reduce the possibility of human error when determining results.

But these tests are also less sensitive than culture or nucleic acid testing, Green said. They have no ability to subtype viruses, and recent studies have shown their sensitivity to be somewhere between 50 percent and 70 percent. According to the Centers for Disease Control and Prevention, their negative predictive value is not high enough for negative results to be trusted without verification with further testing, Green said.

The sensitivity problem of RIDTs came to light during the 2009 H1N1 pandemic. A study at that time found that RIDT performance varies, depending on the level of virus in a given sample. At the lowest concentrations of viral load, the performance of these test is inadequate, Green said. Another study of H3N2 showed some assays can't even detect viral strains when they're present at high levels in a patient sample.

In order to solve this problem, the FDA convened an advisory panel on RIDTs in 2013. After a period of study and public comment, the agency reclassified them from Class I to Class II with special controls. Class I in vitro diagnostics are low risk devices for which general controls, such as registration of manufacturing facilities and quality control procedures, are sufficient, Green said. Class II devices are those that pose a higher risk to patients if their effectiveness is compromised in any way. These devices require special controls in addition to general controls — performance standards, postmarket surveillance, review by the FDA under 510(k) procedures, and so on.

The FDA determined that the special controls for RIDTs would include minimum performance standards, checking of results against reference methods, annual monitoring, and provisions for public health emergencies, according to Green.

To set minimum standards of sensitivity, the agency focused on tests that were cleared within the last five years because they tended to demonstrate somewhat higher sensitivity. It also determined that RIDT manufacturers would need to obtain 510(k) clearance to certify that any new tests meet minimum performance standards. According to the new regulations, old products must be withdrawn from the market by January 2018 or must be modified to meet the new criteria.

The tests must be compared against reference methods such as viral culture or nucleic acid amplification tests, Green said. If compared to viral culture, the RIDT must have a sensitivity of at least 90 percent for detecting Influenza A and 80 percent for detecting Influenza B. When compared to nucleic acid tests, the RIDTs must have sensitivity of at least 80 percent to detect both Influenza A and B.

Further, because of the reclassification, all manufacturers must conduct annual analytical testing of their RIDTs with contemporary strains of the flu, including vaccine strains, with panels provided by the CDC. The protocol involves testing panels of viruses in three different dilutions — in order to claim that an RIDT detects a given strain of the flu, the manufacturer must show that the test can detect at least one dilution of that strain. If it can't, then the labeling must reflect that limitation.

And finally, Green said, manufacturers must make provisions for public health emergencies by testing the reactivity of their RIDTs with novel viruses as soon as they become available. Companies must also provide the FDA with written protocols as to how they plan to deal with such emergencies using their tests.

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Alere Launches the Aler Malaria Ag P.f, the First-Ever Rapid Test to Screen Malaria Infection in Asymptomatic Individuals

Alere Inc., a global leader in rapid diagnostics, announced the launch of its Alere™ Malaria Ag P.f, a major technological breakthrough in high-sensitivity rapid testing versus currently available malaria RDTs (rapid diagnostic tests).

The Alere Malaria Ag P.f offers a greater than tenfold improvement in the detection of histidine rich protein II (HRP-II) antigen of Plasmodium falciparum, which will enable better identification of individuals with very low parasitemia, many of whom may be without evident symptoms of malaria infection. This highly sensitive diagnostic will help healthcare workers screen individuals who are asymptomatic but may be carrying the parasite, and thus can aid the implementation of surveillance and mass screen-and-treat programs that are critical for accelerating malaria elimination.

The development of the Alere Malaria Ag P.f was supported by the Bill & Melinda Gates Foundation, which has identified a test to uncover the asymptomatic reservoir as a key component of its malaria strategy. Alere and the Gates Foundation are partnering to generate field data to demonstrate the public health value of the test, particularly in elimination settings. Clinical evaluation and technical support was also provided by PATH and FIND.

"Breaking the cycle of malaria transmission requires identifying all infected individuals, but until now point-of-care diagnostic tests have not been sensitive enough to reliably detect asymptomatic infections," said Avi Pelossof, Alere Global President of Infectious Disease. "The Alere Malaria Ag P.f represents a game-changing tool to empower healthcare workers to identify asymptomatic individuals in even the most remote settings, contributing to a reduced malaria reservoir in communities – key strategies in eliminating this devastating disease."

"We are excited by the potential for this product to address one of the barriers identified on the path to malaria eradication – the non-acute disease reservoir that complicates efforts to stop transmission," said Dr. Bruno Moonen, Deputy Director of the Malaria program at the Bill & Melinda Gates Foundation. "Until now, we haven't had a tool to tackle this challenge in low-transmission settings. Added to existing RDTs, prevention methods and treatments that reduce disease burden in high- and medium-transmission settings, this test will add a new tool to the fight against malaria."

"Improving our ability to detect and define the transmissible reservoir of malaria in a community is critical to inform evidence-based strategies to eliminate malaria. A test that can produce immediate sensitive results even in rural and remote settings represents a significant advance in our ability to do this for Plasmodium falciparum malaria and as such is a valuable tool for malaria elimination," said Gonzalo Domingo, Scientific Director and lead of malaria diagnostics, PATH. "We now have an opportunity to better understand where and how this test can best impact the reduction of the malaria burden world-wide."

"Elimination of malaria rests on our ability to identify asymptomatic malaria infections," noted Catharina Boehme, CEO of FIND. "This test is a valuable addition to the malaria elimination toolkit. Looking forward, we are excited to see the results of our forthcoming demonstration studies, as these will provide valuable insights into how this test can have the biggest positive impact."

The Alere Malaria Ag P.f was granted CE marking in December 2016, and was evaluated during Round 7 of the WHO FIND Malaria RDT Evaluation Program.

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