The D.C. Forensic Sciences Department’s new glass-and-steel facility has been home to a pair of high-profile testing issues in recent years, including those on DNA and for the Zika virus. (Sarah L. Voisin/The Washington Post)

When Anthony Tran took over the District’s public health lab late last year, he had a feeling something was wrong with its testing for the Zika virus. He had just come from the public health lab in New York City, where technicians had been finding markers for Zika in the blood of arriving travelers almost every day. In the smaller, but still international, city of Washington, the same test was negative — every time.

Soon, U.S. health officials joined in Tran’s concern: Samples supplied by the federal government of the frightening, mosquito-borne virus that were tested in the lab as a control were appearing as if they contained no virus.

“I knew then that something was tremendously wrong,” Tran said late last week in an interview. He halted testing, and with help from analysts at the Centers for Disease Control and Prevention, traced the problem to a mistake that any high school chemistry student could understand.

There were two types of solution the District could have purchased to conduct a phase of the test. One bottle came marked with a “D,” for diluted, and the other with a “U”, for undiluted. D.C. lab workers had purchased the diluted version, Tran said, and then mistakenly watered it down as if it was the more concentrated one, weakening the ability to detect for Zika.

For a public health lab to commit such an error once would be an embarrassment in the high-stakes testing of Zika, which has potentially devastating consequences for pregnant women, scientists and federal health officials say. That the District lab — which is also a first line of defense in screening bioterrorism threats — repeated the mistake daily, and without anyone catching it for more than six months, amounts to a more systemic and worrisome failure, experts said.

Jenifer Smith, director of the D.C. Forensic Sciences Department. (Sarah L. Voisin/The Washington Post)

Interviews and public documents show that the debacle unfolded over a period of months last year in which the District lab was in a state of turmoil — understaffed, lacking permanent leadership, spread thin on competing projects, and relying on new employees to test for the emerging public health threat in Zika.

“A relatively inexperienced staff and a lack of leadership — that’s a bad combination,” said Kelly Wroblewski, director of infectious diseases for the Association of Public Health Laboratories, which works to safeguard the integrity of public health labs nationwide. “The positive here is that the lab does have new leadership and he caught the error quickly, even though the situation is already really, very unfortunate.”

Since D.C. officials announced the error on Feb. 9, public health officials in a network of nearly 50 labs studying Zika have questioned how it could have gone so wrong. It was the only jurisdiction in the country to have mishandled the testing.

In the District, the mishap has shaken public confidence in Zika testing and, attorneys say, could result in millions of dollars in legal claims. And all of that may pale in comparison to the effects that the botched tests may have on a handful of D.C. families.

At least nine pregnant women in the nation’s capital were caught in the faulty testing between July and December of last year, D.C. officials said Thursday. The women were told they did not have Zika when in fact new testing by a CDC lab in Colorado shows that the women did have antibodies that could signal the presence of the virus. Follow-up tests on eight have been inconclusive and one has been confirmed as having a Zika infection.

District officials say they do not know how far along any of the women were in their pregnancies when they were wrongly told they were healthy. The District also does not know if any of the women have since given birth.

Zika is primarily transmitted through the bite of an infected mosquito but it can also be passed through sex, even if the infected person shows no symptoms.

Public health officials say they have relayed the new test results to the women’s health care providers, and they are girding for the number of new Zika patients to rise as retesting is completed for the final third of the 300 pregnant women the District says were inaccurately tested.

According to a study in December, about 6 percent of pregnant women believed to be infected with Zika in the United States had a baby or fetus with at least one birth defect related to the viral infection last year. Most suffered from severe microcephaly, characterized by abnormally small head size and, often, an underdeveloped brain.

But babies infected with Zika are not always obvious. Some problems stemming from infection may not show up until a year after birth or longer and can include hearing loss, irritability, difficulty swallowing, and cognitive, sensory and motor-skill difficulties.

That means the fallout in the District may not be felt for some time.

‘A mad scramble’

Tucked away in the District’s new glass-and-steel forensic sciences building, a couple of blocks south of the National Air and Space Museum, are a group of scientists who quietly toil away on some vitally important work.

At inauguration time, they test the food served at high-dollar balls for food-borne illnesses and contamination. When a suspicious, white powder is found in the District, the lab is one of the first to test the substances for biological weapons. And on more mundane days, the staff of the Public Health Laboratory Division tests water pollution in the Potomac and helps to identify influenza strains that should be included in the next year’s vaccines.

As a division of the District’s Forensic Sciences Department, the lab was rarely in the news.

By comparison, the lab’s sister division, the crime lab, had to halt DNA testing in 2015, after questions surfaced about the accuracy of its work.

The scandal forced out the department’s former director and D.C. Mayor Muriel E. Bowser (D) brought in Jenifer Smith, a former FBI forensics expert, to clean house and restore the lab’s reputation.

According to two officials familiar with Smith’s rebuilding plan, as well as public budget documents from the agency, Smith’s top priority early last year was restarting DNA testing at the crime lab.

In the year before it confronted Zika, the public health lab was shedding its most experienced staff — for a variety of reasons.

Former director Alpha Diallo, a native of Guinea, left to fight Ebola in Africa. The lab’s head of virology, Anicet Dahourou, and its head of microbiology, Morris Blaylock, also resigned and took jobs with the CDC in Atlanta.

In March 2016, with Zika as a looming threat, three of the lab’s six quality assurance jobs were vacant.

Maurice Knuckles, who had directed the office a decade earlier, was brought back from semiretirement by Smith to run things on an interim basis, officials said.

Blaylock, reached in Atlanta, said preparing for Zika in the early months of last year with a thin staff was “a mad scramble.”

The District wanted to join dozens of states that were applying to the CDC for the authority to conduct their own Zika tests instead of shipping them off to the federal agency. To apply, the lab was sent a panel of blind tests by the CDC and had to replicate positive and negative results achieved at a federal lab.

The D.C. health lab successfully did so and got permission in late May to begin its own Zika testing program, Tran said.

Smith said the lab hired four people to conduct the tests and trained them for weeks before Zika testing was launched on July 14.

Part of the preparation process involved setting up quality controls, testing procedures and what Tran described as a small “manufacturing plant” of solutions and coatings to conduct the tests.

In an interview, Smith and Tran said it was unclear if the lab employees had used the appropriate dilution in the spring when the lab passed the CDC test.

But Tran said that it is now clear that by July, workers had begun the lab’s Zika testing with an over-diluted solution.

It was not caught earlier, Tran said, because even with a weakened solution, daily control tests still showed positive results, at least, until a final batch was mixed late last year.

Blaylock said the loss of the lab’s virology director was a major gap, and any sustained campaign of virus testing would have exposed weaknesses in the team’s staffing.

“We could deal with an emergency response, we could manage it for a short time, but anything long-term, it was going to wear down that team because it was so short-staffed,” he said. “I know they were trying to get personnel on board, I do know that was the primary challenge.”

Smith dismissed the idea that the botched tests were the result of a newly hired staff or lack of seasoned leaders in key positions. “This is not an issue of staffing,” she said.

‘Something’s wrong’

The faulty solution wasn’t the failure point that Tran noticed in the District’s Zika testing procedures when he joined the lab late last fall.

Concerned about the high number of negative results, Tran began double-checking the team’s math and found an error, he said. Lab workers had fumbled a formula to calculate test results, skewing some inconclusive ones toward negative results.

“I started to investigate and the first thing I found was a really, a quite basic arithmetic error,” he said.

That discovery led Tran to stop the testing and recalculate the score for the tests done since July.

None of the recalculations resulted in a shift to a clearly positive result, he said. Tran was prepared to resume testing in the lab when a new problem developed with the daily control tests: “Now, we were getting no reaction through the plates and I said, ‘Something’s wrong. . . . We need to do a deep dig and figure out what’s wrong.’ ”

On a conference call with the CDC’s Zika experts in December, Tran was stumped. The analysts had gone over all the other complicated calibrations and procedures and everything seemed right. Then one asked a basic question. What type of solution had the District purchased?

Tran had never thought to check. “That was huge,” he said. “The dilution factor was a problem since the day we began.”

Smith said that no one involved in the mistake still works at the lab, but she declined to say how many employees were affected or if they resigned under pressure. Bowser last week said no one had been fired.

Smith said she thought the District’s trouble would someday be held up as an example of something more than a mistake.

“We are going to learn from this; the field of science will advance,” Smith said. “This is a situation where you had positive controls working, but the test is at a level where it gave a different result. . . . It’s a great example in the grayness of the world we work in. . . . This is not the instant pregnancy test, these are more complicated tests.”

Tran said he knew the lab would attract intense scrutiny over the mistake but never hesitated to go public.

“I knew what was going to be happening, but there was no other decision for me to make,” he said. “You need to give the best care because there’s no cure for this.”

Staff writer Lena H. Sun and researcher Jennifer Jenkins contributed to this report.