Your Comprehensive Guide to Vaginal Health Testing Methods

Last updated 10/31/22

You visit the doctor complaining of symptoms like itching, burning, and discharge, hoping you’ll walk out with a clear picture of what’s causing them and some treatment that will give you relief. But getting a clear picture of what’s going on and finding effective treatment for vaginal health is not always easy. Why? Understanding the problem starts with understanding the standards around how we test our vaginal health today.

It may be surprising to realize that the current standard for diagnosing vaginal health conditions often relies on vague data. Like many other women’s health conditions, vaginal health often relies on “storytelling diagnostics.” In other words, we depend on patients to correctly self-identify their symptoms and stories because we lack the data or tests to reach specific diagnoses.

Given the subjectivity involved in vaginal health testing and the variation in the testing methods themselves, we often end up in confusing situations trying to compare different results that came from different testing mechanisms.

To help with this, we’ve put together a comprehensive overview of the different ways you can check on what’s going on in your vagina with a look at each of their pros & cons.  Next time you go to your doctor, ask them which method they use and then you can review our guide below to learn more about it!

Non-DNA testing methods

Amsel Method (symptoms-based)

What is it:

Amsel criteria or symptom based diagnostics are the most commonly performed vaginitis tests in the doctor's office. This test is named after Dr. Amsel who, in 1983, described a set of criteria for diagnosing “nonspecific vaginitis” aka what we now know as bacterial vaginosis.

How it works:

If a patient comes into the doctor’s office with symptoms consistent with bacterial vaginosis, the doctor can use the Amsel method to determine a BV diagnosis. The diagnosis is considered positive if a patient has three of the four criteria: 

1. Excessive discharge
2. A pH greater than 4.5
3. A positive whiff test (This is when a clinician smells the vaginal discharge after adding potassium hydroxide. If they smell a fishy odor, then the sample has a positive whiff test.) 
4. Clue cells (based on a wet mount, microscope slide)

A vaginal wet mount (also known as vaginal smear or wet prep) is when a doctor looks at a vaginal swab under the microscope without any dyes or processing. In this case, they are looking for "clue cells" — a vaginal epithelial cell (the cells that make up the vaginal wall) covered in bacteria. In the photo above, the larger blobs are epithelial cells, also known as squamous cells. The tiny pinpoint dots are bacteria. The clue cell is the large epithelial cell completely covered in bacteria. 

What does a result look like?

The results of an Amsel test do not come with lab results and instead are delivered with a verbal “positive/negative” from your doctor during your visit. With the Amsel test, your doctor will likely ask you about your symptoms, take a swab from your vagina, leave your exam room, and come back during the same visit to deliver the diagnosis. 

During the time when your doctor is “BRB,” they’re likely looking at your swab under the microscope to conclude whether or not you meet three of the four above Amsel criteria (pH greater than 4.5, excessive discharge, the whiff test, and the presence of clue cells).

How it stacks up:

Pros:

• Fast: can be done at the doctor’s office with immediate results

Cons:

• Unreliable: Not all women suspected of having BV meet all of these criteria
• Imprecise: The Amsel does not provide any information on the types of bacteria present which can make it difficult to provide the correct diagnosis and prescribe the most effective treatment, as different antibiotics work better on certain types of bacteria
• Requires equipment: Requires a doctor to have a microscope in their office and be trained on how to use it

Nugent Score (microscope-based) 

What is it:

Nugent tests are primarily used for scientific research, and rarely used in clinical practice. Similar to the Amsel, a Nugent test also requires a doctor to look at a vaginal sample under the microscope, but in this process, a stain is added (called a Gram stain).

When the stain is added, bacteria either show up purple (Gram positive) or pink (Gram negative). The staining corresponds to how thick the cell wall is. A thicker cell wall = darker purple = Gram positive (e.g. Lactobacillus), a thinner cell wall = lighter pink = Gram negative (e.g. E. coli). Some microbes, like Gardnerella, are small Gram variable cocci (can stain either purple or pink).

How it works:

To do a Nugent test, a clinician must look at a stained vaginal smear under the microscope and count the number of different types of cells present. They then give the sample a score between 1-10. A score of 0-3 indicates “normal” lactobacilli, 4-6 intermediate, and 7-10 indicates high diversity and a positive BV diagnosis. 

Although the Nugent criteria is considered the gold standard by the World Health Organization, it only measures the physical diversity of bacteria within a sample and does not classify the bacteria present. 

If you’re familiar with the vaginal microbiome community state types:

• A Nugent score of 0-3 would likely correspond a Lactobacillus dominant CST: CST1, CST2, or CST5, but it wouldn’t be able to tell you if you have a L. crispatus or an L. jensenii dominant microbial community

• A Nugent score of 4-6 or 7-10 would likely correspond to CST4, but it wouldn’t be able to tell you if you would not be able to tell you had Gardnerella or Streptococcus present. 

However, it can be misleading because some organisms don’t stain like you would expect. For example in the figure below, L. iners (a) can sometimes end up looking more like Gardnerella (c) than they do other lactobacilli (b). Also, the problem with Nugent is that it is not a measurement of BV, but a measurement of diversity. Not all women with high Nugent scores have symptoms.

What does a result look like?

Nugent tests are rarely performed at the doctor’s office and are mostly used for research so it’s unlikely that your doctor will use this method to diagnose you. 

True to its name, the Nugent test will give a Nugent score based on your doctor’s interpretation of the Gram stain—with a lower score indicating lactobacilli dominance and a higher score indicating a diversity of bacteria.  More specifically, the score ranges are: “normal” lactobacilli (as score from 0-3), intermediate (a score of 4-6), and BV (a score of 7-10). 

How it stacks up:

Pros:

• Fast: Can be completed at the doctor’s office by anyone with training, reagents, and a microscope. Note that this method is rarely done in the doctor's office and few clinical labs provide it as a test. Instead, this is primarily used for research purposes.

Cons:

• Unreliable: Given that the clinical definition of BV requires a patient to have symptoms, the Nugent score can produce a result of “positive BV” for people who have a diverse microbiome but no symptoms (and therefore would not have clinically diagnosed BV).
• Imprecise: Does not identify the specific organisms present, making it difficult to provide the correct diagnosis and prescribe the most effective treatment.
• Misclassification: Key vaginal microbes look very similar under the microscope (even if they are very different in terms of their positive or negative implications), so they can be confused for each other when a diagnosis depends on microscopy. Some examples:
  - L. iners can be mistaken for Gardnerella or other Gram negative short rod. This means that someone may be treated for an infection like bacterial vaginosis when they don’t need to be.  
  - Gardnerella, Mycoplasma, and Ureaplasma are all Gram variable - meaning they can show up as either positive or negative on a Gram stain, which can make the results difficult to interpret.

• Requires equipment: Requires a doctor to have a microscope in their office and be trained on how to use it

Culture

What is it:

A culture can be considered the old school method of detecting bacteria. 

How it works:

Culturing involves adding a vaginal sample to a nutrient rich growth-media (the agar or broth the bacteria is grown in) and waiting for something to grow. 

Unfortunately, not all bacteria grow well in cultures. Some fungi and bacteria (like Mycoplasma) are notoriously slow growing or finicky, and it is hard to routinely grow them. This is because bacteria are like plants, and some need very specific conditions to grow. For this reason, microbial culture is generally limited to the “weeds” of the microbial world —  the organisms that can grow anywhere no matter the conditions.

What does a result look like?

Very few clinics do routine vaginal culturing. It is time consuming and many common vaginal pathogens, like Gardnerella and Prevotella, are hard to grow in the lab.

However, culturing for other urogenital disorders, like UTIs, is still quite common. A culture result will normally include the annotation CFU/ml which stands for colony forming units per milliliter, and is a measurement of the amount of bacterial cells within a sample. For example, a positive UTI might read: >100,000 CFU/ml E. coli. 

You might receive this result in the form of paperwork or by notification that something has been uploaded to your medical provider’s portal! 

How it stacks up:

Pros:

• Can enable further testing: Because the doctor has access to a living set of the microbes present in your sample, they can test it for antibiotic susceptibility or other experimental procedures
• Quantitative: Due to the way culturing is done, we can count the number of bacterial colonies that grow in order to determine how many of that specific bacteria were alive in a sample. 

Cons:

• Biased: Not everything can be grown in a lab, so you can miss organisms that are present and may be contributing to your symptoms 
• Slow: Culture is time consuming and requires a lot of manpower by highly skilled professionals. Some organisms take days/weeks to grow, or require special equipment in order to grow. Additionally, once the microbe has grown, culturing requires highly skilled professionals to actually identify which microbe is growing in the petri dish.

DNA-based methodologies

As you may (or may not) remember from high school biology, DNA is the code of life. Every single organism — including bacteria & yeast — has a unique genetic code.

DNA is made up of a long string of 4 base pairs (A, T, C, & G) strung together in a plethora of combinations. With DNA testing, we can read this genetic code and use it to identify which organisms are present in a sample. But, different methods look at the DNA in different ways, and there are pros and cons to each method. 

Before we jump into our explainers, here are definitions for a few terms to know when thinking about DNA testing: 

• ‍Genome: The complete set of DNA in a cell that codes for everything the organism does. Genomes vary in size for example, the human genome is 700 times larger than a microbial genome like E. coli (3.2 billion vs 5 million base pairs long)‍
• Gene: A segment of the genome that codes for one protein. That protein usually has a very specific function within the cell. An average bacterial cell encodes about 5000 different genes. ‍
• Sequencing: A process by which we can extract the DNA from an organism or sample and read its genetic code. Sometimes sequencing uses primers and PCR, and sometimes not. Sometimes it sequences all DNA in a sample (shotgun metagenomics), and sometimes it sequences one specific target gene (16S amplicon sequencing). 

qPCR

What is it:

PCR, which stands for polymerase chain reaction, is a method commonly used at the doctor’s office to test for microbes like Gardnerella, Candida, and Trichomoniasis in a vaginal sample (through commonly ordered tests like the BD Affirm, BD Max, LabCorp NuSwab, and others). qPCR is a version of PCR that allows it to be quantitative which means it allows you to know exactly how many copies of that gene were present in the sample. 

How it works:

PCR is a process where a specific section of DNA is amplified to high enough concentrations that it can be detected. Short segments of DNA, called primers, are used to bind to a specific region of DNA allowing the amplification process to proceed. Primers are proprietary and can range from lab to lab which means different labs may have different accuracy and specificity. 

PCR is very useful when you are trying to detect something very specific. Because it is so targeted, it can literally find the genetic equivalent of a needle in a haystack. Actually, because it is an amplification based process, it can detect an organism even if that bacteria makes up only 1 cell in 10,000 — so forget a needle in a haystack: it is a needle in a whole field of haystacks.  

For example, It is perfect for identifying cancer mutations or COVID-19. But PCR is not going to provide any information about other organisms in the microbiome other than what it was specifically designed to look for. 

In vaginal health, most qPCR tests detect a defined short list of species, such as Lactobacillus crispatus, Candida albicans, Mycoplasma genitalium, and Gardnerella vaginalis. A qPCR panel designed for those microbes will be able to tell you if those four organisms are there, but won't tell you if microbes like Atopobium, L. gasseri, or Prevotella are there or not. 

In other words, it only reports on what it was designed to detect. 

What does a result look like?

A qPCR result is usually a lab report with a list of microbes reported as either negative or positive. You’ll likely receive a copy of this lab report in your doctor’s online medical portal. If your PCR comes back positive, it might also include information about whether the result was low, intermediate, or high. Some tests will then interpret the positive/negative to provide guidance to the clinician about whether or not the positive/negative results are consistent with a yeast infection, BV, STI, or normal flora.

How it stacks up:

Pros:

• Highly specific: It will detect what it was designed to detect even if it is in low concentrations.
• Fast: While the results aren’t provided in the clinic, they are often available within 1-3 days.
• Quantitative: If the test uses qPCR, it can provide the number of copies of a specific gene found in a sample.

Cons:

• Narrow scope: It will only report on exactly what it was told to look for (i.e. Gardnerella or Candida albicans) and will not provide any information about the rest of the microbiome. Therefore it can potentially miss a microbe that is related to vaginal symptoms a patient may be experiencing. 
• Hard to compare across tests: Each lab uses their own proprietary PCR primer and could have different accuracy. So one PCR primer might find one species of Gardnerella but completely miss another.  
• Typically relies on presence/absence of a microbe: Many PCR tests provide presence/absence rather than amount. If this is the case, then you don’t know if the microbe was the majority of the microbiome or just a small player.
• No context on the overall community: With PCR, you don’t get any information on the relative amounts of bacteria present or the overall bacterial community. For example, did Gardnerella make up 10% or 90% of the sample? What did the rest of the sample consist of? This fails to give both the clinician and the patient a full picture of what’s going on.

16S rRNA sequencing

What is it:

16S sequencing is a form of next generation sequencing (or NGS, which is often used to refer to any sequencing technology that was invented in the 2000s). 16S sequencing uses a combination of PCR and NGS sequencing, and it is not commonly used at doctor’s offices today.

How it works:

“16S sequencing” gets its name from the 16S ribosomal sequence, a specific region of the genome that exists in all bacteria. It is a gene required for survival, so it mutates very slowly over evolutionary time. Those slight variations can be used as a molecular clock to help us identify which bacteria is which. 

16S sequencing works by looking for different mutations in the 16S gene to identify the bacteria. Unfortunately, it is not always a great source of truth. We know that certain bacteria are more likely to be detected in 16S sequencing (like Lactobacillus), while others, like Bifidobacterium, are less likely to be detected. Also, yeast/fungi literally do not have a 16S gene, meaning they are not identified by any 16S sequencing. 

Finally, 16S sequencing only sequences a small portion (less than 20%!) of the 16S gene. This means there is limited information to work with for classification, which can make it hard to distinguish between different species of certain microbes. 

Why does this matter? 

This is a problem when you are trying to figure out which microbe might be present alongside specific symptoms. For example, if the test you use can’t identify between something benign, like Staphylococcus epidermidis, and something pathogenic, like Staphylococcus aureus, then it can be hard to understand the full picture of what’s going on. 

Also, scientists are learning that while which microbes are there is important, what they are doing (their function) is also critical. For example, many people with vaginas have a microbiome dominated by L. iners, with some experiencing extreme symptoms and others with no symptoms at all. The key to understanding the difference between those microbiomes could lie in the content of their genomes and comparing their functions. What can one strain of L. iners do that another strain cannot? And how might this help explain why one person with L.iners is experiencing symptoms while the other is not? This type of nuance cannot be detected by 16S. 

What does a result look like?

Results for 16S sequencing are usually a lab report with a list of organisms followed by the representative percentage within the community (relative abundance). Most of the organisms will be reported at the genus level (e.g. Escherichia coli or Mycoplasma hominis would show up as Escherichia or Mycoplasma), with only those with enough specificity in the 16S gene reported at the species level (e.g. Lactobacillus crispatus, L. gasseri, L. iners, & L. jensenii would be separated out).

How it stacks up:

Pros:

• Broad(er): Provides insight into a wider list of organisms than PCR does
• Relatively cheap: It is much more expensive than PCR, but slightly cheaper than other NGS technologies (like metagenomics, described below)

Cons:

• Slow: It often takes 1-3 weeks to report results
• Bacteria only: The 16S gene will not detect yeast. Another test (either a PCR test or something called ITS amplicon sequencing) must be done in order to determine if Candida is present, meaning you cannot compare the abundance of yeast to bacteria with 16S testing.
• Biased: Certain bacteria are more likely to be detected than others. For example, Lactobacillus is very easy to detect, but Streptococcus or Bifidobacterium are not. 
• Non-specific: There is not enough information to classify everything to the species level. Only certain organisms, like Lactobacillus, can be classified to the species level, other organisms like Prevotella or Eschericia are often only reported at the genus level. Additionally, 16S provides no information about the potential function of the microbes present.

Shotgun Metagenomics

What is it:

Shotgun metagenomics is another type of NGS, but one that looks at the whole genome instead of just the 16S variable region. Metagenomic testing is not common at the doctor’s office and Evvy is the only at-home vaginal microbiome test to leverage this technology.

How it works:

To do metagenomics sequencing, an enzyme is added that breaks all DNA into fragments (this is the shotgun part) and then those fragments are sequenced. This means that metagenomic sequencing will sequence random segments of DNA from bacteria, yeast, and viruses — any and all microbial DNA is sequenced.

The trick is then putting all that information together to then identify what organisms make up the community. This computational step is called bioinformatics. It is like taking 10 different jigsaw puzzles, mixing up all the pieces, then picking a piece out at random and trying to figure out which puzzle it went to. Thankfully with modern algorithms, we can fairly easily identify which bacteria are in a particular sample. 

Because this method is non-targeted, we get information about all the organisms present, enabling someone to get a full picture of their microbiome. Not only is that valuable to people looking to better understand their own bodies, but it is critical for furthering our understanding of the vaginal microbiome.

With Evvy, for the first time ever, we can study all the things in the vaginal microbiome that had been previously overlooked, which will hopefully lead to better diagnostics and treatment options for all of us. 

Finally, because metagenomics is untargeted, we get DNA sequences from all over the bacterial genome, not just from one gene. This allows us to more accurately classify microbes at a more specific level, and also to identify genes that might help us discover the cause of a disease or develop gene-specific diagnostics. 

What does a result look like?

Results are often returned in a lab report showing the list of microbes as a percentage of the overall microbiome, called relative abundance. However, unlike 16S, almost all microbes can be classified to the species level. 

Also, as we learn more about the vaginal microbiome with tests like Evvy, you might also start to see sub species identification (strain level research) and information on the bacterial functions (such as how they relate to symptoms) in metagenomic results!

How it stacks up:

Pros:

• Most comprehensive: This type of testing will detect everything in a sample - yeast, bacteria, viruses, everything. We can detect previously overlooked organisms, and help identify important bacteria that have been ignored by science. 
• Most precise: Because the test sequences the whole genome instead of just one variable region like 16S, you’re able to get the most precise information about which species are present in a sample. As the research progresses, this can start to include information on which strains are present and if there are any specific bacterial genes that might be causing symptoms!

Cons:

• Resources: Metagenomics requires additional time and money for high sequencing power and analyzing huge amounts of data
• Turnaround time:  It takes 1-2 weeks to turnaround results 
• A lot of detail: Since metagenomics identifies everything present, the results can feel overwhelming. You may see organisms that you, or your doctor, are unfamiliar with. (PS — this is why Evvy’s results experience comes with transparent information on what science knows so far, in-depth explanations, and links to relevant research!)

Why is it so hard to diagnose vaginal disorders?

PHEW, that was a lot of methods to get through! But wait—if we have so many ways to check on our vaginal health, why do so few people with vaginas feel like they get answers?

Even though DNA technology has progressed rapidly, the amount of research into women’s health (and specifically vaginal health!) has unsurprisingly lagged behind (another reason to focus on closing the gender health gap). 

Frustratingly, when it comes to vaginitis, most clinicians still rely on Amsel or Nugent scores to determine a diagnosis. And if and when they do order a lab test, it’s almost always a limited PCR panel. Remember, a test is only as good as what it was designed to do — and most vaginal health tests were designed to look for a limited number of organisms because of the limited research that exists on vaginal health. 

Given the complexity of the vagina, our goal is that Evvy’s community and platform will encourage more clinicians and people with vaginas to demand a more holistic picture of the vaginal microbiome rather than the simplified approaches commonly used today.

Along the way, we’re laser-focused on leveraging the best technology to provide everyone with a vagina with the most comprehensive, personalized, scientifically rigorous education on their own vaginal health. That means being a part of Evvy means being a part of pushing science forward, and making health care better for all people with vaginas!