Shish Kebabs, Pears and Grapes: All “Clue(s)” to Diagnosing Microbial Infections on Pap Smears 

What is the test?

In the 1920s, the Greek physician Georgios Papanikolaou developed a method of cervical cancer screening, now reliably used and colloquially known as a “Pap smear”. During a Pap smear, a healthcare provider swabs cells from the cervix for further analysis by the lab​¹​, including cytopathologic examination. Regular utility of Pap smears in women aged 21 to 65 has decreased the incidence and mortality of cervical cancer by at least 80% in the United States alone​²​. 95% of cervical cancer cases worldwide are caused by persistent human papilloma virus (HPV) infection of the cervix​³​. However, other sexually transmitted infections (STIs) and common non-STIs with associated morphological changes can also be identified on Pap smears. 

Bacteria

Bacterial vaginosis (BV) is the most common cause of abnormal discharge in young women, characteristically with a “fishy” ammonia-like odor. Although not strictly considered an STI, this infection is associated with severe rare complications including pelvic inflammatory disease (PID), infertility and premature labor. The vagina typically have an abundance of Lactobacillus spp., which is a gram positive bacilli (Figure 1). However, if the normal vaginal flora is disrupted with high abundance of gram negative bacteria such as Gardnerella vaginalis or Mobiluncus, then BV may occur. On pap smears, ‘clue cells’ which are squamous epithelial cells with coccobacilli, appear as dark purple staining and cloudy appearance suggest BV. In cervicovaginal samples typically found in women using intrauterine device usage, clumps of filamentous bacteria suggestive of Actinomyces species may also be visible. For cases pertaining to bacterial etiologies, staining of the rods or filaments usually warrant suspicion of infection. Other the other hand although Chlamydia trachomatis is the most common bacterial STI in the United States, with up to 4 million new cases every year, diagnosis on pap smears is not the definitive diagnosis since the cytopathology is non-specific. The interpretations include visualization of inflammatory exudates and inflammatory cells. Oftentimes, further testing is needed. 

Parasite

Trichomonas vaginalis is the most prevalent non-viral STI in the United States, strongly associated with an increased risk of HIV infection among women​⁴​. Majority of the patients may exhibit symptoms such as burning, itching and vaginal discharge. T. vaginalis is a parasitic flagellated protozoan although in some cases, a flagella may be found. Typically, it is of a pear-shape and staining of a nucleus may be visualized (Figure 2).

Fungus

Yeast infections (vulvovaginal candidiasis) due to Candida infection occur in 75% of women at some point in their lives. Classic clinical presentation includes itching, erythema, and thick white “cottage-cheese” discharge. There is also an increased association with HIV infection, diabetes, and any cause of immunosuppression (e.g. transplant, chemotherapy, steroids). Morphologically, budding yeast forms (conidia, small and oval measuring 3-6 µm) and pseudohyphae (long filamentous spores) are found. The combination of pseudohyphae and yeast forms are referred to as “sticks and stones” and frequently, squamous cells lined up along the pseudohyphae are found referred to as having “shish kebab” appearance (Figure 3)⁵. Note that no true septation is found.

On pap smears, sometimes there could be superficial mucosal infections that may appear associated with enlarged hyperchromatic nuclei with halos, which can be confused with low grade squamous intraepithelial lesions.

Viruses

HPV with its associated risk of cervical cancer remains the most crucial microorganism to detect on Pap smears. Given HPV’s association to cancer development, it is crucial to examine the samples for cervical lesions and their associated pathologies. HPVs in the low-risk category typically is associated with low-grade squamous intraepithelial lesions and HPVs in the high-risk category is associated with high-grade squamous intraepithelial lesions and invasive squamous cell carcinoma.  Other viral etiologies seen in PAP smears include Herpes simplex virus (HSV) and cytomegalovirus (CMV). Infections with HSV can be asymptomatic but the most common symptom is the development of vesiculopustular or small ulcerative lesions on the genitalia. The classic cytopathological findings of HSV infection are the 3 “Ms”: multinucleation, nuclear molding, and margination of chromatin. CMV infections are rare, usually asymptomatic and can be transient. On the pap smears, the infected cells may appear with intranuclear inclusions surrounded by a halo.

Conclusions

While Pap smears are routinely performed on women and can provide a presumptive diagnosis, current developments in molecular technologies (e.g. Nucleic acid amplification tests (NAATs)) is transforming the field. There are several FDA-approved molecular platforms available for clinical diagnostic labs to test for HPV and can even genotype the strain to determine risk levels. Crucially, the remaining sample from liquid-based Pap tests​⁶​ can also be submitted for both NAAT testing and HPV-DNA testing, facilitating a quicker turnaround time and obviating the need for additional patient sampling.  Most recently, a PCR-based test is now on the market (Cepheid, Sunnyvale, CA) that can diagnose all three etiologies, BV, Candidiasis, and Trichomoniasis, within 60 minutes from a single specimen.  In summary, accurate examination of the Pap smear often incidentally provides the first step in the diagnosis and further work-up of all these infectious diseases.

References 

​​1. Pap Smear: MedlinePlus Medical Test [Internet]. [cited 2024 Feb 4]. Available from: https://medlineplus.gov/lab-tests/pap-smear/ 

​2. PDQ® Screening and Prevention Editorial Board. Cervical Cancer Screening (PDQ®): Health Professional Version. National Cancer Institute [Internet]. 2022 [cited 2024 Feb 4];1–26. Available from: https://www.cancer.gov/types/cervical/hp/cervical-screening-pdq 

​3. Lei J, Ploner A, Elfström KM, Wang J, Roth A, Fang F, et al. HPV Vaccination and the Risk of Invasive Cervical Cancer. New England Journal of Medicine. 2020 Oct 1;383(14):1340–8.  

​4. Davis A, Dasgupta A, Goddard-Eckrich D, El-Bassel N. Trichomonas vaginalis and Human Immunodeficiency Virus Coinfection Among Women Under Community Supervision: A Call for Expanded T. vaginalis Screening. Sex Transm Dis [Internet]. 2016 Sep 15 [cited 2024 Feb 5];43(10):617–22. Available from: https://pubmed.ncbi.nlm.nih.gov/27631355/ 

​5. Kamal Meherbano M.  The Pap smear in inflammation and repair. Cytojournal [Internet]. 2022 Apr 30; 19:29. Available from: doi: 10.25259/CMAS_03_08_2021

​6. Hawthorne CM, Farber PJ, Bibbo M. Chlamydia/gonorrhea combo and HR HPV DNA testing in liquid-based pap. Diagn Cytopathol [Internet]. 2005 Sep [cited 2024 Feb 5];33(3):177–80. Available from: https://pubmed.ncbi.nlm.nih.gov/16078250/ 

-Zoon Tariq is a pathology resident at George Washington University. Her interests include surgical pathology and cytopathology.

-Rebecca Yee, PhD, D(ABMM), M(ASCP)^CM is the Chief of Microbiology, Director of Clinical Microbiology and Molecular Microbiology Laboratory at the George Washington University Hospital. Her interests include bacteriology, antimicrobial resistance, and development of infectious disease diagnostics.