Predictive value of neutrophil-lymphocyte ratio and platelet-lymphocyte ratio in patients with vaginitis: nested case-control study

Ali Mohammed Ali Al-Nuaimi

doi:10.3897/folmed.67.e146769

Abstract

Aim: To investigate the value of the hematological indices to differentiate bacterial vaginosis (BV) from vulvovaginal candidiasis (VVC) and predict treatment response.

Materials and methods: This nested case-control study included 390 women divided into three groups: healthy women, women diagnosed with BV, and women diagnosed with VVC. Additionally, two groups (BV and VVC) were treated and followed prospectively until cured. Demographic data, blood indices, and high-sensitivity C-reactive protein (hsCRP) were obtained from the women.

Results: hsCRP and neutrophil-lymphocyte ratio (NLR) showed the best diagnostic utility to predict BV with AUC=0.721 and 0.735, respectively. hsCRP showed the best specificity (99.23%), while NLR showed the best sensitivity (82.31%) to differentiate BV. Regarding percentage change after treatment, NLR showed a higher reduction percentage in the BV group (−27.41±8.151%) than VVC (0.158±5.804%).

Conclusions: NLR is an efficient diagnostic instrument for distinguishing individuals with bacterial vaginosis from those with vulvovaginal candidiasis. Its diagnostic reliability is comparable to more regularly used markers such as WBC and hsCRP. Both NLR and PLR are good predictors of clinical response to treatment in bacterial vaginosis.

Keywords

bacterial vaginosis, neutrophile lymphocyte ratio, treatment response, vulvovaginal candidiasis

Introduction

Vaginitis, characterized by inflammation or infection of the vagina and vaginal epithelium, presents with a range of symptoms comprising vulvovaginal pruritus, burning sensation, irritation, dyspareunia, vaginal “fishy” odor, and abnormal vaginal discharge.^[1] The syndrome is the predominant cause for women to pursue medical attention globally, with the majority of women encountering an episode of vaginitis at least once in their lives.^[2,3] Despite not being designated as serious diseases, vaginal infections or microbial dysbioses resulting in vaginitis symptoms, such as bacterial vaginosis (BV), trichomoniasis, and vulvovaginal candidiasis (VVC), are estimated to affect approximately 20 million women.^[4]

The three predominant etiologies of vaginitis are trichomoniasis, BV, and VVC, which collectively account for around 70% of cases. The remaining 30% may pertain to alternative etiologies of vaginitis, such as atrophic vaginitis, desquamative inflammatory vaginitis, and vaginal erosive illness.^[5] The diagnosis of vaginitis is complex and difficult due to its intricate polymicrobial characteristics and diverse clinical manifestations.^[6] To address these diagnostic challenges, different procedures have been employed, including molecular, enzymatic, and chromatographic techniques.^[7]

The inflammatory response is the coordinated activation of signaling pathways that govern the levels of inflammatory mediators in tissue cells, the inflammatory cells recruited from the bloodstream.^[8,9] Inflammation is a prevalent mechanism underlying numerous diseases like infection, preeclampsia, and diabetes.^[10–14] The inflammatory response mechanisms, while contingent upon the specific characteristics of the original stimulus and its anatomical site, uniformly adhere to a common mechanism, which can be succinctly articulated as follows: 1) Cell surface pattern receptors identify harmful stimuli; 2) Inflammatory pathways are initiated; 3) Inflammatory markers are secreted; and 4) Inflammatory cells are summoned at the site of injury.^[15,16]

Markers are utilized in clinical applications to differentiate between normal and pathological biological processes and evaluate responses to treatment interventions. Inflammatory indicators may serve as predictors of inflammatory disorders.^[17,18] They correlate with the etiologies and ramifications of diverse inflammatory diseases, including cardiovascular disorders, endothelial dysfunctions, and infections.^[19,20] The inflammatory response entails a well-orchestrated network of many cell types. Activated macrophages, monocytes, and other cells facilitate localized responses to tissue injury and infection. At locations of tissue damage, compromised epithelium and endothelial cells secrete substances that initiate the inflammatory cascade, along with chemokines and growth factors that recruit neutrophils and monocytes. The initial cells recruited to an injury site are neutrophils, succeeded by monocytes, lymphocytes, and mast cells.^[21,22] Neutrophils are essential mediators of the inflammatory response, programming antigen-presenting cells to activate T cells and releasing localized factors to attract monocytes and dendritic cells.‌^[23] Numerous studies have shown that platelets influence inflammatory processes, ranging from atherosclerosis to infection. Interactions between platelets and inflammatory cells may facilitate pro-inflammatory effects. The acute phase response is the initial reaction to infection or injury, with some research suggesting that platelets initiate the acute phase response.^[24]

The neutrophil-lymphocyte ratio (NLR) is extensively utilized in nearly all medical fields as a dependable and readily accessible indicator of immunological response to diverse viral and non-infectious stimuli.^[25] NLR indicates the dynamic interaction between the innate immune response (neutrophils) and the adaptive cellular immunological response (lymphocytes) during disease and diverse pathological conditions.^[25] The NLR serves as a dependable and cost-effective marker of active cancer-related inflammation and a viable prognostic predictor for solid tumors.‌^[25] The NLR is a highly sensitive marker for infection, inflammation, and sepsis, corroborated by several researches.^[14,26-28] A clinical study validated the sensitivity of NLR for the diagnosis and stratification of systemic infection, sepsis, and bacteremia, along with its strong predictive and prognostic significance.^[25] Some authors suggest that the NLR should be assessed daily, and its absolute levels and dynamic progression should be monitored in acute disease or critical sickness cases. A large increase in NLR levels indicates the severity of critical illness, stress, and significant inflammation.^[25]

The platelet-lymphocyte ratio (PLR) has become a significant measure indicating changes in platelet and lymphocyte numbers resulting from acute inflammatory and prothrombotic conditions.^[29] PLR has been thoroughly investigated in neoplastic disorders characterized by immune suppression and thrombosis.^[29,30] The significance of PLR as an inflammatory marker escalates when its variations are analyzed in conjunction with other complementing hematologic indices, especially the NLR, which offers more insights into disease activity.^[29]

There is a lack of studies that examine hematological indices in the context of vaginitis and based on the previous promising results seen by PLR and NLR in diseases with high inflammatory status like cancer, autoimmune disease, and infection. We hypothesized that hematological parameters could have diagnostic and prognostic value to differentiate the two main causes of vaginitis, namely bacterial and function, as the former is associated with intense inflammation.

Aim

This study aimed to ascertain whether these basic hematologic markers are modified in individuals with infectious vaginitis and whether anti-infective therapy influences these parameters.

Materials and methods

Study design

We conducted a nested case-control study that included 390 women divided into three groups: healthy women (n=130), women diagnosed with BV (n=130), and women diagnosed with VVC (n=130). Additionally, two groups (BV and VVC) were treated and followed prospectively until cured. The diagnosis and classification of BV from VVC were mostly based on clinical examination, supplemented by microscopic findings.

Distinctive features of vaginal candidiasis encompass the presence of lactobacillus bacteria and inflammatory cells on wet preparation, a negative Whiff test (indicating the absence of a fishy odor upon potassium hydroxide application to the slide), and the presence of budding yeast, hyphae, or pseudohyphae observed microscopically, most readily identified on the 10% KOH preparations.^[31] These criteria were used in the current study to conf

BV diagnosis was established in the current study based on Amsel’s criteria.^[32] This clinical diagnosis necessitates the fulfillment of three out of the following four criteria: Firstly, a vaginal pH exceeding 4.5; secondly, the presence of clue cells in the vaginal secretions; thirdly, a milky, homogeneous vaginal discharge; and finally, the emission of a fishy aroma upon the addition of 10% potassium hydroxide to the vaginal fluid.^[32]

Study settings

The data were collected from a single outpatient clinic in Baghdad province between December 2023 and June 2024.

Eligibility criteria

Nonpregnant women of reproductive age (20–45 years) without chronic diseases were included in the study. The following women were excluded from the study: women on contraception, obese women (BMI≥30 kg/m²), women who received systemic or local antibacterial or antifungal therapy 10 days before entering the study, women who received anti-inflammatory drugs (including non-steroidal anti-inflammatory drugs or steroids), and women who refused to participate in the study.

Data collection

The following data were collected from the participants: age, body mass index (BMI), total white blood cells (WBC), platelet count, neutrophil count, lymphocyte count, and high-sensitivity C-reactive protein (hsCRP).

Plasma hsCRP assessment

Blood was obtained under standard conditions: 5 mL of venous blood in a plastic tube containing ethylenediaminetetraacetic acid (EDTA); after collection, the blood was centrifuged at 5000 rpm for 10 minutes to separate plasma. The hsCRP was measured using an ELISA (enzyme-linked immunosorbent assay) kit (Cat#: MBS494066, MyBioSource, USA). The test concept relies on an enzyme immunoassay, wherein a monoclonal antibody specific to CRP is immobilized on a microwell plate, while another monoclonal antibody targeting a distinct area of CRP is coupled to horseradish peroxidase (HRP). The CRP from the sample and calibrators are let to bind to the plate, followed by washing and subsequent incubation with the HRP conjugate^[33], assay sensitivity=0.01 µg/ml).

Complete blood count analysis

Complete blood count parameters were determined using the ADVIA 2120i (Siemens Healthcare), and all blood components were expressed as their total number obtained.

Sample size

Sample size was determined using G*Power version 3.1.9.7^[34,35], the effect size was 0.20, α-level 0.05, power=95%, F-family test, and the total sample size was 390 (130 in each group)^[36,37].

Ethical approval

The Research Ethical Committee of Gilgamesh University, Baghdad, Iraq, approved the study (approval number: GAU-2023-003, date: 2nd October 2023). Written informed consent was obtained from all women involved in the study.

Statistical analysis

Continuous variables were analyzed using One-way ANOVA (post hoc Tukey test used for pair-wise comparisons). The receiver operator curve (ROC) test was used to assess the diagnostic utility of various markers. Linear correlation analysis was used to determine the relationship between various blood indices presented in a matrix plot. A p-value of less than 0.05 was considered significant. GraphPad Prism version 10.3 and MedCal version 14 were used for statistical analysis.^[38]

Results

The study involved 390 women divided into three groups (each with 130 women). There was no significant difference in age (32.68±7.82, 32.39±7.79, and 31.44±7.12 years, in control, BV, and VVC groups, respectively), and no difference in BMI (24.68±3.41, 24.27±3.15, and 24.30±2.89 in control, BV, and VVC groups, respectively).

Before starting the treatment, BV had no significant difference in the mean WBC, neutrophils, NLR, PLR, and hsCRP compared to the normal control. At the same time, lymphocytes and platelets were significantly lower in BV compared to the control. hsCRP and platelets were significantly higher in VVC compared to the control, as seen in Table 1.

Table 1.

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CSV

XLSX

Assessment of hematological markers before treatment between different groups

Variables	Normal control	BV	VVC	p-value
Number	130	130	130	-
WBC ×10³ (cell/mm³)	7.4±1.7	7.8±1.8	7.4±1.7	0.067
hsCRP (µg/ml)	0.4±0.2	1.2±0.6^#	0.7±0.4^$	<0.001
Platelets ×10³ (cell/mm³)	211.8±34.6	195.5±35.1^#	222.1±36.6^$	<0.001
Neutrophils (cell/mm³)	3,970.5±579.6	4,503.5±896.0^#	3,905.1±596.8	<0.001
Lymphocytes (cell/mm³)	1,998.7±539.7	1,631.2±577.0^#	2,033.7±600.1	<0.001
PLR	116.0±44.3	138.6±63.1^#	120.3±45.3	0.001
NLR	2.2±0.7	3.2±1.6^#	2.1±0.8	<0.001

Before starting the treatment, hsCRP and NLR showed the best diagnostic utility (the highest AUC). In contrast, PLR, neutrophil count, lymphocyte count, and platelet count showed poor ability to predict BV from VVC, as seen in Table 2 and Fig. 1. hsCRP showed the best specificity, while NLR showed the best sensitivity to differentiate BV.

Figure 1.

ROC analysis of hematological markers to predict BV.

Table 2.

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XLSX

Diagnostic utility of the hematological markers to differentiate bacterial vaginosis from VVC

Variables	Cut-off	AUC	SN	SP	PPV	NPV
hsCRP	>1.29	0.721	50.00	99.23	98.5	66.5
Platelet ^#	≤212	0.692	70.00	62.31	65.0	67.5
Neutrophil ^#	>4981	0.690	33.85	100.00	100.0	60.2
Lymphocyte ^#	≤2583	0.684	98.46	27.69	57.7	94.7
PLR	>110.3	0.574	60.77	56.15	58.1	58.9
NLR	>1.9	0.735	82.31	53.85	64.1	75.3

Treatment of vaginal infections affected the hematological markers in varying degrees; WBC count, hsCRP, platelet count, and neutrophil count showed a significant reduction in both BV and VVC groups, while lymphocytes showed a significant increase in both BV and VVC groups. Both PLR and NLR showed a significant reduction in the BV group, while no significant change was seen in VVC, as seen in Fig. 2.

Figure 2.

Effect of treatment on hematological markers according to type of vaginal infection. A) WBC count, B) hsCRP, C) platelet count, D) neutrophil count, E) lymphocyte count, F) PLR, and G) NLR.

Regarding percentage change, the BV group showed a significant reduction in PLR after treatment (−22.69±7.726%) compared to the VVC group (−0.543±6.322%). Similarly, NLR showed a higher reduction percentage after treatment in the BV group (−27.41±8.151%) than VVC (0.158±5.804%), as seen in Fig. 3.

Figure 3.

Percentage change in (A) PLR and (B) NLR after treatment.

PLR and NLR showed a significant direct correlation in BV and VVC groups. Lymphocyte count was inversely correlated with NLR and NLR in both BV and VVC groups. Neutrophil count directly correlated with NLR in both BV and VVC groups. Platelet count showed a direct correlation with PLR in both BV and VVC groups, as seen in Fig. 4.

Figure 4.

Correlation matrix between various hematological markers before treatment. A) in the BV group, B) in the VVC group. WBC: white blood cell; hsCRP: high-sensitivity C-reactive protein; PLR: platelet-lymphocyte ratio, NLR: neutrophil-lymphocyte ratio

Discussion

Clinical and microscopic techniques have originally been employed to detect BV; however, these approaches necessitate proficient personnel and time and exhibit limited sensitivity and specificity. Additionally, these methods suffer some drawbacks, such as being prone to interobserver variability, as evaluating the diagnostic criteria relies on the observer’s proficiency and expertise.^[39,40] Recently, more novel diagnostics approaches have been designed; they encompass highly sensitive and specific point-of-care assays; however, these tests are expansive and require specialized laboratories.^[41] The intricate etiology of BV leads to misdiagnosis, particularly in asymptomatic carriers, resulting in suboptimal treatment and clinical consequences. The ongoing advancement of precise, user-friendly point-of-care testing for BV is essential, especially in resource-constrained environments like those seen in Iraq.

In the current study, we hypothesized that some hematological indices would change due to the increased inflammatory status during the course of BV. In the current study, NLR showed a fair ability to differentiate BV from VVC, in which higher levels predicting BV (with cut-off ≥1.9), with 82.31% sensitivity; NLR also showed a high NPV (75.3%). Moreover, the neutrophil count was significantly higher in BV compared to the other groups, coupled with lower lymphocytes in BV. The increased NLR levels observed in this study may indicate an active immunological response to bacterial infection, leading to neutrophil proliferation and a reduction in lymphocyte numbers.

The NLR is a biomarker that integrates two aspects of the immune system: the innate immune response, primarily mediated by neutrophils, and adaptive immunity, facilitated by lymphocytes.^[42] Neutrophilia and lymphocytopenia are recognized indicators of serious bacterial infection. Some have established that the NLR serves as a readily quantifiable indicator of the degree of systemic inflammation and sepsis in 90 oncology patients.^[43] Furthermore, the NLR is an effective indicator for anticipating bacteremia in emergency medical environments.^[44] Additionally, NLR was shown to discriminate between bacterial and viral infection.‌^[45] In a study involving 1468 patients with suspected bacteremia and septicemia, Gürol et al. determined that the NLR exhibited greater sensitivity than hsCRP and WBC, using procalcitonin as a reference.^[46] They concluded that NLR is a more advantageous measure for infection than CRP, exhibiting high specificity (83.9%) but modest sensitivity for identifying septicemia in critically ill patients.^[46]

The NLR is an affordable and readily accessible parameter that only requires assessment of a single test, i.e., complete blood count for its measurement. The alterations in WBC populations exhibit rapid kinetics, indicating neutrophils’ function during the inflammatory response’s initial phase. Neutrophilia is typically associated with lymphocytopenia, which has also been proposed as an effective predictor of bacteremia.^[47] Lymphocytopenia likely arises from the necessity to inhibit the adaptive immune response to prioritize innate immunity. Studies corroborate this concept by indicating that CD⁴⁺ T cells represent the most significantly changed lymphocyte subset after severe bacterial infections and sepsis.^[48]

In the present study, PLR and NLR correlated well with treatment response. Women with BV significantly reduced PLR and NLR compared to before-treatment values. In contrast, women with VVC did not show a significant reduction in PLR and NLR.

As discussed previously, blood indices correlate with the degree of inflammation in the body; since BV is usually associated with a more severe inflammatory response, this argument explains the study findings. This study is the first to show a good correlation between change in PLR and NLR with BV; this indicates that blood indices are useful tools in diagnosing and predicting BV response to treatment.

Based on the findings of the current results, it is recommended to incorporate these simple, inexpensive tests for initial diagnosis of BV, in which NLR above 1.9 in patients is highly suggestive of differential diagnosis of BV, in addition to a 20% reduction in NLR and PLR, which is highly suggestive of response to antibiotics.

Study limitations

The data was acquired from a single center, raising issues over the generalizability of the study’s results. Our data does not clarify if there is a causal association between NLR and systemic inflammation.

Conclusions

The NLR serves as an efficient diagnostic instrument for distinguishing individuals with BV from those with vulvovaginal candidiasis. Its diagnostic reliability is comparable to more regularly used markers such as WBC and hsCRP. Both NLR and PLR are good predictors of clinical response to treatment in BV.

Conflicts of interest

The author of this work has nothing to disclose.

Ethics approval

The study was approved by the College of Pharmacy, Gilgamesh University Research Ethical Committee (Approval number: GAU-2023-003, date: 2^nd October 2023), and written informed consent was obtained from all participants in the study, per the Helsinki Declaration and its later amendments.

Funding

This study did not receive any funding in any form.

Author contributions

A single author performed the study.

Data availability

Zenodo: Al-Nuaimi, A. M. A. (2024). Hematological indices in vaginitis [Data set]. Zenodo. https://doi.org/10.5281/zenodo.14051788

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgments

I want to express my gratitude to the gynecologist, Dr. Zahraa Mohammed Ali Hussein, for helping her patients with data and to the Eileen Privet Laboratory for helping carry out the laboratory test. Finally, I am grateful to Professor Shatha H. Ali for her guidance.

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