Prevalence of Listeria monocytogenes fecal carriers in HIV-infected and -uninfected pregnant women from Brazil

Abstract

Objective

The aim of this study is to describe the prevalence of Listeria spp. in feces of HIV-infected and -uninfected pregnant women in Brazil.

Methods

Cross-sectional study. Women on their second or third trimester of pregnancy were submitted to a clinical questionnaire and feces collection. The feces were inoculated on selective media and identification by biochemical tests combined with PCR.

Results

A total of 213 pregnant women were enrolled: 73 (34%) HIV-infected and 140 (66%) -non-infected. The prevalence of Listeria spp. and L. monocytogenes in feces of HIV-infected women were 8.2% and 2.7%. In the HIV-uninfected were 8.6% and 2.9% (p-values = 0.98 and 0.66, respectively).

Conclusion

The prevalence of fecal carriers of Listeria spp. and L. monocytogenes was not associated with HIV infection during pregnancy.

Background

Listeria monocytogenes is a foodborne pathogen. Outbreaks of human listeriosis are related to a wide variety of foods: milk products such as cheeses (mainly white or soft cheeses), meat, and ready-to-eat products (processed foods, salads, raw vegetables, sausages) [1, 2]. In humans, L. monocytogenes is mainly a gastrointestinal tract (GI) pathogen; 5 to 10% of the population is asymptomatic GI carriers [3].

Listeriosis usually occurs as a result of immunodeficiency: mainly due to impaired T cell–mediated immune response in the control of L. monocytogenes proliferation in the GI tract, which leads to bacteremia and invasion. In this context, individuals with physiological and pathological immunodeficiency are part of the risk group for listeriosis. Pregnant women, newborns, elderly, and immunocompromised individuals (such as HIV-infected patients or on immunosuppressive treatment) are the most vulnerable groups [4].

Hormonal and immunological changes that occur during pregnancy, especially in the third trimester, contribute to pregnant women being more vulnerable to infection and are up to 20 times more likely to develop listeriosis. Pregnant women with another immunosuppressive characteristic, such as those infected with HIV, may present a potentiated risk for developing listeriosis, although the interaction between pregnancy and HIV infection was not studied [5].

The aim of this study was to assess the prevalence of Listeria spp. and specifically L. monocytogenes in HIV-infected and -uninfected pregnant women.

Materials and methods

Recruitment

This is a cross-sectional study, from 2014 to 1016. Pregnant women in the second or third trimester of pregnancy were approached during their antenatal visits and were invited to participate in this study. The HIV-infected women were recruited at the Instituto de Puericultura e Pediatria Martagão Gesteira (IPPMG), a reference center for the care of HIV-infected pregnant women, affiliated to the Universidade Federal do Rio de Janeiro. The HIV-uninfected pregnant were enrolled in primary care clinics in Petrópolis (a city near Rio de Janeiro). The women who accepted to participate were interviewed and fresh stools (from the day of the interview) were collected. None of the women were using antibiotics at the time of the interview and feces collection.

HIV infection was defined based on the Brazilian Health Ministry algorithm: two different samples from different tests (serology, rapid test, or molecular based test) positive for HIV.

All samples were processed at the Laboratório de Zoonoses Bacterianas (LABZOO) from Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (Fiocruz), during the first 24 h after collection.

Detection and isolation of Listeria by phenotypic methods

A sample of fresh stools (around 5 g) were collected for each woman enrolled in the study. They were collected in sterile vials, and they were transported in low temperature 4–8 °C, and must arrived in the laboratory in less than 24 h after collection. The isolation and identification of Listeria spp. were done following the recommendations of Sauders et al. [6] with minor modifications. Briefly, approximately 1 g of feces was added to 10 ml of University of Vermont Medium Broth I (UVM I) (Oxoid) and incubated for 24 h/37 °C. A total of 0.1 ml of the obtained growth was transferred to 10 ml of UVM II (Oxoid) and incubated for 48 h/37 °C. The growth was seeded on PALCAM Agar (OXOID), incubated for 72 h/37 °C. Suspicious green colonies were selected and inoculated in tubes with a motility test medium (Oxoid) and incubated for 72 h at room temperature (25–28 °C). Colonies with motility characteristic of Listeria spp.; (umbrella motility) were plated on sheep blood agar (5%, Oxoid) to detect hemolytic activity and subjected to sugar fermentation tests according to Rocourt et al. [7]. The UVMII broths were kept refrigerated (4–8 °C) and processed again after 7, 14, 21, and 28 days of cryo-enrichment.

Detection and molecular serotyping of Listeria spp. BY PCR

A duplex PCR using primers specific for the Listeria genus (23S rRNA) and L. monocytogenes specie (hly), according to HUDSON et al. (2001), was utilized to confirm the identification of the isolates in the item (“Detection and isolation of Listeria by phenotypic methods”) as well to detect the presence of Listeria spp. in the UVM II broths.

DNA were extracted from 1 ml aliquots of the UVM II broths with the QIAamp® DNA Stool Mini Kit (Qiagen), after 7, 15, 21, and 28 days of cryo-enrichment, following the manufacturer’s instructions. The isolates positive for Listeria spp. by phenotypic methods were confirmed using the same procedure. The isolates confirmed as L. monocytogenes were analyzed according to Doumith et al. [8] for molecular serotyping.

All PCR reactions were performed in a MG96G thermocycler (Long Gene, China). The products were submitted to gel electrophoresis and visualized with the Image Quant 300 (GE). The standard strains ATCC 19,111 (CLIST 2035), CDC F4976 (CLIST 2032), CDC F6254 (CLIST 2033), and CDC F4555 (CLIST 2045) were used as positive controls for all reactions. Reaction tubes without template DNA were used as negative controls. Samples/colonies positives for 23S rRNA only were considered Listeria spp., with the final identification achieved by the phenotypic method.

Statistical analysis

All data were stored on spreadsheets from Excel 16.0 and exported to the STATA 13.1 (Texas, EUA).

We studied the prevalence of L. monocytogenes fecal carriers (defined as genotyping or phenotyping isolation of L. monocytogenes on a fresh stool sample) in HIV-infected and -non-infected pregnant women. The distribution of the continuous variables was described according to the measures of central tendency (median) and dispersion (interquartile range-IQR), while the categorical variables were summarized by frequency measures. The analysis was performed using Fisher exact test (categorical variables) and Mann–Whitney test (continuous variables).

Results

From 2014 to 2016, a total of 213 pregnant women were included in the study: 73 HIV-infected (all under use of antiretrovirals at the study entry) and 140 not HIV-infected. Their median age was 27 years old (interquartile range (IQR), 22–33), and the median gestational age at the study entry was 26 weeks (IQR = 19–32). All women were asymptomatic at the time of the study inclusion.

Listeria spp. was detected in the stools of 16 pregnant women (7.5%), in both the phenotypic and genotypic methods. L. monocytogenes was detected in five samples (2.4%) with the phenotypic method and in 6 samples (2.8%) with the genotypic method (Table 1). There was no statistical difference on the prevalence of Listeria spp or L. monocytogenes between HIV-infected and non-HIV-infected pregnant women. All samples positive in the phenotypic method were also PCR positive, except for two samples of L. grayi isolated. Three samples were PCR positive, but negative in culture with the selective media utilized.

Table 1.

Prevalence of Listeria spp. and L. monocytogenes from fecal specimens of HIV positive and negative pregnant women

Source	Phenotypic method	P value	Genotypic method	P value
Listeria spp.		0.59		0.91
HIV positive women	4 (5.5%)		6 (8.2%)
HIV negative women	12 (8.6%)		10 (7.2%)
Total	15 (7.5%)		16 (8.0%)
L. monocytogenes		0.66		0.98
HIV-positive women	1 (1.4%)		2 (2.7%)
HIV-negative women	4 (2.9%)		4 (2.9%)
Total	5 (2.4%)		6 (2.8%)

Regarding the weekly attempts to isolate Listeria spp., we successfully recovered isolates from nine samples in all weeks, but in two samples, Listeria spp. were isolated only in the first week and three samples only presented isolates after two weeks of cryo-enrichment.

The species of Listeria non-monocytogenes isolated were: L. innocua (N = 8), L. grayi (N = 2), and L. seeligeri (N = 1). The serotypes found in the five samples of L. monocytogenes detected in culture were 1/2a (N = 2), 4b (N = 2), and 1/2b (N = 1).

Discussion

In this study, we were able to demonstrate that the prevalence of Listeria spp. and L. monocytogenes GI carriers, in both groups was 8% and 3%, respectively. Some samples were positive in the phenotypic method (culture) only after 2 weeks of cryo-enrichment, reinforcing the importance of this procedure before registering the sample as negative (6). There was no difference between HIV-infected and -non-infected pregnant women regarding Listeria spp. prevalence in the fecal samples.

Considering the GI tract carriers, our findings are in agreement with other studies, with prevalence rates ranging from 3.3 to 8% [6, 9]. In this study, we isolated strains of L. innocua, L. grayi, and L. seeligeri which, although very rarely infects humans, were also isolated from human samples by other authors. The isolation of other Listeria spp. may indicate the presence of L. monocytogenes and sometimes other serotypes can overgrow in the culture media, especially L. innocua [9].

During the determination of L. monocytogenes serotypes, it was possible to verify the predominance of 1/2a, 4b, and 1/2b in the fecal samples, corroborating with the results of studies conducted in several countries [2]. These results bring a warning to public health authorities, since these serotypes were also frequently implicated in foodborne outbreaks [4].

We hypothesized that the role of HIV infection on listeriosis, is based on the immunosupression, rather than GI colonization per se, since the majority of HIV-infected women were taking antiretrovirals with high adherence, which might also explain the lack of difference on the L. monocytogenes GI carrier prevalence between HIV-infected and -non-infected pregnant women, although the relative small sample size may play a role.

In conclusion, the prevalence of Listeria spp. or Listeria monocytogenes GI colonization was the same between HIV-infected and -uninfected pregnant women, in a middle-income country as Brazil.

Funding

This work was supported by CNPq—(grant numbers: 306699/2014–1 for EH and 304476/2018–8 for CBH) and CAPES—for IGRF.

Data availability

As per request.

Declarations

Conflicts of interest

The authors declare no competing interests.

Ethical considerations

The study was approved by the Research Ethics Committee of the Instituto de Puericultura e Pediatria Martagão Gesteira (IPPMG) of UFRJ under CAAE no. 608.313.

All patients signed informed consent.