The First Serological Study of Q Fever in Humans in Lebanon

Abstract

Objective:

The aim of this study was to estimate, for the first time, the human seroprevalence of Q fever in Lebanon, by assessing the presence of antibodies against the causative agent, Coxiella burnetii. A total number of 421 serum samples (226 females and 196 males) were collected in February 2015 from hospitals and laboratories dispersed in five Lebanese provinces: Akkar, Bekaa, Mount Lebanon, Nabatieh, and South Lebanon.

Methods:

Serial testing approach was used. Samples were first screened for IgG phase II antibodies against C. burnetii by Enzyme Linked Immunosorbent Assay (ELISA) Kit. Then, both positive and inconclusive sera were reexamined by immunofluorescence assay (IFA) test with the aims to confirm and specify the infection status (past or probably acute infection) by detecting IgG (I/II) and IgM (I/II) in human sera.

Results:

Screening of 421 samples was estimated to be 38.70% (95% CI 34–43.3) positive samples, 5.90% (95% CI 3.7–8.2) suspect samples (as doubtful results), and 55.40% (95% CI 50.7–60.1) negative samples. Furthermore, all positive and suspect samples by ELISA test were retested by immunofluorescence assay test (IFAT), and the prevalence of positive sample was 37% and the infection case was recorded: 23.75% (95% CI 19.7–27.8) samples resulted from past infection, 1.9% (95% CI 0.6–3.2) probably acute infection characterized by several dominance clinical symptoms as: fever, cough, headache, difficulty breathing, and atypical pneumonia, and 0.23% (95% CI 0–0.7) inconclusive sample accompanied by different symptoms as bone metastasis and lung cancer.

Conclusion:

The study records the exposition of 37% of 421 patients to C. burnetii distributed in five Lebanese provinces with the highest seroprevalence in Bekaa and Akkar provinces and the lowest reported in Mount Lebanon. This difference may be due to the presence of high density of livestock production and of major agricultural areas in these two provinces.

Introduction

Q fever or Query fever is a worldwide zoonotic disease caused by Coxiella burnetii, an obligate intracellular Gram-negative bacterium (Maurin and Raoult 1999), present inside phagolysosomes of the host eukaryotic cells (Dupont et al. 1994).

C. burnetii expresses two antigens named phase I and phase II antigens. Phase I is the natural high pathogenic phase present in natural hosts. After subculture in laboratory, modification of the lipopolysaccharides results in an antigenic shift to the less infectious phase II (Williams 1991).

The clinical symptoms of Q fever are characterized by nonpathognomonic signs, about 40% of people infected with C. burnetii show clinical signs (Fournier et al. 1998). The symptoms of acute disease may cause fever, severe headache, general malaise, myalgia, chills, sweats, nonproductive cough, chest pain, atypical pneumonia, myocarditis, nausea, vomiting, diarrhea, abdominal pain, meningitis, and encephalitis (Fournier et al. 1998, Maurin et al. 1999, Knobel et al. 2013). Fever generally lasts 2–3 weeks. Acute Q fever is fatal in less than 2% of cases. In a matter of weeks to years after an acute infection, several symptoms have been observed as aortic aneurysms, granulomatous lesion in bones, liver, or reproductive organs (Maurin et al. 1999, Fenollar et al. 2001). In addition to that, abortion in pregnant women and fetal morbidity may be observed (Nielsen et al. 2013). Even more, clinical diagnosis of Q fever is difficult, due to the unspecific symptoms. Laboratory diagnosis, that is mainly based on serological methods, is a tool to establish the presence of specific antibodies, 7–21 days after the onset of acute symptoms, with the phase II antibody response earlier and higher than the phase I antibody response. Immunoglobulin M (IgM) antibodies to phase II antigen develop during the second week of acute illness, with an increase in phase II IgG occurring almost simultaneously. In contrast, unsuccessfully treated or spontaneously resolving disease, IgG and IgM titers to phase I antigen might continue to increase later in specimens, but typically do not exceed phase II titers (CDC 2013).

Among the commercially available serological assays, enzyme linked immunosorbent assay (ELISA) test gives results comparable with the reference method, immunofluorescence assay test (IFAT), reaching high performance (99% sensitivity and 88% specificity) (Fournier et al. 1998). The interpretation of serologic results must include differential reactivity to Coxiella antigens.

Q fever's presence in the Middle Eastern areas is being reported from Syria (Bottieau et al. 2000), Iraq (Faix et al. 2005), Cyprus (Psaroulaki et al. 2006), and south-Eastern Iran (Esmaeili et al. 2016). Information on the presence of C. burnetii infection in Lebanon is scanty and only related to a survey performed in the last century that recorded the C. burnetii infection between both sick and healthy people (Garabedian et al. 1956).

To assess the prevalence of C. burnetii infection, a serological survey was performed in several provinces of Lebanon.

Serologic tests evaluate antibodies through two distinct antigenic forms of C. burnetii that is referred to as phase I and phase II.

In this study, an ELISA test was used for screening purpose (Peter et al. 1987) followed by IFAT which is considered the reference method for serodiagnosis of Q fever and is able to detect and discriminate IgG and IgM in phases II and I (Angelakis et al. 2011).

Materials and Methods

Study area

Lebanon (10,452 km²) is located on the East Coast of the Mediterranean Sea. The Lebanese population is about 4,132,000 in 2014. This study was focused in five Lebanese provinces: Akkar, Bekaa, Mount Lebanon, Nabatieh, and South Lebanon.

Samples

On February 2015 a survey was performed to obtain information on the presence of Q fever antibodies in Lebanon where this disease is not usually investigated. Based on the availability of samples and the compliance of hospitals and laboratories to participate in the study, 421serum samples (226 females and 196 males) were obtained from hospitals and laboratories distributed in five Lebanese provinces: Akkar, Bekaa, Mount Lebanon, Nabatieh, and South Lebanon. Samples were collected from patients attending hospitals and laboratories and submitted to blood venipunctures, presenting severe clinical symptoms. No anamnesis histories were obtained due to privacy reasons, except for nine people who decided to give information.

Methods

A serial testing approach was performed. Two different serological tests were conducted, ELISA and IFA, to determine the presence of antibodies. For IFA test, samples were previously treated using human IgG sorbent, to avoid both false positive results (by eliminating the rheumatoid factor) and false negative results (due to an excess of IgG antibodies).

A commercial ELISA Kit from Panbio (Queensland, Australia; Cat. No. E-QFB01G) was utilized, as described by the manufacturer, to screen all samples for the detection of IgG anti-phase II antibodies against C. burnetii, using a purified C. burnetii phase II antigen. This class of antibodies appears earlier after exposure to the bacterium.

The optical density (OD) value of each sample was measured by spectrophotometer (BioTek ELX800 or Multiskan EX). The OD of each sample was divided by the cutoff value of the test. The antibody index was calculated accordingly to the formula: (sample OD/cutoff serum) × 10. (The results were expressed in units according to the instructions).

The sample was considered as positive (the presence of IgG II in sera suggests the exposure of patient to C. burnetii) if the antibody index was higher than 11, as equivocal (this case is inconclusive) if the value was between 9 and 11, and if it was less than 9 the sample was considered as negative.

The serological sensitivity of Panbio Kit is between 64.6% and 79.8%, and the serological specificity is between 96.6% and 100%.

Furthermore, any sample which resulted positive or inconclusive by ELISA was tested by IFA using a commercial kit from Vircell (S.L, Granada, Spain; Cat. No. PCOBUI+II). Each slide contains 10 pairs of wells, coated with C. burnetii Nine Mile strain Q (ATCC VR-616) able to find antibodies against IgG and IgM. Each pair of wells contains one well with phase I and other well with phase II.

The IFA test was carried out not only to confirm the positivity of samples for IgG II antibodies in ELISA but also to detect the presence of IgG I and IgM (I and II phases). The cutoff of seropositivity for IgM I and II was ≥1/50, and it was ≥1/64 for both IgG I and II.

Some studies were performed to interpret the results of IFA test as described below:

The patients were considered to have acute Q fever infection when the cutoff points were: for IgM II antibodies ≥1:50 and for IgG II ≥1:256 accompanied with clinical syndrome as fever, pneumonia, hepatitis, and flu-like syndrome (Dupont et al. 1994). Furthermore, the patients were considered to have past Q fever infection when any IgG II ≥1:128 and without clinical symptoms; and the patients were considered seronegative infection if IgM titers <1/50 and IgG titers <1/64.

In Vircell IFA Kit, the serological sensitivity is 93.8%, 97.2%, 100%, and 100% and the serological specificity is 75%, 100%, 53.6%, and 56.3%, respectively, for IgG I, IgG II, IgM I, and IgM II as described by the manufacturer.

Statistical analysis

To compare the prevalence of seropositivity based on IFA results against C. burnetii human infection among the five Lebanese provinces, Pearson's chi-square test was used, with p ≤ 0.05 as the level of significance.

Results

Among the 421 human sera tested by indirect ELISA for IgG II antibody detection, 195 (46.30%) were from male patients and 226 (53.70%) from females. A total number of 163/421 (38.71%; 95% CI 34.0–43.4) samples were positive, 25/421 (5.95%; 95% CI 3.7–8.20) were equivocal, and 233/421 (55.4%; 95% CI 50.7–60.1) were negative (Table 1).

Table 1.

Presence of IgG II Antibodies Against Coxiella burnetii by ELISA Test

Variable	Frequency (n)	Category	Akkar	Bekaa	South Lebanon	Nabatieh	Mount Lebanon
IgG II	—	Positive	—	—	—	—	—
Male	195	84/195 (43.1%)	18/42 (42.86%)	21/39 (53.8%)	8/25 (32%)	29/66 (43.9%)	8/23 (34.78%)
Female	226	79/226 (34.96%)	12/26 (46.15%)	18/41 (43.9%)	14/51 (27.4%)	29/78 (37.15%)	6/30 (20%)
Total	421	163/421 (38.70%)	30/68 (44.11%)	39/80 (48.75%)	22/76 (28.94%)	58/144 (40.27%)	14/53 (26.41%)

To show the distribution of seropositive samples among the five Lebanese provinces, IFA test was performed on the same 188/421 positive and suspect samples by ELISA test to discover both phases of IgG I and II and IgM I and II antibodies (Table 2).

Table 2.

Distribution of Seropositive Samples Against Coxiella burnetii by IFA Test in Five Lebanese Provinces

Variable	Frequency (n)	Positive ^*	Prevalence with CI (95%)	Akkar	Bekaa	South Lebanon	Nabatieh	Mount Lebanon
Male	195	83/195	42.6% (35.7–49.5%)	17/42 (40.47%)	18/39 (46.15%)	11/25 (44.0%)	29/66 (43.9%)	8/23 (34.78%)
Female	226	73/226	32.3% (26.2–38.4%)	11/26 (42.3%)	15/41 (36.6%)	15/51 (29.4%)	27/78 (34.6%)	5/30 (17.0%)
Total	421	156/421	37.0% (32.4–41.6%)	28/68 (41.2%)	33/80 (41.25%)	26/76 (34.2%)	56/144 (39.0%)	13/53 (24.5%)

Positive^*: serum sample with specific antibodies IgG (I and/or II) with or without IgM (I and/or II) against C. burnetii.

IFA, immunofluorescence assay; IgM, immunoglobulin M.

Furthermore, from 188 patients (96 males and 92 females), 32/188 were seronegative (17%; 95% CI 11.6–22.4) and 156/188 (83%; 95% CI 77.6–88.4) were seropositive by IFA test that detected the presence of different phases of IgG and IgM as described in Table 3.

Table 3.

(IgG I and IgG II), (IgM I and IgM II) Detection and Title by IFAT in ELISA IgG II Positive and Suspect Serum Samples and Its Status of Infection

Category	IFAT, IgG I	IFAT, IgG II	IFAT, IgM I	IFAT, IgM II	Number and prevalence of samples	Status ^a of infection
Seronegative	Negative	Negative	Negative	Negative	31/188 (16.8%)	No infection
Seronegative	Negative	Negative	Negative	<1/50	1/188 (0.5%)	No infection
Seropositive by IgG II	Negative	1/64	Negative	≤1/25	40/188 (21.3%)	Exposed to infection
Seropositive by IgG I	1/64	1/32	Negative	Negative	1/188 (0.5%)	Exposed to infection
Seropositive by IgG I and IgG II	1/64	1/64	Negative	Negative	3/188 (1.6%)	Exposed to infection
Seropositive by IgG II and IgM I	Negative	1/64	1/50	Negative	2/188 (1%)	Exposed to infection
Seropositive by IgG II and IgG I	1/512	1/128	Negative	Negative	1/188 (0.5%)	Inconclusive case
Seropositive by IgG I and IgG II	1/128	1/128	Negative	≤1/25	1/188 (0.5%)	Exposed to infection
Seropositive by IgG II	Negative	1/128	≤1/25	≤1/25	56/188 (30%)	Past infection
Seropositive by IgG II	Negative	1/128	Negative	1/25	1/188 (0.5%)	Past infection
Seropositive by IgG II and IgM II	Negative	1/128	Negative	1/50	1/188 (0.5%)	Past infection
Seropositive by IgG II	Negative	1/256	Negative	≤1/25	32/188 (17%)	Past infection
Seropositive by IgG I and IgG II	1/128	1/256	Negative	Negative	10/188 (5.3%)	Past infection
Seropositive by IgG II and IgM I and IgM II	Negative	1/256	1/50	1/50	2/188 (1%)	Probably acute case
Seropositive by IgG I and IgG II and IgM II	1/128	1/256	≤1/25	1/50	1/188 (0.5%)	Probably acute case
Seropositive by IgG II and IgM II	Negative	1/256	Negative	1/50	2/188 (0.5%)	Probably acute case
Seropositive by IgG I and IgG II and IgM II	1/64	1/256	Negative	1/50	1/188 (0.5%)	Probably acute case
Seropositive by IgG II and IgM II	Negative	1/256	Negative	1/100	2/188 (0.5%)	Probably acute case
—	18	156	4	9	188	—

The reference that was used to describe the status of patients is Healy et al. (2011) and Dupont et al. (1994).

IFAT, immunofluorescence assay test.

Compared to ELISA results, IFAT confirmed 149/163 (91.40%) as positive sera; as for the doubtful results, 7/25 (28%) resulted as seropositive. Therefore, the total positivity for IgG II was 156/188 (83%; 95% CI 77.6–88.4).

There were four different infection cases distributed in different Lebanese provinces, as described in Table 4: 100/421 (23.75%; 95% CI 19.7–27.8) was due to past infection distributed among 54 males and 46 females, whereas 47/421 (7.6%; 95% CI 19.7–27.8) exposed to infection (24 males and 23 females) while one patient (male) had the antibody titers: IgG I = 1/512, IgG II = 1/128; IgM (I, II) = 0, and the pathology was: bone metastasis and lung cancer; he passed away some weeks after the test. Finally, eight patients, four males and four females, had IgG II = 1/256 with IgM II ≥1/50, accompanied by different clinical symptoms: fever, cough, headache, difficulty breathing, chest pain, myalgia, diarrhea, and atypical pneumonia, which can be considered as acute case (Table 5).

Table 4.

IFA Results in Males and Females Distributed in Five Lebanese Provinces

Province	Number of past infections	Number of probable acute infections	Number of inconclusive infections	Number of seropositive IgG II	Number of seropositive IgG I	Number of seropositive IgG I and IgG II	Number of seropositive IgG II and IgM I	Prevalence of seropositive and ICg
Akkar	12♂	2♂	1♂	1♂	0♂	1♂	0♂	28/68 (41.2%; IC_95%: 29.5–52.9%)
	4♀	0♀	0♀	7♀	0♀	0♀	0♀
Bekaa	14♂	2♂	0♂	1♂	0♂	0♂	1♂	33/80 (41.25%; IC_95%: 30.5–52%)
	10♀	1♀	0♀	3♀	0♀	1♀	0♀
Mount Lebanon	6♂	0♂	0♂	2♂	0♂	0♂	0♂	13/53 (24.5%; IC_95%: 12.9–36.1%)
	5♀	0♀	0♀	0♀	0♀	0♀	0♀
Nabatieh	15♂	0♂	0♂	11♂	0♂	2♂	1♂	56/144 (39%; IC_95%: 31–47%)
	14♀	3♀	0♀	10♀	0♀	0♀	0♀
South Lebanon	7♂	0♂	0♂	4♂	0♂	0♂	0♂	26/76 (34.2%; IC_95%: 23.5–44.9%)
	13♀	0♀	0♀	1♀	1♀	0♀	0♀
Total: ♂+♀
156	54♂	4♂	1♂	19♂	0♂	3♂	2♂	156/421 (37%; IC_95%: 32.4–41.6%)
	46♀	4♀	0♀	21♀	1♀	1♀	0♀

Table 5.

Clinical Symptoms of Probably Acute Cases Appeared in Eight Patients

Patient No.	1	2	3	4	5	6	7	8
Sex	♂	♂	♂	♂	♀	♀	♀	♀
Age	56	35	35	33	44	30	52	60
Fever	+	+	+	+	−	+	+	+
Cough	+	+	+	+	−	+	+	+
Headache	+	+	+	+	−	+	+	+
Difficulty breathing	+	−	+	+	−	+	−	−
Chest pain	−	−	+	−	+	+	−	−
Myalgia	−	−	−	+	+	−	+	−
Diarrhea	−	+	−	−	−	−	−	−
Atypical pneumonia	+	+	−	+	−	+	−	−

+, presence of clinical symptoms; −, absence of clinical symptoms. 1 and 2—from Akkar Province; 3, 4, and 5—from Bekaa Province; 6, 7, and 8—from Nabatieh Province.

Statistical analysis

The seroprevalence based on IFA test indicated no significant difference between the five Lebanese provinces (p > 0.05) using Pearson's chi-square test (Table 6).

Table 6.

Prevalence of Seropositivity (IgG and/or Without IgM) Against Coxiella burnetii by IFA Test in Human Sera in the Different Lebanese Provinces

Province	Number of positive and negative human sera	Number of seropositive (Prevalence, %)	p
Akkar	68	28 (41.2)	0.24
Mount Lebanon	53	13 (24.5)	0.24
Akkar	68	28 (41.2)	1.00
Bekaa	80	33 (41.25)	1.00
Akkar	68	28 (41.2)	0.94
Nabatieh	144	56 (39)	0.94
Akkar	68	28 (41.2)	0.67
South Lebanon	76	26 (34.2)	0.67
Bekaa	80	33 (41.25)	0.22
Mount Lebanon	53	13 (24.5)	0.22
Bekaa	80	33 (41.25)	0.92
Nabatieh	144	56 (39)	0.92
Bekaa	80	33 (41.25)	0.65
South Lebanon	76	26 (34.2)	0.65
Mount Lebanon	53	13 (24.5)	0.24
Mount Lebanon	144	56 (39)
Nabatieh	53	13 (24.5)
Mount Lebanon South Lebanon	76	26 (34.2)	0.50
Nabatieh	144	56 (39)	0.74
South Lebanon	76	26 (34.2)	0.74

Furthermore, the comparison of IFA seropositive results between female and male recorded no significant difference between the two groups (using chi-square test) (p > 0.05).

The agreement between the two tests ELISA and IFA to detect IgG II was 149/163 (91.40%).

For seropositivity, the highest seroprevalence (41.25%) (33/80) was discovered in Bekaa and (41.20%) (28/68) in Akkar compared to the average (34.8%) of other provinces (95/273), with the odds ratio of being seropositive 1.31 times higher for Bekaa and for Akkar compared to other provinces.

Discussion

The findings of the human infection status confirmed the exposition of patient to C. burnetii with prevalence of 37% and were described as: 100/421 (23.75%) past infection, 47/421 (11.16%) exposed to infection, 1/421 (0.23%) inconclusive (this sample was hampered by the death of patient leading to an inconclusive outcome), and 8/421 (1.9%) indicate a probable acute infection because they were characterized by clinical evidence of infection (CDC 2013). Indeed for the laboratory confirmation, we need to test another sample from these patients after 3–6 weeks (CDC 2013), but it was disserved to receive a request of follow-up serum samples from these patients.

This study shows the presence of IgG and IgM antibodies in both phase I and II against C. burnetii in human sera.

The present IgG II investigation suggests that ELISA had detected IgG II in 163/421 seropositive samples, and among those 163 positive results retested by IFA test only the positivity of 149 was confirmed. The difference between the two tests could also be related to the antigen strains used in the two kits, as ELISA Panbio can give false positive results due to the contamination of serum with other bacteria such as Leptospirosis (as described by the manufacturer of kit) and to high specificity of IFA test.

The ELISA and IFA results obtained from human sera showed the presence of different antibodies IgM and IgG against C. burnetii with the highest seropositive prevalence in Bekaa and Akkar provinces and the lowest was recorded in Mount Lebanon. The difference can be explained by the presence of major agricultural areas in these two provinces that spread by the vegetables mainly Zucchini and eggplants in Bekaa plain and planted by the different kind of trees, mainly citrus and olive, in Akkar. This type of cultivation requires manure; for this reason a high percentage of Lebanese planters purchase the untreated dry manure from small ruminant's farmers distributed in all Lebanese provinces at a low price and use it as fertilizer for the crops. So, this manure can be considered as an important source of environmental contamination and transmission to humans through aerosol route for several months. In addition, these provinces are known by the highest density of livestock production with semiextensive management system for sheep and goats that can play a main role in spreading the bacteria in the environment through the products of infected ruminants as birth products, urine, and feces.

While indeed there aren't any confirmed information about the exposure of these patients to the source of the infection by C. burnetii, there are several conditions that may contribute to human infection such as spreading the contaminated dust by the wind; contact with infected animals as ruminants and pets; living near an infected farm; the presence of different species of ticks in Lebanon (Dabaja et al. 2017) that can transmit this bacteria and spread it by feces for several months in the environment; drinking unpasteurized milk or eating dairy products obtained from raw milk; and working as veterinarians, farmers, planters, or laboratory technicians, because they are in direct contact with animals and their products.

Some previous studies have been recorded in humans using ELISA to detect IgG II, revealing high seropositive rates such as 36% in Bardsir (Southeast of Iran) and in Northern Togo (Khalili et al. 2010, Anna et al. 2013) and 65.22% from humans working and living in farms in Germany (Eibach et al. 2012).

Concerning the IFA, some investigation on the seroprevalence for different types of Immunoglobulin (IgG I, IgG II, and IgM II) has been performed worldwide. For IgG II, the seroprevalence was 5.3% in Barcelona (Northeast of Spain) using the titer 1/40 as cutoff value (Cardeňosa et al. 2006), while the titer 1/32 as cutoff value was reported in different countries, with the following results: 36% of IgG II in Northern Togo (Anna et al. 2013); 23.40% and 30.90% of IgG I and IgG II, respectively, in Rural Western Kenya (Knobel et al. 2013) and 97.60%, 98.90%, and 1.20% of IgG I, IgG II, and IgM II among dairy cattle farmers in Netherlands (Schimmer et al. 2014). Furthermore, the titer 1/16 for IgG I and IgG II was used as cutoff value in Northern and Northwestern Thailand (Blacksell et al. 2015), with 2.40% IgG II and 0.27% IgG I prevalence from hospitalized and medical clinic patients.

This work was the first survey on human sera to screen IgG II by ELISA test and to detect the IgG and IgM antibodies on both phases I and II against C. burnetii of IFA test. Finally, we can conclude the exposition of Lebanese patients to C. burnetii, distributed in Lebanon mainly in the five Lebanese provinces. Anyway, data obtained with the present research implicate the need for a strong improvement of public health awareness about human Q fever in Lebanon as providing the date of onset of disease and referring requested follow-up serum samples to confirm the infection case. Further studies should be performed in humans distributed in all Lebanese provinces. Some prevention should be carried out by the Ministry of agriculture to decrease the source of human infection as putting vaccination programs for ruminants and managing the manure to kill bacteria before subsequent application to agriculture ground.

Footnotes

Acknowledgments

The authors thank the Director General of Lebanese Agricultural Research Institute (LARI) Dr. Michel Afram and our many friends who sent serum samples from different regions of the country. This study was supported by Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”–Italy, Lebanese Agricultural Research Institute and Doctoral School of Sciences and Technology (EDST) at Lebanese University.

Author Disclosure Statement

No competing financial interests exist.

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