Detection of Coxiella burnetii in acute undifferentiated febrile illnesses (AUFIs) in Iran

Introduction

Acute undifferentiated febrile illnesses (AUFI) has many causes in tropical areas, including malaria, dengue fever, scrub typhus, other rickettsioses, leptospirosis, enteric fever and Q fever. ¹ Q fever has previously not been recorded in Iran. Its manifestations in humans are variable, but usually occur as an asymptomatic or mild flu-like disease with spontaneous recovery. A small minority present with more serious effects and occasionally the disease may present years later, even in the absence of primary acute symptoms. ² Bacterial DNA can now be detected by polymerase chain reaction (PCR) prior to the antibody response, thereby curtailing the diagnostic delay. A positive PCR is indicative of infection, but a negative result is inconclusive. ³ Previous studies have highlighted the hyper-endemic pattern of C. burnetii in domestic animals and human serologic studies in Iran. ⁴ This survey aimed to detect C. burnetii in UFIs by PCR in Eastern Iran.

Materials and Methods

Adult patients (aged ≥ 18 years) who were hospitalised at emergency division of Imam Reza Hospital in Mashhad from January 2014 to January 2015 with fever or recent history of a fever were included in this study. AUFI was defined as any febrile illness for ≤2 weeks and no indication of an organ-specific disease after full history and complete physical examination. Those in whom the presence of IS111 in multiple copies of the C. burnetii genome detected by nested PCR assay, as previously described ⁵ were deemed as testing positive for Q fever (Table 1).

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Table 1.

Characteristics and sequences of trans-PCR primers for C. burnetii detection.

Protocol	Primer	Sequence	Gen	Amplicon	Reference
Trans-PCR	Trans1	5-TATGTATCCACCGTAGCCAGTC-3	IS1111	687	5
	Trans2	5-CCCAACAACACCTCCTTATTC-3
nested PCR	261F	5-GAGCGAACCATTGGTATCG-3	IS1111	203	5
	463R	5-CTTTAACAGCGCTTGAACGT-3

Results

DNA was detected in seven out of 92 patient samples by nested PCR (7.36%). (Figure 1). These included five men and two women. They all lived in rural areas and their ages were in the range of 19–61 years (mean age, 39.3 ± 11.8 years). While, the longest duration of symptoms was 14 days in one case, in most cases the symptoms started 7–8 days before admission. One patient was a pregnant house wife and another a soldier who worked in his father’s livestock farm. All the others were ranchers. A history of consumption of unpasteurised milk and dairy products was noted in all. Three patients had participated in sheep or cattle delivery a few weeks before the onset of their symptoms. Physical examination revealed no organ-specific sign. However, bilateral cervical lymphadenopathy was found in one patient who also had multiple intra-abdominal and mesenteric adenopathy found on ultrasonographic examination. Bilateral interstitial infiltration on a chest radiograph was seen in the pregnant woman. Mild to moderate leucocytosis (11.2 to 16.6 × 10⁹/L) was detected in four cases, elevated total bilirubin level (42.7 umol/L) in one and abnormal liver transaminases (AST = 85 IU/L, ALT = 59 IU/L) in another case. No patient had anaemia or thrombocytopenia except one in whom Burkitt’s lymphoma was also diagnosed (haemoglobin = 7.5 g/dL, platelet count = 54 × 10⁹/L, LDH = 2179 U/L, ESR = 31). Brucellosis was ruled out in all cases. Interestingly, the man who presented with severe headache and nausea was found to have aseptic meningitis (CSF pleocytosis [WBC = 0.1 × 10⁹/L, Lym = 88%, PMN = 12%]), mild hypoglycorrhachia (CSF glucose = 2.6 mmol/L) and mildly elevated CSF protein level (7.1 mg/L). His CSF Gram stain and cultures were negative for rapid growing bacteria, fungi, mycobacterium, cryptococcal antigen and PCR for TB and HSV. His CSF cytology was reported as normal. OPEN IN VIEWER

Discussion

Q fever has been recognised as an emerging cause of AUFI in Spain, France, the UK, Guyana and Mali, with more severe cases commonly manifesting as pneumonia and/or hepatitis.^6,7 A history of contact with animals (namely cats, goats, sheep, rabbits and pigeons) prior to the onset of clinical symptoms was detected in six patients (85.7%) This has previously been documented in other studies.^8,9 A history of direct contact through work with animals or with delivery products in recent weeks was detected in five (71.4%) and 27 cases (31.8%) in the other group (without any history contact with animals) (P = 0.03). The acquisition of infection through inhalation of aerosols produced during delivery of infected sheep has been suggested as an important route of transmission of C. burnetii. ¹⁰

As in our study, the clinical picture of acute Q fever as pyrexia with flu-like symptoms is well recognised. ⁹ Other studies where common features were pneumonia (25.8%) and hepatitis (9%) ¹¹ may well be because less seriously ill patients did not have tests done for Q fever. Some of the highest rates of prevalence for Q fever have been reported in the Middle East and studies have been conducted in parallel in both human and animal populations. Recent surveys in Iran demonstrated high rates of seropositivity in both humans and livestock, including goats and sheep.^12,13 Clinical investigation of febrile illnesses of unknown origin in Saudi Arabia showed that out of 51 patients who had febrile illness in the past 4–8 weeks, 35% were seropositive for Q fever compared to just 4% of the control participants. ¹⁴ We have previously demonstrated in a cohort study that 36% of febrile patients were positive for antibody responses against phase I and II antigens, respectively, highlighting the endemicity of Q fever in the Middle East. ¹⁵ Only two previous studies used detection of anti-C. burnetii antibodies in febrile patients in Iran, and both reported high seropositivity of Q fever in south-eastern Iran.^15,16

In this study, we analysed acute-phase blood samples from patients with AUFIs, because no convalescent-phase serum samples were available. Another limitation in our study was the low number of cases. Notwithstanding these limitations, we confirm, for the first time, C. burnetii in febrile patients in Iran.