Test of 259 serums from patients with arthritis or neurological symptoms confirmed existence of Lyme disease in Hainan province, China

Abstract

Indirect Fluorescent-Antibody Test (IFA), Western Blot (WB) and Nested-PCR were applied to identify the Borrelia burgdorferi in human serum samples in Hainan province. A total of 259 serum samples were collected from Sanya Peoples’ Hospital, Hainan province. These samples were examined for the presence of B. burgdorferi serologically and etiologically by the two tier tests (IFA and WB) and Nested-PCR. 43 in total of 259 serum samples were tested positive by IFA assay, the positive rate was 16.6%. Among 43 IFA-positive samples, 6 were identified positive by WB. Nested-PCR were also used to test B. burgdorferi DNA in 259 serum samples at the same time, 27 samples were tested positive with positive rate of 10.42%. It is the first time to confirm that there are Lyme patients in Hainan province of China. The study suggested that Lyme disease should be commonly considered by clinicians with the patients who had correlated symptoms with lyme disease in Hainan.

Introduction

Lyme disease (LD), caused by the tick-borne spirochete Borrelia burgdorferi, was initially recognized in the United States in 1976 because of a geographic clustering of children who had Lyme arthritis [1,2]. Human Lyme disease generally occurs in stages, from early localized stage of erythema migrans (EM), fatigue, chills and fever and so on, to large disseminated stage of intermittent bouts of arthritis with severe joint pain and swelling, neurological symptoms, etc. [3].

While culture or visualization of B. burgdorferi from patient specimens is difficult and single serologic test is partially hampered by the occurrence stage of antibodies detecting, leading to insufficient results, so that patients may still be seronegative in early stages of infection [4]. Nowadays, the diagnosis of Lyme disease by serologic tests were recommended by CDC including screening test (Immunofluorescence Assay, IFA) and confirmation test (Western Blot, WB). Along with the development of molecular biology, PCR becomes useful way to detect the objective gene. Nowadays, Nested-PCR assay is a widely accepted way of detecting B. burgdorferi [5-7].

In China, Lyme disease has been studied since the 1980s. Until now, 29 provinces were found infection of B. burgdorferi in human beings. The investigation showed that LD is widely distributed in China with an average infection rate of 5.06% in the human population [8]. While most of the investigations concentrate on the forest area in the northeast China and Inner Mongolia. Nowadays, more and more Lyme disease in human beings were discovered in the south of China [8]. Hainan (also Hainan Island) is located in the South China Sea, separated from Guangdong’s Leizhou Peninsula to the north by Qiongzhou Strait (Figure 1). The island is renowned for its tropical climate which is totally different from northeast China and Inner Mongolia. Other researches have showed tick-borne diseases such as spotted fever group rickettsiae was a great problem in Hainan province [9,10]. However there is no report on Lyme disease before in Hainan province. In this research, we applied IFA, WB and Nested-PCR to examine the presence of B. burgdorferi in patients with arthritis or neurological symptoms in Hainan province.

Figure 1.

Distribution of 6 WB confirmed positive patients in Hainan province.

Material and methods

Standard strains and serum collection

Standard Borrelia burgdorferi sensu lato (B. valaisiana, QX-S13) was used as our positive comparison in the nested-PCR assay, which was presented by China CDC. B. burgdirferi QX-S13 was lysed by treatment with ammonium hydroxide (100°C for 15 minutes).

We collected 259 serums of patients with arthritis or neurological symptoms from Sanya Peoples’ Hospital in Sanya, Hainan Province. 5 ml of venous blood was taken from the elbow vein of each participant in the morning prior to breakfast. A total of 259 blood samples from patients with clinical diagnosis of arthritis or nervous system disease suspected of Lyme disease. We collected the serum samples with age interval 3 to 89 years and sex rate of 129/130 (male/female) from March to April in 2013. This study was approved by the Ethical Review Committee of National Institute for Communicable Disease Control and Prevention (ICDC), Chinese Center for Disease Control and Prevention (China CDC). Participants also provide their written informed consent to participate in this study.

DNA extractions from serum samples

The serum sample DNA was extracted by the DNeasy Blood & Tissue Kit (QIAGEN Group) according to manufacturer’s instructions.

Serological test

We used Two-tier testing CDC suggested to properly test for Lyme disease [11]. IFA assay was applied for screening test and WB for confirmation test.

Immunofluorescent assay (IFA)

IFA method was used to screen serum samples for anti-Bb (Borrelia burgdorferi) antibodies by PD91 antigen slides as a surrogate antigen. Sera were initially screened at dilutions of 1:128 for IgG antibody and 1:64 for IgM antibody. Titers of positive serum samples were subsequently determined to end point.

Antigen used to test anti-B. burgdorferi s. l. IgG antibodies was prepared from a Chinese human B. garinii isolate, PD 91. The PD91 isolate was cultivated in BSK-media at 33°C for a week, harvested and washed in phosphate-buffered saline. Antigen was spotted onto the wells of microtiter slides and fixed with acetone. A titer of ≥1/128 was considered positive [12].

SDS-PAGE and Western Blot (WB)

With the positive or equivocal results of the first test yielded, we conducted both the IgG and IgM Western Blot (WB) assay for further confirmation.

We dissolved the antigen using SDS-PAGE in an equal volume of lysing buffer (2× protein electrophoresis on sample short-term buffer RT209 TANGEN), and heated at 100°C for 10 min. Protein was run on polyacrylamide gels (12 acrylamide/bisacrylamide ratio 29:1; 12 cm by 14 cm by 16 mm) at 120 to 150 V for 7 hours at room temperature. The protein then were transferred to nitrocellulose membrane (16 cm×16 cm chemical, factory in Beijing) for 2 hours at 24 V and 200 mA. After the transfer, the membrane was blocked with nonfat dried milk diluted in PBST (0.01 mol/L, pH 7.0, PBS: Tween 20=2000:1) at 4°C for 24 hours. It was washed with PBS, dried and cut into strips 3 mm in width, and stored at 4°C until used.

Antigen strips were put into the seropositive samples by IFA, which diluted with PBS-T by 1:25 for both IgG and IgM, then incubated at room temperature in Orbital Shaker for 4 hours. After five washes of at least 10 min each, the strips then were incubated over night with horseradish peroxidase-conjugated rabbit antihuman IgG (1:1500) and IgM (1:2500) antibodies (Sigma), respectively. After washing for 10 min with PBST, we added 4-Chloro-1-Naphthol, cold CH₃OH, H₂O₂, and PBS to develop the color to identify the bands. The color development was stopped by purred water when the positive control serum sample reached a defined intensity.

Three pathogenic genotypes, which are B. garinii, B. afzelii and Borrelia burgdorferi sensu stricto causing Lyme disease exist in China. According to their different genomic species, the United States [13,14] and Europe [15] separately established criteria for a positive WB result. In China, criteria for a positive diagnosis of Lyme disease were established in 2010 as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, P66 and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17 and P41 in the IgM test [16,17].

Nested-PCR

All of 259 blood samples were detected B. burgdorferi by nested-PCR specific for 5S-23S rRNA intergenic spacer gene as previously described [18]. The primary PCR primers used in this research were nucleotides (5’-cgaccttcttcgccttaaagc-3’) and (5’-taagctgactaatactaattaccc-3’). The nested PCR primers were nucleotides (5’-tcctaggcattcaccata-3’) and (5’-gagttcgcgggaga-3’). The thermocycling steps were programmed to 35 cycles using a denaturing temperature of 94°C for 45 seconds, and annealing temperature of 53°C for 45 seconds, and extension temperature of 72°C for 45 seconds, and each sample (0.5 µl) was transferred to a second PCR reaction mix in which the starting primers were replaced with the nested primers, and carried out for 35 cycles at the same conditions except annealing temperature of 55°C for 45 seconds.

DNA sequencing

Positive nested PCR products were sequenced by TSINGKE biological company, China. Sequences were analyzed to validated B. burgdorferi sensu lato using the Basic Local Alignment Search Tool (BLAST) in National Center for Biotechnology Information website (NCBI).

Results

A total of 259 serum samples from patients with arthritis or nervous system disease were examined for the Borrilia burgdorferi by the Two-tier Testing and nested-PCR.

All 259 samples were screened by IFA assay which tested for antibodies in blood, and 43 were positive (IFA titer, 128 for IgG and 64 for IgM), therefore an overall infection rate was 16.6% (Table 1). The median age of the 43 patients was 56.2 years (range, 3-85 years) and 54.55% were male.

Table 1.

Results of 259 samples detected of B. burgdorferi by IFA and nested-PCR in Hainan

	Total	Positive (Positive rate, %)
IFA	259	43 (16.6)
Nested-PCR	259	27 (10.42)
Total	259	67(25.87)

All 43 IFA -positives were tested by a standardized Western immunoblot. The results showed that only 6 IFA positive samples were confirmed (Figure 1). 6 samples were all from patients with arthritis (Table 2).

Table 2.

Six in 43 IFA-positives were confirmed by WB assay

NO.	WB belts	Sex	age	Symptom	Career	Location
NO. 38	IgG: P83/100, P39 and P30	Female	14	Arthritis	Student	Ledong county
NO. 45	IgG: OspA (P31)	Male	50	Arthritis	Worker	Wanning
NO. 100	IgG: P66 IgM: P83/100	Male	85	Arthritis	Retired	Sanya
NO. 117	IgG: P83 IgM: P75	Male	51	Arthritis	Farmer	Ledong county
NO. 179	IgG: OspB (P34-35)	Male	73	Arthritis	Retired	Sanya
NO. 212	IgG: OspA (P31) IgM: OspA (P31)	Female	60	Arthritis	Farmer	Ledong county

All 259 serum samples were also tested by nested-PCR (Figure 2). 27 were determined positive with the positive rate of 10.42% (Table 1). DNA sequences of 5S-23S rRNA of 27 PCR- positive patients were compared with sequences collected in NCBI. The results showed that 20 sequences of patients had 98-100% similarities with standard sequences of B. garinii available from NCBI, and 5 sequences of patients had 98-100% similarities with standard sequences of B. afzelii, 2 sequences of patients had 100% similarities with standard sequences of B. valaisiana-related species. Moreover, 3 IFA- positive samples were determined by nested-PCR. None of the 6 WB confirmed samples were tested positive by nested-PCR assay.

Figure 2.

Results of part blood samples tested by nested-PCR. M: 100~1000 bp marker; 1-20: blood samples; PC: positive control; NC: negative control.

Discussion

Lyme disease has been studied for at least 30 years since 1980s in China. In our investigation, 43 were tested positive by IFA, suggested these patients were suspected infection with LD. The total of 6 positives identified by WB confirmed that the presence of LD in Hainan province. This is the first report of Lyme disease in Hainan province in China.

According to this survey, the results showed that the infection rate of Lyme disease was 16.6% (43 tested positive by IFA assay) in Hainan province. The age interval was 3 to 85 with 19 males and 24 females. Among them there were 7 patients with neurological symptoms and 36 with arthritis. While using WB assay, we confirmed 6 positives including 4 males and 2 females (14-85 years). All of the 6 patients were suffered from arthritis. As well as the serological diagnosis of all 259 suspected patients, we conducted the nested-PCR and 27 were tested positive. The median age of the 27 patients was 56.4 years (range, 18-86 years) with 5 unknown and 54.55% were male. 4 in 27 patients had neurological symptom and the rest 23 patients had arthritis. The results showed Lyme disease existed in patients with arthritis or nervous symptoms in Hainan, and this disease should be commonly considered by clinicians with the patients who had correlated symptoms.

Moreover, we used nested-PCR to test B. burgdorferi in serum samples of patients. We identified three genospecies sequences from patients with arthritis and nervous system disease according to the 5S-23S rRNA gene. Those were B. garinii, B. afzelii and B. valaisiana-related spirochaetes. B. valaisiana-related spirochaetes were firstly discovered in humans recently in China [19]. This might raise the possibility of further study of the diverse genospecies of Borrelia burgdorferi in Hainan province.

With final results, none of the 6 WB confirmed positives were found B. burgdorferi by nested-PCR. This may be explained by the period between the appearance of the symptoms and first medical visit in our study. Human Lyme disease generally occurs in stages, from early localized stage of erythema migrans (EM), fatigue, chills and fever and so on, to lage disseminated stage of intermittent bouts of arthritis with severe joint pain and swelling. Different stage of infection could lead to tests with various levels of sensitivity and specificity.

In our investigation, we use IFA assay as the screening test and WB assay as the confirmation test. The IFA method used in this survey was one of the standard serologic tests for LD, which can provide a useful result but its specificity remains unsatisfactory. It is therefore conducted WB assay to serologically confirm the positive samples. For its sensitivity, WB had the deficiency in use of testing of different stages of infection. Nested-PCR showed that even the WB-negative samples could be tested positive. Therefore, using etiological assay such as nested-PCR can compensate for reduced sensitivity of serological methods in some degree. So we suggested serological methods and etiological methods should be used at the same time in diagnosis of LD patients.

Disclosure of conflict of interest

None.