From May 1 to November 30, 2009, 463 paired samples of EDTA-anticoagulated venous blood and venous blood without additives from patients suspected of having Lyme disease were received by the Milford Hospital-affiliated Milford Medical Laboratory to be tested for Lyme disease.
Of these 463 pairs of blood samples, 130 were collected on the order of the physicians working in the hospital emergency room (ER) and walk-in clinic (WALKIN) because clinical manifestations of the patients were suggestive of Lyme disease with or without the history of a recent tick bite. Milford is a suburban town in Connecticut in which Lyme disease is endemic.
Milford Hospital is a community hospital. Its ER and WALKIN have about 40,000 patient visits a year. The local residents and practicing physicians are aware that Lyme borreliosis should always be a differential diagnosis during the months from spring to fall when a patient presents with a recent onset of fatigue, skin rash, fever, muscle aches, neck pain, joint pains or lymphadenopathy, without a clear etiology. These symptoms and signs which may vary from patient to patient are recognized as common clinical presentations in early Lyme disease in the United States .
The remaining 333 pairs of blood samples were from patients referred by their primary care private physicians in the area for possible Lyme disease.
The 130 ER/WALKIN patients had an age range between 14 and 84 years old with a median age of 42. In comparison, the 333 patients referred from the private physicians' offices had an age range between 11 and 89 with a median age of 51.
For every pair of the blood samples received, the plasma was separated from the EDTA-blood for nested PCR/DNA sequencing for the detection of B. burgdorferi 16S rDNA, which was performed at the Milford Medical Laboratory, a clinical laboratory approved by the Department of Public Health, State of Connecticut, under the Clinical Laboratory Improvement Act of 1988 to perform high-complexity laboratory testing, including PCR and DNA sequencing for the molecular identification of Borrelia burgdorferi. The latter methodology was published elsewhere . Briefly, a 100 μL aliquot of the patient plasma was mixed with 200 μL 0.7 M ammonium hydroxide in a 1.5 mL Eppendorf tube for DNA extraction. The mixture was heated at 95-98°C for 5 min with closed cap, followed by 10 min with open cap. After the tube was cooled to room temperature, 700 μL of 95% ethanol and 30 μL of 3 M sodium acetate were added to the mixture. The mixture was centrifuged at 13,000 rpm (~16,000 g) for 5 min and the supernatant discarded. The precipitate was re-suspended in 1 mL of cold 70% ethanol. Then the suspension was centrifuged at 13,000 rpm for 5 min. After all liquid was discarded, the pellet was air-dried and re-suspended in 100 μL TE buffer with heating at 95-98°C for 5 min. The heated suspension was finally centrifuged at 13,000 rpm for 5 min. One μL of the supernatant was used for primary PCR to be followed by nested PCR amplification without further purification, using a ready-to-use HiFi® DNA polymerase LoTemp® PCR mix (HiFi DNA Tech, LLC, Trumbull, CT) in a total volume of 25 μL. A trace of the primary PCR products without purification was transferred by a micro glass rod to another 25 μL LoTemp® PCR mix containing a pair of heminested (nested) primers for nested PCR amplification.
The primary PCR primers used were nucleotides LD1 (5'-ATGCACACTTGGTGTTAACTA) and LD2 (5'-GACTTATCACCGGCAGTCTTA) . The nested PCR primers were nucleotides TEC1 (5'-CTGGGGAGTATGCTCGCA AGA)  and LD2 . The thermocycling steps were programmed to 30-cycles at 85°C for 30 seconds, 50°C for 30 seconds, and 65°C for 1 minute after an initial heating for 10 minutes at 85°C, with a final extension at 65°C for 10 minutes for both primary and nested PCR in a TC-412 Thermal Cycler (Techne Incorporated, Burlington, NJ). All positive nested PCR products showing a band of expected target size on gel electrophoresis were subjected to direct automated DNA sequencing, using TEC1 nucleotide as the sequencing primer.
The serum sample was submitted for Lyme disease antibody screen by the 2-tier immunoglobulin M (IgM) and immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and Western Blot for the detection of antibodies against sonicated whole-cell B. burgdorferi by Quest Diagnostics Incorporated, Wallingford, CT, a recognized commercial reference clinical laboratory, according to the CDC guidelines .
Publication of general analytical data extracted from hospital records with concealed patient identities was approved by the Milford Hospital Institutional Review Board.