Raymond W. Ryan

Persistent Parasitemia after Acute Babesiosis

BACKGROUND Babesiosis, a zoonosis caused by the protozoan Babesia microti, is usually not treated when the symptoms are mild, because the parasitemia appears to be transient. However, the microscopical methods used to diagnose this infection are insensitive, and few infected people have been followed longitudinally. We compared the duration of parasitemia in people who had received specific antibabesial therapy with that in silently infected people who had not been treated. METHODS Forty-six babesia-infected subjects were identified from 1991 through 1996 in a prospective, community-based study designed to detect episodes of illness and of seroconversion among the residents of southeastern Connecticut and Block Island, Rhode Island. Subjects with acute babesial illness were monitored every 3 months for up to 27 months by means of thin blood smears, Bab. microti polymerase-chain-reaction assays, serologic tests, and questionnaires. RESULTS Babesial DNA persisted in the blood for a mean of 82 days in 24 infected subjects without specific symptoms who received no specific therapy. Babesial DNA persisted for 16 days in 22 acutely ill subjects who received clindamycin and quinine therapy (P=0.03), of whom 9 had side effects from the treatment. Among the subjects who did not receive specific therapy, symptoms of babesiosis persisted for a mean of 114 days in five subjects with babesial DNA present for 3 or more months and for only 15 days in seven others in whom the DNA was detectable for less than 3 months (P<0.05); one subject had recrudescent disease after two years. CONCLUSIONS When left untreated, silent babesial infection may persist for months or even years. Although treatment with clindamycin and quinine reduces the duration of parasitemia, infection may still persist and recrudesce and side effects are common. Improved treatments are needed.

Evaluation of Two-Test Serodiagnostic Method for Early Lyme Disease in Clinical Practice

The Centers for Disease Control and Prevention (CDC) recommend a two-test approach for the serodiagnosis of Lyme disease (LD), with EIA testing followed by Western immunoblotting (WB) of EIA-equivocal and -positive specimens. This approach was compared with a simplified two-test approach (WB of EIA equivocals only) and WB alone for early LD. Case-patients with erythema migrans (EM) rash >/=5 cm were recruited from three primary-care practices in LD-endemic areas to provide acute- (S1) and convalescent-phase serum specimens (S2). The simplified approach had the highest sensitivity when either S1 or S2 samples were tested, nearly doubling when S2 were tested, while decreasing slightly for the other two approaches. Accordingly, the simplified approach had the lowest negative likelihood ratio for either S1 or S2. For early LD with EM, the simplified approach performed well and was less costly than the other testing approaches since less WB is required.

Single dose azithromycin treatment of gonorrhea and infections caused by C. trachomatis and U. urealyticum in men

BACKGROUND AND OBJECTIVES Single dose regimens have advantages in the treatment of STD. Azithromycin has unique pharmacokinetics that may make single dose regimens feasible. Treatment with a single 1 g dose of azithromycin was compared to 100 mg doxycycline twice daily for seven days. STUDY DESIGN This was a randomized third-party blinded study on 183 male patients, 176 of whom could be evaluated for efficacy. RESULTS Chlamydia trachomatis was cultured from 148 patients, 79 receiving azithromycin and 69 receiving doxycycline. Six patients receiving azithromycin had positive cultures on follow-up, four were known to have had sexual intercourse with infected partners. Fifty-one patients had gonorrhea; 28 were treated with azithromycin and 23 with doxycycline. Neisseria gonorrhoeae was eradicated from all patients except one receiving azithromycin. He denied sexual exposure during follow-up. Sixty patients were infected with Ureaplasma urealyticum, 35 were treated with azithromycin and 25 with doxycycline. Five patients in each group had positive cultures on follow up. Three patients receiving azithromycin and two receiving doxycycline were known to have had sexual exposure during follow-up. CONCLUSION A single dose of azithromycin showed similar effectiveness as a 7-day regimen of doxycycline.

Clinical and ecological characteristics of Vibrio vulnificus in the northeastern United States.

Multiple seawater sites in the northeastern United States, particularly Long Island Sound, and shellfish from Long Island Sound were sampled from April to November for 3 successive yr, 1983-1985. Hospitals in coastal and metropolitan areas of Connecticut were surveyed for the same 3-yr period, Vibrio vulnificus can be found in these waters during the summer months. The appearance of these virulent bacteria in both seawater and shellfish are a function of the water temperature; no V. vulnificus could be isolated until the temperature was approximately 17 degrees C. Although the risk of infection is small, as shown by isolation of this organism from patients, certain high-risk groups exist. Consumption of raw shell fish during the summer months should be discouraged in people with liver disease or patients on immunosuppressive therapy.

Confirmation of Tick Bite by Detection of Antibody to Ixodes Calreticulin Salivary Protein

ABSTRACT Ticks introduce a variety of pharmacologically active molecules into their host during attachment and feeding in order to obtain a blood meal. People who are repeatedly exposed to ticks may develop an immune response to tick salivary proteins. Despite this response, people usually are unaware of having been bitten, especially if they are not repeatedly exposed to ticks. In order to develop a laboratory marker of tick exposure that would be useful in understanding the epidemiology of tick-borne infection and the immune response to tick bite, we developed an enzyme-linked immunosorbent assay (ELISA) to detect antibody to a recombinant form of calreticulin protein found in the salivary glands of Ixodes scapularis, a member of a complex of Ixodes ticks that serve as the vectors for Lyme disease, human babesiosis, and human granulocytic anaplasmosis. Using this assay, we tested sera obtained from C3H/HeN and BALB/c mice before and after experimental deer tick infestation. These mice developed antibody to Ixodes calreticulin antigen after infestation. We then used the same assay to test sera obtained from people before and after they experienced deer tick bite(s). People experiencing deer tick bite(s) developed Ixodes calreticulin-specific antibody responses that persisted for up to 17 months. This Ixodes recombinant calreticulin ELISA provides objective evidence of deer tick exposure in people.

Quantitative PCR for detection of babesia microti in ixodes scapularis ticks and in human blood

Babesia microti, the primary cause of human babesiosis in the United States, is transmitted by Ixodes scapularis ticks; transmission may also occur through blood transfusion and transplacentally. Most infected people experience a viral-like illness that resolves without complication, but those who are immunocompromised may develop a serious and prolonged illness that is sometimes fatal. The geographic expansion and increasing incidence of human babesiosis in the northeastern and midwestern United States highlight the need for high-throughput sensitive and specific assays to detect parasites in both ticks and humans with the goals of improving epidemiological surveillance, diagnosis of acute infections, and screening of the blood supply. Accordingly, we developed a B. microti-specific quantitative PCR (qPCR) assay (named BabMq18) designed to detect B. microti DNA in tick and human blood samples using a primer and probe combination that targets the 18S rRNA gene of B. microti. This qPCR assay was compared with two nonquantitative B. microti PCR assays by testing tick samples and was found to exhibit higher sensitivity for detection of B. microti DNA. The BabMq18 assay has a detection threshold of 10 copies per reaction and does not amplify DNA in I. scapularis ticks infected with Babesia odocoilei, Borrelia burgdorferi, Borrelia miyamotoi, or Anaplasma phagocytophilum. This highly sensitive and specific qPCR assay can be used for detection of B. microti DNA in both tick and human samples. Finally, we report the prevalence of B. microti infection in field-collected I. scapularis nymphs from three locations in southern New England that present disparate incidences of human babesiosis.

Diagnosis of babesiosis using an immunoblot serologic test.

ABSTRACT Although the current indirect immunofluorescent assay (IFA) diagnostic antibody test for human babesiosis is sensitive and specific, an immunoblot antibody test may be easier to standardize and to perform. Our objective, therefore, was to determine the efficacy of and develop interpretive criteria for an immunoblot antibody test for diagnosing acute human babesiosis using a Babesia microtiwhole-cell lysate as the antigen. We compared the reactivity of sera to a B. microti immunoblot assay in 24 human subjects experiencing symptoms and expressing laboratory evidence of babesiosis, 28 subjects who experienced Lyme disease, 12 subjects who experienced human granulocytic ehrlichiosis, and 51 subjects who reported no history of any of these diseases and whose sera did not react againstB. microti antigen in an IFA test. Immunoblot strips were impregnated with proteins derived from the GI strain of B. microti that had been electrophoresed in an acrylamide sodium dodecyl sulfate gel, followed by electroblotting onto nitrocellulose membranes. The sera of all subjects who experienced babesiosis reacted against the B. microti antigen in the IFA and against at least one of nine immunoblot protein bands specific to B. microti. In contrast, none of the sera from people who appeared not to have experienced this infection reacted against the B. microti antigen in the IFA (compared to 4% in the immunoblot assay). When two reactive bands were considered as definitive, immunoblot test sensitivity was 96%, while specificity was 99% and predictive positivity and predictive negativity were 96 and 99%, respectively. Our B. microti immunoblot procedure shows promise as a sensitive, specific, and reproducible assay for routine clinical diagnosis of acute babesiosis.

DNA probe versus culture for detection of Mycoplasma pneumoniae in clinical specimens

The laboratory diagnosis of Mycoplasma pneumoniae is often difficult because of lengthy and complicated cultural methods and serological tests that may be both insensitive and nonspecific. In this study, 82 patients suspected of Mycoplasma pneumonia were cultured for M. pneumoniae, and their respiratory secretions were tested by a DNA probe for M. pneumoniae. The probe test was 100% sensitive and 98% specific compared to culture. This DNA probe, then, is an effective alternative method for the detection of M. pneumoniae in respiratory specimens.

Rapid detection of Chlamydia trachomatis by an enzyme immunoassay method

Chlamydia trachomatis has been shown to be a major cause of sexually transmitted diseases in the United States. An enzyme immunoassay (Abbot Laboratories) has been developed that detects chlamydial antigen directly in the urogenital specimens of patients. We have evaluated specimens from 1,074 patients belonging to one of three risk groups. Three swabs were collected from each patient--one each for Neisseria gonorrhoeae, chlamydia cell culture, and enzyme immunoassay. When compared with cell culture, the sensitivity and specificity of the enzyme immunoassay for symptomatic males and females attending a sexually transmitted disease clinic was 82% and 100%, and 91.3% and 95.0%, respectively. A moderate risk group, consisting of female patients seen at either urology or gynecology clinics for genitourinary symptoms was also evaluated. The sensitivity and specificity of the test on this group was 96% and 96.7%. A population of females at low risk were also screened for chlamydial infection. In this group, the sensitivity and specificity of the enzyme immunoassay was 89.3% and 93.2%, respectively. This rapid test is a highly specific and sensitive procedure for the detection of chlamydial antigen in genital specimens from high risk female patients as well as symptomatic males.

Kell blood group activity of gram‐negative bacteria

To understand better the relationships between blood‐group antigens and bacterial constituents, examples of 23 gram‐negative bacteria (representing the 10 genera Citrobacter, Edwardsiella, Enterobacter, Escherichia, Klebsiella, Proteus, Pseudomonas, Salmonella, Serratia, and Shigella) were tested for the presence of Kl‐like antigens by hemagglutination‐inhibition (HAI) assays against both IgG and IgM anti‐ Kl. Saline‐suspended whole organisms, cell‐free culture media, and disrupted organisms were used to test for such antigens in, on, and secreted by the microorganisms examined. Disrupted organisms of an isolate of Shigella sonnei nonspecifically inhibited IgG anti‐Kl as well as IgG antibodies of the specificities Kpb, Fya, S, and c. However, only Escherichia coli 0125:B15, subtype 12808, had specific K1‐ like activity (no activity with other IgG [(k, Kpb, Jka, Fya, S, c] and IgM [A, B, M, P1] antibodies). Disrupted organisms inhibited IgM but not IgG anti‐K1 in the HAI assay. A second subtype, E. coli 0125:B15, subtype 12809, exhibited no K1‐like activity. These findings support the report of K1 activity in cell‐free broth cultures of E. coli 0125:B15 (subtype unspecified). Thus, although not all E. coli 0125:B15 possesses K1‐like activity, the finding of such activity in at least one E. coli subtype confirms the idea that bacterial components may play a role in the production of naturally occurring antibodies directed against non‐ABO red cell antigens.