Introduction of an in-house PCR for routine identification of M. tuberculosis in a low-income country.

SETTING: National Tuberculosis (TB) Treatment Centre, Makerere University Medical School and Joint Clinical Research Centre, Kampala, Uganda. OBJECTIVE: To evaluate the introduction of a polymerase chain reaction (PCR) based assay for identification of the Mycobacterium tuberculosis complex (MTC) into routine practice. DESIGN: Routine diagnostic specimens were processed and inoculated into Bactec 12B vials and monitored daily. At a growth index (GI) > or =10, 0.5 ml of the 12B broth was removed and assayed with PCR. The same 12B vial was analyzed using the Bactec NAP method at GI > or =500. Vials at various levels of GI were included. Recurrent cost and time required to perform PCR and NAP were compared. RESULTS: Initially, 71 specimens were analyzed; of these, 68 were NAP-positive while 69 were PCR-positive for MTC. PCR resulted in a 75% reduction in cost for a single test compared with Bactec NAP. PCR has been successfully incorporated into routine practice, and 432 samples have been analyzed. In addition, isolates from solid media were also well identified by PCR. With PCR, more samples can be analyzed at a time, it is faster and is less labor intensive. CONCLUSION: PCR is a reliable and cheaper alternative for the identification of MTC.

Resolution of HSV corneal infection in the absence of delayed-type hypersensitivity.

The role of delayed-type hypersensitivity (DTH) in the resolution of herpes simplex virus type 1 (HSV-1) ocular infection was examined. Infection of Balb/c mice on the sacrificed cornea with HSV-1 resulted in sensitization for DTH. This response, demonstrable by swelling of the ear following inoculation with ultraviolet-irradiated virus, was optimal 7 days postinfection. The reaction was immunologically specific and characterized histologically by a predominately mononuclear cell infiltrate. DTH responsiveness could be completely abrogated if the mice were inoculated intravenously with an attenuated strain of HSV-17 days before corneal infection. DTH-unresponsive mice were, nevertheless, resistant to corneal challenge with sublethal or lethal doses of HSV-1. Resistance was accompanied by a greater than 30-fold reduction in infectious virus in the eye 24 hr post challenge. A cellular infiltrate characteristic of a DTH response was not observed within the cornea during virus clearance. Tolerance was restricted to DTH, as antibodies to HSV antigens could be readily demonstrated 6-7 days after intravenous virus immunization. These antibodies may have contributed to the resistance observed. The results establish that neither a systemic nor local DTH response is required by the host to resist HSV-1 ocular infection.

Eosinophilia-induced vascular and airway remodeling and hyperresponsiveness in rat lungs.

We evaluated the effects of pulmonary infiltration of eosinophils without exogenous activators on airway and vascular hyperresponsiveness to muscarinic challenge in the lungs of rats infected with Toxocara [correction of Toxicara] canis, the canine round worm. Bronchoalveolar lavage of infected lungs produced 4.26 x 10(7) cells with 85% eosinophils, 15% mononuclear cells, and essentially no neutrophils. Eosinophils were present in the air spaces and interstitial spaces surrounding airways and vessels. The smooth muscle thickness increased about fourfold in large airways and vessels, and medium and small vessels were muscularized in infected lungs. In the T. canis-infected lungs, baseline airway resistance increased 288%, total vascular resistance (RT) increased 202%, and capillary filtration coefficient increased 208% compared with uninfected control lungs. Lung compliance was 56% of control. The concentration of acetylcholine that produced 50% of maximal response was 18.4 times greater for airway resistance and 18.7 times greater for RT in uninfected controls than in infected lungs. Isoproterenol (10(-4) M) decreased RT and peak airway pressure by 21% in infected lungs but had no significant effect on controls. We conclude that pulmonary interstitial infiltrates of eosinophils cause airway and vascular remodeling and increase baseline resistances and muscarinic reactivities of airways and vessels in rat lungs infected with T. canis.

Oxygen radical scavengers protect against eosinophil-induced injury in isolated perfused rat lungs.

The protective effect of oxygen radical scavengers on lung injury induced by activated eosinophils was examined in isolated perfused rat lungs. Eosinophils were obtained by bronchoalveolar lavage from rats infected with Toxocara canis and activated with phorbol myristate acetate (PMA). There were no changes in pulmonary vascular (RT) and airway (Raw) resistances and only minimal changes in vascular permeability assessed using the capillary filtration coefficient (Kf,c) in PMA control lungs and nonactivated eosinophil-treated lungs. In lungs receiving 3 x 10(6) PMA-activated eosinophils, there were significant increases from baseline of 7.3-fold in RT at 30 min, primarily due to the constriction of small arteries and veins; 3.6-fold in Kf,c at 90 and 130 min; and 2.5-fold in Raw. The lungs also became markedly edematous. Both superoxide dismutase and catalase pretreatment prevented the significant increase in Kf,c and lung wet-to-dry weight ratios and partially attenuated the increase in Raw, but did not significantly inhibit the increase in RT induced by activated eosinophils. Heat-inactivated catalase did not attenuate the eosinophil-induced increases in Kf,c, Raw, or RT. Thus, activated eosinophils acutely increased microvascular permeability primarily through production of oxygen free radicals. The free radical scavengers superoxide dismutase and catalase partially attenuated the bronchoconstriction but had no significant effect on the vasoconstriction induced by activated eosinophils.

Effects of phorbol myristate acetate-stimulated human leukocytes on rat lung.

Human blood was separated into polymorphonuclear (PMN) and mononuclear (MN) leukocyte fractions, and 3 x 10(7) cells (PMN or MN) were added to isolated rat lungs perfused with 5% human albumin in buffer and stimulated with phorbol myristate acetate (PMA). Lungs perfused with either albumin alone, PMN, or MN but not stimulated with PMA showed no change in vascular resistance or endothelial permeability measured as the capillary filtration coefficient (Kf,c). Lungs that were stimulated with PMA with no cells showed no change in Kf,c (0.34 +/- 0.07 vs. 0.37 +/- 0.7), but vascular resistance increased in all segments of the circulation. Capillary pressure, the major force responsible for edema formation, nearly doubled in the absence of cells 40 min after PMA. Lungs perfused with either PMN or MN and stimulated with PMA were injured. Kf,c increased from 0.41 +/- 0.03 to 0.87 +/- 0.10 (PMN) and from 0.36 +/- 0.07 to 0.81 +/- 0.23 (MN) 90 min after PMA. In addition to the increased endothelial permeability, vascular resistances and pressures also increased in the cell-perfused PMA-stimulated lungs. These results demonstrate that cells other than granulocytes are capable of producing severe acute lung injury and cannot be ignored when the effects of PMA on neutrophil-depleted lungs are studied.

Effect of inoculum size and length of infection on the distribution of Toxocara canis larvae in the mouse.

The effect of inoculum size and time on the distribution of Toxocara canis larvae in the mouse was investigated by recovering larvae from various body regions 7, 14, 28, and 56 days after administration of either 200, 600 or 1,000 infective eggs to groups of ten male mice. An analysis of variance of larval recoveries from the carcass, liver, brain and cardiopulmonary system suggests that inoculum size was a significant factor determining the proportional recovery for each of these sites. Length of infection was significant in relation to numbers of larvae in the anterior carcass, genitourinary system, brain and heart plus lungs, while length of infection and inoculum size acting in concert influenced the numbers of larvae recovered from the carcass, liver, brain, heart and lungs. Crowding effects, manifested as altered dispersion rates, were seen in the heavier infections.

Spleen cell responses in experimental murine toxocariasis.

Splenic responses of CBA/J mice infected with 250 embryonated ova of the nematode, Toxocara canis were characterized during the first month post-inoculation (P.I.). By the 6th day of infection, spleen to body weight ratios were over 3.5 times greater in infected mice than uninfected controls. This ratio peaked at 5.0 on day 14 and declined slightly by day 36 P.I. Lymphocyte transformation studies were carried out using the T cell mitogens, Con A and PHA and the B mitogen, LPS. The responses of infected mice to all of the mitogens were greater than or equal to the responses of uninfected mice at all times studied. Unstimulated lymphocytes from infected mice spontaneously incorporated approximately 10-fold more 3H-TdR than did uninfected control mice during the first 2 weeks of infection. Antigen-specific lymphocyte transformation was also performed using a crude extract of homogenized embryonated ova (TEE) as the antigen. Positive responses were obtained at all times examined but were greatest during the first 2 weeks of infection. The T cell basis of this response was demonstrated by Anti-Thy 1.2 plus complement treatment prior to the initiation of the lymphocyte transformation assay. Collectively, these results suggest that in mice, T. canis elicits a striking splenomegaly as early as 1 week after oral inoculation. The lymphocyte transformation studies suggest that the splenomegaly may be due in large part to proliferation of the white pulp based on the amount of spontaneous labelling observed. Increased mitogenic responses indicate that T. canis does not elicit nonspecific immunosuppression and the positive response to the TEE antigen suggests that at least some of the lymphoproliferation is antigen specific.

Eosinophil peroxidase levels in hearts and lungs of mice infected with Toxocara canis.

Toxocara canis infection of abnormal hosts results in a condition in which infective larvae migrate through the soft tissues of the body, exclusive of the skin. This condition is known as visceral larva migrans (VLM) and causes a syndrome characterized by hepatosplenomegaly, hyperglobulinemia, hypereosinophilia, and transient pulmonary infiltrates. Because of the known association between hypereosinophilia and eosinophilic heart disease, we have been studying the hearts of mice infected with T. canis for evidence of myocardial damage and have previously described a severe eosinophilic myocarditis that leads to a marked myocardial fibrosis. We have measured eosinophil peroxidase (EPO) levels (a marker enzyme for specific granules of eosinophils) in homogenized lungs, homogenized hearts, and eosinophils recovered from the lungs of mice infected with T. canis over a 6-wk period. A marked accumulation of EPO was observed in the lungs of infected mice from day 14 postinfection (PI) to at least 6 wk of infection. Most of the EPO was associated with eosinophils that comprise the bulk of the pulmonary infiltrates associated with the VLM syndrome. However, following bronchoalveolar lavage, cytochemical localization of EPO activity in lungs from infected mice suggested that eosinophil degranulation had resulted in this marker enzyme being deposited within the pulmonary parenchyma. Peak levels of EPO were found in the myocardium by day 14 PI and declined over the 6-wk period. These levels equaled about 1/3 of the levels seen in the lungs of the same mice. These studies suggest that in mice infected with T. canis, the presence of increased numbers of eosinophils may lead to marked peroxidatic cardiopulmonary damage.

Pulmonary granuloma formation in murine toxocariasis: transfer of granulomatous hypersensitivity using bronchoalveolar lavage cells.

A major drawback to studying granuloma formation in murine toxocariasis is the ability of the second-stage larva of Toxocara canis to escape from a developing granuloma, migrate elsewhere, and initiate granuloma formation anew. In an attempt to circumvent this difficulty, 2 different T. canis-derived antigenic preparations were covalently attached to Sepharose 4B beads and embolized into the microvasculature of the lungs of CBA/J mice that had been infected 10 days previously with 25 T. canis ova. Both T. canis egg extract (TEE) and T. canis exoantigens (TEX) were able to elicit antigen-specific granulomas 6 days postembolization as determined by both histologic and morphologic criteria. Histologically, the eosinophil-rich granulomas forming around antigen-coated beads embolized into infected mice resembled the developing granuloma previously described forming around the second-stage larva. Attempts to transfer granulomatous reactivity in this model using either immune spleen cells or immune serum were unsuccessful. Successful transfer of granulomatous hypersensitivity was achieved using cells obtained by bronchoalveolar lavage of previously infected mice. The results suggest the feasibility of using this embolic model of granuloma formation in murine toxocariasis.

Overproduction of SM28GST in a baculovirus expression vector and its use to evaluate the in vivo immune responses of mice vaccinated against Schistosoma mansoni with naked DNA encoding the SM28GST gene.

To evaluate the immune responses of mice vaccinated intramuscularly with naked DNA encoding a single parasite-derived gene, sufficient quantities of protein are necessary for use in the immunological assays. A plasmid carrying the cDNA encoding the entire sequence for the 28-kDa Schistosoma mansoni glutathione S-transferase (Sm28GST) was used as a source of naked DNA to vaccinate mice. Using polymerase chain reaction employing custom primers to add Eco RI and Hind III restriction sites at the 5' and 3' ends, respectively, a 651-bp fragment was amplified from the vaccine plasmid. This product was isolated, ligated into the pFastBac HTb donor plasmid containing a 6X histidine (6X-his) tag, and transposed into the baculovirus expression vector system. Following blue white selection screening, high molecular weight DNA was isolated and transfected in Sf21 insect ovary cells using a liposomal preparation. Culture medium containing infective virus particles was used to infect a series of Sf21 cultures and the cells were lysed after 3-5 days. The lysates were subjected to immobilized metal (Ni-NTA) affinity chromatography from which the 6X-his-tagged recombinant Sm28GST was eluted in 250 mM imidazole. The eluted protein was probed with a polyclonal rabbit antibody specific for the Sm28GST and subsequently recognized using a monoclonal antibody specific for the 6X-his tag following concentration of the pooled fractions. Mice were vaccinated intramuscularly with purified plasmid DNA encoding either the Sm28GST or firefly luciferase. Skin tests performed using recombinant Sm28GST were positive in only those mice vaccinated with naked DNA encoding the Sm28GST gene. In a different group of experimental mice, only sera from mice vaccinated with naked DNA encoding Sm28GST contained IgG-specific anti-Sm28GST antibodies at 14 days postvaccination, and at 42 days the levels were suggestive of an anamnestic response. These results suggest that naked DNA vaccination of mice is capable of inducing both antigen-specific cell-mediated and humoral immune responses against Sm28GST and further strengthen the case for this antigen being a vaccine candidate.

Human anisakiasis transmitted by marine food products.

Seafood-transmitted parasitic diseases represent an emerging area of interest to the U.S. Food and Drug Administration. Human infections with marine parasites are generally the result of ingesting uncooked seafood products. Over 50 species of helminthic parasites are known to infect humans worldwide. Recently, the number of infections with one of these helminths, the juvenile stage of the marine nematode, Anisakis simplex, has increased in the United States. Raw fish dishes such as lomi lomi salmon and sashimi are known to transmit the parasite to unsuspecting citizens and the most frequently implicated fish in the transmission of this zoonotic disease is the Pacific salmon (Oncorhynchus spp.). The risk of infection from fishes caught in Hawaiian waters is slight; however, a juvenile Anisakis simplex infected one patient from either locally caught aku or ahi. We report 4 new cases, which brings the total number of known cases in Hawaii to 7. Five of the 7 cases were diagnosed and treated by means of an endoscope and biopsy forceps. Serological profiles are presented in several of these cases. One case represents the first known instance of reinfection; the initial infection occurred 2 years prior. The second infection gave an opportunity to compare the human response to a challenge infection and to investigate the validity of the "double hit" theory. Increased awareness by physicians to the clinical features of this disease is warranted. The zoonotic disease, anisakiasis, should be considered in patients presenting with intense abdominal pain, if these patients admit they have recently eaten raw or undercooked seafoods.

Nonspecific allergic granulomatosis in the lungs of mice infected with large but not small inocula of the canine ascarid, Toxocara canis.

Infection of CBA/J mice with 250 ova of Toxocara canis results in development of splenic lymphocytes hyperergic to nonspecific stimuli. These studies were performed to see if the granulomatous response to a nonspecific stimulus was exaggerated. Using eosinophil counts and MBSA ear thickening it was demonstrated that concurrent infection with the parasite and immunization with methylated bovine serum albumin (MBSA) elicit no immunologic cross reactions. Eosinophil counts were proportional to the worm burden and unaffected by concurrent MBSA immunization. Embolization of Sepharose 4B beads conjugated to MBSA into the pulmonary vasculature of MBSA-immune mice, elicited large, florid macrophage-rich granulomata while in MBSA-naive mice infected with 10 T. canis ova, the beads elicited small, foreign body reactions. In MBSA-naive mice infected with 250 T. canis ova MBSA-coated beads elicited eosinophil-rich granulomata that were as large or larger than the granulomata in MBSA-immune mice. In mice immune and infected, lesions contained eosinophils in proportion to larval worm burdens. Because MBSA beads elicited large, eosinophilic granulomata in T. canis infected, MBSA-naive mice it is concluded that the lung is hyperergic and the disease process appears similar to other kinds of eosinophilic pneumonitis such as bronchial asthma or Loeffler's pneumonia.

Human toxocariasis and the visceral larva migrans syndrome: correlative immunopathology.

The surface of T. canis is now recognized as a dynamic structure which turns over quite rapidly and serves as a renewable source of large quantities of antigen(s). The major host responses to these antigens include a marked eosinophilia and hyperglobulinemia. Both of these responses are apparently ineffective at ridding the body of infective larvae. Both eosinphils and IgE antibodies are manifestations of the Th2 subset of T helper cells and the cytokines that they secrete. Further, there is reason to believe that the antigens released from T. canis larvae favor the induction of this cellular population. Finally, there is mounting evidence that the chronic production of parasite antigen and its continued stimulation of the host immune system with a concomitant production of eosinophils can lead to a permanent alteration of the normal organization of the cardiopulmonary system. In the absence of any well-documented drugs capable of killing infective larvae, it would seem that immunological intervention may offer the only way to minimize or neutralize this 'gift from man's best friend'. This chapter was not intended to be an exhaustive review of the literature pertaining to toxocariasis. Several other recent publications will hopefully fulfill the need for more detailed information on the biology of this organism and the clinical spectrum of the disease it produces [16, 138-140]. Finally, a MEDLARS search of the current medical literature should bring anyone up to speed in a very short time.

Depletion of eosinophils by anti-IL-5 monoclonal antibody treatment of mice infected with Trichinella spiralis does not alter parasite burden or immunologic resistance to reinfection.

Mechanisms of parasite killing by eosinophils are widely studied and are often implicated in mediating resistance to parasitic infection, especially in conjunction with specific antibodies. Evidence for the eosinophil as an anti-parasite killer cell in vivo is limited and may not justify the belief that eosinophils engage and/or kill infective helminths. We reexamined this question in a mouse model of trichinosis in which antisera to eosinophils were previously used to show the requirement for eosinophils in resistance to this nematode. The current studies used mAb to IL-5 to suppress eosinophil levels in CF1 mice infected with Trichinella spiralis. In mice given a primary infection and injected with an isotype control mAb or left untreated, the medullary and peripheral blood eosinophil numbers peaked at 3 wk postinfection (PI) and returned to baseline levels by 4 wk PI. Peripheral blood eosinophil numbers in infected mice injected with anti-IL-5 were maintained at levels below those of uninfected normal mice through 4 wk of infection. Histologically, there was a prominent eosinophil accumulation in infected, untreated, or control-mAb-treated mice associated with nurse cell complexes containing infective juveniles in skeletal muscle at 3 and 4 wk PI. This was largely eliminated in mice treated with anti-IL-5 mAb. However, the number of muscle stage juvenile worms recovered 3 and 4 wk PI after acid pepsin digestion was unaffected by eosinophil depletion. Challenge infections, in which mice were infected at day 0 with 125 muscle stage worms and challenged at day 28 PI with 350 muscle stage worms, developed peak eosinophil numbers in bone marrow and peripheral blood 3 wk after primary infection and 2 wk after challenge infection in mice receiving either no treatment or control mAb. In challenged mice receiving anti-IL-5 mAb, medullary and peripheral blood eosinophil numbers remained at or below those of uninfected animals. Although all groups exhibited significant resistance measured as muscle stage worm burdens 56 days PI, eosinophil depletion did not affect resistance of muscle worm recovery. These results suggest that eosinophils are not essential in the control of T. spiralis in either primary or challenge infections of CF1 mice. This in vivo study illustrates the questionable value of in vitro killing assays to assign effector function to any single inflammatory cell type.

Immunoregulation in experimental schistosomiasis: in vitro induction and assay of spleen cell suppressor activity.

Spleen cells from normal CBA/J mice or mice infected with Schistosoma mansoni were exposed for 48 to 72 hr to either concanavalin A (Con A), soluble egg antigen (SEA), or soluble worm antigenic preparation (SWAP), treated with mitomycin C to prevent further DNA synthesis, and admixed with either normal or sensitized syngeneic spleen cells exposed to a concentration gradient of phytohemagglutinin (PHA) or SEA, respectively. Both nonspecific (by Con A) and "antigen-specific" (by SEA and SWAP in infected mice only) induction of suppression was observed when using PHA-induced blastogenesis as the final assay. The number of mice with inducible splenic suppressive activity and the degree of PHA suppression induced by exposure to SEA appeared to decline between 8 and 20 weeks of infection. In contrast, when the response of spleen cells from mice infected for 8 weeks to SEA served as the final assay, strong suppressive activity was induced from the spleen cells of all chronically infected mice (20 weeks of infection). This model permits parallel analysis of the induction of suppressor activity by nonspecific and schistosome antigen-specific signals during the course of this chronic, immunoregulated condition, schistosomiasis mansoni.

Immunoregulation in experimental schistosomiasis. II. Soluble egg antigen-induced chronic spleen cell augmentation of baseline lymphocyte reactivity.

Spleen cells from mice harboring infections of Schistosoma mansoni for 20 weeks exposed to Con A or to soluble schistosome egg antigenic preparation (SEA), treated with Mitomycin C (Mc), and cocultured with spleen cells from either normal or infected mice caused an augmented baseline [3H]TdR incorporation by the otherwise unstimulated responder cells. This regulation required an in vitro induction phase. SEA-exposed, Mc-treated normal spleen cells had no effect on responder cell cultures. SEA-stimulated, Mc-treated chronic spleen cell augmentation was effective on responder cell populations from either normal mice or mice infected with S. mansoni for 8 weeks. Augmentation was most pronounced when assayed on cells from infected mice assayed over a 5-day incubation. In addition, it is demonstrated that these Con A- and SEA-elicited activities are mediated by soluble mediators which lack H-2 restriction.

Immunoregulation in experimental Schistosomiasis. III. Role of macrophages in soluble egg antigen-induced chronic spleen cell augmentation of baseline lymphocyte reactivity.

Spleen cells from mice infected for 20 weeks with Schistosoma mansoni, exposed in vitro to soluble schistosomal egg antigens (SEA), treated with mitomycin C (Mc), and cocultured with syngeneic responder spleen cells increased the baseline proliferation of the otherwise unstimulated responder cells in cocultures. The role of macrophages in this "spontaneous" thymidine incorporation was studied directly by removal of macrophages on Sephadex G-10 columns. Removal of esterase-positive, Sephadex G-10-adherent cells (macrophages) greatly reduced the amount of SEA-induced, chronically infected spleen cell-mediated stimulation observed in cocultures. It also reduced an elevated background of spontaneous DNA synthesis seen with control cultures of spleen cells from infected animals. Depletion of T lymphocytes from chronic spleen cell populations by treatment with anti-Thy 1.2 serum and complement prior to exposure to SEA partially abrogated the augmentation effect. Comparison of these results with mitogen (concanavalin A)-induced spleen cell-mediated stimulation (which is elevated, rather than reduced, by macrophage removal) and with known alterations in splenic T- and B-lymphocyte ratios in chronic murine schistosomiasis suggests that antigen-stimulated, chronically infected splenic macrophage-dependent baseline augmentation may depend on specific T-lymphocyte-derived lymphokine induction. These results may reflect a general mechanism whereby animals harboring a persistent, chronic infection can respond quickly to a second or challenge infection or a flareup of the primary infection.

Development of the granulomatous response in murine toxocariasis. Initial events.

The cellular evolution of the persisting, muscle-associated granuloma in murine toxocariasis (visceral larva migrans) was chronicled for 11 weeks by light and electron microscopy. The initial granuloma consisted primarily of eosinophils and appeared to develop from the acute inflammatory infiltrate. During the ensuing 48 hours, most of the eosinophils appeared to loose their granules and disintegrate. The resulting cellular debris was then taken up by newly arrived macrophages which become the predominant mononuclear cell in the lesion by 28 days of infection. By 11 weeks, the granuloma had become a fibrotically encapsulated epithelioid granuloma surrounding the inciting larva. This histologic reaction is compared with the liver granulomatous response to Toxocara and to the well-characterized schistosome egg granuloma. A possible delayed hypersensitive etiology for the Toxocara granuloma is suggested.