Natural history of HIV infection in Filipino female commercial sex workers.

A prospective follow-up study of the progression of HIV infection, from seroconversion to onset of opportunistic infections (OI) indicative of immune deficiency and to death, was performed in a cohort of 54 HIV-1 antibody positive Filipino female commercial sex workers (FCSW). The cumulative probability of having a CD4+ T cell count of < 200/mm3 and/or an OI indicative of severe immune deficiency was 52.9% within 5 years and 73.8% within 6 years after seroconversion. The cumulative probability of death was 52.1% within 6.5 years following seroconversion and 52.7% within 1.5 years after a depressed (< 200/mm3) CD4+ T cell or onset of an OI. Although several OI associated with immune impairment were observed, a CD4+ cell count of < 200/mm3 was the initial indicator of a failing immune system in more than 50% of the patients. Mycobacterium tuberculosis or unidentified acid fast bacilli (presumed to be M. tuberculosis) and Pneumocystis carinii pneumonia were the initial indicators of immune deficiency in the remaining patients.

Allelic variants of the Plasmodium falciparum merozoite surface antigen 2 (MSA-2) in a geographically restricted area of Irian Jaya.

Blood samples were collected from 12 residents of 4 villages in the Oksibil area of Irian Jaya. Eleven patients were positive for Plasmodium falciparum infection as evidenced by successful amplification of the MSA-2 gene by the polymerase chain reaction. Two patients showed evidence of infection by 2 strains of Plasmodium falciparum. All MSA-2 genes were completely sequenced and all could be assigned to one of the two major allelic families of MSA-2, however all MSA-2 gene sequences differed from previously described alleles. Five new allelic forms were identified, one of which was present in 8 of the 11 patients. Within small natural populations of P. falciparum, it appears that variation in MSA-2 approximates that seen world-wide. All samples were also analysed by hybridisation of amplified DNA to family specific probes and all samples hybridised to known probes. Our results demonstrate that there is a degree of microheterogeneity of MSA-2 that is undetectable by hybridisation studies alone.

Use of micro-ELISA for quantitating antibody to Trypanosoma cruzi and Trypanosoma rangeli.

Of 229 residents of a Panamanian village where both Trypanosoma cruzi and Trypanosoma rangeli are endemic, 52% had antibody to one or both species by the enzyme-linked immunosorbent assay (ELISA), 26% were positive by complement fixation, 17% were positive by direct agglutination, and 32% were positive on the basis of clinical impression. Although the sensitivity of ELISA makes it the procedure of choice for sero-epidemiologic studies, there does appear to be some serologic cross-reactivity between the two species. Sera from 55 Panamanians and 33 Bolivians were reactive with antigens of T. cruzi and T. rangeli, thus making definitive diagnosis by serologic methods impossible. Although the presence of antibodies to both species suggests serologic cross-reactivity, the possibility of dual infection must be considered also. Fifty-four Panamanians and 20 Bolivians had antibody to only one species.

Antigenic differentiation of Trypanosoma cruzi and Trypanosoma rangeli by means of monoclonal-hybridoma antibodies.

Sera from Balb/c mice, hyperimmunized with ruptured epimastigotes of either Trypanosoma cruzi or Trypanosoma rangeli, lacked species-specificity when assayed for antibodies by indirect immunofluorescent microscopy and by the enzyme-linked immunosorbent assay. However, when plasma cells from those mice were fused with syngeneic mouse plasmacytoma cells, many of the resultant hybridomas synthesized antibodies which were species-specific. Four clones are synthesizing antibodies specific for antigens of T. rangeli. These antigens are associated with the cytoplasm, plasma membrane and flagellum. One clone is synthesizing a specific anti-T. rangeli antibody which appears to be reactive with the entire surface of the epimastigote. Another clone is reactive with a subpopulation of epimastigotes, thus suggesting some antigenic variability among cultured forms. Two clones are synthesizing antibody specific for antigens of T. cruzi, one of which is confined to the cytoplasm.

Micro enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of New World leishmaniasis.

Of 21 confirmed cases of New World leishmaniasis, 16 exhibited antibody to antigens of the promastigote of Leishmania braziliensis panamensis by the enzyme-linked immunosorbent assay (ELISA). Comparison of antibody titers obtained by ELISA with titers obtained by indirect immunofluorescence using an amastigote substrate confirmed that the sensitivities of the two techniques were within the same range (r = 0.80). Although sera from patients with New World leishmaniasis failed to react with antigens extracted from epimastigotes of Trypanosoma cruzi, sera from 39 cases of Chagas' disease were reactive with promastigotes of L. braziliensis panamensis. This apparent unidirectional cross-reactivity has been attributed to differences in potency of the antigenic stimulus presented in the two diseases.

Rapid detection of Leishmania amastigotes in fluid aspirates and biopsies of human tissues.

When using a genus-specific monoclonal antibody (83-J3D2) as the primary reagent in an indirect immunofluorescent antibody assay (IFA), intracellular amastigotes of Leishmania were easily identified in 9 of 9 biopsies and in 11 of 12 needle aspirates taken from human lesions. In contrast, only 5 of the biopsies and 4 of the aspirates yielded promastigotes upon culture in vitro. Similarly, all but 2 of the aspirates and one-half of the biopsies were reported as negative for parasites when stained with Wright's and hematoxylin-eosin, respectively. Serum antibody titers, ranging from 1:8 to 1:128, corroborated the results of the amastigote detection assays when histopathology and isolation were negative. These findings support the practicality of using the genus-specific monoclonal IFA in those field situations where it becomes necessary to differentiate leishmaniasis from other skin infections.

Subcellular and taxonomic specificity of monoclonal antibodies to New World Leishmania.

Murine monoclonal antibodies to flagellar, surface membrane and cytoplasmic antigens of New World Leishmania were assessed for their taxonomic specificity in enzyme-linked immunosorbent assays with three genera of the family Trypanosomatidae and three species and seven subspecies of the genus Leishmania. Antibodies exhibiting exclusive reactivity with either the flagellum, flagellar pocket, kinetoplast, or nucleus lacked specificity at all phylogenetic levels and, in fact, recognized epitopes common to cultured mammalian cells. Monoclonals to intracellular antigens were capable of distinguishing Leishmania from Trypanosoma and Endotrypanum. Antibodies reactive at the surface membrane could separate six isolates of L. braziliensis from three isolates of L. mexicana but the differences in antigen expression were frequently quantitative rather than qualitative. Antigenic variability within species and/or subspecies often exceeded that which was observed between species and/or subspecies. At least one monoclonal antibody was specific for a surface antigen peculiar to a subpopulation of promastigotes of an L. braziliensis panamensis isolate.

Heightened transmission of stable malaria in an isolated population in the highlands of Irian Jaya, Indonesia.

Malaria at an elevation of 1,500 meters is uncommon and is usually unstable when it occurs. To confirm reports of a recent increase in transmission of stable malaria in the Oksibil Valley, which is at an elevation of 1,250-1,500 meters in the Jayawijaya Mountains of Irian Jaya, Indonesia, five malariometric surveys were conducted in four villages between May 1990 and July 1991. A total of 3,380 blood smears, representative of 1,949 persons, was examined. Prevalence rates over the survey period were consistent in each of the four villages, averaging 10% for infants, 50% for children 1-4 years of age, 35% for those 5-9 years old, 28% for those 10-14 years old, and 16% for adults (greater than 15 years old). The spleen rate for the those less than five years old was 96%, with an average enlarged spleen score of 2.32. Plasmodium falciparum accounted for 55% of the infections in the valley, but P. vivax was the predominant species in those less than 10 years old. In the village of Kutdol at an elevation of 1,500 meters, P. malariae was identified in 43% of the positive smears. Four cases were diagnosed as P. ovale. Infection with P. falciparum without obvious clinical symptoms was common in both adults and children. Entomologic and epidemiologic data suggested that the recent upsurge in transmission coincided with the replacement of traditional village huts with the more modern social housing. This replacement required the extensive construction of drainage ditches, which inadvertently also served as additional vector breeding sites. We suspect that this manipulation of the environment, in an effort to improve the quality of life, created conditions conductive for heightened transmission of stable malaria.

PIP: Malaria at an elevation of 1500 meters is uncommon and is usually unstable when it occurs. To confirm reports of a recent increase in the transmission of stable malaria in the Oksibil Valley, at an elevation of 1250-1500 meters in the Jayawijaya Mountains of Irian Jaya, Indonesia, 5 malariometric surveys were conducted in 4 villages between May 1990 and July 1991. A total of 3380 blood smears from 1949 people was examined. Prevalence rates over the survey period were consistent in each of the 4 villages,with averages of 10% for infants, 50% for children 1-4 years old, 35% for those 5-9 years old, 28% for those 10-14 years old, and 16% for adults (over 15 years of age). The spleen rate for those less than 5 years old was 96%, with an average enlarged spleen score of 2.32. Plasmodium falciparum accounted for 55% of the infections in the valley, but P. vivax was the predominant species in those less than 10 years old. In the village of Kutdol, at an elevation of 1500 meters, P. Malariae was identified in 43% of the positive smears. 4 cases were diagnosed as P. ovale. Infection with P. falciparum without obvious clinical symptoms was common in both adults and children. Entomologic and epidemiologic data suggested that the recent upsurge in transmission coincided with the replacement of traditional village huts with more modern social housing. This replacement required the extensive construction of drainage ditches, which inadvertently also served as additional vector breeding sites. The authors suspect that this manipulation of the environment, while attempting to improve the quality of life, created conditions which were conducive for the increased transmission of stable malaria.

Prevalence of serum antibodies to human T lymphotropic virus-1 in an isolated tribe in the highlands of Irian Jaya, Indonesia.

There have been several recent reports on the high prevalence of serum antibodies to human T lymphotropic virus-1 (HTLV-1) in isolated populations residing in the coastal areas and highlands of Papua New Guinea. In the absence of significant cases of clinical disease, it has been surmised that this reactivity might be the consequence of serologic recognition of yet undefined human retroviruses or parasite antigens. These observations prompted an investigation of the prevalence of anti-HTLV-1 antibodies among members of the Ngalum tribe that dwells in a secluded highland valley in the eastern Jayawijaya Mountains of Irian Jaya, Indonesian New Guinea. Of 165 tribespeople, 85 (52%) were positive for IgG antibodies to HTLV-1 in an indirect enzyme-linked immunosorbent assay. Eighty-two were more than 10 years of age. On the Western blot, all positive sera reacted strongly with the p19 core antigen, but recognition of the envelope antigens, gp46 and gp21, was conspicuously absent. Thirty-four of the 85 villagers with these indeterminant blots had active Plasmodium falciparum infections, but antibody absorption studies with HTLV-1 and P. falciparum erythrocytic stage antigens failed to confirm suspected serologic cross-reactivities. Thirty-three others had acute malaria and/or high titers of anti-malaria antibodies but were seronegative for HTLV-1. We suspect that indeterminant Western blots for HTLV-1 reflect antibody responses to related latent retroviruses that are activated as a consequence of immunosuppression following malaria infection and chloroquine therapy.

Serodiagnosis of New World leishmaniasis by using a genus-specific antigen in enzyme linked immunosorbent assays.

A genus-specific monoclonal antibody (83L-5G9), generated against promastigotes of Leishmania braziliensis panamensis (WRAIR-470), has been used as a ligand in immunochromatography columns for the recovery and purification of the genus-specific antigen. When the purified polypeptide, actually a doublet comprised of a 58 kd and a 31 kd moiety, was used as the antigen in enzyme linked immunosorbent assays (ELISA), it was reactive with 36 of 85 sera from Leishmania patients and with 1 of 49 sera from confirmed cases of Chagas' disease. An additional 25 sera from an unexposed population and five specimens from individuals seropositive for Toxoplasma gondii were nonreactive.

Isolation of hybridomas secreting monoclonal antibodies against Physalia physalis (Portuguese man-o'war) nematocyst venom.

Balb/C mice were immunized with crude Portuguese Man-O'War (Physalia physalis) nematocyst venom and their spleen immunocytes were fused with plasmacytoma cells. Nine hybridomas which produced IgG specific for Man-O'War venom were identified using a specific ELISA technique. Ammonium sulfate and DEAE cellulose-purified monoclonal anti-venom antibody had an ELISA titer of 1:4000 and an ability to neutralize the lethal activity (4 LD50/0.6 ml ascites fluid) of an i.v. challenge of crude venom. Indirect immunofluorescence testing demonstrated that the monoclonal antibody isolated in these experiments reacted against a venom component located in the nematocyst wall and thread.

Longitudinal studies of children born to HIV-1 antibody positive Filipino commercial sex workers (CSW): diagnostic dilemmas.

Fifteen term babies born to 12 HIV-1 antibody positive Filipino CSW have been monitored for signs and symptoms of HIV-1 infection. Eleven babies were enrolled in the study within the first 6 months after birth; 4 others were enrolled at 4, 9, 11 and 21 months of age respectively. Every 3 months after enrolment, each baby received a physical examination, serum was tested for HIV-1 antibodies and p24 antigen and peripheral blood mononuclear cells were cultured for isolation of virus. After a mean follow-up period of 39.3 months (range 7-72 months), virus isolation and serum p24 antigen assays confirmed that 2 babies have been infected with HIV-1. If the 4 babies less than 18 months of age were excluded, the vertical transmission rate was 18.2%. Seven babies who have been monitored for a minimum of 25 months (range 31-60 months) lost their maternal antibodies but 6 of them subsequently developed indeterminant Western blots (WB); reactivity to p24 and/or gp120/ 160 but no reactivity to gp41. Of the remaining 6 babies, still less than 25 months of age (range 7-24 months), 2 lost their maternal antibodies within one year. The other 4 continued to recognize either p24 or gp120/160 well after the accepted 15-month period for loss of maternal antibody. Although a diagnosis could not be established upon the basis of these laboratory findings, clinical observations (failure to thrive, anergy, persistent generalized lymphadenopathy and recurrent pneumonias) mimicked HIV-1 infection. However, because these clinical features are common among many babies in the developing world, their usefulness in supporting a diagnosis of perinatal HIV-1 infection is limited.

PIP: Clinicians monitored 15 full-term infants born to 12 HIV-1 seropositive commercial sex workers in the Philippines for signs and symptoms of HIV-1 infection. They performed a physical examination and HIV test on each infant every 3 months. The mean follow-up period was 39.3 months (range 7-72 months). Two infants tested positive for HIV-1 infection. When the researchers excluded the four infants under 18 months of age, the HIV-1 perinatal transmission rate stood at 18.2%. The maternal antibodies in seven infants who were monitored for at least 25 months disappeared; yet six subsequently developed indeterminant Western blots (i.e., reactivity to p24 and/or gp120/160, but no reactivity to gp41). The maternal antibodies in two of the six remaining infants who were under 25 months old disappeared within 1 year. The Western blots still recognized either p24 or gp120/160 in the other four infants beyond the accepted 15-month period for loss of maternal antibody. The clinicians could not establish a diagnosis based on these laboratory findings. They did diagnose signs and symptoms that may indicate HIV-1 infection (i.e., failure to thrive, anergy, persistent generalized lymphadenopathy, and recurrent pneumonias). Yet these clinical features are prevalent in many infants in developing countries, thereby making their usefulness in diagnosing perinatal HIV-1 infection limited.

Use of a passive hemagglutination inhibition test for monitoring levels of serum carcinoembryonic antigen following surgical therapy.

A technique of passive hemagglutination inhibition (PHI) has been used to monitor levels of carcinoembryonic antigen (CEA) in human sera following surgical therapy. CEA was coupled to human type O-negative erythrocytes in the presence of bis-diazotized benzidine. Pre-operative and post-operative sera from 11 patients with primary adenocarcinomas of the gastrointestinal tract and from one patient with ulcerative colitis were then tested for their capacity to inhibit the agglutination of the sensitized cells in the presence of a predetermined amount of goat anti-CEA serum. Positive sera were defined as those which inhibited agglutination at dilutions of greater than 1:8. The pre-operative sera from 11 of the 12 patients inhibited agglutination at dilutions of 1:16 or greater. The one negative serum was from a patient with primary adenocarcinoma of the colon in the stage of Dukes' C. At one month post-resection, the PHI titer of six patients with colon cancer and of the patient with ulcerative colitis was less than or equal to 1:8. However, by 4 months post-resection, all but 3 of the patients had PHI titers in the positive range. These elevated titers were accompanied by recurrence of tumor growth and/or metastatic dissemination. A radioimmunoassay was used to quantitate CEA in 22 of the sera which had been tested by PHI. When positive sera were defined as those which inhibited agglutination at dilutions of greater than 1:8 and contained CEA in excess of 5 ng per ml, the results of the two procedures were in agreement for 17 of the 22 specimens. Five sera, representative of 2 patients with colon cancer, were false negative by PHI.

On-site diagnosis of Plasmodium falciparum, P. vivax, and P. malariae by using the Quantitative Buffy Coat system.

The Quantitative Buffy Coat (QBC) system was used for the detection and identification of malaria parasites in blood specimens from 570 residents of Oksibil, an isolated highland valley in the eastern Jayawijaya Mountains of Irian Jaya (Indonesian New Guinea). The availability of a battery-powered centrifuge and a fiberoptic Paralens enabled us to complete and interpret the assay in this remote environment. Of 322 QBC tubes examined for 2-4 min each, results of 295 (92%) concurred with findings on the matched Giemsa-stained thick smear (GTS). The 27 discrepant results included 13 QBC+/GTS- that, upon reexamination, were found to be GTS+. When using the corrected GTS results as the standard, the sensitivity and specificity of the QBC were 94% and 96%, respectively. Because electricity was available only 3 hr per day, it was decided to decrease the examination for an additional 248 QBC to a maximum of 90 sec per tube. This shortened inspection time resulted in a reduction of sensitivity to 53% but specificity was preserved at 89%. Forty-two of 45 conflicting results, QBC-/GTS+ from cases of light Plasmodium falciparum infections with < 1 trophozoite or gametocyte per field, were resolved by reexamination of the QBC in the laboratory. Tubes held at 4 C could be reexamined, without noticeable loss of fluorescence, for at least 6 wk after collection. Despite some difficulty in the identification of Plasmodium species, it was concluded that the QBC is an easy, sensitive method for the rapid diagnosis of malaria in the field and that it provides the inexperienced microscopist with an additional means for on-site identification of individuals needing treatment.

AIDS related attitudes and sexual practices of the Jakarta WARIA (male transvestites).

As part of a community based educational campaign to convey the risk of HIV infection and AIDS to commercial sex workers in Jakarta, over 600 male transvestites (WARIA) were questioned about their sexual behavior patterns and their knowledge and attitude towards HIV infection and AIDS. Most expressed a genuine fear about AIDS, but they stated they did not have sufficient information to determine if their risk of infection, at this time, was great enough to command a change in their sexual conduct. Among the group who felt they were not at risk for HIV infection, 40% had 8 or more different sex partners per week. Low risk receptive oral sex, thigh massage (simulated vaginal sex) and masturbation of the client were routine activities but high risk receptive anal sex without condoms was most common. Despite such high risk behavior, the WARIA community remains free of HIV infections. However, if they refuse to accept the endorsement of their peer leaders and they fail to alter their sexual behavior because of an unawareness of their risk, that status will change soon.

Isolation of HIV-1 from Filipino commercial sex workers (CSWs).

Peripheral blood mononuclear cells (PBMC) from 36 HIV-1 antibody positive Filipino female commercial sex workers (CSWs) were co-cultivated at a 1:1 ratio with phytohemagglutinin-P activated PBMC from healthy, HIV-1 antibody negative donors. After 3-18 (mean 7.2) days of incubation at 37 degrees C in 5% CO2, 29 cultures showed evidence of replication of HIV-1: increasing concentrations of p24 antigen in the growth medium and the appearance of multinucleated giant cells. Although the length of incubation required for the appearance of cytopathogenic effect for each particular isolate was essentially the same when either 6 microwell plates were seeded with 3.0 x 10(6) cells/well or 24 well plates were seeded with 1.5 x 10(6) cells/well, the 24 well format was more sensitive. The ability to isolate HIV-1 from PBMC did not appear to be associated with the progression of disease or the presence or absence of any specific clinical findings. However, if the PBMC were from individuals with a concomitant p24 antigenemia, the incubation time required for isolation was significantly shorter (mean 3.8 days). The absolute CD4+ lymphocyte count was also slightly reduced in the culture positive, p24 antigenemic patients (range 302-813 cells/mm3, mean 502 cells/mm3) compared to the culture positive, p24 serum negative cases (range 311-1,511 cells/mm3, mean 830 cells/mm3). The p24 serum negative cases with CD4+ counts of < 500 cells/mm3 had positive PBMC cultures by 6 days of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

A scanning electron microscopic investigation of accessory foramina in the furcation and pulp chamber floor of molar teeth.

Observations of the pulp chamber floor and furcation surface of human maxillary and mandibular first and second molars were made with the scanning electron microscopic to determine the incidence, size, and location of patent accessory foramina. Accessory foramina on both the pulp chamber floor and the furcation surface were found in 36% of maxillary first molars, 12% of maxillary second molars, 32% of mandibular first molars, and 24% of mandibular second molars. Mandibular teeth had a higher incidence (56%) of foramina involving both the pulp chamber floor and furcation surface than did the maxillary teeth (48%).

Antigenic differentiation of the kinetoplastids Leishmania braziliensis and Trypanosoma cruzi by means of monoclonal antibodies.

BALB/c mice were hyperimmunized with non-infectious extracts of either Leishmania braziliensis promastigotes or Trypanosoma cruzi epimastigotes. When spleen cells from these mice were fused with P3X63Ag8 plasmacytoma cells, the resultant hybridomas synthesized monoclonal antibodies which displayed specific reactivity by indirect immunofluorescence with distinct subcellular components of the parasites. These studies revealed that antigens associated with the flagellum and with a nongranular component of the cytoplasm would account for much of the serologic cross-reactivity observed between the two species. Conversely, antigens associated with surface and/or cytoplasmic granules and with an intracellular organelle believed to be the kinetoplast appeared to be species-specific.

Characterization and quantitation of membrane antigens of New World Leishmania species by using monoclonal antibodies in Western blot and flow microfluorometric assays.

Membrane-specific monoclonal antibodies generated against promastigotes of New World Leishmania species were used in Western blot, ELISA, and flow microfluorometric assays to characterize their antigen specificity and to determine the external surface distribution of the reactive epitopes. Three major membrane antigens of molecular weight 72 KD, 55 KD, and 42 KD were identified as well as a dominant antigen that migrated as a broad band on SDS-PAGE, corresponding to a molecular weight of 10-15 KD. By dot-ELISA this antigen was also found to be excreted by promastigotes into their culture medium. One minor membrane antigen of 25 KD and a triplet component of 66, 58, and 56 KD were also identified. While assays performed on air-dried promastigotes revealed the almost ubiquitous presence of the 72 KD and 55 KD antigens, indirect immunofluorescent staining of live promastigotes followed by flow cytometric analysis revealed that these antigens had no external exposure. Antibodies binding the 55 KD component were also reactive toward purified mammalian tubulin. The remaining antigens had a variable distribution on the eight isolates utilized, and these quantitative differences could be used to distinguish isolates of the Leishmania mexicana complex from those belonging to the Leishmania braziliensis complex.

Flow cytometric analysis of the effects exerted by monoclonal antibodies on binding and uptake of Leishmania mexicana subsp. mexicana promastigotes by murine peritoneal macrophages.

A flow cytometric assay was developed to quantitate the binding kinetics of Leishmania mexicana subsp. mexicana promastigotes to murine peritoneal macrophages and to determine if selected membrane-specific monoclonal antibodies would exert an effect on the binding process. A total of three monoclonal antibodies, all reactive with a similar 42-kilodalton surface membrane component by Western blot analysis, enhanced parasite-macrophage binding at levels greater than 45%. An additional three monoclonal antibodies that identified low-molecular-weight antigens of the promastigote (15 to 20 kilodaltons) had no effect on the binding process. Of these antibodies, however, one did appear to inhibit the internalization of the parasites after it attached to the macrophage membrane.