Simian T-lymphotropic virus type I infection among wild-caught Indonesian pig-tailed macaques (Macaca nemestrina).

Evidence for the presence of simian T-lymphotropic viruses (STLV-I) was identified in live-caught pig-tailed macaques from two locations in southern Sumatra, Indonesia. Of 60 animals tested, 13.3% of the animals showed seroreactivity to HTLV-I/II enzyme-linked immunosorbent assay (ELISA) antigens. Of these, 75% showed indeterminate reactivity and 25% showed positive reactivity to HTLV-I/II Western blot antigens. Polymerase chain reaction (PCR) analysis of 6 of 8 seroreactive monkeys' peripheral blood mononuclear cell (PBMC) DNA showed production of proper size molecular weight product that hybridized specifically to an STLV-I tax gene-specific probe. Phylogenic analyses of tax gene fragment sequences from the PCR products of two samples, 930287 and 930306, indicated that these animals were infected with retroviruses related to those of the Asian STLV-I clade.

Genetic, antigenic and serologic characterization of human immunodeficiency virus type 1 from Indonesia.

To examine the genetic and antigenic characteristics of HIV-1 in Indonesia, samples from 19 HIV-positive volunteers were studied. By a combination of PCR typing and DNA sequence analysis, 12 of the 19 volunteers were determined to be infected with HIV-1 clade B and seven with clade E. Six of the seven Indonesian clade E isolates were from volunteers associated with the Indonesian Military during a peacekeeping mission in Cambodia. Infectivity reduction neutralization assays showed that the Indonesian E viruses were effectively neutralized by Thailand clade E HIV-1 antisera but not by U.S. clade B antisera. The Indonesian clade B virus tested was neutralized by U.S. clade B antisera and not by the Thailand E antisera. Using a previously described serologic typing ELISA based on clade B and E V3 peptides, genetic clade was accurately determined in eight of eight sera tested. This is the first report of the genetic and antigenic analysis of HIV-1 isolates from Indonesia. The data indicate that at least two genetic and antigenic HIV-1 clades (clade E and B) circulate in Indonesia.

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.

Malaria transmission by Anopheles punctulatus in the highlands of Irian Jaya, Indonesia.

A 21-month (1991-1992) assessment of malaria and the relative contribution of Anopheles punctulatus and An. koliensis to its transmission was conducted in Oksibil, a highland community in Irian Jaya, Indonesia. Nearly all (98%) of the 2577 Anopheles from indoor or outdoor human-landing collections were An. punctulatus. Nineteen (0.75%) of the 2518 An. punctulatus tested were positive when tested for malaria circumsporozoite protein (CSP) by ELISA: nine (45% of positives) for Plasmodium vivax-210, six (30%) for Plasmodium falciparum, three (15%) for P. vivax-247 and two (10%) for P. malariae. One mosquito was positive for both P. vivax 210 and P. falciparum. All 59 An. koliensis tested were negative. All mosquitoes positive for CSP were from indoor collections, 89% from the main houses and the remaining 11% from the traditional dwellings (bokams). These findings are discussed with respect to highland malaria epidemiology and transmission in the Oksibil Valley.

PIP: Perennial malaria is a major health problem for most lowland and coastal populations of Irian Jaya, Indonesia. The highlands, however, are generally thought to be free of autochonthous malaria despite the presence of potential vectors up to 2250 m. The authors measured the prevalence of Anopheles punctulatus and A. koliensis during January 1991 to September 1992 in Oksibil, a highland community in Irian Jaya, and their relative contribution to the transmission of malaria. 98% of the 2577 Anopheles from indoor and outdoor human-landing collections were A. punctulatus. 19 (0.75%) of the 2518 A. punctulatus tested were positive when tested for malaria circumsporozoite protein (CSP) by ELISA: nine for Plasmodium vivax-210, six for P. falciparum, three for P. vivax-247, and two for P. malariae. One mosquito was positive for both P. vivax-210 and P. falciparum. All 59 A. koliensis tested were negative. All mosquitoes positive for CSP were from indoor collections, 89% from the main houses and the remaining 11% from the traditional dwellings. Study findings are discussed with respect to highland malaria epidemiology and transmission in Oksibil Valley.

Intestinal parasites of humans in a highland community of Irian Jaya, Indonesia.

An indigenous, highland community in the Oksibil Valley of Irian Jaya, Indonesia, was examined on two occasions to determine the prevalence of intestinal parasites. Overall, 478 subjects from three villages in the study area, about 10% of the available population, were sampled. Using a standard, wet-mount technique, 15 different species of parasites were found. Hookworm was predominant in all age groups, with prevalences of 51%-86%, followed by Ascaris lumbricoides (46%-57%) and Trichuris trichiura (15%-25%). Strongyloides stercoralis was only seen infrequently (< 1%). In general, intestinal protozoa were uncommon. Entamoeba histolytica (14%-20%) and Entamoeba coli (12%-17%) were the most frequent. Sex- and age-specific differences were not dramatic but prevalences and species diversity both increased with age up to 6-15 years, then decreased slightly into adulthood. Rarely were faecal samples free of detectable parasites and some adult subjects had as many as six species. The results are discussed with respect to previous studies in the highlands of New Guinea and the local transmission of soil- and water-borne parasites.

Mapping of a human T-lymphotropic virus type I gag protein epitope that cross-reacts with anti-Plasmodium falciparum antibodies.

Studies in Palawan, Philippines, and Irian Jaya, Indonesia, showed that indeterminate human T-lymphotropic virus type I (HTLV-I) Western blot immunoreactivity is due to cross-reacting anti-Plasmodium falciparum antibodies. To further define this immunoreactivity, mapping studies were conducted using the HTLV-I p19 protein to identify the precise epitope that reacts with these antibodies. Anti-P. falciparum antibody-positive sera from Palawan, Philippines, and Irian Jaya, Indonesia, were studied using overlapping synthetic peptides. Immunoreactivity was localized to residues 108-120 of p19. Further analysis of the sera with 5 biotinylated synthetic peptides showed that the cross-reactive epitope consists of the sequence PDSDPQI (amino acid residues 110-116), which was shown to be homologous to a 7 amino acid sequence on the Exp-1 protein of the P. falciparum blood stage parasite. This is the first study that identifies a specific HTLV-I protein epitope that cross-reacts with malaria antibodies.

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.

Plasmodium falciparum: a region of polymorphism in the 3' end of the gene for the ring-infected erythrocyte surface antigen.

A clone expressing the 3' end of the RESA gene of the Palo Alto isolate of Plasmodium falciparum was isolated by screening a lambda expression library of Mung bean nuclease-digested genomic DNA fragments with antibodies from malaria patients. The DNA sequence of the RESA gene of this isolate has seven single-base changes compared to that seen in the Papua New Guinean isolate FC27. Six of the seven mutations cause amino acid substitutions. The same amino acid sequence as the Palo Alto sequence is found in the Ghanaian isolate NF7. To determine whether these are the only changes in this region of the molecule, we performed PCR analyses on 12 blood samples from malaria patients from Indonesia. The Palo Alto form of the RESA gene was detected in 10 of the samples and the FC27 form of the gene was detected in the other 2 samples. No other forms of the RESA gene were found, suggesting that RESA exists in two alleles for the examined region of the gene. These results indicate that there are two major allelic forms of RESA present in this part of Indonesia.

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)

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.

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.

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.

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.

Long-term culture of hamster duodenal explants and cells.

Explants from adult Syrian hamster duodenum have been maintained in organ culture on gelatin sponges using CMRL 1066 serum supplemented media for 30 days. There was necrosis of the tall villi architecture during the 1st week in culture while columnar and mucous cells survived, migrated, and replicated along portions of the explant basement membrane and in the gelatin sponge matrix. Cells in the sponge multiplied and formed epithelial sheets which showed villus projections and cyst configurations. The cells in these epithelial structures were attached to one another by junctional complexes. Epithelial cells were isolated from the sponge matrix by collagenase digestion and were successfully grown in culture. These duodenal explants and cells show potential for use as in vitro models for experimental studies, involving acute and chronic response of cells to injury or carcinogen-induced transformation.

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.

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.

Immunohistochemistry of the cytoskeleton of human prostatic epithelium. Evidence for disturbed organization in neoplasia.

An indirect immunoperoxidase technique was used to evaluate keratin, actin, tubulin, and calmodulin immunoreactivity in histologic sections of normal, hyperplastic, and neoplastic human prostate. Polyclonal as well as monoclonal keratin antibodies produced equivalent and intense staining of normal epithelium. The immunoreactivity of normal prostate with keratin antibodies was more pronounced than with antibodies to the other components of the cytoskeleton. Variation in staining for components of the cytoskeleton was minimal. The same findings applied to hyperplastic prostate. The immunoreactivity of prostate tumors with antibodies to these cytoskeletal proteins differed markedly from normal prostate. Prostatic carcinomas showed reduced keratin immunoreactivity with a panepithelial antibody, but unaltered or enhanced immunoreactivity with tubulin, actin, and calmodulin antibodies. Many tumors were unreactive with a monoclonal keratin antibody that was strongly reactive with tissues that contained cytokeratin 18 (45-kd) and which intensely stained normal and hyperplastic prostate. In addition, prostate carcinomas often yielded heterogeneous patterns of staining with actin, tubulin, and calmodulin antibodies in contrast to normal and hyperplastic prostate, which showed uniform staining. The results suggest that a disturbance in the organization of the cytoskeleton may accompany neoplastic transformation of human prostate.