Indirect haemagglutination (IHA) test in the serodiagnosis of amoebiasis

Among 72 patients clinically suspected of Entamoeba histolytica (E. histolytica) infections, 39 positive cases (54%) were detected serologically by the indirect hemagglutination (IHA) test. Parasitologically, microscopic examination of three consecutive stool specimens from all these patients indicated positivity for E. histolytica cysts and or trophozoites in 10 of the patients with IHA antibody titers greater than or equal to 1:128, which is of clinical significance. Another 2 patients were parasitologically positive but showed low IHA antibody titres (1:32-1:64); follow up indicated response to treatment with metronidazole. The highest serological positivity (100%) were detected in patients with liver abscess, all were clinically proven cases of extra-intestinal amoebiasis. IHA antibody levels of clinical significance were seen in all four patients with chronic dysentery with parasitological evidence of E. histolytica in their stools. In a group of patients with abdominal pain nine positives were detected serologically, four of which were positively diagnosed concurrently by parasitology; the remaining five patient's sera showed high IHA antibody titres with absence of cysts or trophozoites in stools, indicative possibly of persistence of antibodies from past infection. The serologic determination of E. histolytica IHA antibodies in a control group consisting of normal healthy school children and adults of both sexes without any clinical evidence of amoebiasis showed the absence of any positive titres of clinical significance; low titres (1:32-1:64) were detected in 5.2% of 232 sera tested. Parasitological examination of three consecutive stool specimens from all individuals in the control group showed the presence of cysts of E. histolytica in just two among 232 tested (0.9%).     

Intestinal parasitic infections in homosexual men: prevalence,symptoms and factors in transmission.

In a controlled study 67.5% of 200 homosexual men but only 16% of 100 heterosexual men were found to be infected with intestinal parasites. Entamoeba histolytica was isolated from 27% of the homosexual and 1% of the heterosexual men, and Giardia lamblia was isolated from 13% of the homosexual and 3% of the heterosexual men. The presence of symptoms could not be correlated with infection except when the infection was caused by more than one organism, including G. lamblia. Symptoms were much more common in both infected and uninfected homosexuals than in heterosexuals. Among the homosexual men recent foreign travel, living in a homosexual household and promiscuity were not correlated with intestinal parasitic infection, but cleansing of the anus before and sex was associated with a significantly lower prevalence of infection. These findings suggest that the male homosexual community may be an important reservoir of potentially pathogenic protozoa.     

Impaired responsiveness of homosexual men with HIV antibodies to plasma derived hepatitis B vaccine.

Thirty five homosexual men (17 positive for antibody to the human immunodeficiency virus (HIV) and 18 consistently negative) were vaccinated against hepatitis B virus infection. Eight of the 17 seropositive patients failed to develop detectable hepatitis B surface antibody within three months of the third injection compared with only one of the 18 seronegative patients (p less than 0.01). HIV infection is prevalent in the developed world in groups at risk for hepatitis B infection and in certain Third World countries where widespread vaccination programmes exist. This study shows the impact that coincident HIV infection may have on an otherwise efficacious vaccine. The efficacy of this and other vaccines in patients infected with HIV needs to be studied urgently.     

Evaluation of antigen detection and polymerase chain reaction for diagnosis of amoebic liver abscess in patients on anti-amoebic treatment

ABSTRACT: BACKGROUND: Diagnosis of amoebic liver abscess (ALA) in patients on anti-amoebic drugs is difficult. There is scanty data on this issue using Entamoeba histolytica (E. histolytica) lectin antigen and polymerase chain reaction (PCR). We studied lectin antigen, PCR, and IgG antibody in liver abscess patients. Liver aspirate of 200 patients, of which 170 had anti-amoebic drug prior to drainage, was tested for E. histolytica lectin antigen by ELISA, PCR, bacterial culture, and serum IgG antibody by ELISA. Classification of abscesses was based on result of anti-amoebic IgG antibody and bacterial culture, E. histolytica PCR and bacterial culture, and E. histolytica lectin antigen and bacterial culture was evaluated. FINDINGS: Using anti-amoebic IgG antibody and bacterial culture, 136/200 (68.0%) were classified as ALA, 12/200 (6.0%) as pyogenic liver abscess (PLA), 29/200 (14.5%) as mixed infection, and 23/200 (11.5%) remained unclassified. Using amoebic PCR and bacterial culture 151/200 (75.5%) were classified as ALA, 25/200 (12.5%) as PLA, 16/200 (8.0%) as mixed infection, and 8/200 (4.0%) remained unclassified. With E. histolytica lectin antigen and bacterial culture, 22/200 (11.0%) patients were classified as ALA, 39/200 (19.5%) as PLA, 2/200 (1.0%) as mixed infection, and 137/200 (68.5%) remained unclassified. CONCLUSIONS: E. histolytica lectin antigen was not suitable for classification of patients who had prior anti-amoebic treatment. However, PCR may be used as alternative test to anti-amoebic antibody in diagnosis of ALA.     

HIV infection in patients attending clinics for sexually transmitted diseases in England and Wales. Collaborative Study by Consultants in Genitourinary Medicine and the Public Health Laboratory Service.

A national study of the prevalence of HIV antibody designed to monitor sexual spread of HIV infection in England and Wales was made of homosexual and heterosexual patients attending sexually transmitted disease clinics in four districts in 1985, seven in 1986, and 14 in 1987. Patients were invited to participate and were counselled. Among homosexual men in two clinics in south east England, HIV antibody was found in 92 (12.9%) of 711 in 1985, 65 (15.2%) of 428 in 1986, and 81 (14.6%) of 556 in 1987: corresponding findings in the other regions were 16 (5.0%) of 321, 41 (6.3%) of 654, and 21 (3.1%) of 678. The prevalence of HIV antibody was higher in homosexual than bisexual men, in patients aged 25 years or more, or with one or more specified minor complaints. Among heterosexual patients in the south east in 1986, HIV antibody was found in seven (3.0%) of 230 men and three (1.3%) of 233 women and in 1987 in 10 (1.0%) of 962 men and seven (0.7%) of 949 women. In other areas corresponding findings in 1986 were two (0.2%) of 950 men and three (0.4%) of 752 women and in 1987 were three (0.06%) of 5312 men and one (0.02%) of 4778 women. All but one of the heterosexual patients with the antibody were intravenous drug abusers or had had sexual contacts in or were from an area abroad with a high prevalence of AIDS. Failure to identify a heterosexual patient with HIV antibody not in a risk group (other than that of being an attender at the clinic) or who did not have a sexual partner in a risk group suggests that their prevalence in the patient population of the clinics in the south east is less than one in 700 and in the other regions less than one in 3000. Refusals to participate increased during the study but comparisons of patients who agreed and refused in terms of age, the presence of symptoms suggesting AIDS, travel abroad, and number of sexual partners a month showed little evidence of selective bias.     

The Vancouver Lymphadenopathy-AIDS Study: 5. Antecedent behavioural,clinical and laboratory findings in patients with AIDS and HIV-seropositive controls.

In a group of homosexual men in Vancouver studied prospectively since November 1982, 26 cases of acquired immune deficiency syndrome (AIDS) have arisen. To identify behavioural, clinical and laboratory findings that might predict the development of AIDS in people with antibody to human immunodeficiency virus (HIV), we compared data for 25 patients with AIDS with corresponding data for 80 controls serologically positive for HIV selected from the cohort. The clinical and laboratory data for the patients with AIDS preceded the diagnosis of the syndrome by a mean of 17.5 months. The controls had been both seropositive and AIDS-free for a mean of 16.7 months after acquisition of their data. We detected significant differences between the patients with AIDS and the controls in IgG and IgA levels, absolute number of helper T cells and ratio of helper to suppressor T cells but not in lifetime number of male sexual partners, frequency of receptive anal intercourse or receptive fisting, illicit drug use or history of infectious disease. We also detected an increased risk of AIDS among those who had an elevated number of sexual contacts in AIDS-endemic areas in the 5 years before enrollment. A history of increased early sexual contact in AIDS-endemic areas is likely to be associated with early infection and with an increased risk of AIDS among men with HIV infection of unknown duration. Thus, although our analysis had limited statistical power, we conclude that most lifestyle variables appear to act as exposure factors in HIV infection but not as cofactors in the development of AIDS.     

Entamoeba histolytica: rapid identification and differentiation of Indian isolates by riboprinting

Entamoeba histolytica infection still remains one of the major public health problem for developing countries like India. A rapid and accurate detection of this parasite is essential for prevention and control of amoebiasis. In this study, using the method of 'riboprinting' (PCR-RFLP of rRNA genes from amoeba) we have analysed 15 stool samples from symptomatic patients of amoebiasis. All 15 patients of clinical amoebiasis had E. histolytica in their stool and two of the samples also showed mixed infection of E. dispar. Apart from the known restriction enzyme sites within the amoeba SSU-rRNA genes, a new Sau3A site having a discriminatory value is identified in these E. histolytica isolates from India. Hence, it is possible to rapidly identify E. histolytica DNA and differentiating it from E. dispar using minute amounts of clinical stool samples, thus eliminating the laborious parasite culturing process. Thus, riboprinting is advantageous for clearcut identification of E. histolytica in order to decide an effective antiamoebic therapy.     

Human immunodeficiency viruses in patients attending a sexually transmitted disease clinic in London, 1982-7.

OBJECTIVE--To determine the prevalence of infection with the human immunodeficiency virus (HIV) in all patients attending a London sexually transmitted disease clinic over four weeks at the end of 1987 and to see how it varied from that in similar samples studied between 1982 and 1986. DESIGN--Anonymous testing of serum samples from consecutive heterosexual and homosexual patients having routine serological investigations for syphilis. Testing was for anti-HIV-I, anti-HIV-II, and hepatitis B core antibody (anti-HBc) and P24 antigen. Age, nationality, sexual orientation, and past sexually transmitted diseases were recorded for each patient. Gonorrhoea rates by quarters were analysed among homosexual and bisexual men and heterosexual men and women from 1981 to 1987. SETTING--Outpatient department of genitourinary medicine. PATIENTS--A total of 1074 patients attending consecutively for syphilis serology. Thirty five homosexual and bisexual men were excluded (these were regular attenders as part of a prospective study of the natural course of HIV infection). MEASUREMENTS AND MAIN RESULTS--The prevalence of anti-HIV-I in homosexual and bisexual men in 1987 was 25.6% (64/250). Results in the same clinic population between 1982 and 1984 had shown a rise in prevalence, which flattened out in 1985-6 and continued at that level. Among heterosexual attenders in 1987 the prevalence of anti-HIV-I was 1% (women 4/412; men 4/377), which contrasted with a prevalence of 0.5% (women 2/395; men 3/757) in January 1986. One homosexual man was seropositive for anti-HIV-II and seronegative for anti-HIV-I. Among homosexual and bisexual men the rate of gonorrhoea had declined by an average of 2.7% a year since 1981, such that by 1987--and for the first time in the clinic--there was no significant difference in the rates between these men and heterosexual men and women. CONCLUSIONS--The appearance of HIV-I infection among heterosexuals indicates a need for more aggressive education programmes and intervention strategies along the lines adopted for homosexual men. Surveillance for HIV-II infection is needed to provide information for future policy in national screening programmes.     

Molecular differentiation of Entamoeba histolytica and Entamoeba dispar from Tunisian food handlers with amoeba infection initially diagnosed by microscopy

The purpose of the study was to obtain more reliable epidemiological data concerning Entamoeba (E.) histolytica infection in Tunisian food handlers using established molecular tools able to differentiate E. histolytica from E. dispar. From 2002 to 2005, 4,266 fresh stools specimens received in the setting of the National program of food handlers' control were analysed by optical microscopy. Twelve (2.8 per thousand) were positive for the presence of four nuclei cysts identified as E. histolytica/E. dispar. Extraction of DNA from the 12 samples, followed by specific amplifications of E. histolytica and E. dispar SSU rDNA, showed that 11 samples (92%) were positive for E. dispar and negative for E. histolytica. Sequencing analysis of 8 PCR products permitted to verify the results obtained with conventional PCR. The remaining sample was negative by PCR amplifying E. histolytica DNA or E. dispar DNA specifically, although it did not show any inhibition. It probably contains protozoan cysts genetically distinct from these two species but morphological similar. Estimation of relative proportions between E. histolytica and E. dispar in cyst carriers showed that all explored individuals harboured the non pathogenic E. dispar strains. This result highlights the need of use in this population of complementary tests that allow specific diagnosis and obviate unnecessary chemotherapy.     

Entamoeba histolytica: acute granulomatous intestinal lesions in normal and neutrophil-depleted mice

To study the role of neutrophils in the innate resistance to Entamoeba histolytica intestinal infection in mice, animals were treated with anti-neutrophil monoclonal antibodies prior to intracecal parasite inoculation and the resulting lesions were compared with normal mice that had been equally infected. In contrast to our previous finding that neutrophils are critical in eliminating E. histolytica infection in the liver, we show here that neutrophils are not absolutely required to eliminate E. histolytica infection from the intestine. Although the neutrophils are not critical for resolution of the E. histolytica infection, neutrophils do appear to provide some measure of protection as the intestinal amoeba burden was higher at early timepoints after infection in the neutropenic animals. In addition, we found that while both the normal and the neutrophil-depleted mice developed ulcerative lesions in the colon, the neutropenic mice had an increased frequency of granulomas that formed around the amoeba. Thus, our findings appear to be the first evidence showing that granulomatous inflammation can occur after intestinal infection in mice using axenically cultured amoeba.     

Seminal fluid excretion of cytomegalovirus related to immunosuppression in homosexual men.

Seminal fluid samples from 84 Danish homosexual men were successfully cultured to determine the prevalence of cytomegalovirus excretion. Ten (15%) out of 66 men positive for the antibody were found to be excreting the virus. Although the proportion excreting was inversely related to age (p less than 0.01), three men aged over 30 and with many years of homosexual experience excreted the virus. In addition, a 50 year old man with Kaposi''s sarcoma excreted the virus. A further study of the ratio of T cell helpers to suppressors in the men aged over 30 and a series of age matched non-excreting homosexual control or heterosexual men showed that those excreting cytomegalovirus in their seminal fluid had statistically lower ratios (all less than 0.77) than the controls (p less than 0.05). Excretion of cytomegalovirus may be related to re-emergence of latent infection in immunosuppressed homosexual men.     

Entamoeba histolytica: specific antigen recognized by a monoclonal antibody

Specific antigenic determinants on the membrane surface of Entamoeba histolytica that distinguish it from other Entamoeba species were demonstrated. Evidence for these antigenic determinants was obtained with a monoclonal antibody to E. histolytica which showed not only specificity but also sensitivity as demonstrated in enzyme linked immunosorbent assay. Immunofluorescence microscopy showed that the monoclonal antibody recognized an epitope present on the membrane surface of E. histolytica trophozoites. The epitope detected by the monoclonal antibody was present in three components of different molecular weight. These components may have a common precursor or may be the result of enzymatic degradation under the conditions tested.     

Amebiasis in HIV-1-infected Japanese men: clinical features and response to therapy

Invasive amebic diseases caused by Entamoeba histolytica are increasing among men who have sex with men and co-infection of ameba and HIV-1 is an emerging problem in developed East Asian countries. To characterize the clinical and epidemiological features of invasive amebiasis in HIV-1 patients, the medical records of 170 co-infected cases were analyzed retrospectively, and E. histolytica genotype was assayed in 14 cases. In this series of HIV-1-infected patients, clinical presentation of invasive amebiasis was similar to that described in the normal host. High fever, leukocytosis and high CRP were associated with extraluminal amebic diseases. Two cases died from amebic colitis (resulting in intestinal perforation in one and gastrointestinal bleeding in one), and three cases died from causes unrelated to amebiasis. Treatment with metronidazole or tinidazole was successful in the other 165 cases. Luminal treatment was provided to 83 patients following metronidazole or tinidazole treatment. However, amebiasis recurred in 6 of these, a frequency similar to that seen in patients who did not receive luminal treatment. Recurrence was more frequent in HCV-antibody positive individuals and those who acquired syphilis during the follow-up period. Various genotypes of E. histolytica were identified in 14 patients but there was no correlation between genotype and clinical features. The outcome of metronidazole and tinidazole treatment of uncomplicated amebiasis was excellent even in HIV-1-infected individuals. Luminal treatment following metronidazole or tinidazole treatment does not reduce recurrence of amebiasis in high risk populations probably due to amebic re-infection.     

Sexually transmitted diseases and HIV-1 infection among homosexual men in England and Wales.

OBJECTIVE--To examine surveillance data for evidence of changing sexual behaviour and continuing transmission of HIV-1 among men who have sex with men. DESIGN--Analytic study of surveillance data on sexually transmitted diseases. SETTING--England and Wales. MAIN OUTCOME MEASURES--Number of cases of rectal gonorrhoea and newly diagnosed HIV infection in homosexual men. RESULTS--New cases of gonorrhoea among men attending genitourinary medicine clinics increased by 7.7% in 1989 and by 4.2% in 1990. Reports of rectal isolates of Neisseria gonorrhoeae also rose and the male to female ratio for patients with rectal gonorrhoea changed from 0.3:1 during 1988-9 to 2.6:1 in 1990-1. Although the overall number of cases of acute hepatitis B fell during 1988-91, 81 and 82 homosexual men were infected in 1990 and 1991 respectively compared with 50 and 42 in 1988 and 1989. 1526 men had HIV-1 infection diagnosed in 1991, the largest number since 1987. Twenty eight of the 97 (29%) men who seroconverted between January 1989 and December 1991 were aged less than 25. The proportion of men aged 15-19 who were found to be infected with HIV-1 at their first test increased from an average of 2.4% up to 1990 to 4.7% in the first nine months of 1991. The prevalence of HIV infection in men under 25 attending genitourinary medicine clinics in London was 17% compared with 7.8% outside London. CONCLUSION--Unsafe sexual behaviour and HIV transmissions have increased among homosexual men after a period of decline. Recent HIV transmissions may disproportionately affect younger men.     

Tissue invasion by Entamoeba histolytica: evidence of genetic selection and/or DNA reorganization events in organ tropism

Entamoeba histolytica infection may have various clinical manifestations. Nine out of ten E. histolytica infections remain asymptomatic, while the remainder become invasive and cause disease. The most common form of invasive infection is amebic diarrhea and colitis, whereas the most common extra-intestinal disease is amebic liver abscess. The underlying reasons for the different outcomes are unclear, but a recent study has shown that the parasite genotype is a contributor. To investigate this link further we have examined the genotypes of E. histolytica in stool- and liver abscess-derived samples from the same patients. Analysis of all 18 paired samples (16 from Bangladesh, one from the United States of America, and one from Italy) revealed that the intestinal and liver abscess amebae are genetically distinct. The results suggest either that E. histolytica subpopulations in the same infection show varying organ tropism, or that a DNA reorganization event takes place prior to or during metastasis from intestine to liver.     

Development of simple and stable ELISA for detection of copro antigen in intestinal amoebiasis

A simple, sensitive and stable ELISA (enzyme linked immunosorbent assay) was developed using rabbit antibody to fractionated Entamoeba histolytica antigen for the detection of copro antigen in the faeces of individuals with intestinal amoebiasis. In this test none of the healthy individuals, all trophozoite positive, 40% cyst passers and 6% individuals living in endemic area showed the presence of copro antigen. ELISA using polyclonal rabbit antibody could detect 1-5 trophozoites/well and 20-50 ng protein per well of NIH-200 strain of E. histolytica and the sensitivity of the test was comparable with that using monoclonal antibody. Cross reaction was observed only with E. invadens when faeces having other parasites were screened. The reagents of ELISA were stabilized and found to be stable for more than 6 months when stored at 4 degrees C.     

Amebic liver abscess: report of three cases

Amebic abscess is a common manifestation of extraintestinal amebiasis and it is associated with relatively high morbidity and mortality. We present three cases seen in Bari, Southern Italy, one of which was autochthonous and the other two were not. Diagnosis was performed by elevated antibody titre for E. histolytica through immunofluorescence assay and positive antigen determination by ELISA in stools and in abscess aspirate. Fever often accompanied by chills, abdominal pain, weight loss and hepatomegaly were present. Laboratory findings also revealed leukocytosis with neutrophilia. Pleural effusion was observed in two patients. In all our patients multiple abscesses were observed. All the patients were treated with metronidazole and two of them also underwent the aspiration of the amoebic abscess. In all of them there was improvement of the clinical picture, as demonstrated by computerized tomography.     

Continuing transmission of sexually transmitted diseases among patients infected with HIV-1 attending genitourinary medicine clinics in England and Wales.

OBJECTIVE--To determine whether those who are aware of being infected with HIV continue to adopt behaviours that place others at risk of HIV infection. DESIGN--Ongoing survey of current diagnosis of sexually transmitted disease and awareness of HIV infection among patients attending genitourinary medicine clinics. SETTING--Six genitourinary medicine clinics in England and Wales (two in London and four outside) participating in unlinked anonymous HIV serosurveillance during 1990-3. SUBJECTS--All attenders having blood drawn for syphilis serology for the first time during the calendar quarter of attendance. MAIN OUTCOME MEASURES--The proportion of syphilis serology specimens with antibody to HIV-1 detected by unlinked anonymous testing of the residue. The proportion of attenders infected with HIV-1 who remained clinically undetected, and the proportion who had another recently acquired sexually transmitted disease. RESULTS--Of 85441 specimens tested, 2328 (2.7%) were positive for antibodies to HIV-1. About 30% of these specimens were from attenders whose HIV-1 infection remained clinically undetected. HIV-1 infection was found to coexist with another recently acquired sexually transmitted disease in 651 attenders, of whom 522 were homosexual or bisexual men. Of these, 245 (47%) already knew themselves to be infected with HIV-1. This proportion increased between 1990 and 1993. CONCLUSIONS--A considerable proportion of patients infected with HIV-1 are not identified by voluntary confidential HIV testing in genitourinary medicine clinics. Substantial numbers of homosexual or bisexual men attending genitourinary medicine clinics continue to practise unsafe sex despite being aware of their infection with HIV-1.     

Entamoeba histolytica and/or Entamoeba dispar: infection frequency in HIV+/AIDS patients in Mexico city

The objective of this work was to evaluate the frequency of Entamoeba histolytica/Entamoeba dispar intestinal infection in HIV+/AIDS subjects and their HIV- close relatives or sexual partners. Enteric parasites were investigated in stool samples by microscopic examination and E. histolytica and E. dispar were identified by PCR. We found by microscopic analysis in HIV+/AIDS group that the E. histolytica/E. dispar complex was present in 5.9% of the members, while in the HIV- group was 2.9%. With PCR we found that the E. histolytica prevalence was 25.3% in the HIV+/AIDS group and 18.5% in the HIV-group. The difference in the results obtained with the microscopic and PCR is due to the different sensibility of the procedures. Besides, we found patients who were infected with E. histolytica in both groups were asymptomatic cyst passers. Our results suggest that E. histolytica strains prevalent in the studied community appear to be of low pathogenic potential.     

Risk factors for infection with human immmunodeficiency virus among European expatriates in Africa.

The pattern of cases of AIDS in Belgium suggests that Europeans infected with human immunodeficiency virus (HIV) acquired the infection in Africa. The prevalence of infection was assessed in Belgian advisers and European expatriates and risk factors for infection defined in a case-control study of expatriate men. Fifteen (1.1%) of 1401 Belgian advisers working in Africa and 41 (0.9%) of 4564 European expatriates living in Africa, were positive for antibody to HIV in a voluntary screening programme in Belgium. Among subjects with antibody to HIV the ratio of men to women was 3:1. These subjects did not have a history of intravenous drug abuse or blood transfusion and only one was homosexual. In a case-control study of 33 expatriate men who had antibody to HIV and 119 controls the men with antibody reported significantly more female sexual partners, who were more commonly local; and significantly more sexual contact with prostitutes in Africa. They had a significantly higher prevalence of history of sexually transmitted disease and had received significantly more injections by unqualified staff in Africa during the previous five years. No specific sexual practices were associated with having antibody to HIV. After multivariate analysis sexual contact with local women (adjusted odds ratio 14.7; 95% confidence interval 2.81 to 76.9), sexual contact with prostitutes (adjusted odds ratio 10.8 (1.6 to 71.9), and injections by unqualified staff (adjusted odds ratio 13.5 (3.7 to 49.8) remained independent risk factors for infection. European expatriates in Africa were at increased risk from infection with HIV and were a means of introducing HIV into the heterosexual population in Europe. Transmission from women to men by vaginal intercourse seemed to be the most probable route of infection.