Seroprevalence of Entamoeba histolytica in the context of HIV and AIDS: the case of Vhembe district, in South Africa's Limpopo province.

In a recent study in northern South Africa, the seroprevalence of Entamoeba histolytica infection among 257 HIV-positive and 117 HIV-negative individuals was determined, using an ELISA for the detection of antibodies reacting with the parasite's galactose/-acetyl-D-galactosamine(Gal/GalNAc)-inhibitable adherence lectin. Overall, 34.0% of the 374 participants (36.1% of the females and 28.1% of the males) were found seropositive for E. histolytica. Although all age-groups were affected by the amoebic pathogen, the subjects aged 50-59 years had the highest seroprevalence (69.2%). The seroprevalence of E. histolytica was also significantly higher among the HIV-positive subjects than among the HIV-negative (42.8% v. 14.5%; chi(2)=28.65; P<0.0001). Among the HIV-positive subjects, those with fewer than 200 CD4+ cells/microl were relatively more likely to be seropositive for E. histolytica (60.3% v. 43.8%; chi(2)=4.016; P=0.045). This is the first report indicating a positive association between E. histolytica infection and HIV in South Africa. Further studies, for example to determine the occurrence of diarrhoea or liver abscess in the study area, in relation to seropositivity for E. histolytica and/or HIV, are now needed.

A rapid test for infectious and inflammatory enteritis.

BACKGROUND: Inflammatory illnesses are an indication for specific diagnostic studies and possible antimicrobial therapy. The presence of fecal leukocytes has been used as a marker of inflammatory diarrhea; however, microscopic examination of the fecal smear is unreliable if the specimen is transported, refrigerated, frozen, or collected by swab. OBJECTIVE: To evaluate a rapid, sensitive, semiquantitative test for detection of fecal leukocytes using antilactoferrin latex bead agglutination (LFLA), a test that remains sensitive even after specimens are refrigerated, frozen, or stored on swabs. METHODS: LFLA titers were determined in stool specimens from previously healthy volunteers before and after experimental infection with different enteric pathogens and from patients with nosocomial diarrhea caused by Clostridium difficile. RESULTS: Healthy controls and subjects with noninflammatory diarrhea caused by Vibrio cholerae consistently demonstrated LFLA titers less than 1:50. In contrast, subjects with inflammatory diarrhea caused by Shigella species and C difficile had markedly elevated titers. Titers for subjects with experimental shigellosis ranged from 1:50 to 1:3200, with seven (78%) of nine at 1:400 or greater. Titers for patients with C difficile enteritis ranged as high as 1:1200, with six (50%) of 12 at 1:400 or greater. Subjects with experimental enteropathogenic Escherichia coli infection also had elevated titers, ranging from 1:100 to 1:1600, with three (43%) of seven at 1:400 or greater. Titers for subjects with experimental enterotoxigenic E coli infection were moderately elevated, with nine (53%) of 17 ranging from 1:50 to 1:200 (only one [6%] of 17 was > or = 1:400), suggesting a mild inflammatory process. CONCLUSIONS: The fecal LFLA assay distinguishes inflammatory from noninflammatory diarrhea, may provide new information on mildly inflammatory processes, and may be a useful, rapid test in a diagnostic algorithm for acute, infectious diarrheal illnesses.

Human immunodeficiency virus type-1 reverse transcriptase and ribonuclease H as substrates of the viral protease.

A study has been made of the susceptibility of recombinant constructs of reverse transcriptase (RT) and ribonuclease H (RNase H) from human immunodeficiency virus type 1 (HIV-1) to digestion by the HIV-1 protease. At neutral pH, the protease attacks a single peptide bond, Phe440-Tyr441, in one of the protomers of the folded, active RT/RNase H (p66/p66) homodimer to give a stable, active heterodimer (p66/p51) that is resistant to further hydrolysis (Chattopadhyay, D., et al., 1992, J. Biol. Chem. 267, 14227-14232). The COOH-terminal p15 fragment released in the process, however, is rapidly degraded by the protease by cleavage at Tyr483-Leu484 and Tyr532-Leu533. In marked contrast to this p15 segment, both p66/p51 and a folded RNase H construct are stable to breakdown by the protease at neutral pH. It is only at pH values around 4 that these latter proteins appear to unfold and, under these conditions, the heterodimer undergoes extensive proteolysis. RNase H is also hydrolyzed at low pH, but cleavage takes place primarily at Gly436-Ala437 and at Phe440-Tyr441, and only much more slowly at residues 483, 494, and 532. This observation can be reconciled by inspection of crystallographic models of RNase H, which show that residues 483, 494, and 532 are relatively inaccessible in comparison to Gly436 and Phe440. Our results fit a model in which the p66/p66 homodimer exists in a conformation that mirrors that of the heterodimer, but with a p15 segment on one of the protomers that is structurally disordered to the extent that all of its potential HIV protease cleavage sites are accessible for hydrolysis.

Potential role of adherence traits of Escherichia coli in persistent diarrhea in an urban Brazilian slum.

We examined stools from 40 children with persistent diarrhea (duration, 14 days or more), from 50 children with acute diarrhea and from 38 control children to determine infectious etiologies for persistent diarrhea in Goncalves Dias, an urban favela (slum) in Fortaleza, Ceara, Brazil. Children with persistent diarrhea and children with acute diarrhea had similar rates of isolation of routine viral, bacterial and parasitic enteric pathogens. Routine pathogens were identified in at least 20% of cultures done more than 14 days into the diarrheal illness. We examined Escherichia coli isolated from these stools for adherence potential. Enteroaggregative E. coli were isolated significantly more often from children with persistent diarrhea than from control children or children with acute diarrhea (P less than 0.05). E. coli with hemagglutination patterns suggestive of adherence pili were also isolated more often from children with persistent diarrhea than from children with acute diarrhea (38% vs. 18%; P less than 0.05). Enterotoxigenic E. coli were isolated in combination with rotavirus more often from children with persistent diarrhea than from children with acute diarrhea. E. coli which were hydrophobic or exhibited hemagglutination were also seen more often in association with Giardia in children with persistent diarrhea. These findings suggest that the etiology of persistent diarrhea in children is complex and that the aggregative E. coli are associated with prolonged diarrheal illness. Although routine diarrheal pathogens may be present for more than 14 days, combinations of pathogens, including E. coli with adherence potential, may also contribute to prolonged diarrheal disease.

Association of torovirus with acute and persistent diarrhea in children.

OBJECTIVE: To study the etiologic role of toroviruses as a cause of gastroenteritis in humans. METHODS: The design was a case-control study. We compared the rate of torovirus detection in fecal specimens from a selection of children with acute or persistent diarrhea and controls without diarrhea from a study of childhood diarrhea in an urban Brazilian slum. Stool samples were coded and tested in a blinded fashion for the presence of torovirus antigen by enzyme-linked immunosorbent assay, other enteropathogens, toxins and fecal leukocytes. RESULTS: Thirty-three children with acute diarrhea, 41 children with persistent diarrhea and 17 controls were enlisted in the study. Torovirus antigen was detected in 9 (27%) samples from children with acute diarrhea, 11 (27%) samples from children with persistent diarrhea and none of the samples from controls (P < 0.05). In addition the presence of enteroaggregative E. coli was associated with persistent diarrhea and the presence of Cryptosporidium oocysts was common although not significant (P = 0.08); torovirus and Cryptosporidium occurred in different subsets of samples, whereas torovirus and enteroaggregative Escherichia coli were commonly found in combination. CONCLUSIONS: These data indicate that toroviruses, alone or in combination with enteroaggregative E. coli, may play a pathogenic role in acute and possibly persistent diarrhea. Further studies are warranted to determine the etiologic role of toroviruses in gastroenteritis.

Seroepidemiologic study of Cryptosporidium infection in children from rural communities of Anhui, China and Fortaleza, Brazil.

A cluster-sampling, cross-sectional study was conducted for assessing the prevalence of Cryptosporidium infection in children less than 16 years of age from three villages, Dondian, Linshan, and Fuziyin, in rural Anhui in eastern China. Among 320 apparently healthy children less than 10 years of age from Dondian who had stool specimens collected, cryptosporidial oocysts were found in stools of three children from Dondian, and no positive specimens were found in 239 children studied from Linshan. In addition, a total of 610 serum samples from children in these three villages were tested for specific IgG antibody to Cryptosporidium with an enzyme-linked immunosorbent assay (ELISA) and the prevalence rates were 42.3%, 51.7%, and 57.5%, respectively, in Dondian, Linshan, and Fuziyin. Seroprevalence increased progressively with age. No detectable antibody was found in infants between two and six months of age, and seropositivity steadily increased after one year of age. Among 36 sera from adults 15-60 years of age without diarrheal illness in Huanglu villages of rural Chaohu, 50% (18 of 36) were positive. As expected, a good correlation was found in the specific IgG antibody between the paired serum specimens from 30 matched mother-neonates who showed transplacental transfer of IgG. However, little or no IgM antibody was seen in the neonates even though several mothers had a positive anticryptosporidial IgM enzyme-linked immunoassay result. Forty randomly selected serum samples from children less than four years of age in a similarly impoverished semiurban community in Fortaleza, Brazil, where the majority of households also have pit toilets and shared community water supplies and 172 serum samples from patients one month to 29 years of age admitted to the University of Virginia Hospital without diarrhea were also examined. In Fortaleza, almost all children acquired antibody by their second year of life, demonstrating the high prevalence of this infection. In rural Anhui, only about half the children were infected by 5-7 years of age. The overall prevalence rate (16.9%) of seropositivity among children and young adults in Virginia was much lower than in China and Brazil. These results indicate that cryptosporidial infection is ubiquitous, and is highly endemic in these impoverished communities. The difference between China and Brazil may reflect earlier weaning, hygiene practices, poorer water or sanitation, multiple siblings in family and geographic environment in Brazil.

Prevalence of Campylobacter species, Helicobacter pylori and Arcobacter species in stool samples from the Venda region, Limpopo, South Africa: studies using molecular diagnostic methods.

OBJECTIVES: This study determined the prevalence of Campylobacter spp., Helicobacter pylori and Arcobacter spp. in stool samples from Venda in relation to diarrhea, intestinal inflammation and HIV status using specific molecular methods. METHODS: Stool samples were collected from hospital patients (255) and primary school children (67). Genomic DNA was extracted from the stools and molecular methods including PCR, PCR followed by restriction analysis and multiplex PCR were used to test for the different organisms. The lactoferrin content of the stools was determined using commercial kits from TechLab (Blacksburg, VA, USA). RESULTS: The prevalence of the different organisms was 50.6% for H. pylori, 10.2% for C. jejuni, 6.2% for A. butzleri, 6.5% for C. coli, 3.1% for C. concisus, 2.8% for A. cryaerophilus and 1.9% for A. skirrowii. Of all the organisms, only C. jejuni was significantly associated with diarrhea (84.8%) (chi2=21.025, P<0.001) and elevated levels of lactoferrin (78.8%) (chi2=16.919, P<0.005) and was an important pathogen associated with diarrhea among HIV positive individuals (22.8%). CONCLUSIONS: Campylobacter infections are common causes of gastroenteritis in Venda. Non-C. jejuni/coli Campylobacters such as C. concisus as well as A. butzleri and H. pylori may be involved in gastrointestinal diseases in the region but further studies are needed to confirm this hypothesis.

Urea protects Helicobacter (Campylobacter) pylori from the bactericidal effect of acid.

Colonization of the stomach with Helicobacter (Campylobacter) pylori is common in patients with duodenal ulcer disease, which is known for its high acid secretion. Although the bacterium is usually isolated by culture of a gastric biopsy specimen, viable organisms may sometimes be found in the acidic gastric juice. It was postulated that urease, by generating ammonia, protected H. pylori from acid. To test this hypothesis, the pH susceptibility of H. pylori, Proteus mirabilis, and the urease-negative Campylobacter jejuni was examined in the presence and absence of urea. It was found that without urea the three bacteria were all highly susceptible to acid. In striking contrast, the addition of 5 mmol/L of urea completely protected H. pylori but not P. mirabilis or C. jejuni from pH values as low as 1.5. Furthermore, the protective effect of urea on H. pylori was found with urea concentrations as low as 0.05 mmol/L. It is concluded that the high urease activity of H. pylori enables it to survive in gastric acid.

Susceptibility of Campylobacter jejuni to strain-specific bactericidal activity in sera of infected patients.

Campylobacter jejuni is a common cause of inflammatory enteritis, which in normal hosts is usually self-limited and resolves without antibiotic therapy. C. jejuni bacteremia is very rare. We examined sera for bactericidal activity that might be important in limiting the extent of C. jejuni infection in man. We studied the ability of nonimmune sera and homologous and heterologous immune sera from infected patients to kill different fresh case isolates of C. jejuni in vitro. The reduction of the log10 concentration of viable C. jejuni (log10 killing) by fresh sera from nonimmune donors was only 0.2. Log10 killing by homologous acute sera varied from 0 to 3.8 (mean, 1.8). Convalescent sera showed remarkable log10 killing of only homologous C. jejuni, with values of 2.7 to 4.4 (mean, 3.7). The bactericidal effects of acute and convalescent sera were abrogated by heat and EDTA chelation, indicating mediation by complement. The role of classical complement pathway activation was supported by chelating sera with magnesium EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N, N, N', N'-tetraacetic acid] and by reconstitution experiments with heat-inactivated sera, C2-deficient serum, and purified C2. The requirement of specific antibody for the serum bactericidal effect was indicated by the loss of bactericidal activity when immune sera were absorbed with homologous but not heterologous whole C. jejuni isolates. The presence of specific antibodies was further documented by agglutination of only homologous C. jejuni suspensions by heat-inactivated immune sera. Studies with polymorphonuclear leukocytes suggested that ingestion and killing of two C. jejuni strains were modest and variable in the presence of heat-inactivated homologous serum. In summary, the data document a potent serum bactericidal effect that develops rapidly and specifically during C. jejuni enteritis and may be an important factor in host defense against C. jejuni.

Correlation of lactoferrin with neutrophilic inflammation in body fluids.

We have reported that lactoferrin, a 77-kDa iron-binding glycoprotein found in secondary neutrophil granules, provides a useful marker of fecal leukocytes in fecal specimens from patients with inflammatory diarrhea (R. L. Guerrant, V. Araujo, E. Soares, K. Kotloff, A. A. M. Lima, W. H. Cooper, and A. G. Lee, J. Clin. Microbiol. 30:1238-1242, 1992). In order to determine the usefulness of this marker of neutrophilic inflammation in different body fluids, we examined blood, gingival swabs, sputum, and saliva using antilactoferrin antibodies (lactoferrin latex agglutination [LFLA]). LFLA titers in whole blood samples were < or = 1:4 in all eight samples from patients with neutropenia (absolute neutrophil count [ANC] = < 150 polymorphonuclear cells [PMNs] per microliter), < or = 1:8 in samples from 13 individuals with moderate leukocyte counts (ANC = 150 to 8,000), and 1:8 to 1:32 in samples from six patients with neutrophilia (ANC > 8,000). While the overlap precludes a useful role in the identification of neutropenia, these data confirm that lactoferrin titers of > 1:100 indeed indicate inflammation in fluid specimens. On quantitative elution of lactoferrin from gingival swabs, all 7 patients with dental plaque had titers of 1:200 to 1:400; 9 of 12 patients with clinical gingivitis had LFLA titers of 1:200 to 1:1,600, while all 7 individuals with healthy gums and teeth and 4 edentulous patients had LFLA titers of < or = 1:100. Eight purulent sputum samples had titers of > or = 1:400 (7 were 1:1,600) while 11 normal saliva samples showed titers of < or = 1:100. Lactoferrin titers in sputum, gingival swabs, and whole blood correlate with the presence of neutrophils or inflammation in these specimens and may offer a convenient rapid test for inflammatory processes.

Metronidazole susceptibility testing for Helicobacter pylori: comparison of disk, broth, and agar dilution methods and their clinical relevance.

Since the methods for metronidazole susceptibility testing of Helicobacter pylori have not been standardized or validated, we compared three methods that are used to test the metronidazole susceptibilities of 25 isolates of H. pylori. Specifically, we examined the methods of Steer's replicator agar dilution, tube broth microdilution, and modified Kirby-Bauer disk diffusion. The metronidazole disk zone sizes obtained by the disk diffusion method correlated well (r = 0.74) with the MICs obtained by the agar dilution method. Afterward, the disk diffusion method was used to characterize the metronidazole susceptibilities of 44 isolates of H. pylori. Dual therapy (bismuth and metronidazole) proved to be highly effective against metronidazole-susceptible strains (81.6% eradication rate) but fared poorly against resistant strains (16.7% eradication rate; P < 0.01). Using agar dilution testing, we validated the modified Kirby-Bauer disk diffusion method for metronidazole susceptibility testing of H. pylori and conclude that it is practical, accurate, and clinically applicable.

Production of a unique cytotoxin by Campylobacter jejuni.

Campylobacter jejuni is an important diarrheal pathogen worldwide; the mechanisms by which it causes disease remain unclear. Because of its association with inflammatory diarrhea, we postulated that C. jejuni might produce a cytotoxin similar to that produced by Shigella sp., enterohemorrhagic Escherichia coli O157, or Clostridium difficile. Filtrates of 12 polymyxin-treated isolates of C. jejuni were placed on HeLa cells (sensitive to Shiga toxin cytotoxicity) and Chinese hamster ovary (CHO) cells. Of 12 isolates of C. jejuni tested, 5 killed 50% of the cells at greater than or equal to 1:4 dilutions of filtered suspensions of 10(9) bacteria per ml; killing was similar in HeLa and CHO cells (the CHO cells being insensitive to Shiga cytotoxin). One isolate produced a titer of 1:32 to 1:128. The relative potency in HeLa cells was comparable to that of E. coli strains that produce intermediate amounts of Shiga-like toxin. The other seven strains showed no cytotoxic effect, nor did the control diluents, polymyxin B, or supernatants of C. jejuni not treated with polymyxin B. Sonication also released active cytotoxin, but slightly less well than did polymyxin. The cytotoxic effect was dose dependent. Concentration of the C. jejuni in suspension by 10-fold before treatment with polymyxin B resulted in a 10-fold increase in the 50% cytotoxic dose. The cytotoxin effect was not neutralized by Shiga toxin immune serum against either Shiga-like toxin I or II or by anti-Clostridium difficile antiserum. The C jejuni cytotoxin was partially labile to trypsin (0.25%) and to heating to greater than or equal to 60 degrees C. Cytotoxicity was retained in Scientific Products dialysis tubing D1615-1 (Mr cutoff, 12,000 to 14,000). Some isolates of C. jejuni release a substance lethal to HeLa or CHO cells in vitro that is distinct from Shiga-like or Clostridium difficile toxin. This cytotoxin may contribute to the colonic mucosal invasive process that characterizes C. jejuni enteritis.

Concurrence of Clostridium difficile toxin A enzyme-linked immunosorbent assay, fecal lactoferrin assay, and clinical criteria with C. difficile cytotoxin titer in two patient cohorts.

The accurate and sensitive diagnosis of Clostridium difficile-related diarrhea, normally treated with vancomycin, has become increasingly important in light of the emergence of dangerous new strains of vancomycin-resistant enterococci. In order to improve the threshold for C. difficile diagnosis and treatment, a number of commonly used assays for the diagnosis of C. difficile diarrhea were examined. These included an enzyme-linked immunosorbent assay for C. difficile toxin A (ToxA), a CHO cell culture assay for fecal C. difficile (cyto)toxin B, and a lactoferrin latex agglutination assay for fecal lactoferrin (LFLA). We studied 722 fecal specimens submitted by physicians for C. difficile toxin testing at the Salem, Va., Veterans' Affairs Hospital and at the University of Virginia Medical Center in Charlottesville. Charts were reviewed from 123 Veterans' Hospital patients and 114 University of Virginia patients for clinical criteria indicative of C. difficile diarrhea. An increasing titer of CHO cell cytotoxicity was correlated with an increasing likelihood of ToxA positivity (5 to 90%), LFLA positivity (39 to 77%), and clinical agreement (28 to 85%). However, some data indicate that the CHO cell cytotoxicity assay may be nonspecific when positive only at low titers. When the CHO assay result is positive at high titers, it remains the best diagnostic tool. Yet, when it is positive at a low titer, careful interpretation of the results in conjunction with other assays and the clinical setting is warranted, especially in light of new drug-resistant strains of microorganisms.

Early treatment of Campylobacter jejuni enteritis.

The bacteriologic and clinical effects of early antibiotic treatment of Campylobacter jejuni enteritis were studied. Erythromycin rapidly eliminated C. jejuni from stools, whereas trimethoprim-sulfamethoxazole did not. Despite its bacteriologic effectiveness, erythromycin did not reduce the duration or severity of diarrhea, abdominal pain, or other symptoms.

Mucosal injury and disruption of intestinal barrier function in HIV-infected individuals with and without diarrhea and cryptosporidiosis in northeast Brazil.

OBJECTIVES: To determine the relative effects of AIDS-related diarrhea with or without cryptosporidiosis and microsporidiosis on intestinal function and injury. METHODS: We studied 40 HIV-infected patients (20 with and 20 without diarrhea) and 13 healthy volunteers, using the differential urinary excretion of ingested lactulose and mannitol as respective markers of barrier disruption and overall villous surface area. We also examined them for fecal leukocytes, lactoferrin, and alpha 1-antitrypsin. Fasting subjects drank test solution containing lactulose (5 g) and mannitol (1 g). Urine was collected for 5 h and tested for sugars by high-performance liquid chromatography with pulsed amperometric detection. RESULTS: HIV-positive patients with diarrhea had a 2.8-fold higher lactulose:mannitol excretion ratio (L:M) than HIV-positive patients without diarrhea (p = 0.01) and 10.4-fold higher than healthy volunteers (p = 0.004). This was accounted for by a 1.5- to 3.1-fold higher rate of lactulose excretion by HIV patients with diarrhea than by those without diarrhea or by healthy volunteers. Mannitol excretion was 32-55% less in patients with diarrhea than in those without diarrhea or in healthy volunteers. Patients with cryptosporidial diarrhea had a nearly 6-fold higher L:M ratio than those without diarrhea (p < 0.001) and nearly 3-fold higher than those with non-cryptosporidial diarrhea (p = 0.02). One patient with microsporidial infection had a nearly 3-fold higher L:M ratio than controls without diarrhea. Alpha 1-Antitrypsin was positive in 40% of HIV-positive patients with cryptosporidial infections and none of 12 HIV-positive patients with non-cryptosporidial diarrhea. Fecal lactoferrin or leukocytes were increased in all HIV patients with diarrhea. CONCLUSION: HIV infection is associated with intestinal dysfunction and injury, even in patients who do not have diarrhea. However, those with diarrhea, especially with cryptosporidiosis or microsporidiosis, have even greater disruption of intestinal barrier function with potentially important nutritional consequences.

PIP: The effects of AIDS-related diarrhea--with and without cryptosporidiosis and microsporidiosis--on intestinal function and injury were studied in 40 AIDS patients and 13 healthy volunteers from Fortaleza, Brazil. The differential urinary excretion of ingested lactulose and mannitol was used as a marker of barrier disruption and overall villous surface area. HIV-infected patients with diarrhea had a 2.8-fold higher lactulose to mannitol excretion ratio than HIV-positive patients without diarrhea and a 10.4-fold higher ratio than healthy volunteers. Moreover, those with crypotosporidial infection had a lactulose to mannitol ratio almost 6-fold greater than those without diarrhea and nearly 3-fold higher than those with non-cryptosporidial diarrhea. This effect involved both decreased mannitol excretion (decreased intestinal absorptive area) and increased lactulose excretion (mucosal barrier disruption). The single patient with microsporidial infection had a nearly 3-fold higher ratio than healthy volunteers. Alpha1-antitrypsin tests were positive in two of five (40%) HIV-positive patients with cryptosporidial infections compared with none of 12 HIV-infected patients with non-cryptosporidial diarrhea. These findings confirm that HIV infection is associated with profound intestinal dysfunction and injury, even in those without diarrhea. Disruption of the intestinal barrier is even greater, however, in HIV-infected patients with cryptosporidial diarrhea, with potential nutritional consequences.