The sensitivity of the histological assessment of H. pylori infection was considerably lower in Mozambique than in Portugal, which may be explained by a lower density of colonization among Mozambican patients.
In Portuguese samples, DNA was extracted from frozen antral biopsies and used for PCR, followed by reverse hybridization onto specific probes, whereas in Mozambican samples, DNA was extracted from formalin-fixed, paraffin-embedded biopsies and used for PCR, followed by electrophoresis in agarose gels stained with ethidium bromide. Visual inspection of PCR products on agarose gels provides only limited reliability, as compared to hybridization to allele-specific probes. However, it has been previously shown that H. pylori genotypes obtained from paraffin sections matched those corresponding cultured strains . Nevertheless, even if PCR results for Mozambican samples were underestimated, this would mean that the sensitivity of histological assessment for this population would even be lower, and more discrepant than those obtained for the Portuguese samples.
Co-infections with other Helicobacter species have been described in African populations , but the PCR analysis that we have used is specific for H. pylori (we have used primers directed to the vacA gene which is only present in this species). Primer sequences used in this study were analyzed for specificity by using the Blast program at the National Institute of Health Data libraries .
The histological identification of H. pylori in tissue sections may be affected by biopsy and observer-related factors. Sensitivity is higher when at least samples from the antrum and the body are analyzed, but the proportion of infected subjects did not vary meaningfully when we considered the histological assessment of infection status using biopsies from the three locations, which is in accordance with the fact that the antrum biopsies tend to yield a higher prevalence of infection than the other locations , strengthening the validity of our conclusions. Moreover, no meaningful increase in the prevalence of infection when assessed by PCR was to be expected, as it was well above 90% when only one biopsy is used for this purpose.
Staining methods such as modified Giemsa are highly sensitive, but histological examination relies on the experience of the histopathologist to recognize the typical morphology of the organism, and small numbers of organisms may be missed [14, 15], while PCR methods have been described as more sensitive and not dependent on the level of experience of the pathologist [16, 17].
In our study, samples from both settings were collected and processed following similar methods and were evaluated by the same experienced pathologists. It is unlikely that differences in technical aspects or inter-observer and laboratory variations explain the differences in the results obtained in Portuguese and Mozambican samples. The same applies to the grading of the density of colonization, which was conducted following the same criteria in both samples.
Although coccoid forms (suggesting possible degenerative forms of H. pylori) could have been found in the negative specimens, and would contribute to the low perceived sensitivity of histological assessment of infection, the pathologists where aware of this possibility. Even considering the difficulties in classifying correctly these uncharacteristic forms in the absence of findings of structures with the characteristic H. pylori shape, these were seldom observed and the differences observed in sensitivity or in the density of H. pylori colonization cannot be ascribed to such phenomenon.
A lower sensitivity of histology for the assessment of H. pylori infection has been recognized after partially effective eradication therapy [15, 16], as low levels of recurrent infection can be easily missed by biopsy, leading to overestimation of therapeutic efficacy . The proportion of Mozambican subjects reporting a previous eradication treatment was surprisingly high, which could explain the lower density of colonization among these patients, but no information was obtained regarding the success of eradication or time since eradication. No significant differences were found in infection status or density of H. pylori colonization according to self-reported eradication treatment, probably reflecting the large potential for erroneous reporting of such a specific therapy, and sensitivity was similar when only subjects not reporting an eradication treatment were considered. Similarly, the previous use of PPI/H2 receptor blockers was also self-reported and no information was collected regarding current treatment or time since last treatment course, but sensitivity was similar when only subjects not reporting the use of PPI/H2 receptor blockers were considered.
Gastric atrophy and intestinal metaplasia lead to a pH increase in the stomach, which can create an unfavorable environment for H. pylori survival, and could contribute to lower density of colonization [19, 20]. The density of H. pylori may be increased in macroscopic erosions . However, only 14.7% of the Mozambican patients had chronic atrophic gastritis (5.9%) or intestinal metaplasia (8.8%), in comparison with 43.9% in the Portuguese subjects (chronic atrophic gastritis: 6.5%; intestinal metaplasia: 37.4%), and erosions/ulcerations were observed in one Portuguese and four Mozambicans. Therefore, the unequal distribution of these lesions could not explain the differences in the prevalence of infection detected by histology.
Although the samples were evaluated in different time periods and present different characteristics regarding the participants' ethnicity and gender, our analysis only included subjects testing positive for H. pylori infection by PCR and the above differences between the Portuguese and the Mozambican sample are not likely to account for our conclusions.
In studies conducted in dyspeptic patients from different African populations the overall prevalence of H. pylori infection, assessed by histology, was approximately 72%, varying from 25% in Uganda to 97% in Ghana , although a high prevalence of infection is expected given its strong association with low socioeconomic status. The heterogeneity of estimates across African settings, as well as unexpectedly low figures, is likely to be explained both by observer-related factors and differences in the density of colonization by H. pylori.
Mozambique and other African countries have a low frequency of gastric cancer and gastric precancerous lesions, despite the high prevalence of infection [23, 24], reflecting what has been called the African enigma. In addition to providing an example of how spectrum effects can affect the sensitivity of histological assessment of H. pylori infection, our results add a piece of information to our current understanding of the "enigma", suggesting that despite the absence of differences in the frequency of infection with the more virulent strains in Portugal and Mozambique , the higher prevalence of mild infections in Africa may be associated with a lower incidence of cancer.