Guillermo Vázquez-Rosales

Genotypic testing for HIV-1 drug resistance using dried blood samples

In third-world countries, dried blood samples (DBS) are a convenient alternative to plasma for monitoring viral load during HIV-1 therapy. In this study, we evaluated the feasibility of using DBS to perform HIV-1 drug resistance genotyping in a ViroSeq assay in which the protease and reverse transcriptase regions of the pol gene are analyzed. Fifty-seven antiretroviral genotypes from plasma samples were tested, and drug resistance genotypes were determined. Only 38.6% paired DBS samples were sequenced. Failure to amplify DNA from DBS samples generally correlated with plasma viral loads below log10 5.1. The majority of the mutations identified in plasma pol sequences were also found in their DBS counterpart, with a concordance in genotype interpretation of 96.4%. Several factors were identified that could potentially improve both the sensitivity and the quality of genotype data, such as sample storage conditions and sequence analysis. Therefore, DBS sampling is useful to determine viral load and drug resistance genotypes in HIV patients.

Hepatitis C Virus RNA (HCV-RNA) in Blood Donors and Family Members Seropositive for Anti-HCV Antibodies

BACKGROUND Non-A, non-B virus is responsible for 75-90% of all cases of blood transfusion-related hepatitis. The aim of this work was to determine hepatitis C virus RNA (HCV-RNA) in a group of blood donors and their household contacts. Serotype and genotype of the isolates were also studied. METHODS HCV antibodies were investigated in 44,588 blood donors with a commercial immunoassay. Forty-four seropositive donors and 72 household members were further studied. Quantitative analysis of viral RNA was performed with Amplicor HCV 2.0 test, while genotype was determined by INNO-LiPA test and serotype with Murex HCV test. RESULTS Among the 44,588 donors studied, 333 (0.74%) were positive for anti-HCV. Viral RNA was found in 35 (80%) of the 44 seropositive cases studied. Among the 72 household members, HCV antibodies were detected in six (8.3%) and HCV-RNA in four of these individuals. Serotype 1 and genotype 1 were the most frequent types detected (48 and 64%, respectively). The genotype in the blood donor matched that of his seropositive family member in four of six cases. CONCLUSIONS Our results suggest that intrafamilial transmission of HCV may occur and we stress the need to study household members of seropositive blood donors, as they have a high risk of infection. In this community, genotype 1 is the most prevalent type in blood donors and family members.

Critical Analysis of Deaths Due to Atypical Pneumonia during the Onset of the Influenza A (H1N1) Virus Epidemic

BACKGROUND The ongoing influenza A (H1N1) pandemic stroked Mexico and posed a huge challenge to the medical care and public health systems. This report analyzes the clinical course and process of care of patients who died due to atypical pneumonia and fulfilled the clinical criteria of suspected case of novel influenza A (H1N1) virus infection. METHODS We conducted a retrospective analysis of a series of 38 patients who died between April 7 and April 28, 2009 at Instituto Mexicano del Seguro Social (IMSS) hospitals due to severe pneumonia and respiratory distress. These cases coincided with the beginning of the outbreak, so patients did not undergo laboratory testing to diagnose influenza. According to IMSS and CDC criteria, post-hoc analysis allowed considering the presumptive diagnosis of S-OIV infection. A multidisciplinary group analyzed the information from the clinical charts, laboratory tests, radiographic studies and death certificates, using descriptive statistics. RESULTS Most cases were middle-aged (mean 33 years, range: 4-62 years) and previously healthy; 18.4% had an underlying chronic disease, 23.7% were obese and 7.9% were current smokers. None had received the seasonal influenza vaccine; they had cough (92%), fever (86.8%), and malaise (73.7%). The median time from disease onset to hospital admission was 6 days (range 0-8 days). All were admitted to the intensive care unit with pneumonia and/or respiratory distress. Average time from disease onset to death was 8 days (range 4-18 days). CONCLUSIONS An increased number of severe cases of atypical pneumonia in previously healthy adults highlight the importance of the availability of a timely surveillance system able to identify sudden increases in the number of cases or presentation of apparently known diseases.

Blood culture and respiratory syncytial virus identification in acute lower respiratory tract infection

Even though the incidence of pneumonia in developed and developing countries is similar, the mortality is five times higher in developing countries. This study aimed to determine the prevalence of bacteremia in children with acute lower respiratory tract infection (LRTI) and relative contribution of respiratory syncytial virus (RSV). One hundred and one children under five years of age who attended a primary care level clinic with diagnosis of acute LRTI, were enrolled. Diagnosis and management of pneumonia were done according to the WHO guidelines. Two blood cultures were drawn at the time of admission. A nasopharyngeal sample was taken for detection of RSV by indirect immunofluorescence.Blood cultures were positive for pathogenic bacteria (Streptococcus pneumoniae, Haemophilus influenzae andStaphylococcus aureus) in three patients. The detection for RSV was positive in 24 patients (23.7%). The clinical and radiographic presentations were not significantly different between patients with and without RSV (p > 0.05).RSV is a common cause of LRTI in children younger than five years old. Blood cultures are not commonly positive in outpatients with acute LRTI. The practice of obtaining blood cultures in primary and secondary care clinics is not useful to guide the treatment of patients with communityacquired pneumonia.

Occult hepatitis B virus infection among Mexican human immunodeficiency virus-1-infected patients

AIM To determine the frequency of occult hepatitis B infection (OHBI) in a group of human immunodeficiency virus (HIV)-1+/ hepatitis B surface antigen negative (HBsAg)- patients from Mexico. METHODS We investigated the presence of OHBI in 49 HIV-1+/HBsAg- patients. Hepatitis B virus (HBV) DNA was analyzed using nested PCR to amplify the Core (C) region and by real-time PCR to amplify a region of the S and X genes. The possible associations between the variables and OHBI were investigated using Pearsons χ(2) and/or Fishers exact test. RESULTS We found that the frequency of OHBI was 49% among the group of 49 HIV-1+/HBsAg- patients studied. The presence of OHBI was significantly associated with the HIV-1 RNA viral load [odds ratio (OR) = 8.75; P = 0.001; 95%CI: 2.26-33.79] and with HIV-antiretroviral treatment with drugs that interfere with HBV replication (lamivudine, tenofovir or emtricitabine) (OR = 0.25; P = 0.05; 95%CI: 0.08-1.05). CONCLUSION The OHBI frequency is high among 49 Mexican HIV-1+/HBsAg- patients and it was more frequent in patients with detectable HIV RNA, and less frequent in patients who are undergoing HIV-ARV treatment with drugs active against HBV.

Use of Dried Plasma Spots for HIV-1 Viral Load Determination and Drug Resistance Genotyping in Mexican Patients.

Monitoring antiretroviral therapy using measurements of viral load (VL) and the genotyping of resistance mutations is not routinely performed in low- to middle-income countries because of the high costs of the commercial assays that are used. The analysis of dried plasma spot (DPS) samples on filter paper may represent an alternative for resource-limited settings. Therefore, we evaluated the usefulness of analyzing DPS samples to determine VL and identify drug resistance mutations (DRM) in a group of HIV-1 patients. The VL was measured from 22 paired plasma and DPS samples. In these samples, the average VL was 4.7 log10 copies/mL in liquid plasma and 4.1 log10 copies/mL in DPS, with a correlation coefficient of R = 0.83. A 1.1 kb fragment of HIV pol could be amplified in 14/22 (63.6%) of the DPS samples and the same value was amplified in plasma samples. A collection of ten paired DPS and liquid plasma samples was evaluated for the presence of DRM; an excellent correlation was found in the identification of DRM between the paired samples. All HIV-1 pol sequences that were obtained corresponded to HIV subtype B. The analysis of DPS samples offers an attractive alternative for monitoring ARV therapy in resource-limited settings.

Serum Dried Samples to Detect Dengue Antibodies: A Field Study

Background Dried blood and serum samples are useful resources for detecting antiviral antibodies. The conditions for elution of the sample need to be optimized for each disease. Dengue is a widespread disease in Mexico which requires continuous surveillance. In this study, we standardized and validated a protocol for the specific detection of dengue antibodies from dried serum spots (DSSs). Methods Paired serum and DSS samples from 66 suspected cases of dengue were collected in a clinic in Veracruz, Mexico. Samples were sent to our laboratory, where the conditions for optimal elution of DSSs were established. The presence of anti-dengue antibodies was determined in the paired samples. Results DSS elution conditions were standardized as follows: 1 h at 4°C in 200 µl of DNase-, RNase-, and protease-free PBS (1x). The optimal volume of DSS eluate to be used in the IgG assay was 40 µl. Sensitivity of 94%, specificity of 93.3%, and kappa concordance of 0.87 were obtained when comparing the antidengue reactivity between DSSs and serum samples. Conclusion DSS samples are useful for detecting anti-dengue IgG antibodies in the field.

Antibody responses to influenza viruses in paediatric patients and their contacts at the onset of the 2009 pandemic in Mexico

INTRODUCTION On April 2009, the Mexican Ministry of Health received notification of cases of severe pneumonia mostly affecting young healthy people; this was the beginning of the first influenza pandemic of the 21st century. The nature of the immune response to the influenza A(H1N1)2009 pandemic strain in Mexico at the beginning of the pandemic outbreak has not been completely defined. We describe the serological response to the 2009 pandemic influenza virus in paediatric patients with influenza-like illness, their household contacts (HHCs), and exposed health-care workers (HCWs) at the beginning of the pandemic outbreak in Mexico City. METHODOLOGY thirty pre-epidemic and 129 epidemic samples were collected and serum antibodies were measured against A(H1N1)2009 pandemic virus and two non-pandemic swine influenza viruses by an haemagglutination inhibition assay . RESULTS 91% (29/32) of the convalescence samples from confirmed patients had an antibody titre ≥ 10 (GMT 25), 63% (41/65) of the HHCs (GMT 12), 41% of HCWs (GMT 6) and 13% (4/30) of pre-epidemic samples (GMT 6) for the pandemic influenza virus. Of the 32 confirmed cases, 60% had an antibody titre ≥ 40 for the pandemic strain, 53% for the A/swine/Iowa(H1N1) virus (GMT 62) and 43% for the A/swine/Texas(H3N2) virus (GMT 66). CONCLUSION The antibody response to 2009 pandemic influenza virus was widespread in convalescence samples from patients with confirmed pandemic influenza infection but the GMT was below the protective titre. There was no evidence that antibodies to the swine influenza viruses had cross-protective effect against the 2009 pandemic influenza virus.